Dicer interacting proteins and uses therefor

Abstract

Dicer (e.g., DCR-1) interactors are disclosed as are methods to positively or negatively modulate Dicer activity. Uses of Dicer interactors as drug targets are featured. Also featured are uses of Dicer interactors and modulators of same to modulate various Dicer functions in vitro, in cell cultures, and in vivo.

Description

STATEMENT AS TO SPONSORED RESEARCH

Funding for the work described herein was, at least in part, supported by grants from the National Institutes of Health (R01 GM058800; R21 ES012021-02).

BACKGROUND OF THE INVENTION

RNA-mediated gene silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Dicer is an RNase III-family enzyme characterized by its ribonuclease activity and dsRNA-binding properties. The enzyme generates nucleotide products from dsRNA of approximately 21-23. Processing of microRNAs, for example the let-7 precursor, by Dicer has also been observed. Dicer includes a dsRNA-binding domain located at the C-terminus of the enzyme.

Given the important role of Dicer in the generation of RNA-mediated gene silencing agents, the identification of proteins that interact with and/or regulate Dicer will help improve our understanding of RNA silencing and other Dicer-related processes. Moreover, Dicer-interacting and/or Dicer-regulating proteins are useful for the identification of a variety of modulatory agents for use in regulating RNA-mediated gene silencing.

SUMMARY OF THE INVENTION

Important in the RNAi pathway of most organisms is the ribonuclease III enzyme Dicer. In particular, Dicer has been shown to play a key role in the processing of RNA precursors triggering the activation of both endogenous and exogenous pathogen responses (i.e., RNAi) and of small RNAs active as developmental regulators called microRNAs. The enzyme and its ancillary components have been poorly characterized to date. The instant invention is based, at least in part, on the identification of numerous interacting components of the enzyme Dicer, in particular, proteins previously unknown to interact with this critical protein. Moreover, the invention provides an assay for the identification of other components of this and related enzymes. Importantly, the invention demonstrates that the identified interactors of Dicer are capable of modulating its function in, for example RNAi. Still further, the identified C. elegans proteins have related homologs in vertebrates, for example, the mouse and humans, and therefore have application in the development of human diagnostic and therapeutic agents.

Accordingly, the invention has several advantages, which include, but are not limited to, the following:

providing interacting proteins of Dicer and there use in modulating Dicer function;

methods for identifying further interactors of Dicer and their structural and functional characteristics;

method for regulating Dicer activity though the use of Dicer interactors;

methods for improving the in vitro or in vivo processing of Dicer proteins or for use as targets for pharmaceutical intervention in order to modulate the properties of Dicer in vivo for improved RNAi; and

methods for stabilizing RNAi agents/compositions comprising Dicer by the addition of stabilizing interactor proteins or the same for use in purifying Dicer and other Dicer components.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic of major components of the RNAi pathway, the role of Dicer, and Dicer interacting proteins, which have roles in microRNA maturation, RNAi initiation, and as enhancers of RNAi.

FIGS. 2A-C depicts biochemical fractionation and immunoprecipitations of DCR-1 from C. elegans embryos, and adults using the coupled HA monoclonal method. dcr−/− 8×HA rescue fractions and IP were realized using a complex array rescued strain of dcr-1 (ok247) with a transgene driving a 8×HA fusion.

FIGS. 3A-C depict the molecular architecture of the eri genes.

FIGS. 4A-B depict results regarding RNAi sensitivity, enhancement, and developmental defects of the eri genes. A. N2(WT) or eri mutants were fed on unc-73 (rnai) feeding strain for a generation and F1 broods of animals were scored for their exhibition of the associated phenotype: uncoordination, twisted morphology and limited movement (see lower panel). In the upper panel, results are shown for n=15, depicted error bars are shown for a confidence interval p=0.05. B. Brood sizes of the eri mutants at 15° C. (blue) and 25° C. (purple) are shown (upper panel). WT brood size is restored at 25° C. for all the eri mutations when crossing in with N2(wt) males (see lower panel). For all the broods, n=10, depicted error bars are shown for a confidence interval p=0.05.

FIGS. 5A-E depict small RNA defects in depletions for the DCR-1 interactors. In addition to dcr-1 and drh-3, the k02e2.6 locus also required the eri genes for accumulation, and the siRNAs were also absent from the eri genes RNA preparations from animals grown at 15° C. (5A-D). The lack of small RNAs in k02e2.6 in the eri mutants correlated with an upregulation of its mRNA, as quantified by real time PCR (5E). See the Materials and Methods for further detail.

FIG. 6 depicts a schematic showing that multiple silencing pathways are initiated by DCR-1, the eri gene products, and DRH-3. Distinct subsets of DCR-1 interactions are responsible for initiation of multiple small RNA silencing pathways. Shown here are the ‘classical’ RNAi pathway involving the RDE-1, RDE-4 and the DRH-1/2 proteins, the eri ‘endo’ RNAi pathway relying on the eri gene products, and the broader drh-3 dependent endo siRNA pathway.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery of previously unrecognized activity of several proteins as Dicer-interacting proteins (i.e., Dicer-interactors) and/or Dicer modulatory proteins (e.g., positive and/or negative regulatory proteins), see Tables 1 and 3. The invention features the defining of Dicer (DCR-1) interactions with an array of proteins involved in a variety of functions in C. elegans or other species, and the usage and alteration of these interactors and/or interactions to modulate or modify the different functions or activities of Dicer. The invention also features methods for efficient Dicer purification and identification of further interactors and/or interactions. This invention features methods for more efficient in vitro Dicer processing and materials for use in said methods, e.g., by the addition of a Dicer interacting protein that enhances Dicer activity. Knowledge of these Dicer interactors and/or interactions allows for the development of drug screening and/or targeting strategies or rationales, e.g., screening and/or targeting of Dicer and/or Dicer interactors in C. elegans, as well as in other species having homologous genes, to activate or antagonize Dicer's different functions and activities or to modulate its specificity toward its different proteins.

Accordingly, the present invention features Dicer interactors and methods of use of said interactors. In certain aspects, the invention provides methods for identifying a Dicer modulator, RNAi modulator and/or gene silencing modulator, including contacting a composition comprising, or a cell or organism that expresses Dicer or a bioactive fragment thereof and a Dicer interactor or a bioactive fragment thereof with a test compound and determining the ability of the test compound to modulate interaction (e.g., binding) of Dicer or the Dicer bioactive fragment to the Dicer interactor or the Dicer interactor bioactive fragment, such that the Dicer modulator, RNAi modulator and/or gene silencing modulator is identified.

In other aspects, the present invention provides methods for identifying a Dicer modulator, RNAi modulator and/or gene silencing modulator, including contacting a composition comprising, or a cell or organism that expresses Dicer or a bioactive fragment thereof and a Dicer interactor or a bioactive fragment thereof with a test compound and determining the ability of the test compound to modulate an activity of Dicer or the Dicer bioactive fragment, such that an the modulator is identified.

In certain embodiments, the activity of Dicer or the bioactive fragment thereof may be selected from the group consisting of: (1) processing of miRNA precursors; (2) processing of siRNA precursors; (3) mediating mRNA cleavage; (4) mediating assembly of RISC (e.g., via siRNAs); (5) directing translation repression (e.g., via miRNAs); (6) a ribonuclease activity (e.g., cleavage of dsRNA); and (7) initiation of RNAi.

In other aspects, the invention provides methods for identifying a Dicer modulator, RNAi modulator and/or gene silencing modulator, including contacting a composition comprising, or a cell or organism that expresses Dicer or a bioactive fragment thereof and a Dicer interactor or a bioactive fragment thereof with a test compound and determining the ability of the test compound to modulate an activity of the protein or the protein bioactive fragment, such that the Dicer modulator, RNAi modulator and/or gene silencing modulator is identified. In various embodiments of the preceding aspects the modulator identified may be a positive modulator or a negative modulator.

In various embodiments of the preceding aspects of the invention, the Dicer interactor may be selected from the proteins described in subsections IIIA-IIIMM, infra. In other embodiments, the Dicer is either Dicer1 or Dicer2. A Dicer bioactive fragment is any fragment of Dicer having sufficient size and structure to carry out at least one activity (e.g., biological activity) of the corresponding full-length Dicer protein. Similarly, a Dicer interactor bioactive fragment is any fragment of the Dicer interactor having sufficient size and structure to carry out at least one activity (e.g., biological activity) of the corresponding full-length Dicer interactor protein. Exemplary bioactive fragments include, but are not limited to, enzymatic domains, protein binding and/or interaction domains, and nucleic acid binding domains. Preferred bioactive fragments include regions or domains as described in detail in subsections IIIA-IIIMM, infra. The Dicer, Dicer bioactive fragment, Dicer interactor or the interactor bioactive fragment may be detectably labeled, radioactively labeled, or fluorescently labeled. Furthermore, in other embodiments, the interaction or activity may be compared to an appropriate control. In addition, at least one of the Dicer, Dicer bioactive fragment, Dicer interactor or protein bioactive fragment may be immobilized.

In various embodiments, the activity of the Dicer interactor or protein bioactive fragment is an activity set forth in subsections IIIA-IIIMM, infra. Bioactive fragments and/or fragment activities (and accordingly, Dicer interactor activities) are further described in detail in the references cited throughout subsections IIIA-IIIMM, infra. The entire content of these references is incorporated herein by reference.

In the aspects of the present invention, where the method involves a cell or organism, the cell or organism may overexpress the Dicer interactor or the bioactive fragment thereof, Dicer or the bioactive fragment thereof, or both the Dicer interactor (or protein bioactive fragment) and Dicer (or Dicer bioactive fragment).

In another aspect, the invention provides a modulator as identified by any of the preceding claims. The invention also provides for a pharmaceutical composition including the modulator.

In one aspect, the invention provides a method for identifying a Dicer:Dicer interactor modulator, including contacting a cell or organism expressing, or a composition comprising, Dicer or a bioactive fragment thereof and a Dicer interactor or a bioactive fragment thereof with a test compound and determining the ability of the test compound to affect interaction (e.g., binding) of the Dicer or the bioactive fragment thereof to the Dicer interactor or the bioactive fragment thereof, such that the modulator is identified.

In another aspect, the invention provides a method for identifying a Dicer:Dicer interactor modulator, including contacting a cell or organism expressing, or a composition comprising, Dicer or a bioactive fragment thereof and a Dicer interactor or a bioactive fragment thereof with a test compound and determining the ability of the test compound to affect activity of the Dicer or the bioactive fragment thereof, such that the modulator is identified.

In another aspect, the invention provides a method for identifying a Dicer:Dicer interactor modulator, including contacting a cell or organism expressing, or a composition comprising, Dicer or a bioactive fragment thereof and a Dicer interactor or a bioactive fragment thereof with a test compound and determining the ability of the test compound to affect activity of the Dicer interactor protein or the bioactive fragment thereof, such that the modulator is identified.

In yet another aspect, the invention provides a method for identifying a Dicer:Dicer interactor modulator, including contacting a cell or organism expressing, or a composition comprising, Dicer or a bioactive fragment thereof and a Dicer interactor or a bioactive fragment thereof with a test compound and determining the ability of the test compound to affect the phosphorylation state of the Dicer interactor or the bioactive fragment thereof, such that the modulator is identified.

In certain embodiments of the preceding aspects, the ability of the test compound to affect, for example, an interaction or activity includes the ability of the test compound to either enhance or inhibit such an interaction or activity. The Dicer may be Dicer1 or Dicer2.

In certain embodiments, the present invention provides methods of modulating Dicer, RNAi or gene silencing in a subject including administering to the subject a Dicer modulator, RNAi modulator and/or gene silencing modulator identified according to any of the above methods.

In another aspect, the invention provides an antibody that specifically binds to Dicer, a Dicer-interacting protein, or fragment thereof, wherein the antibody is capable of identifying, altering, or interfering with a Dicer:Dicer interactor interaction. In a related embodiment, the invention provides an antibody capable of binding an epitope within amino acid residue positions 1145 to 1347 of Dicer (DCR-1), or corresponding residues of a homolog thereof. The invention also provides polypeptides comprising Dicer epitopes suitable for raising such antibodies, e.g., for use as immunogens or screening polypeptides. In one embodiment, the epitope is within amino acid residue positions 1145 to 1347 of Dicer (DCR-1), or corresponding residues of a homolog thereof. The invention further provides for a pharmaceutical composition including the antibody.

In another aspect, the present invention provides a pharmaceutical composition including a Dicer-interacting protein. In yet another aspect, the present invention provides a pharmaceutical composition including a Dicer interacting protein domain of a Dicer protein or a Dicer interacting domain of a Dicer interacting protein, wherein either or both domains are capable of interfering with a Dicer:Dicer interacting protein interaction.

In yet another aspect, the invention provides a modulator of Dicer activity suitable for enhancing an RNAi therapy, and pharmaceutical compositions comprising such a modulator.

In other aspects, the present invention provides methods for treating an disease or disorder including administering any of the pharmaceutical compositions described above.

Various aspects of the invention are described in further detail in the following subsections:

I. Definitions

So that the invention may be more readily understood, certain terms are first defined.

As used herein, a “Dicer interacting protein” or “Dicer interactor” includes polypeptides having the amino acid sequences set forth in subsections IV, infra, as well as homologs, paralogs, and/or orthologs of such polypeptides, i.e. polypeptides having sufficient sequence identity to function in the same manner as the described polypeptides. Such polypeptides can interact directly, for example, physically bind with Dicer or a bioactive fragment thereof, and/or interact indirectly, for example, as measured by affecting a change in Dicer activity either in vitro or in vivo.

The term “Dicer” includes polypeptides having the amino acid sequences set forth in subsections III, infra, as well as homologs, paralogs, and/or orthologs of such polypeptides, i.e. polypeptides having sufficient sequence identity to function in the same manner as the described polypeptides.

The term “Dicer activity” includes any of the following properties or functions that can be ascribed to a Dicer protein such as: protein:protein binding activity (e.g., direct association with a Dicer interacting protein), miRNA maturation activity, RNAi initiation activity, RNAi enhancer activity, helicase activity, RISC activity, target recognition activity, and/or target gene cleavage activity.

The term “modulator of Dicer activity” includes agents capable of affecting a change in Dicer activity. Modulator agents include small molecules, nucleic acids (e.g., RNAi agents, siRNAs, shRNAs), peptides, and polypeptides. Dicer interacting proteins can be modulators of Dicer either directly or indirectly, for example, by physically interacting with Dicer or by affecting a change in Dicer activity. Thus, a modulator of a Dicer interacting protein which results in a change in Dicer activity can be considered a modulator of Dicer activity, albeit indirectly.

The term “derived from” includes partial, synthetic, recombinant, or genetically engineered nucleic acids or polypeptides that encode or represent a gene product substantially similar to a gene product from a particular source, for example, a nucleic acid source, a cell, or organismal source, from, for example, a nematode, fruit fly, rat, mouse, primate, or human.

The terms “homolog,” “paralog,” “ortholog,” includes their art recognized meaning. Typically, a homolog of a given gene product is one of functional similarity as well as sequence similarity. If the homolog is derived from a different organism, the homolog may be referred to as the ortholog. If several homologs exist in a given organism, the homolog may be referred to as a paralog. Typically, the sequence similarity/identity between homologs is at least about 40%, 50%, 60%, 70%, 80%, 90%, or more (or a percentage falling within any interval or range of the foregoing). Methods for determining such similarity/identity are described, infra. Motifs conserved between homologs can have a sequence similarity/identity of at least about 70%, 80%, 90%, or more. It is understood that when comparing gene product sequence between diverse organisms, for example, nematodes and humans, sequence similarity between given homologs across the entire protein sequence may be low. However, if functional complementarity exists, and in addition, if conserved motifs exist, e.g., protein; protein interaction motifs, e.g., motifs involved in Dicer activity or Dicer:Dicer interacting protein interactions, then the gene products being compared can be considered homologs and thus selected as compositions for use in the methods of the invention, as described herein.

The phrase “introducing into the cell or organism” includes any art recognized method for introducing genetic information into an cell extract, cell, or organism. Typical modes of such transfer of genetic information include the contacting, transfection, microinjection and/or feeding of nucleic acid agents or expression vectors to an extract, cell, or organism. Other methods include cell fusion, pronuclear injection, genetic crosses/mutagenesis, and the like.

The term “bioactive fragment” includes any portion (e.g., a segment of contiguous amino acids) of a Dicer interactor or Dicer protein sufficient to exhibit or exert at least one Dicer protein- or Dicer-associated activity including, for example, the ability to bind to Dicer or Dicer interactor protein, respectively.

The phrase “encodes a gene product” includes the generation of a RNA molecule from a DNA molecule (i.e., a complementary RNA molecule generated from the DNA molecule by the process of transcription) and/or the generation of a polypeptide or protein molecule from an RNA (i.e., by the processes of transcription and translation).

The term “kit” is any manufacture (e.g. a package or container) comprising at least one reagent or component, e.g. a construct, molecule, and/or compound, the manufacture being promoted, distributed, or sold as a unit for performing the methods of the invention.

The term “target gene” includes a gene intended for downregulation via RNA interference (“RNAi”). The term “target gene product” or “target protein” refers to a gene product, e.g., a nucleic acid or protein, intended for downregulation via RNAi. The term “target RNA” refers to an RNA molecule intended for degradation by RNAi, e.g., by nucleic acid cleavage. An exemplary “target RNA” is a coding RNA molecule (e.g., an RNA molecule encoding a gene product, e.g., an mRNA and protein so encoded therefrom).

The term “expression” of a gene or nucleic acid encompasses not only cellular gene expression, but also the transcription and translation of nucleic acid(s) in cloning systems and in any other context.

The term “RNA interference” or “RNAi”, as used herein, refers generally to a sequence-specific or selective process by which a target molecule (e.g., a target gene, protein, or RNA) is downregulated. In specific embodiments, the process of “RNA interference” or “RNAi” features degradation of RNA molecules, e.g., RNA molecules within a cell, the degradation being triggered by an RNAi agent. Degradation is catalyzed by an enzymatic, RNA-induced silencing complex (RISC). RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by the hand of man, for example, to silence the expression of target genes.

The term “RNAi agent”, includes an RNA (or analog thereof), comprising a sequence having sufficient complementarity to a target RNA (i.e., the RNA being degraded) to direct RNAi. A sequence having a “sufficiently complementary to a target RNA sequence to direct RNAi” means that the RNAi agent has a sequence sufficient to trigger the destruction of the target RNA by the RNAI machinery (e.g., the RISC complex) or process. The term RNA agent or RNAi agent includes small interfering RNA (siRNA) (also referred to in the art as short interfering RNAs) as well as small hairpin RNA or shRNA.

The term “small interfering RNA,” “siRNA,” or “short interfering RNAs” includes a double-stranded RNA agent, which is capable of directing or mediating RNA interference. Naturally occurring siRNAs are generated from longer dsRNA molecules (e.g., >25 nucleotides in length) by a cell's RNAi machinery (e.g., the RISC complex).

The term “small hairpin RNA” or “shRNA” (also referred to in the art as “short hairpin RNA”), includes an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.

The term “subject”, as used herein, includes living organisms at risk for or having a cellular, neurological, e.g. neurodegenerative disease, or disorder. Examples of subjects include humans, monkeys, cows, sheep, goats, dogs, cats, mice, rats, and transgenic species thereof. Administration of the compositions of the present invention to a subject to be treated can be carried out using known procedures, at dosages and for periods of time effective to modulate RNAi in the subject as further described herein.

The term “treatment”, as used herein, is defined as the application or administration of a therapeutic agent to a subject, or application or administration of a therapeutic agent to an isolated tissue or cell line from a subject, who has a disease or disorder, a symptom of a disease or disorder, or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward a disease or disorder. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes, antisense oligonucleotides, RNAi agents, chemotherapeutic agents, and radiation.

The term “effective amount”, as used here in, is defined as that amount necessary or sufficient to treat or prevent a disorder, e.g. a neurological or a neurodegenerative disease or disorder. The effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular agent being administered. One of ordinary skill in the art would be able to study the aforementioned factors and make the determination regarding the effective amount of the agent without undue experimentation.

The term “pharmaceutical composition” as used herein, refers to an agent formulated with one or more compatible solid or liquid filler diluents or encapsulating substances, which are suitable for administration to a human or lower animal.

The phrase “a gene involved” in a disorder includes a gene, the normal or aberrant expression or function of which effects or causes a disease or disorder or at least one symptom of said disease or disorder.

The phrase “examining the function of a gene in a cell or organism” refers to examining or studying the expression, activity, function, or phenotype arising therefrom.

A “suitable control” or “appropriate control” refers to any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein. For example, a Dicer activity, a RISC level of activity or amount, target gene level or target gene degradation level, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing a nucleic acid or test compound of the invention into a cell extract, cell, or organism.

The term “cell” refers to any eukaryotic cell which exhibits RNAi activity and includes, e.g., animal cells (e.g., mammalian cells, e.g., human or murine cells), nematode cells, plant cells, and yeast. The term includes cell lines, e.g., mammalian cell lines such as HeLa cells as well as embryonic cells, e.g., embryonic stem cells and collections of cells in the form of, e.g., a tissue.

The term “cell extract” refers to a lysate or acellular preparation of a cell as defined above and can be a crude extract or partially purified as well as comprise additional agents such as recombinant polypeptides, nucleic acids, and/or buffers or stabilizers.

The term “organism” refers to multicellular organisms such as, e.g., C. elegans, Drosophila, mouse, and human.

The term “vector” refers to a nucleic acid molecule (either DNA or RNA) capable of conferring the expression of a gene product when introduced into a host cell or host cell extract. In one embodiment, the vector allows for temporal or conditional expression of one or more nucleic acids of the invention, e.g., a single strand, RNA agent, siRNA, or shRNA. The vector may be episomal or chromosomally (e.g., transgenically) integrated into a host cell genome.

The terms used herein are not intended to be limiting of the invention.

II. Overview

Dicer, a ribonuclease III/DExH-box helicase (DCR-1 in C. elegans) plays a central role in a variety of small RNA-directed gene silencing mechanisms for a large range of organisms (see FIGS. 1 & 6).

Its best characterized activity is the processing of double-stranded RNAs into smaller RNA hybrid species of 21 to 25 nucleotides (nt) in length with staggered 2 nucleotides overhangs at the 3′ ends of the duplex, and a 5′ phosphate group; both of which determinants have been shown to be required for efficient silencing.

This Dicer activity was first shown to act in the initiation phase of two modes of post-transcriptional gene silencing. In RNA interference (RNAi) and in the microRNA-dependent silencing, Dicer recognizes a double-stranded RNA (dsRNA) trigger to direct a potent, and sequence-specific gene silencing response. This process requires the assembly of the small RNA product in a downstream complex called RISC, for which Argonaute proteins are a central component. This complex is responsible for a cognate mRNA search, and for the subsequent silencing of the complementary transcript.

Dicer is responsible for the integration of a variety of RNA signals with distinct biological outcomes. Dicer also initiates other RNA-dependent silencing pathways such as chromosome folding and the like. Therefore a key problem to address is how some specific classes of dsRNAs are recognized and recruited to be processed by Dicer, and how RNA triggers of distinct origins potentiate different silencing responses.

The present invention provides methods and compositions for conducting in vitro and in vivo assays for identifying Dicer interacting proteins, in particular, Dicer interacting proteins that can affect Dicer activity, and modulators thereof.

III. Dicer Interacting Proteins or Dicer Interactors

According to the invention, several proteins have been identified as interacting with and/or regulating Dicer, e.g., Dicer activity. These Dicer interactors are described in detail below under subsections IIIA through IIIMM. Using methods described in the present disclosure, use of any one of these proteins, or cognate orthologs or paralogs, in appropriate screening assays would provide for the identification of Dicer modulators and/or RNAi-modulators, and/or gene silencing modulators.

IIIA. RDE-4

LOCUS      NP_499265    385 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION RNAi Defective RDE-4, RNA interference
           promoting factor with double-stranded
           RNA binding motif (43.4 kD) (rde-4)
           [Caenorhabditis elegans].
ACCESSION  NP_499265
VERSION    NP_499265.1 GI: 17555186
DBSOURCE   REFSEQ: accession NM 066864.2
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida;
                    Rhabditoidea; Rhabditidae;
                    Peloderinae; Caenorhabditis.
REFERENCE  1 (residues 1 to 385)
           AUTHORS  Walhout,A. J., Reboul,J., Shtanko,O.,
                    Bertin,N., Vaglio,P., Ge,H., Lee,H.,
                    Doucette-Stamm,L., Gunsalus,K. C.,
                    Schetter,A. J., Morton,D. G.,
                    Kemphues,K. J., Reinke,V., Kim,S. K.,
                    Piano,F. and Vidal, M.
           TITLE    Integrating interactome, phenome, and
                    transcriptome mapping data for the C.
                    elegans germline
           JOURNAL  Curr. Biol. 12 (22), 1952-1958 (2002)
           MEDLINE  22335532
           PUBMED   12445390
REFERENCE  2 (residues 1 to 385)
           AUTHORS  Tabara,H., Yigit,E., Siomi,H. and
                    Mello,C. C.
           TITLE    The dsRNA binding protein RDE-4 inter-
                    acts with RDE-1, DCR-1, and a DExH-box
                    helicase to direct RNAi in C. elegans
           JOURNAL  Cell 109 (7), 861-871 (2002)
           MEDLINE  22105477
           PUBMED   12110183
REFERENCE  3 (residues 1 to 385)
           AUTHORS  Tijsterman,M., Ketting,R. F.,
                    Okihara,K. L., Sijen,T. and
                    Plasterk,R. H.
           TITLE    RNA helicase MUT-14-dependent gene
                    silencing triggered in C. elegans by
                    short antisense RNAs
           JOURNAL  Science 295 (5555), 694-697 (2002)
           MEDLINE  21669321
           PUBMED   11809977
REFERENCE  4 (residues 1 to 385)
           AUTHORS  Parrish,S. and Fire,A.
           TITLE    Distinct roles for RDE-1 and RDE-4
                    during RNA interference in
                    Caenorhabditis elegans
           JOURNAL  RNA 7 (10), 1397-1402 (2001)
           MEDLINE  21535543
           PUBMED   11680844
REFERENCE  5 (residues 1 to 385)
           AUTHORS  Grishok,A., Tabara,H. and Mello,C. C.
           TITLE    Genetic requirements for inheritance of
                    RNAi in C. elegans
           JOURNAL  Science 287 (5462), 2494-2497 (2000)
           MEDLINE  20207007
           PUBMED   10741970
REFERENCE  6 (residues 1 to 385)
           AUTHORS  Tabara,H., Sarkissian,M., Kelly,W. G.,
                    Fleenor,J., Grishok,A., Timmons,L.,
                    Fire,A. and Mello,C. C.
           TITLE    The rde-1 gene, RNA interference, and
                    transposon silencing in C. elegans
           JOURNAL  Cell 99 (2), 123-132 (1999)
           MEDLINE  20004389
           PUBMED   10535731
COMMENT    REVIEWED REFSEQ: This record has been
           curated by NCBI staff. This record is
           derived from an annotated genomic se-
           quence (NC_003281). The reference se-
           quence was derived from AY071926.1.
           Summary: This gene rde-4, also known as
           T20G5.11, 3L306 or YK5801, maps at
           (III; +1.89). Its phenotype is rnai
           defective. It encodes a RNA interfer-
           ence promoting factor with double-
           stranded RNA binding motif. From Pfam
           homology, the product would have
           double-stranded RNA binding activity
           and would localize in intracellular.
           According to the Worm Transcriptome
           Project, it is well expressed at all
           stages of development [Kohara cDNAs].
           Its sequence is defined by 10 cDNA
           clones.
           Phenotype
           WM49 rde-4 (ne301) III [Craig Mello,
           Tabara/Mello, mut-6] RNAi deficient.
           RNA interference results:
           [T. Hyman 2000] No obvious phenotype
           (by injecting genomic PCR product
           TH: T20G5.11).
           [J. Ahringer 2003] No obvious phenotype
           (by feeding genomic PCR product
           JA: T20G5.11).
           [F. Piano 2002] No P0 sterility de-
           tected. No postembryonic phenotypes
           observed among progeny. No obvious
           phenotype.
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 47%, L1 or L2 larvae 29%,
           L3 to adult 25%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           Pattern germline enriched [Piano,
           2002].
           This complete mRNA is 1747 bp long. Its
           sequence exactly matches the genome.
           The premessenger has 4 exons. It covers
           1.89 kb on the WS97 genome. It is tran-
           spliced to SL1. It has a very long 3′
           UTR. The protein (385 aa, 43.4 kDa, pI
           5.2) contains 2 Double-stranded RNA
           binding (DsRBD) domain motifs. It also
           contains a coil coil stretch [(Psort2].
           Taxblast (threshold 10{circumflex over ( )}-3) tracks an-
           cestors down to caenorhabditis elegans.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 385
                    /organism = “Caenorhabditis
                    elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “III”
                    /map = “III; +1.89 cM (interpolated
                    genetic position)”
                    /map = “III; covering 1888 bp, from
                    base 10218484 to 10216597 on genome
                    release WS97”
                    /clone = “Primers to amplify the
                    CDS (3468 bp, Stop included):
                    ATGGATTTAACCAAACTAACGTTTGAA (T =
                    55.9), TCAATCCGTGAAATCATAGGTGT
                    (T = 56.6). Complete CDS clones:
                    AY071926, yk832c2. Recommended
                    clone (from the Kohara collection):
                    yk832c2. Other clone(s): yk627d6,
                    yk333g4, yk596c11, yk565d11,
                    yk469h7, yk1429h2, yk1706h7,
                    yk1726d4.
                    for edited clone sequences see
                    www.wormgenes.org”
                    /clone_lib = “Kohara embryonic
                    lambda gt11 library: yk627d6,
                    yk333g4, yk596c11, yk565d11,
                    yk469h7; Kohara Sugano L1 larvae
                    cap-selected library: yk832c2;
                    Kohara Sugano L2 larvae cap-
                    selected library: yk1706h7,
                    yk1726d4; Kohara Sugano L4 larvae
                    cap-selected library: yk1429h2; gb:
                    AY071926”
           Protein  1 . . . 385
                    /product = “RNAi DEfective RDE-4,
                    RNA interference promoting factor
                    with double-stranded RNA binding
                    motif (43.4 kD) (rde-4)”
           Region   41 . . . 104
                    /region_name = “[Pfam/InterPro
                    description] double-stranded RNA
                    binding (DsRBD) domain”
                    /db_xref = “CDD: pfam00035”
           Region   116 . . . 149
                    /region_name = “[PSORT] coil coil
                    domain:
                    PGTTKEEALSNIDQISDKAEELKRSTSDAVQDND”
           Region   170 . . . 232
                    /region_name = “[Pfam/InterPro
                    description] double-stranded RNA
                    binding (DsRBD) domain”
                    /db_xref = “CDD: pfam00035”
           CDS      1 . . . 385
                    /gene = “rde-4”
                    /locus_tag = “3L306”
                    /coded_by =
                    “NM_066864.2: 1 . . . 1158”
                    /db_xref = “AceView/WormGenes:
                    rde-4”
                    /db_xref = “GeneID: 176438”
                    /db_xref = “LocusID: 176438”
                    /db_xref = “WormBase: T20G5.11”
ORIGIN
1          mdltkltfes vfggsdvpmk psrsednktp
           rnrtdlemfl kktplmvlee aakavyqktp
61         twgtvelpeg femtlilnei tvkgqatskk
           aarqkaavey lrkvvekgkh eiffipgttk
121        eealsnidqi sdkaeelkrs tsdavqdndn
           ddsiptsaef ppgisptenw vgklqeksqk
181        sklqapiyed sknerterfl victmcnqkt
           rgirskkkda knlaawlmwk aledgiesle
241        sydmvdvien leeaehllei qdqaskikdk
           hsalidilsd kkrfsdysmd fnvlsvstmg
301        ihqvlleisf rrlvspdpdd lemgaehtqt
           eeimkataek eklrkknmpd sqplvfaghg
361        ssaeeakqca cksaiihfnt ydftd
//

IIIB. ALG-1

ALG-1 is a homolog of rde-1 that is involved in RNA interference and affects developmental timing along with alg-2 and dcr-1 by regulating expression of the lin-4 and let-7 small temporal RNAs. The ALG-1 protein contains regions of similarity to Pfam domains PF02170 (PAZ domain, Residues 377-514), PF02171 (Piwi domain, Residues 660-961). The protein has been implicated in embryonic development, inferred from mutant phenotype Grishok, A. et al., Cell 2001 106:23-34. Homologs include H. sapiens eukaryotic translation initiation factor 2C 4, C. elegans gene T07D3.7a, M. musculus Argonaute 1 protein (Fragment), R. norvegicus eukaryotic translation initiation factor 2C 2 (eIF2C 2) (eIF-2C 2)s(Golgi ER protein 95 kDa) (GERp95) and D. melanogaster AGO1.

LOCUS      NP_510322    1002 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION argonaute (plant)-Like Gene (110.9 kD)
           (alg-1) [Caenorhabditis elegans].
ACCESSION  NP_510322
VERSION    NP_510322.2 GI: 25148113
DBSOURCE   REFSEQ: accession NM 077921.2
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 1002)
           AUTHORS  Kamath,R. S., Fraser,A. G., Dong,Y.,
                    Poulin,G., Durbin,R., Gotta,M.,
                    Kanapin,A., Le Bot,N., Moreno,S.,
                    Sohrmann,M., Welchman,D. P.,
                    Zipperlen,P. and Ahringer,J.
           TITLE    Systematic functional analysis of the
                    Caenorhabditis elegans genome using
                    RNAi
           JOURNAL  Nature 421 (6920), 231-237 (2003)
           MEDLINE  22417569
           PUBMED   12529635
REFERENCE  2 (residues 1 to 1002)
           AUTHORS  Morel,J. B., Godon,C., Mourrain,P.,
                    Beclin,C., Boutet,S., Feuerbach,F.,
                    Proux,F. and Vaucheret,H.
           TITLE    Fertile hypomorphic ARGONAUTE (agol)
                    mutants impaired in post-transcription-
                    al gene silencing and virus resistance
           JOURNAL  Plant Cell 14 (3), 629-639 (2002)
           MEDLINE  21907852
           PUBMED   11910010
REFERENCE  3 (residues 1 to 1002)
           AUTHORS  Grishok,A., Pasquinelli,A. E.,
                    Conte,D., Li,N., Parrish,S., Ha, I.,
                    Baillie,D. L., Fire,A., Ruvkun,G. and
                    Mello,C. C.
           TITLE    Genes and mechanisms related to RNA
                    interference regulate expression of the
                    small temporal RNAs that control C.
                    elegans developmental timing
           JOURNAL  Cell 106 (1), 23-34 (2001)
           MEDLINE  21354308
           PUBMED   11461699
COMMENT    VALIDATED REFSEQ: This record has un-
           dergone preliminary review of the se-
           quence, but has not yet been subject to
           final review. This record is derived
           from an annotated genomic sequence
           (NC_003284). The reference sequence was
           derived from WormBase CDS: F48F7.1. On
           Nov. 21, 2002 this sequence version re-
           placed gi: 17549901.
           Summary: This gene alg-1, also known as
           F48F7.1, XO573 or YK3586, maps at (X;
           +14.45). Its phenotype is clear, trans-
           lucent appearance, uncoordinated loco-
           motion, protruding vulva. It encodes an
           argonaute (plant)-Like Gene.
           According to the Worm Transcriptome
           Project, it is well expressed at all
           stages of development [Kohara cDNAs].
           Its sequence is fully supported by 17
           cDNA clones.
           RNA interference results
           [J. Ahringer 2003] Clear, uncoordina-
           ted, protruding vulva (by feeding geno-
           mic PCR product JA: F48F7.1).
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 26%, L1 or L2 larvae 27%,
           L3 to adult 48%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           The CDS has 6 exons. It covers 3.42 kb
           on the WS97 genome. The protein (1002
           aa, 110.9 kDa, pI 9.3) contains one
           Argonaute and Dicer protein, PAZ motif,
           one stem cell self-renewal protein Piwi
           motif. It also contains a 2nd peroximal
           domain Psort2]. Taxblast (threshold
           10{circumflex over ( )}-3) tracks ancestors down to
           eukaryota.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 1002
                    /organism = “Caenorhabditis
                    elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “X”
                    /map = “X; +14.45 cM (interpolated
                    genetic position)”
                    /map = “X; covering 4989 bp, from
                    base 13941769 to 13946759 on genome
                    release WS97”
                    /clone_lib = “Kohara embryonic
                    lambda gt11 library: yk60e5,
                    yk403g7, yk142f4, yk375c8,
                    yk481b11, yk245e6; Kohara Sugano L1
                    larvae cap-selected library:
                    yk759f4, yk889c6, yk1013a7,
                    yk1108b4, yk1164h8; Kohara Sugano
                    L2 larvae cap-selected library:
                    yk1609d8; Kohara Sugano
                    L4 larvae cap-selected
                    library: yk1427e7; Kohara mixed
                    stage library, from him-8 strain,
                    containing 15-30% males: yk100d5,
                    yk545h7, yk369b2; Kohara mixed
                    stage library, from him-8 strain,
                    containing 15-30% males: yk286h2”
           Protein  1 . . . 1002
                    /product = “argonaute (plant)-Like
                    Gene (110.9 kD) (alg-1)”
           Region   377 . . . 514
                    /region_name = “[Pfam/InterPro
                    description] argonaute and Dicer
                    protein, PAZ”
                    /db_xref = “CDD: pfam02170”
           Region   460 . . . 468
                    /region name = “[PSORT] 2nd perox-
                    imal domain: RIQLKYPHL”
           Region   660 . . . 961
                    /region name = “[Pfam/InterPro
                    description] stem cell self-renewal
                    protein Piwi”
                    /db_xref = “CDD: pfam02171”
           CDS      1 . . . 1002
                    /gene = “alg-1”
                    /locus_tag = “XO573”
                    /coded_by =
                    “NM_077921.2: 1 . . . 3009”
                    /db_xref = “AceView/WormGenes:
                    alg-1”
                    /db_xref = “GeneID: 181504”
                    /db_xref = “LocusID: 181504”
                    /db_xref = “WormBase: F48F7.1”
ORIGIN
1          msggpqylpg vmnstiqqqp qsatssflps
           qpisststss qvvptsgatq qppfpsaqaa
61         astalqndle eifnspptqp qtfsdvpqrq
           agslapgvpi gntsvsiqep antlqqglpg
121        gapgqlpggn qsgiqfqcpr rpnhgvegrs
           illranhfav ripggtiqhy qvdvtpdkcp
181        rrvnreiisc lisafskyft nirpvydgkr
           nmytreplpi grermdfdvt lpgdsaverq
241        fsvslkwvgq vslstledam egrvrqvpfe
           avqamdvilr hlpslkytpv grsffsppvp
301        nasgvmagsc ppqasgavag gahsagqyha
           esklgggrev wfgfhqsvrp sqwkmmlnid
361        vsatafyrsm pviefiaevl elpvqalaer
           ralsdaqrvk ftkeirglki eithcgqmrr
421        kyrvcnvtrr paqtqtfplq letgqtiect
           vakyfydkyr iqlkyphlpc lqvgqeqkht
481        ylppevcniv pqqrcikklt dvqtstmika
           tarsaperer eisnlvrkae fsadpfahef
541        gitinpamte vkgrvlsapk llyggrtrat
           alpnqgvwdm rgkqfhtgid vrvwaiacfa
601        qqqhvkendl rmftnqlqri sndaqmpivg
           npcfckyavg veqvepmfky lkqnysgiql
661        vvvvlpgktp vyaevkrvgd tvlgiatqcv
           qaknairttp qtlsnlclkm nvklggvnsi
721        llpnvrprif nepviffgcd ithppagdsr
           kpsiaavvgs mdahpsryaa tvrvqqhrqe
781        iisdltymvr ellvqfyrnt rfkparivvy
           rdgvsegqff nvlqyelrai reacmmlerg
841        yqpgitfiav qkrhhtrlfa vdkkdqvgka
           ynippgttvd vgithptefd fylcshagiq
901        gtsrpshyhv lwddnnltad elqqltyqmc
           htyvrctrsv sipapayyah ivafraryhl
961        vdrehdsgeg sqpsgtsedt tlsnmaravq
           vhpdannvmy fa
//

IIIC. ALG-2

LOCUS      NP_871992    910 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION argonaute (plant)-Like Gene (101.6 kD)
           (alg-2) [Caenorhabditis elegans].
ACCESSION  NP_871992
VERSION    NP_871992.1 GI: 32564644
DBSOURCE   REFSEQ: accession NM 182192.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida;
                    Rhabditoidea; Rhabditidae;
                    Peloderinae; Caenorhabditis.
REFERENCE  1 (residues 1 to 910)
           AUTHORS  Morel,J. B., Godon,C., Mourrain,P.,
                    Beclin,C., Boutet,S., Feuerbach, F.,
                    Proux, F. and Vaucheret,H.
           TITLE    Fertile hypomorphic ARGONAUTE (agol)
                    mutants impaired in post-transcription-
                    al gene silencing and virus resistance
           JOURNAL  Plant Cell 14 (3), 629-639 (2002)
           MEDLINE  21907852
           PUBMED   11910010
REFERENCE  2 (residues 1 to 910)
           AUTHORS  Grishok,A., Pasquinelli,A. E.,
                    Conte,D., Li,N., Parrish,S., Ha,I.,
                    Baillie,D. L., Fire,A., Ruvkun,G. and
                    Mello,C. C.
           TITLE    Genes and mechanisms related to RNA in-
                    terference regulate expression of the
                    small temporal RNAs that control C.
                    elegans developmental timing
           JOURNAL  Cell 106 (1), 23-34 (2001)
           MEDLINE  21354308
           PUBMED   11461699
REFERENCE  3 (residues 1 to 910)
           AUTHORS  Missotten,M., Nichols,A., Rieger,K. and
                    Sadoul,R.
           TITLE    Alix, a novel mouse protein undergoing
                    calcium-dependent interaction with the
                    apoptosis-linked-gene 2 (ALG-2) protein
           JOURNAL  Cell Death Differ. 6 (2), 124-129
                    (1999)
           MEDLINE  99218669
           PUBMED   10200558
COMMENT    VALIDATED REFSEQ: This record has un-
           dergone preliminary review of the se-
           quence, but has not yet been subject to
           final review. This record is derived
           from an annotated genomic sequence
           (NC_003280). The reference sequence was
           derived from WormBase CDS: T07D3.7a.
           Summary: This gene alg-2, also known as
           T07D3.7, 2B167 or YK2467, maps at (II;
           −13.80). It encodes an argonaute
           (plant)-Like Gene. According to the
           Worm Transcriptome Project, it is ex-
           pressed at high level mainly in embryos
           and some in L1 larvae [Kohara cDNAs].
           Its sequence is fully supported by 29
           cDNA clones and produces, by alterna-
           tive splicing, 2 different transcripts
           a, b altogether encoding 2 different
           protein isoforms.
           Phenotype
           Knock-out allele, deletion obtained by
           the Gene Knockout Consortium ok215,
           ok304 (strain RB574) [R Barstead,
           Oklahoma MRF, USA]. Selected strain
           available from the CGC.
           RB574 alg-2 (ok304) II [Robert
           Barstead, OMRF Knockout Group/Barstead,
           UV/TMP] [Craig Mello description]
           Homozygous viable, contains an out of
           frame deletion removing nucleotides
           encoding amino acids 34-374. [R
           Barstead] Homozygous. Outer Left Se-
           quence: tctgagtttggctcgatgtg. Outer
           Right Sequence: atgttccttggataccagcg.
           Inner Left Sequence:
           agcccagaactgggaaactt. Inner Right Se-
           quence: aagtcgaattccgttggatg. Inner
           Primer PCR Product: 3297. Deletion
           length: 1378 bp. Deletion breakpoints:
           Flanking positions are T07D3 coordi-
           nates 2397/3776. Sequence read at break
           from ok304 internal left primer:
           TCTAATTTTCCAATTTTCAG/
           GATATTGTTCCAGGACAGCG. Breakpoint data
           provided by the Vancouver Gene Knockout
           Lab (URL: www.zoology.ubc.ca/kogeno-
           mics/kowebpge.html).www.mutantfac-
           tory.ouhsc.edu/.
           RNA interference results:
           [J. Ahringer 2003] No obvious phenotype
           (by feeding genomic PCR product
           JA: T07D3.7).
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 75%, L1 or L2 larvae 16%,
           L3 to adult 9%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           The CDS has 7 exons. It covers 5.26 kb
           on the WS97 genome. The protein (910
           aa, 101.6 kDa, pI 9.2) contains one
           Argonaute and Dicer protein, PAZ motif,
           one stem cell self-renewal protein Piwi
           motif. It also contains a 2nd peroximal
           domain [Psort2]. It is predicted to
           localise in the cytoplasm [Psort2].
           Taxblast (threshold 10{circumflex over ( )}-3) tracks an-
           cestors down to eukaryota.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 910
                    /organism = “Caenorhabditis elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “II”
                    /map = “II; −13.80 cM (interpolated
                    genetic position)”
                    /map = “II; covering 6702 bp, from
                    base 873182 to 879885 on genome
                    release WS97”
           Protein  1 . . . 910
                    /product = “argonaute (plant)-Like
                    Gene (101.6 kD) (alg-2)”
           Region   282 . . . 419
                    /region name = “[Pfam/InterPro
                    description] argonaute and Dicer
                    protein, PAZ”
                    /db_xref = “CDD: pfam02170”
           Region   365 . . . 373
                    /region_name = “[PSORT] 2nd
                    peroximal domain: RIQLKYPHL”
           Region   566 . . . 867
                    /region_name = “[Pfam/InterPro
                    description] stem cell self-renewal
                    protein Piwi”
                    /db_xref = “CDD: pfam02171”
           CDS      1 . . . 910
                    /gene = “alg-2”
                    /locus_tag = “2B167”
                    /coded_by = “NM_182192.1:
                    1 . . . 2733”
                    /db_xref = “AceView/WormGenes:
                    alg-2”
                    /db_xref = “GeneID: 173468”
                    /db_xref = “LocusID: 173468”
                    /db_xref = “WormBase: T07D3.7a”
ORIGIN
1          mfplpvhngp rlgklsifem pgdsltsssf
           mpdggaetss ssqlggsahg aigtkpdagv
61         qfqcpvrpnh gvegrsillr anhfavripg
           gsvqhyqidv fpdkcprrvn revigcliss
121        fskyftnirp vydgkrnmyt replpigtep
           mnfevtlpgd saverkfsvt mkwigqvcls
181        alddamegrv rqvpheavqs idvilrhlps
           lkytpvgrsf ftppgvmkpg mqmhqesklg
241        ggrevwfgfh qsvrpsqwkm mlnidvsata
           fyrampvief vaevlelpvq alaerralsd
301        aqrvkftkei rglkieithc qavrrkyrvc
           nvtrrpaqtq tfplqletgq tiectvakyf
361        fdkyriqlky phlpclqvgq eqkhtylppe
           vcdivpqqrc lkkltdvqts tmikatarsa
421        perereickl vskaelsadp fahefqitin
           pamtevkqrv lsapkllygg rhrattalpn
481        qgvwdmrgkq fhtgmevrtw aiacfaqqsh
           vkendlrmft tqlqristda gmpiigtpmf
541        ckyasgveqv epmfkylkqt ysaiqlivvv
           lpgktpiyae vkrvqdtvlg iatqcvqakn
601        airttpqtls nlclkmnvkl qqvnsillpn
           vrprifnepv iflgcdithp aagdtrkpsi
661        aavvgsmdah psryaatvrv qqhrqeiitd
           ltymvrellv qfyrntrfkp arivvyrdgv
721        segqlfnvlq yelraireac vmlesgyqpg
           itfiavqkrh htrlfaadka dqvgkafnip
781        pqttvdvgit hptefdfflc shagiqgtsr
           pshyhvlwdd ndltadelqq ltyqmchtyv
841        rctrsvsipa payyahlvaf raryhlvdrd
           hgsgeegsqp sgtssedttl ssmakavqvh
901        pdsnnvmyfa
//

