Patent Application
20100305186
The present invention is concerned with methods for enhancing gene suppression in cells and in particular it is concerned with improved methods for enhancing RNAi-mediated gene silencing by manipulation of factors associated with RNAi. The present invention is also concerned with methods for identifying factors which down-regulate as well as those which up-regulate RNAi. It is also concerned with genetic constructs useful for enhancing or modulating gene silencing and cells harbouring such constructs.
The use of double-stranded RNA (dsRNA) to specifically interfere with gene expression has received considerable attention because of its demonstrated potency in a range of organisms, including some which have so far been genetically intractable. Termed RNA interference (RNAi), it has been implicated in viral defense, control of transpositional elements, genetic imprinting and endogenous gene regulation. It has been hypothesised to be the central mechanism in post-transcriptional gene silencing (PTGS), co-suppression, quelling, and antisense RNA-mediated gene suppression. One model that has been proposed is that dsRNA is fragmented into 21-25 nt species by dsRNA-specific nucleases, amplified by RNA-dependent RNA polymerase, and then dissociated and free to attack homologous mRNA by RNA nuclease-mediated degradation. The application of this technique will greatly facilitate the dissection of gene function and the validation of genes involved in disease states.
Recently at least two different strategies have been undertaken to identify the cellular proteins composing a proposed multi-protein complex involved in the recognition of dsRNA and the activation of dsRNA-mediated gene interference. The first involves the use of classical chemical mutagenesis or insertional mutagenesis to isolate mutants completely defective for RNAi and cloning of the relevant genes using complementation. These studies have been carried out in genetically tractable organisms such as plants, worms and fungi. The genetic screens described involve the use of RNAi systems in which the degree of suppression is complete. The mutagenesis produces mutants in which the RNAi effect is completely reversed indicating the loss of a cellular factor (function) required for the RNAi effect. Thus these genetic screens would most likely miss factors that have subtle effects or rate limiting or rate determining roles in RNAi.
The second strategy for finding key players in RNAi has involved the use of cell free assays. These in vitro reconstitution assays, on the other hand, identify cellular factors that impact on RNAi outside of the cellular context and therefore the cellular role of these factors must always be tested.
However, the major disadvantages of these strategies are that genes will not be identified if they are essential to the organism, nor will they directly identify gene activities which will enhance RNAi when overexpressed.
Thus there is a need for models which can demonstrate a range of RNAi efficacies, with both increasing and decreasing quantitative activities being selectable. This would enable the identification of factors which can enhance or reduce the gene silencing effect.
It is therefore an object of the present invention to overcome or at least ameliorate one or more disadvantages of the prior art, or provide a useful alternative.
Through the use of a fission yeast model for the study of dsRNA-mediated gene silencing and in the search for factors involved in this process, it was surprisingly found that the natural level of RNAi activity can be enhanced by manipulating factors associated with RNAi activity or efficacy. Thus, it has been found that by increasing the steady-state levels of a target nucleic acid sequence in the presence of the same pool of the corresponding antisense sequence, or a part thereof, the antisense-mediated suppression was not only maintained, but enhanced. This is indicative of an RNAi-like mechanism of gene suppression. It has also been found that overexpression of certain sequences, named herein RNAi enhancing sequences (res), (also referred to herein as anti-sense enhancing sequences—aes), also had the ability to enhance RNAi.
This ability of RNAi activity to be enhanced in S. pombe provides a model system which enables the analysis of RNAi processes and the identification and study of factors which either up-regulate or down-regulate its activity. The system has been further used to identify RNAi enhancing gene sequences which increase PTGS efficacy when their resulting protein activities are augmented in vivo. The model used to exemplify the present invention and the methods described are also applicable to treatment of disorders in which gene expression requires more efficient modulation or silencing.
According to a first aspect there is provided a method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of
(i) elevating in the cell the level of an RNAi factor, and
(ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.
According to a second aspect there is provided a method of increasing cellular susceptibility to anti-sense-mediated inhibition of target nucleic acid expression, which method comprises elevating the level of an RNAi factor in a cell that expresses said target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.
According to a third aspect there is provided a method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of
(i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid is expressed, and
(ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.
According to a fourth aspect there is provided a method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of
(i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and
(ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.
According to a fifth aspect there is provided a method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of
(i) elevating the level of an RNAi factor in a cell whose phenotype correlates with that of a cell from a subject having the disorder;
(ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and
(iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.
In a preferred embodiment the target nucleic acid is an endogenous nucleic acid or a part thereof, but it may also be an exogenous sequence or part thereof.
Preferably the level of the RNAi factor is elevated by introducing into the cell additional copies of, or agents which give rise to, the RNAi factor. It will be understood therefore that up-regulating the expression of an endogenous RNAi factor will also achieve the same result and is contemplated herein as part of the invention.
