PUF proteins are post-transcriptional repressors that control diverse processes such as development and differentiation, mitochondrial function, cellular growth, and lifespan. These sequence specific RNA-binding proteins bind to regulatory sequences in the 3' untranslated regions (3'UTRs) of target mRNAs to repress gene expression. The mechanism of repression by PUF proteins is not known. Our working hypothesis is that PUF proteins bind to the 3'UTR of an mRNA and recruit the basal mRNA turnover machinery to degrade that mRNA. We have chosen the yeast PUF5 protein as a model system for determining how PUF proteins function. Puf5p binds to and represses expression of the HO endonuclease mRNA, which is involved in mating type switching. We propose to identify protein-protein interactions between the Puf5p and yeast proteins using a two-hybrid screen. Also, biochemical purification of Puf5p protein complexes and protein microsequencing will be used to identify Puf5p-associated protein complexes. We will identify the components of the regulatory protein complex that assembles on the 3'UTR of the HO mRNA with Puf5p by using a three-hybrid protein-RNA interaction screen. An in vivo functional assay will be used to identify genes that are required for Puf5p-mediated repression and to identify the pathway of mRNA turnover that is controlled by Puf5p. Finally, an in vitro mRNA turnover assay will used to further dissect the mechanism of Puf5p repression.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM067427-02
Application #
6703657
Study Section
Special Emphasis Panel (ZRG1-F05 (20))
Program Officer
Tompkins, Laurie
Project Start
2003-02-01
Project End
2005-01-31
Budget Start
2004-02-01
Budget End
2005-01-31
Support Year
2
Fiscal Year
2004
Total Cost
$47,296
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715