IIID. DRH-1

LOCUS      NP_501018    1037 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION Dicer-Related Helicase, a DExH-box
           helicase (119.2 kD) (drh-1)
           [Caenorhabditis elegans].
ACCESSION  NP_501018
VERSION    NP_501018.1 GI: 17539846
DBSOURCE   REFSEQ: accession NM 068617.2
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 1037)
           AUTHORS  Tabara,H., Yigit,E., Siomi,H. and
                    Mello,C. C.
           TITLE    The dsRNA binding protein RDE-4 inter-
                    acts with RDE-1, DCR-1, and a DExH-box
                    helicase to direct RNAi in C. elegans
           JOURNAL  Cell 109 (7), 861-871 (2002)
           MEDLINE  22105477
           PUBMED   12110183
REFERENCE  2 (residues 1 to 1037)
           AUTHORS  Marcotte,E. M., Xenarios,I., van Der
                    Bliek,A. M. and Eisenberg,D.
           TITLE    Localizing proteins in the cell from
                    their phylogenetic profiles
           JOURNAL  Proc. Natl. Acad. Sci. U.S.A. 97 (22),
                    12115-12120 (2000)
           MEDLINE  20504472
           PUBMED   11035803
COMMENT    REVIEWED REFSEQ: This record has been
           curated by NCBI staff. This record is
           derived from an annotated genomic se-
           quence (NC_003282). The reference se-
           quence was derived from AU205212,
           AF480439.1 and AU217173.
           Summary: This gene drh-1, also known as
           F15B10.2, 4H372 or YK7673, maps at (IV;
           +3.32). It encodes a Dicer-Related
           Helicase, a DExH-box helicase. From
           Pfam homology, the product would have
           ATP binding, nucleic acid binding, ATP
           dependent helicase, helicase
           activities.
           According to the Worm Transcriptome
           Project, it is well expressed at all
           stages of development [Kohara cDNAs].
           Its sequence is defined by 19 cDNA
           clones.
           RNA interference results:
           [A. Sugimoto 2000] No obvious phenotype
           (by injecting cDNA clone SA: yk317d8).
           [J. Ahringer 2003] No obvious phenotype
           (by feeding genomic PCR product JA:
           F15B10.2).
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 6%, L1 or L2 larvae 19%, L3
           to adult 74%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           Pattern [pm11035803] predicted
           mitochondrial.
           This complete mRNA is 3298 bp long. Its
           sequence exactly matches the genome.
           The premessenger has 20 exons. It
           covers 5.98 kb on the WS97 genome. It
           is transpliced to SL1 or SL2. The
           protein (1037 aa, 119.2 kDa, pI 6.3)
           contains one DEAD/DEAH box helicase
           motif, one helicase, C-terminal motif.
           Taxblast (threshold 10{circumflex over ( )}-3) tracks an-
           cestors down to archaea and bacteria
           and eukaryota.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 1037
                    /organism = “Caenorhabditis
                    elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome= “IV”
                    /map = “IV; +3.32 cM (interpolated
                    genetic position)”
                    /map = “IV; covering 5976 bp, from
                    base 6613343 to 6607368 on genome
                    release WS97”
                    /clone = “Primers to amplify the
                    CDS (9336 bp, Stop included):
                    ATGAGGAAAAAGCAGTGTTCTTCAATA (T =
                    57.4), TTATGCTTCTCTGATTAAATTGACTAC
                    (T = 55.9). Complete CDS clones:
                    AF480439, yk850g8, yk1388a5,
                    yk1414c1, yk1627h8. Recommended
                    clone (from the Kohara collection):
                    yk850g8. Other clone(s): yk1716a1,
                    yk447b12, yk296e5, yk6g7, yk317d10,
                    yk354h4, yk240c5, yk134d4,
                    yk606h12, yk317d8, yk225b1,
                    yk207d7, yk219b1, yk1752c2. for
                    edited clone sequences see
                    www.wormgenes.org”
                    /clone_lib = “Kohara embryonic
                    lambda gt11 library: yk447b12,
                    yk606h12; Kohara Sugano L2 larvae
                    cap-selected library: yk1716a1,
                    yk1388a5, yk1414c1, yk1627h8,
                    yk1752c2; Kohara Sugano L4 larvae
                    cap-selected library: yk850g8;
                    Kohara mixed stage library, from
                    him-8 strain, containing 15-30%
                    males: yk207d7; Kohara mixed stage
                    library, from him-8 strain, con-
                    taining 15-30% males: yk296e5,
                    yk6g7, yk317d10, yk354h4, yk240c5,
                    yk134d4, yk317d8, yk225b1, yk219b1;
                    gb: AP480439”
           Protein  1 . . . 1037
                    /product = “Dicer-Related Helicase,
                    a DExH-box helicase (119.2 kD)
                    (drh-1)”
           Region   283 . . . 510
                    /region_name = “[Pfam/InterPro
                    description] DEAD/DEAN box
                    helicase”
                    /db_xref = “CDD: pfam00270”
           Region   723 . . . 810
                    /region_name = “[Pfam/InterPro
                    description] helicase, C-terminal”
                    /db_xref = “CDD: pfam00271”
           CDS      1 . . . 1037
                    /gene = “drh-1”
                    /locus_tag = “4H372”
                    /coded_by = “NM_068617.2:
                    6 . . . 3119”
                    /db_xref = “AceView/WormGenes:
                    drh-1”
                    /db_xref = “GeneID: 177425”
                    /db_xref = “LocusID: 177425”
                    /db_xref = “WormBase: F15B10.2”
ORIGIN
1          mrkkqcssil slydkeiilc lepiyrdpek
           gdgfsellpl gridelkiqs enaqefskql
61         yhdlknsils nadderlykd imtylqtylp
           kctvhkllnc snrevklsdf hyildhfegf
121        lrfiepkvvl ayldsypqyi davavlrkei
           erneednqds dfikklilrt vpllgeqavy
181        dimytiseks snnldveakq fiakvlrlkn
           dgflrfyqii nasrrqlngr iyicpvhesa
241        temmvylgta alntnryrmi nirvdnivqe
           nstprlvies vrqrihrqrq lclrnyqeel
301        cqvalqgknt ivtaptgsgk tviaaniike
           hfesrssegk rfkalfmtpn smilnqqaas
361        issyldhvyh tqiiqgsdnv ptrnviqskd
           livatpqmiv nlcnehrnsl ddesrldqff
421        lstftiiffd echntvknsp ysnimreyhy
           lknmgnmpeg hslpqiiglt aslgtgdknd
481        clqvrnyiag lcasmdvkdl sivkdnleel
           rgyspivpdk vllcerstdg pigmftnrlt
541        lmmqevegli rtalrnehig ieqrrqiett
           erdfrpdssf ldppadkeha gyqnwvcnqm
601        nlvsgtsfre tgtrtiinea ldvlkecfct
           lsyninfhpe valnylkdem eyrtpnftvn
661        miriweryhn qlvgtgsaen pmisktvqyi
           veqnlqrads rtiifvrtry eatilnkvln
721        sneellmlgi ksewmsglnk stassadisa
           skqkqmeklk mfadgeiril vstsvaeegl
781        dvpecslvik ynyatneiah vqrrgrgral
           nsecvlitns ialrdqesnn rdkeslmset
841        isliqnspae frkcvdeesn kiwprilred
           tdkaqkieeq inrnivykii ckkceailct
901        skdirsrntq ylvcdpgfws lvrktrltde
           qqalikynat gsincrrenc glklgqliev
961        ntvdlpclsa lsivllvegt dkriivkkwk
           nildkyftpt eirqldvqtm rdadqartpm
1021       vfehhangev vnlirea
//

IIIE. DRH-2

LOCUS       NP_501019    620 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION  Dicer-Related Helicase (71.3 kD)
            (drh-2) [Caenorhabditis elegans].
ACCESSION   NP_501019
VERSION     NP_501019.2 GI: 25145329
DBSOURCE    REFSEQ: accession NM 068618.2
KEYWORDS    .
SOURCE      Caenorhabditis elegans
            ORGANISM Caenorhabditis elegans
                     Eukaryota; Metazoa; Nematoda;
                     Chromadorea; Rhabditida; Rhabditoidea;
                     Rhabditidae; Peloderinae;
                     Caenorhabditis.
REFERENCE 1 (residues 1 to 620)
            AUTHORS  Tabara,H., Yigit,E., Siomi,H. and
                     Mello,C. C.
            TITLE    The dsRNA binding protein RDE-4
                     interacts with RDE-1, DCR-1, and a
                     DExH-box helicase to direct RNAi in C.
                     elegans
            JOURNAL  Cell 109 (7), 861-871 (2002)
            MEDLINE  22105477
            PUBMED   12110183
COMMENT     REVIEWED REFSEQ: This record has been
            curated by NCBI staff. This record is
            derived from an annotated genomic se-
            quence (NC_003282). The reference se-
            quence was derived from AF480440.1 and
            D33924.1. On Nov. 21, 2002 this sequence
            version replaced gi: 17538344.
            Summary: This gene drh-2, also known as
            C01B10.1, 4H380 or YK1203, maps at (IV;
            +3.33). It encodes a Dicer-Related
            Helicase. From Pfam homology, the pro-
            duct would have ATP binding, nucleic
            acid binding, helicase activities.
            According to the Worm Transcriptome
            Project, it is well expressed mostly
            from L1 larvae to adult [Kohara cDNAs].
            Its sequence is defined by 10 cDNA
            clones.
            RNA interference results:
            [A. Sugimoto 2000] No obvious phenotype
            (by injecting cDNA clone SA: yk272f7).
            [J. Ahringer 2003] No obvious phenotype
            (by feeding genomic PCR product JA:
            C01B10.1).
            Expression
            The expression profile for the gene,
            derived from the proportion of animals
            at each stage in each Kohara library
            is: embryos 2%, L1 or L2 larvae 27%, L3
            to adult 70%.
            In situ hybridisation pictures to all
            stages of development are available
            from Kohara NextDB.
            This complete CDS mRNA is 3277 bp long.
            Its sequence exactly matches the
            genome. The premessenger has 19 exons.
            It covers 4.76 kb on the WS97 genome.
            It has a very long 5′ UTR. The protein
            (620 aa, 71.3 kDa, pI 6.2) contains one
            helicase, C-terminal motif. Taxblast
            (threshold 10{circumflex over ( )}-3) tracks ancestors down
            to archaea and viruses and bacteria and
            eukaryota.
            COMPLETENESS: full length.
            Method: conceptual translation.
FEATURES    Location/Qualifiers
            source   1 . . . 620
                     /organism = “Caenorhabditis
                     elegans”
                     /db_xref = “taxon: 6239”
                     /chromosome = “IV”
                     /map = “IV; +3.33 cM (interpolated
                     genetic position)”
                     /map = “IV; covering 4758 bp, from
                     base 6618488 to 6613731 on genome
                     release WS97”
                     /clone = “Primers to amplify the
                     CDS (5583 bp, Stop included):
                     ATGATTGTAAATCTTTGCAATGAGCAC (T =
                     57.4),
                     TTATGCTTGTCTAATTACATTGATTACTT (T =
                     55.0). Complete CDS clones:
                     AF480440, yk38c3, yk226c6,
                     yk1564a4, yk1605b6. Recommended
                     clone (from the Kohara collection):
                     yk226c6. Other clone(s): yk315f1,
                     yk1017f9, yk1007f1, yk1080b12,
                     yk272f7. Anomalous clones:
                     yk1080b12 (Suspected internal dele-
                     tion) for edited clone sequences
                     see www.wormgenes.org”
                     /clone_lib = “Kohara Sugano L1
                     larvae cap-selected library:
                     yk1017f9, yk1007f1, yk1080b12;
                     Kohara Sugano L2 larvae cap-
                     selected library: yk1605b6; Kohara
                     Sugano L4 larvae cap-selected
                     library: yk1564a4; Kohara mixed
                     stage library, from him-8 strain,
                     containing 15-30% males: yk226c6,
                     yk38c3, yk315f1, yk272f7; gb:
                     AF480440”
            Protein  1 . . . 620
                     /product = “Dicer-Related Helicase
                     (71.3 kD) (drh-2)”
            Region   305 . . . 392
                     /region_name = “[Pfam/InterPro de-
                     scription] helicase, C-terminal”
                     /db_xref = “CDD: pfam00271”
            CDS      1 . . . 620
                     /gene = “drh-2”
                     /locus_tag = “4H380”
                     /coded_by = “NM_068618.2:
                     1238 . . . 3100”
                     /db_xref = “AceView/WormGenes:
                     drh-2”
                     /db_xref = “GeneID: 177426”
                     /db_xref = “LocusID: 177426”
                     /db_xref = “WormBase: C01B10.1”
ORIGIN
1           mivnlcnehr dplddeyppe qfflstftii
            ffdechntvk nspysnvmre yhylknmqnm
61          peghsfpqii gltaslgtgd kkncmqvrsy
            iaglcanmdv kelsivkdnl eelldhnpfv
121         tdqvsfcers ndgpiemftk rlkqmmqeve
            dlirttlkne ptvkyeippt dkehnryenw
181         isnqrncvsl agsrnktlii evldvlkdcf
            yalsyninfn pevalkkyle kelgperirn
241         ftdnmnliwd nchrelvgig saenpmiart
            vqfildqneq tsdfraiifv rtkkeadfln
301         yvlndrlhel giksdwmsgq kkstassadi
            saskqkqmek lkmfadgenq ilvstsvaee
361         qldipecslv ikynyatnet ahvqrrgrar
            arnskcvlit nsialhvqes nnlakenlmt
421         etisliqnsp gefrqcvdee snkvwpriqr
            edtdkaqrik eqinrnivyk ivcmkcdtvl
481         ctnkdirskn tqyivcnpgf wslvrriplp
            leqrasnkfn stgsieclge rcgsklgqli
541         dvntvnlpcl kvksilllie stnerilvkq
            wknildehft pttlkqrdvq tmkdadygra
601         piefehhtan gevinvirga
//

IIIF. Helicase Homologous to DCR-2 (DRH-3)

DCR-2 has been officially renamed DRH-3 and is a paralog of DRH-1 and DRH-2 which are essential for RNAi. Importantly, the human ortholog for DRH-3 is melanoma differentiation associated protein-5.

MQPTAIRLEDYDKSKLRLPFESPYFPAYFRLLKWKFLDVCVESTRNNDIG
YFKLFESLFPPGKLEEIARMIIDEPTPVSHDPDMIKIRNADLDVKIRKQA
ETYVTLRHAHQQKVQRRRFSECFLNTVLFDEKGLRIADEVMFNYDKELYG
YSHWEDLPDGWLTAETFKNKFYDEEEVTNNPEGYQKLDRVAGAARGMIIM
KHLKSNPRCVSETTILAFEVFNKGNHQLSTDLVEDLLTEGPAFELKIENG
EEKKYAVKKWSLHKTLTMFLAIIGFKSNDKKEKNEHEEWYYGFIDAMKND
PANRAALYFLDKNWPEELEEREKERDRIRLTLLKSQRTNEEAVGEDVCTT
IRPQPKDSGYNPDAVVTELVLRTYQEELVQPALEGKNCVIVAPTGSGKTE
VAIYAALKHIEERTSQGKPSRVVLLVPKIPLVGQQKDRFLKYCNGMYEVN
GFHGSESSVSGTGRRDEVIATHVSVMTPQILINMLQSVRQNERLYVSDFS
MMIFDEVHKAAKNHPYVLINQMVQEWKYEKPQIIGLTASLSVKVDGQKDE
NQMLNDIYNMLALINAPHLSTITRQSSIDELNEHVGKPDDSVELCLPAKE
NILRDYIERYLNHAHGKFLEELASMSKSTGRNNTIPPNMINTFKKNQPKN
YEYYDSLLQGIIQELNKLNVPEKWNSQTWAKYMKVYLEARGIVDLMPAMV
AFKYMEKAIGKLNESHSETVEYSTFIKDHDTLKQTIQSVEPEIVLRLKKY
THQSVPHQFGNYGEQMVGYVLGTNKQGAVQQTSQEQQLTLDKFNNGRLKV
IVATSVVEEGLDVTACNLIIKYNCSSGSAIQLVQQRGRARAKNSRSVLLS
VKSSINETETNALISEKYMRLCVKKITENGEKQLAAEVKRVAELNAAERK
RNLEEQLNLRLRHENKIYKLMCSNCSKEFCKSIYIKKVFSNYMVFDPSVW
RFLHVESVETFIKCLKITWKCRIADYQIAEFPNFAFRQLTFRLFLCNFQM
FQKRKVSKYLSEDNQPLSDIKCFHCKLDVGRAYKIRGTYLPQLSVKALTF
VQESDYSSMTKAKWSDVEQDLFYISEAIEDDFRIMLNALSDTEENIEKKI
VLDLDSRQHNKQLEMKRFHIQQEPPTKGVAPEAQ

Also see the C. elegans Protein Database: Wormpep at http://www.sanger.ac.uk/Projects/C_—elegans/wormpep/; Accesion No. ce09069.

IIIG. Double Helicase

MADELARIQQYEYRQNSNLVLSVDYNLTDRRGREEPTGEVLPITDKEMRK
MKMGDRAIKGKAPVQDQKKKRKKKDDEKAQQFGRNVLVDNNELMGAYKPR
TQETKQTYEVILSFILDALGDVPREVLCGAADEVLLTLKNDKFRDKEKKK
EVEALLGPLTDDRIAVLINLSKKISDFSIEEENKPEGDGDIYENEGVNVQ
FDSDEEEDDGGMVNEIKGDSEEESEEEEGVDTDYTATLKGDGHLTEDEQK
ARGILHPRDIDAHWIQRSLAKYFKDPLIAQQKQTEVIGILKNAADDRDAE
NQLVLLLGFDQFEFIKCLRQNRLMILYCTLLRQANEKERLQIEDDMRSRP
ELHPILALLQETDEGSVVQVEKSKRDAEKSKKAATAANEAISAGQWQAGR
KMLDLNDLTFSQGSHLMSNKRCELPDGSYRRQKKSYEEIHVPALKPRPFA
EGEKLVSVSELPKWAQPAFDGYKSLNRIQSRLCDSALRSKEHLLLCAPTG
AGKTNVALLTMLQEIGNHLAEDGSVKLDEFKIVYIAPMKSLVQEMVGSFS
KRLAPFGITVGEMTGDAQMSKEQFMATQVIVCTPEKYDVVTRKGGERAYN
QMVRLLIIDEIHLLHDDRGPVLESIVVRTIRQMEQNHDECRLVGLSATLP
NYQDVATFLRVKPEHLHFPDNSYRPVPLEQQYIGVTEKKALKRFQAMNEV
VYDKIMEHAGKSQVLVFVHSRKETAKTAKAIRDACLEKDTLSAFMREGSA
STEILRTEAEQAKNLDLKDLLPYGFAIHHAGMNRVDRTLVEDLFADRHIQ
VLFSTATLAWGVNLPAHTVIIKGTQIYNPEKGRWTELGALDIMQMLGRAG
RPQYDDRGEGILITNHSELQYYLSLMNQQLPVESQMVSRLTDMLNAEVVL
GTVSSVSEATNWLGYTFLFVRMLKNPTLYGITHEQARADPLLEQRRADLI
HTACVLLDKAGLIKYDKRSGIIQATELGRIASHFYCTYESMQTYNKLLVE
TCSDIDLFRIFSMSSEFKLLSVRDEEKLELQKMAEHAPIPIKENLDEASA
KTNVLLQAYISQLKLEGFALQADMVFVAQSAGRLFRALFEIVLWRGWAGL
AQKVLTLCKMVTQRQWGSLNPLHQFKKIPSEVVRSIDKKNYSFDRLYDLD
QHQLGDLIKMPKMGKPLFKFIRQFPKLEMTTLIQPITRTTMRIELTITPD
FKWDEKVHGSAEGFWIFIEDTDGEKILHHEFFLLKQKFCSDEHVVKMIVP
MFDPMPPLYYVRIVSDRWIGAETVLPISFRHLILPEKYPPPTELLDLQPL
PISAVTNKEFQTVFAESGFKVFNPIQTQVFRTVFESNENVIVCAPNGSGK
TAIAELAVLRHFENTPEAKAVYITPMEDMATKVYADWKRRLEPAIGHTIV
LLTGEQTMDLKLAQRGQLIISTPERWDNISRRWKQRKSVQNVKLFIADDL
HMIGASNGAVFEVVCSRTRYISSQLESAVRVVALSSSLTNARDLGMWLGC
SASATFNFMPSTRPVPLDLEIKSFNLSHNASRFAAMERPVYQAICRHAGK
LEPKPALVFVPVRRQTRPVAVALLTMALADGAPKRFLRLAEHDDTFQALL
ADIEDESLRESVSCGVGFLHEGTAPKDVHIVQQLFESNAIQVCVVPRGMC
YQIEMSAYLVVVMDTQFYNGKYHVYEDYPIADMLHMVGLANRPILDSDAK
CVVMCQTSKRAYYKKFLCDPLPVESHLDHCLHDHFNAEIVTKTIENKQDA
IDYLTWTLLYRRMTQNPNYYNLQGTTHRHLSDALSELVELTLKDLENSKC
IAVKDEMDTVSLNLGMIASYYYISYQTIELFSMSLKEKTKTRALIEIISA
SSEFGNVPMRHKEDVILRQLAERLPGQLKNQKFTDPHVKVNLLIHAHLSR
VKLTAELNKDTELIVLRACRLVQACVDVLSSNGWLSPAIHAMELSQMLTQ
AMYSNEPYLKQLPHCSAALLERAKAKEVTSVFELLELENDDRSDILQMEG
AELADVARFCNHYPSIEVATELENDVVTSNDNLMLAVSLERDNDIDGLAP
PVVAPLFPQKRKEEGWWLVIGDSESNALLTIKRLVINEKSSVQLDFAAPR
PGHHKFKLFFISDSYLGADQEFDVAFKVEEPGRSNRKRKHEKEED

Also see the C. elegans Protein Database: Wormpep at http://www.sanger.ac.uk/Projects/C_—elegans/wormpep/; Accesion No. ce21971.

IIIH. EFT-2, EF-Tu Family GTP Binding Protein

LOCUS      NP_492457    852 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION translation Elongation FacTor (94.8 kD)
           (eft-2) [Caenorhabditis elegans].
ACCESSION  NP_492457
VERSION    NP_492457.1 GI: 17506493
DBSOURCE   REFSEQ: accession NM 060056.2
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 852)
           AUTHORS  Fraser,A. G., Kamath,R. S.,
                    Zipperlen,P., Martinez-Campos,M.,
                    Sohrmann,M. and Ahringer,J.
           TITLE    Functional genomic analysis of C.
                    elegans chromosome I by systematic RNA
                    interference
           JOURNAL  Nature 408 (6810), 325-330 (2000)
           MEDLINE  20548709
           PUBMED   11099033
REFERENCE  2 (residues 1 to 852)
           AUTHORS  Ofulue,E. N. and Candido,E. P.
           TITLE    Isolation and characterization of
                    eft-1, an elongation factor 2-like gene
                    on chromosome III of Caenorhabditis
                    elegans
           JOURNAL  DNA Cell Biol. 11 (1), 71-82 (1992)
           MEDLINE  92153310
           PUBMED   1739435
REFERENCE  3 (residues 1 to 852)
           AUTHORS  Ofulue,E. N. and Candido,E. P.
           TITLE    Molecular cloning and characterization
                    of the Caenorhabditis elegans elonga-
                    tion factor 2 gene (eft-2)
           JOURNAL  DNA Cell Biol. 10 (8), 603-611 (1991)
           MEDLINE  92029622
           PUBMED   1930695
COMMENT    REVIEWED REFSEQ: This record has been
           curated by NCBI staff. This record is
           derived from an annotated genomic se-
           quence (NC_003279). The reference se-
           quence was derived from BJ105642.1,
           AU205829, M86959 and AU218565.
           Summary: This essential gene eft-2,
           also known as F25H5.4, 1J741 or YK6,
           maps at (I; +3.37). Its phenotype is
           embryonic lethal, protruding vulva. It
           encodes a translation Elongation
           FacTor. From Pfam homology, the product
           would have GTP binding, translation
           elongation factor activities, would be
           involved in translational elongation.
           According to the Worm Transcriptome
           Project, it is expressed at very high
           level at all stages of development
           except in embryos [Kohara cDNAs]. Its
           sequence is defined by 1015 cDNA
           clones.
           RNA interference results
           [J. Ahringer 2000] embryonic lethal
           (100%), protruding vulva (by feeding
           genomic PCR product JA: F25H5.4).
           Function
           Protein properties: [C.elegansII] NMK.
           Encodes protein with >80% similarity to
           elongation factor EF-2 from yeast,
           Drosophila, human. [Ofolue and Candido
           1992].
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 3%, L1 or L2 larvae 13%,
           L3 to adult 34%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           For a detailed expression pattern de-
           scription, see Wormbase Expr1390.
           This complete mRNA is 2819 bp long. Its
           sequence exactly matches the genome.
           The premessenger has 6 exons. It covers
           3.23 kb on the WS97 genome. It is
           transpliced to SL1. The protein (852
           aa, 94.8 kDa, pI 6.1) contains one
           Elongation factor, GTP-binding motif,
           one Elongation factor Tu, domain 2
           motif, one Elongation factor G, domain
           IV motif, one Elongation factor G, C-
           terminal motif. It also contains a coil
           coil stretch, an ER membrane domain
           [Psort2]. Taxblast (threshold 10{circumflex over ( )}-3)
           tracks ancestors down to archaea and
           bacteria and eukaryota.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 852
                    /organism = “Caenorhabditis
                    elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “I”
                    /map = “I; +3.37 cM (interpolated
                    genetic position)”
                    /map = “I; covering 3303 bp, from
                    base 9171586 to 9174890 on genome
                    release WS97”
           Protein  1 . . . 852
                    /product = “translation Elongation
                    FacTor (94.8 kD) (eft-2)”
           Region   17 . . . 356
                    /region_name = “[Pfam/InterPro de-
                    scription] elongation factor, GTP-
                    binding”
                    /db_xref = “CDD: pfam00009”
           Region   176 . . . 177
                    /region_name = “[PSORT] dileucine
                    domain: LL”
           Region   298 . . . 325
                    /region_name = “[PSORT] coil coil
                    domain:
                    VMNIKKDKTAALVEKLGIKLANDEKDLE”
           Region   401 . . . 480
                    /region_name = “[Pfam/InterPro
                    description] elongation factor Tu,
                    domain 2”
                    /db_xrefr = “CDD: pfam03144”
           Region   614 . . . 731
                    /region_name = “[Pfam/InterPro de-
                    scription] elongation factor G,
                    domain IV”
                    /db_xref = “CDD: pfam03764”
           Region   655 . . . 656
                    /region_name = “[PSORT] dileucine
                    domain: LL”
           Region   733 . . . 821
                    /region_name = “[Pfam/InterPro de-
                    scription] elongation factor G,
                    C-terminal”
                    /db_xref = “CDD: pfam00679”
           Region   734 . . . 735
                    /region_name = “[PSORT] dileucine
                    domain: LL”
           Region   833 . . . 836
                    /region_name = “[PSORT] nuclear
                    localization domain: RKRK”
           Region   848 . . . 851
                    /region_name = “[PSORT] ER membrane
                    domain: YLDK”
           CDS      1 . . . 852
                    /gene = “eft-2”
                    /locus_tag = “1J741”
                    /coded_by = “NM_060056.2:
                    124 . . . 2682”
                    /db_xref = “AceView/WormGenes:
                    eft-2”
                    /db_xref = “GeneID: 172743”
                    /db_xref = “LocusID: 172743”
                    /db_xref = “WormBase: F25H5.4”
ORIGIN
1          mvnftvdeir almdrkrnir nmsviahvdh
           gkstltdslv skagiiaqsk agetrftdtr
61         kdeqerciti kstaislffe lekkdlefvk
           genqfetvev dgkkekyngf linlidspgh
121        vdfssevtaa lrvtdgalvv vdcvsgvcvq
           tetvlrqaia erikpvlfmn kmdrallelq
181        lgaeelfqtf qriveninvi iatygdddgp
           mqpimvdpsi gnvgfgsqlh gwaftlkqfa
241        emyagkfgvq vdklmknlwg drffdlktkk
           wsstqtdesk rgfcqfvldp ifmvfdavmn
301        ikkdktaalv eklgikland ekdlegkplm
           kvfmrkwlpa gdtmlqmiaf hlpspvtaqk
361        yrmemlyegp hddeaavaik tcdpngplmm
           yiskmvptsd kgrfyafgrv fsgkvatgmk
421        ariqgpnyvp gkkedlyekt iqrtilmmgr
           fiepiedips gniaglvgvd qylvkggtit
481        tykdahnmrv mkfsvspvvr vaveaknpad
           lpklveglkr laksdpmvqc ifeesgehii
541        agagelhlei clkdleedha ciplkksdpv
           vsyretvqse snqiclsksp nkhnrlhcta
601        qpmpdgladd ieggtvnard efkarakila
           ekyeydvtea rkiwcfgpdg tgpnllmdvt
661        kgvqylneik dsvvagfqwa tregvlsden
           mrgvrfnvhd vtlhadaihr gggqiiptar
721        rvfyasvlta eprllepvyl veiqcpeaav
           ggiygvlnrr rghvfeesqv tgtpmfvvka
781        ylpvnesfgf tadlrsntgg qafpqcvfdh
           wqvlpgdple agtkpnqivl dtrkrkglke
841        gvpaldnyld km
//

III. EFT-4 (eIF1 alpha)

LOCUS      NP_509323    463 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION translation Elongation FacTor (50.7 kD)
           (eft-4) [Caenorhabditis elegans].
ACCESSION  NP_509323
VERSION    NP_509323.1 GI: 17569207
DBSOURCE   REFSEQ: accession NM 076922.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 463)
           AUTHORS  Kamath,R. S., Fraser,A. G., Dong,Y.,
                    Poulin,G., Durbin,R., Gotta,M.,
                    Kanapin,A., Le Bot,N., Moreno,S.,
                    Sohrmann,M., Welchman,D. P.,
                    Zipperlen,P. and Ahringer,J.
           TITLE    Systematic functional analysis of the
                    Caenorhabditis elegans genome using
                    RNAi
           JOURNAL  Nature 421 (6920), 231-237 (2003)
           MEDLINE  22417569
           PUBMED   12529635
COMMENT    VALIDATED REFSEQ: This record has un-
           dergone preliminary review of the se-
           quence, but has not yet been subject to
           final review. This record is derived
           from an annotated genomic sequence
           (NC_003284). The reference sequence was
           derived from WormBase CDS: R03G5.1a.
           Summary: This essential gene eft-4,
           also known as eln-2, R03G5.1, XI443 or
           YK211, maps at (X; −0.81). Its pheno-
           type is embryonic lethal, partial, slow
           growth. It encodes a translation Elon-
           gation FacTor. From Pfam homology, the
           products would have GTP binding, trans-
           lation elongation factor activities,
           would be involved in translational
           elongation.
           According to the Worm Transcriptome
           Project, it is expressed at very high
           level at all stages of development
           [Kohara cDNAs]. Its sequence is fully
           supported by 406 cDNA clones and pro-
           duces, by alternative splicing, 4 dif-
           ferent transcripts a, b, c, d alto-
           gether encoding 4 different protein
           isoforms.
           RNA interference results
           [J. Ahringer 2003] Embryonic lethal
           (40%), slow growth (by feeding genomic
           PCR product JA: R03G5.1).
           Function
           Protein properties: [C.elegansII] NMK.
           Encodes EF1 alpha protein, aa sequence
           identical to eft-3. [FK].
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 6%, L1 or L2 larvae 58%,
           L3 to adult 37%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           The CDS has 3 exons. It covers 1.59 kb
           on the WS97 genome. The protein (463
           aa, 50.7 kDa, pI 9.1) contains one
           Elongation factor, GTP-binding motif,
           one Elongation factor Tu, domain 2
           motif, one Elongation factor Tu, C-
           terminal motif. It also contains an ER
           membrane domain [Psort2]. Taxblast
           (threshold 10{circumflex over ( )}-3) tracks ancestors down
           to eukaryota.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 463
                    /organism = “Caenorhabditis
                    elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “X”
                    /map = “X; −0.81 cM (interpolated
                    genetic position)”
                    /map = “X; covering 2129 bp, from
                    base 7814176 to 7816306 on genome
                    release WS97”
           Protein  1 . . . 463
                    /product = “translation Elongation
                    FacTor (50.7 kD) (eft-4)”
           Region   5 . . . 239
                    /region_name = “[Pfam/InterPro de-
                    scription] elongation factor, GTP-
                    binding”
                    /db_xref = “CDD: pfam00009”
           Region   258 . . . 327
                    /region_name = “[Pfam/InterPro de-
                    scription] elongation factor Tu,
                    domain 2”
                    /db_xref = “CDD: pfam03144”
           Region   333 . . . 442
                    /region_name = “[Pfam/InterPro de-
                    scription] elongation factor Tu, C-
                    terminal”
                    /db_xref = “CDD: pfam03143”
           Region   459 . . . 462
                    /region name = “[PSORT] ER membrane
                    domain: APKK”
           Region   460 . . . 463
                    /region_name = “[PSORT] nuclear lo-
                    calization domain: PKKK”
           CDS      1 . . . 463
                    /gene = “eft-4”
                    /locus_tag = “XI443”
                    /coded_by = “NM_076922.1:
                    1 . . . 1392”
                    /db_xref = “AceView/WormGenes:
                    eft-4”
                    /db_xref = “GeneID: 181044”
                    /db_xref = “LocusID: 181044”
ORIGIN
1          mgkekvhini vvighvdsgk stttghliyk
           cggidkrtie kfekeaqemg kgsfkyawvl
61         dklkaererg itidialwkf etakyyitii
           dapghrdfik nmitgtsQad cavlvvacgt
121        gefeagiskn gqtrehalla qtlgvkqliv
           acnkmdstep pfsearftei tnevsgfikk
181        igynpkavpf vpisgfngdn mlevssnmpw
           fkgwaverke gnasgktlle aldsiippqr
241        ptdrplrlpl qdvykiggig tvpvgrvetg
           iikpgmvvtf apqnvttevk svemhheslp
301        eavpgdnvgf nvknvsvkdi rrgsvcsdsk
           qdpakeartf haqviimnhp gQisngytpv
361        ldchtahiac kfnelkekvd rrtgkkvedf
           pkflksgdag iveliptkpl cvesftdyap
421        lgrfavrdmr qtvavgviks veksdgssgk
           vtksaqkaap kkk
//

IIIJ. GAP/RAN-GAP Family

SWSGDKLAWLQTWRRVISLVDPYTNSSAHVAIDCMSLTIENLLLVNLHPL
AHWLACRLVTVPPILLPRCVPALSAILNESTIRRPPPLLSANILLCFIRL
MQSKEQLVVPAICGLSAHELSIVAPRALEHLPKMLQAAKSSKDTKVSSNS
LKLFSMLASSYPGAEQILLDQLVNTDVSENAVVIVNSLAILIVQKAQIDL
VLTALKTIETHQFAMRLIPLFCSSIASLAQFSSTTLLQALLPAASLLRDE
RTRTEIEWQMVKLCMQWPQPQMPLVIRGILADRHMVLHGELVTLGGQYPV
RGFEVQRWSSAGAPPLQGEDKTVYINRQSAIISVSRKDFHAKSPCEITSR
TVVGRHIWDLDTHEDVRKPATNVTNWLRKEALKGKRPGRESQGILGAMDD
PFDDLPDYPPSRGSPSPVDGAAQFTSMIETSRRQPQPLGTSSAAHDHLPA
FTPNAKLLEWRSLSASLGFVPLVSQVHANFPRDLKHLDQTSSREVHKVAV
IYVGESQEDRASILSNTTASASAQFDSFTSELGWEVKVGRGHDGYTGGLP
VETRAPYFADAEAEVIFHVSTMLNGDVQQKWKHIGNDEVHVVWTENTRKV
YTRETIATKFCDVLIVLEQVGDKMVRVRVDTASALEFGPLFDGALVTMSE
LSQLVRLTVINASRAYRLARVEHSRPLRHREEVFCNEALAHMKPMPLAQS
INHLYVPTI

Also see the C. elegans Protein Database: Wormpep at http://www.sanger.ac.uk/Projects/C_—elegans/wormpep/; Accesion No. ce21437.

IIIK. HMG-I/Y DNA Binding Protein

MVEGDVDESASGTSGTNKKILFTKKPSVWKDFDNWINDEPENRYDLFQVV
KSAMLLQSGYTTILMDQVTDNGADELRISLEYSNFIKIVNSTKLVVGKEQ
CPPSNVFTLLAEIFANTPGNTSEVGRISTWLTSHLGALLHNDVIWKIHFF
DPDLFRSVYWQLIFTLKLAPGDTENLEEDENYAKLLFSCFITAVMVALWH
DHEMSFNSICPDYLKPETASEYMVMLISSPPFRSLSQFFLFGLHLLGKYQ
SEGGCVVVREEAYIAEIRQNDEEKRQSIETRTNLISDDMVYDDGEDLLEQ
IDRVQQLHEAHCIVLLKKGFLKAPDGFKIVQKGGRPRKYPASATKKRKKK
TPRSSPKKKMSKESPINHQKEPIDEQKPSTSLPIYSVATLKPRRKVVKTA
DEVGLGAPIFVMQSELLKKFREEVQRRYAEGSSASDQERVRNMVYEAYDN
IYHINRLSANEGPRILTSDQKLVMQQYKTTFRQGPTFAEETESDVEEEEE
KKVVEVVTAKVIKGSAKSSKKFKRRY

Also see the C. elegans Protein Database: Wormpep at http://www.sanger.ac.uk/Projects/C_—elegans/wormpep/; Accesion No. ce08872.

IIIL. HMG-I/Y DNA Binding PB1 Domain

Also see the C. elegans Protein Database: Wormpep at http://www.sanger.ac.uk/Projects/C_—elegans/wormpep/; Accesion No. ce20336.

IIIM. SNR-2 SM Protein

Member of the Small Nuclear Ribonucleoprotein gene class.

MTISKNNKNM AHLNYRMKII LQDGRTFIGF FKAFDKHMNI
LLAECEEHRQ IKPKAGKKTD
GEEKRILGLV LVRGEHIVSM TVDGPPPRDD DSVRLAKAGG
AGGVGQAKPG GRGMPAMPGM
PGMPPGGAPG GLSGAMRGHG GPGMAANQPG
YGGPPGGRPF

Also see the C. elegans Protein Database: Wormpep at http://www.sanger.ac.uk/Projects/C_—elegans/wormpep/; Accesion No. ce14704.

Homologs include Swiss-Prot. TrEMBL Accession No. Q15182 H. sapiens and TrEMBL Accession No. 070499, M. musculus Small nuclear ribonucleoprotein N.

IIIN. SNR-3 SM Protein

The SNR-3 SM protein is a member of the Small Nuclear Ribonucleoprotein SMD1 gene class. A homolog for this gene product is human SMD1.

—————————————.
MKLVRFLMKL SHETVNIELK NGTQVSGTIM GVDVAMNTHL
RAVSMTVKNK EPVKLDTLSI
RGNNIRYIIL PDPLALDTLL IDDEPRKKAR AARAGASRGR
GRGGMRGGRG GRGRGRGGPR GGGPRR

Also see the C. elegans Protein Database: Wormpep at http://www.sanger.ac.uk/Projects/C_—elegans/wormpep/; Accesion No. ce02065.

IIIO. Dual Specificity Phosphatase

MPEPRCTAIV NFLNLSHSIL ISIFSVSVMS NYHHNHNYQH
RPRGYERLPG KRLPDRWNIY
DNVGRDIDGT RFVPEKTPLD SSFFDGKNMP VELQFGVKTL
ISLAQQANKQ IGLVIDLTNT
DRYYKKTEWA DHGVKYLKLN CPGHEVNERE DLVQDFINAV
KEEVNDKEND GKLIGVHCTH
GLNRTGYLIC RYMIDVDNYS ASDAISMFEY YRGHPMEREH
YKKSLYEAER KKKYGKSSGK
SSGNSADSTI SSEQLHRNNS Q

Homologs include, for example, Swiss Prot. Accession No. 075319, H. sapiens Dual specificity protein phosphatase 11 and TrEMLB Accession No. Q8BTR4, similar to dual specificity protein phosphatase 11.

Also see the C. elegans Protein Database: Wormpep at http://www.sanger.ac.uk/Projects/C_—elegans/wormpep/; Accesion No. ce03706.

