METABOLISM OF YEAST mRNAS THAT LACK A STOP CODON

描述（由申请人提供）：mRNA降解是基因表达的一个重要方面。现在很清楚，相同的酶降解稳定和不稳定的mRNA。因此，理解差异mRNA降解的关键是理解特定mRNA与基础机制的相互作用。这里提出的实验旨在从分子细节上了解一种特定的mRNA如何与mRNA衰变机制相互作用，以及这如何导致其快速降解。这个建议集中在酵母mRNA的非常快速的降解，缺乏一个终止密码子（“不停止衰变”）的四个原因。首先，不间断的mRNA是酵母中最不稳定的mRNA。第二，不间断mRNA的降解是基因表达的重要质量控制方面。第三，不间断衰减的机制对于确保mRNA完全降解可能很重要。第四，不停的酵母mRNA被3'核酸外切酶（外泌体）的复合物降解。外泌体在酵母和哺乳动物之间是保守的，并且具有许多功能。这些功能可能包括重要哺乳动物mRNA的衰变。了解酵母中的不间断衰变应该会增加我们对其他外泌体功能的理解。在目前的mRNA不间断降解模型中，当核糖体到达其3'末端时，mRNA被认为是异常的。该核糖体通过核糖体A位点被Ski7p识别，这导致外泌体的募集。这项建议旨在测试和扩大这一模式。目的1是识别识别和降解不间断mRNA的细胞机制的所有部分。目的2是表征这些部分在体内的作用。初步结果表明，蛋白酶体可以降解由不间断mRNA编码的蛋白质。第三步是检验这个假设。第四个目的是详细描述Ski7p的功能，Ski7p是基础mRNA衰减机制的不间断mRNA识别和募集的关键蛋白。这些目标应该导致在分子细节的识别和不间断的mRNA的衰变的理解。由于正常的mRNA被相同的酶降解，这些实验也应该增加我们对正常细胞mRNA降解的理解。

DESCRIPTION (provided by applicant): mRNA degradation is an important aspect of gene expression. It is now clear that the same enzymes degrade both stable and unstable mRNAs. Thus, the key to understanding differential mRNA degradation is to understand the interactions of a particular mRNA with the basal machinery. The experiments proposed here are aimed at understanding in molecular detail how one particular mRNA interacts with the mRNA decay machinery and how this causes its rapid degradation. This proposal is focused on the extremely rapid degradation of yeast mRNAs that lack a stop codon ("nonstop decay") for four reasons. First, nonstop mRNAs are the least stable mRNAs in yeast. Second, degradation of nonstop mRNAs is an important quality control aspect of gene expression. Third, the mechanism of nonstop decay is likely important to assure that mRNAs are completely degraded. Fourth, nonstop yeast mRNAs are degraded by a complex of 3' exonucleases (the exosome). The exosome is conserved between yeast and mammals and has many functions. These functions may include the decay of important mammalian mRNAs. Understanding nonstop decay in yeast should increase our understanding of other exosome functions. In the current model for nonstop mRNA degradation an mRNA is recognized as aberrant when a ribosome reaches its 3' end. This ribosome is recognized by Ski7p through the ribosomal A-site, which results in recruitment of the exosome. This proposal is aimed at testing and expanding this model. Aim 1 is to identify all parts of the cellular machinery for recognition and decay of nonstop mRNAs. Aim 2 is to characterize the role of these parts in vivo. Preliminary results suggest that the proteasome may degrade proteins encoded by nonstop mRNAs. Aim 3 is to test this hypothesis. The fourth aim is to characterize the function of Ski7p in detail, which is the key protein in nonstop mRNA recognition and recruitment of the basal mRNA decay machinery. These aims should result in an understanding of the recognition and decay of nonstop mRNAs in molecular detail. Since normal mRNAs are degraded by the same enzymes, these experiments should also increase our understanding of the degradation of normal cellular mRNAs.