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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061096-03
Application #
6520208
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Rhoades, Marcus M
Project Start
2000-04-01
Project End
2004-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
3
Fiscal Year
2002
Total Cost
$245,478
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Genetics
Type
Schools of Medicine
DUNS #
660735098
City
Worcester
State
MA
Country
United States
Zip Code
01655
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Mangus, David A; Evans, Matthew C; Agrin, Nathan S et al. (2004) Positive and negative regulation of poly(A) nuclease. Mol Cell Biol 24:5521-33
Parker, Kenneth C; Patterson, Dale; Williamson, Brian et al. (2004) Depth of proteome issues: a yeast isotope-coded affinity tag reagent study. Mol Cell Proteomics 3:625-59
Mangus, David A; Evans, Matthew C; Jacobson, Allan (2003) Poly(A)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression. Genome Biol 4:223
Duvel, Katrin; Valerius, Oliver; Mangus, David A et al. (2002) Replacement of the yeast TRP4 3' untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail. RNA 8:336-44
Sachs, Matthew S; Wang, Zhong; Gaba, Anthony et al. (2002) Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs. Methods 26:105-14