IIIP. LIN-41

A homolog of LIN-41 is the human tripartite motif protein 2 (RING finger protein 86)

LOCUS      NP_492487    1143 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION abnormal cell LINeage LIN-41, heterochronic gene; Drosophila
           dappled/vertebrate TRipartite Motif protein related; B-box
           zinc finger, Filamin and NHL repeat containing protein
           (123.8 kD) (lin-41) [Caenorhabditis elegans].
ACCESSION  NP_492487
VERSION    NP_492487.2 GI: 25149908
DBSOURCE   REFSEQ: accession NM 060086.2
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda; Chromadorea; Rhabditida;
                    Rhabditoidea; Rhabditidae; Peloderinae; Caenorhabditis.
REFERENCE  1 (residues 1 to 1143)
           AUTHORS  Lin,S. Y., Johnson,S. M., Abraham,M., Vella,M. C.,
                    Pasquinelli, A., Gamberi,C., Gottlieb,E. and Slack,F. J.
           TITLE    The C elegans hunchback homolog, hbl-1, controls temporal
                    patterning and is a probable microRNA target
           JOURNAL  Dev. Cell 4 (5), 639-650 (2003)
           MEDLINE  22623382
           PUBMED   12737800
REFERENCE  2 (residues 1 to 1143)
           AUTHORS  Grosshans,H. and Slack,F. J.
           TITLE    Micro-RNAs: small is plentiful
           JOURNAL  J. Cell Biol. 156 (1), 17-21 (2002)
           MEDLINE  21640444
           PUBMED   11781331
REFERENCE  3 (residues 1 to 1143)
           AUTHORS  Ketting,R. F., Fischer,S. E., Bernstein,E., Sijen,T.,
                    Hannon,G. J. and Plasterk, R. H.
           TITLE    Dicer functions in RNA interference and in synthesis of small
                    RNA involved in developmental timing in C. elegans
           JOURNAL  Genes Dev. 15 (20), 2654-2659 (2001)
           MEDLINE  21521222
           PUBMED   11641272
REFERENCE  4 (residues 1 to 1143)
           AUTHORS  Sonoda,J. and Wharton,R. P.
           TITLE    Drosophila Brain Tumor is a translational repressor
           JOURNAL  Genes Dev. 15 (6), 762-773 (2001)
           MEDLINE  21172744
           PUBMED   11274060
REFERENCE  5 (residues 1 to 1143)
           AUTHORS  Slack,F. J., Basson,M., Liu,Z., Ambros,V., Horvitz,H. R. and
                    Ruvkun, G.
           TITLE    The lin-41 RBCC gene acts in the C. elegans heterochronic
                    pathway between the let-7 regulatory RNA and the LIN-29
                    transcription factor
           JOURNAL  Mol. Cell 5 (4), 659-669 (2000)
           MEDLINE  20337950
           PUBMED   10882102
REFERENCE  6 (residues 1 to 1143)
           AUTHORS  Reinhart,B. J., Slack,F. J., Basson,M., Pasquinelli,A. E.,
                    Bettinger,J. C., Rougvie,A. E., Horvitz,H. R. and Ruvkun,G.
           TITLE    The 21-nucleotide let-7 RNA regulates developmental timing in
                    Caenorhabditis elegans
           JOURNAL  Nature 403 (6772), 901-906 (2000)
           MEDLINE  20168806
           PUBMED   10706289
COMMENT    REVIEWED REFSEQ: This record has been curated by NCBI staff.
           This record is derived from an annotated genomic sequence
           (NC_003279). The reference sequence was derived from AF195610.
           On Nov. 21, 2002 this sequence version replaced gi: 17508265.
           Summary: This gene lin-41, also known as C12C8.3, 1J912 or
           YK872, maps at (I; +3.53). Its phenotype is abnormal cell
           lineage, heterochronic. It encodes a heterochronic gene;
           Drosophila dappled/vertebrate TRipartite Motif protein related;
           B-box zinc finger, Filamin and NHL repeat containing protein.
           From Pfam homology, the products would have zinc binding
           activity and would localize in intracellular.
           According to the Worm Transcriptome Project, it is well
           expressed mostly from L1 larvae to adult [Kohara cDNAs]. Its
           sequence is defined by 11 cDNA clones and produces, by alterna-
           tive splicing, at least 2 different transcripts b, a altogether
           encoding 2 different protein isoforms. The transcripts appear to
           differ by common exons with different boundaries.
           Phenotype
           [from C. elegans II book] Allele ma104: heterochronic defect
           in L4 larvae to adult switch. [Victor Ambros].
           Selected strains available from the CGC.
           CT8 lin-41 (ma104) I [Frank Slack, V. Ambros, mutator TR679]
           Dpy. Precocious heterochronic. Reduced brood size. There may be
           a linked Dpy mutation in this strain.
           MT7897 lin-41 (n2914)/unc-29 (e1072) lin-11 (n1281) I [Bob
           Horvitz, M. Basson, EMS] Heterozygotes are WT and segregate WT,
           UncVul and lin-41 (Dpy, Scrawny and Sterile).
           RNA interference results:
           [J. Ahringer 2000] No obvious phenotype (by feeding genomic PCR
           product JA: C12C8.3).
           Expression
           The expression profile for the gene, derived from the
           proportion of animals at each stage in each Kohara library is:
           embryos 3%, L1 or L2 larvae 50%, L3 to adult 47%.
           In situ hybridisation pictures to all stages of development
           are available from Kohara NextDB.
           The report below describes variant a.
           This complete mRNA is 4797 bp long. It is supported by 2 cDNA
           clones. Its sequence exactly matches the genome. The
           premessenger has 16 exons. It covers 7.70 kb on the WS97 genome.
           It is transpliced to SL1. It has a very long 3′ UTR. The protein
           (1143 aa, 123.8 kDa, pI 6.1) contains 2 Zn-finger, B-box motifs,
           one Filamin/ABP280 repeat motif, 6 NHL repeat motifs. It also
           contains a coil coil stretch [Psort2]. Taxblast (threshold 10{circumflex over ( )}-3)
           tracks ancestors down to archaea and bacteria and eukaryota.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 1143
                    /organism = “Caenorhabditis elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “I”
                    /map = “I; +3.53 cM (interpolated genetic position)”
                    /map = “I; +3.75 cM (measured genetic position)”
                    /map = “I; covering 7702 bp, from base 9350549 to
                    9342848 on genome release WS97”
                    /clone = “Primers to amplify the CDS (10290 bp, Stop
                    included): ATGGCGACCATCGTGCCATGCT (T = 63.8),
                    CTAGAAGACACGGATGCAATTGTTTCCGAA (T = 63.4). Clone
                    specific of this variant is AF195610. Complete CDS clones:
                    yk1728d7. Recommended clone (from the Kohara collection):
                    yk1728d7. for edited clone sequences see www.wormgenes.org”
                    /clone_lib = “Kohara Sugano L2 larvae cap-selected
                    library: yk1728d7; gb: AF195610”
           Protein  1 . . . 1143
                    /product = “abnormal cell LINeage LIN-41, heterochronic
                    gene; Drosophila dappled/vertebrate TRipartite Motif
                    protein related; B-box zinc finger, Filamin and NHL
                    repeat containing protein (123.8 kD) (lin-41)”
           Region   366 . . . 412
                    /region name = “[Pfam/InterPro description] zn-finger,
                    B-box”
                    /db_xref = “CDD: pfam00643”
           Region   470 . . . 512
                    region_name = “[Pfam/InterPro description] zn-finger,
                    B-box”
                    /db_xref = “CDD: pfam00643”
           Region   553 . . . 617
                    /region_name = “[PSORT] coil coil domain:
                    TAENEIRAAFDTHVNALEERRKELLKRVETVKNLKLSVLISQAESLQSKQIDLQQAIQ
                    TATKLMD”
           Region   688 . . . 810
                    /region_name = “[Pfam/Interpro description]
                    Filamin/ABP280 repeat”
                    /db_xref = “CDD: pfam00630”
           Region   841 . . . 868
                    /region_name = “[Pfam/InterPro description] NHL repeat”
                    /db_xref = “CDD: pfam01436”
           Region   888 . . . 915
                    /region_name = “[Pfam/InterPro description] NHL repeat”
                    /db_xref = “CDD: pfam01436”
           Region   935 . . . 962
                    /region_name = “[Pfam/InterPro description] NHL repeat”
                    /db_xref = “CDD: pfam01436”
           Region   983 . . . 1010
                    /region_name = “[Pfam/Interpro description] NHL repeat”
                    /db_xref = “CDD: pfam01436”
           Region   1031 . . . 1058
                    /region_name = “[Pfam/InterPro description] NHL repeat”
                    /db_xref = “CDD: pfam01436”
           Region   1116 . . . 1143
                    /region_name = “[Pfam/Interpro description] NHL repeat”
                    /db_xref = “CDD: pfam01436”
           CDS      1 . . . 1143
                    /gene = “lin-41”
                    locus_tag = “1J912”
                    /coded_by = “NM_060086.2: 196 . . . 3627”
                    /db_xref = “AceView/WormGenes: lin-41”
                    /db_xref = “GeneID: 172760”
                    /db_xref = “LocusID: 172760”
                    /db_xref = “WormBase: C12C8.3a”
ORIGIN
1          mativpcsle keegapsgpr rlqteidvda ndsgnelsmg gsssegdsms
           hhrgehspnh
61         hhqdnhlgsg ppppqftgsl fdtppsmiqs pqqqpqfqfn tgfglglpqd
           sfrcsvcsks
121        stigvlpfvc ahktcqscyq mtpssydrra cklcgavsta tanftsqmyl
           sptlpspprg
181        almsdcstpt mnnhinsstp lhqprafsfs isqmpgspsp vmqarmpssa
           gglmrnrpigf
241        pdsdssltsw splqqpsqls innlssiggh qqqspmlmqn vfdslavndd
           tpvfsplspt
301        ntsmhmppsl maspdvpkhs atiapprnsm cstprlqlat pmssqsqqtf
           pipsplqsqp
361        qqqqpmgpiq cqgceskisf aycmqcqeal cihcvqahqr vratkqhafv
           elqqlmatlm
421        sravqpqqaq qytqnvqgsv rqalgsvgsg dghvsgvend sigsgesspr
           sssvcgthds
481        viigicencp hsvllcaicv aqhpgkhrvq plgdirvavg evvnesqllq
           wqcektgdti
541        kqiidgivtn attaeneira afdthvnale errkellkrv etvknlklsv
           lisqaeslqs
601        kqidlqqaiq tatklmdssd cdemvlrqvf eklascqingn eqtepnnnil
           nvlmlacqvn
661        eddrlkftap qdgillnkar qfgniesgpc aknssivgds fkkairerqt
           viyvqlrdac
721        gdllsssiaa tqptsqallp hqephshleq amptsdvqaf vispdgstve
           vtmtprengi
781        valsyypsie gsytlnilvk qtpisgcptt mdirrgrnyd eiaakgpilt
           fgkegsgdge
841        lcrpwgicvd qrgrvivadr snnrvqifdk dgnfiskfgt sgnrpgqfdr
           pagittnsln
901        nivvadkdnh rvqvfdengm fllkfgdrgr avgyfnypwg vatnshnaia
           vsdtrnhrvq
961        iftpqgqfvr kcgfdsayff knldsprglc ylpdgqllit dfnnhrlavl
           sprnmsemkv
1021       ygsegdgdgm fvrpqgvvid peghilvcds rnnrvqvfas ddmrfigsfg
           lgpvpnsgfq
1081       mpqelpapys slggpfgapa fssaptpltp sprqlldrpt dlavgpdgri
           yvvdfgnnci
1141       rvf
//
LOCUS      NP_492488    1147 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION abnormal cell LINeage LIN-41, heterochronic gene; Drosophila
           dappled/vertebrate TRipartite Motif protein related; B-box
           zinc finger, Filamin and NHL repeat containing protein
           (124.2 kD) (lin-41) [Caenorhabditis elegans].
ACCESSION  NP_492488
VERSION    NP_492488.2 GI: 25149913
DESOURCE   REFSEQ: accession NM 060087.2
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda; Chromadorea; Rhabditida;
                    Rhabditoidea; Rhabditidae; Peloderinae; Caenorhabditis.
REFERENCE  1 (residues 1 to 1147)
           AUTHORS  Lin,S. Y., Johnson,S. M., Abraham,M., Vella,M. C.,
                    Pasquinelli, A. Gamberi,C., Gottlieb,E. and Slack,F. J.
           TITLE    The C elegans hunchback homolog, hbl-1, controls temporal
                    patterning and is a probable microRNA target
           JOURNAL  Dev. Cell 4 (5), 639-650 (2003)
           MEDLINE  22623382
           PUBMED   12737800
REFERENCE  2 (residues 1 to 1147)
           AUTHORS  Grosshans,H. and Slack,F. J.
           TITLE    Micro-RNAs: small is plentiful
           JOURNAL  J. Cell Biol. 156 (1), 17-21 (2002)
           MEDLINE  21640444
           PUBMED   11781331
REFERENCE  3 (residues 1 to 1147)
           AUTHORS  Ketting,R. F., Fischer,S. E., Bernstein,E., Sijen,T.,
                    Hannon,G. J. and Plasterk, R. H.
           TITLE    Dicer functions in RNA interference and in synthesis of small
                    RNA involved in developmental timing in C. elegans
           JOURNAL  Genes Dev. 15 (20), 2654-2659 (2001)
           MEDLINE  21521222
           PUBMED   11641272
REFERENCE  4 (residues 1 to 1147)
           AUTHORS  Sonoda,J. and Wharton,R. P.
           TITLE    Drosophila Brain Tumor is a translational repressor
           JOURNAL  Genes Dev. 15 (6), 762-773 (2001)
           MEDLINE  21172744
           PUBMED   11274060
REFERENCE  5 (residues 1 to 1147)
           AUTHORS  Slack,F. J., Basson,M., Liu,Z., Arnbros,V., Horvitz,H. R. and
                    Ruvkun, G.
           TITLE    The lin-41 RBCC gene acts in the C. elegans heterochronic
                    pathway between the let-7 regulatory RNA and the LIN-29
                    transcription factor
           JOURNAL  Mol. Cell 5 (4), 659-669 (2000)
           MEDLINE  20337950
           PUBMED   10882102
REFERENCE  6 (residues 1 to 1147)
           AUTHORS  Reinhart,B. J., Slack,F. J., Basson,M., Pasquinelli,A. E.,
                    Bettinger,J. C., Rougvie,A. E., Horvitz,H. R. and Ruvkun,G.
           TITLE    The 21-nucleotide let-7 RNA regulates developmental timing in
                    Caenorhabditis elegans
           JOURNAL  Nature 403 (6772), 901-906 (2000)
           MEDLINE  20168806
           PUBMED   10706289
COMMENT    REVIEWED REFSEQ: This record has been curated by NCBI staff.
           This record is derived from an annotated genomic sequence
           (NC_003279). The reference sequence was derived from AF195611.
           On Nov. 21, 2002 this sequence version replaced gi: 17508263.
           Summary: This gene lin-41, also known as C12C8.3, 1J912 or
           YK872, maps at (I; +3.53). Its phenotype is abnormal cell
           lineage, heterochronic. It encodes a heterochronic gene;
           Drosophila dappled/vertebrate TRipartite Motif protein related;
           B-box zinc finger, Filamin and NHL repeat containing protein.
           From Pfam homology, the products would have zinc binding activity
           and would localize in intracellular.
           According to the Worm Transcriptome Project, it is well
           expressed mostly from L1 larvae to adult [Kohara cDNAs]. Its
           sequence is defined by 11 cDNA clones and produces, by alternative
           splicing, at least 2 different transcripts b, a altogether encoding
           2 different exons protein isoforms. The transcripts appear to differ
           by common with different boundaries.
           Phenotype
           [from C. elegans II book] Allele ma104: heterochronic defect
           in L4 larvae to adult switch. [Victor Ambros].
           Selected strains available from the CGC.
           CT8 lin-41 (ma104) I [Frank Slack, V. Ambros, mutator TR679]
           Dpy. Precocious heterochronic. Reduced brood size. There may be a
           linked Dpy mutation in this strain.
           MT7897 lin-41 (n2914)/unc-29 (e1072) lin-11 (n1281) I [Bob
           Horvitz, M. Basson, EMS] Heterozygotes are WT and segregate WT,
           UncVul and lin-41 (Dpy, Scrawny and Sterile).
           RNA interference results:
           [J. Ahringer 2000] No obvious phenotype (by feeding genomic PCR
           product JA: C12C8.3).
           Expression
           The expression profile for the gene, derived from the
           proportion of animals at each stage in each Kohara library is:
           embryos 3%, L1 or L2 larvae 50%, L3 to adult 47%. The expression
           profile for the gene, derived from the proportion of animals at each
           stage in each Kohara library is: embryos 3%, L1 or L2 larvae 45%, L3
           to adult 51%.
           In situ hybridisation pictures to all stages of development
           are available from Kohara NextDB.
           The report below describes variant b.
           This complete mRNA is 4809 bp long. It is supported by 8 cDNA
           clones, 7 of which match only this alternative variant. Its
           sequence exactly matches the genome. The premessenger has 16
           exons. It covers 7.70 kb on the WS97 genome. It is transpliced to
           SL1. It has a very long 3′ UTR. The protein (1147 aa, 124.2 kDa, pI
           6.1) contains 2 Zn-finger, B-box motifs, one Filamin/ABP280 repeat
           motif, 6 NHL repeat motifs. It also contains a coil coil
           stretch [Psort2]. Taxblast (threshold 10{circumflex over ( )}-3) tracks ancestors down
           to archaea and bacteria and eukaryota.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 1147
                    /organism = “Caenorhabditis elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “I”
                    /map = “I; +3.53 cM (interpolated genetic position)”
                    /map = “I; +3.75 cM (measured genetic position)”
                    /map = “I; covering 7702 bp, from base 9350549 to
                    9342848 on genome release WS97”
                    /clone = “Primers to amplify the CDS (10326 bp, Stop
                    included): ATGGCGACCATCGTGCCATGCT (T = 63.8),
                    CTAGAAGACACGGATGCAATTGTTTCCGAA (T = 63.4). Clones
                    specific of this variant are AF195611, yk20b11,
                    yk307c10, yk1100f6, yk1102h6, yk1111g2, yk1223b8.
                    Complete CDS clones: yk1728d7. Recommended clone
                    (from the Kohara collection): yk1728d7. for edited
                    clone sequences see www.wormgenes.org”
                    /clone_lib = “Kohara Sugano L1 larvae cap-selected
                    library: yk1111g2, yk1100f6, yk1102h6, yk1223b8;
                    Kohara Sugano L2 larvae cap-selected library:
                    yk1728d7; Kohara mixed stage library, from him-8
                    strain, containing 15-30% males: yk307c10; Kohara
                    mixed stage library, from him-8 strain, containing
                    15-30% males: yk20b11; gb: AF195611”
           Protein  1 . . . 1147
                    /product = “abnormal cell LINeage LIN-41, heterochronic
                    gene; Drosophila dappled/vertebrate TRipartite Motif
                    protein related; B-box zinc finger, Filamin and NHL
                    repeat containing protein (124.2 kD) (lin-41)”
           Region   366 . . . 412
                    /region_name = “[Pfam/InterPro description] zn-finger,
                    B-box”
                    /db_xref = “CDD: pfam00643”
           Region   474 . . . 516
                    /region_name = “[Pfam/Interpro description] zn-finger,
                    B-box”
                    /db_xref = “CDD: pfam00643”
           Region   557 . . . 621
                    /region_name = “[PSORT] coil coil domain:
                    TAENEIRAAFDTHVNALEERRKELLKRVETVKNLKLSVLISQAESLQSKQIDLQQAIQ
                    TATKLMD”
           Region   692 . . . 814
                    /region_name = “[Pfam/InterPro description]
                    Filamin/ABP280 repeat”
                    /db_xref = “CDD: pfam00630”
           Region   845 . . . 872
                    /region_name = “[Pfam/InterPro description] NHL repeat”
                    /db_xref = “CDD: pfam01436”
           Region   892 . . . 919
                    /region_name = “[Pfam/InterPro description] NHL repeat”
                    /db_xref = “CDD: pfam01436”
           Region   939 . . . 966
                    /region_name = “[Pfam/Interpro description] NHL repeat”
                    /db_xref = “CDD: pfam01436”
           Region   987 . . . 1014
                    /region_name = “[Pfam/InterPro description] NHL repeat”
                    /db_xref = “CDD: pfam01436”
           Region   1035 . . . 1062
                    /region_name = “[Pfam/InterPro description] NHL repeat”
                    /db_xref = “CDD: pfam01436”
           Region   1120 . . . 1147
                    /region_name = “[Pfam/InterPro description] NHL repeat”
                    /db_xref = “CDD: pfam01436”
           CDS      1 . . . 1147
                    /gene = “lin-41”
                    /locus_tag = “1J912”
                    /coded_by = “NM_060087.2: 196 . . . 3639”
                    /db_xref = “AceView/WormGenes: lin-41”
                    /db_xref = “GeneID: 172760”
                    /db_xref = “LocusID: 172760”
ORIGIN
1          mativpcsle keegapsgpr rlqteidvda ndsgnelsmg gsssegdsms
           hhrgehspnh
61         hhqdnhlgsg ppppqftgsl fdtppsmiqs pqqqpqfqfn tgfglglpqd
           sfrcsvcsks
121        stigvlpfvc ahktcqscyq mtpssydrra cklcgavsta tanftsqmyl
           sptlpspprg
181        almsdcstpt mnnhinsstp lhqprafsfs lsgmpgspsp vmgarmpssa
           gglmmrpigf
241        pdsdssltsw splqqpsqls innlssiggh qqqspmlmqn vfdslavndd
           tpvfsplspt
301        ntsmhmppsl maspdvpkhs atiapprnsm cstprlqlat pmssqsqqtf
           pipsplgsqp
361        qqqqpmgpiq cqgceskisf aycmqcqeal cihcvqahqr vratkqhafv
           elqqlmatlm
421        sravqpqqaq qytqnvggsv rqalgsvgsg dvffsghvsg vendsigsge
           ssprsssvcg
481        thdsviigic encphsvllc aicvaqhpgk hrvqplgdir vavgevvnes
           qllqwqcekt
541        gdtikqiidg ivtnattaen eiraafdthv naleerrkel lkrvetvknl
           klsvlisqae
601        slqskqidlq qaiqtatklm dssdcdemvl rqvfeklasc qmgnegtepn
           nnilnvlmla
661        cqvneddrlk ftapqdgill nkarqfgnie sgpcaknssi vgdsfkkair
           erqtviyvql
721        rdacqdllss siaatqptsq allphqephs hleqamptsd vqafvispdg
           stvevtmtpr
781        engivalsyy psiegsytln ilvkqtpisg cpttmdirrg rnydeiaakg
           piltfgkegs
841        gdgelcrpwg icvdqrgrvi vadrsnnrvq ifdkdgnfis kfgtsgnrpg
           qfdrpaqitt
901        nslnnivvad kdnhrvqvfd engmfllkfg drgravgyfn ypwgvatnsh
           naiavsdtrn
961        hrvqiftpqg qfvrkcgfds ayffknldsp rglcylpdgq llitdfnnhr
           lavlsprnms
1021       emkvyqsegd gdgmfvrpqg vvidpeghil vcdsrnnrvq vfasddmrfi
           gsfglgpvpn
1081       sgfqmpqelp apysslggpf gapafssapt pltpsprqll drptdlavgp
           dgriyvvdfg
1141       nncirvf
//

LIN-41 homologs include H. sapiens gi|37550026|ref|XP_—067369.3|[37550026], M. musculus gi|38090144|ref|XP_—356199.1|[38090144] and R. norvegicus gi|34866457|ref|XP_—236676.2|[34866457].

IIIQ. Low Homology MADS Box Protein, Novel

Also see the C. elegans Protein Database: Wormpep at http://www.sanger.ac.uk/Projects/C_—elegans/wormpep/; Accesion No. ce01506.

IIIR. RPN-9 Proteasome Subunit

MTAQDYLNGK LAAANGPLAD DWKNLKELWE KKLWHQLTVL
TRSLVKKPQF VASTDMHEFY
RLFVAEWELR VNPLQLVEIC ISIAQNIATK DKQKSMEFLS
KIGNVINKDK IAVARLHTGE
IEARLENKDK NGQIIDLKSI RTQIDSTQHE VDSLVGVTEV
HAPFYRVSSL YLREVGDFAG
YYREALRYLG VEDANNLTTE QKQVHAVLLG FAALLGENVH
NFGELLAHPI LKSLEGTRER
WIVDVLLAFN SGDLTRFFSL EGDWGGWDDL KKQKDFLTAK
IRLMAVMELA VSRPTKARSV
SFKEIATKCQ IPFDEVEFLV MKALSKDLIR GDINQVEQVV
YVTWVQPRVL DNPQIMQMAT
RISAWRNDVN SMEGIVSKEA REILTQN

Homologs include, for example, Swiss-Prot. Accession No. Q9WVJ2, M. musculus 26S proteasome non-ATPase regulatory subunit 13 (26S proteasomesregulatory subunit S11) (26S proteasome regulatory subunit p40.5). Swiss-Prot. Q9UNM6, H. sapiens 26S proteasome non-ATPase regulatory subunit and Swiss Prot. Accession No. Q04062, S. cerevisiae Regulatory Particle Non-ATPase.

IIIS. TAF 6.1

The TAF 6.1 is part of an operon with w09b6.3 (an enhancer of RNAi) and expressed as a polypeptide fusion. This protein is well conserved and the human ortholog is Transcription initiation factor TFIID subunit 6.

LOCUS      NP_493919    470 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION TBP-Associated transcription Factor
           family member (52.7 kD) (taf-6.1)
           [Caenorhabditis eleqans].
ACCESSION  NP_493919
VERSION    NP_493919.1 GI: 17536589
DBSOURCE   REFSEQ: accession NM 061518.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
COMMENT    VALIDATED REFSEQ: This record has un-
           dergone preliminary review of the se-
           quence, but has not yet been subject to
           final review. This record is derived
           from an annotated genomic sequence
           (NC_003280). The reference sequence was
           derived from WormBase CDS: W09B6.2.
           Summary: This gene taf-6.1, also known
           as W09B6.2, 2B421 or YK5540, maps at
           (II; −12.83). It encodes a TBP-Associ-
           ated transcription Factor family
           member.
           According to the Worm Transcriptome
           Project, it is well expressed mostly
           in embryos, and some at all stages of
           development [Kohara cDNAs]. Its se-
           quence is fully supported by 8 cDNA
           clones.
           RNA interference results:
           [J. Ahringer 2003] No obvious phenotype
           (by feeding genomic PCR product JA:
           W09B6.2). Warning: this double stranded
           RNA may also interfere with gene 2B417.
           Function
           Protein properties: used to be called
           taf-3.
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 56%, L1 or L2 larvae 21%,
           L3 to adult 24%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           The CDS has 8 exons. It covers 4.31 kb
           on the WS97 genome. The protein (470
           aa, 52.7 kDa, pI 8.7) contains no Pfam
           motif. It is predicted to localise in
           the mitochondria [Psort2]. Taxblast
           (threshold 10{circumflex over ( )}-3) tracks ancestors down
           to eukaryota.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 470
                    /organism = “Caenorhabditis
                    elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “II”
                    /map = “II; −12.83 cM (inter-
                    polated genetic position)”
                    /map = “II; covering 4373 bp, from
                    base 1127981 to 1132355 on genome
                    release WS97”
                    /clone_lib = “Kohara embryonic
                    lambda gt11 library: yk314a6,
                    yk502e6, yk649h1, yk650b11,
                    yk670h10; Kohara Sugano L1 larvae
                    cap-selected library: yk1035e11,
                    yk1330h4; Kohara Sugano L4 larvae
                    cap-selected library: yk850e10”
           Protein  1 . . . 470
                    /product = “TBP-Associated trans-
                    cription Factor family member (52.7
                    kD) (taf-6.1)”
           Region   247 . . . 250
                    /region_name = “[PSORT] nuclear
                    localization domain: KKRH”
           CDS      1 . . . 470
                    /gene = “taf-6.1”
                    locus_tag = “2B421”
                    /coded_by = “NM_061518.1:
                    1 . . . 1413”
                    /db_xref = “AceView/WormGenes:
                    taf-6.1”
                    /db_xref = “GeneID: 173498”
                    /db_xref = “LocusID: 173498”
                    /db_xref = “WormBase: W09B6.2”
ORIGIN
1          msktvtirrp sptktseepa ahqtpiftqt
           aaemlgitsl nteaaellef lsreklkeiv
61         rlsakwtqks arrrmavadv ehairstrqc
           gglnissvdt lnlgiqqlqp iqgtstgiys
121        flkssadvdv dkedtetfik iprdlrvisy
           plvnegqpvq seytvnvded dgnffekivp
181        evmtmipekn tpsssttssl qmfrdavkta
           kidqkvglkp stieiltveq qifmkdiitv
241        cmgqddkkrh ealytletda glqvflphlt
           ericksisan isqrclslii yagrvlrsls
301        hnkacdmtvt lhhvlpalls ccvqrnmclr
           petdnhwalr dfsaktlvgl vrdqvdkhda
361        grtarrlfdf shrifrdtgs sfsmiygtvh
           ilqefvagpk kaawlltelg etnarckshi
421        esgsrigasq lsiqeaqkln qqilkcensi
           rnrynlqqqa pgvpinrrfh
//

Also see the C. elegans Protein Database: Wormpep at http://www.sanger.ac.uk/Projects/C_—elegans/wormpep/; Accesion No. ce05915.

IIIU. RRM Protein

MATSFYTGGGEDGDGFNPRVHARIAEREGFQLASGSEDPRTLFVANLDPA
ITDEFLATLFNQIGAVMKAKIIFEGLNDPYAFVEFSDHNQATLALQSHNG
RELLEKEMHVTWAFEPREPGENRSKPETSRHFHVFVGDLCSEIDSTKLRE
AFVKFGEVSEAKIIRDNNTNKGKGYGFVSYPRREDAERAIDEMNGAWLGR
RTIRTNWATRKPDEDGERGGDRGDRRGGGGGGRDRYHNQSEKTYDEIFNQ
AAADNTSVYVGNIANLGEDEIRRAFDRFGPINEVRTFKIQGYAFVKEETK
ESAARAIVQMNNADIGGQIVRCSWGKSGDSGKPSERGSGGGGGSGNYGYG
YGNSGGGGGSGGPGNSQFSNFNQRPPPSGNGGSGGGSGGQNNQYWQYYSQ
YYNNPHLMQQWNNYWQKDGPPPPPAAAASSTGGN

Also see the C. elegans Protein Database: Wormpep at http://www.sanger.ac.uk/Projects/C_—elegans/wormpep/; Accesion No. ce21988.

IIIV. Worm Unique/Novel

MQPVLVNSRPLRVKSHESESKLNLIEQEDQFEGANYSSSSGVIICYSNGT
GEVITQEAFDDSGIHFIFSKATCIQYPSNFDPIGVGSVVQIFWSRSFERV
VRGNHIIVQIEKMEVYKCCAMLREQVFVTFNSPSTAGVAIGVTERNITVA
FHPNGSPVIRYETLKAHSIGRTEFEIKDVREFEFSNGKNRHRENTNRMVD
VILAAVPFRVEIHGNVDKIPFFVIEKCRNSPGRSGAAVITKIMKNHFMEA
NFLQNSESIYFDSTSCHSNILEKVSIGSLINVLADPTFATSSYKWYGYDV
TLCNNYLAHASTQRSFVLENNEILQNCKKLEKSPEEAETTTKNDLRFVPP
QPEKGEVKKNELPEREAKSIINSYFIDRLAEGIKIEKIDKNWRTFGEILP
KTPKKYSESLKKSIQNVLEPFGLNKPEKAAETPKIVEYFPKNPKKRVEIV
EKPTVDEIRELFGALMDAEGFALNQRVKPHFVLPDTRWKPTERRYIGIYD
DVQWTFMSTFCPKIEENSENRPLAGGWWYRRTVPRDHPVEIVQKMETRRN
IIKDCTESPFIE

Also see the C. elegans Protein Database: Wormpep at http://www.sanger.ac.uk/Projects/C_—elegans/wormpep/; Accesion No. ce27223.

IIIW. TBB-4

LOCUS      NP_509585    444 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION tubulin, Beta (49.8 kD) (tbb-4)
           [Caenorhabditis elegans]
ACCESSION  NP_509585
VERSION    NP_509585.1 GI: 17549915
DBSOURCE   REFSEQ: accession NM 077184.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans.
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 444)
           AUTHORS  Maeda,I., Kohara,Y., Yamamoto,M. and
                    Sugimoto,A.
           TITLE    Large-scale analysis of gene function
                    in Caenorhabditis elegans by high-
                    throughput RNAi
           JOURNAL  Curr. Biol. 11 (3), 171-176 (2001)
           MEDLINE  21154836
           PUBMED   11231151
COMMENT    VALIDATED REFSEQ: This record has un-
           dergone preliminary review of the se-
           quence, but has not yet been subject to
           final review. This record is derived
           from an annotated genomic sequence
           (NC_003284). The reference sequence was
           derived from WormBase CDS: B0272.1.
           Summary: This essential gene tbb-4,
           also known as B0272.1, XK54 or YK4801,
           maps at (X; +1.30). Its phenotype is
           embryonic lethal, partial. It encodes a
           tubulin, Beta.
           According to the Worm Transcriptome
           Project, it is well expressed mainly in
           embryos and some in L1 and L2 larvae
           [Kohara cDNAs]. Its sequence is fully
           supported by 7 cDNA clones.
           RNA interference results:
           [J. Ahringer 2003] No obvious phenotype
           (by feeding genomic PCR product JA:
           B0272.1). [A. Sugimoto 2002] Embryonic
           lethal (20%) (by injecting cDNA clone
           SA: yk313f12).
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 82%, L1 or L2 larvae 14%,
           L3 to adult 4%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           The CDS has 7 exons. It covers 2.21 kb
           on the WS97 genome. The protein (444
           aa, 49.8 kDa, pI 4.8) contains one
           Tubulin/FtsZ protein motif, one
           Tubulin/FtsZ protein motif. It also
           contains a coil coil stretch [Psort2].
           It is predicted to localise in the
           cytoskeleton [Psort2]. Taxblast
           (threshold 10{circumflex over ( )}-3) tracks ancestors
           down to eukaryota.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 444
                    /organism = “Caenorhabditis
                    elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “X”
                    /map = “X; +1.30 cM (interpolated
                    genetic position)”
                    /map = “X; covering 2444 bp, from
                    base 9427407 to 9424964 on genome
                    release WS97”
                    /clone_lib = “Kohara embryonic
                    lambda gt11 library: yk230e11,
                    yk313f12, yk646g2, yk671e8,
                    yk674c9, yk80b7; Kohara Sugano L2
                    larvae cap-selected library:
                    yk1730e2”
           Protein  1 . . . 444
                    /product = “tubulin, Beta (49.8 kD)
                    (tbb-4)”
           Region   45 . . . 244
                    /region_name = “[Pfam/InterPro de-
                    scription] Tubulin/FtsZ protein”
                    /db_xref = “CDD: pfam00091”
           Region   246 . . . 383
                    /region_name = “[Pfam/InterPro de-
                    scription] Tubulin/FtsZ protein”
                    /db_xref = “CDD: pfam03953”
           Region   402 . . . 438
                    /region_name = “[PSORT] coil coil
                    domain:
                    GMDEMEFTEAESNMNDLVSEYQQYQEATADDEGEFDE”
           CDS      1 . . . 444
                    /gene = “tbb-4”
                    /locus_tag = “XK54”
                    /coded_by = “NM_077184.1:
                    1 . . . 1335”
                    /db_xref = “AceView/WormGenes: tbb-4”
                    /db_xref = “GeneID: 181170”
                    /db_xref = “LocusID: 181170“
                    /db_xref = “WormBase: B0272.1”
ORIGIN
1          mreivhiqag qcgnqigakf wevisdehgi
           dptgayngds dlqlerinvy yneasggkyv
61         praclvdlep qtmdsvragp fgqlfrpdnf
           vfgqsgagnn wakghytega elvdnvldvv
121        rkeaescdcl qgfqmthslg ggtgsgmgtl
           liskireeyp drimmtfsvv pspkvsdtvv
181        epynatlsvh qlventdetf cidnealydi
           cfrtlklttp tygdlnhlvs mtmsgvttcl
241        rfpgqlnadl rklavnmvpf prlhffmpgf
           apltsrgsqq yrsltvpelt qqmfdaknmm
301        aacdprhgry ltvaamfrgr msmkevdegm
           lnvqnknssy fvewipnnvk tavcdipprg
361        vkmaatfvgn staiqelfkr iseqftamfr
           rkaflhwytg egmdemefte aesnmndlvs
421        eyqqyqeata ddegefdehd qdve
//

IIIX. RPS-14

LOCUS      NP_498572    152 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION ribosomal Protein, Small subunit (16.2
           kD) (rps-14) [Caenorhabditis elegans].
ACCESSION  NP_498572
VERSION    NP_498572.1 GI: 17554776
DBSOURCE   REFSEQ: accession NM 066171.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 152)
           AUTHORS  Kamath,R. S., Fraser,A. G., Dong,Y.,
                    Poulin,G., Durbin,R., Gotta, M.
                    Kanapin,A., Le Bot,N., Moreno,S.,
                    Sohrmann,M., Welchman,D. P.,
                    Zipperlen,P. and Ahringer,J.
           TITLE    Systematic functional analysis of the
                    Caenorhabditis elegans genome using
                    RNAi
           JOURNAL  Nature 421 (6920), 231-237 (2003)
           MEDLINE  22417569
           PUBMED   12529635
REFERENCE  2 (residues 1 to 152)
           AUTHORS  Gonczy,P., Echeverri,C., Oegema,K.,
                    Coulson,A., Jones,S. J., Copley,R. R.,
                    Duperon,J., Oegema,J., Brehm,M.,
                    Cassin,E., Hannak,E., Kirkham,M.,
                    Pichler,S., Flohrs,K., Goessen,A.,
                    Leidel,S., Alleaume,A. M., Martin,C.,
                    Ozlu,N., Bork,P. and Hyman,A. A.
           TITLE    Functional genomic analysis of cell
                    division in C. elegans using RNAi of
                    genes on chromosome III
           JOURNAL  Nature 408 (6810), 331-336 (2000)
           MEDLINE  20548710
           PUBMED   11099034
COMMENT    VALIDATED REFSEQ: This record has un-
           dergone preliminary review of the se-
           quence, but has not yet been subject to
           final review. This record is derived
           from an annotated genomic sequence
           (NC_003281). The reference sequence was
           derived from WormBase CDS: F37C12.9.
           Summary: This essential gene rps-14,
           also known as F37C12.9, 3I268 or
           YK9313, maps at (III; −0.77). Pheno-
           types and affected processes are ab-
           normal cytoplasmic appearance, em-
           bryonic lethal, sterile adult, un-
           healthy, abnormal pseudocleavage. It
           encodes a ribosomal Protein, Small sub-
           unit. The product would be involved in
           pseudocleavage (sensu Nematoda). Ac-
           cording to the Worm Transcriptome
           Project, it is expressed at very high
           level at all stages of development
           [Kohara cDNAs]. Its sequence is fully
           supported by 144 cDNA clones.
           RNA interference results
           [T. Hyman; 2000] All embryos dead. DIC
           phenotype -- Multiple female pronuclei;
           irregular cytoplasmic appearance;
           karyomeres in daughter blastomeres;
           nuclei in AB are off-center for a
           while, nuclei in P1 stay close to
           posterior cortex for a while. Phenotype
           comment -- Semi-sterile. Phenotype con-
           firmed with independent dsRNA
           (F37C12.9-RNA2; similar phenotype) (by
           injecting genomic PCR product TH:
           330a9).
           Same description as TH: 330a9 (by in-
           jecting genomic PCR product TH: 340d4).
           [J. Ahringer 2003] Sterile, sick (by
           feeding genomic PCR product JA:
           F37C12.9).
           Function
           Protein properties: Orthologous to
           yeast (S.cerevisiae) ribosomal protein
           rps14 using blastP.
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 23%, L1 or L2 larvae 49%,
           L3 to adult 27%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           The CDS has 3 exons. It covers 0.55 kb
           on the WS97 genome. The protein (152
           aa, 16.2 kDa, pI 10.4) contains one
           ribosomal protein S11 motif. It is pre-
           dicted to localise in the cytoplasm
           [Psort2]. Taxblast (threshold 10{circumflex over ( )}-3)
           tracks ancestors down to archaea and
           bacteria and eukaryota.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 152
                    /organism = “Caenorhabditis elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “III”
                    /map = “III; −0.77 cM (interpolated
                    genetic position)”
                    /map = “III; covering 615 bp, from
                    base 7179511 to 7178897 on genome
                    release WS97”
           Protein  1 . . . 152
                    /product = “ribosomal Protein, Small
                    subunit (16.2 kD) (rps-14)”
           Region   3 . . . 9
                    /region_name = “[PSORT] nuclear lo-
                    calization domain: PARKGKA”
           Region   30 . . . 148
                    /region_name = “[Pfam/Interpro de-
                    scription] ribosomal protein S11”
                    /db_xref = “CDD: pfam00411”
           CDS      1 . . . 152
                    /gene = “rps-14”
                    /locus_tag = “31268”
                    /coded_by = “NM_066171.1:
                    1 . . . 459”
                    /db_xref = “AceView/WormGenes:
                    rps-14”
                    /db_xref = “GeneID: 176006”
                    /db_xref = “LocusID: 176006”
                    /db_xref = “WormBase: F37C12.9”
ORIGIN
1          maparkgkak eeqavvslgp qakegelifg
           vahifasfnd tfvhitdisg retivrvtgg
61         mkvkadrdes spyaamlaaq dvadrckqlg
           inalhiklra tggtrtktpg pgaqsalral
121        aragmkigri edvtpipsdc trrkggrrgr
           rl
//

IIIZ. RPS-13

LOCUS      NP_498393    151 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION ribosomal Protein, Small subunit (17.3
           kD) (rps-13) [Caenorhabditis elegans].
ACCESSION  NP_498393
VERSION    NP_498393.1 GI: 17554774
DBSOURCE   REFSEQ: accession NM 065992.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 151)
           AUTHORS  Kamath,R. S., Fraser,A. G., Dong,Y.,
                    Poulin,G., Durbin,R., Gotta,M.,
                    Kanapin,A., Le Bot,N., Moreno,S.,
                    Sohrmann,M., Welchman,D. P.,
                    Zipperlen,P. and Ahringer,J.
           TITLE    Systematic functional analysis of the
                    Caenorhabditis elegans genome using
                    RNAi
           JOURNAL  Nature 421 (6920), 231-237 (2003)
           MEDLINE  22417569
           PUBMED   12529635
REFERENCE  2 (residues 1 to 151)
           AUTHORS  Gonczy,P., Echeverri,C., Oegema,K.,
                    Coulson,A., Jones,S. J., Copley,R. R.,
                    Duperon,J., Oegema,J., Brehm,M.,
                    Cassin,E., Hannak, E., Kirkham,M.,
                    Pichler,S., Flohrs,K., Goessen,A.,
                    Leidel,S., Alleaume,A. M., Martin,C.,
                    Ozlu,N., Bork,P. and Hyman,A. A.
           TITLE    Functional genomic analysis of cell
                    division in C. elegans using RNAi of
                    genes on chromosome III
           JOURNAL  Nature 408 (6810), 331-336 (2000)
           MEDLINE  20548710
           PUBMED   11099034
COMMENT    VALIDATED REFSEQ: This record has un-
           dergone preliminary review of the se-
           quence, but has not yet been subject to
           final review. This record is derived
           from an annotated genomic sequence
           (NC_003281). The reference sequence was
           derived from WormBase CDS: C16A3.9.
           Summary: This essential gene rps-13,
           also known as C16A3.9, 3H464 or YK2267,
           maps at (III; −1.25). Its phenotype is
           embryonic lethal, sterile adult, abnor-
           mal pseudocleavage. It encodes a ribo-
           somal Protein, Small subunit. The pro-
           duct would be involved in pseudo-
           cleavage (sensu Nematoda). According to
           the Worm Transcriptome Project, it is
           expressed at high level in L3, L4,
           adult and culminating in embryos
           [Kohara cDNAs]. Its sequence is fully
           supported by 34 cDNA clones.
           RNA interference results
           [T. Hyman; 2000] All embryos dead. DIC
           phenotype -- Multiple female pronuclei;
           irregular cytoplasmic appearance;
           aberrant pseudocleavage stage; karyo-
           meres in daughter blastomeres; nuclei
           in AB are off-center for a while,
           nuclei in P1 stay close to posterior
           cortex for a while (by injecting geno-
           mic PCR product TH: 309g1). Movies are
           available on Hyman's site.
           [J. Ahringer 2003] Sterile (by feeding
           genomic PCR product JA: C16A3.9).
           Function
           Protein properties: Orthologous to
           yeast (S.cerevisiae) ribosomal protein
           rps13 using blastP.
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 81%, L1 or L2 larvae 1%, L3
           to adult 17%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           The CDS has 3 exons. It covers 0.85 kb
           on the WS97 genome. The protein (151
           aa, 17.3 kDa, pI 10.7) contains one
           Ribosomal protein S15 motif. It is pre-
           dicted to localise in the cytoplasm
           [Psort2]. Taxblast (threshold 10{circumflex over ( )}-3)
           tracks ancestors down to archaea and
           eukaryota.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 151
                    /organism = “Caenorhabditis elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “III”
                    /map = “III; −1.25 cM (interpolated
                    genetic position)”
                    /map = “III; covering 909 bp, from
                    base 6374934 to 6374026 on genome
                    release WS97”
                    /clone_lib = “Kohara embryonic
                    lambda gt11 library: yk74d8, yk96e4,
                    yk139e1, yk141e12, yk196b12,
                    yk269e10, yk319c12, yk329h11,
                    yk332c6, yk390g10, yk418c7, yk418e3,
                    yk432h1, yk433b12, yk436g3, yk467c6,
                    yk474h7, yk479b1, yk502h5, yk508h12,
                    yk533f12, yk538g7, yk572a6, yk623c2,
                    yk628b6, yk631e5, yk641b4, yk641h7,
                    yk666c1, yk668a5, yk627h1; mixed
                    stage, Stratagene library
                    [PMID1302005]: CEMSA36, CEMSH68;
                    Kohara mixed stage library, from
                    him-8 strain, containing 15-30%
                    males: yk304b4”
           Protein  1 . . . 151
                    /product = “ribosomal Protein, Small
                    subunit (17.3 kD) (rps-13)”
           Region   61 . . . 151
                    /region name = “[Pfam/InterPro de-
                    scription] ribosomal protein S15”
                    /db_xref = “CDD: pfam00312”
           CDS      1 . . . 151
                    /gene = “rps-13”
                    /locus_tag = “3H464”
                    /coded_by = “NM_065992.1:
                    1 . . . 456”
                    /db_xref = “AceView/WormGenes:
                    rps-13”
                    /db_xref = “GeneID: 175901”
                    /db_xref = “LocusID: 175901”
                    /db_xref = “WormBase: C16A3.9”
ORIGIN
1          mgrmhnpqkg maksaipyrr svpswqkmta
           eevqdqivkm akkglrpsqi gvilrdshgv
61         gqvrrlagnk ifrilkskgm apelpedlyh
           lvkkavairk hlersrkdid skyrlilves
121        rihrlaryyk tkrqlpptwk yesgtaaslv
           s
//

