My long-term goal is an understanding of the molecular mechanisms that regulate mRNA stability in the yeast Saccharomyces cerevisiae. Recent work in my laboratory has been focused on nonsense mediated mRNA decay (NMD), the destabilization of an otherwise stable mRNA by a premature termination codon. We have defined the set of endogenous NMD substrates, demonstrated that they are available for NMD at every round of translation, and showed that premature termination and normal termination are not equivalent biochemical events. Our experiments demonstrate that premature termination is aberrant and that its NMD-stimulating defects are likely to be attributable to the ribosome's failure to terminate adjacent to a specific RNP structure or set of factors localized 3' to the stop codon. In the experiments of this proposal, we seek to follow up on these observations, test aspects of a new model, and, ultimately, arrive at a more definitive understanding of the link between translation termination and mRNA decay. In addition, we will also pursue two provocative observations, respectively indicating that the failure to complete the peptide hydrolysis step of termination may induce a specific endonucleolytic cleavage and that the 5' to 3' mRNA decay pathway might play only a minor role in yeast mRNA decay. To these ends, I plan to: a) define in detail the in vitro and in vivo events that distinguish premature (aberrant) termination from normal termination, b) elucidate the mechanistic connections between premature translation termination and the triggering of mRNA decay, c) identify the specific mRNAs associated with the critical NMD factors, Upflp and Nmd2p, d) characterize the putative endonucleolytic cleavage products of the can1-100 and his4-38 mRNAs, and e) compare the genome-wide expression profiles of total mRNA to those of poly(A)+ mRNA in wild-type cells and in cells defective in distinct steps of the 5' to 3' and 3' to 5' mRNA decay pathways.
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