Research Project
2881812
The heterogeneous nuclear ribonucleoprotein (hnRNP) C family, are one of the most abundant nuclear proteins in vertebrate nuclei, and consist of hnRNP C1, C2, and a newly identified member designated in humans as p542. p542 has been placed in the C family of hnRNP proteins based on its conservation of several structural motifs which have been characterized in hnRNP C1. These motifs include an amino terminal RNA recognition motif, a proline rich region that has been shown to mediate interaction between C1 and proteins containing Src homology 3 (SH3) domains, a basic leucine zipper motif (bZLM) that in C1 binds RNA with high affinity, and a nuclear retention signal (NRS) that has been shown to restrict C1 to the nucleus. hnRNP C1 and p542 are 290 and 291 amino acids in length, respectively, and the structural domains are colinear between the two proteins. Though the proteins have a high degree of sequence identity, it is clear that they are not functionally redundant in that mice lacking a functional homologue of p542 (Raly) fail to develop beyond the blastocyst stage. It is the focus of this proposal to analyze the structural determinants in both proteins that generate functional convergence or divergence. To achieve this objective, extensive equilibrium binding studies will be carried out on wild-type p542, selected deletion mutants of the protein, and p542/C1 chimeras. These studies will determine if the conserved regions in p542 are functionally equivalent to those in hnRNP C1 as well as identify those determinants that confer functional identity on both proteins. High affinity p542 RNA ligands will be evaluated from selection amplification experiments, and the molecular determinants that confer this specificity will be evaluated from similar experiments using p542 deletion mutants or p542/C1 chimeras. p542 specific protein-protein interactions will be evaluated using a yeast two hybrid assay. These studies will provide important insight into understanding the function of p542 through a comparative analysis of data on a well characterized protein, hnRNP C1, as well as, gaining new insight into the determinants that govern RNA recognition, sequence specificity, and protein-protein interaction. Because pre-mRNA processing occurs in the context of an hnRNP occluded complex, a detailed understanding of pre-mRNA processing is dependent upon characterization of the active components involved.