IIIAA. RPL-24

LOCUS      NP_491399    159 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION ribosomal Protein, Large subunit (17.8
           kD) (rpl-24.1) [Caenorhabditis elegans].
ACCESSION  NP_491399
VERSION    NP_491399.1 GI: 17506331
DESOURCE   REFSEQ: accession NM 058998.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 159)
           AUTHORS  Fraser,A. G., Kamath,R. S.,
                    Zipperlen,P., Martinez-Campos,M.,
                    Sohrmann,M. and Ahringer,J.
           TITLE    Functional genomic analysis of C.
                    elegans chromosome I by systematic RNA
                    interference
           JOURNAL  Nature 408 (6810), 325-330 (2000)
           MEDLINE  20548709
           PUBMED   11099033
REFERENCE  2 (residues 1 to 159)
           AUTHORS  Walhout,A. J., Sordella,R., Lu,X.,
                    Hartley,J. L., Temple,G. F.,
                    Brasch,M. A., Thierry-Mieg,N. and
                    Vidal,M.
           TITLE    Protein interaction mapping in C.
                    elegans using proteins involved in
                    vulval development
           JOURNAL  Science 287 (5450), 116-122 (2000)
           MEDLINE  20082953
           PUBMED   10615043
COMMENT    VALIDATED REFSEQ: This record has un-
           dergone preliminary review of the se-
           quence, but has not yet been subject to
           final review. This record is derived
           from an annotated genomic sequence
           (NC_003279). The reference sequence was
           derived from WormBase CDS: D1007.12.
           Summary: This essential gene rpl-24.1,
           also known as D1007.12, 1F153 or
           YK1971, maps at (I; −1.08). Its pheno-
           type is embryonic lethal, sterile
           adult. It encodes a ribosomal Protein,
           Large subunit. From Pfam homology, the
           product would be involved in protein
           biosynthesis and would localize in
           intracellular, ribosome.
           According to the Worm Transcriptome
           Project, it is expressed at very high
           level at all stages of development
           [Kohara cDNAs]. Its sequence is fully
           supported by 124 cDNA clones.
           RNA interference results
           [J. Ahringer 2000] embryonic lethal
           (100%), larval arrest, sterile (ma-
           ternal brood size 1 to 5) (by feeding
           genomic PCR product JA: D1007.12).
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 24%, L1 or L2 larvae 44%,
           L3 to adult 31%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           Interactions
           The protein encoded by this gene inter-
           acts with: protein LIN-15A: [Vidal M,
           pm10615043] interaction seen in a 2-
           hybrid screen, with lin-15a as bait.
           The CDS has 4 exons. It covers 1.00 kb
           on the WS97 genome. The protein (159
           aa, 17.8 kDa, pI 11.3) contains one
           Ribosomal protein L24E motif. It also
           contains a coil coil stretch, an ER
           membrane domain [Psort2]. It is pre-
           dicted to localise in the nucleus
           [Psort2]. Taxblast (threshold 10{circumflex over ( )}-3)
           tracks ancestors down to archaea and
           eukaryota.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 159
                    /organism = “Caenorhabditis elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome-“I”
                    /map = “I; −1.08 cM (interpolated
                    genetic position)”
                    /map = “I; covering 1100 bp, from
                    base 4585116 to 4586217 on genome
                    release WS97”
                    /clone_lib = “Kohara embryonic
                    lambda gt11 library: yk62c11,
                    yk63a1, yk74c6, yk78g2, yk79a4,
                    yk79g4, yk81c9, yk83e5, yk89b7,
                    yk96d9, yk103d8, yk138g11, yk172a2,
                    yk210c12, yk216g11, yk325h1,
                    yk375g5, yk401h10, yk424d6, yk449h5,
                    yk458b1, yk469h3, yk473a2, yk476f10,
                    yk479b3, yk483f11, yk486g10,
                    yk489g9, yk502b6, yk533d12,
                    yk602a11, yk606e6, yk667g3, yk505c9,
                    yk175d3, yk460a5; Kohara Sugano L1
                    larvae cap-selected library:
                    yk771e9, yk796g4, yk831f10,
                    yk1104c7, yk1310a12, yk874a12,
                    yk877b6, yk878b6, yk1006c9,
                    yk1072h3, yk1087d2, yk1098e12,
                    yk1129g2, yk1149e2, yk1165b8,
                    yk1166d2, yk1168d9, yk1181f2,
                    yk1193a10, yk1204f1, yk1219a9,
                    yk1235a12, yk1272d7, yk1298e3,
                    yk1320g11, yk1352a8, yk890c5,
                    yk1081e12, yk1067g3; Kohara Sugano
                    L2 larvae cap-selected library:
                    yk818c3, yk775h12, yk810g8,
                    yk816b10, yk1377d10, yk1386h10,
                    yk1407b1, yk1418d5, yk1579d7,
                    yk1583a8, yk1590g3, yk1600a2,
                    yk1608h5, yk1670f3, yk1365h1,
                    yk1381b7, yk1386b10, yk1390d12,
                    yk1401g11, yk1420a1, yk1510f8,
                    yk1517c6, yk1518f1, yk1578b5,
                    yk1581a9, yk1587g3, yk1592d5,
                    yk1610e10, yk1638c2, yk1667a11,
                    yk1699g11, yk1719h1, yk1720e12,
                    yk1722a8, yk1742e12, yk1756f2,
                    yk1493f7, yk1360e6; Kohara Sugano L4
                    larvae cap-selected library:
                    yk1437a7, yk1541g9, yk1685h11;
                    Kohara Sugano mixed stage cap-
                    selected library: yk732a10; mixed
                    stage, Stratagene library
                    [PMID1302005]: CEMSC16; Kohara mixed
                    stage library, from him-8 strain,
                    containing 15-30% males: yk70e3,
                    yk71b8, yk71g7, yk99e8, yk170h5,
                    yk206h1, yk361a7, yk379c9, yk545f5,
                    yk547e1, yk557f9, yk545f11; Marc
                    Vidal 2 hybrid library: mv508,
                    mv1325, mv1525, mv1326”
           Protein  1 . . . 159
                    /product = “ribosomal Protein, Large
                    subunit (17.8 kD) (rpl-24.1)”
           Region   1 . . . 71
                    /region_name = “[Pfam/InterPro de-
                    scription] ribosomal protein L24E”
                    /db_xref = “CDD: pfam01246”
           Region   60 . . . 76
                    /region_name = “[PSORT] nuclear
                    localization domain:
                    KKGTHGQEQVTRKKTKK”
           Region   104 . . . 136
                    /region_name = “[PSORT] coil coil
                    domain:
                    RRQQREQAAKIAKDANKAVRAAKAAANKEKKAS"
           Region   155 . . . 158
                    /region_name = “[PSORT] ER membrane
                    domain: VGGK”
           CDS      1 . . . 159
                    /gene = “rpl-24.1”
                    /locus_tag = “1F153”
                    /coded_by = “NM_058998.1:
                    1 . . . 480”
                    /db_xref = “AceView/WormGenes:
                    rpl-24.1”
                    /db_xref = “GeneID: 172062”
                    /db_xref = “LocusID: 172062”
                    /db_xref = “WormBase: D1007.12”
ORIGIN
1          mkvetcvysg ykihpghgkr lvrtdgkvqi
           flsgkalkga klrrnprdir wtvlyriknk
61         kgthgqeqvt rkktkksvqv vnravaglsl
           dailakrnqt edfrrqqreq aakiakdank
121        avraakaaan kekkasqpkt qqktaknvkt
           aaprvggkr
//
LOCUS      NP_492572    162 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION ribosomal Protein, Large subunit (18.8
           kD) (rpl-24.2) [Caenorhabditis
           elegans].
ACCESSION  NP_492572
VERSION    NP_492572.1 GI: 17505458
DBSOURCE   REFSEQ: accession NM 060171.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 162)
           AUTHORS  Fraser,A. G., Kamath,R. S.,
                    Zipperlen,P., Martinez-Campos,M.,
                    Sohrmann,M. and Ahringer,J.
           TITLE    Functional genomic analysis of C.
                    elegans chromosome I by systematic RNA
                    interference
           JOURNAL  Nature 408 (6810), 325-330 (2000)
           MEDLINE  20548709
           PUBMED   11099033
COMMENT    VALIDATED REFSEQ: This record has un-
           dergone preliminary review of the se-
           quence, but has not yet been subject to
           final review. This record is derived
           from an annotated genomic sequence
           (NC_003279).
           The reference sequence was derived from
           WormBase CDS: C03D6.8.
           Summary: This gene rpl-24.2, also known
           as C03D6.8, 1K245 or YK5780, maps at
           (I; +3.90). It encodes a ribosomal
           Protein, Large subunit. From Pfam
           homology, the product would be involved
           in protein biosynthesis and would lo-
           calize in intracellular, ribosome.
           According to the Worm Transcriptome
           Project, it is expressed at high level
           at all stages of development [Kohara
           cDNAs]. Its sequence is fully supported
           by 22 cDNA clones.
           RNA interference results
           [J. Ahringer 2000] slow growth (by
           feeding genomic PCR product JA:
           C03D6.1). Warning: this double stranded
           RNA may also interfere with gene 1K244.
           [J. Ahringer 2000] slow growth (by
           feeding genomic PCR product JA:
           C03D6.8). Warning: this double stranded
           RNA may also interfere with gene 1K244.
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 18%, L1 or L2 larvae 61%,
           L3 to adult 22%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           The CDS has 3 exons. It covers 0.59 kb
           on the WS97 genome. The protein (162
           aa, 18.8 kDa, pI 10.6) contains one
           Ribosomal protein L24E motif. It is
           predicted to localise in the nucleus
           [Psort2]. Taxblast (threshold 10{circumflex over ( )}-3)
           tracks ancestors down to archaea and
           eukaryota.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 162
                    /organism = “Caenorhabditis elegans”
                    /db_xrefr = “taxon: 6239”
                    /chromosome = “I”
                    /map = “I; +3.90 cM (interpolated
                    genetic position)”
                    /map = “I; covering 698 bp, from
                    base 9677465 to 9678164 on genome
                    release WS97”
                    /clone_lib = “Kohara embryonic
                    lambda gt11 library: yk331a1,
                    yk512c8, yk663g11, yk176h7; Kohara
                    Sugano L1 larvae cap-selected
                    library: yk753c12, yk772h12,
                    yk900d1, yk1127c1, yk1299f7,
                    yk1304b7, yk1057e1, yk1255f6,
                    yk1214c9, yk1159g10, yk1291g4,
                    yk871c5; Kohara Sugano L2 larvae
                    cap-selected library: yk1527g1,
                    yk1569d5, yk1605a7, yk1668g2; Kohara
                    mixed stage library, from him-8
                    strain, containing 15-30% males:
                    yk361d3, yk582d11”
           Protein  1 . . . 162
                    /product = “ribosomal Protein, Large
                    subunit (18.8 kD)
                    (rpl-24.2)”
           Region   1 . . . 71
                    /region_name = “[Pfam/InterPro de-
                    scription] ribosomal protein L24E”
                    /db_xref = “CDD: pfam01246”
           Region   41 . . . 57
                    /region_name = “[PSORT] nuclear lo-
                    calization domain:
                    KKKKNPRKLRFTKAARR”
           Region   43 . . . 59
                    /region_name = “[PSORT] nuclear lo-
                    calization domain:
                    KKNPRKLRFTKAARRAR”
           CDS      1 . . . 162
                    /gene = “rpl-24.2”
                    /locus_tag = “1K245”
                    /coded_by = “NM_060171.1:
                    1 . . . 489”
                    /db_xref = “AceView/WormGenes:
                    rpl-24.2”
                    /db_xref = “GeneID: 172815”
                    /db_xref = “LocusID: 172815”
                    /db_xref = “WormBase: C03D6.8”
ORIGIN
1          mriekcyfcs spiypghgiq fvrndstvfk
           fcrsrcnklf kkkknprklr ftkaarrarg
61         kelindatql leqrrdepvk yeramfqkti
           eaaktisalk tkrygnhlrk rlqpgkivqk
121        kgllakvdkk mhlirapvan rdgvktraaa
           kekktaesme tn
//

IIIBB. RPS-11

LOCUS      NP_502186    155 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION ribosomal Protein, Small subunit (17.7
           kD) (rps-11) [Caenorhabditis elegans].
ACCESSION  NP_502186
VERSION    NP_502186.1 GI: 17542016
DBSOURCE   REFSEQ: accession NM 069785.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 155)
           AUTHORS  Kamath,R. S., Fraser,A. G., Dong,Y.,
                    Poulin,G., Durbin,R., Gotta,M.,
                    Kanapin,A., Le Bot,N., Moreno,S.,
                    Sohrmann,M., Welchman,D. P.,
                    Zipperlen,P. and Ahringer,J.
           TITLE    Systematic functional analysis of the
                    Caenorhabditis elegans genome using
                    RNAi
           JOURNAL  Nature 421 (6920), 231-237 (2003)
           MEDLINE  22417569
           PUBMED   12529635
REFERENCE  2 (residues 1 to 155)
           AUTHORS  Piano,F., Schetter,A. J., Mangone,M.,
                    Stein,L. and Kemphues, K. J.
           TITLE    RNAi analysis of genes expressed in the
                    ovary of Caenorhabditis elegans
           JOURNAL  Curr. Biol. 10 (24), 1619-1622 (2000)
           MEDLINE  21065924
           PUBMED   11137018
COMMENT    VALIDATED REFSEQ: This record has un-
           dergone preliminary review of the se-
           quence, but has not yet been subject to
           final review. This record is derived
           from an annotated genomic sequence
           (NC_003282). The reference sequence was
           derived from WormBase CDS: F40F11.1.
           Summary: This essential gene rps-11,
           also known as F40F11.1, 4M367 or
           YK2226, maps at (IV; +5.45). Its pheno-
           type is sterile adult, unhealthy,
           catastrophic one cell arrest. It
           encodes a ribosomal Protein, Small sub-
           unit. From Pfam homology, the product
           would be involved in protein biosyn-
           thesis and would localize in intra-
           cellular, ribosome.
           According to the Worm Transcriptome
           Project, it is expressed at very high
           level at all stages of development
           [Kohara cDNAs]. Its sequence is
           fully supported by 87 cDNA clones.
           RNA interference results
           [F. Piano 2000] Embryonic lethal; egg
           production ceases in injected animal;
           catastrophic one-cell arrest (by in-
           jecting cDNA clone FP: SP13H3).
           [J. Ahringer 2003] Sterile, sick (by
           feeding genomic PCR product JA:
           F40F11.1).
           Function
           Protein properties: Orthologous to
           yeast (S.cerevisiae) ribosomal protein
           rps11 using blastP.
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 27%, L1 or L2 larvae 44%,
           L3 to adult 29%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           Pattern in ovary [F Piano, 2000].
           The CDS has 3 exons. It covers 0.57 kb
           on the WS97 genome. The protein (155
           aa, 17.7 kDa, pI 10.5) contains one
           Ribosomal protein S17 motif. It also
           contains a peroxisomal domain, an ER
           membrane domain [Psort2]. It is pre-
           dicted to localise in the cytoplasm
           [Psort2]. Taxblast (threshold 10{circumflex over (12 )}-3)
           tracks ancestors down to archaea and
           bacteria and eukaryota.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 155
                    /organism = “Caenorhabditis elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “IV”
                    /map = “IV; +5.45 cM (interpolated
                    genetic position)”
                    /map = “IV; covering 651 bp, from
                    base 11602617 to 11603269 on genome
                    release WS97”
                    /clone_lib = “Kohara embryonic
                    lambda gt11 library: yk67b1, yk89c6,
                    yk106b9, yk138a8, yk172e3, yk173e4,
                    yk258c6, yk258d2, yk273e4, yk290h1,
                    yk327b12, yk400d7, yk468h9, yk471b5,
                    yk477h6, yk485b4, yk500e2, yk521e9,
                    yk572f9, yk616b11, yk629c3,
                    yk639h12, yk644g7, yk646g7, yk647f7,
                    yk681e7, yk325e1, yk678h4; Kohara
                    Sugano L1 larvae cap-selected li-
                    brary: yk752e4, yk759c3, yk1292a10,
                    yk753e4, yk1019c9, yk883b2, yk892b7,
                    yk898a5, yk1011f1, yk1028c1,
                    yk1106c9, yk1304c12, yk1326e8,
                    yk1356h8, yk1207c9, yk871f6,
                    yk1169b3, yk1298a10, yk1246f10;
                    Kohara Sugano L2 larvae cap-selected
                    library: yk778e12, yk1636d2,
                    yk1593e1, yk1639a9, yk1576e5,
                    yk1674h10, yk1691b5, yk1359c2,
                    yk1414d7, yk1417e8, yk1417f11,
                    yk1418f7, yk1489g4, yk1520d6,
                    yk1521a3, yk1531b12, yk1567h6,
                    yk1572a9, yk1577g10, yk1639h8,
                    yk1650d4, yk1660f1, yk1671f12,
                    yk1718h2, yk1727a4, yk1741b8,
                    yk1706a1, yk1750b7; Kohara Sugano L4
                    larvae cap-selected library:
                    yk785e9, yk834a6, yk1439e1,
                    yk1545a1, yk1555g5, yk1442b4,
                    yk1552h12; Kohara mixed stage li-
                    brary, from him-8 strain, con-
                    taining 15-30% males: yk145g6,
                    yk205e11, yk361b5, yk380g2; Piano
                    ovary library: BE228125”
           Protein  1 . . . 155
                    /product = “ribosomal Protein, Small
                    subunit (17.7 kD) (rps-11)”
           Region   72 . . . 142
                    /region_name = “[Pfam/InterPro de-
                    scription] ribosomal protein S17”
                    /db_xref = “CDD: pfam00366”
           Region   86 . . . 102
                    /region_name = “[PSORT] nuclear lo-
                    calization domain:
                    RRDYLHYIKKYRRYEKR”
           Region   101 . . . 104
                    /region_name = “[PSORT] nuclear lo-
                    calization domain: KRHK”
           Region   151 . . . 154
                    /region_name = “[PSORT] ER membrane
                    domain: GFSK”
           Region   153 . . . 155
                    /region_name = “[PSORT] peroxisomal
                    domain: SKF”
           CDS      1 . . . 155
                    /gene = “rps-11”
                    /locus_tag = “4M367”
                    /coded_by = “NM_069785.1:
                    1 . . . 468”
                    /db_xref = “AceView/WormGenes:
                    rps-11”
                    /db_xref = “GeneID: 178083”
                    /db_xref = “LocusID: 178083”
                    /db_xref = “WormBase: P40F11.1”
ORIGIN
1          mseqterafl kqptvnlnnk arilagskkt
           pryirevglg fkaprdaveg tyidkkcpwa
61         gnvpirgmil tgvvlknkmt rtivvrrdyl
           hyikkyrrye krhknvpahc spafrdihpg
121        dlvtiqecrp lsktvrfnvl kvnksgtskk
           gfskf
//

IIICC. Agglutinin

MTTVRKTYRFCVFSSCLSVSCALVTQVHSSSLPIYSSPFVEKVFLHSSIY
VRLCGDMYEQWPTLEFSDLNSSILDLFTKATSQSVASSLLYELTRSDADE
NGGSIRLNNEEHLKWCMQVLNHSLTLSFATSREYETLKGAVRIYLHWLRA
LCDTPDNNIPTPLLATPEKYFRNIIDALRWIFCRREDDFDTTVGGQVPRG
LAIERQSIEIDMVLDSLKYLTRNSSRKYQDEVWARSISFLLNSSDILLSE
PNATEEMGTRTCVRVADTLFDMWLNAVLNEHIPSLTYWSSLATLARRWRH
NVPIIECWAKKILGLSTLVCRKMYGDDFLKIDIVDESVLPFENVPMTAEE
DENEVHLLYRTWFNMLCLFDSPAKILNHDATRNLCLNGNSPRRTTSSISM
SNFELASSSAAQGVSFFLAAVTLQRMVDLFYGDSRVKIDLRNYPVPDGKT
APNTRTASVLTDNHSHHTNRTSSTTGDSSRYVSLGGAVGQIIVDDHQVSM
SSGSTASGKTSTATGTSSTHTISSEIRRDQRIMSVNDRSRDPSHRTVSVT
DSVNISNQSRYSEQTSSTLTYKSAPIPETANENGHGESISQLVSNSTVSA
PVGGAGNDLTLKAGVHPSEMKIGRSSGVIGSAQHNNFYADTTSPYRSAQR
FVTNFLTANQATMPYVGGKRPKTDRMLNLVGDWLFAIVNSPTNSPRVTGN
DHSGHHKKNNDGVSDVSFISHHFVFTLLSAITTEVISIYICVSMISLTGL
NKHHLRIGIIDDETVCTSECPFSPFFAKFTITDGVDFLNNEADSKTTPTS
FDFDDFDSFHKFRFQHIYTSK

Also see the C. elegans Protein Database: Wormpep at http://www.sanger.ac.uk/Projects/C_—elegans/wormpep/; Accesion No. ce03050.

IIIDD. SIP-1 (hsp20)

Member of the Stress Induced Protein gene class.
MSSLCPYTGR PTGLFRDFED MMPYWAQRHS MLNNFNNIVP
QQLNEVENTA QKFCVKLDVA
AFKPEELKVN LEGHVLTIEG HHEVKTEHGF SKRSFTRQFT
LPKDVDLAHI HTVINKEGQM
TIDAPKTGSN TTVRALPIHT SAGHAVTQKP SSTTTTGKH

Homologs include, for example, Swiss-Prot. Accession No. P02511 , H. sapiens Alpha crystallin B chain.

IIIEE. CCT-6 (chaperonin)

LOCUS      NP_741153    539 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION chaperonin Containing TCP-1 (58.9 kD)
           (cct-6) [Caenorhabditis elegans].
ACCESSION  NP_741153
VERSION    NP_741153.1 GI: 25144678
DBSOURCE   REFSEQ: accession NM 171135.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 539)
           AUTHORS  Kamath,R. S., Fraser,A. G., Dong,Y.,
                    Poulin,G., Durbin,R., Gotta,M.,
                    Kanapin,A., Le Bot,N., Moreno,S.,
                    Sohrmann,M., Welchman,D. P.,
                    Zipperlen,P. and Ahringer,J.
           TITLE    Systematic functional analysis of the
                    Caenorhabditis elegans genome using
                    RNAi
           JOURNAL  Nature 421 (6920), 231-237 (2003)
           MEDLINE  22417569
           PUBMED   12529635
REFERENCE  2 (residues 1 to 539)
           AUTHORS  Gonczy,P., Echeverri,C., Oegema,K.,
                    Coulson,A., Jones,S. J., Copley,R. R.,
                    Duperon,J., Oegema,J., Brehm,M.,
                    Cassin,E., Hannak, E., Kirkham,M.,
                    Pichler,S., Flohrs,K., Goessen,A.,
                    Leidel,S., Alleaume,A. M., Martin,C.,
                    Ozlu,N., Bork,P. and Hyman,A. A.
           TITLE    Functional genomic analysis of cell di-
                    vision in C. elegans using RNAi of
                    genes on chromosome III
           JOURNAL  Nature 408 (6810), 331-336 (2000)
           MEDLINE  20548710
           PUBMED   11099034
REFERENCE  3 (residues 1 to 539)
           AUTHORS  Leroux,M. R. and Candido,E. P.
           TITLE    Characterization of four new tcp-1-
                    related cct genes from the nematode
                    Caenorhabditis elegans
           JOURNAL  DNA Cell Biol. 14 (11), 951-960 (1995)
           MEDLINE  96069542
           PUBMED   7576182
COMMENT    VALIDATED REFSEQ: This record has un-
           dergone preliminary review of the se-
           quence, but has not yet been subject to
           final review. This record is derived
           from an annotated genomic sequence
           (NC_003281). The reference sequence was
           derived from WormBase CDS: F01F1.8a.
           Summary: This essential gene cct-6,
           also known as F01F1.8, 3G944 or YK828,
           maps at (III; −1.53). Phenotypes and
           affected processes are embryonic
           lethal, sterile adult, unhealthy,
           clear, translucent appearance, pro-
           truding vulva, small embryos, slow
           embryonic cell division, cytokinesis
           defect, abnormal cytoplasmic appear-
           ance. It encodes a chaperonin Contain-
           ing TCP-1.
           According to the Worm Transcriptome
           Project, it is expressed at very high
           level at all stages of development
           [Kohara cDNAs]. Its sequence is fully
           supported by 122 cDNA clones and pro-
           duces, by alternative splicing, 2 dif-
           ferent transcripts a, b altogether en-
           coding 2 different protein isoforms.
           RNA interference results
           [T. Hyman; 2000] All embryos dead. DIC
           phenotype -- Semi-sterile; complex DIC
           phenotype; many embryos loose struc-
           tural integrity upon dissection; areas
           lacking yolk granules; failure in dif-
           ferent microtubule-based processes
           (centration/rotation, spindle assembly,
           chromosome segregation). DIC phenotype
           comment -- see also results from
           C07G2.3. Phenotype comment -- Confirmed
           with independent dsRNA (F01F1.8-RNA2;
           similar phenotype) (by injecting
           genomic PCR product TH: 304C1). Movies
           are available on Hyman's site.
           Same description as TH: 304C1 (by in-
           jecting genomic PCR product TH: 341B5).
           [J. Ahringer 2003] Embryonic lethal
           (100%), sterile, sick, clear, pro-
           truding vulva (by feeding genomic PCR
           product JA: F01P1.8).
           Function
           Protein properties: [C.elegansII] NMK.
           Encodes one of 7-9 related subunits of
           eukaryotic cytosolic chaperonin
           CCT.Ortholog of mouse Cctz (67% aa se-
           quence identity) [PC].
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 5%, L1 or L2 larvae 53%,
           L3 to adult 41%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           For a detailed expression pattern de-
           scription, see Wormbase Expr2045.
           The CDS has 6 exons. It covers 1.87 kb
           on the WS97 genome. The protein (539
           aa, 58.9 kDa, pI 5.9) contains one
           chaperonin Cpn60/TCP-1 motif. It is
           predicted to localise in the cytoplasm
           [Psort2]. Taxblast (threshold 10{circumflex over ( )}-3)
           tracks ancestors down to archaea and
           eukaryota.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 539
                    /organism = “Caenorhabditis elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “III”
                    /map = “III; −1.53 cM (interpolated
                    genetic position)”
                    /map = “III; covering 2163 bp, from
                    base 5855637 to 5853475 on genome
                    release WS97”
           Protein  1 . . . 539
                    /product = “chaperonin Containing
                    TCP-1 (58.9 kD) (cct-6)”
           Region   30 . . . 530
                    /region_name = “[Pfam/InterPro de-
                    scription] chaperonin Cpn60/TCP-1”
                    /db_xref = “CDD: pfam00118”
           CDS      1 . . . 539
                    /gene = “cct-6”
                    /locus_tag = “3G944”
                    /coded_by = “NM_171135.1:
                    1 . . . 1620”
                    /db_xref = “AceView/WormGenes:
                    cct-6”
                    /db_xref = “GeneID: 175819”
                    /db_xref = “LocusID: 175819”
                    /db_xref = “WormBase: F01F1.8a”
ORIGIN
1          mssiqclnpk aelarhaaal elnisgargl
           qdvmrsnlgp kgtlkmlvsg agdikltkdg
61         nvllhemaiq hptasmiaka staqddvtgd
           gttstvllig ellkqaeslv leglhprivt
121        egfewantkt lellekfkke apverdllve
           vcrtalrtkl hqkladhite cvvdavlair
181        rdgeepdlhm vekmemhhds dmdttlvrgl
           vldhgarhpd mprhvkdayi ltcnvsleye
241        ktevnsglfy ktakereall aaerefitrr
           vhkiielkkk vidnspdgkn kgfvvinqkg
301        idppsldlla segilalrra krrnmerlql
           avggeavnsv ddltpedlgw aglvyehslg
361        eekytfieec rapksvtlli kgpnkhtitq
           ikdaihdglr avfntivdka vlpgaaafei
421        aayvmlkkdv enlkgraklg aeafaqallv
           ipktlavngg ydaqetlvkl ieektaagpd
481        iavgldletg gavepqgiwd nvtvkknsis
           satvlacnll lvdevmragm tnlkqpqpe
//
LOCUS      NP_741154    429 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION chaperonin Containing TCP-1 (cct-6)
           [Caenorhabditis elegans].
ACCESSION  NP_741154
VERSION    NP_741154.1 GI: 25144680
DBSOURCE   REFSEQ: accession NM 171136.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 429)
           AUTHORS  Kamath,R. S., Fraser,A. G., Dong,Y.,
                    Poulin,G., Durbin,R., Gotta,M.,
                    Kanapin,A., Le Bot,N., Moreno,S.,
                    Sohrmann,M., Welchman,D. P.,
                    Zipperlen,P. and Ahringer,J.
           TITLE    Systematic functional analysis of the
                    Caenorhabditis elegans genome using
                    RNAi
           JOURNAL  Nature 421 (6920), 231-237 (2003)
           MEDLINE  22417569
           PUBMED   12529635
REFERENCE  2 (residues 1 to 429)
           AUTHORS  Gonczy,P., Echeverri,C., Oegema,K.,
                    Coulson,A., Jones,S. J., Copley,R. R.,
                    Duperon,J., Oegema,J., Brehm,M.,
                    Cassin,E., Hannak, E., Kirkham,M.,
                    Pichler,S., Flohrs,K., Goessen,A.,
                    Leidel,S., Alleaume,A. M., Martin,C.,
                    Ozlu,N., Bork,P. and Hyman,A. A.
           TITLE    Functional genomic analysis of cell di-
                    vision in C. elegans using RNAi of
                    genes on chromosome III
           JOURNAL  Nature 408 (6810), 331-336 (2000)
           MEDLINE  20548710
           PUBMED   11099034
REFERENCE  3 (residues 1 to 429)
           AUTHORS  Leroux,M. R. and Candido,E. P.
           TITLE    Characterization of four new tcp-1-
                    related cct genes from the nematode
                    Caenorhabditis elegans
           JOURNAL  DNA Cell Biol. 14 (11), 951-960 (1995)
           MEDLINE  96069542
           PUBMED   7576182
COMMENT    PROVISIONAL REFSEQ: This record has not
           yet been subject to final NCBI review.
           This record is derived from an an-
           notated genomic sequence (NC_003281).
           The reference sequence was derived from
           WormBase CDS: F01F1.8b.
           Summary: This essential gene cct-6,
           also known as F01F1.8, 3G944 or YK828,
           maps at (III; −1.53). Phenotypes and
           affected processes are embryonic
           lethal, sterile adult, unhealthy,
           clear, translucent appearance, pro-
           truding vulva, small embryos, slow
           embryonic cell division, cytokinesis
           defect, abnormal cytoplasmic appear-
           ance. It encodes a chaperonin
           Containing TCP-1.
           According to the Worm Transcriptome
           Project, it is expressed at very high
           level at all stages of development
           [Kohara cDNAs]. Its existence, but not
           its exact sequence, derived here from
           the genome sequencing consortium an-
           notation, is supported by 122 cDNA
           clones. It would produce, by alterna-
           tive splicing, 2 different transcripts
           a, b altogether encoding 2 different
           protein isoforms.
           RNA interference results
           [T. Hyman; 2000] All embryos dead. DIC
           phenotype -- Semi-sterile; complex DIC
           phenotype; many embryos loose struc-
           tural integrity upon dissection; areas
           lacking yolk granules; failure in dif-
           ferent microtubule-based processes
           (centration/rotation, spindle assembly,
           chromosome segregation). DIC phenotype
           comment -- see also results from
           C07G2.3. Phenotype comment -- Confirmed
           with independent dsRNA (F01F1.8-RNA2;
           similar phenotype) (by injecting geno-
           mic PCR product TH: 304C1). Movies are
           available on Hyman's site.
           Same description as TH: 304C1 (by in-
           jecting genomic PCR product TH: 341B5).
           [J. Ahringer 2003] Embryonic lethal
           (100%), sterile, sick, clear, pro-
           truding vulva (by feeding genomic PCR
           product JA: F01F1.8).
           Function
           Protein properties: [C.elegansII] NMK.
           Encodes one of 7-9 related subunits of
           eukaryotic cytosolic chaperonin
           CCT.Ortholog of mouse Cctz (67% aa
           sequence identity) [PC].
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 5%, L1 or L2 larvae 53%,
           L3 to adult 41%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           For a detailed expression pattern de-
           scription, see Wormbase Expr2045.
           The predicted CDS has 6 exons. It
           covers 1.54 kb on the WS97 genome. The
           protein (429 aa, 47.6 kDa, pI 6.3)
           contains one chaperonin Cpn60/TCP-1
           motif. It also contains an ER membrane
           domain [Psort2]. It is predicted to
           localise in the cytoplasm [Psort2].
           Taxblast (threshold 10{circumflex over ( )}-3) tracks
           ancestors down to archaea and
           eukaryota.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 429
                    /organism = “Caenorhabditis elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “III”
                    /map = “III; −1.53 cM (interpolated
                    genetic position)”
                    /map = “III; covering 2163 bp, from
                    base 5855637 to 5853475 on genome
                    release WS97”
           Protein  1 . . . 429
                    /product = “chaperonin Containing
                    TCP-1 (cct-6)”
           Region   30 . . . 428
                    /region_name = “[Pfam/InterPro de-
                    scription] chaperonin Cpn60/TCP-1”
                    /db_xref = “CDD: pfam00118”
           Region   425 . . . 428
                    /region_name = “[PSORT] ER membrane
                    domain: VEKR”
           CDS      1 . . . 429
                    /gene = “cct-6”
                    /locus_tag = “3G944”
                    /coded_by = “NM_171136.1:
                    1 . . . 1290”
                    /db_xref = “AceView/WormGenes:
                    cct-6”
                    /db_xref = “GeneID: 175819”
                    /db_xref = “LocusID: 175819”
ORIGIN
1          mssiqclnpk aelarhaaal elnisqargl
           qdvmrsnlgp kgtlkmlvsg agdikltkdg
61         nvllhemaiq hptasmiaka staqddvtgd
           gttstvllig ellkqaeslv leqlhprivt
121        egfewantkt lellekfkke apverdllve
           vcrtalrtkl hqkladhite cvvdavlair
181        rdgeepdlhm vekmemhhds dmdttlvrgl
           vldhgarhpd mprhvkdayi ltcnvsleye
241        ktevnsqlfy ktakereall aaerefitrr
           vhkiielkkk vidnspdgkn kgfvvinqkg
301        idppsldlla segilalrra krrnmerlql
           avggeavnsv ddltpedlgw aglvyehslg
361        eekytfieec rapksvtlli kgpnkhtitq
           ikdaihdglr avfntivdsc spwsccfrnc
421        clrdvekrc

IIIFF. RDE-4

The RDE-4 protein is structurally related to drosophila R2D2 and the human TAR binding protein with conservation in the dsRBDs motifs.

LOCUS      CAA83012    385 aa    linear  INV 23-
FEBRUARY
2005
DEFINITION Hypothetical protein T20G5.11
           [Caenorhabditis elegans].
ACCESSION  CAA83012
VERSION    CAA83012.1 GI: 458490
DESOURCE   embl locus CET20G5, accession Z30423.2
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 385)
           AUTHORS  .
           CONSRTM  WormBase Consortium
           TITLE    Genome sequence of the nematode C.
                    elegans: a platform for investigating
                    biology
           JOURNAL  Science 282 (5396), 2012-2018 (1998)
           PUBMED   9851916
REFERENCE  2 (residues 1 to 385)
           AUTHORS  Berks,M., Lloyd,C. R. and Smith,A.
           TITLE    Direct Submission
           JOURNAL  Submitted (07-MAR-1994) Nematode Se-
                    quencing Project, Sanger Institute,
                    Hinxton, Cambridge CB10 1SA, England
                    and Department of Genetics, Washington
                    University, St. Louis, MO 63110, USA.
                    E-mail: worm@sanger.ac.uk
COMMENT    Coding sequences below are predicted
           from computer analysis, using predic-
           tions from Genefinder (P. Green, U.
           Washington), and other available in-
           formation.
           Current sequence finishing criteria for
           the C. elegans genome sequencing con-
           sortium are that all bases are either
           sequenced unambiguously on both
           strands, or on a single strand with
           both a dye primer and dye terminator
           reaction, from distinct subclones.
           Exceptions are indicated by an explicit
           note.
           IMPORTANT: This sequence is NOT neces-
           sarily the entire insert of the spec-
           ified clone. It may be shorter because
           we only sequence overlapping sections
           once, or longer because we arrange for
           a small overlap between neighbouring
           submissions.
           This sequence is the entire insert of
           clone T20G5. The start of this sequence
           (1 . . . 100) overlaps with the end of
           sequence Z30974. The end of this se-
           quence (47996 . . . 48095) overlaps
           with the start of sequence AL032660.
           For a graphical representation of this
           sequence and its analysis see:-
           http://www.wormbase.org/perl/ace/
           elegans/seq/sequence?
name = ZK1321; class = Sequence.
FEATURES   Location/Qualifiers
           source   1 . . . 385
                    /organism = “Caenorhabditis elegans”
                    /strain = “Bristol N2”
                    /db_xref = “taxon: 6239”
                    /chromosome = “III”
                    /clone = “T20G5”
           Protein  1 . . . 385
                    /product = “Hypothetical protein
                    T20G5.11”
           CDS      1 . . . 385
                    /gene = “rde-4”
                    /locus_tag = “T20G5.11”
                    /standard_name = “T20G5.11”
                    /coded_by = “complement(join
                    (Z30423.2: 45951 . . . 46375,
                    Z30423.2: 46424 . . . 46544,
                    Z30423.2: 46589 . . . 46820,
                    Z30423.2: 46870 . . . 47249))”
                    /note = “C. elegans RDE-4 protein;
                    contains similarity to Pfam domain
                    PF00035 (Double-stranded RNA binding
                    motif)”
                    /db_xref = “GOA: Q22617”
                    /db_xref = “InterPro: IPR001159”
                    /db_xref = “UniProt/TrEMBL: Q22617”
ORIGIN
1          mdltkltfes vfggsdvpmk psrsednktp
           rnrtdlemfl kktplmvlee aakavyqktp
61         twgtvelpeg femtlilnei tvkgqatskk
           aarqkaavey lrkvvekgkh eiffipgttk
121        eealsnidqi sdkaeelkrs tsdavqdndn
           ddsiptsaef ppgisptenw vgklqeksqk
181        sklqapiyed sknerterfl victmcnqkt
           rgirskkkda knlaawlmwk aledgiesle
241        sydmvdvien leeaehllei qdqaskikdk
           hsalidilsd kkrfsdysmd fnvlsvstmg
301        ihqvlleisf rrlvspdpdd lemgaehtqt
           eeimkataek eklrkknmpd sgplvfaghg
361        ssaeeakqca cksaiihfnt ydftd

IIIGG. DRH-3 (D2005.5)

The DRH-3 protein now has been officially renamed DRH-3, this protein is a paralog of DRH-1 and DRH-2 which are essential for RNAi and have a human ortholog: melanoma differentiation associated protein-5.

LOCUSCAB02082    1119 aa    linear  INV 23-FEBRUARY2005DEFINITIONHypothetical protein D2005.5[Caenorhabditis elegans].ACCESSIONCAB02082VERSIONCAB02082.3 GI: 38422755DBSOURCEembl locus CED2005, accession Z79752.2KEYWORDS.SOURCECaenorhabditis elegansORGANISMCaenorhabditis elegansEukaryota; Metazoa; Nematoda;Chromadorea; Rhabditida; Rhabditoidea;Rhabditidae; Peloderinae;Caenorhabditis.REFERENCE1 (residues 1 to 1119)AUTHORS.CONSRTMWormBase ConsortiumTITLEGenome sequence of the nematode C.elegans: a platform for investigatingbiologyJOURNALScience 282 (5396), 2012-2018 (1998) PUBMED9851916REFERENCE2 (residues 1 to 1119)AUTHORSWilkinson, J.TITLEDirect SubmissionJOURNALSubmitted (04-SEP-1996) Nematode Se-quencing Project, Sanger Institute,Hinxton, Cambridge CB10 1SA, Englandand Department of Genetics, WashingtonUniversity, St. Louis, MO 63110, USA.E-mail: worm@sanger.ac.ukCOMMENTOn Nov. 18, 2003 this sequence versionreplaced gi: 21615449. Coding sequencesbelow are predicted from computeranalysis, using predictions from Gene-finder (P. Green, U. Washington), andother available information.Current sequence finishing criteria forthe C. elegans genome sequencing con-sortium are that all bases are eithersequenced unambiguously on bothstrands, or on a single strand withboth a dye primer and dye terminatorreaction, from distinct subclones.Exceptions are indicated by anexplicit note.IMPORTANT: This sequence is NOT neces-sarily the entire insert of the spec-ified clone. It may be shorter becausewe only sequence overlapping sectionsonce, or longer because we arrange fora small overlap between neighbouringsubmissions.IMPORTANT: This sequence is not theentire insert of clone D2005. It maybe shorter because we only sequenceoverlapping sections once, or longerbecause we arrange for a small overlapbetween neighbouring submissions.The true left end of clone D2005 is at1 in this sequence. The true right endof clone D2005 is at 104 in sequenceZ81073. The true left end of cloneF30F8 is at 43337 in this sequence.The start of this sequence(1 . . . 104) overlaps with the end ofsequence AL033124. The end of thissequence (43337 . . . 43440) overlapswith the start of sequence Z81073.For a graphical representation of thissequence and its analysis see:-http://www.wormbase.org/perl/ace/elegans/seq/sequence?name = ZK1321; class = Sequence.FEATURESLocation/Qualifierssource1 . . . 1119/organism = “Caenorhabditis elegans”/strain = “Bristol N2”/db_xref = “taxon: 6239”/chromosome = “I”/clone = “D2005”Protein1 . . . 1119/product = “Hypothetical proteinD2005.5”CDS1 . . . 1119/locus_tag = “D2005.5”/standard_name = “D2005.5”/coded_by = “join(Z79752.2:37322 . . . 37419, Z79752.2:37479 . . . 37652, Z79752.2:37906 . . . 37981, Z79752.2:38029 . . . 38110, Z79752.2:38156 . . . 38680, Z79752.2:38868 . . . 38993, Z79752.2:39040 . . . 39221, Z79752.2:39303 . . . 39484, Z79752.2:39682 . . . 40007, Z79752.2:40444 . . . 40724, Z79752.2:40768 . . . 41676, Z79752.2:42116 . . . 42216, Z79752.2:42273 . . . 42411, Z79752.2:42459 . . . 42565, Z79752.2:42668 . . .42719)”/note = “contains similarity to Pfamdomains PF00270 (DEAD and DEAH boxhelicases), PF00271 (Helicases con-served C-terminal domain)”/db_xref = “GOA: Q93413”/db_xref = “InterPro: IPR001410”/db_xref = “InterPro: IPR001650”/db_xref = “InterPro: IPR011545”/db_xref = “UniProt/TrEMBL: Q93413”ORIGIN  1mqptairled ydksklrlpf espyfpayfrllkwkfldvc vestrnndig yfklfeslfp 61pgkleeiarm iideptpvsh dpdmikirnadldvkirkqa etyvtlrhah qqkvqrrrfs 121ecflntvlfd ekglriadev mfnydkelygyshwedlpdg wltaetfknk fydeeevtnn 181pfgyqkldrv agaargmiim khlksnprcvsettilafev fnkgnhqlst dlvedllteg 241pafelkieng eekkyavkkw slhktltmflaiigfksndk kekneheewy ygfidamknd 301panraalyfl dknwpeelee rekerdrirltllksqrtne eavgedvctt irpqpkdsgy 361npdavvtelv lrtyqeelvq palegkncvivaptgsgkte vaiyaalkhi eertsqgkps 421rvvllvpkip lvgqqkdrfl kycngmyevngfhgsessvs gtgrrdevia thvsvmtpqi 481linmlqsvrq nerlyvsdfs mmifdevhkaaknhpyvlin qmvqewkyek pqiigltasl 541svkvdgqkde nqmlndiynm lalinaphlstitrqsside lnehvgkpdd svelclpake 601nilrdyiery lnhahgkfle elasmskstgrnntippnmi ntfkknqpkn yeyydsllqg 661iiqelnklnv pekwnsqtwa kymkvyleargivdlmpamv afkymekaig klneshsetv 721eystfikdhd tlkqtiqsve peivlrlkntltnqfhvape srviifvtqr staqrvsdfl 781neskvldqfg nygeqmvgyv lgtnkqgavqqtsqeqqltl dkfnngrlkv ivatsvveeg 841ldvtacnlii kyncssgsai qlvqqrgraraknsrsvlls vkssinetet nalisekymr 901lcvkkiteng ekqlaaevkr vaelnaaerkrnleeqlnlr lrhenkiykl mcsncskefc 961ksiyikkvfs nymvfdpsvw rflhveskrkvskylsednq plsdikcfhc kldvgrayki1021rgtylpqlsv kaltfvqesd yssmtkakwsdveqdlfyis eaieddfrim lnalsdteen1081iekkivldld srqhnkqlem krfhiqqepptkgvapeaq

IIIHH. ERI-1

The ERI-1 protein is conserved and enhances RNAi and has a human homolog: AAH35279.

LOCUS      T32581    562 aa    linear  INV 18-
NOVEMBER
2002
DEFINITION hypothetical protein T07A9.10 -
           Caenorhabditis elegans.
ACCESSION  T32581
VERSION    T32581 GI: 7507339
DBSOURCE   pir: locus T32581;
           summary: #length 562 #molecular-weight
           64656 #checksum 867;
           genetic: #gene CESP: T07A9.10
           #map_position 4 #introns 9/1; 54/1;
           218/1; 258/3; 349/1; 432/3; 516/1;
           superfamily: vacuolar protein sorting
           protein VPS45;
           PIR dates: 29-Oct-1999 #sequence—
           revision 29-Oct-1999 #text_change
           18-Nov-2 002.
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 562)
           AUTHORS  Scheet,P. and Maggi,L.
           TITLE    Direct Submission
           JOURNAL  Submitted (??-DEC-1997) to the EMBL
                    Data Library
FEATURES   Location/Qualifiers
           source   1 . . . 562
                    /organism = “Caenorhabditis elegans”
                    /db_xref = “taxon: 6239”
           Protein  1 . . . 562
                    /product = “hypothetical protein
                    T07A9.10”
ORIGIN
1          mlrelvkkqi ienilrpqny dsklghrkfs
           vlvldksamv vvnsclslne vfeegvtlve
61         dltrnrepmp smdaiyiisp vaesidilin
           dfsrktkfnp gnsyrsahif fldpccdelf
121        eklskspavk wiktlkelnl nlkpvesqif
           tvnsqfrgdm tktadgivsl catlnihptl
181        rfqsdfaqss eicqrveqkl kefgnegmgt
           daelvvldrs fdlvspllhe vtlqamvvdv
241        tafkdgvyry teagdskeiv ldekdqnwld
           lrhkllpevm ksvnkmvkdf kntnktepen
301        iknqsskdfs ttvrtlqpyl kmkakmaayi
           slteecrsky fdslekiial eqdmavehtp
361        ehvritdsqa vgrlstfilp aiptetrlrl
           ilifmltigk dkdeqyfnrl lhhtdipese
421        fqiikrmliw rdktqksqfq hrrpppeder
           fiasrwdpki knlieeiyer rlderefkva
481        gkkstsdfrp aasarygsgl agkprekrki
           iifvvggity semrvayels kktnttvilg
541        sdeiltpssf leslrdrntv nc

III. RRF-3

This protein is also conserved in S. pombe and many plants.

