Messenger RNA Metabolism in Yeast

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My long-term goal is an understanding of the molecular mechanisms that regulate nonsense-mediated mRNA decay (NMD) in the the yeast Saccharomyces cerevisiae. Our recent experiments have led us to formulate the faux UTR model for NMD in yeast, and independent studies in higher organisms have provided Strong support for the general applicability of this model to all eukaryotes. Our data Indicate that premature and normal termination differ mechanistically, with premature termination being a relatively inefficient process that leads to the ribosomal recruitment of at least three key proteins (Upf1, Upf2yNmd2, and Upf3). These factors subsequently function in the dissociation of the premature termination complex and in the recruitment ofthe decapping enzyme responsible for initiating decay ofthe transcript. In the experiments of this proposal, we will define the critical Interactions underlying these observations and elucidate the mechanisms by which ribosomes are recycled subsequent to premature and normal termination, and by which decapping of NMD substrates is triggered. With the goal of further testing the faux UTR model for NMD, I plan to: a) define the timing and interaction-dependencies of Upf1, Upf2/Nmd2, and Upf3 association with prematurely terminating ribosomes, b) delineate the protein:protein interactions that link the decapping enzyme to the UPF/NMD factors, c) evaluate the regulatory role of ribosome recycling in cis subsequent to normal or premature termination, and d) characterize the Upf1 activity that dissociates and/or remodels ribosomal subunits at premature termination codons.