METABOLISM OF YEAST mRNAS THAT LACK A STOP CODON

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DESCRIPTION (provided by applicant): mRNA degradation is an important aspect of gene expression. It is now clear that the same enzymes degrade both stable and unstable mRNAs. Thus, the key to understanding differential mRNA degradation is to understand the interactions of a particular mRNA with the basal machinery. The experiments proposed here are aimed at understanding in molecular detail how one particular mRNA interacts with the mRNA decay machinery and how this causes its rapid degradation. This proposal is focused on the extremely rapid degradation of yeast mRNAs that lack a stop codon ("nonstop decay") for four reasons. First, nonstop mRNAs are the least stable mRNAs in yeast. Second, degradation of nonstop mRNAs is an important quality control aspect of gene expression. Third, the mechanism of nonstop decay is likely important to assure that mRNAs are completely degraded. Fourth, nonstop yeast mRNAs are degraded by a complex of 3' exonucleases (the exosome). The exosome is conserved between yeast and mammals and has many functions. These functions may include the decay of important mammalian mRNAs. Understanding nonstop decay in yeast should increase our understanding of other exosome functions. In the current model for nonstop mRNA degradation an mRNA is recognized as aberrant when a ribosome reaches its 3' end. This ribosome is recognized by Ski7p through the ribosomal A-site, which results in recruitment of the exosome. This proposal is aimed at testing and expanding this model. Aim 1 is to identify all parts of the cellular machinery for recognition and decay of nonstop mRNAs. Aim 2 is to characterize the role of these parts in vivo. Preliminary results suggest that the proteasome may degrade proteins encoded by nonstop mRNAs. Aim 3 is to test this hypothesis. The fourth aim is to characterize the function of Ski7p in detail, which is the key protein in nonstop mRNA recognition and recruitment of the basal mRNA decay machinery. These aims should result in an understanding of the recognition and decay of nonstop mRNAs in molecular detail. Since normal mRNAs are degraded by the same enzymes, these experiments should also increase our understanding of the degradation of normal cellular mRNAs.