Project Summary/Abstract This proposal addresses the translation and degradation of mRNAs subject to premature translation termination. Such mRNAs are usually thought to be derived from genes harboring nonsense mutations, but also include cytoplasmic products of failed or alternative splicing, transcripts of pseudogenes or unproductive gene rearrangements, and mRNAs with upstream open reading frames, as well as mRNAs arising from non-standard transcription initiation or undergoing out-of-frame translation initiation or unexpected frameshifting. Translational elongation on these transcripts generally leads to ribosomal recognition of a premature termination codon (PTC) and to the triggering of accelerated mRNA decay in a process known as nonsense-mediated mRNA decay (NMD). The central regulators of this mRNA quality control pathway are the three Upf proteins (Upf1, Upf2, and Upf3). Although NMD has been studied extensively in multiple eukaryotes, the precise mechanisms by which the Upf proteins recognize a ribosome undergoing an atypical termination event and respond to it by triggering accelerated degradation of the associated mRNA remain unknown. In several recent studies we have begun to gain significant mechanistic insight into NMD in the yeast model system and to discern what appear to be at least four definable steps during which: 1) Upf1 binds stochastically to elongating ribosomes while monitoring translational termination, 2) Upf1 association with a ribosome engaged in premature termination is stabilized and a commitment to NMD is promoted by Upf2 and Upf3 binding, 3) activation of the Upf1 helicase activity by ATP hydrolysis promotes dissociation of the termination complex and recruitment of the mRNA decapping enzyme, and 4) post-decapping, the body of the mRNA is digested exonucleolytically by Xrn1. In the experiments of this proposal we will use the tools of yeast genetics, molecular biology, and cryo-electron microscopy to pursue several critical aspects of this model by addressing three goals that seek to: elucidate the mechanism of decapping activation of NMD substrates by Upf1, determine the roles and functional order for factors that link premature termination to mRNA decapping, and define the basis for Pab1 enhancement of translation termination efficiency, likely a key step for distinguishing premature from normal termination. At the conclusion of this study we anticipate being able to formulate a more integrated model detailing the atypical aspects of premature translation termination, the mechanism by which premature termination is targeted by the three Upf proteins, and the molecular events linking the functions of Upf proteins to targeted mRNA decay. We expect that our refined model for yeast NMD will be generally applicable to the molecular events of NMD in higher organisms and will provide insights to improving therapeutic approaches to human diseases caused by nonsense mutations.