LOCUS      CAA88315    1780 aa    linear  INV 22-
MARCH 2005
DEFINITION Hypothetical protein F10B5.7
           [Caenorhabditis elegans].
ACCESSION  CAA88315
VERSION    CAA88315.1 GI: 3875716
DESOURCE   embl locus CEF10B5, accession Z48334.1
           embl locus CET05C12, accession Z66500.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE 1 (residues 1 to 1780)
           AUTHORS  .
           CONSRTM  C. elegans Sequencing Consortium
           TITLE    Genome sequence of the nematode C.
                    elegans: a platform for investigating biology
           JOURNAL  Science 282 (5396), 2012-2018 (1998)
           PUBMED   9851916
REFERENCE  2 (residues 1 to 1780)
           AUTHORS  Sims,M. A.
           TITLE    Direct Submission
           JOURNAL  Submitted (16-FEB-1995) Nematode Se-
                    quencing Project, Sanger Institute,
                    Hinxton, Cambridge CB10 1SA, England
                    and Department of Genetics, Washington
                    University, St. Louis, MO 63110, USA.
                    E-mail: worm@sanger.ac.uk
COMMENT    Coding sequences below are predicted
           from computer analysis, using predic-
           tions from Genefinder (P. Green, U.
           Washington), and other available
           information.
           Current sequence finishing criteria for
           the C. elegans genome sequencing con-
           sortium are that all bases are either
           sequenced unambiguously on both
           strands, or on a single strand with
           both a dye primer and dye terminator
           reaction, from distinct subclones.
           Exceptions are indicated by an
           explicit note.
           IMPORTANT: This sequence is NOT neces-
           sarily the entire insert of the speci-
           fied clone. It may be shorter because
           we only sequence overlapping sections
           once, or longer because we arrange for
           a small overlap between neighbouring
           submissions.
           IMPORTANT: This sequence is not the
           entire insert of clone F10B5. It may be
           shorter because we only sequence over-
           lapping sections once, or longer be-
           cause we arrange for a small overlap
           between neighbouring submissions.
           The true left end of clone F10B5 is at
           1 in this sequence. The true right end
           of clone F10B5 is at 15182 in sequence
           Z66500. The true left end of clone
           T05C12 is at 29032 in this sequence.
           The true right end of clone C41C4 is
           at 2219 in this sequence. The start of
           this sequence (1 . . . 99) overlaps
           with the end of sequence Z48045.
           The end of this sequence
           (29032 . . . 29132) overlaps with the
           start of sequence Z66500. For a
           graphical representation of this
           sequence and its analysis see:-
           http:.//www.wormbase.org/perl/ace/
           elegans/seq/sequence?
           name = ZK1321; class = Sequence.
FEATURES   Location/Qualifiers
           source   1 . . . 1780
                    /organism = “Caenorhabditis elegans”
                    /strain = “Bristol N2”
                    /db_xref = “taxon: 6239”
                    /chromosome = “II”
                    /clone = “F10B5”
           Protein  1 . . . 1780
                    /product = “Hypothetical protein
                    F10B5.7”
           CDS      1 . . . 1780
                    /gene = “rrf-3”
                    /locus_tag = “F10B5.7”
                    /standard_name = “F10B5.7”
                    /coded_by = “join(Z48334.1:
                    23435 . . . 23502, Z48334.1:
                    23558 . . . 24167, Z48334.1:
                    24214 . . . 24449, Z48334.1:
                    24497 . . . 24610, Z48334.1:
                    24661 . . . 25018, Z48334.1:
                    25064 . . . 25883, Z48334.1:
                    25931 . . . 26489, Z48334.1:
                    26532 . . . 26743, Z48334.1:
                    26790 . . . 27477, Z48334.1:
                    27526 . . . 28249, Z48334.1:
                    28294 . . . 28751, Z48334.1:
                    28797 . . . 28902, Z48334.1:
                    28954 . . . 29132, Z66500.1:
                    102 . . . 114, Z66500.1:
                    161 . . . 358)”
                    /note = “C. elegans RRF-3 protein;
                    contains similarity to Pfam domain
                    PF05183 (RNA dependent RNA
                    polymerase)”
                    /db_xref = “InterPro: IPR007855”
                    /db_xref = “UniProt/TrEMBL: Q19285”
ORIGIN
1          mlpfdnddss ddattsvrpk hprgvpqsqs
           tfprgrsnfs sgtlpnrkte ctpvntltig
61         hsnkmllttf rmdrnsksks evdvqeqpvh
           ssssafpgnh lnnfsypvnr gylrdyllqs
121        qrpstskpvd csvlkrhslp sthilyektk
           hrggvnieeq eklvrmlwaa aeesetvakt
181        rqfskkqaie lnfdakligs mnndcfgycr
           ahmenikdvl kthlklskvd evnwikvgmv
241        praayedksy vidahlvltp ngevedenel
           fsefassfts ritgmlhdqv flevpkmhtl
301        ftkitpqhmd inisaiaign cpnsglflvr
           gdfisqentv csvklqshhn adasrenssf
361        kvagsnkyls yarfehdkrl avvyfgvrla
           efaddgldha gfrlnlyynl fvrivvdmsh
421        ettnsiyiqm knpphlwegi pkntifhpsk
           skvlnmetct ewtrvlswpg daegrgvgct
481        seafsqsswi rltmrkdddn dsvsstqlmd
           ivtrlsarsk akvmfgsifs irrklapspa
541        fhslgsfran yalqalitrg svftdqlfda
           tdenipssdn dndedddddv ddtkkpmelv
601        heplflklvr rgmkecsqat eetleqllna
           fderrqidvv tafttmyqsr kiqyerllkg
661        eslqdvglak plpkncvsva kvivtpsril
           lmapevmmvn rvvrrfgpdy alrcvfrddn
721        lgrlairdfs innidhmsni vtegiyltlk
           nqiqvadrvy sflgwsnsqm rdqqcylyap
781        rvnaltgevt gtvedirvwm gdfrdaisvp
           kmmsrmgqcf tqaqptvyss vknihiveni
841        qvrlerhhwi vepdieggve nkycfsdgcg
           risiklathi skilqlkevp acfqvrfkgf
901        kgilvidpti ddiinmpkvi frksqqkfge
           qggelqdeyi evvkyampsp vclnrpfiti
961        ldqvsekqsa sshrritnrv hyylerelcs
           lsnmlinenq aaeelvnrtn laidwnaask
1021       ragfelsvdp lirdmlfsiy ryniihhisk
           akiflppslq rsmyqvvdet gllqyqqvfi
1081       qyspsirqts nrpilktgkv litknpchvp
           gdvrvfdavw qpalahlvdv vvfpqhgprp
1141       hpdemagsdl dqdeysiiwd qemlldynee
           amvfpsssaa eedkepttdd mvefflrylq
1201       qdsigrmsha hlayadlhgl fhenchaial
           kcavavdfpk sgvpaeplss feqcemtpdy
1261       mmsggkpmyy strlngqlhr karkveevle
           efetrgsvfe reydklicpe dvdvffgnei
1321       klvqtltlrd eyvdrmqqll deygiedeas
           vvsghaasik rlagmerddy sfyhtdkvve
1381       lryeklyavf rakffeefgg eeiniendgk
           ntrlkctkam hekirqwyfv ayvqpkinka
1441       grcigqslpw vawdalcdlr rqlmldknda
           vlrgkypiaa rleeeiensi erqfdkflkl
1501       kdlieshkda lflrryvyfy gdqiikmlfi
           lkvwlerenv lpssvlsiwq lgrllirlgl
1561       gdllgnptid yeksllmptt mfqqwiskke
           dadeaplirn fdmgtmmlef lrylasqsfa
1621       saesislrvf yekdivepil tksaqwmplh
           liayrtfhsi avsgrfdalh lddedavdqi
1681       teskdpilvn eslfssrnyn ddypisrsri
           lqslkdwsgv keiipreitg trksdmiyvt
1741       svgtvlarqr larlilisge tirdaiannv
           vpnevrdefl

IIIJJ. ERI-3 (W09B6.3)

This protein is expressed as an operon with TAF-6.1 and expressed as a fusion protein and enhances RNAi when mutated.

LOCUS      NP_493918    578 aa    linear  INV 26-
JANUARY
2005
DEFINITION putative protein (66.4 kD) (2B417)
           [Caenorhabditis elegans].
ACCESSION  NP_493918
VERSION    NP_493918.2 GI: 32565182
DBSOURCE   REFSEQ: accession NM 061517.2
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
COMMENT    VALIDATED REFSEQ: This record has un-
           dergone preliminary review of the se-
           quence, but has not yet been subject
           to final review. This record is derived
           from an annotated genomic sequence
           (NC_003280). The reference sequence was
           derived from WormBase CDS: W09B6.3. On
           Jul. 12, 2003 this sequence version re-
           placed gi: 17536803.
           Summary: This gene 2B417, also known as
           W09B6.3 or YK7122, maps at (II;
           −12.85). It encodes a putative protein.
           According to the Worm Transcriptome
           Project, it is well expressed at all
           stages of development [Kohara cDNAs].
           Its sequence is fully supported by 7
           cDNA clones.
           RNA interference results:
           [J. Ahringer 2003] No obvious phenotype
           (by feeding genomic PCR product JA:
           W09B6.3).
           [J. Ahringer 2003] No obvious phenotype
           (by feeding genomic PCR product JA:
           W09B6.2). Warning: this double stranded
           RNA may also interfere with gene
           taf-6.1.
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 21%, L1 or L2 larvae 31%,
           L3 to adult (including dauer) 48%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           The CDS has 11 exons. It covers 4.20
           kb on the WS97 genome. The protein
           (578 aa, 66.4 kDa, pI 8.5) contains no
           Pfam motif. Taxblast (threshold 10{circumflex over ( )}-3)
           tracks ancestors down to caenorhabditis
           elegans.
           COMPLETENESS: full length.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 578
                    /organism = “Caenorhabditis elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “II”
                    /map = “II; −12.85 cM (interpolated
                    genetic position)”
                    /map = “II; covering 4252 bp, from
                    base 1123539 to 1127792 on genome
                    release WS97”
                    /clone_lib = “Kohara embryonic
                    lambda gt11 library: yk516c2,
                    yk590b10; Kohara Sugano L1 larvae
                    cap-selected library: yk1341c6,
                    yk1341d5; Kohara Sugano L2 larvae
                    cap-selected library: yk1378a11;
                    Kohara Sugano mixed stage cap-
                    selected library: yk724f12; Kohara
                    mixed stage library, from him-8
                    strain, containing 15-30% males:
                    yk379e7”
           Protein  1 . . . 578
                    /product = “putative protein (66.4
                    kD) (2B417)”
           Region   347 . . . 350
                    /region_name = “[PSORT] nuclear
                    localization domain: KKKK”
           Region   414 . . . 417
                    /region_name = “[PSORT] vacuolar
                    domain: ILPK”
           Region   456 . . . 462
                    /region_name = “[PSORT] nuclear
                    localization domain: PKNPKKR”
           Region   459 . . . 465
                    /region_name = “[PSORT] nuclear
                    localization domain: PKKRVEI”
           CDS      1 . . . 578
                    /gene = “2B417”
                    /locus_tag = “2B417”
                    /coded_by = “NM_061517.2:
                    1 . . . 1737”
                    /db_xref = “AceView/WormGenes:
                    2B417”
                    /db_xref = “GeneID: 173497”
                    /db_xref = “WormBase: W09B6.3”
ORIGIN
1          mqpvlvnsrp lrvksheses klnlieqedq
           feganyssss gviicysngt gevitqeafd
61         dsgihfifsk atciqypsnf dpigvgsvvq
           ifwsrsferv vrgnhiivqi ekmevykcca
121        mlreqvfvtf nspstagvai gvternitva
           fhpncspvir yetlkahsig rtefeikdrh
181        rentnrmvdv ilaavpfrve ihgnvdkipf
           fviekcrnsp grsgaavitk imknhfmean
241        flqnsesiyf dstschsnil ekvsigslin
           vladptfats sykwygydvt lcnnylahas
301        tqrsfvlenn eilqnckkle kspeeaettt
           kndlrfvppq pekgevkkkk mtnclkfnsk
361        saqfklrhli ldrcfselpe reaksiinsy
           fidrlaegik iekidknwrt fgeilpktpk
421        kyseslkksi qnvlepfgln kpekaaetpk
           iveyfpknpk krveivekpt vdeirelfga
481        lmdaegfaln qrvkphfvlp dtrwkpterr
           yigiyddvqw tfmstfcpki eensenrpla
541        ggwwyrrtvp rdhpveivqk metrrniikd
           ctespfie

IIIKK. ERI-5 (Y38F2AR.1)

This protein has homologs in multiple species, with conservation found in the TUDOR domain. The paralog f22d6.6 plays a role in other small RNA silencing pathways in C. elegans.

LOCUS      NP_500199    458 aa    linear  INV 26-
JANUARY
2005
DEFINITION maternal tudor protein (4D159)
           [Caenorhabditis elegans].
ACCESSION  NP_500199
VERSION    NP_500199.1 GI: 17543178
DBSOURCE   REFSEQ: accession NM 067798.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
COMMENT    PROVISIONAL REFSEQ: This record has not
           yet been subject to final NCBI review.
           This record is derived from an an-
           notated genomic sequence (NC_003282).
           The reference sequence was derived
           from WormBase CDS: Y38F2AR.1.
           Summary: This gene 4D159, also known as
           Y38F2AR.1 or YK7605, maps at (IV;
           −9.66). It encodes a maternal tudor
           protein.
           According to the Worm Transcriptome
           Project, it is moderately expressed in
           embryos, L1, L2 and L3 larvae [Kohara
           cDNAs]. Its existence, but not its
           exact sequence, derived here from the
           genome sequencing consortium annota-
           tion, is supported by 5 cDNA clones.
           RNA interference results:
           [J. Ahringer 2003] No obvious phenotype
           (by feeding genomic PCR product JA:
           Y38F2A_6126.j).
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 16%, L1 or L2 larvae 66%,
           L3 to adult 18%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           The predicted CDS has 6 exons. It
           covers 5.00 kb on the WS97 genome. The
           protein (458 aa, 53.0 kDa, pI 4.7)
           contains one maternal tudor protein
           motif. It also contains an ER membrane
           domain [Psort2]. Taxblast (threshold
           10{circumflex over ( )}-3) tracks ancestors down to
           caenorhabditis elegans.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 458
                    /organism = “Caenorhabditis elegans”
                    /db_xref = “taxon: 6239”
                    /chromosome = “IV”
                    /map = “IV; −9.66 cM (interpolated
                    genetic position)”
                    /map = “IV; covering 5438 bp, from
                    base 2390825 to 2396264 on genome
                    release WS97”
                    /clone_lib = “Kohara embryonic
                    lambda gt11 library: yk592e2;
                    Kohara Sugano L2 larvae cap-selected
                    library: yk818d10, yk1502b5,
                    yk1498b6, yk1503h5”
           Protein  1 . . . 458
                    /product = “maternal tudor protein
                    (4D159)”
           Region   13 . . . 65
                    /region_name = “[Pfam/InterPro de-
                    scription] maternal tudor protein”
                    /db_xref = “CDD: pfam00567”
           Region   454 . . . 457
                    /region_name = “[PSORT] ER membrane
                    domain: DKDS”
           CDS      1 . . . 458
                    /gene = “4D159”
                    /locus_tag = “4D159”
                    /coded_by = “NM_067798.1:
                    1 . . . 1377”
                    /db_xref = “AceView/WormGenes:
                    4D159”
                    /db_xref = “GeneID: 177029”
                    /db_xref = “WormBase: Y38F2AR.1”
ORIGIN
1          mamaplrprv farclilknl elieaariff
           idsavtanvs wkclfqiden lkfhpwqamh
61         ctlgrlvhls dswtdtqcte frnivskfak
           fqitanqcdv dfrsdrpsll vnlyglpngt
121        eidkkvaiee icavsmqnvm vsqfptnfmv
           npkleeldke qdhldville efrrdlpadw
181        aheppadyre ddadwdilqc hvaewndtal
           eqfrradgsf wamlepsctv spwemhvtpi
241        lapekmsdne hwifeqlvkn senqqkiddf
           ysnlknqrpl emeeikfalq tgrtyvmati
301        knrqkssaqw lrceiidflp nanvalryvd
           lgtrgilklk nlhrmhieht kiapacieig
361        rfldddlsma dsemewnthf wreivpydvp
           ivvgpdmefl etgklqfsqi rvagdedden
421        lldkipspsp fftersddlr tqkeddddgn
           vsddkdsg

IIILL. PIR-1 (T23G7.5)

This gene is an ortholog of the well conserved PIR-1 from human and mouse and required for RNAi in C. elegans. An ortholog is the human dual specificity phosphatase 11 (DUSP11).

LOCUS      CAA92703    261 aa    linear  INV 23-
FEBRUARY
2005
DEFINITION Hypothetical protein T23G7.5
           [Caenorhabditis elegans].
ACCESSION  CAA92703
VERSION    CAA92703.1 GI: 3880145
DBSOURCE   embl locus CET23G7, accession Z68319.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 261)
           AUTHORS  .
           CONSRTM  WormBase Consortium
           TITLE    Genome sequence of the nematode C.
                    elegans: a platform for investigating
                    biology
           JOURNAL  Science 282 (5396), 2012-2018 (1998)
           PUBMED   9851916
REFERENCE  2 (residues 1 to 261)
           AUTHORS  Barlow,K.
           TITLE    Direct Submission
           JOURNAL  Submitted (22-DEC-1995) Nematode Se-
                    quencing Project, Sanger Institute,
                    Hinxton, Cambridge CB10 1SA, England
                    and Department of Genetics, Washington
                    University, St. Louis, MO 63110, USA.
                    E-mail: worm@sanger.ac.uk
COMMENT    Coding sequences below are predicted
           from computer analysis, using predic-
           tions from Genefinder (P. Green, U.
           Washington), and other available
           information.
           Current sequence finishing criteria for
           the C. elegans genome sequencing con-
           sortium are that all bases are either
           sequenced unambiguously on both
           strands, or on a single strand with
           both a dye primer and dye terminator
           reaction, from distinct subclones.
           Exceptions are indicated by an explicit
           note.
           IMPORTANT: This sequence is NOT neces-
           sarily the entire insert of the speci-
           fied clone. It may be shorter because
           we only sequence overlapping sections
           once, or longer because we arrange for
           a small overlap between neighbouring
           submissions.
           951009: yk82b3.3 delimits the 3′ end of
           ZK1067.6 960305: T23G7 deleted in union
           with ZK1067.6
           IMPORTANT: This sequence is not the
           entire insert of clone T23G7. It may be
           shorter because we only sequence over-
           lapping sections once, or longer be-
           cause we arrange for a small overlap
           between neighbouring submissions. The
           true left end of clone T23G7 is at 1
           in this sequence. The true right end
           of clone T23G7 is at 16033 in sequence
           Z70038. The true left end of clone
           ZK1067 is at 19833 in this sequence.
           The true right end of clone W07A12
           is at 6609 in this sequence. The start
           of this sequence (1 . . . 104) overlaps
           with the end of sequence Z68320. The
           end of this sequence
           (19833 . . . 19934) overlaps with the
           start of sequence Z70038.
           For a graphical representation of this
           sequence and its analysis see:-
           http://www.wormbase.org/perl/ace/
           elegans/seq/sequence?
           name = ZK1321; class = Sequence.
FEATURES   Location/Qualifiers
           source   1 . . . 261
                    /organism = “Caenorhabditis elegans”
                    /strain = “Bristol N2”
                    /db_xref = “taxon: 6239”
                    /chromosome = “II”
                    /clone = “T23G7”
           Protein  1 . . . 261
                    /product = “Hypotheticai protein
                    T23G7.5”
           CDS      1 . . . 261
                    /locus_tag = “T23G7.5”
                    /standard_name = “T23G7.5”
                    /coded_by = “join (Z68319.1:
                    12488 . . . 12654, Z68319.1:
                    12851 . . . 13093, Z68319.1:
                    13144 . . . 13241, Z68319.1:
                    13297 . . . 13407, Z68319.1:
                    13455 . . .13621)”
                    /note = “contains similarity to Pfam
                    domain PF00782 (Dual specificity
                    phosphatase, catalytic domain)”
                    /db_xref = “GOA: Q22707”
                    /db_xref = “InterPro: IPR000340”
                    /db_xref = “InterPro: IPR000387”
                    /dbx_ref = “UniProt/TrEMBL: Q22707”
ORIGIN
1          mpeprctaiv nflnlshsil isifsvsvms
           nyhhnhnyqh rprgyerlpg krlpdrwniy
61         dnvgrdidgt rfvpfktpld ssffdgknmp
           velqfgvktl islaqqankq iglvidltnt
121        dryykktewa dhgvkylkln cpghevnere
           dlvqdfinav kefvndkend gkligvhcth
181        glnrtgylic rymidvdnys asdaismfey
           yrghpmereh ykkslyeaer kkkygkssgk
241        ssgnsadsti sseqlhrnns q

IIIMM. C32A3.2

LOCUS      CAA88285    346 aa    linear  INV 23-
FEBRUARY
2005
DEFINITION Hypothetical protein C32A3.2
           [Caenorhabditis elegans].
ACCESSION  CAA88285
VERSION    CAA88285.1 GI: 3874617
DBSOURCE   embl locus CEC32A3, accession Z48241.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM Caenorhabditis elegans
                    Eukaryota; Metazoa; Nematoda;
                    Chromadorea; Rhabditida; Rhabditoidea;
                    Rhabditidae; Peloderinae;
                    Caenorhabditis.
REFERENCE  1 (residues 1 to 346)
           AUTHORS  .
           CONSRTM  WormBase Consortium
           TITLE    Genome sequence of the nematode C.
                    elegans: a platform for investigating
                    biology
           JOURNAL  Science 282 (5396), 2012-2018 (1998)
           PUBMED   9851916
REFERENCE  2 (residues 1 to 346)
           AUTHORS  Thomas,K.
           TITLE    Direct Submission
           JOURNAL  Submitted (14-FEB-1995) Nematode Se-
                    quencing Project, Sanger Institute,
                    Hinxton, Cambridge CB10 1SA, England
                    and Department of Genetics, Washington
                    University, St. Louis, MO 63110, USA.
                    E-mail: worm@sanger.ac.uk
COMMENT    Coding sequences below are predicted
           from computer analysis, using predic-
           tions from Genefinder (P. Green, U.
           Washington), and other available
           information.
           Current sequence finishing criteria for
           the C. elegans genome sequencing
           consortium are that all bases are
           either sequenced unambiguously on both
           strands, or on a single strand with
           both a dye primer and dye terminator
           reaction, from distinct subclones.
           Exceptions are indicated by an explicit
           note.
           IMPORTANT: This sequence is NOT neces-
           sarily the entire insert of the speci-
           fied clone. It may be shorter because
           we only sequence overlapping sections
           once, or longer because we arrange for
           a small overlap between neighbouring
           submissions.
           IMPORTANT: This sequence is not the
           entire insert of clone C32A3. It may be
           shorter because we only sequence over-
           lapping sections once, or longer be-
           cause we arrange for a small overlap
           between neighbouring submissions.
           The true left end of clone C32A3 is at
           1 in this sequence. The true right end
           of clone C32A3 is at 44660 in this
           sequence. The true left end of clone
           C46F11 is at 45409 in this sequence.
           The true right end of clone C48D5 is
           at 4074 in this sequence. The start of
           this sequence (1 . . . 102) overlaps
           with the end of sequence Z36237.
           The end of this sequence
           (45409 . . . 45510) overlaps with the
           start of sequence Z81449. For a
           graphical representation of this se-
           quence and its analysis see:-
           http://www.wormbase.org/perl/ace/
           elegans/seq/sequence?
           name = ZK1321; class = Sequence.
FEATURES   Location/Qualifiers
           source   1 . . . 346
                    /organism = “Caenorhabditis elegans”
                    /strain = “Bristol N2”
                    /db_xref = “taxon: 6239”
                    /chromosome = “III”
                    /clone = “C32A3”
           Protein  1 . . . 346
                    /product = “Hypothetical protein
                    C32A3.2”
           CDS      1 . . . 346
                    /locus_tag = “C32A3.2”
                    /standard_name = “C32A3.2”
                    /coded_by = “complement(join
                    (Z48241.1: 31596 . . . 31840,
                    Z48241.1: 32812 . . . 33113,
                    Z48241.1: 33160 . . . 33321,
                    Z48241.1: 33366 . . . 33460,
                    Z48241.1: 33508 . . . 33603,
                    Z48241.1: 33657 . . . 33797))”
                    /note = “contains similarity to Homo
                    sapiens Kinesin-like protein KTF14;
                    ENSEMBL: ENSP00000236917”
                    /db_xref = “Uniprot/Swiss-Prot:
                    Q09261”
ORIGIN
1          mqadgekkkk ktnpersthd dtpksrtrvl
           fsqyfflsfs lffraifmlr slcsiavrlg
61         garqprllss aasgdgndgk gakdaidedl
           lnaiegvann ihpqngsekk slkntlinrl
121        vanekasfda aaasasseml ddqaliglla
           dvagdakvek klppksaqlr qekrglvllr
181        keifyqavqs gftteearvk setivneaqi
           klqeqrkall ndvrekveqe eveetersek
241        dqklftmale fmekiykddl issavqfpta
           hsdqqilskn ksngqqkenn gniqsimssk
301        wamnrmfhsl ityswrdiyh hwvsrnlvql
           lilciwfvlv yprihi

Selected Human Homologs

Under this subsection, selected human homologs referred to above, are described in further detail.

Human Melanoma Differentiation Associated Protein-5

LOCUS      NP_071451    1025 aa    linear  PRI 02-
MARCH 2005
DEFINITION melanoma differentiation associated
           protein-5 [Homo sapiens].
ACCESSION  NP_071451
VERSION    NP_071451.2 GI: 27886568
DBSOURCE   REFSEQ: accession NM 022168.2
KEYWORDS   .
SOURCE     Homo sapiens (human)
           ORGANISM Homo sapiens
                    Eukaryota; Metazoa; Chordata; Craniata;
                    Vertebrata; Euteleostomi; Mammalia;
                    Eutheria; Fuarchontoglires; Primates;
                    Catarrhini; Hominidae; Homo.
REFERENCE  1 (residues 1 to 1025)
           AUTHORS  Andrejeva,J., Childs,K. S.,
                    Young,D. F., Carlos,T. S., Stock,N.,
                    Goodbourn,S. and Randall,R. E.
           TITLE    The V proteins of paramyxoviruses bind
                    the IFN-inducible RNA helicase, mda-5,
                    and inhibit its activation of the IFN-
                    beta promoter
           JOURNAL  Proc. Natl. Acad. Sci. U.S.A. 101 (49),
                    17264-17269 (2004)
           PUBMED   15563593
           REMARK   GeneRIF: mda-5 plays a central role in
                    an intracellular signal transduction
                    pathway that can lead to the activation
                    of the IFN-beta promoter, and that the
                    V proteins of paramyxoviruses interact
                    with mda-5 to block its activity.
REFERENCE  2 (residues 1 to 1025)
           AUTHORS  Kanq,D. C., Gopalkrishnan,R. V.,
                    Lin,L., Randolph,A., Valerie, K.,
                    Pestka,S. and Fisher,P. B.
           TITLE    Expression analysis and genomic char-
                    acterization of human melanoma dif-
                    ferentiation associated gene-5, mda-5:
                    a novel type I interferon-responsive
                    apoptosis-inducing gene
           JOURNAL  Oncogene 23 (9), 1789-1800 (2004)
           PUBMED   14676839
           REMARK   GeneRIF: mda-5 is a novel type I IFN-
                    inducible gene, which may contribute to
                    apoptosis induction during terminal
                    differentiation and during IFN treat-
                    ment
REFERENCE  3 (residues 1 to 1025)
           AUTHORS  Kang,D. C., Gopalkrishnan,R. V., Wu,Q.,
                    Jankowsky,E., Pyle,A. M. and Fisher,
                    P.B.
           TITLE    mda-5: An interferon-inducible putative
                    RNA helicase with double-stranded RNA-
                    dependent ATPase activity and melanoma
                    growth-suppressive properties
           JOURNAL  Proc. Natl. Acad. Sci. U.S.A. 99 (2),
                    637-642 (2002)
           PUBMED   11805321
           REMARK   GeneRIF: mda-5: An interferon-inducible
                    putative RNA helicase with double-
                    stranded RNA-dependent ATPase activity
                    and melanoma growth-suppressive
                    properties
COMMENT    REVIEWED REFSEQ: This record has been
           curated by NCBI staff. The reference
           sequence was derived from AF095844.1
           and BU902097.1. On Jan. 24, 2003 this
           sequence version replaced gi: 11545922.
           Summary: DEAD box proteins, character-
           ized by the conserved motif Asp-Glu-
           Ala-Asp (DEAD), are putative RNA
           helicases. They are implicated in a
           number of cellular processes involving
           alteration of RNA secondary structure
           such as translation initiation, nu-
           clear and mitochondrial splicing, and
           ribosome and spliceosome assembly.
           Based on their distribution patterns,
           some members of this family are be-
           lieved to be involved in embryogenesis,
           spermatogenesis, and cellular growth
           and division. This gene encodes a DEAD
           box protein that is upregulated in re-
           sponse to treatment with beta-inter-
           feron (IFNB) and a protein kinase C-
           activating compound, mezerein (MEZ).
           Irreversible reprogramming of melanomas
           can be achieved by treatment with both
           these agents; treatment with either
           agent alone only achieves reversible
           differentiation.
FEATURES   Location/Qualifiers
           source   1 . . . 1025
                    /organism = “Homo sapiens”
                    /db_xrefr = “taxon: 9606”
                    /chromosome = “2”
                    /map = “2p24.3-q24.3”
           Protein  1 . . . 1025
                    /product = “melanoma differentiation
                    associated protein-5”
                    /note = “DEAD/H (Asp-Glu-Ala-Asp/His)
                    box polypeptide”
           CDS      1 . . . 1025
                    /gene = “IFIH1”
                    /coded_by = “NM_022168.2:
                    223 . . . 3300”
                    /db_xref = “CCDS: CCD52217.1”
                    /db_xref = “GeneID: 64135”
                    /db_xref = “MIM: 606951”
ORIGIN
1          msngystden fryliscfra rvkmyiqvep
           vldyltflpa evkeqiqrtv atsgnmqave
61         lllstlekgv whlgwtrefv ealrrtgspl
           aarymnpelt dlpspsfena hdeylqllnl
121        lqptlvdkll vrdvldkcme eelltiedrn
           riaaaenngn esgvrellkr ivqkenwfsa
181        flnvlrqtgn nelvqeltgs dcsesnaeie
           nlsqvdgpqv eeqllsttvq pnlekevwgm
241        ennssessfa dssvvsesdt slaegsvscl
           deslghnsnm gsdsgtmgsd sdeenvaara
301        spepelqlrp yqmevaqpal egkniiiclp
           tgsgktrvav yiakdhldkk kkasepqkvi
361        vlvnkvllve qlfrkefqpf lkkwyrvigl
           sgdtqlkisf pevvkscdii istaqilens
421        linlengeda gvqlsdfsli iidechhtnk
           eavynnimrh ylmqklknnr lkkenkpvip
481        lpqilgltas pgvggatkqa kaeehilklc
           anldaftikt vkenldqlkn qiqepckkfa
541        iadatredpf keklleimtr iqtycgmspm
           sdfgtqpyeq waiqmekkaa kegnrkervc
601        aehlrkynea lqindtirmi daythletfy
           neekdkkfav ieddsdeggd deycdgdede
661        ddlkkplkld etdrflmtlf fennkmlkrl
           aenpeyenek ltklrntime qytrteesar
721        giiftktrqs ayalsqwite nekfaevgvk
           ahhligaghs sefkpmtqne qkeviskfrt
781        gkinlliatt vaeegldike cniviryglv
           tneiamvqar graradesty vlvahsgsgv
841        iehetvndfr ekmmykaihc vqnmkpeeya
           hkilelqmqs imekkmktkr niakhyknnp
901        slitflcknc svlacsgedi hviekmhhvn
           mtpefkelyi vrenkalqkk cadyqingei
961        ickcgqawgt mmvhkgldlp clkirnfvvv
           fknnstkkqy kkwvelpitf pnldyseccl
1021       fsded
//

Human SMD1

LOCUS      NP_008869    119 aa    linear  PRI 26-
OCTOBER
2004
DEFINITION small nuclear ribonucleoprotein D1
           polypeptide 16 kDa [Homo sapiens].
ACCESSION  NP_008869
VERSION    NP_008869.1 GI: 5902102
DBSOURCE   REFSEQ: accession NM 006938.2
KEYWORDS   .
SOURCE     Homo sapiens (human)
           ORGANISM Homo sapiens
                    Eukaryota; Metazoa; Chordata; Craniata;
                    Vertebrata; Euteleostomi; Mammalia;
                    Eutheria; Euarchontoglires; Primates;
                    Catarrhini; Hominidae; Homo.
REFERENCE  1 (residues 1 to 119)
           AUTHORS  Fong,Y. W. and Zhou,Q.
           TITLE    Stimulatory effect of splicing factors
                    on transcriptional elongation
           JOURNAL  Nature 414 (6866), 929-933 (2001)
           PUBMED   11780068
REFERENCE  2 (residues 1 to 119)
           AUTHORS  Sun,D., Ou,Y. C. and Hoch,S. O.
           TITLE    Analysis of genes for human snRNP
                    Sm-D1 protein and identification of the
                    promoter sequence which shows segmental
                    homology to the promoters of Sm-E and
                    U1 snRNA genes
           JOURNAL  Gene 189 (2), 245-254 (1997)
           PUBMED   9168134
REFERENCE  3 (residues 1 to 119)
           AUTHORS  Lehmeier,T., Raker,V., Hermann, H. and
                    Luhrmann,R.
           TITLE    cDNA cloning of the Sm proteins D2 and
                    D3 from human small nuclear ribonucleo-
                    proteins: evidence for a direct
                    D1-D2 interaction
           JOURNAL  Proc. Natl. Acad. Sci. U.S.A. 91 (25),
                    12317-12321 (1994)
           PUBMED   7527560
REFERENCE  4 (residues 1 to 119)
           AUTHORS  Lehmeier,T., Foulaki,K. and Luhrmann,R.
           TITLE    Evidence for three distinct D proteins,
                    which react differentially with anti-Sm
                    autoantibodies, in the cores of the
                    major snRNPs U1, U2, U4/U6 and U5
           JOURNAL  Nucleic Acids Res. 18 (22), 6475-6484
                    (1990)
           PUBMED   1701240
REFERENCE  5 (residues 1 to 119)
           AUTHORS  Rokeach,L. A., Haselby,J. A. and
                    Hoch,S. O.
           TITLE    Molecular cloning of a cDNA encoding
                    the human Sm-D autoantigen
           JOURNAL  Proc. Natl. Acad. Sci. U.S.A. 85 (13),
                    4832-4836 (1988)
           PUBMED   3260384
COMMENT    REVIEWED REFSEQ: This record has been
           curated by NCBI staff. The reference
           sequence was derived from J03798.1.
           Summary: This gene encodes a small
           nuclear ribonucleoprotein that belongs
           to the SNRNP core protein family. The
           protein may act as a charged protein
           scaffold to promote SNRNP assembly or
           strengthen SNRNP—SNRNP interactions
           through nonspecific electrostatic
           contacts with RNA.
FEATURES   Location/Qualifiers
           source   1 . . . 119
                    /organism = “Homo sapiens”
                    /db_xref = “taxon: 9606”
                    /chromosome = “18”
                    /map = “18q11.2”
           Protein  1 . . . 119
                    /product = “small nuclear ribonu-
                    cleoprotein D1 polypeptide 16 kDa”
                    /note = “snRNP core protein D1;
                    Sm-D autoantigen; small nuclear ri-
                    bonucleoprotein D1 polypeptide
                    (16 kD)”
           CDS      1 . . . 119
                    /gene = “SNRPD1”
                    /coded_by = “NM_006938.2:
                    132 . . . 491”
                    /db_xref = “GeneID: 6632”
                    /dbxref = “MIM: 601063”
ORIGIN
1          mklvrflmkl shetvtielk ngtqvhgtit
           gvdvsmnthl kavkmtlknr epvqletlsi
61         rgnniryfil pdslpldtll vdvepkvksk
           kreavagrgr grgrgrgrgr grgrggprr
//

Human Tripartite Motif Protein 2 (RING Finger Protein 86)

LOCUS      Q90040    744 aa    linear  PRI 01-
MAY 2005
DEFINITION Tripartite motif protein 2 (RING finger
           protein 86).
ACCESSION  Q9C040
VERSION    Q9C040 GI: 21363034
DBSOURCE   swissprot: locus TRIM2_HUMAN, accession
           Q9C040;
           class: standard.
           extra accessions: O60272,Q9BSI9,Q9UFZ1,
           created: Feb. 28, 2003.
           sequence updated: Feb. 28, 2003.
           annotation updated: May 1, 2005.
           xrefs: AF220018.1, AAG53472.1,
           AB011089.1, BAA25443.1, BC005016.1,
           AAH05016.1, BC011052.1, AAH11052.1,
           AL110234.1, CAB53687.2, T00082 xrefs
           (non-sequence databases): HSSPP28990,
           EnsemblENSG00000109654,
           GenewHGNC: 15974, H-InvDBHIX0004577,
           GO0005737, GO0017022, GO0008270,
           InterProIPR01044, InterProIPR003649,
           InterProIPR001298, InterProIPR001258,
           InterProIPR000315, InterProIPR001841,
           PfamPF00630, PfamPF01436, PfamPF00643,
           PfamPF00097, PRINTSPR01406,
           SMARTSM00502, SMARTSM00336,
           SMARTSM00557, SMARTSM00184,
           PROSITEPS50194, PROSITEPS50119,
           PROSITEPS00518, PROSITEPS50089
KEYWORDS   Metal-binding; Repeat; Zinc;
           Zinc-finger.
SOURCE     Homo sapiens (human)
           ORGANISM Homo sapiens
                    Eukaryota; Metazoa; Chordata; Craniata;
                    Vertebrata; Euteleostomi; Mammalia;
                    Eutheria; Euarchontoglires; Primates;
                    Catarrhini; Hominidae; Homo.
REFERENCE  1 (residues 1 to 744)
           AUTHORS  Reymond,A., Meroni,G., Fantozzi,A.,
                    Merla,G., Cairo,S., Luzi, L.,
                    Riganelli,D., Zanaria,E., Messali,S.,
                    Cainarca,S., Guffanti,A., Minucci,S.,
                    Pelicci,P. G. and Ballabio,A.
           TITLE    The tripartite motif family identifies
                    cell compartments
           JOURNAL  EMBO J. 20 (9), 214014 2151 (2001)
           PUBMED   11331580
           REMARK   NUCLEOTIDE SEQUENCE.
REFERENCE  2 (residues 1 to 744)
           AUTHORS  Nagase,T., Ishikawa,K., Miyajima,N.,
                    Tanaka,A., Kotani,H., Nomura,N. and
                    Ohara,O.
           TITLE    Prediction of the coding sequences of
                    unidentified human genes. IX. The com-
                    plete sequences of 100 new cDNA clones
                    from brain which can code for large
                    proteins in vitro
           JOURNAL  DNA Res. 5 (1), 31-39 (1998)
           PUBMED   9628581
           REMARK   NUCLEOTIDE SEQUENCE [LARGE SCALE MRNA].
                    TISSUE = Brain
REFERENCE  3 (residues 1 to 744)
           AUTHORS  Strauserg,R. L., Feingold,E. A.,
                    Grouse,L. H., Derge,J. G., Klausner,R.
                    D., Collins,F. S., Wagner,L.,
                    Shenmen,C. M., Schuler, G. D.,
                    Altschul,S. F., Zeeberg,B.,
                    Buetow,K. H., Schaefer,C. F.,
                    Bhat, N. K., Hopkins,R. F.,
                    Jordan,H., Moore,T., Max,S. I.,
                    Wang,J., Hsieh, F., Diatchenko,L.,
                    Marusina,K., Farmer,A. A., Rubin,G. M.,
                    Hong,L., Stapleton,M., Soares,M. B.,
                    Bonaldo,M. F., Casavant,T. L.,
                    Scheetz,T. E., Brownstein,M. J.,
                    Usdin,T. B., Toshiyuki,S., Carninci,P.,
                    Prange,C., Raha,S. S., Loquellano,N.
                    A., Peters,G. J., Abramson,R. D.,
                    Mullahy,S. J., Bosak,S. A., McEwan,P.
                    J., McKernan,K. J., Malek,J. A.,
                    Gunaratne,P. H., Richards,S., Worley,K.
                    C., Hale,S., Garcia,A. M., Gay,L. J.,
                    Hulyk,S. W., Villalon,D. K., Muzny,D.
                    M., Sodergren,E. J., Lu,X., Gibbs,R.
                    A., Fahey,J., Helton,E., Ketteman,M.,
                    Madan,A., Rodrigues,S., Sanchez,A.,
                    Whiting,M., Madan,A., Young,A. C.,
                    Shevchenko,Y., Bouffard,G. G.,
                    Blakesley,R. W., Touchman,J. W.,
                    Green,E. D., Dickson,M. C.,
                    Rodriguez,A. C., Grimwood,J.,
                    Schmutz,J., Myers,R. M., Butterfield,Y.
                    S., Krzywinski,M. I., Skalska,U.,
                    Smailus,D. E., Schnerch,A., Schein,J.
                    E., Jones,S. J. and Marra,M. A.
           CONSRTM  Mammalian Gene Collection Program Team
           TITLE    Generation and initial analysis of more
                    than 15,000 full-length human and mouse
                    cDNA sequences
           JOURNAL  Proc. Natl. Acad. Sci. U.S.A. 99 (26),
                    16899-16903 (2002)
           PUBMED   12477932
           REMARK   NUCLEOTIDE SEQUENCE [LARGE SCALE MRNA].
                    TISSUE = Brain, and Placenta
REFERENCE  4 (residues 1 to 744)
           AUTHORS  .
           CONSRTM  The German cDNA consortium
           TITLE    Direct Submission
           JOURNAL  Submitted (??-AUG-1999)
           REMARK   NUCLEOTIDE SEQUENCE [LARGE SCALE MRNA]
                    OF 515-744.
                    TISSUE = Kidney
COMMENT    On Mar. 15, 2005 this sequence ver-
           sion replaced gi: 7513001. [FUNCTION]
           May contribute to the alteration of
           neural cellular mechanisms (By sim-
           ilarity).
           [SUBUNIT] Interacts with myosin V (By
           similarity).
           [SUBCELLULAR LOCATION] Cytoplasmic
           (By similarity).
           [DOMAIN] The interaction with myosin V
           is dependent upon its NHL repeats,
           which form a beta-propeller (NHL)
           domain containing six blades (By
           similarity).
           [SIMILARITY] Belongs to the TRIM/RBCC
           family.
           [SIMILARITY] Contains 1 B box-type zinc
           finger.
           [SIMILARITY] Contains 1 filamin repeat.
           [SIMILARITY] Contains 6 NHL repeats.
           [SIMILARITY] Contains 1 RING-type zinc finger.
FEATURES   Location/Qualifiers
           source   1 . . . 744
                    /organism = “Homo sapiens”
                    /db_xref = “taxon: 9606”
           gene     1 . . . 744
                    /gene = “TRIM2”
                    /note = “synonyms: KIAA0517, RNF86”
           Protein  1 . . . 744
                    /gene = “TRIM2”
                    /product = “Tripartite motif
                    protein 2”
           Region   23 . . . 64
                    /gene = “TRIM2”
                    /region_name = “Zinc finger region”
                    /note = “RING-type.”
                    /evidence = experimental
           Region   113 . . . 154
                    /gene = “TRIM2”
                    /region_name = “Zinc finger region”
                    /note = “B box-type.”
                    /evidence = experimental
           Region   320 . . . 421
                    /gene = “TRIM2”
                    /region_name = “Repetitive region”
                    /note = “Filamin.”
                    /evidence = experimental
           Region   486 . . . 513
                    /gene = “TRIM2”
                    /region_name = “Repetitive region”
                    /note = “NHL 1.”
                    /evidence = experimental
           Region   515
                    /gene = “TRIM2”
                    /region_name = “Conflict”
                    /note = “N −> G (in REF. 4).”
                    /evidence = “experimental
           Region   533 . . . 560
                    /gene = “TRIM2”
                    /region_name = “Repetitive region”
                    /note = “NHL 2.”
                    /evidence = experimental
           Region   575 . . . 602
                    /gene = “TRIM2”
                    /region_name = “Repetitive region”
                    /note = “NHL 3.”
                    /evidence = experimental
           Region   622 . . . 649
                    /gene = “TRIM2”
                    /region_name = “Repetitive region”
                    /note = “NHL 4.”
                    /evidence = experimental
           Region   669 . . . 696
                    /gene = “TRIM2”
                    /region_name = “Repetitive region”
                    /note = “NHL 5.”
                    /evidence = experimental
           Region   713 . . . 740
                    /gene = “TRIM2”
                    /region_name = “Repetitive region”
                    /note = “NHL 6.”
                    /evidence = experimental
           Region   737 . . . 744
                    /gene = “TRIM2”
                    /region_name = “Conflict”
                    /note = “FKVYRYLQ −>
                    LILIYSRHLFFYESKC (in REF. 3;
                    AAH05016).”
                    /evidence = experimental
ORIGIN
1          masegtnips pvvrqidkqf licsiclery
           knpkvlpclh tfcerclqny ipahsltlsc
61         pvcrqtsilp ekgvaalqnn ffitnlmdvl
           qrtpgsnaee ssiletvtav aagkplscpn
121        hdgnvmefyc qscetamcre ctegehaehp
           tvplkdvveq hkaslqvgld avnkrlpeid
181        salqfiseii hqltnqkasi vddihstfde
           lqktlnvrks vllmelevny glkhkvlqsq
241        ldtllqgqes ikscsnftaq alnhgtetev
           llvkkqmsek lneladqdfp lhprendqld
301        fiveteglkk sihnlgtilt tnavasetva
           tgeglrqtii gqpmsvtitt kdkdgelckt
361        gnayltaels tpdgsvadge ildnkngtye
           flytvqkegd ftlslrlydq hirgspfklk
421        virsadvspt tegvkrrvks pgsghvkqka
           vkrpasmyst gkrkenpied dlifrvgtkg
481        rnkgeftnlq gvaastngki liadsnnqcv
           qifsndgqfk srfgirgrsp gqlqrptgva
541        vhpsgdiiia dydnkwvsif ssdgkfktki
           gsgklmgpkg vsvdrnghii vvdnkaccvf
601        ifqpngkivt rfgsrgngdr qfagphfaav
           nsnneiiitd fhnhsvkvfn qegefmlkfg
661        sngegngqfn aptgvavdsn gniivadwgn
           sriqvfdgsg sflsyintsa dplygpqgla
721        ltsdghvvva dsgnhcfkvy rylq
//