Preferably the factor is selected from the group consisting of a gene, cDNA, RNA or a protein. More preferred is a factor selected from the group consisting of a transcriptional activator of the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery. Even more preferred is an RNAi factor which is an res sequence.
Also for preference the factor can be selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.
Further preferred factors are represented by the res sequences which are obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.
Preferably the res sequence is represented by any one of Seq ID Nos 1 to 4.
The preferred cell is a eukaryotic cell and even more preferred is a mammalian cell. In certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.
Preferably the antisense nucleic acid corresponds to a part only of the target nucleic acid.
According to a sixth aspect there is provided a pharmaceutical composition for use in performing the method of any one of the previous aspects comprising
(i) an expressible nucleic acid encoding, or capable of increasing or decreasing the expression of, an RNAi factor;
(ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and
(iii) a pharmaceutically acceptable carrier,
wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.
According to a seventh aspect there is provided a pharmaceutical composition for use in performing the method of any one of claims 2 to 17 comprising
(i) an nucleic acid which is the target nucleic acid or a part thereof, or an expressible nucleic acid encoding a factor capable of elevating the intracellular level of the target nucleic acid;
(ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and
(iii) a pharmaceutically acceptable carrier,
wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.
According to an eighth aspect there is provided a cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses a target nucleic acid and (ii) comprises an elevated level of an RNAi factor, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.
For preference the cell is a eukaryotic cell but more preferred is a mammalian cell. As indicated above, in certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.
According to a ninth aspect there is provided a method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of
(i) augmenting the level of the target nucleic acid or a part thereof in the cell, and
(ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of said target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of said target nucleic acid.
According to a tenth aspect there is provided a method of increasing cellular susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which method comprises augmenting the level of the target nucleic acid or a part thereof in a cell expressing the target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.
According to an eleventh aspect there is provided a method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of
(i) augmenting the level of said target nucleic acid or a part thereof in the subject's cells where the target nucleic acid is expressed, and
(ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.
According to a twelfth aspect there is provided a method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of
(i) augmenting the level of the target nucleic acid or a part thereof in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and
(ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.
According to a thirteenth aspect there is provided a method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of
(i) augmenting the level of the target nucleic acid in a cell whose phenotype correlates with that of a cell from a subject having the disorder;
(ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and
(iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.
In a preferred embodiment the target nucleic acid is an endogenous nucleic acid or a part thereof, but it may also be an exogenous sequence or part thereof. Preferably the level of the target nucleic acid is augmented by introducing into the cell additional copies of, or agents which are capable of inducing intracellular over-expression of, the target nucleic acid. Over-expression can be achieved for an endogenous as well as an exogenous target nucleic acid. Preferably the nucleic acid used for augmenting content of the target nucleic acid is a fragment, derivative or analogue of the target nucleic acid. However it will be understood that the entire native sequence of the target nucleic acid may be employed.
Conveniently, the target nucleic acid may be coupled to a selectable marker.
The preferred cell is a eukaryotic cell and even more preferred is a mammalian cell. In certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.
Preferably the antisense nucleic acid corresponds to a part only of the target nucleic acid.
According to a fourteenth aspect there is provided a cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses said target nucleic acid and (ii) comprises an elevated level of said target nucleic acid, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.
The preferred cell is a eukaryotic cell and even more preferred is a Schizosaccharomyces pombe cell.
According to a fifteenth aspect there is provided a method of identifying a cellular factor capable of effecting and/or modulating expression of a target nucleic acid in a cell having the target nucleic acid and a nucleic acid which is an antisense of the target nucleic acid or part thereof, which method comprises over-expressing said factor in the cell and wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.
According to a sixteenth aspect there is provided a factor identified by the method of the fifteenth aspect.
The preferred factors can be selected from the group consisting of a gene, cDNA, RNA or a protein. More preferred are factors selected from the group consisting of a transcriptional activator or the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery. Even more preferred is a factor having an res sequence.
Preferred factors can also be selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.
According to a seventeenth aspect there is provided an RNAi factor which is an res sequence obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.
According to an eighteenth aspect there is provided an RNAi factor which is an res sequence represented by Seq ID Nos 1 to 4.
According to a nineteenth aspect there is provided a Schizosaccharomyces pombe cell having a target nucleic acid or a part thereof and a antisense nucleic acid or a part thereof which corresponds to the target nucleic acid or a part thereof, wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.
The term “inhibiting expression of a target nucleic acid” as used in the context of the present invention is intended to encompass, but not be limited to, reduction or elimination of gene expression, whether or not the target nucleic acid is a gene, or a part thereof, introduced into the cell or it is an endogenous gene.
The term “RNAi factor” is intended to include in its scope any naturally occurring, modified or synthetic molecule capable of enhancing RNAi activity. This definition includes in its scope the factors referred to herein as RNAi enhancing sequences (res). The term res may be used herein interchangeably with the term aes (anti-sense enhancing sequences).
