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.