Human TFIID Subunit 6

LOCUS      P49848    677 aa    linear  PRI 01-
MAY 2005
DEFINITION Transcription initiation factor TFIID
           subunit 6 (Transcription initiation
           factor TFIID 70 kDa subunit)
           (TAF(II)70) (TAFII-70) (TAFII-80)
           (TAFII80).
ACCESSION  P49848
VERSION    P49848 GI: 1729810
DBSOURCE   swissprot: locus TAF6_HUMAN, accession
           P49848;
           class: standard.
           created: Oct. 1, 1996.
           sequence updated: Oct. 1, 1996.
           annotation updated: May 1, 2005.
           xrefs: L25444.1, AAA63643.1, U31659.1,
           AAA84390.1, AY149894.1, AAN10295.1,
           BC018115.1, AAH18115.1
           xrefs (non-sequence databases):
           HSSPP49847, TRANSFACT00783,
           TRANSFACT02208, GenewHGNC: 11540,
           H-InvDBHIX0006909, ReactomeP49848,
           MIM 602955, GO0005669, GO0005673,
           GO0016251, GO0005515,
           InterProIPR007124, InterProIPR009072,
           InterProIPR004823, PfamPF02969
KEYWORDS   Direct protein sequencing; Nuclear
           protein; Polymorphism; Transcription;
           Transcription regulation.
SOURCE     Homo sapiens (human)
           ORGANISM Homo sapiens
                    Eukaryota; Metazoa; Chordata; Craniata;
                    Vertebrata; Euteleostomi; Mammalia;
                    Eutheria; Euarchontoglires; Primates;
                    Catarrhini; Hominidae; Homo.
REFERENCE  1 (residues 1 to 677)
           AUTHORS  Weinzierl,R. O., Ruppert,S., Dynlacht,
                    B. D., Tanese,N. and Tjian, R.
           TITLE    Cloning and expression of Drosophila
                    TAFII60 and human TAFII70 reveal con-
                    served interactions with other subunits
                    of TFIID
           JOURNAL  EMBO J. 12 (13), 5303-5309 (1993)
           PUBMED   8262073
           REMARK   NUCLEOTIDE SEQUENCE, AND PARTIAL
                    PROTEIN SEQUENCE.
REFERENCE  2 (residues 1 to 677)
           AUTHORS  Hisatake,K., Ohta,T., Takada,R.,
                    Guermah,M., Horikoshi,M., Nakatani,Y.
                    and Roeder,R. G.
           TITLE    Evolutionary conservation of human
                    TATA-binding-polypeptide-associated
                    factors TAFII31 and TAFII80 and in-
                    teractions of TAFII80 with other TAFs
                    and with general transcription factors
           JOURNAL  Proc. Natl. Acad. Sci. U.S.A. 92 (18),
                    8195-8199 (1995)
           PUBMED   7667268
           REMARK   NUCLEOTIDE SEQUENCE.
                    TISSUE = Placenta
REFERENCE  3 (residues 1 to 677)
           AUTHORS  Rieder,M. J., Livingston,R. J.,
                    Daniels,M. R., Montoya,M. A., Chung,M.
                    -W., Miyamoto,K. E., Nguyen,C. P.,
                    Nguyen,D. A., Poel,C.L., Robertson,
                    P. D., Schackwitz,W. S., Sherwood,
                    J. K., Witrak,L. A. and Nickerson,
                    D. A.
           TITLE    Direct Submission
           JOURNAL  Submitted (??-SEP-2002)
           REMARK   NUCLEOTIDE SEQUENCE, AND VARIANT
                    SER-36.
REFERENCE  4 (residues 1 to 677)
           AUTHORS  Strausberg,R. L., Feingold,E. A.,
                    Grouse,L. H., Derge,J. G., Klausner,
                    R. D., Collins,F. S., Wagner,L.,
                    Shenmen,C. M., Schuler,G. D.,
                    Altschul,S. F., Zeeberg,B., Buetow,
                    K. H., Schaefer,C. F., Bhat, N. K.,
                    Hopkins,R. F., Jordan,H., Moore,T.,
                    Max,S. I., Wang,J., Hsieh, F.,
                    Diatchenko,L., Marusina,K., Farmer,A.
                    A., Rubin,G. M., Hong,L., Stapleton,M.,
                    Soares,M. B., Bonaldo,M. F., Casavant,
                    T. L., Scheetz,T. E., Brownstein,M. J.,
                    Usdin,T. B., Toshiyuki,S., Carninci,P.,
                    Prange,C., Raha,S. S., Loquellano,N.
                    A., Peters, G. J., Abramson,R. D.,
                    Mullahy,S. J., Bosak,S. A., McEwan,P.
                    J., McKernan,K. J., Malek,J. A.,
                    Gunaratne,P. H., Richards,S., Worley,
                    K. C., Hale,S., Garcia,A. M., Gay,L.
                    J., Hulyk,S. W., Villalon,D. K.,
                    Muzny,D. M., Sodergren,E. J., Lu,X.,
                    Gibbs,R. A., Fahey,J., Helton,E.,
                    Ketteman,M., Madan,A., Rodrigues,S.,
                    Sanchez,A., Whiting,M., Madan,A.,
                    Young,A. C., Shevchenko,Y., Bouffard,
                    G. G., Blakesley,R. W., Touchman,J.
                    W., Green,E. D., Dickson,M. C.,
                    Rodriguez,A. C., Grimwood,J., Schmutz,
                    J., Myers,R. M., Butterfield,Y. S.,
                    Krzywinski,M. I., Skalska,U., Smailus,
                    D. E., Schnerch,A., Schein,J. E.,
                    Jones,S. J. and Marra,M. A.
           CONSRTM  Mammalian Gene Collection Program Team
           TITLE    Generation and initial analysis of more
                    than 15,000 full-length human and mouse
                    cDNA sequences
           JOURNAL  Proc. Natl. Acad. Sci. U.S.A. 99 (26),
                    16899-16903 (2002)
           PUBMED   12477932
           REMARK   NUCLEOTIDE SEQUENCE [LARGE SCALE MRNA].
                    TISSUE = Pancreas
COMMENT    [FUNCTION] TAFs are components of the
           transcription factor IID (TFIID) com-
           plex, PCAF histone acetylase complex
           and TBP-free TAFII complex (TFTC).
           TIIFD is multimeric protein complex
           that plays a central role in mediating
           promoter responses to various
           activators and repressors.
           [SUBUNIT] TFIID and PCAF are composed
           of TATA binding protein (TBP) and a
           number of TBP-associated factors
           (TAFs). TBP is not part of TFTC. Binds
           tightly to TAFII-250 and also directly
           interacts with TAFII-40.
           [SUBCELLULAR LOCATION] Nuclear.
           [SIMILARITY] Belongs to the TAF6
           family.
FEATURES   Location/Qualifiers
           source   1 . . . 677
                    /organism = “Homo sapiens”
                    /db_xref = “taxon: 9606”
           gene     1 . . . 677
                    /gene = “TAF6”
                    /note = “synonyms: TAF2E, TAFII70”
           Protein  1 . . . 677
                    /gene = “TAF6”
                    /product = “Transcription initiation
                    factor TFIID subunit 6”
           Region   36
                    /gene = “TAF6”
                    /region_name = “Variant”
                    /note = “C −> S./FTId =
                    VAR_0143492.”
                    /evidence = experimental
ORIGIN
1          maeekklkls ntvlpsesmk vvaesmgiaq
           iqeetcqllt devsyrikei aqdalkfmhm
61         qkrqklttsd idyalklknv eplygfhaqe
           fipfrfasgg grelyfyeek evdlsdiint
121        plprvpldvc lkahwlsieg cqpaipenpp
           papkeqqkae ateplksakp gqeedgplkg
181        kgqgattadg kgkekkappl legaplrlkp
           rsihelsveq qlyykeitea cvgsceakra
241        ealqsiatdp glyqmlprfs tfisegvrvn
           vvqnnlalli ylmrmvkalm dnptlyleky
301        vhelipavmt civsrqlclr pdvdnhwalr
           dfaarlvaqi ckhfstttnn iqsritktft
361        kswvdektpw ttrygsiagl aelghdvikt
           lilprlqqeg erirsvldgp vlsnidriga
421        dhvqslllkh capvlaklrp ppdnqdayra
           efgslgpllc sqvvkaraqa alqaqqvnrt
481        tltitqprpt ltlsqapqpg prtpgllkvp
           gsialpvqtl vsaraaappq psppptkfiv
541        msssssapst qqvlslstsa pgsgstttsp
           vtttvpsvqp ivklvstatt appstapsgp
601        gsvqkyivvs lpptgegkgg ptshpspvpp
           passpsplsg salcggkqea gdspppapgt
661        pkangsqpns gspqpap
//

Human TAR-Binding Protein

LOCUS      NP_000958    403 aa    linear  PRI 02-
MARCH 2005
DEFINITION ribosomal protein L3 [Homo sapiens].
ACCESSION  NP_000958
VERSION    NP_000958.1 GI: 4506649
DBSOURCE   REFSEQ: accession NM 000967.2
KEYWORDS   .
SOURCE     Homo sapiens (human)
           ORGANISM Homo sapiens
                    Eukaryota; Metazoa; Chordata; Craniata;
                    Vertebrata; Euteleostomi; Mammalia;
                    Eutheria; Euarchontoglires; Primates;
                    Catarrhini; Hominidae; Homo.
REFERENCE  1 (residues 1 to 403)
           AUTHORS  Collins,J. E., Wright,C. L., Edwards,C.
                    A., Davis,M. P., Grinham, J. A., Cole,
                    C. G., Goward, M.E., Aguado,B., Mallya,
                    M., Mokrab,Y., Huckle,E. J., Beare,D.
                    M. and Dunham,I.
           TITLE    A genome annotation-driven approach to
                    cloning the human ORFeome
           JOURNAL  Genome Biol. 5 (10), R84 (2004)
           PUBMED   15461802
REFERENCE  2 (residues 1 to 403)
           AUTHORS  Uechi,T., Tanaka,T. and Kenmochi,N.
           TITLE    A complete map of the human ribosomal
                    protein genes: assignment of 80 genes
                    to the cytogenetic map and implications
                    for human disorders
           JOURNAL  Genomics 72 (3), 223-230 (2001)
           PUBMED   11401437
REFERENCE  3 (residues 1 to 403)
           AUTHORS  Duga,S., Asselta,R., Malcovati,M.,
                    Tenchini,M. L., Ronchi,S. and
                    Simonic, T.
           TITLE    The intron-containing L3 ribosomal pro-
                    tein gene (RPL3): sequence analysis and
                    identification of U43 and of two novel
                    intronic small nucleolar RNAs
           JOURNAL  Biochim. Biophys. Acta 1490 (3),
                    225-236 (2000)
           PUBMED   10684968
REFERENCE  4 (residues 1 to 403)
           AUTHORS  Kenmochi,N., Kawaguchi,T., Rozen,S.,
                    Davis,E., Goodman,N., Hudson,T. J.,
                    Tanaka,T. and Page,D. C.
           TITLE    A map of 75 human ribosomal protein
                    genes
           JOURNAL  Genome Res. 8 (5), 509-523 (1998)
           PUBMED   9582194
REFERENCE  5 (residues 1 to 403)
           AUTHORS  Wool,I. G., Chan,Y. L. and Gluck,A.
           TITLE    Structure and evolution of mammalian
                    ribosomal proteins
           JOURNAL  Biochem. Cell Biol. 73 (11-12),
                    933-947 (1995)
           PUBMED   8722009
           REMARK   Review article
REFERENCE  6 (residues 1 to 403)
           AUTHORS  Reddy,T. R., Suhasini,M., Rappaport,J.,
                    Looney,D. J., Kraus,G. and Wong-Staal,
                    F.
           TITLE    Molecular cloning and characterization
                    of a TAR-binding nuclear factor from T
                    cells
           JOURNAL  AIDS Res. Hum. Retroviruses 11 (6),
                    663-669 (1995)
           PUBMED   7576925
REFERENCE  7 (residues 1 to 403)
           AUTHORS  Matoba,R., Okubo,K., Hori,N.,
                    Fukushima,A. and Matsubara,K.
           TITLE    The addition of 5′-coding information
                    to a 3′-directed cDNA library improves
                    analysis of gene expression
           JOURNAL  Gene 146 (2), 199-207 (1994)
           PUBMED   8076819
COMMENT    REVIEWED REFSEQ: This record has been
           curated by NCBI staff. The reference
           sequence was derived from BC012146.1
           and BC008492.1.
           Summary: Ribosomes, the organelles that
           catalyze protein synthesis, consist of
           a small 40S subunit and a large 60S
           subunit. Together these subunits are
           composed of 4 RNA species and approxi-
           mately 80 structurally distinct pro-
           teins. This gene encodes a ribosomal
           protein that is a component of the 60S
           subunit. The protein belongs to the L3P
           family of ribosomal proteins. It is
           located in the cytoplasm. The protein
           can bind to the HIV-1 TAR mRNA, and it
           has been suggested that the protein
           contributes to tat-mediated trans-
           activation. This gene is co-transcribed
           with the small nucleolar RNA genes U43,
           U86, U83a, and U83b, which are located
           in its first, third, fifth, and seventh
           introns, respectively. As is typical
           for genes encoding ribosomal proteins,
           there are multiple processed pseudo-
           genes of this gene dispersed through
           the genome.
FEATURES   Location/Qualifiers
           source   1 . . . 403
                    /organism = “Homo sapiens”
                    /db_xref = “taxon: 9606”
                    /chromosome = “22”
                    /map = “22q13”
           Protein  1 . . . 403
                    /product = “ribosomal protein L3”
                    /note = “60S ribosomal protein L3;
                    HIV-1 TAR RNA-binding protein B“
           CDS      1 . . . 403
                    /gene = “RPL3“
                    /coded_by = “NM_000967.2:
                    27 . . . 1238”
                    /db_xref = “CCDS: CCDS13988.1”
                    /db_xref = “GeneID: 6122”
                    /db_xref = “MIM: 604163”
ORIGIN
1          mshrkfsapr hgslgflprk rssrhrgkvk
           sfpkddpskp vhltaflgyk agmthivrev
61         drpgskvnkk evveavtive tppmvvvgiv
           gyvetprglr tfktvfaehi sdeckrrfyk
121        nwhkskkkaf tkyckkwqde dgkkqlekdf
           ssmkkycqvi rviahtgmrl lplrqkkahl
181        meiqvnggtv aekldwarer leqqvpvnqv
           fgqdemidvi gvtkgkgykg vtsrwhtkkl
241        prkthrglrk vacigawhpa rvafsvarag
           qkgyhhrtei nkkiykigqg ylikdgklik
301        nnastdydls dksinplggf vhygevtndf
           vmlkgcvvgt kkrvltlrks llvqtkrral
361        ekidlkfidt tskfghgrfq tmeekkafmg
           plkkdriake ega
//

Human ERI-1 (AAH35279)

LOCUS      AAH35279    349 aa    linear  PRI 05-
APRIL 2005
DEFINITION Histone mRNA 3′ end-specific
           exonuclease [Homo sapiens].
ACCESSION  AAH35279
VERSION    AAH35279.1 GI: 23271401
DBSOURCE   accession BC035279.1
KEYWORDS   MGC.
SOURCE     Homo sapiens (human)
           ORGANISM Homo sapiens
                    Eukaryota; Metazoa; Chordata; Craniata;
                    Vertebrata; Euteleostomi; Mammalia;
                    Eutheria; Euarchontoglires; Primates;
                    Catarrhini; Hominidae; Homo.
REFERENCE  1 (residues 1 to 349)
           AUTHORS  Strausberg,R. L., Feingold,E. A.,
                    Grouse,L. H., Derge,J. G., Klausner,
                    R. D., Collins,F. S., Wagner,L.,
                    Shenmen,C. M., Schuler,G. D.,
                    Altschul,S. F., Zeeberg,B., Buetow,
                    K. H., Schaefer,C. F., Bhat, N. K.,
                    Hopkins,R. F., Jordan,H., Moore,T.,
                    Max,S. I., Wang,J., Hsieh, F.,
                    Diatchenko,L., Marusina,K., Farmer,A.
                    A., Rubin,G. M., Hong,L., Stapleton,
                    M., Soares,M. B., Bonaldo,M. F.,
                    Casavant,T. L., Scheetz,T. E.,
                    Brownstein,M. J., Usdin,T. B.,
                    Toshiyuki,S., Carninci,P., Prange,C.,
                    Raha,S. S., Loquellano,N. A., Peters,
                    G. J. Abramson,R. D., Muilahy,S. J.,
                    Bosak,S. A., McEwan,P. J., McKernan,
                    K. J., Malek,J. A., Gunaratne,P. H.,
                    Richards,S., Worley,K. C., Hale,S.,
                    Garcia,A. M., Gay,L. J., Hulyk,S. W.,
                    Villalon,D. K., Muzny,D. M.,
                    Sodergren,E. J., Lu,X., Gibbs,R. A.,
                    Fahey,J., Helton,E., Ketteman,M.,
                    Madan,A., Rodrigues,S., Sanchez,A.,
                    Whiting,M., Madan,A., Young,A. C.,
                    Shevchenko,Y., Bouffard,G. G.,
                    Blakesley,R. W., Touchman,J. W.,
                    Green,E. D., Dickson,M. C.,
                    Rodriguez,A. C., Grimwood,J.,
                    Schmutz,J., Myers, R. M., Butterfield,
                    Y. S., Krzywinski,M. I., Skalska,U.,
                    Smailus,D. E., Schnerch,A., Schein,
                    J. E., Jones,S. J. and Marra,M. A.
           CONSRTM  Mammalian Gene Collection Program
                    Team
           TITLE    Generation and initial analysis of
                    more than 15,000 full-length human
                    and mouse cDNA sequences
           JOURNAL  Proc. Natl. Acad. Sci. U.S.A. 99
                    (26), 16899-16903 (2002)
           PUBMED   12477932
REFERENCE  2 (residues 1 to 349)
           AUTHORS  .
           CONSRTM  NIH MGC Project
           TITLE    Direct Submission
           JOURNAL  Submitted (31-JUL-2002) National
                    Institutes of Health, Mammalian
                    Gene Collection (MGC), Bethesda,
                    MD 20892-2590, USA
           REMARK   NIH-MGC Project URL:
                    http://mgc.nci.nih.gov
COMMENT    Contact: MGC help desk
           Email: cgapbs-r@mail.nih.gov
           Tissue Procurement: Life Technologies,
           Inc.
           cDNA Library Preparation: Life
           Technologies, Inc.
           cDNA Library Arrayed by: The I.M.A.G.E.
           Consortium (LLNL) DNA
           Sequencing by: Baylor College of Med-
           icine Human Genome Sequencing Center
           Center code: BCM-HGSC
           Web site:
           http://www.hgsc.bcm.tmc.edu/cdna/
           Contact: amg@bcm.tmc.edu
           Gunaratne, P. H., Garcia, A. M., Lu,
           X., Hulyk, S. W., Loulseged, H., Kowis,
           C. R., Sneed, A. J., Martin, R. G.,
           Muzny, D. M., Nanavati, A. N., Gibbs,
           R. A.
           Clone distribution: MGC clone distribu-
           tion information can be found through
           the I.M.A.G.E. Consortium/LLNL at:
           http://image.llnl.gov
           Series: IRAK Plate: 50 Row: g
           Column: 1
           This clone was selected for full length
           sequencing because it passed the fol-
           lowing selection criteria: matched
           mRNA gi: 31543183.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source   1 . . . 349
                    /organism = “Homo sapiens”
                    /db_xref = “taxon: 9606”
                    /clone = “MGC: 35395 IMAGE: 5186320'
                    /tissue_type = “Colon, Kidney,
                    Stomach, adult, whole pooled”
                    /clone_lib = “NIH_MGC_116”
                    /lab_host = “DH10B”
                    /note = “Vector: pCMV-SPORT6”
           Protein  1 . . . 349
                    /product = “histone mRNA 3′ end-
                    specific exonuclease”
           CDS      1 . . . 349
                    /gene = “3′ HEXO”
                    /coded_by = “BC035279.1:
                    125 . . . 1174”
                    /db_xref = “GeneID: 90459”
ORIGIN
1          medpqskepa geavalalle sprpeggeep
           prpspeetqq ckfdgqetkg skfitssasd
61         fsdpvykeia itngcinrms keelraklse
           fkletrgvkd vlkkrlknyy kkqklmlkes
121        nfadsyydyi ciidfeatce egnppefvhe
           iiefpvvlln thtleiedtf qqyvrpeint
181        qlsdfcislt gitqdqvdra dtfpqvlkkv
           idwmklkelg tkykyslltd gswdmskfln
241        iqcqlsrlky ppfakkwini rksygnfykv
           prsqtkltim leklgmdydg rphcglddsk
301        niariavrml qdgcelrine kmhagqlmsv
           ssslpiegtp ppqmphfrk
//

Human TUDOR Protein

LOCUS      Q9BXT4    777 aa    linear  PRI 01-
MAY 2005
DEFINITION Tudor domain containing protein 1.
ACCESSION  Q9BXT4
VERSION    Q9BXT4 GI: 17368689
DBSOURCE   swissprot: locus TDRD1_HUMAN,
           accession Q9BXT4;
           class: standard.
           extra accessions: Q9H7B3, created:
           Feb. 28, 2003.
           sequence updated: Feb. 28, 2003.
           annotation updated: May 1, 2005.
           xrefs: AF285606.1, AAK31985.1,
           AK024735.1, BAB14982.1
           xrefs (non-sequence databases):
           GenewHGNC: 11712, MIM 605796, Inter-
           ProIPR008191, InterProIPR002999,
           PfamPF00567, SMARTSM00333,
           PROSITEPS50304
KEYWORDS   Repeat.
SOURCE     Homo sapiens (human)
           ORGANISM Homo sapiens
                    Eukaryota; Metazoa; Chordata; Craniata;
                    Vertebrata; Euteleostomi; Mammalia;
                    Eutheria; Euarchontoglires; Primates;
                    Catarrhini; Hominidae; Homo.
REFERENCE  1 (residues 1 to 777)
           AUTHORS  Wang,P. J., McCarrey,J. R., Yang,F. and
                    Page,D. C.
           TITLE    An abundance of X-linked genes ex-
                    pressed in spermatogonia
           JOURNAL  Nat. Genet. 27 (4), 422-426 (2001)
           PUBMED   11279525
           REMARK   NUCLEOTIDE SEQUENCE.
                    TISSUE = Testis
REFERENCE  2 (residues 1 to 777)
           AUTHORS  Ota,T., Suzuki,Y., Nishikawa,T.,
                    Otsuki,T., Sugiyama,T., Irie, R.,
                    Wakamatsu,A., Hayashi,K., Sato,H.,
                    Nagai,K., Kimura,K., Makita, H.
                    Sekine,M., Obayashi,M., Nishi,T.,
                    Shibahara,T., Tanaka,T., Ishii,S.,
                    Yamamoto,J., Saito,K., Kawai,Y.,
                    Isono,Y., Nakamura, Y., Nagahari,K.,
                    Murakami,K., Yasuda,T., Iwayanagi,T.,
                    Wagatsuma,M., Shiratori,A., Sudo,H.,
                    Hosoiri,T., Kaku,Y., Kodaira,H.,
                    Kondo, H., Sugawara,M., Takahashi,M.,
                    Kanda,K., Yokoi,T., Furuya,T., Kikkawa,
                    E., Omura,Y., Abe,K., Kamihara,K.,
                    Katsuta,N., Sato, K., Tanikawa,M.,
                    Yamazaki,M., Ninomiya,K., Ishibashi,T.,
                    Yamashita, H., Murakawa,K., Fujimori,
                    K., Tanai,H., Kimata,M., Watanabe,M.,
                    Hiraoka,S., Chiba,Y., Ishida,S., Ono,
                    Y., Takiguchi,S., Watanabe, S., Yosida,
                    M., Hotuta,T., Kusano,J., Kanehori,K.,
                    Takahashi-Fujii, A. Hara,H., Tanase,T.
                    O., Nomura,Y., Togiya,S., Komai,F.,
                    Hara,R., Takeuchi,K., Arita,M., Imose,
                    N., Musashino,K., Yuuki,H., Oshima,A.,
                    Sasaki,N., Aotsuka,S., Yoshikawa,Y.,
                    Matsunawa,H., Ichihara,T., Shiohata,N.,
                    Sano,S., Moriya,S., Momiyama,H.,
                    Satoh,N., Takami, S., Terashima,Y.,
                    Suzuki,O., Nakagawa,S., Senoh,A.,
                    Mizoguchi,H., Goto,Y., Shimizu,F.,
                    Wakebe,H., Hishigaki,H., Watanabe,T.,
                    Sugiyama,A., Takemoto,M., Kawakami,B.,
                    Yamazaki,M., Watanabe, K., Kumagai,A.,
                    Itakura,S., Fukuzumi,Y., Fujimori,Y.,
                    Komiyama,M., Tashiro,H., Tanigami,A.,
                    Fujiwara,T., Ono,T., Yamada,K., Fujii,
                    Y., Ozaki,K., Hirao,M., Ohmori,Y.,
                    Kawabata,A., Hikiji,T., Kobatake,N.,
                    Inagaki,H., Ikema,Y., Okamoto,S.,
                    Okitani,R., Kawakami,T., Noguchi,S.,
                    Itoh,T., Shigeta,K., Senba,T.,
                    Matsumura,K., Nakajima,Y., Mizuno,T.,
                    Morinaga,M., Sasaki,M., Togashi,T.,
                    Oyama,M., Hata,H., Watanabe,M.,
                    Komatsu,T., Mizushima-Sugano, J.,
                    Satoh,T., Shirai,Y., Takahashi,Y.,
                    Nakagawa,K., Okumura,K., Nagase,T.,
                    Nomura,N., Kikuchi,H., Masuho,Y.,
                    Yamashita,R., Nakai,K., Yada,T.,
                    Nakamura,Y., Ohara,O., Isogai,T. and
                    Sugano, S.
           TITLE    Complete sequencing and characteriza-
                    tion of 21,243 full-length human cDNAs
           JOURNAL  Nat. Genet. 36 (1), 40-45 (2004)
           PUBMED   14702039
           REMARK   NUCLEOTIDE SEQUENCE [LARGE SCALE MRNA]
                    OF 67-777.
COMMENT    [TISSUE SPECIFICITY] Testis and ovary
           specific. [SIMILARITY] Contains 3 Tudor
           domains.
FEATURES   Location/Qualifiers
           source   1 . . . 777
                    /organism = “Homo sapiens”
                    /db_xref = “taxon: 9606”
           gene     1 . . . 777
                    /gene = “TDRD1”
           Protein  1 . . . 777
                    /gene = “TDRD1”
                    /product = “Tudor domain containing
                    protein 1”
           Region   138 . . . 197
                    /gene = “TDRD1”
                    /region_name = “Domain”
                    /note = “Tudor 1.”
                    /evidence = experimental
           Region   359 . . . 418
                    /gene = “TDRD1”
                    /region_name = “Domain”
                    /note = “Tudor 2.”
                    /evidence = experimental
           Region   587 . . . 645
                    /gene = “TDRD1”
                    /region_name = “Domain”
                    /note = “Tudor 3.”
                    /evidence = experimental
           Region   737
                    /gene = “TDRD1”
                    /region_name = “Conflict”
                    /note = “T −> M (in REF. 2).”
                    /evidence = experimental
           Region   775 . . . 777
                    /gene = “TDRD1”
                    /region_name = “Conflict”
                    /note = “VKS −> KKKKK (in REF. 2).”
                    /evidence = experimental
ORIGIN
1          meqycsikiv dileeevvtf avevelpnsg
           klldhvliem gyglkpsgqd skkenadqsd
61         pedvgkmtte nnivvdksdl ipkvltlnvg
           defcgvvahi qtpedffcqq lqsgrklael
121        qaslskycdq lpprsdfypa igdiccaqfs
           eddqwyrasv layaseesvl vgyvdygnfe
181        ilslmrlcpi ipkllelpmq aikcvlagvk
           pslgiwtpea iclmkklvqn kiitvkvvdk
241        lensslveli dksetphvsv skvlldagfa
           vgeqsmvtdk psdvketsvp lgvegkvnpl
301        ewtwvelgvd qtvdvvvcvi yspgefychv
           lkedalkkln dlnkslaehc qqklpngfka
361        eigqpccaff agdgswyral vkeilpnghv
           kvhfvdygni eevtadelrm isstflnlpf
421        qgircqladi qsrnkhwsee aitrfgmcva
           giklqarvve vtengigvel tdlstcypri
481        isdvlidehl vlksasphkd lpndrlvnkh
           elqvhvqglq atssaeqwkt ielpvdktiq
541        anvleiispn ifyalpkgmp enqeklcmlt
           aelleycnap ksrppyrpri gdaccakyts
601        ddfwyravvl gtsdtdvevl yadygnietl
           plcrvqpits shlalpfqii rcsleglmel
661        ngsssqliim llknfmlnqn vmlsvkgitk
           nvhtvsvekc sengtvdvad klvtfglakn
721        itpqrqsaln tekmyrtncc ctelqkqvek
           hehillflln nstnqnkfie mkklvks
//

Human Dual Specificity Phosphatase II (DUSPII)

LOCUS      NP_003575    330 aa    linear  PRI 02-
MARCH 2005
DEFINITION dual specificity phosphatase 11 [Homo
           sapiens].
ACCESSION  NP_003575
VERSION    NP_003575.1 GI: 4503415
DBSOURCE   REFSEQ: accession NM 003584.1
KEYWORDS   .
SOURCE     Homo sapiens (human)
           ORGANISM Homo sapiens
                    Eukaryota; Metazoa; Chordata; Craniata;
                    Vertebrata; Euteleostomi; Mammalia;
                    Eutheria; Euarchontoglires; Primates;
                    Catarrhini; Hominidae; Homo.
REFERENCE  1 (residues 1 to 330)
           AUTHORS  Yuan,Y., Li,D. M. and Sun,H.
           TITLE    PIR1, a novel phosphatase that exhibits
                    high affinity to RNA ribonucleoprotein
                    complexes
           JOURNAL  J. Biol. Chem. 273 (32), 20347-20353
                    (1998)
           PUBMED   9685386
COMMENT    REVIEWED REFSEQ: This record has been
           curated by NCBI staff. The reference
           sequence was derived from AF023917.1.
           Summary: The protein encoded by this
           gene is a member of the dual specific-
           ity protein phosphatase subfamily.
           These phosphatases inactivate their
           target kinases by dephosphorylating
           both the phosphoserine/threonine and
           phosphotyrosine residues. They neg-
           atively regulate members of the mito-
           gen-activated protein (MAP) kinase
           superfamily (MAPK/ERK, SAPK/JNK, p38),
           which is associated with cellular
           proliferation and differentiation.
           Different members of the family of
           dual specificity phosphatases show
           distinct substrate specificities for
           various MAP kinases, different tissue
           distribution and subcellular localiza-
           tion, and different modes of inducibil-
           ity of their expression by extracel-
           lular stimuli. This gene product is
           localized to the nucleus, and is novel
           in that it binds directly to RNA and
           splicing factors, and thus suggested
           to participate in nuclear mRNA meta-
           bolism.
FEATURES   Location/Qualifiers
           source   1 . . . 330
                    /organism = “Homo sapiens”
                    /db_xref = “taxon: 9606”
                    /chromosome = “2”
                    /map = “2p13.1”
           Protein  1 . . . 330
                    /product = “dual specificity phos-
                    phatase 11”
                    /EC_number = “3.1.3.16”
                    /EC_number = “3.1.3.48”
                    /note = “serine/threonine specific
                    protein phosphatase; RNA/RNP com-
                    plex-interacting phosphatase”
           CDS      1 . . . 330
                    /gene = “DUSP11”
                    /coded_by = “NM_003584.1:
                    125 . . . 1117”
                    /note = “go_component: nucleus
                    [goid 0005634] [evidence TAS]
                    [pmid 9685386];
                    go_function: RNA binding [goid
                    0003723] [evidence TAS]
                    [pmid 9685386];
                    go_function: hydrolase activity
                    [goid 0016787] [evidence IEA];
                    go_function: protein tyrosine
                    phosphatase activity [goid
                    0004725] [evidence TAS]
                    [pmid 9685386];
                    go_process: RNA processing [goid
                    0006396] [evidence TAS]
                    [pmid 9685386];
                    go_process: protein amino acid
                    dephosphorylation [goid 0006470]
                    [evidence IEA]”
                    /db_xref = “CCDS: CCDS1928.1”
                    /db_xref = “GeneID: 8446”
                    /db_xref = “MIM: 603092”
ORIGIN
1          msqwhhprsg wgrrrdfsgr ssakkkggnh
           iperwkdylp vgqrmpgtrf iafkvplqks
61         tekklapeec fspldlfnki reqneelgli
           idltytqryy kpedlpetvp ylkiftvghq
121        vpddetifkf khavngflke nkdndkligv
           hcthglnrtg ylicrylidv egvrpddaie
181        lfnrcrqhcl erqnyiedlq ngpirknwns
           svprssdfed sahlmqpvhn kpvkqgpryn
241        lhqiqghsap rhfhtqtqsl qqsvrkfsen
           phvyqrhhlp ppgppgedys hrryswnvkp
301        nasraaqdrr rwypynysrl sypacwewtq
//

IV. Dicer

Dicer proteins for use in the present invention can be from any suitable source. Preferred sources include C. elegans, H. sapeins and M. musculus, as depicted infia, although the skilled artisan will appreciate that other sources can readily be used based on the significant conservation exhibited between Dicer homologs. For example, Dicer homologs from D. melanogaster, Rattus norvegicus, and primate are useful (see, e.g., Accession Nos. gi:51316117; gi:34867687; and gi:55641327, respectively).

