Research Project
6807072
DESCRIPTION (provided by applicant): p542 is an auto-antigen found in individuals suffering from a range of auto-immune disorders including systemic lupus erethymatosus, rheumatoid arthritis, and Chrohn's disease suggesting that impairment of p542 function may be associated with the molecular pathology of these illnesses. P542 conserves important primary structural and functional motifs that have been identified in the heterogeneous nuclear ribonucleoproteins C1 and C2 (hnRNP C), two very abundant nuclear proteins associated with pre-mRNA processing, regulation of mRNA stability, and translation. Though on the surface it appears that p542 is just a structural variant of hnRNP C, reciprocal hnRNP C and p542 gene ablation studies, as well as our recent findings suggest overlapping but non-redundant functions for both proteins. We have demonstrated that the RNA binding properties of p542 are indistinguishable from hnRNP C. We have also shown that p542 binds specifically to the double and single-stranded telomere repeat sequence suggesting a role for p542 in telomere architecture. Using the yeast two hybrid assay we have shown that hnRNP C and p542 interact with microtubule associated protein 1A, a protein recently proposed to function in the attachment of mRNA to microtubules, and nischarin, a protein reported to alter the actin component of the cytoskeleton through a specific interaction with integrin alpha5. The attachment of mRNA to the cytoskeletal network is linked to mRNA localization, mRNA translation, and regulation of mRNA stability, p542 and hnRNP C's interaction with MAP 1A may provide an initial foundation for uncovering the molecular basis for hnRNP C's role in IRES mediated translation and mRNA turnover. We also have shown that p542 but not hnRNP C, interacts with CD81, a member of the tetraspanin family of membrane proteins associated with the regulation of integrins. The focus of the proposal outlined here is to gain additional insight into the function of p542, by using genomic SELEX to identify in-vivo p542 specific RNA ligands. Furthermore, since hnRNP C and p542 functionally diverge at the point of protein-protein interactions, we will use immunoprecipitation studies to identify p542 in-vivo protein associates in both the nucleus and the cytoplasm. Specifically, we are interested in doing a comparative analysis of hnRNP C and p542 nuclear immunoprecipitates, to determine if p542, like hnRNP C is involved in RNA polymerase II transcript processing or perhaps another class of RNA processing. An analysis of cytoplasmic immunoprecipitates are aimed at verifying the cellular interaction of p542 with MAP IA as well as identifying other protein components associated with the complex. Finally, understanding the molecular architecture of telomeres is critical to our understanding of carcinogenesis, since cancer cell have evolved and altered telomere structure that protects them from cellular senescence that results from telomere attrition following repeated cell division. The in-vitro telomere binding specificity of p542, parallels inferences linking hnRNP C to telomeres, and prompts our investigations into the in vivo association of p542 with telomeric DNA.
