Nonsense-mediated mRNA decay (NMD), the destabilization of an otherwise stable mRNA by premature translation termination, is a conserved quality control pathway that exemplifies the interdependence of mRNA decay and protein synthesis. NMD has been extensively studied in multiple eukaryotes, particularly with respect to the nature of its substrates and the structures and interactions of its central regulators, Upf1, 2, and 3. In spite of these efforts many key mechanistic questions about this important regulatory pathway remain to be resolved. We have yet to understand the detailed differences between normal and premature termination or the molecular events by which the Upf proteins selectively target translating mRNAs containing premature termination codons (PTCs), promote mRNA destabilization, or enhance the disassembly of a poorly dissociable premature termination complex. In part, an understanding of these problems required new approaches that take into account the important roles played by components of the protein synthesis apparatus in implementing NMD. Using the yeast Saccharomyces cerevisiae as a model system, we have now established such approaches. We developed a selective ribosome profiling procedure that allows delineation of the specificity and timing of Upf factor association with translating ribosomes, combined the selective purification of Upf1-associated ribosomes with cryo-electron microscopy to localize Upf1 to a specific ribosomal domain, combined mass spectrometry and efficient purification of full-length proteins derived from nonsense codon readthrough to elucidate details of aberrant translation termination, and identified and characterized novel negative and positive regulatory elements, including two Upf1-binding sites, in the previously uncharacterized C- terminal domain of Dcp2, the catalytic component of the mRNA decapping enzyme. In the experiments of this proposal, we will follow up on these developments, addressing four aims that seek to: i) determine when each of the three Upf proteins associates with ribosomes and whether they specifically target NMD substrates and prematurely terminating ribosomes; ii) characterize function-dependent Upf binding sites and conformations on the 80S ribosome; iii) identify qualitative and quantitative differences between premature and normal translation termination; and iv) elucidate the mechanisms by which decapping activator proteins, including Upf1, interact with the Dcp2 C-terminal domain to regulate decapping of specific substrates. At the conclusion of these studies we anticipate being able to formulate an integrated model detailing the molecular events linking premature translational termination to targeted mRNA decay.
The long-term goal of this project is an understanding of the molecular mechanisms that regulate nonsense-mediated mRNA decay (NMD), a cellular quality control mechanism that is triggered by premature termination of the process of protein synthesis. An understanding of NMD is particularly pertinent to public health because approximately 20% of all inherited disorders are attributable to nonsense mutations, i.e., genetic alterations which give rise to premature termination of protein synthesis. Drugs aimed at bypassing the effects of nonsense mutations are currently being evaluated in patients and our experiments have the potential to lead to improvements in these novel therapeutic modalities.
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