LOCUS      NP_498761    1845 aa    linear  INV 21-
NOVEMBER
2003
DEFINITION DiCer Related, LEThal LET-740 (dcr-1)
           [Caenorhabditis elegans].
ACCESSION  NP_498761
VERSION    NP_498761.1 GI: 17552834
DBSOURCE   REFSEQ: accession NM 066360.1
KEYWORDS   .
SOURCE     Caenorhabditis elegans
           ORGANISM  Caenorhabditis elegans
                     Eukaryota; Metazoa; Nematoda;
                     Chromadorea; Rhabditida; Rhabditoidea;
                     Rhabditidae; Peloderinae;
                     Caenorhabditis.
REFERENCE  1 (residues 1 to 1845)
           AUTHORS   Dillin,A., Hsu,A. L., Arantes-Oliveira,
                     N., Lehrer-Graiwer,J., Hsin,H., Fraser,
                     A. G., Kamath,R. S., Ahringer,J. and
                     Kenyon,C.
           TITLE     Rates of behavior and aging specified
                     by mitochondrial function during
                     development
           JOURNAL   Science 298 (5602), 2398-2401 (2002)
           MEDLINE   22382053
           PUBMED    12471266
REFERENCE  2 (residues 1 to 1845)
           AUTHORS   Piano,F., Schetter,A. J., Morton,D. G.,
                     Gunsalus,K. C., Reinke, V. Kim,S. K.
                     and Kemphues,K. J.
           TITLE     Gene clustering based on RNAi pheno-
                     types of ovary-enriched genes in
                     C. elegans
           JOURNAL   Curr. Biol. 12 (22), 1959-1964 (2002)
           MEDLINE   22335533
           PUBMED    12445391
REFERENCE  3 (residues 1 to 1845)
           AUTHORS   Walhout,A. J., Reboul,J., Shtanko,O.,
                     Bertin,N., Vaglio,P., Ge, H., Lee,H.,
                     Doucette-Stamin,L., Gunsalus,K. C.,
                     Schetter,A. J., Morton,D. G., Kemphues,
                     K. J., Reinke,V., Kim,S. K., Piano,F.
                     and Vidal, M.
           TITLE     Integrating interactome, phenome, and
                     transcriptome mapping data for the
                     C. elegans germline
           JOURNAL   Curr. Biol. 12 (22), 1952-1958 (2002)
           MEDLINE   22335532
           PUBMED    12445390
REFERENCE  4 (residues 1 to 1845)
           AUTHORS   Tabara,H., Yigit,E., Siomi,H. and
                     Mello,C.C.
           TITLE     The dsRNA binding protein RDE-4 inter-
                     acts with RDE-1, DCR-1, and a DExH-box
                     helicase to direct RNAi in C. elegans
           JOURNAL   Cell 109 (7), 861-871 (2002)
           MEDLINE   22105477
           PUBMED    12110183
2REFERENCE 5 (residues 1 to 1845)
           AUTHORS   Banerjee,O. and Slack,F.
           TITLE     Control of developmental timing by
                     small temporal RNAs: a paradigm for
                     RNA-mediated regulation of gene
                     expression
           JOURNAL   Bioessays 24 (2), 119-129 (2002)
           MEDLINE   21823375
           PUBMED    11835276
REFERENCE  6 (residues 1 to 1845)
           AUTHORS   Ketting,R. F., Fischer,S. E.,
                     Bernstein,E., Sijen,T., Hannon,G. J.
                     and Plasterk, R. H.
           TITLE     Dicer functions in RNA interference and
                     in synthesis of small RNA involved in
                     developmental timing in C. elegans
           JOURNAL   Genes Dev. 15 (20), 2654-2659 (2001)
           MEDLINE   21521222
           PUBMED    11641272
REFERENCE  7 (residues 1 to 1845)
           AUTHORS   Knight,S. W. and Bass,B. L.
           TITLE     A role for the RNase III enzyme DCR-1
                     in RNA interference and germ line de-
                     velopment in Caenorhabditis elegans
           JOURNAL   Science 293 (5538), 2269-2271 (2001)
           MEDLINE   21451181
           PUBMED    11486053
REFERENCE  8 (residues 1 to 1845)
           AUTHORS   Jones,S. J., Riddle,O. L., Pouzyrev,A.
                     T., Velculescu,V. E., Hillier,L., Eddy,
                     S. R., Stricklin,S. L., Baillie,D. L.,
                     Waterston, R. and Marra,M. A.
           TITLE     Changes in gene expression associated
                     with developmental arrest and longevity
                     in Caenorhabditis elegans
           JOURNAL   Genome Res. 11 (8), 1346-1352 (2001)
           MEDLINE   21376140
           PUBMED    11483575
REFERENCE  9 (residues 1 to 1845)
           AUTHORS   Grishok,A., Pasquinelli,A. E., Conte,
                     D., Li,N., Parrish,S., Ha, I., Baillie,
                     D. L., Fire,A., Ruvkun,G. and Mello,
                     C. C.
           TITLE     Genes and mechanisms related to RNA in-
                     terference regulate expression of the
                     small temporal RNAs that control C.
                     elegans developmental timing
           JOURNAL   Cell 106 (1), 23-34 (2001)
           MEDLINE   21354308
           PUBMED    11461699
REFERENCE  10 (residues 1 to 1845)
           AUTHORS   Stewart,H. I., O'Neil,N. J., Janke,D.
                     L., Franz,N. W., Chamberlin,H. M.,
                     Howell,A. M., Gilchrist,E. J., Ha,T.
                     T., Kuervers,L. M., Vatcher,G. P.,
                     Danielson,J. L. and Baillie,D. L.
           TITLE     Lethal mutations defining 112 comple-
                     mentation groups in a 4.5 Mb sequenced
                     region of Caenorhabditis elegans
                     chromosome III
           JOURNAL   Mol. Gen. Genet. 260 (2-3), 280-288
                     (1998)
           MEDLINE   99077298
           PUBMED    9862482
COMMENT    PROVISIONAL REFSEQ: This record has not
           yet been subject to final NCBI review.
           This record is derived from an anno-
           tated genomic sequence (NC_003281).
           The reference sequence was derived
           from WormBase CDS: K12H4.8.
           Summary: This essential gene dcr-1,
           also known as let-740, K12H4.8,
           3J162 or YK334, maps at (III; −0.30).
           Phenotypes and affected processes are
           required for RNA interference, required
           for synthesis of microrna, sterile
           adult, lethal. It encodes a DiCer Re-
           lated. From Pfam homology, the product
           would have ATP binding, nucleic acid
           binding, ATP dependent helicase, heli-
           case, RNA binding, double-stranded RNA
           binding, ribonuclease III activities,
           would be involved in RNA processing
           and would localize in intracellular.
           According to the Worm Transcriptome
           Project, it is expressed at high level
           at all stages of development [Kohara
           cDNAs], except dauers [SAGE]. Its
           existence, but not its exact sequence,
           derived here from the genome sequenc-
           ing consortium annotation, is sup-
           ported by 26 cDNA clones.
           Phenotype
           [WormBase] dcr-1 is required both for
           RNA interference and for synthesis of
           small developmental RNAs. Fertilization
           of dcr-1 oocytes does not occur. While
           this fertilization defect can be res-
           cued by a dcr-1(+) transgene, fertil-
           ized eggs fail to hatch, and mothers
           are defective in egg-laying. Whereas
           wild-type oocytes normally do not un-
           dergo cell division in the gonad, dcr-
           1 (pk1531) oocytes undergo such divi-
           sion frequently. dcr-1 mutations also
           cause postembryonic defects: alae are
           absent in 60%, and a burst vulva is
           observed in 80%, of dcr-1 (pk1531)
           homozygotes. The postembryonic defects
           are consistent with the hypothesis that
           dcr-1 mutants hyperactivate lin-41 in
           vivo because they are unable to form
           active let-7 stRNA; in vitro assays of
           DCR-1 protein confirm that it can gen-
           erate let-7 stRNA from a double-
           stranded let-7 precursor. [Ann Rose,
           1998, pm9862482] let-704 homozygous
           s2624 and s2795 each develop into
           sterile adults. Knock-out allele, de-
           letion obtained by the Gene Knockout
           Consortium ok247 (strain BB1) [R
           Barstead, Oklahoma MRF, USA]. Selected
           strains available from the CGC. BC4825
           [David Baillie]. NL687 dcr-1 (pk1351)/+
           III [Ronald Plasterk, Fischer/Thijssen,
           UV/TMP] Heterozygotes are WT and segre-
           gate WT and animals with protruding
           vulvas (dcr-1 homozygotes). PD8753 dcr-1
           (ok247) III/hT2[qIs48] (I; III) [Andrew
           Fire, Barstead/Moulder] [Brenda Bass
           description] Heterozygotes are WT and
           segregate WT, Uncs, and Steriles. [B
           Barstead] dcr-1 homozygotes are com-
           pletely sterile. qIs48 is an insertion
           of ccEx9747 with markers: myo-2: :GFP
           expressed brightly in the pharynx
           throughout development, pes-10: :GFP
           expressed in embryos, and a gut pro-
           moter driving GFP in the intestine.
           Segregates WT glowing hets, non-glowing
           steriles, very rare homozygous hT2
           glowing animals, and dead eggs.
           BB1.
           RNA interference results:
           [T. Hyman 2000] No obvious phenotype
           (by injecting genomic PCR product TH:
           K12H4.8). [J. Ahringer 2003] No obvious
           phenotype (by feeding genomic PCR pro-
           duct JA: K12H4.8). [F. Piano 2002] No
           P0 sterility detected. Pleiotropic
           phenotypes (may include abnormal trans-
           lucence, Dpy, Egl, Gon, Muv, Pvl, Sma)
           observed in <10% of progeny. No obvious
           phenotype.
           Function
           Protein properties: [Wormbase] biden-
           tate ribonuclease, contains a helicase
           domain, a PAZ domain, two RNAse III
           domains, and a double-stranded RNA-
           binding domain.
           Expression
           The expression profile for the gene,
           derived from the proportion of animals
           at each stage in each Kohara library
           is: embryos 7%, L1 or L2 larvae 19%,
           L3 to adult 75%.
           In situ hybridisation pictures to all
           stages of development are available
           from Kohara NextDB.
           Pattern [pm11483575] From SAGE compar-
           ative analysis of dauer and mixed
           stages, this gene is one of 533 whose
           expression is lowered in dauer larvae,
           a facultative developmentally arrested
           and long lived stage in C. elegans life
           cycle. germline enriched [Piano, 2002].
           The predicted CDS has 26 exons. It
           covers 8.17 kb on the WS97 genome. The
           protein (1845 aa, 210.9 kDa, pI 5.6)
           contains one DEAD/DEAH box helicase
           motif, one helicase, C-terminal motif,
           one Protein of unknown function DUF283
           motif, one Argonaute and Dicer protein,
           PAZ motif, 2 Ribonuclease III family
           motifs, one Double-stranded RNA binding
           (DsRBD) domain motif. It also contains
           3 coil coil stretch [Psort2]. It is
           predicted to localise in the cytoplasm
           [Psort2]. Taxblast (threshold 10{circumflex over ( )}-3)
           tracks ancestors down to archaea and
           viruses and bacteria and eukaryota.
           Method: conceptual translation.
FEATURES   Location/Qualifiers
           source    1 . . . 1845
                     /organism = “Caenorhabditis elegans”
                     /db_xref = “taxon: 6239”
                     /chromosome = “III”
                     /map = “III; −0.30 cM (interpolated
                     genetic position)”
                     /map = “III; covering 6084 bp, from
                     base 8077912 to 8071829 on genome
                     release WS97”
                     /clone_lib = “Kohara embryonic
                     lambda gt11 library: yk571d8,
                     yk675c6; Kohara Sugano L1 larvae
                     cap-selected library: yk1080g6,
                     yk1084b3, yk1086f1, yk1249b10,
                     yk1271d8; Kohara Sugano L2 larvae
                     cap-selected library: yk1627e3,
                     yk1734b12; Kohara Sugano L4 larvae
                     cap-selected library: yk1448b2,
                     yk1548a2, yk1554a2; Kohara mixed
                     stage library, from him-8 strain,
                     containing 15-30% males: yk11h10,
                     yk18g7, yk24e10, yk86c11, yk181d7,
                     yk192e1, yk243c2, yk249e11, yk318d2,
                     yk355e9, yk355h8, yk419h11,
                     yk154a11; early embryos, Stratagene
                     library [PMID1302005]: T02268”
           Protein   1 . . . 1845
                     /product = “DiCer Related, LEThal
                     LET-740 (dcr-1)”
           Region    3 . . . 218
                     /region_name = “[Pfam/InterPro
                     description] DEAD/DEAH box helicase”
                     /db_xref = “CDD: pfam00270”
           Region    190 . . . 218
                     /region_name = “[PSORT] coil coil 4:
                     PEKLMEQLKKLESAMDSVIETASDLVSLS”
           Region    339 . . . 345
                     /region_name = “[PSORT] nuclear lo-
                     calization domain: PEMKKIK”
           Region    427 . . . 498
                     /region_name = “[Pfam/InterPro de-
                     scription] helicase, C-terminal”
                     /db_xref = “CDD: pfam00271”
           Region    503 . . . 602
                     /region_name = “[Pfam/InterPro de-
                     scription] protein of unknown
                     function DUF283”
                     /db_xref = “CDD: pfam03368”
           Region    669 . . . 675
                     /region_name = “[PSORT] nuclear lo-
                     calization domain: PKRRKFE”
           Region    764 . . . 770
                     /region_name = “[PSORT] nuclear lo-
                     calization domain: PLNKRKD”
           Region    782 . . . 961
                     /region_name = “[Pfam/InterPro de-
                     scription] argonaute and Dicer pro-
                     tein, PAZ”
                     /db_xref = “CDD: pfam02170”
           Region    891 . . . 897
                     /region_name = “[PSORT] nuclear lo-
                     calization domain: PRRSRTV”
           Region    1008 . . . 1036
                     /region_name = “[PSORT] coil coil 4:
                     IQQLRDLNQKSIEDQERETRENDKIDDGE”
           Region    1179 . . . 1214
                     /region_name = “[PSORT] coil coil 4:
                     PKQLTKEEEQFKKLQNDLLKQAKERLEALEMSEDME”
           Region    1215 . . . 1218
                     /region_name = “[PSORT] nuclear lo-
                     calization domain: KPRR”
           Region    1348 . . . 1524
                     /region_name = “[Pfam/InterPro
                     description] ribonuclease III
                     family”
                     /db_xref = “CDD: pfam00636”
           Region    1614 . . . 1740
                     /region_name = “[Pfam/InterPro
                     description] ribonuclease III
                     family”
                     /db_xref = “CDD: pfam00636”
           Region    1769 . . . 1829
                     /region_name = “[Pfam/InterPro de-
                     scription] double-stranded RNA
                     binding (DsRBD) domain”
                     /db_xref = “CDD: pfam00035”
           CDS       1 . . . 1845
                     /gene = “dcr-1”
                     /locus_tag = “3J162”
                     /coded_by = “NM_066360.1:
                     1 . . . 5538”
                     /db_xref = “AceView/WormGenes: dcr-1”
                     /db_xref = “GeneID: 176138”
                     /db_xref = “LocusID: 176138”
                     /db_xref = “WormBase: K12H4.8”
ORIGIN
1          mvrvradlqc fnprdyqvel ldkatkknti
           vqlgtgsgkt fiavlllkey gvqlfapldq
61         ggkraffvve kvnlveqqai hievhtsfkv
           gqvhgqtssg lwdskeqcdq fmkrhhvvvi
121        taqclldlir haylkiedmc vlifdechha
           lgsqhpyrsi mvdykllkkd kpvprvlglt
181        aslikakvap eklmeqlkkl esamdsviet
           asdlvslsky gakpyevvii ckdfeigclg
241        ipnfdtviei fdetvafvnt ttefhpdldl
           dprrpikdsl kttravfrql gpwaawrtaq
301        vwekelgkii ksqvlpdktl rflnmaktsm
           itikrllepe mkkiksieal rpyvpqrvir
361        lfeiletfnp efqkermkle kaehlsaiif
           vdqryiaysl llmmrhiksw epkfkfvnpd
421        yvvgasgrnl assdsqglhk rqtevlrrfh
           rneincliat svleegvdvk qcnlvikfdr
481        pldmrsyvqs kgrarragsr yvitveekdt
           aaycsklpsd iftrlvphnq iipieengvt
541        kycaelllpi nspikhaivl knpmpnkkta
           qmavaleacr qlhlegeldd nllpkgresi
601        akllehidee pdeyapgiaa kvgsskrkql
           ydkkiaraln esfveadkec fiyafelerf
661        reaeltlnpk rrkfedpfny eycfgflsak
           eipkippfpv flrqgnmkvr livapkkttv
721        taaqlqeiql fhnylftqvl qmcktgnlef
           dgtsnaplnt livplnkrkd dmsytinmky
781        vsevvanmen mpripkdevr rqykfnaedy
           kdaivmpwyr nleqpvfyyv aeilpewrps
841        skfpdthfet fneyfikkyk leiydqnqsl
           ldvdftstrl nhlqpriqnq prrsrtvsns
901        stsnipqasa sdskesntsv phssqrqilv
           pelmdihpis atlwnviaal psifyrvnql
961        lltdelreti lvkafgkekt klddnvewns
           layateyeek qtiivkkiqq lrdlnqksie
1021       dqeretrend kiddgeelfn igvwdpeeav
           rigveissrd drmdgedqdt vgltqglhdg
1081       nisdeddelp fvmhdytarl tsnrngigaw
           sgsesivpsg wgdwdgpepd nspmpfqilg
1141       gpgglnvqal madvgrvfdp stassslsqt
           vqestvsppk qltkeeeqfk klqndllkqa
1201       kerlealems edmekprrle dtvnledygd
           dqenqedent ptnfpktide eieelsigar
1261       kkqeiddnaa ktdvlerenc evlpvainek
           srsfsfekes kaingrlirq rseeyvshid
1321       sdiglgvspc llltalttsn aadgmslerf
           etigdsflkf attdylyhtl ldqhegklsf
1381       arskevsncn lyrlgkklgi pqlivankfd
           ahdswlppcy iptcdfkapn tddaeekdne
1441       ierildgqvi eekpenktgw diggdvskst
           tdgietitfp kqarvgnddi splpynlltq
1501       qhisdksiad avealigvhl ltlgpnptlk
           vmnwmglkvi qkdqksdvps pllrfidtpt
1561       npnaslnfln nlwqqfqftq leekigyrfk
           eraylvqaft hasyinnrvt gcyqrleflg
1621       davldymitr ylfedsrqys pgvltdlrsa
           lvnntifasl avkfefqkhf iamcpglyhm
1681       iekfvklcse rnfdtnfnae mymvtteeei
           degqeediev pkamgdifes vagaiyldsg
1741       rnldttwqvi fhmmrgtiel ccanpprspi
           relmefeqsk vrfskmeril esgkvrvtve
1801       vvnnmrftgm grnyriakat aakralkylh
           qieqqrrqsp slttv
//
LOCUS      NP_803187    1922 aa    linear  PRI 22-
DECEMBER
2003
DEFINITION dicerl; helicase-mol; K12H4.8-LIKE;
           helicase with RNAse motif [Homo
           sapiens].
ACCESSION  NP_803187
VERSION    NP_803187.1 GI: 29294651
DESOURCE   REFSEQ: accession NM 177438.1
KEYWORDS   .
SOURCE     Homo sapiens (human)
           ORGANISM  Homo sapiens
                     Eukaryota; Metazoa; Chordata; Craniata;
                     Vertebrata; Euteleostomi; Mammalia;
                     Eutheria; Primates; Catarrhini;
                     Hominidae; Homo.
REFERENCE  1 (residues 1 to 1922)
           AUTHORS   Handa,V., Saha,T. and Usdin,K.
           TITLE     The fragile X syndrome repeats form RNA
                     hairpins that do not activate the in-
                     terferon-inducible protein kinase, PKR,
                     but are cut by Dicer
           JOURNAL   Nucleic Acids Res. 31 (21), 6243-6248
                     (2003)
           PUBMED    14576312
           REMARK    GeneRIF: fragile X syndrome CGG repeats
                     readily form RNA hairpins and is di-
                     gested by the human Dicer enzyme, a
                     step central to the RNA interference
                     effect on gene expression
REFERENCE  2 (residues 1 to 1922)
           AUTHORS   Kawasaki,H., Suyama,E., Iyo,M. and
                     Taira,K.
           TITLE     siRNAs generated by recombinant human
                     Dicer induce specific and significant
                     but target site-independent gene si-
                     lencing in human cells
           JOURNAL   Nucleic Acids Res. 31 (3), 981-987
                     (2003)
           PUBMED    12560494
REFERENCE  3 (residues 1 to 1922)
           AUTHORS   Doi,N., Zenno,S., Ueda,R., Ohki-
                     Hamazaki,H., Ui-Tei,K. and Saigo, K.
           TITLE     Short-interfering-RNA-mediated gene
                     silencing in mammalian cells requires
                     Dicer and eIF2C translation initiation
                     factors
           JOURNAL   Curr. Biol. 13 (1), 41-46 (2003)
           PUBMED    12526743
REFERENCE  4 (residues 1 to 1922)
           AUTHORS   Zhang,H., Kolb,F. A., Brondani,V.,
                     Billy,E. and Filipowicz,W.
           TITLE     Human Dicer preferentially cleaves
                     dsRNAs at their termini without a
                     requirement for ATP
           JOURNAL   EMBO J. 21 (21), 5875-5885 (2002)
           PUBMED    12411505
           REMARK    GeneRIF: purification and properties of
                     a recombinant human Dicer
REFERENCE  5 (residues 1 to 1922)
           AUTHORS   Provost,P., Dishart,D., Doucet,J.,
                     Frendewey,D., Samuelsson,B. and
                     Radinark, O.
           TITLE     Ribonuclease activity and RNA binding
                     of recombinant human Dicer
           JOURNAL   EMBO J. 21 (21), 5864-5874 (2002)
           PUBMED    12411504
           REMARK    GeneRIF: cloning and expression of the
                     218 kDa human Dicer, and characteriza-
                     tion of its ribonuclease activity and
                     dsRNA-binding properties
REFERENCE  6 (residues 1 to 1922)
           AUTHORS   Matsuda,S., Ichigotani,Y., Okuda,T.,
                     Irimura,T., Nakatsugawa, S. and
                     Hamaguchi, M.
           TITLE     Molecular cloning and characterization
                     of a novel human gene (HERNA) which
                     encodes a putative RNA-helicase
           JOURNAL   Biochim. Biophys. Acta 1490 (1-2),
                     163-169 (2000)
           PUBMED    10786632
REFERENCE  7 (residues 1 to 1922)
           AUTHORS   Provost,P., Samuelsson,B. and
                     Radmark,O.
           TITLE     Interaction of 5-lipoxygenase with
                     cellular proteins
           JOURNAL   Proc. Natl. Acad. Sci. U.S.A. 96 (5),
                     1881-1885 (1999)
           PUBMED    10051563
COMMENT    REVIEWED REFSEQ: This record has been
           curated by NCBI staff. The reference
           sequence was derived from AB023145.2,
           AB028449.1, AK091094.1, AW297296.1,
           BI913232.1 and BQ937506.1.
           Summary: This gene encodes a protein
           possessing an RNA helicase motif con-
           taining a DEXH box in its amino
           terminus and an RNA motif in the car-
           boxy terminus. The encoded protein
           functions as a ribonuclease and is re-
           quired by the RNA interference and
           small temporal RNA (stRNA) pathways to
           produce the active small RNA component
           that represses gene expression. Two
           transcript variants encoding the same
           protein have been identified for this
           gene.
           Transcript Variant: This variant (1)
           represents the longer transcript.
           Variants 1 and 2 encode the same
           isoform.
FEATURES   Location/Qualifiers
           source    1 . . . 1922
                     /organism = “Homo sapiens”
                     /db_xref = “taxon: 9606”
                     /chromosome = “14”
                     /map = “14q32.2”
           Protein   1 . . . 1922
                     /product = “dicer1”
                     /EC_number = “3.1.26.-”
                     /note = “helicase-moi; K12H4.8-LIKE;
                     helicase with RNAse motif”
           Region    37 . . . >208
                     /region_name = “ERCC4-like helicases
                     [DNA replication, recombination, and
                     repair]”
                     /note = “MPH1”
                     /db_xref = “CDD: 10833”
           Region    40 . . . 211
                     /region_name = “DEAD-like helicases
                     superfamily”
                     /note = “DEXDc”
                     /db_xref = “CDD: 22813”
           Region    107 . . . 1899
                     /region_name = “dsRNA-specific
                     nuclease Dicer and related ribo-
                     nucleases [RNA processing and
                     modification]”
                     /note = “KOG0701”
                     /db_xref = “CDD: 18495'
           variation 257
                     /replace = “*”
                     /replace = “C”
                     /db_xref = “dbSNP: 12432511”
           Region    <499 . . . 553
                     /region_name = “Helicase conserved
                     C-terminal domain”
                     /note = “helicase_C”
                     /db_xref = “CDD: 22962”
           variation 499
                     /replace = “R”
                     /replace = “T”
                     /db_xref = “dbSNP: 4566088”
           Region    625 . . . 722
                     /region_name = “Domain of unknown
                     function”
                     /note = “DUF283”
                     /db_xref = “CDD: 5126”
           Region    895 . . . 1064
                     /region_name = “PAZ domain”
                     /note = “PAZ”
                     /db_xref = “CDD: 17101”
           Region    1296 . . . >1387
                     /region_name = “Ribonuclease III
                     family”
                     /note = “RIBOc”
                     /db_xref = “CDD: 22830”
           Region    1682 . . . 1846
                     /region_name = “Ribonuclease III
                     family”
                     /note = “RIBOc”
                     /db_xref = “CDD: 22830”
           CDS       1 . . . 1922
                     /gene = “DICER1”
                     /coded_by = “NM_177438.1:
                     239 . . . 6007”
                     /note = “go_component: intracellular
                     [goid 0005622] [evidence NAS]
                     [pmid 12560494];
                     go_function: double-stranded RNA
                     binding [goid 0003725] [evidence
                     IDA] [pmid 12411504];
                     go_function: endonuclease activity
                     [goid 0004519] [evidence IEA];
                     go_function: ATP binding [goid
                     0005524] [evidence IEA];
                     go_function: ribonuclease III
                     activity [goid 0004525] [evidence
                     IDA] [pmid 12560494];
                     go_function: ATP dependent helicase
                     activity [goid 0008026] [evidence
                     IEA];
                     go_function: hydrolase activity
                     [goid 0016787] [evidence IEA];
                     go_process: RNA processing [goid
                     0006396] [evidence IEA];
                     go_process: RNA interference,
                     targeting of mRNA for destruction
                     [goid 0030423] [evidence IEP] [pmid
                     12560494]”
                     /db_xref = “GeneID: 23405”
                     /db_xref = “LocusID: 23405”
                     /db_xref = “MIM: 606241”
ORIGIN
1          mkspalqpls maglqlmtpa sspmgpffgl
           pwqqeaihdn iytprkyqve lleaaldhnt
61         ivclntgsgk tfiavlltke lsyqirgdfs
           rngkrtvflv nsanqvaqqv savrthsdlk
121        vgeysnlevn aswtkerwnq eftkhqvlim
           tcyvalnvlk ngylslsdin llvfdechla
181        ildhpyreim klcencpscp rilgltasil
           ngkcdpeele ekiqklekil ksnaetatdl
241        vvldrytsqp ceivvdcgpf tdrsglyerl
           lmeleealnf indcnisvhs kerdstlisk
301        qilsdcravl vvlgpwcadk vagmmvrelq
           kyikheqeel hrkfllftdt flrkihalce
361        ehfspasldl kfvtpkvikl leilrkykpy
           erqqfesvew ynnrnqdnyv swsdseddde
421        deeieekekp etnfpspftn ilcgiifver
           rytavvlnrl ikeagkqdpe layissnfit
481        ghgigknqpr nkqmeaefrk qeevlrkfra
           hetnlliats iveegvdipk cnlvvrfdlp
541        teyrsyvqsk grarapisny imladtdkik
           sfeedlktyk aiekilrnkc sksvdtgetd
601        idpvmddddv fppyvlrpdd ggprvtinta
           ighinrycar lpsdpfthla pkcrtrelpd
661        gtfystlylp insplrasiv gppmscvrla
           ervvalicce klhkigeldd hlmpvgketv
721        kyeeeldlhd eeetsvpgrp gstkrrqcyp
           kaipeclrds yprpdqpcyl yvigmvlttp
781        lpdelnfrrr klyppedttr cfgiltakpi
           pqiphfpvyt rsgevtisie lkksgfmlsl
841        qmlelitrlh qyifshilrl ekpalefkpt
           dadsaycvlp lnvvndsstl didfkfmedi
901        eksearigip stkytketpf vfkledyqda
           viipryrnfd qphrfyvadv ytdltplskf
961        pspeyetfae yyktkynldl tnlnqplldv
           dhtssrlnll tprhlnqkgk alplssaekr
1021       kakweslqnk qilvpelcai hpipaslwrk
           avclpsilyr lhclltaeel raqtasdagv
1081       gvrslpadfr ypnldfgwkk sidsksfisi
           snsssaendn yckhstivpe naahqganrt
1141       sslenhdqms vncrtllses pgklhvevsa
           dltainglsy nqnlangsyd lanrdfcqgn
1201       qlnyykqeip vqpttsysiq nlysyenqpq
           psdectllsn kyldgnanks tsdgspvmav
1261       mpgttdtiqv lkgrmdseqs psigyssrtl
           gpnpglilqa ltlsnasdgf nlerlemlgd
1321       sflkhaitty lfctypdahe grlsymrskk
           vsncnlyrlg kkkglpsrmv vsifdppvnw
1381       lppgyvvnqd ksntdkwekd emtkdcmlan
           gkldedyeee deeeeslmwr apkeeadyed
1441       dfleydqehi rfidnmlmgs gafvkkisls
           pfsttdsaye wkmpkksslg smpfssdfed
1501       fdysswdamc yldpskavee ddfvvgfwnp
           seencgvdtg kqsisydlht eqciadksia
1561       dcveallgcy ltscgeraaq lflcslglkv
           lpvikrtdre kalcptrenf nsqqknlsvs
1621       caaasvassr ssvlkdseyg clkipprcmf
           dhpdadktln hlisgfenfe kkinyrfknk
1681       ayllqaftha syhyntitdc yqrleflgda
           ildylitkhl yedprqhspg vltdlrsalv
1741       nntifaslav kydyhkyfka vspelfhvid
           dfvqfqlekn emqgmdselr rseedeekee
1801       dievpkamgd ifeslagaiy mdsgmsletv
           wqvyypmmrp liekfsanvp rspvrellem
1861       epetakfspa ertydgkvrv tvevvgkgkf
           kgvgrsyria ksaaarralr slkanqpqvp
1921       ns
//
LOCUS      NP_683750    1917 aa    linear  ROD 16-
MARCH 2004
DEFINITION dicer1; endoribonuclease Dicer [Mus
           musculus].
ACCESSION  NP_683750
VERSION    NP_683750.1 GI: 22507359
DBSOURCE   REFSEQ: accession NM 148948.1
KEYWORDS   .
SOURCE     Mus musculus (house mouse)
           ORGANISM  Mus musculus
                     Eukaryota; Metazoa; Chordata; Craniata;
                     Vertebrata; Euteleostomi; Mammalia;
                     Eutheria; Rodentia; Sciurognathi;
                     Muridae; Murinae; Mus.
REFERENCE  1 (residues 1 to 1917)
           AUTHORS   Bernstein,E., Kim,S. Y., Carmeli,M. A.,
                     Murchison,E. P., Alcorn, H., Li,M. Z.,
                     Mills,A. A., Elledge,S. J., Anderson,
                     K. V. and Hannon, G. J.
           TITLE     Dicer is essential for mouse
                     development
           JOURNAL   Nat. Genet. 35 (3), 215-217 (2003)
           PUBMED    14528307
           REMARK    GeneRIF: role in lethality early in
                     development
REFERENCE  2 (residues 1 to 1917)
           AUTHORS   Okazaki,N., Kikuno,R., Ohara,R.,
                     Inamoto,S., Koseki,H., Hiraoka, S.,
                     Saga,Y., Nagase,T., Ohara,O. and
                     Koga, H.
           TITLE     Prediction of the coding sequences of
                     mouse homologues of KIAA gene: III. the
                     complete nucleotide sequences of 500
                     mouse KIAA-homologous cDNAs identified
                     by screening of terminal sequences of
                     cDNA clones randomly sampled from size-
                     fractionated libraries
           JOURNAL   DNA Res. 10 (4), 167-180 (2003)
           PUBMED    14621295
REFERENCE  3 (residues 1 to 1917)
           AUTHORS   Doi,N., Zenno,S., Ueda,R., Ohki-
                     Hamazaki,H., Ui-Tei,K. and Saigo, K.
           TITLE     Short-interfering-RNA-mediated gene
                     silencing in mammalian cells requires
                     Dicer and eIF2C translation initiation
                     factors
           JOURNAL   Curr. Biol. 13 (1), 41-46 (2003)
           PUBMED    12526743
REFERENCE  4 (residues 1 to 1917)
           AUTHORS   Nicholson,R. H. and Nicholson,A. W.
           TITLE     Molecular characterization of a mouse
                     cDNA encoding Dicer, a ribonuclease III
                     ortholog involved in RNA interference
           JOURNAL   Mamm. Genome 13 (2), 67-73 (2002)
           PUBMED    11889553
COMMENT    PROVISIONAL REFSEQ: This record has not
           yet been subject to final NCBI review.
           The reference sequence was derived from
           AF430845.1.
FEATURES   Location/Qualifiers
           source    1 . . . 1917
                     /organism = “Mus musculus”
                     /strain = “CZECHII”
                     /db_xref = “taxon: 10090”
                     /chromosome = “12”
                     /map = “12F1”
           Protein   1 . . . 1917
                     /product = “dicer1”
                     /note = “endoribonuclease Dicer”
           Region    38 . . . >226
                     /region_name = “ERCC4-like helicases
                     [DNA replication, recombination, and
                     repair]”
                     /note = “MPH1”
                     /db_xref = “CDD: 10833”
           Region    41 . . . 242
                     /region_name = “DEAD-like helicases
                     superfamily”
                     /note = “DEXDc”
                     /db_xref = “CDD: 24291”
           Region    109 . . . 1894
                     /region_name = “dsRNA-specific
                     nuclease Dicer and related ribo-
                     nucleases [RNA processing and
                     modification]”
                     /note = “KOG0701”
                     /db_xref = “CDD: 18495”
           Region    <500 . . . 554
                     /region_name = “Helicase conserved
                     C-terminal domain”
                     /note = “Helicase_C”
                     /db_xref = “CDD: 24402”
           Region    631 . . . 723
                     /region_name = “Domain of unknown
                     function”
                     /note = “DUF283”
                     /db_xref = “CDD: 26059”
           Region    <926 . . . >1039
                     /region_name = “Germ-line stem cell
                     division protein Hiwi/Piwi”
                     /note = “KOG1042”
                     /db_xref = “CDD: 18835”
           Region    1297 . . . >1388
                     /region_name = “Ribonuclease III
                     family”
                     /note = “RIBOc”
                     /db_xref = “CDD: 22830”
           Region    1677 . . . 1841
                     /region_name = “Ribonuclease III
                     family”
                     /note = “RIBOc”
                     /db_xref = “CDD: 22830”
           CDS       1 . . . 1917
                     /gene = “Dicer1”
                     /coded_by = “NM_148948.1:
                     255 . . . 6008”
                     /note = “go_component: cellular—
                     component unknown [goid 0008372]
                     [evidence ND];
                     go_component: intracellular [goid
                     0005622] [evidence ISS] [pmid
                     12466851];
                     go_function: ribonuclease III
                     activity [goid 0004525] [evidence
                     IDA] [pmid 14528307];
                     go_function: nuclease activity
                     [goid 0004518] [evidence IEA];
                     go_function: RNA binding [goid
                     0003723] [evidence IEA];
                     go_function: helicase activity [goid
                     0004386] [evidence IEA];
                     go_function: endonuclease activity
                     [goid 0004519] [evidence IEA];
                     go_function: ATP binding [goid
                     0005524] [evidence IEA];
                     go_function: ATP-dependent helicase
                     activity [goid 0008026] [evidence
                     IEA];
                     go_function: hydrolase activity
                     [goid 0016787] [evidence IEA];
                     go_function: nucleic acid binding
                     [goid 0003676] [evidence IEA];
                     go_function: double-stranded RNA
                     binding [goid 0003725] [evidence
                     ISS] [pmid 12466851];
                     go_process: biological_process un-
                     known [goid 0000004] [evidence ND];
                     go_process: RNA processing [goid
                     0006396] [evidence IDA] [pmid
                     14528307];
                     go_process: stem cell maintenance
                     [goid 0019827] [evidence IMP] [pmid
                     14528307];
                     go_process: RNA interference,
                     production of guide RNAs [goid
                     0030422] [evidence IDA] [pmid
                     14528307]”
                     /db_xref = “GeneID: 192119”
                     /db_xref = “LocusID: 192119”
                     /db_xref = “MGI: 2177178”
ORIGIN
1          mnekpcfaal smaglqlmtp asspmgpffg
           lpwqqeaihd niytprkyqv elleaaldhn
61         tivclntgsg ktfiavlltk elahqirgdl
           nphakrtvfl vnsanqvcqq vsavrthsdl
121        kvgeysdlev naswtkerws qeftkhqvli
           mtcyvaltvl kngylslsdi nllvfdechl
181        aildhpyrei mklcescpsc prilqltasi
           lngkcdpeel eekiqkleri lrsdaetatd
241        lvvldrytsq pceivvdcgp ftdrsglyer
           llmeleaald findcnvavy skerdstlis
301        kqilsdcrav lvvlgpwcad kvagmmvrel
           qkyikheqee lhrkfllftd tllrkihalc
361        eeyfspasld lkyvtpkvmk lleilrkykp
           yerqqfesve wynnrnqdny vswsdseddd
421        ddeeieekek petnfpspft nilcgiifve
           rrytavvlnr likeagkqdp elayissnfi
481        tghqigknqp rskqmeaefr kqeevlrkfr
           ahetnlliat svveegvdip kcnlvvrfdl
541        pteyrsyvqs kgrarapisn yvmladtdki
           ksfeedlkty kaiekilrnk csksadgaea
601        dvhagvdded afppyvlrpd dggprvtint
           aighinryca rlpsdpfthl apkcrtrelp
661        dgtfystlyl pinsplrasi vgppmdsvrl
           aervvalicc eklhkigeld ehlmpvqket
721        vkyeeeldlh deeetsvpgr pgstkrrqcy
           pkaipeclrd sypkpdqpcy lyvigmvltt
781        plpdelnfrr rklyppedtt rcfgiltakp
           ipqiphfpvy trsgevtisi elkksgfils
841        qqmlelitrl hqyifshilr lekpalefkp
           tgaesaycvl plnvvndsgt ldidfkfmed
901        ieksearigi pstkysketp fvfkledyqd
           aviipryrnf dqphrfyvad vytdltplsk
961        fpspeyetfa eyyktkynld ltnlnqplld
           vdhtssrlnl ltprhlnqkg kalplssaek
1021       rkakweslqn kqilvpelca ihpipaslwr
           kavclpsily rlhclltaee lraqtasdag
1081       vgvrslpvdf rypnldfgwk ksidsksfis
           tcnsslaesd nyckhsttvv pehaahqgat
1141       rpslenhdqm svnckrlpae spaklqsevs
           tdltaingls ynknlangsy dlvnrdfcqg
1201       nqlnyfkqei pvqpttsypi qnlynyenqp
           kpsnecplls ntyldgnant stsdgspavs
1261       tmpammnavk alkdrmdseq spsvgyssrt
           lgpnpglilq altlsnasdg fnlerlemlg
1321       dsflkhaitt ylfctypdah egrlsymrsk
           kvsncnlyrl gkkkglpsrm vvsifdppvn
1381       wlppgyvvnq dksnsekwek demtkdclla
           ngklgeacee eedltwrapk eeaededdfl
1441       eydqehiqfi dsmlmgsgaf vrkislspfs
           asdsayewkm pkkaslgsmp fasqledfdy
1501       sswdamcyld pskaveeddf vvgfwnpsee
           ncgvdtgkqs isydlhteqc iadksiadcv
1561       eallgcylts cgeraaqlfl cslglkvlpv
           ikrtsrekal dpaqengssq qkslsgscaa
1621       pvgprssagk dleygclkip prcmfdhpda
           ektlnhlisg fetfekkiny rfknkayllq
1681       afthasyhyn titdcyqrle flgdaildyl
           itkhlyedpr qhspgvltdl rsalvnntif
1741       aslavkydyh kyfkavspel fhviddfvkf
           qleknemqgm dselrrseed eekeedievp
1801       kamgdifesl agaiymdsgm slevvwqvyy
           pmmqpliekf sanvprspvr ellemepeta
1861       kfspaertyd gkvrvtvevv gkgkfkgvgr
           syriaksaaa rralrslkan qpqvpns

V. Screening Assays

According to the invention, the following assays may be used to identify compounds that modulate interaction (e.g., binding) of Dicer or bioactive fragments thereof with Dicer interactors or bioactive fragments thereof, or modulate a Dicer activity or Dicer interactor activity, and hence, modulators of gene silencing or RNAi. Such modulators are particularly useful in regulation of (1) processing of miRNA precursors; (2) processing of siRNA precursors; (3) mediating mRNA cleavage; (4) mediating assembly of RISC (e.g., via siRNAs); (5) directing translation repression (e.g., via miRNAs); (6) a ribonuclease activity (e.g., cleavage of dsRNA); and (7) initiation of RNAi. The assays feature identifying modulators of the activity of Dicer interactors or bioactive fragments thereof, including, but not limited to, those activities identified in supra.

The assays of the present invention are used to identify modulators of the activity of Dicer or bioactive fragments thereof or Dicer interactors or bioactive fragments thereof. The modulators of the present invention are particularly useful in modulating Dicer and/or RNAi related activities but can also affect non-RNAi related activities.

VA. Cell Free Assays

In one embodiment, an assay of the present invention is a cell-free assay in which a composition comprising assay reagents (e.g., a Dicer interactor polypeptide, Dicer polypeptide or biologically active portions thereof), is contacted with a test compound and the ability of the test compound to modulate binding of the Dicer interactor polypeptide to the Dicer polypeptide (or bioactive fragments thereof) is determined. Binding of the Dicer interactor or Dicer (or bioactive fragments thereof) can be accomplished, for example, by coupling the polypeptide or fragment with a radioisotope or enzymatic label such that binding of polypeptide reagents can be determined by detecting the labeled compound or polypeptide in a complex. For example, test compounds or polypeptides can be labeled with ¹²⁵I, ³⁵S, ³³P, ³²P, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, polypeptides can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate protein to product.

Determination of binding of reagents can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein, “BIA” is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

In a preferred embodiment, the assay includes contacting Dicer polypeptide or biologically active portion thereof with a Dicer target molecule, e.g., a Dicer interactor or a bioactive fragment thereof to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the Dicer polypeptide, wherein determining the ability of the test compound to interact with the Dicer polypeptide comprises determining the ability of the test compound to preferentially bind to Dicer or the bioactive portion thereof as compared to the Dicer target molecule (e.g., a Dicer protein). In another embodiment, the assay includes contacting the Dicer interactor polypeptide or biologically active portion thereof with a Dicer interactor target molecule, e.g., Dicer or a bioactive fragment thereof to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to modulate binding between the Dicer interactor polypeptide and the Dicer polypeptide.

In another embodiment, the assay is a cell-free assay in which a composition comprising a Dicer polypeptide and a Dicer interactor polypeptide (or bioactive portions thereof) is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the Dicer polypeptide or Dicer interactor polypeptide (or biologically active portions thereof) is determined.

Determining the ability of the test compound to modulate the activity of a Dicer or a Dicer interactor polypeptide can be accomplished, for example, by determining the ability of the Dicer interactor polypeptide to modulate the activity of a downstream binding partner or target molecule by one of the methods described herein for cell or organism-based assays. For example, the catalytic/enzymatic activity of the target molecule on an appropriate downstream protein can be determined as previously described.

In yet another embodiment, the cell-free assay involves contacting a Dicer interactor polypeptide or biologically active portion thereof with a Dicer interactor target molecule that binds the Dicer interactor polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound (e.g., Dicer) to preferentially modulate the activity of a Dicer interactor binding partner or target molecule, as compared to the Dicer protein.

In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either the Dicer interactor or Dicer (or target molecules) to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. The ability of a test compound to modulate Dicer interactor polypeptide activity, Dicer polypeptide activity, interaction of a Dicer interactor polypeptide with a Dicer polypeptide (or target interaction or activity) in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided so as to add a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/Dicer interactor fusion proteins, glutathione-S-transferase/Dicer fusion proteins, or target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed Dicer polypeptide or Dicer interactor polypeptide (or target polypeptide), and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of Dicer interactor binding or activity or Dicer binding or activity (or target binding or activity) determined using standard techniques.

Additional exemplary Dicer and/or Dicer interactor fusion proteins (or target fusion proteins) include, but are not limited to, chitin binding domain (CBD) fusion proteins, hemagglutinin epitope tagged (HA)-fusion proteins, His fusion proteins (e.g., His₆ tagged proteins), FLAG tagged fusion proteins, AU1 tagged proteins, and the like.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a Dicer polypeptide, a Dicer interactor polypeptide or target polypeptide can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated Dicer polypeptide, Dicer interactor polypeptide or target polypeptide can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with Dicer polypeptide, Dicer interactor polypeptide or target polypeptide but which do not interfere with binding of the Dicer interactor polypeptide to Dicer polypeptide (or protein to target binding) can be derivatized to the wells of the plate, and unbound Dicer or Dicer interactor polypeptide (or target) trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the Dicer interactor polypeptide, Dicer polypeptide or target polypeptide, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the Dicer interactor polypeptide, Dicer polypeptide or target polypeptide.

In one aspect of the invention, the Dicer interactor or Dicer polypeptides can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with Dicer interactor or Dicer (“binding proteins” or “target molecules”) and are involved in Dicer interactor or Dicer activity. Such target molecules are also likely to be involved in the regulation of cellular activities modulated by the Dicer interactor polypeptides or Dicer polypeptides.

At least one exemplary two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a first polypeptide (the “bait” polypeptide, e.g., Dicer or Dicer protein) is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein that interacts with the bait polypeptide.

Another exemplary two-hybrid system, referred to in the art as the CytoTrap™ system, is based in the modular nature of molecules of the Ras signal transduction cascade. Briefly, the assay features a fusion protein comprising the “bait” protein and Son-of-Sevenless (SOS) and the cDNAs for unidentified proteins (the “prey”) in a vector that encodes myristylated target proteins. Expression of an appropriate bait-prey combination results in translocation of SOS to the cell membrane where it activates Ras. Cytoplasmic reconstitution of the Ras signaling pathway allows identification of proteins that interact with the bait protein of interest, for example, a Dicer or Dicer interactor protein. Additional mammalian two hybrid systems are also known in the art and can be utilized to identify Dicer or Dicer interactor interacting proteins. Moreover, at least one of the above-described assays can be utilized to identify Dicer-interacting domains or regions of the Dicer interactor protein or alternatively, to identify Dicer protein-interacting domain or regions of the Dicer protein.

VB. Cell or Organism Based Assays

In one embodiment, an assay is a cell or organism-based assay in which a cell or organism capable of expressing a Dicer interactor polypeptide, or biologically active portion thereof, is contacted with a test compound and the ability of the test compound to modulate the expression of the Dicer interactor polypeptide, or biologically active portion thereof, determined. In another embodiment, an assay is a cell or organism-based assay in which a cell or organism which expresses a Dicer interactor polypeptide or Dicer polypeptide (or biologically active portions thereof) is contacted with a test compound and the ability of the test compound to modulate the activity of the Dicer interactor polypeptide or Dicer polypeptide (or biologically active portions thereof) determined. The cell, for example, can be of mammalian origin or a yeast cell. The organism can be a nematode, for example, C. elegans or C. briggsae or D. melanogaster. The polypeptides, for example, can be expressed heterologously or native to the cell or organism. Determining the ability of the test compound to modulate the activity of a Dicer interactor or Dicer polypeptide (or biologically active portions thereof) can be accomplished by assaying for any of the activities of a Dicer interactor or Dicer polypeptide described herein. Determining the ability of the test compound to modulate the activity of a Dicer interactor polypeptide or Dicer polypeptide (or biologically active portions thereof) can also be accomplished by assaying for the activity of a Dicer downstream molecule. In one embodiment, determining the ability of the test compound to modulate the activity of a Dicer interactor polypeptide, or biologically active portion thereof, is accomplished by assaying for the ability to bind Dicer or a bioactive portion thereof. In another embodiment, determining the ability of the test compound to modulate the activity of a Dicer interactor polypeptide, or biologically active portion thereof, is accomplished by assaying for the activity of the Dicer interactor polypeptide. In a preferred embodiment, the cell or organism overexpresses the Dicer interactor polypeptide, or biologically active portion thereof, and optionally, overexpresses Dicer, or biologically active portion thereof. In another preferred embodiment, the cell or organism expresses Dicer, or biologically active portion thereof. In yet another preferred example, the cell or organism is contacted with a compound that stimulates a Dicer protein-associated activity or Dicer-associated activity and the ability of a test compound to modulate the Dicer protein-associated activity is determined.

As used herein, the term “bioactive” fragment includes any portion (e.g., a segment of contiguous amino acids) of a Dicer interactor or Dicer protein sufficient to exhibit or exert at least one Dicer protein- or Dicer-associated activity including, for example, the ability to bind to Dicer or Dicer protein, respectively. In various embodiments, the Dicer may be one of two isoforms, Dicer1 or Dicer2. In another embodiment, the bioactive peptide is derived from the amino acid sequence of Dicer. In another embodiment, the bioactive peptide corresponds to a fragment or domain as set forth in subsections IA-IEE, supra or a smaller bioactive fragment thereof. In another embodiment, the bioactive peptide is derived from a Dicer interactor and can include, for example, amino acid residues sufficient to effect enzymatic or nucleic acid-binding activity.

According to the cell or organism-based assays of the present invention, determining the ability of the test compound to modulate the activity of the Dicer polypeptide or biologically active portion thereof, can be determined by assaying for any of the native activities of a Dicer polypeptide as described herein. Moreover, the activity of Dicer, can be determined by assaying for an indirect activity which is coincident to the activity of Dicer. Furthermore, determining the ability of the test compound to modulate the activity of the Dicer and/or Dicer interactor polypeptide or biologically active portion thereof, can be determined by assaying for an activity which is not native to the Dicer interactor or Dicer polypeptide, but for which the cell or organism has been recombinantly engineered. For example, the cell or organism can be engineered to express a target molecule which is a recombinant protein comprising a bioactive portion of Dicer operatively linked to a non-Dicer polypeptide or a bioactive portion of a Dicer interactor operatively linked to a non-Dicer interactor polypeptide. It is also intended that in preferred embodiments, the cell or organism-based assays of the present invention comprise a final step of identifying the compound as a modulator of Dicer interactor activity or Dicer activity.

VI. Assay Reagents

VIA. Test Compounds

The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner supra.).

In a preferred embodiment, the library is a natural product library.

VIB. Antibodies Bioactive Fragments and Fusion Proteins

Preferred aspects of the invention feature Dicer polypeptides, Dicer interactor polypeptides and biologically active portions (i.e., bioactive fragments) of Dicer polypeptides or Dicer interactor polypeptides, including polypeptide fragments suitable for use in making Dicer interactor or Dicer fusion proteins. In one embodiment, Dicer polypeptides or Dicer interactor polypeptides can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. Dicer polypeptide or Dicer interactor polypeptides can be further derived from said isolated polypeptides using standard enzymatic techniques. In another embodiment, Dicer interactor polypeptides, Dicer polypeptides or bioactive fragments thereof are produced by recombinant DNA techniques. Alternative to recombinant expression, Dicer interactor polypeptides, Dicer polypeptides or bioactive fragments thereof can be synthesized chemically using standard peptide synthesis techniques.

Polypeptides of the invention are preferably “isolated” or “purified”. The terms “isolated” and “purified” are used interchangeably herein. “Isolated” or “purified” means that the protein or polypeptide is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the polypeptide is derived, substantially free of other protein fragments, for example, non-desired fragments in a digestion mixture, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations in which the polypeptide is separated from other components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of polypeptide having less than about 30% (by dry weight) of non-Dicer interactor or non-Dicer polypeptide (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-Dicer interactor or non-Dicer polypeptide, still more preferably less than about 10% of non-Dicer interactor or non-Dicer polypeptide, and most preferably less than about 5% non-Dicer interactor or non-Dicer polypeptide. When the polypeptide or protein is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the polypeptide preparation. When the polypeptide or protein is produced by, for example, chemical or enzymatic processing from isolated or purified Dicer interactor or Dicer protein, the preparation is preferably free of enzyme reaction components or chemical reaction components and is free of non-desired Dicer interactor or Dicer fragments, i.e., the desired polypeptide represents at least 75% (by dry weight) of the preparation, preferably at least 80%, more preferably at least 85%, and even more preferably at least 90%, 95%, 99% or more or the preparation.

The language “substantially free of chemical precursors or other chemicals” includes preparations of polypeptide in which the polypeptide is separated from chemical precursors or other chemicals which are involved in the synthesis of the polypeptide. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations having less than about 30% (by dry weight) of chemical precursors or reagents, more preferably less than about 20% chemical precursors or reagents, still more preferably less than about 10% chemical precursors or reagents, and most preferably less than about 5% chemical precursors or reagents.

Bioactive fragments of Dicer interactor or Dicer include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the Dicer interactor protein or the Dicer protein, respectively, which include less amino acids than the full length protein, and exhibit at least one biological activity of the full-length protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the full-length protein. A biologically active portion of a Dicer interactor or Dicer polypeptide can be a polypeptide which is, for example, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more amino acids in length. In a preferred embodiment, a bioactive portion of a Dicer protein comprises a portion comprising a Dicer interactor interacting domain. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native Dicer interactor or Dicer protein. Mutants of Dicer and/or Dicer interactors can also be utilized as assay reagents, for example, mutants having reduced, enhanced or otherwise altered biological properties identified according to one of the activity assays described herein.

As defined herein, a Dicer polypeptide or Dicer interactor polypeptide of the invention includes polypeptides having the amino acid sequences set forth in subsections IA-IMM or II, infra, as well as homologs an/or orthologs of said polypeptides, i.e. polypeptides having sufficient sequence identity to function in the same manner as the described polypeptides. To determine the percent identity of two amino acid sequences (or of two nucleotide or amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), optionally penalizing the score for the number of gaps introduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (i.e., a local alignment). A preferred, non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST alignments can be generated and percent identity calculated using BLAST protein searches (e.g., the XBLAST program) using Dicer protein, Dicer or a portion thereof as a query, score=50, wordlength=3.

In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Research 25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment). A preferred, non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

The invention also provides Dicer interactors and Dicer chimeric or fusion proteins. As used herein, a Dicer interactor or Dicer “chimeric protein” or “fusion protein” comprises a Dicer interactor or Dicer polypeptide operatively linked to a non-Dicer interactor polypeptide or non-Dicer polypeptide, respectively. A “Dicer interactor polypeptide” or “Dicer polypeptide” refers to a polypeptide having an amino acid sequence corresponding to the Dicer interactor or Dicer protein, respectively, whereas a “non-Dicer interactor polypeptide” or “non-Dicer polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially identical to the Dicer interactor protein or Dicer protein. Within a fusion protein the Dicer interactor or Dicer polypeptide can correspond to all or a portion of a Dicer interactor or Dicer protein. In a preferred embodiment, a Dicer interactor or Dicer fusion protein comprises at least one biologically active portion of a Dicer interactor or Dicer protein, respectively. In another preferred embodiment, a Dicer interactor or Dicer fusion protein comprises at least two biologically active portions of a Dicer interactor or Dicer protein, respectively. In yet another preferred embodiment, a fusion protein can comprise Dicer protein, or a bioactive portion thereof, operatively linked to Dicer, or a bioactive portion thereof, such that Dicer interactor and Dicer, or their respective bioactive portions are brought into close proximity. Within the fusion protein, the term “operatively linked” is intended to indicate that the Dicer interactor or Dicer polypeptide and the non-Dicer interactor polypeptide or non-Dicer polypeptide are fused in-frame to each other. The non-Dicer interactor polypeptide or non-Dicer polypeptide can be fused to the N-terminus or C-terminus of the Dicer interactor polypeptide or Dicer polypeptide, respectively.

For example, in one embodiment, the fusion protein is a GST-fusion protein in which the Dicer interactor or Dicer sequences are fused to the C-terminus of the GST sequences. In another embodiment, the fusion protein is a chitin binding domain (CBD) fusion protein in which the Dicer interactor or Dicer sequences are fused to the N-terminus of chitin binding domain (CBD) sequences. Such fusion proteins can facilitate the purification of recombinant Dicer interactor or Dicer.

Preferably, a chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety. A Dicer protein- or Dicer-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the Dicer interactor or Dicer polypeptide.

A Dicer interactor polypeptide or Dicer polypeptide, or a portion or fragment of Dicer interactor or Dicer, can also be used as an immunogen to generate antibodies that bind Dicer interactor or Dicer or that block Dicer protein/Dicer binding using standard techniques for polyclonal and monoclonal antibody preparation. A full-length polypeptide can be used or, alternatively, the invention provides antigenic peptide fragments for use as immunogens. Preferably, an antigenic fragment comprises at least 8 amino acid residues of the amino acid sequence of a Dicer interactor or Dicer and encompasses an epitope of Dicer interactor or Dicer such that an antibody raised against the peptide forms a specific immune complex with Dicer interactor or Dicer, respectively. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of Dicer interactor or Dicer that are located on the surface of the protein, e.g., hydrophilic regions. Antigenic determinants at the termini of Dicer interactor are preferred for the development of antibodies that do not interfere with the Dicer protein:Dicer interaction. Alternatively, interfering antibodies can be generated towards antigenic determinants located within the Dicer interacting domain of Dicer protein. The latter are preferred for therapeutic purposes.

A Dicer interactor or Dicer immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed Dicer interactor or Dicer polypeptide or a chemically synthesized Dicer interactor or Dicer polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic Dicer interactor or Dicer preparation induces a polyclonal anti-Dicer interactor or anti-Dicer antibody response, respectively.

Accordingly, another aspect of the invention pertains to anti-Dicer interactor or anti-Dicer antibodies. The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as Dicer interactor or Dicer. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab+)₂ fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind Dicer protein. The term “monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of Dicer interactor or Dicer. A monoclonal antibody composition thus typically displays a single binding affinity for a particular Dicer interactor or Dicer polypeptide with which it immunoreacts.

Polyclonal anti-Dicer interactor or anti-Dicer antibodies can be prepared as described above by immunizing a suitable subject with a Dicer interactor or Dicer immunogen, respectively. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized Dicer interactor or Dicer. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-Dicer interactor or anti-Dicer antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a Dicer interactor or Dicer immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds Dicer interactor or Dicer, respectively.

Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-Dicer interactor monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (“PEG”). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind Dicer interactor or Dicer, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-Dicer interactor or anti-Dicer antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with Dicer interactor or Dicer to thereby isolate immunoglobulin library members that bind Dicer interactor or Dicer, respectively. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No. WO 92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner et al. PCT International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137; Barbas et al. (1991) PNAS 88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

An anti-Dicer interactor or anti-Dicer antibody (e.g., monoclonal antibody) can be used to isolate Dicer interactor or Dicer, bioactive portions thereof, or fusion proteins by standard techniques, such as affinity chromatography or immunoprecipitation. Anti-Dicer antibodies or anti-Dicer interactor antibodies made according to any of the above-described techniques can be used to detect protein levels in donor or acceptor fractions as part of certain assay methodologies described herein. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

VIC. Recombinant Expression Vectors and Assay Cells or Organisms

Another aspect of the invention pertains to vectors, preferably expression vectors, for producing the proteins reagents of the instant invention. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A preferred vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.

The recombinant expression vectors of the invention comprise a nucleic acid that encodes, for example protein or Dicer or a bioactive fragment or Dicer interactor or bioactive fragment, in a form suitable for expression of the nucleic acid in a host cell or organism, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells or organisms to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell or organism when the vector is introduced into the host cell or organism). The term “regulatory sequence” is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). The expression vectors can be introduced into host cell or organisms to thereby produce proteins, including fusion proteins or peptides. Alternatively, retroviral expression vectors and/or adenoviral expression vectors can be utilized to express the proteins of the present invention.

