This proposal addresses a fundamental aspect of post-transcriptional control in eukaryotes, the molecular mechanisms that regulate mRNA decay. At least three different types of events trigger the initiation of mRNA decay, including poly(A) shortening, endonucleolytic cleavage, and the arrest of translation at a premature nonsense codon. Using the yeast Saccharomyces cerevisiae as a model system, work in my laboratory has shown that the latter mechanism, nonsense-mediated mRNA decay, is dependent on the activity of the interacting UPF/NMD factors. These proteins not only regulate mRNA decay but are also involved in translation termination. Three novel quantitative assays that we have developed recently have been used to show that upf/nmd mutations promote nonsense codon readthrough, physical changes in the complex associated with the termination codon, and alterations in the ability of a released ribosome to be recycled for a subsequent round of translation. In the experiments of this proposal, I will exploit these assays, and several genetic approaches, to test the hypotheses that an aberrant event in translation termination triggers mRNA decay, that the sequence downstream of a nonsense codon plays an important role in regulating the termination event, and that the components of the NMD pathway affect a broad range of regulatory circuits in the cell. More specifically, we will: 1) define the events that promote nonsense suppression in cells harboring upf/nmd mutations; 2) characterize the defects in ribosome release and reutilization that are attributable to upf/nmd mutations; 3) determine the relationships between events at translation termination and the triggering of mRNA decay; and 4) elucidate the biological ramifications of the NMD pathway.
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