It will be understood by those skilled in the art that “elevating RNAi factor level” can be achieved by transfection, upregulation or other means known in the art.
The term “anti-sense nucleic acid molecule” as used in the context of the present invention is intended to encompass RNA or DNA and contain region(s) related to a specific target RNA transcript or its gene. It is also intended to include molecules giving rise to antisense sequences, including inverted repeat, sense RNA, etc.
The term “conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid molecule inhibits expression of the gene” is intended to include the concept of inhibition of gene expression which is high enough to be effective, but not so high as to harm the cell. The effective concentration range can be determined using routine methodology known in the art.
Reference to a “subject” is intended to include human, animal (mouse), plant, or other life forms.
Reference to a “cell” is intended to encompass eukaryotic cells such as yeast, mammalian, plant, etc
The term “disorder” will be understood to include viral infection (HIV, HPV, etc), cancer, autoimmune disease and other chronic and acute diseases.
The term “phenotype” as used in the context of the present invention is intended to include cell staining, morphology, growth, and similar characteristics.
Using the lacZ fission yeast model to investigate features of antisense-RNA technology in vivo (1-4), it has been shown that the degree of target gene suppression is dependent on the level of antisense RNA (FIG. 2A)(1). During the course of developing this model it was surprisingly found that by increasing the steady-state levels of the lacZ target mRNA 20-fold in the presence of the same pool of antisense RNA that antisense-mediated suppression was not only maintained, but enhanced (
To test the ability of dsRNA to specifically interfere with another target sequence in fission yeast, sense and antisense c-myc sequences were co-expressed in a strain containing an integrated c-myc-lacZ fusion cassette (3). A 792 bp antisense c-myc fragment from exon 2 of the human c-myc gene was previously found to suppress β-galactosidase activity within the c-myc-lacZ fusion target strain by 47% (3). β-galactosidase assays demonstrated that co-expression of the antisense and sense c-myc constructs in the target strain enhanced c-myc suppression compared with the antisense c-myc vector alone (
Without wishing to be bound by any particular mechanism, it is probable that a multi-protein complex exists which mediates the post-transcriptional degradation of target mRNA in RNAi. Several genes which are involved in the RNAi phenomenon have been identified through genetic screens in Neurospora and nematodes. These genes include an RNA-dependent RNA polymerase (qde-1, ego-1) a RecQ DNA helicase (qde-3), an RNase D homologue (mut-7), and a putative translation initiation factor (rde-1, qde-2). The genes mut-2, rde-2, rde-4, and rde-7 have also been identified as being involved in either the initiation or maintenance of RNAi. Additionally, Drosophila cell free assays have shown that RNAi is mediated by nuclease degradation of the targeted mRNA while a 21-25 nt RNA species appears to be integral to specific post-transcriptional genetic interference. RNAi has also been shown to be dependent on ATP which may be required for strand dissociation of dsRNA. However, a missing component of the proposed multi-protein complex in the RNAi pathway is the implied RNA helicase.
The dose-dependency of dsRNA-mediated gene silencing in fission yeast described above has allowed us to use an over-expression strategy to identify genes involved in RNAi. In comparison to mutagenesis strategies, over-expression can enable the identification of genes which are otherwise essential for cell viability. Also, cellular factors that quantitatively enhance or reduce RNAi activity can be determined. The first gene that was tested in mediating RNAi activity in the present model was the nmt1 transcription factor thi1. This gene has been shown to specifically up regulate nmt1 expression when overexpressed in fission yeast. As we have previously shown that antisense RNA-mediated gene suppression is dose dependent in S. pombe (1) it was hypothesized that over-expression of thi1 would result in increased production of nmt1-driven antisense lacZ RNA and a consequent enhancement in target gene suppression. The thi1 open reading frame was PCR amplified and subcloned into pREP4 as a BamHI fragment. This vector was then transformed into RB3-2/pGT2 and β-galactosidase assays performed. As predicted, lacZ suppression was enhanced when compared to a strain expressing antisense RNA alone (
The second gene investigated has been the S. pombe ATP-dependent RNA helicase gene, ded1. Ded1 is an essential gene which has previously been characterized as a suppressor of sterility, a suppressor of checkpoint and stress response, and a general translation initiation factor. According to current models of dsRNA-mediated gene regulation an ATP-dependent RNA helicase may be required in conjunction with a dsRNA-dependent RNA polymerase for the formation of short single-stranded RNA fragments which specifically degrades target mRNA. It was therefore reasoned that over-expression of this gene in fission yeast could enhance the efficiency of dsRNA-mediated gene silencing by stimulating the unwinding of dsRNA. Co-expression of the ded1 vector with the antisense lacZ vector significantly enhanced dsRNA-mediated lacZ inhibition by a further 50% compared to the antisense expressing strain (
By over-expressing a cDNA library in a dsRNA fission yeast model we have identified four novel, genes with potential roles in the RNAi pathway. All of the known genes identified are essential and have natural associations with DNA, RNA or nucleic acid binding proteins, consistent with the expectation of RNAi cellular factors. In addition, two of the three known cDNAs encoded proteins involved in the process of translation. These findings along with our previous identification of an ATP-dependent RNA helicase (Raponi & Arndt, submitted) and reports of the involvement of a translation initiation factor in RNAi and the co-purification of ribonuclease activity with ribosomal fractions, suggests that this may be one site at which RNAi functions. Alternatively, the identification of such varied proteins as DNA helicases and translational components as part of the RNAi machinery implies that particular cellular proteins may be recruited into more than one multiprotein complex. Under the latter conditions, over-expression of these specific proteins may result in the generation of an RNAi complex that recognises dsRNA and mediates target gene suppression. Certain of these proteins may be rate-limiting or rate-determining in RNAi and only through supplementation of these factors are their roles in RNAi uncovered. It is proposed, without wishing to be bound by any particular mechanism, that in addition to the previously identified core proteins in the RNAi multiprotein complex(es), additional factors such as EF Tu, L7a, sna41 and res1 may be enlisted for a role in dsRNA-mediated gene suppression.