The recombinant expression vectors of the invention can be designed for expression of Dicer interactor or Dicer polypeptides in prokaryotic or eukaryotic cells. For example, Dicer interactor or Dicer polypeptides can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Purified fusion proteins are particularly useful in the cell-free assay methodologies of the present invention.

In yet another embodiment, a protein or Dicer-encoding or Dicer-protein-encoding nucleic acid is expressed in mammalian cells, for example, for use in the cell or organism-based assays described herein. When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).

Another aspect of the invention pertains to assay cells into which a recombinant expression vector has been introduced. An assay cell can be prokaryotic or eukaryotic, but preferably is eukaryotic. Cell lines are cultured according to art-recognized techniques. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals. An assay cell of the invention, can be contacted with a test compound and assayed for any Dicer interactor and/or Dicer biological activity in order to identify the compound as a modulator. Biological activities that can further be assayed as part of the methodologies of the present invention include, but are not limited to, (1) processing of miRNA precursors; (2) processing of siRNA precursors; (3) mediating mRNA cleavage; (4) mediating assembly of RISC (e.g., via siRNAs); (5) directing translation repression (e.g., via miRNAs); (6) a ribonuclease activity (e.g., cleavage of dsRNA); and (7) initiation of RNAi. In addition, other biological activities which may be assayed for include those listed in Table 1 and/or subsections IA-IMM and II, supra.

VII. Pharmaceutical Compositions

This invention further pertains to modulators identified by the above-described screening assays. Modulators identified by the above-described screening assays can be tested in an appropriate animal model. For example, a Dicer modulator, RNAi modulator and/or gene silencing modulator identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such a modulator. Alternatively, a modulator identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of modulators identified by the above-described screening assays for therapeutic treatments as described infra.

Accordingly, the modulators of the present invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, antibody, or modulatory compound and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

VIII. Methods of Treatment

The present invention also features methods of treatment or therapeutic methods. In one embodiment, the invention features a method of treating a subject (e.g., a human subject in need thereof) with a modulatory compound identified according to the present invention, such that a desired therapeutic effect is achieved. In another embodiment, the method involves administering to an isolated tissue or cell line from the subject a modulatory compound identified according to the methodology described herein, such that a desired therapeutic effect is achieved. In a preferred embodiment, the invention features a method of treating a subject having a disease or disorder characterized by overexpression or aberrant expression of a particular protein. For example, positive modulators of Dicer and/or RNAi can be used to enhance RNAi of deleterious proteins. Likewise, negative modulators of Dicer and/or RNAi can be used to alleviate symptoms resulting from the RNAi pathway. Desired therapeutic effects include a modulation of any Dicer protein-, Dicer- or Dicer protein/Dicer-associated activity, as described herein. Desired therapeutic effects also include, but are not limited to curing or healing the subject, alleviating, relieving, altering or ameliorating a disease or disorder in the subject or at least one symptom of said disease or disorder in the subject, or otherwise improving or affecting the health of the subject. A preferred aspect of the invention pertains to methods of modulating Dicer protein/Dicer interactions for therapeutic purposes.

The modulators identified by the methods disclosed herein may be used in a subject to modulate (1) processing of miRNA precursors; (2) processing of siRNA precursors; (3) mediating mRNA cleavage; (4) mediating assembly of RISC (e.g., via siRNAs); (5) directing translation repression (e.g., via miRNAs); (6) a ribonuclease activity (e.g., cleavage of dsRNA); and/or (7) initiation of RNAi.

The effectiveness of treatment of a subject with a Dicer modulator, RNAi modulator and/or gene silencing modulator can be accomplished by (i) detecting the level of activity in the subject prior to treating with an appropriate modulator; (ii) detecting the level of activity in the subject post treatment with the modulator; (iii) comparing the levels pre-administration and post administration; and (iv) altering the administration of the modulator to the subject accordingly. For example, increased administration of the modulator may be desirable if the subject continues to demonstrate undesireable symptoms of the disease or disorder being treated.

IX. Diagnostic Assays

The present invention also features diagnostic assays, for determining aberrant Dicer protein:Dicer interaction, expression or activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder associated with said aberrancy or is at risk of developing such a disorder. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing such a disorder (e.g., a disorder associated with aberrant Dicer interactor expression or activity). Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disease or disorder. A preferred agent for detecting a Dicer interactor or Dicer protein is an antibody capable of binding to protein or Dicer, respectively, preferably an antibody with a detectable label. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. The invention also encompasses kits for the detection of aberrant Dicer protein:Dicer interaction, expression or activity in a biological sample. For example, the kit can comprise a labeled compound or agent capable of detecting Dicer interactor and/or Dicer in a biological sample; means for determining the amount of Dicer interactor and/or Dicer in the sample; and/or means for comparing the amount of Dicer interactor in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit.

X. Uses

The invention has several further advantageous uses which include, but are not limited to, the following: providing interacting proteins of Dicer and there use in modulating Dicer function; methods for identifying further interactors of Dicer and their structural and functional characteristics; method for regulating Dicer activity though the use of Dicer interactors; methods for improving the in vitro or in vivo processing of Dicer proteins or for as targets for pharmaceutical intervention in order to modulate the properties of Dicer in vivo for improved RNAi; and methods for stabilizing RNAi agents/compositions comprising Dicer by the addition of stabilizing interactor proteins or the same for use in purifying Dicer and other Dicer components.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.

Exemplification

Throughout the examples, the following materials and methods were used unless otherwise stated.

Materials and Methods

In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of nucleic acid chemistry, recombinant DNA technology, molecular biology, biochemistry, cell biology and transgenic animal biology. See, e.g., DNA Cloning, Vols. 1 and 2, (D. N. Glover, Ed. 1985); Oligonucleotide Synthesis (M. J. Gait, Ed. 1984); Oxford Handbook of Nucleic Acid Structure, Neidle, Ed., Oxford Univ Press (1999); RNA Interference: The Nuts & Bolts of siRNA Technology, by D. Engelke, DNA Press, (2003); Gene Silencing by RNA Interference: Technology and Application, by M. Sohail, CRC Press (2004); Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); and Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons (1992), which are incorporated in their entireties by reference herein.

C. elegans Strains

Typical C. elegans strains for carrying out the invention as described herein include, for example, N2; alg-2 strain (ok304); dcr-1 for rescue; dcr-1 counterselectable; f20 counterselectable; drh-3 counterselectable; bn-2 (glp-4); rde-4 ne337; eri-1 (mg366); rrf-3 (pk); eri-3 (tm); and eri-5 (mg, tm).

Antibody Development and Purification

Antisera against C. elegans Dicer, i.e., DCR-1, were raised in rabbits as described by Capralogics services (Capralogics, Hardwick, Mass., USA). The antisera used for the somatic purifications, and for the immunoblot analyses were developed using a fragment encoded by residues 1145 to 1347 of the protein fused to the pCal-KC (Stratagene) encoded fusion. For their affinity purification, another fragment encoding residues 966 to 1347 was expressed as a pET-42a (Novagen) fusion, purified under denaturing conditions, using Guanidine HCl (Ultra grade, FLUKA) 6M/150 mM NaCl/HEPES 25 mM pH 8.0 as a lysis, binding and washing buffer. The purified fusion was eluted in Guanidine HCl 6M/150 mM NaCl/MES 25 mM pH4.8 and added directly to the Affigel 10 (Biorad) and allowed to rock O/N for covalent coupling of the fusion.

The matrix was then washed in coupling buffer (5 column volumes) in Tris (10 column volumes) and remaining active sites were blocked using triethanolamine/HCl for 2 h at 4 degrees. The matrix was then rinsed extensively in PBS and used for affinity-purification of the antisera. The sera (4 ml per batch) were diluted 1:5 in PBS, filtered sterile and loaded directly on the prepared affinity matrixes. After batch binding, the beads were washed extensively with PBS in a column and, the antibodies were eluted (8 column volumes) using glycine 0.2M pH2.2, while harvesting the fractions if 3:7 volumes of Potassium Phosphate solution at pH10, to neutralize the fractions. Consecutive purifications (3) were realized with the same serum batch with similar antibody recovery.

Fractions were then examined by SDS-PAGE, and quantified by comparison with BSA standards. The fractions containing the antibodies were dialyzed against PBS/5% glycerol, and concentrated to ˜1 microgram per microliter using the Centricon 10 centrifuge dialysis system (Millipore). The concentrated antibodies were frozen at −80 until used.

Dicer (Dcr-1) Transgenic Rescue

A fragment encoding the 3′ portion of the Dicer (dcr-1) gene was cloned into Bluescript SK (Stratagene) and a Not I site was inserted prior to its stop codon. A NotI cassette encoding 8 copies of HA, and the yeast sup4o gene embedded in an artificial C. elegans intron was then inserted in frame at the 3′ portion of the recombination cassette. This fusion was then prepared by PCR and used to transform a yeast strain bearing the YAC Y97B3. The strain was then selected on URA-/LYS- for YAC recombinants.

Confirmed recombinants, were screened by PCR and a genomic preparation of the strain was realized. A C. elegans strain bearing dcr-1 (ok247), and the dpy-13 lesions balanced by the sDp-3 free duplication was used for rescue. The genomic DNA was injected in the balanced animals germline at 200 ng per microliter with an additional 50 ng per microliter of sur-5::gfp expressing vector, as a secondary marker for transformation. Mosaics (F1) were selected on the basis of their GFP signal, singled out, and transmitting lines were identified with regard of the GFP signal of the F2s.

Genomic DNA was then prepared from 2-5 animals of such lines, and examined for the presence of the recombinant YAC DNA by PCR. 4 out of 22 independent transgenic lines had the YAC, and of such, 2 of the strains consistently produced dpy animals with fertile progeny. Single picks from such animals led to dpy populations, in which only GFP+ animals were fertile thereby indicating that the rescue was due to the recombinant YAC.

Fractionation and Immunoblot Analysis

For the somatic purifications, and the RNA analysis, the C. elegans strains were grown in standard conditions, as synchronous populations and harvested as adults with a single row of embryos, or allowed to grow 12 h after the L4 to young adult transition, in the case of sterile animals. Animals were rinsed in M9 twice and floated on sucrose if gravid adults were used. Animals were allowed to rock in M9 for 30 min at RT to allow digestion of the gut bacterial load. For embryonic preparations, gravid adults were hypochlorided as previously described, rinsed in M9 three times, and further rinsed in cold water. The animals were then pelleted using a table-top falcon centrifuge, and frozen at −80° C. as a compacted pellet after all the supernatant was drained.

Preparations where further processed using one volume of hypotonic buffer, 10 mM HEPES KOAc pH 7.5; 10 mM K(OAc); 2 mM Mg(OAC)₂; 1 mM DTT with 4× concentration complete protease inhibitors and RNase inhibitors. The suspension was then transferred to a cold Dounce homogenizer, and stroked 20-30 times, on ice. The resulting slurry was then transferred to an Eppendorf, and the recovered volume was adjusted to 110 mM KCl (yields Isotonic buffer), vortexed and allowed to sit on ice for 10 minutes.

The nuclear fraction was prepared in the following manner. The slurry was first centrifugation at 1500×g for 30 sec at 4°. The supernatant was recovered and adjusted to 10% glycerol, 0.01% Triton X-100, and vortexed and allowed to sit on ice for 10 min. The slurry was then loaded on a sucrose cushion (10 mM HEPES pH7.5; 10 mM KCl; 1M sucrose; 10% glycerol; 1 mM EDTA), and centrifuged at 20000×g for 10 min at 4°. The pellet yields the nuclear fraction.

The S100 and P100 fractions were prepared as follows. The supernatant from a short 1500×g centrifugation was further centrifuged at 10000×g for 10 min at 4°, and the supernatant was recovered (S10 fraction). This fraction was then loaded in a Beckman microfuge polyallomer tube and further spun 1 h 4° C. in a TLA100.3 to yield the S100 and P100 fractions. Equivalent volumes of each fraction was precipitated in 2 volumes acetone and resuspended in 1× SDS-PAGE buffer for the fraction analyses.

Immunoblots were realized using PBS/0.1% tween/5% milk for blocking and blotting, and PBS/0.1% tween for washings. Primary antibodies were incubated at RT for lh, and the corresponding HRP-coupled secondaries were used at 1:5000 for 1 h before 3× 5 min washes and ECL development (Pierce).

Purifications and Immunoprecipitations

Immunoprecipitation matrixes were prepared by DimethylPimeliimidate (DMP) (Sigma) covalent coupling to rProtA-agarose beads (Pierce) in sodium borate pH9.0 buffer. The beads were then stripped and blocked in 0.2M glycine pH2.2, rinsed extensively in PBS and kept until use at 4° C. with thymerosal as antibacterial agent. For typical preparations, 1 mg purified polyclonal antibodies were covalently coupled to 200 ul rProtA beads. In the case of embryonic purifications, agarose coupled matrixes from both antibody clones were used.

For the Dicer (DCR-1) purifications, the S100 fraction was further quantified and diluted to 3 mg per ml concentration in 1% Triton X-100 supplemented Isotonic buffer before the suitable buffer-equilibrated matrixes (30 ul bead volume per 2 ml IP) were added to the mixtures. Immunoprecipitations were carried out at 4° C. for 1 h, and beads were then washed 3 times in the immunoprecipitation buffer.

Immunoprecipitates were then treated with 20 ug per ml RNaseA for 30 minutes on ice in the same buffer, then washed three more times. The beads were washed one more time in cold PBS, and all the supernatant was drained. Bound proteins were eluted in 8M urea/50 mM HEPES pH7.5, and acetone-precipitated. ⅕^th the elution volume was kept and monitored on silver stain and/or by immunoblot for a qualitative evaluation of the immunoprecipitation process.

RNA Interferences

Feeding and microinjection RNAi was carried out as previously reported by Conte Jr. D. and Mello, C. C. 2003. RNA interference in Caenorhabditis Elegans. In Current Protocols in Molecular Biology.

Northern Blotting and Real Time PCR

Small RNAs were prepared from N2(wt), bn2(glp4), which lack a germline tissue, and mutants for rde-4(ne337), rrf-3(pk1426), eri-1(mg366), eri-3(tm1361), and eri-5(mg370), all at 25° C. Homozygous dcr-1(ok247), f20d12.1, and drh-3(tm]217) sterile adult animals were isolated using the counterselectable genetic balancer method. For alg-1 and alg-2 depleted preparations, alg-2(ok204) L1 animals were exposed to an alg-1(rnai) feeding strain, and the bursting young adults were harvested and used for small RNA preparations. The isolated small RNA preparations were typically examined by Northern blotting for a variety of endogenous small RNAs as well as miR58, tncR7, and the X chromosome locus contig of small RNAs described in the art. Real time PCR was performed with primer pairs having efficiencies validated for a multiple 10 fold dilution range around the N2(wt) level, and fold changes were calculated using the delta delta Ct method.

Imaging and Video Microscopy

DAPI staining of intact animals was done as described in the art. Endomitotic (Emo) phenotype was scored by intense and irregular DAPI staining or expression of histoneH2::gfp in germ cell nuclei. Nematode gonads were dissected as described in the art with slight modifications. Briefly, young adult worms were placed in a drop of PBS containing 0.15 mM of levamisole on a glass slide for gonad extrusion. The dissected gonads were then fixed in 4% paraformaldehyde in PBS for 5 minutes, followed by three washes of PBS before staining with DAPI for 5 minutes. Gonads were then mounted for imaging after 3 washes with PBS. DIC or fluorescence images were collected by a Hamamatsu Ocre-ER digital camera mounted on a Zeiss Axioplan 2 under the control of Openlab 3.0 software. In time-lapse video microscopy experiments, young adult animals expressing a histoneH2::gfp fusion in the germline (AZ212) were cut open in M9 solution and embryos were mounted on 2% agarose pads in M9 solution for recording by a Leica TCS SP2 confocal microscope system. Movies were processed on a Macintosh computer using the public domain Image J 10.2 program (developed at the U.S. National Institutes of Health.

Multi Dimensional Protein Identification Technology

The MudPIT assays were performed essentially as described in Graumann et al., Mol Cell Proteomics, 3(3):226-37 (2004) and Liu et al., Biotechniques. 32(4):898, 900, 902 passim (2002).

EXAMPLE 1

Methods for Identifying Dicer Interacting Proteins

To identify Dicer interacting proteins a protenomic approach was employed. In particular, a combined and comparative proteomic approach was designed and used to identify novel factors implicated in molecular interactions with Dicer (DCR-1) in the nematode C. elegans. The approach featured a combined transgenic and immuno-biochemical purification scheme with an innovative Mass Spectrometry technology called MudPit (Multi-dimensional protein identification technology) in order to identify proteins interacting with DCR-1 in the embryo and in the adult of the animal and compared with the interactors identified in parallel as being interactors of the RDE-4 and RDE-1 proteins. The MudPit technology has been previously described (see, e.g., Graumann et al., Mol Cell Proteomics, 3(3):226-37 (2004); Liu et al., Biotechniques. 32(4):898, 900, 902 passim (2002)).

Using this approach, several interactions were identified which have important significance as to how DCR-1 can be up- or down-regulated and how DCR-1 is implicated in different functions. Table 1 lists the DCR-1 interactors identified using the above approach. Table 2 shows the corresponding protein interaction data obtained for each interactor. Many of these interactors are widely conserved and have homologs in other species such as human or mouse. These interactors are implicated as activators or inhibitors of the DCR-1 activity, specificity, and/or stability and can be utilized for improved in vitro processing of a variety of DCR-1 proteins. The interactors can also be used as part of a rationale or also as targets for pharmaceutical intervention in order to modulate the properties of DCR-1 in vivo.

TABLE 1
List of Dicer Interacting Proteins Identified in Pilot Scale
	Protein
§	CE#	Description	Phenotype
IIA		RDE-4	RDE
IIB		ALG-1	EXP
IIC		ALG-2	EXP
IID		DRH-1	RDE
IIE		DRH-2	RDE
IIF	ce09069	helicase	ND
		homologous to DCR-2
IIG	ce21971	Double helicase	EMB
IIH		EFT-2 EF-Tu family	EMB; Pvl; Ste; Lva
		GTP binding
III		EFT-4 (elF1 alpha)	EMB; Gro; Lva;
			Unc; Ste
IIJ	ce21437	GAP/RAN-GAP family	ND
IIK	ce08872	HMG-I/Y DNA	WT
		binding
IIL	ce20336	HMG-I/Y DNA	Dpy; EMB; Lvl;
		binding PB1 domain	Ste; Unc; Lva
IIM	ce14704	SNR-2 SM protein	EMB; Ste; EXP; Lva
IIN	ce02065	SNR-3 SM protein	EMB; Clr; Sck; Lva
IIO	ce03706	Dual specificity	EMB; EXP
		phosphatase
IIP		LIN-41
IIQ	ce001506	low homology	GRO
		MADS box, novel
IIR		RPN-9 proteasome	EMB; BMD, Unc,
		subunits	Gro, Lva
IIS	ce14736	TAF 6.1	WT (ND)
IIT	ce05915	T54 homology	Unc Stp Gro
IIU	ce21988	RRM protein (3	ND
		domains)
IIV	ce27223	Worm	WT (ND)
		unique/Novel
IIW	ce00850	TBB-4	EMB
IIX		RPS-14
IIZ		RPS-13
IIAA		RPL-24
IIBB		RPS-11
IICC	ce03050	Agglutinin
IIDD		SIP-1 (hsp20)	WT
IIEE		CCT-6
		(chaperonin)

TABLE 2
List of Dicer: Dicer Interacting Protein Interaction Results
		IP					IP 1001(2) -	R4 (2) -
§	description	Controls	IP 1	IP 2	IP 3	IP 4	ctls	ctrl
IIA	RDE-4	NP	P	P	P	P	P	P
IIB	ALG-1	NP		P	P	P	P
IIC	ALG-2	NP		P	P	P	P
IID	DRH-1	NP	P	P	P	P	P	p
IIE	DRH-2	NP	P	P	P	P	P	P
IIF	helicase homologous	NP			P	P	P
	to DCR-2
IIG	Double helicase	NP	P	P	P	P
IIH	EFT-2 EF-Tu family	NP		P	P	P	P	P
	GTP binding
III	EFT-4 (elF1 alpha)	NP		P	P	P	P
IIJ	GAP/RAN-GAP family	NP		P	P	P
IIK	HMG-I/Y dna binding	NP		P	P	P
IIL	HMG-I/Y dna binding	NP			P	P
	PB1 domain
IIM	SNR-2 SM protein	NP			P	P
IIN	SNR-3 SM protein	NP			P	P	P
IIO	Dual specificity	NP		P	P	P	P
	phosphatase
IIP	LIN-41	NP			P	P	P
IIQ	low homology MADS	NP	P	P	P	P	P
	box, novel
IIR	RPN-9 proteasome	NP		P	P	P
	subunits
IIS	TAF 6.1	NP			P	P	P
IIT	T54 homology	NP			P	P
IIU	RRM protein (3	NP			P	P
	domains)
IIV	Worm unique/Novel	NP			P	P	P
IIW	TBB-4	NP	P	P	P	P		P
IIX	RPS-14	NP		P	P	P
IIZ	RPS-13	NP	P	P	P	P
IIAA	RPL-24	NP			P	P
IIBB	RPS-11	NP	P	P	P	P
IICC	Agglutinin	NP			P	P
IIDD	SIP-1 (hsp20)	NP		P	P	P
IIEE	CCT-6 (chaperonin)	NP		P	P	P
NP = not present;
P = present;
IP = immunoprecipitation;
1 to 4 corresponds to four independent purifications with that affinity matrix;
1001 = second antibody matrix;
R4 (2) - ctls = interactors found in two independent purifications of RDE-4, absent from the controls, and also present in the DCR-1 purifications.

EXAMPLE 2

Methods for Conducting a Whole Organism Search for Dicer Interactions

In order to identify Dicer interacting proteins in a whole organism, strategies to affinity-purify Dicer (DCR-1) by multiple independent matrixes, both from embryos and gravid adults C. elegans, were developed. Fractionation analysis showed that most, if not all the C. elegans Dicer protein can be found in the S100 fraction (FIG. 2).

For the adult purification, rabbit polyclonal sera having efficient immunoprecipitation capacity for the Dicer protein were identified. The antisera were affinity-purified against their respective antigen, coupled covalently to agarose matrixes, and used for batch immuno-affinity purifications. Controls included preparations from extracts genetically null for any Dicer expression (dcr-1 deletion allele (ok247)), or mock purification comprising neutralized affinity beads.

For the embryonic purifications, a transgenic dcr-1::8×HA genomic fusion driven by its own promoter, was used. The transgene allowed the sterility phenotype of dcr-1−/− to be rescued, and a robust expression in young embryos indicating it can support the functions of DCR-1 in the germline. Purification of DCR-1::8×HA fusion protein was carried out using two distinct monoclonal HA-directed affinity matrixes, and used the non-transgenic WT (N2) embryos as a control.

The purified proteins were eluted and analyzed using the Multi-Dimensional Protein Identification Technology (MudPIT). Interacting proteins were identified by comparison of the detected peptides with both the predicted and confirmed ORF library of the C. elegans genome. Protein candidates were not investigated further if they were also found in the depleted control, or in the mock purification (uncoupled matrix only). Chaperones, and two structural proteins, which were found in multiple non-related purifications, and known to be common non-specific interactions, were intentionally excluded. A high confidence set of interactions for proteins that could be detected in multiple purifications, with at least two independent matrixes, was defined. Using this strategy, 16 proteins were shown to interact with DCR-1. Table 3 depicts the interactions that were detected using this criteria.

TABLE 3
List of Dicer Interacting Proteins
	gene	DCR-1	structural
	name	purification	description	Phenotype
1FF	RDE-1	E* W	Piwi/PAZ domain	Rde
1A	RDE-4	E A** W	dsRBD	Rde
1D	DRH-1	E A W	DEAH/D box	Rde
1E	DRH-2	E A W	DEAH/D box	Rde
1GG	D2005.5	E A	DEAH/D box	early
	(DRH-3)			embryonic
				arrest, sterile
1HH	ERI-1	E	SAP domain,	ts sterile, eri
			Exonuclease
1II	RRF-3	E	rdrp	ts sterile, eri
1JJ	W09B6.3	E A	Novel (operon	ts sterile, eri
	(eri-3)		and fusion
			with TAF-6.1)
1KK	Y38F2AR.1	E A	TUDOR domain	ts sterile, eri
	(eri-5)
1S	TAF-6.1	E A	TATA box binding	ND; eri
			protein associated
			factor (operon
			and fusion with
			eri-3)
1B	ALG-1	A W	Piwi/PAZ domain	heterochronic
1C	ALG-2	A W	Piwi/PAZ domain	heterochronic
1P	LIN-41	A	RBCC (NHL	heterochronic,
			family)	pleiotropic
1LL	T23G7.5	E A	Phosphatase	rde, L4
	(PIR-1)			developmental
				arrest
1H	EFT-2	A	EFT-2 family	lethal,
			GTPases	pleiotropic
1N	SNR-3	A	SM domain	lethal,
				pleiotropic
1MM	C32A3.2	A	Novel	WT
Abbreviations are as follows:
E: embryonic purification,
A: gravid adult purification,
W: western detection,
rde: required for RNAi;
eri: enhancer of RNAi;
*weak peptide coverage only,
**due to the robust interaction, weak peptide coverage of RDE-4 is also detected in the dcr counterselected allele, likely due to interaction with the maternal load.

Proteins known to be involved in the initiation step of RNAi were found in all the DCR-1 purifications. The double-stranded binding protein RDE-4 was shown to interact with DCR-1. RDE-4 was also shown to interact with the argonaute family protein RDE-1, and the helicase DRH. In addition, RDE-4, DRH-1 and DRH-2 proteins were detected as interactors when pulling down with DCR-1.

In addition to the proteins involved in initiation of RNAi, other proteins having characterized functions that relate to small RNAs, were detected. Two argonaute proteins, ALG-1 and ALG-2, were also detected in the adult DCR-1 purifications. These paralog proteins are required for the efficient processing of a variety of miRNA precursors, but were heretofore unknown to interact physically with DCR-1.

Interactions with the rdrp RRF-3, and the SAP domain ERI-1 nuclease were also detected in the embryonic purifications. Interestingly when the genes coding for these proteins were inactivated, an enhancement of the classical RNAi response is observed (eri phenotype) indicating that rrf-3 and eri-1 encode negative regulators of RNAi.

In addition, the protein D2005.5 was detected which did not have a characterized small RNA-related function, but is a paralog of the dicer-related helicases drh-1 and drh-2.

For eight other proteins, no previous link with DCR-1 functions, or with small RNA-mediated silencing was known. Four detected proteins have known functions: snRNP core protein D1 (SNR-3), the translation elongation factor 2 (EFT-2), the NHL family ring finger-B box-Coiled coil translational repressor LIN-41, and subunit TAF6 of the transcription initiation factor TFIID (TAF-6.1). Finally four others have unknown functions. This subgroup includes T23G7.1 (an ortholog of the mammalian PIR1), the novel proteins C32A3.2, W09B6.3 (expressed as an operon with TAF-6.1), and the TUDOR domain protein Y38F2AR.1 (FIG. 3)

EXAMPLE 3

Methods for Determining In Vivo Activity of Dicer Interacting Proteins

To address the possible functions of these proteins in DCR-1-related mechanisms, the phenotypes of the rnai knock down for their corresponding genes (Table 3) was examined. The genes snr-3 and eft-2 (rnai) demonstrate pleiotropic phenotypes and growth defects. For the remaining Dicer interacting proteins, deletion alleles for d2005.5 (tm1217), t23g7.5 (tm1496), c32a3.2(tm1314), w09b6.3(tm1361) and y38j2ar.1(tm1705) were generated.

In addition, because two interacting protein were encoded by eri genes, the location of the genes encoding the interacting proteins with the mapping intervals of alleles generated in a screen for mutants that increase sensitivity of a neuronal gfp reporter to gfp rnai, were generated. Using this strategy three genes, TAF-6.1, w09b6.3 (part of a common operon), and y38f2ar.1 were mapped within the intervals of the eri-3 and eri-5 mutations, respectively. These three gene sequences were found to comprise nonsense point mutations. In addition, another enhancer (eri-4 (mg375)) mapped in proximity to DCR-1, and a point mutation (glycine 351 to arginine) was discovered in the C-terminus extremity of the conserved C-terminus sub-domain of its helicase domain.

EXAMPLE 3

Methods for Determining the In Vivo RNAi Activity of Dicer Interacting Proteins

The potency of the RNAi activity in whole animals, either for enhancement or deficiency (Table 2), was examined. First, their response in a high sensitivity unc-22 (rnai) somatic (Po) assay, was determined. This assay revealed that the interactor T23G7.5 allele exhibited a drastically reduced sensitivity to rnai when assayed in the soma, both for endogenous unc-22 (rnai) silencing and for gfp (rnai) silencing of a transgenic reporter. Possibly due to the maternal load the effect on RNAi was important, but partial. The mutant on itself also presents developmental defects: the homozygous null grows normally and suddenly arrests at the L4 stage, never reaching adulthood. A generalized loss of gene expression could not be responsible for the lack of RNAi response, as the arrested animals could still transcribe and translate a reporter de novo to a WT level. This protein encodes a conserved RNA phosphatase with homology to a family of capping enzymes, and associates with RNP particles in mammalian culture cells. Its enzymatic activity was shown to have specificity toward the removal of the β- and γ-phosphate residues on the 5′ end of triphosphate RNA substrates. This interaction was consistently detected both in the adult and embryonic purifications of DCR-1, and indicating its role in RNAi mechanisms. Thus, T23G7.5 was determined to be essential for development and RNAi

EXAMPLE 4

Methods for Determining the In Vivo RNAi Enhancer Activity of Dicer Interacting Proteins

The Dicer interacting proteins, w09b6.3 and y38f2ar.1 were determined enhancers of RNAi. Briefly, mutants using rnai targets, which do not exhibit a phenotype, or exhibit a very low penetrance in the WT (N2) genetic background, were assayed to test the possibility that these genes encode enhancers of rnai (eri). As observed, unc-73 (rnai) does not usually exhibit a strong penetrance when wt (N2) animals are exposed (4+−4%). As previously observed, eri-1(mg366) and rrf-3(pk1426) gave a very penetrant effect when exposed to unc-73 E. coli feeding strain (98+−2%). Similarly, a drastically higher penetrance was observed in the w09b6.3 (tm1361), y38f2ar.1 (mg392), and DCR-1(mg375) alleles (100%, 82.5+−11%, and 100%, respectively). Injection assays for lin-1 (rnai), and feeding assays for dpy-13 (rnai), hmr-1(mai), or gfp(mai) also supported these observations (not shown). Thus, it was concluded that the w09b6.3, and y38f2ar.1 mutations are enhancers of rnai (eri).

EXAMPLE 5

Methods for Determining the In Vivo Developmental Effects of Dicer Interacting Proteins

The Dicer interacting proteins eri-3, eri-4 and eri-5 were determined to have similar developmental defects. In addition to the similar effect on rnai, the rrf-3 and eri-1 genes were previously shown to have indistinguishable developmental defects and to act in the same genetic pathway. Known developmental defects include a strong sterility phenotype at 25° C., which is rescued at 15° C., or by crossing with WT males, suggestive of sperm defects. Mutant animals also exhibit spontaneous silencing of some simple transgenic arrays in the soma and a low incidence of X chromosome non-disjunction, visible by a High Incidence of Males (HIM) phenotype.

To test the idea that eri-3, eri-4 and eri-5 were acting on the same developmental mechanism, the defects associated with their corresponding alleles, were examined. Akin to alleles of rrf-3 and eri-1, mutations in these two genes led to mean brood sizes of 0±1 for w09b6.3 and of 1±1 for y38f2ar.1 −/− animals at 25° C. In contrast, at 15° C. the same alleles gave mean brood sizes of 155±12 for w09b6.3 and 167±20 for y38f2ar.1. Interest what was observed for rrf-3 and eri-1 alleles, the temperature sensitive sterility phenotype of w09b6.3 and y38f2ar.1 can be rescued by crossing with wt males, and therefore believed to be defective in sperm function. Additionally, a 3 to 5 fold increase in incidence of males was also observed in the corresponding mutants, compared to the WT(N2) spontaneous incidence (˜0.1%). Altogether, the unique, and specific combination of defects observed in the eri-3, eri-4 and eri-5 mutants indicates their involvement in a common pathway with eri-1 and rrf-3 (FIG. 4)

EXAMPLE 6

Methods for Determining the In Vivo Helicase/Chromosomal Effects of Dicer Interacting Proteins

The Dicer interacting protein DRH-3, when depleted, was determined to cause sterility and chromosome segregation defects. Mutations in the gene encoding the DCR-1-interacting protein D2005.5 also led to dramatic fertility defects. Because it encodes a paralog of the DEAX/D box helicase drh-1 and drh-2, this Dicer interacting protein was renamed drh-3. In contrast, despite the close homology, drh-3 is not required for initiation of the classical RNAi pathway, at least in the soma where it could be examined (see Table 3). Instead, while the drh-3(tm1217) allele animals were sterile as homozygous and examination of a pie-1::his-3::gfp transgenic strain revealed abnormally shaped oocytes with proximal mitosis, and occasional occurrence of multinucleation, rnai depletion led to a slow onset, but penetrant early embryonic arrest (Table 3). Although the terminal phenotype of this arrest was variable, the observed embryonic arrest was progressively earlier in embryos laid in periods extending two or three days after the adult injection. Interestingly and consistently with the phenotype exhibited in the deletion mutant, earlier injection of Po animals (L3 or L4 animals) also led to sterility. The earliest defects in the affected embryos using time-lapse videomicroscopy were also characterized. As observed, the first cell division was abnormal, and chromosomes lagging on the mitotic spindle could be observed at metaphase. Chromosome segregation later resulted in abnormally shaped nuclei.

Thus, it was noted that this initial developmental defect resembled the observed defects described in S. pombe for mutants in the RNAi machinery.

EXAMPLE 7

Methods for Determining the In Vivo Effects of Dicer Interacting Proteins on the Accumulation of Endogenous Small RNAs

The Dicer interacting protein drh-3 and the eri are required for the accumulation of classes of endogenous small RNAs.

Because divergent phenotypes were observed in many genes encoding the different DCR-1 interacting proteins, different phenotypic groups would be reflected by defects in accumulation and/or processing of different classes of small RNA. To test this hypothesis, the status of 5 classes of small RNAs known to require dcr-1 for efficient production, in the mutants generated, was examined. Sensitivity to exogenous dsRNA-triggering was used as a functional output for involvement in the classical RNAi pathway. Also examined, was the processing of miRNA precursors, the accumulation of the tncR, and small RNA-derived from an X chromosome-derived contig. Finally, accumulation of endogenous siRNAs (endo siRNAs) for a variety of loci previously shown to naturally produce these small RNAs, was examined

Because dcr-1 −/−, and drh-3−/− are sterile, a counter-selectable balancer strategy to select for nulls within large populations of animals, was employed. The maternal load of the two gene products was sufficient to lead the animals through early development and sterile adults could be studied. To look for an alg-1/alg-2 depletion effect on small RNAs, animals depleted of alg-1 by rnai in an alg-2−/− (ok304) animal background, were used.

A variety of miRNA were examined and no defects in the mature form accumulation nor in precursor processing was observed in the rde-4, the eri, nor in the drh-3 mutants. In contrast, a moderate to strong miRNA precursor accumulation was visible in alg-1/2, and dcr-1 depleted animals. These results indicate that these two proteins are crucial to most, if not all the miRNA maturation. However, the effect observed here on the precursor accumulation depends on the timing of the miRNA transcription and export relative to the depletion of ALG-1 protein by RNAi or the turnover of the maternal load of DCR-1 in the counterseleted F1 nulls.

Examination of the small RNA populations in the drh-3 counter-selectable nulls revealed that, while this protein is dispensable for normal accumulation of miRNAs, X-derived small RNAs, or the examined tncRs, it is required for the accumulation of all the examined ORF-derived endo-siRNA. Acting as controls, alg-depleted animals, and another counterselectable sterile mutant j20d12.1 did not show such defects in accumulation of these small RNAs. While most of the endo-siRNAs detected were only detected in the germline, drh-3 was also required for the production of soma-derived endo-siRNA k02e2.6. This result implicates the DCR-1-interacting protein DRH-3 specifically in the production and/or stabilization of a broad range of the ORF-derived endogenous-siRNAs.

The five eri mutants exhibited very consistent molecular defects in the accumulation of the mature small RNAs. While they enhanced the classical RNAi response (when triggered from exogenous sources of dsRNA), their mutation prevented accumulation of the examined tncR, and of the X locus-derived small RNAs. Interestingly, rde-4 was also required for the accumulation of the X locus-derived small RNAs, but dispensable for the tncRs or the ORF-derived endo-siRNAs, showing the modulatory nature of the contribution of the DCR-1 interactors for production of diverse small RNA classes.

The eri mutations did not affect the accumulation of most of the endo-siRNAs. However, surprisingly, the eri mutants also failed to accumulate endo-siRNAs from a very restricted number of genes. Interestingly, the eri genes also exhibited this defect at the permissive temperature in gravid adults, showing that the developmental process involving the eri genes, and not their function in endo-siRNAs accumulation is a temperature-sensitive process. This observation, and the presence of these endo-siRNAs in germline-less animals (FIG. 6, bn2(glp-4)) rules out the idea that the eri genes fail to show these endo-siRNAs because they lack the tissue where they are produced.

These results support the idea that different combinations of DCR-1-interacting proteins are required for efficient accumulation of distinct classes of endogenous small RNAs. These results support a function for the DCR-1-interacting ERI proteins in the initiation of a variety of distinct endogenous small RNA-mediated silencing mechanisms (FIG. 6).

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.

Claims

1. A method of identifying a Dicer interacting protein comprising, contacting a composition having a candidate Dicer interacting protein or bioactive fragment thereof with a Dicer protein or a bioactive fragment thereof, and determining the presence of one or more proteins capable of interacting with Dicer, as compared to an appropriate control, such that a Dicer interacting protein, is identified.

2. A method of identifying a modulator of Dicer activity comprising, contacting a composition comprising Dicer or a bioactive fragment thereof and a Dicer interacting protein or a bioactive fragment thereof with a test compound, and determining the ability of the test compound to modulate activity between Dicer or bioactive fragment thereof and the Dicer interacting protein or bioactive fragment thereof, such that a modulator of Dicer activity, is identified.

3. The method of claims 1 or 2, wherein Dicer or bioactive fragment thereof is derived from an organism selected from the group consisting of nematode, fruit fly, mouse, rat, and human.

4. The method of claims 1 or 2, wherein the Dicer interacting protein or bioactive fragment thereof is derived from an organism selected from the group consisting of nematode, fruit fly, mouse, rat, primate, and human.

5. The method of claim 2, wherein the Dicer activity is selected from the group consisting of protein:protein binding activity, miRNA maturation activity, RNAi initiation activity, RNAi enhancer activity, helicase activity, RISC activity, target recognition activity, and target gene cleavage activity.

6. The method of claims 1 or 2, wherein the composition comprises an extract selected from the group consisting of a cellular extract, nuclear extract, cytoplasmic extract, protein extract, S100 fraction, partially purified protein extract, and purified protein extract.

7. The method of claims 1 or 2, wherein the Dicer interacting protein is selected from the group consisting of RDE-4, ALG-1, ALG-2, DRH-1, DRH-2, helicase homologous to DCR-2 (DRH-3), double helicase, EFT-2 EF-Tu family GTP binding, EFT-4 (eIF1 alpha), GAP/RAN-GAP family, HMG-I/Y DNA binding, HMG-I/Y DNA, binding PB1 domain, SNR-2 SM protein, SNR-3 SM protein, Dual specificity phosphatase, LIN-41, low homology MADS box, novel, RPN-9 proteasome subunits, TAF 6.1, T54 homology, RRM protein (3 domains), Worm unique/Novel (ce27223), TBB-4, RPS-14, RPS-13, RPL-24, RPS-11, Agglutinin, SIP-1 (hsp20), CCT-6 (chaperonin), RDE-1, DRH-3, ERI-1, RRF-3, ERI-3, ERI-5, PIR-1, C32A3.2, and orthologs, paralogs, or bioactive fragments thereof

8. The method of claims 1 or 2, wherein the Dicer interacting proteins are further subjected to a multidimensional protein interaction technology (MudPIT).

9. A method of identifying a modulator of Dicer activity, comprising contacting a cell or cell extract having Dicer or a bioactive fragment and a Dicer interacting protein or bioactive fragment thereof, with a test compound and determining the ability of the test compound to modulate an activity selected from the group consisting of protein:protein binding activity, miRNA maturation activity, RNAi initiation activity, RNAi enhancer activity, helicase activity, RISC activity, target recognition activity, and target gene cleavage activity.

10. The method of claim 9, wherein said cell or cell extract comprises recombinantly expressed Dicer.

11. The method of claim 9, wherein said cell or cell extract comprises a Dicer interacting protein selected from the group consisting of RDE-4, ALG-1, ALG-2, DRH-1, DRH-2, helicase homologous to DCR-2 (DRH-3), double helicase, EFT-2 EF-Tu family GTP binding, EFT-4 (eIF1 alpha), GAP/RAN-GAP family, HMG-I/Y DNA binding, HMG-I/Y DNA, binding PB1 domain, SNR-2 SM protein, SNR-3 SM protein, Dual specificity phosphatase, LIN-41, low homology MADS box, novel, RPN-9 proteasome subunits, TAF 6.1, T54 homology, RRM protein (3 domains), Worm unique/Novel (ce27223), TBB-4, RPS-14, RPS-13, RPL-24, RPS-11, Agglutinin, SIP-1 (hsp20), CCT-6 (chaperonin), RDE-1, DRH-3, ERI-1, RRF-3, ERI-3, ERI-5, PIR-1, and C32A3.2.

12. The method of claim 9, wherein the ability of the test compound to modulate enhancers of RNAi is determined.

13. The method of claim 9, wherein the Dicer or bioactive fragment thereof complexed with a Dicer interacting protein is isolated by immunoaffinity chromatography.

14. The method of claim 13, wherein the Dicer interacting protein or bioactive fragment thereof is subjected to MudPIT.

15. The method of claim 9, wherein a detectable label is associated with a component selected from the group consisting of Dicer, a Dicer interacting protein, and test compound.

16. A modulator identified by any one of the preceding claims.

17. A method of identifying a modulator of Dicer comprising, contacting a S100 fraction comprising Dicer or a bioactive fragment thereof and a Dicer interacting protein with a test compound, and determining the activity of Dicer or bioactive fragment in the presence of the test compound as compared to an appropriate control, wherein the test compound is a potential modulator of Dicer based on its ability to affect the activity of Dicer or a bioactive fragment thereof.

18. An antibody that specifically binds to a component selected from the group consisting of Dicer, Dicer interacting protein, and Dicer:Dicer interacting protein complex.

19. The antibody of claim 18, wherein the Dicer interacting protein is selected from the group consisting of RDE-4, ALG-1, ALG-2, DRH-1, DRH-2, helicase homologous to DCR-2 (DRH-3), double helicases, EFT-2 EF-Tu family GTP binding, EFT-4 (eIF1 alpha), GAP/RAN-GAP family, HMG-I/Y DNA binding, HMG-I/Y DNA, binding PB1 domain, SNR-2 SM protein, SNR-3 SM protein, Dual specificity phosphatase, LIN-41, low homology MADS box, novel, RPN-9 proteasome subunits, TAF 6.1, T54 homology, RRM protein (3 domains), Worm unique/Novel (ce27223), TBB-4, RPS-14, RPS-13, RPL-24, RPS-11, Agglutinin, SIP-1 (hsp20), CCT-6 (chaperonin), RDE-1, DRH-3, ERI-1, RRF-3, ERI-3, ERI-5, PIR-1, and C32A3.2.

20. A pharmaceutical composition comprising the modulator of claim 17.

21. A method of activating target-specific RNA interference (RNAi) in an organism comprising, administering to the organism an RNAi agent and a modulator of Dicer activity, wherein the modulator is administered in an amount sufficient for enhancing the activity of the RNAi agent, thereby achieving degradation of a target mRNA in the organism.

22. The method of claim 21, wherein the target mRNA encodes a gene product involved or predicted to be involved in a human disease or disorder.

23. A method of treating a disease or disorder associated with the activity of a gene product encoded by a target mRNA in a subject comprising, administering to the subject an RNAi agent, and a modulator of Dicer activity, wherein said modulator is administered in an amount sufficient for enhancing the activity of the RNAi agent, thereby treating the disease or disorder associated with a gene product encoded by the target mRNA.

24. A method for deriving information about the function of a gene in a cell or organism comprising, introducing into the cell or organism a Dicer interacting protein or an RNAi agent specific therefore, and maintaining the cell or organism under target-specific RNAi conditions, determining a characteristic or property of the cell or organism, and comparing the characteristic or property to a suitable control, the comparison yielding information about the function of the gene.

25. A method of deriving information about the function of a Dicer interacting protein in a extract, cell, or organism comprising, exposing an extract, cell, or organism capable of expressing Dicer and a Dicer interacting protein to an RNAi agent, maintaining the lysate, cell, or organism under conditions such that target-specific RNAi can occur, determining a characteristic or property of the extract, cell, or organism, and comparing the characteristic or property to a suitable control, the comparison yielding information about the function of the gene.

26. A method of validating a candidate Dicer interacting protein as a suitable target for drug discovery comprising, introducing into a cell or organism a Dicer interacting protein or an RNAi agent specific therefore, and maintaining the cell or organism under target-specific RNAi conditions, determining a characteristic or property of the cell or organism, and comparing the characteristic or property to a suitable control, the comparison yielding information about whether the candidate protein is a suitable target for drug discovery.

27. The method of claims 24, 25, or 26, wherein the organism is selected from the group consisting of nematode, fruit fly, mouse, rat, primate, and human.

28. A method of treating a disease or disorder associated with the activity of a gene product encoded by a target RNA in a subject comprising, administering to the subject an agent sufficient to modulate Dicer activity in one or more cells and administering an RNAi agent in an amount sufficient for degradation of the target RNA to occur, thereby treating the disease or disorder associated with the gene product encoded by the target gene.

29. The method of claim 28, wherein the subject is a human patient.

30. A kit comprising a reagent for activating target-specific RNA interference (RNAi) in a cell or organism, the kit comprising: a component selected from the group consisting of a molecule encoding a Dicer interacting protein, an RNAi agent specific for a Dicer interacting protein, a modulator of Dicer activity, and a modulator of a Dicer interacting protein, and instructions for use.