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，同样的酶可以降解稳定的和不稳定的mrna。因此，了解差异mRNA降解的关键是了解特定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理解一个特定的mRNA如何与mRNA衰变机制相互作用，以及这是如何导致其快速降解的。这一建议的重点是酵母mrna的极快降解，缺乏停止密码子（“不间断衰变”），原因有四个。首先，不间断mrna是酵母中最不稳定的mrna。其次，不间断mrna的降解是基因表达质量控制的一个重要方面。第三，不间断衰变的机制可能对确保mrna完全降解很重要。第四，不间断酵母mrna被3'外切酶复合物（外泌体）降解。外泌体在酵母和哺乳动物之间是保守的，具有许多功能。这些功能可能包括重要的哺乳动物mrna的衰变。了解酵母的不间断衰变应该增加我们对其他外泌体功能的理解。在目前的不间断mRNA降解模型中，当核糖体到达其3'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，从而导致外泌体的招募。本提案旨在测试和扩展该模型。目的1是鉴定细胞机制的所有部分，以识别和降解不间断mrna。目的2是描述这些部分在体内的作用。初步结果表明，蛋白酶体可能降解由不间断mrna编码的蛋白质。目的3是检验这一假设。第四个目标是详细描述Ski7p的功能，Ski7p是不间断mRNA识别和基础mRNA衰变机制募集的关键蛋白。这些目标应该导致在分子细节上对不间断mrna的识别和衰变的理解。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.