The over-expression strategy described overcomes some limitations associated with mutagenesis by identifying essential genes with a role in RNAi. In addition, this strategy complements these other systems by allowing the isolation of cellular factors that modify the efficacy of RNAi in vivo. The roles of these modulators of RNAi may be varied and include recognition and amplification of the dsRNA, delivery of the small 21-25 nt dsRNAs to the target mRNA, association between the antisense and target mRNA strands, and RNAi complex formation. Alternatively, these modulators may control the rate of RNAi or the formation of different complexes within cell types or for different forms of post-transcriptional gene silencing. The fact that over-expression of specific RNAi modulators enhanced dsRNA-mediated gene regulation in fission yeast indicates that a similar approach could be used to identify RNAi modulators in other organisms. In addition, the co-expression of these factors with different forms of post-transcriptional gene silencing including co-suppression, quelling, and antisense RNA could be one way of enhancing the efficacy of these methods. This may be especially important for application of RNAi to mammalian cells and tissues or to genes which have been somewhat recalcitrant to this form of regulation.
The present invention demonstrates for the first time the intrinsic involvement of an ATP-dependent RNA helicase as a key component in RNAi. Further, it can be rate limiting, as the over-expression leads to increased RNAi activity in this system. This ability of the ded1-encoded RNA helicase is consistent with its activities as a member of the DEAD box family of helicases, with their three core domains of ATPase, RNA helicase, and RNA binding activities. These could allow the enzyme to enhance gene suppression as follows: (i) in a dissociative mechanism it could mediate either the unwinding of dsRNA to generate a cRNA in conjunction with an RNA-dependent RNA polymerase or strand separation of fragmented dsRNA to enhance binding to homologous transcripts, and/or (ii) in an associative mechanism it could catalyse the ATP-dependent exchange of the sense strand of the short dsRNA with the target mRNA. The presence of ded1 homologues in other organisms displaying RNAi further supports the general involvement of this component in the RNAi machinery.
The present invention also provides for the first time a novel and quantitative genetic system based on S.pombe, for rapidly identifying essential cellular factors involved in RNAi. The use of this model has enabled verification of RNA helicase activity as a critical contributor to efficient RNAi activity and isolated novel RNAi factors including EF Tu, L7a, sna41, and an unidentified gene res1. The present invention also demonstrates that gene silencing may be enhanced by concomitant expression of such RNAi factors.
The invention will now be described more particularly with reference to non-limiting examples of certain preferred embodiments of the invention.
S. pombe Media and Manipulations.
All yeast strains were maintained on standard YES or EMM media (6). Repression of nmt1 transcription was achieved by the addition of thiamine to EMM media at a final concentration of 4 μM (7). Yeast cells were transformed with plasmid DNA by electroporation (8) and stable integrants were identified as previously described (6). A glass bead procedure (9) was used to isolate genomic DNA which was used for Southern analysis and PCR diagnosis. Total RNA was extracted as previously described (10).
S. pombe Strain and Plasmid Construction.
Construction of the low expressing lacZ strain, KC4-6 (h−, ura4::SV40-lacZ, leu1-32), has been previously described (2). The strain RB3-2 (h−, ura4::adh1-lacZ, leu1-32) which expresses higher levels β-galactosidase has also been previously described (1). The target strain which contains the c-myc:lacZ fusion has previously been described (3).
