Tuesday, April 23, 2024
4/23/2024
DESCRIPTION (provided by applicant): Regulation of mRNA synthesis by RNA polymerase II (pol II) is critical for proper development, homeostasis and functioning of organisms; misregulation contributes to serious diseases including cancer. Pol II is a scaffold upon which other factors important for transcription and post-transcriptional processing of mRNAs assemble, including the general transcription factors, TATA-binding protein and its associated factors, the Mediator complex, the capping enzyme complex and the cleavage/polyadenylation factors. In addition to these factors, the Paf1 complex (Paf1C), associated with pol II at the promoter and in coding regions of genes, is emerging as an important player orchestrating the binding and activity of transcription and post- transcriptional factors. Containing Paf1, Ctr9, Rtf1, Leo1 and Cdc73, the yeast Paf1C is now linked genetically and physically to initiation and elongation factors and to post-transcriptional processing of mRNA. Mutation of yeast Paf1 factors Is not lethal, but causes severe, pleiotropic phenotypes, and results in changes in polyadenylation and the abundance of a subset of mRNAs. Mutation of the human Cdc73 homolog (parafibromin) results in human parathyroid and jaw tumors defining the Paf1C as a human tumor suppressor. We still do not know the interconnections of the Paf1C components or how the complex associates with pol II. Nor do we have a definition of the direct protein/protein contacts made with other transcription and RNA processing factors. Two genome scale analyses of yeast protein-protein interactions have not identified any of these interactions, due in part to failure to include the correct coding regions for the Paf1C factors in the screens. This project proposes a directed test of protein-protein interactions of the members of the Paf1C, with each other, with pol II subunits, and with other functionally linked factors. The yeast two-hybrid technique will be used to detect interactions of proteins in vivo. A complementary in vitro approach will use yeast strains with pairs of tagged proteins to look for co-immunoprecipitation of proteins in both wild-type and Paf1C factor mutant yeast strains. Our results will build a model of the organization of the yeast Paf1C and its interactions critical for understanding how this important complex functions as a tumor suppressor in humans. The simplicity of the model system and techniques to be used assure that under- graduates will be able to make significant contributions to the important questions asked in this proposal.
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