Messenger RNA metabolism in yeast

描述（由申请人提供）： 我的长期目标是了解调节酿酒酵母中mRNA稳定性的分子机制。在我的实验室最近的工作一直集中在无义介导的mRNA衰变（NMD），不稳定的，否则稳定的mRNA的提前终止密码子。我们已经定义了一组内源性NMD底物，证明了它们在每一轮翻译中都可用于NMD，并表明提前终止和正常终止不是等同的生化事件。我们的实验表明，过早终止是异常的，其NMD刺激缺陷可能是由于核糖体的失败终止邻近特定的RNP结构或一组因子定位3'到终止密码子。在这个提议的实验中，我们试图跟进这些观察结果，测试新模型的各个方面，并最终对翻译终止和mRNA衰变之间的联系达成更明确的理解。此外，我们还将进行两个挑衅性的观察，分别表明未能完成终止的肽水解步骤可能会诱导特异性内切核酸裂解，以及5'至3' mRNA衰变途径可能在酵母mRNA衰变中仅起次要作用。为此，我计划：a）详细定义区分早产儿的体外和体内事件，从正常终止（异常）终止，B）阐明过早翻译终止和触发mRNA衰变之间的机制联系，c）鉴定与关键NMD因子Upflp和Nmd 2 p相关的特异性mRNA，d）表征can 1 -100和his 4 -38 mRNA的推定的核酸内切切割产物，和e）将野生型中总mRNA的全基因组表达谱与poly（A）+ mRNA的全基因组表达谱进行比较。型细胞和在5'至3'和3'至5' mRNA衰变途径的不同步骤中有缺陷的细胞中。

DESCRIPTION (provided by applicant): My long-term goal is an understanding of the molecular mechanisms that regulate mRNA stability in the yeast Saccharomyces cerevisiae. Recent work in my laboratory has been focused on nonsense mediated mRNA decay (NMD), the destabilization of an otherwise stable mRNA by a premature termination codon. We have defined the set of endogenous NMD substrates, demonstrated that they are available for NMD at every round of translation, and showed that premature termination and normal termination are not equivalent biochemical events. Our experiments demonstrate that premature termination is aberrant and that its NMD-stimulating defects are likely to be attributable to the ribosome's failure to terminate adjacent to a specific RNP structure or set of factors localized 3' to the stop codon. In the experiments of this proposal, we seek to follow up on these observations, test aspects of a new model, and, ultimately, arrive at a more definitive understanding of the link between translation termination and mRNA decay. In addition, we will also pursue two provocative observations, respectively indicating that the failure to complete the peptide hydrolysis step of termination may induce a specific endonucleolytic cleavage and that the 5' to 3' mRNA decay pathway might play only a minor role in yeast mRNA decay. To these ends, I plan to: a) define in detail the in vitro and in vivo events that distinguish premature (aberrant) termination from normal termination, b) elucidate the mechanistic connections between premature translation termination and the triggering of mRNA decay, c) identify the specific mRNAs associated with the critical NMD factors, Upflp and Nmd2p, d) characterize the putative endonucleolytic cleavage products of the can1-100 and his4-38 mRNAs, and e) compare the genome-wide expression profiles of total mRNA to those of poly(A)+ mRNA in wild-type cells and in cells defective in distinct steps of the 5' to 3' and 3' to 5' mRNA decay pathways.