The construction of the long lacZ antisense containing episomal plasmid, pGT2, and corresponding control plasmids have been described (2). Plasmid pREP4-As was generated by subcloning the lacZ BamHI fragment contained in pGT2 into the plasmid pREP4 (11). pREP4 is identical to pREP1 except that the S. cerevisiae LEU2 gene has been replaced with the S. pombe ura4 selectable marker. To decrease the steady-state level of episomally expressed antisense lacZ, plasmids pREP42-As and pREP82-As were constructed by subcloning the lacZ BamHI fragment from pGT2 into pREP42 and pREP82, respectively. These plasmids are derivatives of pREP4 with mutations in the TATA box of the nmt1 promoter (12). The crippled lacZ vector, pGT62, was generated by end-filling the ClaI site of pGT2 and re-ligating (2). This frameshifted fragment was then subcloned into the BamHI site of pREP4 to generate the plasmid pM54-3.
The lacZ panhandle integration vector, pM30-8, was generated by first introducing a NotI site into the XmaI site of pRIP1/s (11) using the self-complementary linker 5′ CCG GGC GGC CGC 3′ to generate pL121-14. The 2.5 kb sequence of the 5′ end of the frameshifted lacZ gene (2) was then PCR-amplified to give it NotI ends using the forward primer 5′ ATGCGGCCGCAATTCCCGGGGATCGAAAGA 3′ and reverse primer 5′ ATGCGGCCGCAATGGGGTCGCTTCACTTA 3′. This product was cloned using a TA cloning kit (TOPO: Invitrogen, San Diego, Calif., USA) and then subcloned into the NotI site of pL121-14 in the antisense orientation. The integrating vector was introduced into target strains in single copy by using the sup3-5/ade6-704 complementation system (13). The full-length frameshifted lacZ fragment was then introduced into the BamHI site of this vector in the sense orientation upstream of the 2.5 kb antisense fragment to generate pM30-8. The episomal version of this vector (pM53-1) was made by removing the Pst1 sup3-5 fragment and introducing the autonomous replicating sequence as an EcoRI fragment (11). For testing the ability of the panhandle construct to form dsRNA in vivo, the frameshifted lacZ fragment was replaced with the functional lacZ fragment in the vectors pM54-3 and pM53-1, to generate the vectors pM85-1 and pM81-2, respectively. The vector pM91-1, which is unable to form a lacZ panhandle transcript, was generated by removing the 2.5 kb NotI lacZ fragment from pM81-2 and reintroducing it in the sense orientation.
Generation of the c-myc antisense construct, pCM-17, has been described elsewhere (3). The 792 by BglII c-myc fragment from pCM-17 was subcloned into the BamHI site of pREP4 in the sense orientation to generate pN12-1.
The ded1, and thi1, open reading frames were amplified from fission yeast genomic DNA (strain 1913). ded1 was amplified to give it BamHI ends using the forward primer 5′ ATGGGATCCCAACCAAACACTTCAACTCAG 3′ and the reverse primer 5′ ATGGGATCCTCAGAAGCCTGTGCATAACAC 3′. thi1 was amplified to give it BglII ends using the forward primer 5′ ATGAGATCTGTGGTTGGTATTCTAGAGAGA 3′ and the reverse primer 5′ ATGAGATCTAACAAAGACCTGCAAAAAACC 3′. PCR products were purified (Qiagen PCR purification kit), digested with either BamHI or BglII, gel-purified (Qiagen), and subcloned into the BamHI site of pREP4 in the sense orientation.
Nucleic acid electrophoresis and membrane transfer was performed as described (14). Southern and Northern blots were hybridized using ExpressHyb solution according to the manufacturer's instructions (Clontech Laboratories). DNA probes were 32P-labelled using the Megaprime labelling kit (Amersham). Probes included a 960 bp BamHI/ClaI lacZ fragment from pI2-1 (1), a 570 by HindIII/EcoRI ura4-3′ fragment from pGT113 (15), and a 2.2 kb PstI/SacI nmt1 fragment from pRIP1/s. Radioactive signals were detected by autoradiography and quantitated by phosphorimage analysis (ImageQuant; Molecular Dynamics).
Plasmid co-transformants of strain RB3-2 were grown under selective conditions to a cell density of 1-2×107 cells/ml. A serial dilution of each culture was performed and cells plated for single colonies in triplicate onto each of YES, EMM, EMM+leucine, and EMM+uracil agar media. The number of colonies grown on YES was taken as the total number of viable cells, while colonies growing on EMM represented the cells in the sampled population that contained both the ura4-containing (pREP4-based) and the LEU2-containing (pREP1-based) plasmids. Cells containing either the ura4-containing plasmid or the LEU2-containing plasmid were identified from the EMM+leucine and EMM+uracil plates respectively. The ratio of the number of colonies grown on selective media to the total number of viable colonies was used as the quantitative measure of the proportion of cells in the population, grown under selection, which contained plasmids.
β-galactosidase Assays.
The expression of the lacZ gene-encoded product, β-galactosidase, was quantitated using a cell permeabilization protocol as previously described (Raponi et al., 2000). A semi-quantitative overlay assay was also employed for rapid screening of yeast transformants (3).
