We have developed a genetic system in the yeast Saccharomyces cerevisiae that will enable us to determine how mRNA translation and mRNA turnover are related. Four genes have been identified that potentially mediate translation-dependent mRNA turn-over. Two of these genes, called UPF1 and UPF3, have already been shown to be required for rapid turnover associated with premature translational termination. The experiments in this proposal focus on how specific gene products mediate mRNA turnover in a translation-dependent manner, how the signals from translation to turnover are transduced, and what cellular purpose might be served by the existence of translation-dependent turnover systems. For each gene and gene product of interest, we plan to provide a complete characterization, including DNA sequence analysis, gene disruption and expression analysis, copy number and mapping analysis, transcript analysis, amino acid sequence comparison, immunofluorescent localization, biochemical localization, and fractionation. We will also seek to identify potential interactions with ribosomes, ribosomal subunits, ribosomal proteins, ribosomal RNA, mRNA, or other RNAs. UPF2 and UPF4 will be characterized to determine whether these gene products also affect mRNA turnover. In addition, we will screen for mutations in other genes whose products function in a translation-dependent manner. The genetic screens are such that we might also uncover genes that affect mRNA turnover in a manner independent of translation. We present a model in which translation-dependent mRNA turnover is mediated by several different pathways. We will define these pathways by determining for each gene product whether its function depends on translation, read-through of premature termination, and/or the presence of 3'-poly(A) mRNA tail. Potential interactions between the pathways will be examined. Finally, we will attempt to establish the cellular purpose of translation-dependent turnover pathways. We have found that UPF1 controls the stability of PPR1 mRNA, which contains an upstream open reading frame (URF) in the leader region. Experiments will be conducted to determine if the URF targets the mRNA for UPF1-mediated degradation, and if so, what special features of the URF might be required. If the URF is involved in targeting, we will search for other wild-type mRNAs whose stability is controlled by UPF1.
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