Yeast Upf1 Associates With RibosomesTranslating mRNA Coding Sequences Upstream of Normal Termination Codons: A Dissertation
Authors
Min, Ei EiFaculty Advisor
Allan Jacobson, PhDAcademic Program
Interdisciplinary Graduate ProgramUMass Chan Affiliations
Microbiology and Physiological SystemsDocument Type
Doctoral DissertationPublication Date
2015-04-15Keywords
Dissertations, UMMSCodon, Nonsense
Codon, Terminator
Nonsense Mediated mRNA Decay
Trans-Activators
RNA, Messenger
Ribosomes
Saccharomyces cerevisiae
Nonsense Codon
Terminator Codon
Nonsense Mediated mRNA Decay
Trans-Activators
Messenger RNA
Ribosomes
Saccharomyces cerevisiae
Genetics and Genomics
Nucleic Acids, Nucleotides, and Nucleosides
Metadata
Show full item recordAbstract
Nonsense-mediated mRNA decay (NMD) specifically targets mRNAs with premature translation termination codons for rapid degradation. NMD is a highly conserved translation-dependent mRNA decay pathway, and its core Upf factors are thought to be recruited to prematurely terminating mRNP complexes, possibly through the release factors that orchestrate translation termination. Upf1 is the central regulator of NMD and recent studies have challenged the notion that this protein is specifically targeted to aberrant, nonsense-containing mRNAs. Rather, it has been proposed that Upf1 binds to most mRNAs in a translation-independent manner. In this thesis, I investigated the nature of Upf1 association with its substrates in the yeast Saccharomyces cerevisiae. Using biochemical and genetic approaches, the basis for Upf1 interaction with ribosomes was evaluated to determine the specificity of Upf1 association with ribosomes, and the extent to which such binding is dependent on prior association of Upf1’s interacting partners. I discovered that Upf1 is specifically associated with Rps26 of the 40S ribosomal subunit, and that this association requires the N-terminal Upf1 CH domain. In addition, using selective ribosome profiling, I investigated when during translation Upf1 associates with ribosomes and showed that Upf1 binding was not limited to polyribosomes that were engaged in translating NMD substrate mRNAs. Rather, Upf1 associated with translating ribosomes on most mRNAs, binding preferentially as ribosomes approached the 3’ ends of open reading frames. Collectively, these studies provide new mechanistic insights into NMD and the dynamics of Upf1 during translation.DOI
10.13028/M2259WPermanent Link to this Item
http://hdl.handle.net/20.500.14038/32146Rights
Copyright is held by the author, with all rights reserved.ae974a485f413a2113503eed53cd6c53
10.13028/M2259W
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Poly(A)-Binding Protein Regulates the Efficiency of Translation TerminationWu, Chan; Roy, Bijoyita; He, Feng; Yan, Kevin; Jacobson, Allan (2020-11-17)Multiple factors influence translation termination efficiency, including nonsense codon identity and immediate context. To determine whether the relative position of a nonsense codon within an open reading frame (ORF) influences termination efficiency, we quantitate the production of prematurely terminated and/or readthrough polypeptides from 26 nonsense alleles of 3 genes expressed in yeast. The accumulation of premature termination products and the extent of readthrough for the respective premature termination codons (PTCs) manifest a marked dependence on PTC proximity to the mRNA 3' end. Premature termination products increase in relative abundance, whereas readthrough efficiencies decrease progressively across different ORFs, and readthrough efficiencies for a PTC increase in response to 3' UTR lengthening. These effects are eliminated and overall translation termination efficiency decreases considerably in cells harboring pab1 mutations. Our results support a critical role for poly(A)-binding protein in the regulation of translation termination and also suggest that inefficient termination is a trigger for nonsense-mediated mRNA decay (NMD).