Isolation of S. pombe cDNA Clones That Alter Antisense RNA Efficacy.
The S. pombe cDNA library was originally constructed in pREP3Xho by Bruce Edgar and Chris Norbury (5). The vector pREP3Xho is derived from pREP3 which contains the LEU2 marker and inserts are under control of the nmt1 promoter (11). A total of 5 μg of library DNA was transformed into the strain RB3-2 containing the episomal antisense lacZ vector pREP4-lacZAS and grown in EMM liquid media to the mid-logarithmic phase. Transformants were then plated on EMM solid media and grown at 30° C. for 3 days. Colonies were over-layed with medium containing 0.5 M sodium phosphate, 0.5% agarose, 2% dimethylformamide, 0.01% SDS, and 500 μg/ml X-GAL (Progen, Australia). Plates were then incubated at 37° C. for 3 hrs, colonies of interest recovered and assayed for β-galactosidase activity.
Strains were plated on EMM containing limiting uracil and 1 mg/ml 5-fluoroorotic acid (6). Strains were then replica plated on both selective and non-selective media. Those colonies that did not grow on selective media were identified as having lost the ura4-containing antisense plasmid. To determine the mitotic stability of pREP-based plasmids we used the plasmid segregation method previously described (16). This method indicated that up to 30% of the cells grown in selective media did not contain the resident plasmid.
To determine the role of target mRNA steady-state levels in antisense-mediated gene suppression, we investigated the ability of a long lacZ antisense RNA (2) to regulate lacZ target genes under control of both weak and strong constitutive promoters. The low-level expressing strain, KC4-6, contained the lacZ gene driven by the SV40 early promoter integrated at the ura4 locus in chromosome III (
It has previously, been demonstrated that a stably integrated antisense lacZ gene acts in a dose dependent fashion with the steady-state levels of antisense RNA being dependent on genomic position effects and transgene copy number (1). Here the role of the steady-state level of episomally expressed antisense RNA in both the low lacZ expressing target strain, KC4-6, and the high lacZ expressing target strain, RB3-2, was investigated. The expression of lacZ antisense RNA was increased by co-transforming KC4-6 and RB3-2 with plasmids pGT2 and pREP4-As, each of which contains the lacZ antisense gene under control of the nmt1 promoter, but different selectable markers. To decrease the steady-state level of antisense RNA, the plasmids pREP42-As and pREP82-AS were employed. The nmt1 promoter in these vectors contain deletions in the TATA box sequence which affect the level of transcription, but have no impact on the site of transcription initiation or thiamine repressibility (12). Each of the different antisense gene-containing plasmids was co-transformed with a control plasmid to complement auxotrophy where appropriate. This resulted in a set of co-transformants of both RB3-2 and KC4-6 each containing the same lacZ antisense gene, but with different promoter capacities. Each co-transformant was analyzed for antisense RNA steady-state levels and β-galactosidase activity. Table 1 indicates that with both strains the degree of target suppression is enhanced with the increase of antisense lacZ RNA expression.
aThe % suppression of β-galactosidase activity was determined by expressing each β-galactosidase activity as a percentage of the activity found in the pREP2, pREP4 co-transformant.
bWithin each strain the steady-state level of long lacZ antisense RNA was normalized to nmt1 mRNA and then expressed relative to the level observed in the pREP2, pREP4-As co-transformant.
These data agree with the previous report that antisense RNA-mediated gene inhibition is dose dependent in S. pombe (1). However, for each co-transformant the degree of β-galactosidase suppression was greater by 10-15% in the high lacZ expressing strain, RB3-2, than the low lacZ expressing strain, KC4-6, again demonstrating that low levels of target mRNA can limit antisense efficacy.
To determine whether the increase in gene suppression was due to formation of an antisense RNA:target mRNA hybrid or an antisense RNA:sense RNA hybrid, a version of lacZ which is unable to be translated into functional β-galactosidase was co-expressed in strains expressing the lacZ antisense gene. If antisense RNA was required to hybridize to target mRNA for inhibition of the gene expression pathway, then over-expression of the sense RNA would compete with the target for the available antisense molecules and a decrease in lacZ gene suppression would result. Initially, both antisense and sense lacZ vectors were integrated in single copy into separate target strains and then crossed with each other. In strains containing the antisense gene alone, β-galactosidase activity was reduced by approximately 40% (
To further increase the potential formation of intracellular dsRNA an episomal sense lacZ plasmid (pM54-3;
One explanation for the absence of higher levels of target gene suppression is plasmid segregation. S. pombe undergoes asymmetric segregation, with the result that mitosis produces a daughter cell which lacks the segregating plasmid (17). Therefore, when the 65% suppression level seen in the total population is corrected for only those cells containing the ura4-based and LEU2-based plasmids the level of suppression approaches approximately 100% (data not shown). Overall these data suggest that increasing the potential formation of dsRNA, but not necessarily an antisense RNA:target mRNA hybrid, is required for efficient interference of target gene expression in S. pombe. However, unlike the phenomenon of RNAi seen in plants and nematodes, the dsRNA-mediated gene suppression demonstrated in fission yeast seems to be dependent on the concentration of intracellular dsRNA or a threshold level of dsRNA is required to invoke potent gene silencing.
