This proposal addresses a fundamental, but poorly understood aspect of post-transcriptional control in eukaryotes: the molecular mechanisms that regulate cytoplasmic mRNA decay. Using the yeast Saccharomyces cerevisiae as a model system, we have developed a simple and reliable procedure for the determination of mRNA decay rates, identified stable and unstable mRNAs, and focused our efforts on the basis of instability of two types of unstable mRNAs: a) mRNAs that are inherently unstable (i.e., mRNAs whose primary sequence or overall RNP structure ensures that they will decay rapidly under essentially all conditions examined) and b) mRNAs that become unstable only as a consequence of specific nonsense mutations. For both types of unstable mRNA, we have found that rapid decay is dependent on specific cis-acting coding region sequences (instability elements) and ongoing translation. Rapid decay of nonsense-containing mRNAs is also dependent on Upf1p, the polysome-associated trans-acting factor encoded by the UPF1 gene. The involvement of coding sequences, a requirement for ongoing protein synthesis, the association of Upf1p with polysomes, and several other observations all point to an intimate link between translation and turnover. In this proposal, I seek to define the functions of the cis-acting elements, to characterize further the role of UPF1, to identify other trans-acting factors required for rapid mRNA decay, and to understand the nature of the relationship between mRNA turnover and translation. These goals will be addressed by: a) completing the development of a yeast in vitro mRNA decay system that accurately mimics in Vivo mRNA decay, b) determining the function of the PGK1 coding region element essential for nonsense-mediated mRNA decay, c) characterizing the gene products that either appear to interact with Upf1p or that compensate for its absence, d) developing a new methodology to identify the cis-acting coding region sequences that destabilize the inherently unstable HIS3 and URA3 mRNAs, and e) isolating and characterizing mutants or high copy suppressors that selectively stabilize mRNAs containing either the HIS3 or URA3 coding region instability element.
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