NUCLEAR ROLE OF YEAST POLY(A)-BINDING PROTEIN

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DESCRIPTION (Adapted from applicant's abstract): The proposed research seeks to understand the diverse functions of the poly(A)-binding protein that is associated with the poly(A) tail of most eukaryotic mRNAs. There is ample evidence indicating that the presence of the poly(A) tail and its bound protein has profound consequences on the expression of genes. This RNA/protein complex stimulates initiation of translation and promotes mRNA stabilization in the cytoplasm. In the nucleus, poly(A)-binding protein is required for mRNA 3'-end formation and export of the nascent mRNA from to the cytoplasm. This proposal, using the yeast Saccharomyces cerevisiae as a model system, addresses the nuclear functions of poly(A)-binding protein (Pab1p). Factors have been identified that, in concert with Pab1p, regulate the extent of polyadenylation. If the gene encoding the most well-characterized of these factors, PBP1, is disrupted, there is a substantial reduction in the lengths of poly(A) tails synthesized in cell-free extracts and inhibits the in vivo utilization of an early polyadenylation site in a HIS4 reporter. Disruption of PBP1 also suppresses the lethality of a PAB1 deletion without directly effecting mRNA stability or translation, which stands in contrast to the phenotypes of previously isolated pab1 suppressors. Two-hybrid analyses suggest that Pbp1p may be one of several regulatory factors recruited to the RNA processing apparatus. To study the role of Pbp1 and related factors in the regulation of Pab1p's function in 3'-end processing the PI proposes to: 1) analyze the biochemical activities of Pbp1p and the other Pbps, including an assessment of Pbp1p's ability to regulate the activity of poly(A) nuclease or poly(A) polymerase; 2) identify mRNAs whose expression is dependent on the relative extent of polyadenylation using oligonucleotide probe arrays; and 3) analyze the genetic relationships of PAB1 and PBP1 by determining their interactions with other genes encoding cleavage and/or polyadenylation factors by characterizing high copy suppressors of the growth defects resulting from overexpression of these genes.