To further investigate the ability of dsRNA to inhibit gene expression in fission yeast we generated a vector containing the full-length 3.5 kb frameshifted lacZ sequence with a 2.5 kb inverted repeat. This construct generates a transcript of approximately 7 kb in length with 2.5 kb of self-complementarity which, predictably, will form a strong intramolecular RNA duplex. This gene was initially integrated into a fission yeast strain in single copy and then crossed with the strain RB3-2. The resulting strain which contained the single copy inverted repeat gene and target lacZ showed no reduction in β-galactosidase activity when transcription of the inverted repeat was activated. Southern analysis confirmed that the cassette was intact (data not shown) while RNA analysis indicated that the 7 kb transcript was being generated but approximately 10-fold less than episomally expressed antisense lacZ (
To confirm that gene inhibition was due to this construct forming an RNA duplex, an in vivo assay for dsRNA was developed. To this end, a series of vectors were generated which contained functional lacZ sequences including lacZ alone (pM85-1), a lacZ inverted repeat (pM81-2), and the lacZ repeat with both sequences in the sense orientation (pM91-1) (
To test the ability of dsRNA to specifically interfere with other target sequences in fission yeast, sense and antisense c-myc sequences were co-expressed in a strain containing an integrated c-myc-lacZ fusion cassette (
The dose-dependency of dsRNA-mediated gene silencing in fission yeast described above has allowed us to use an over-expression strategy to identify genes involved in RNAi. In comparison to mutagenesis strategies, over-expression can enable the identification of genes which are otherwise essential for cell viability. Also, cellular factors that quantitatively enhance or reduce RNAi activity can be determined. The first gene that we have tested in mediating RNAi activity in the present model has been the S. pombe ATP-dependent RNA helicase gene, ded1. Ded1 is an essential gene which has previously been characterized as a suppressor of sterility, a suppressor of checkpoint and stress response, and a general translation initiation factor. According to current models of dsRNA-mediated gene regulation an ATP-dependent RNA helicase may be required in conjunction with a dsRNA-dependent RNA polymerase for the formation of short single-stranded RNA fragments which specifically degrades target mRNA. We therefore reasoned that over-expression of this gene in fission yeast could enhance the efficiency of dsRNA-mediated gene silencing by stimulating the unwinding of dsRNA. Co-expression of the ded1 vector with the antisense lacZ vector significantly enhanced dsRNA-mediated lacZ inhibition by a further 50% compared to the antisense expressing strain (
The second gene investigated was the nmt1 transcription factor thi1. This gene has been shown to specifically up regulate nmt1 expression when overexpressed in fission yeast. As we have previously shown that antisense RNA-mediated gene suppression is dose-dependent in S. pombe (1) it was hypothesized that over-expression of thi1 would result in increased production of nmt1-driven antisense lacZ RNA and a consequent enhancement in target gene suppression. The thi1 open reading frame was PCR-amplified and subcloned into pREP4 as a BamHI fragment. This vector was then transformed into RB3-2/pGT2 and β-galactosidase assays performed. As predicted, lacZ suppression was enhanced when compared to a strain expressing antisense RNA alone (
Over-expression of a fission yeast cDNA library (5) in an antisense lacZ expressing strain, revealed a series of transformants in which β-galactosidase activity was significantly reduced from that demonstrated in antisense RNA-expressing strains alone. The screening strategy is shown in
The library plasmids were recovered from the aes-containing strains (also referred to as res-containing strains) and their cDNA inserts sequenced. BLASTN and BLASTP analysis identified clones W18, W20, and W30 (named aes2) as homologues of domains 2 and 3 of the mitochondrial elongation factor Tu (EF Tu). EF Tu is an essential protein which plays a role in transporting tRNA to the A site in the ribosome for peptide elongation. The cDNA in transformants W21, W23, and W32 (named aes3) was homologous to a putative protein that was identified in a screen for fission yeast ORFs. Interestingly, the cDNA insert was also homologous to the antisense strand of the 3′ UTR of the fission yeast gene sna41 which has previously been shown to be involved in DNA replication. It is thus possible that aes3 may operate through more than one mechanism in enhancing antisense RNA activity. The cDNA in transformant W47 (named aes4) was homologous to the antisense strand of the ribosomal protein L7a, a component of the 60S ribosomal subunit. aes4 also contained a small ORF of unknown biological function. The inserts in transformants W27 and W28 (named aes1) shared homology with a putative protein from C. albicans that was identified in a screen for genes essential for cell growth. A tertiary quantitative β-galactosidase assay was performed to obtain accurate levels of gene silencing augmentation in the antisense lacZ strains co-expressing these unique factors (
aes1 shared 43% identity with amino acids 4 to 202 of a C. albicans hypothetical protein (AJ390519). aes2 shared 99% identity with nucleotides 10452 to 9484 of the translation elongation factor EF Tu (AL049769). aes3 shared 94% identity with nucleotides 776 to 1145 of D89239 S. pombe ORF (D89239) and 93% identity with nucleotides 3246 to 2876 of the antisense strand of the DNA replication factor sna41 (AB001379). aes3 also contained a 220 nt stretch of a GA repeat sequence at its 3′ end. aes4 shared 99% identity with nucleotides 9678 to 8897 of the antisense strand of ribosomal protein L7a (AJ001133) and 99% identity with nucleoStides 1365 to 584 of the antisense strand of ribosomal protein L4 (AB005750). The reference numerals in brackets refer to accession numbers in the GeneBank database (the GeneBank database can be accessed from the following web site: http://www.ncbi.nlm.nih.gov/).
EF Tu is analogous to the eukaryotic EF1α and acts by transporting tRNA to the A site in the ribosome for peptide elongation. EF1α is an essential protein which has also been implicated in a large array of cellular activities including actin binding, microtubule severing, cellular transformation, cell senescence, protein ubiquitination, and protein folding. Detailed analysis of the EF Tu-expressing strain showed that it enhanced antisense RNA-mediated lacZ silencing by an additional 15% (
The protein encoded by sna41 has previously been shown to be involved in DNA replication. sna41 has low homology with CDC45 and might have DNA helicase properties which could facilitate the expression of complementary sequences. It is conceivable that the antisense plasmid and target DNA sequences may ectopically pair by intermolecular complementarity. Such pairing may inhibit RNA expression and consequently reduce the level of intracellular dsRNA. Similarly the intramolecular pairing of inverted repeat DNA sequences may also interfere with RNA expression. The over-expression of a protein with DNA helicase properties could facilitate the generation of more dsRNA which could in turn enhance the RNAi effect. Furthermore, CDC45 mutants show an increased rate of plasmid segregation. If plasmid loss is inhibited by over-expression of sna41 then more dsRNA may be generated leading to more effective RNAi in this system. In this light it is not unreasonable to expect that other proteins normally involved in DNA and/or RNA metabolism and function could also have a role in RNAi modulation and/or enhancement.
The L7a protein is part of the 60s ribosomal sub-unit. Without wishing to be bound by any particular mechanism of action, RNAi augmentation by over-expression of this protein is reasonable as it is hypothesised that the short dsRNA species may undergo strand displacement with target mRNA at the ribosome. The L7a ribosomal protein may act in RNAi by i) mediating docking of dsRNA or its unwound form into the A site of the ribosome, assisting in association of the antisense strand with the target mRNA, and/or shuttling of dsRNA to the ribosomal complex.
Nucleotide sequences representative of the aes factors referred to above are provided below and identified as Seq ID Nos 1 to 4:
With recent studies on PTGS suggesting that antisense RNA, co-suppression, and dsRNA-mediated interference may share similar mechanisms, we wanted to determine whether over-expression of an aes factor would also enhance dsRNA-mediated regulation. Having demonstrated that dsRNA could mediate gene suppression in this fission yeast model, the effect of an antisense enhancing sequence on dsRNA-mediated regulation was tested. To this end, the aes2 gene was co-expressed with the lacZ panhandle construct in a yeast strain containing the lacZ target gene. Under these conditions, this transformant displayed an additional 30% suppression of β-galactosidase activity when compared to the transformant expressing only the panhandle lacZ RNA (see
Further it was shown that these two forms of regulation are related by over-expressing the ATP-dependent RNA helicase ded1 in the presence of lacZ antisense RNA and showing that this helicase enhanced gene suppression by a further 50% compared to the control strain (example 6). ded1 was tested on both active and inactive antisense plasmids and demonstrated that ded1 augmentation of gene silencing was dependent on an active antisense RNA (see
The methods of the present invention have utility in demonstrating a range of RNAi efficacies, in identifying new factors which enhance or reduce gene silencing, in inhibiting gene expression or increasing sensitivity to antisense inhibition of gene expression, in the treatment or prevention of disorders which require inhibition or down-regulation of gene expression.
Although the present invention has been described with reference to specific examples and preferred embodiments it will be clear to those skilled in the art that variations and modifications which do not depart from the concept and the spirit of the invention described herein are also contemplated as being within the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| PQ7830 | May 2000 | AU | national |
| PQ9246 | Aug 2000 | AU | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/AU2001/000627 | 5/29/2001 | WO | 00 | 7/5/2006 |
