Yeast PUF3 control of mRNA expression

描述（申请人提供）：本研究的重点是表征酵母PUF蛋白控制mRNA表达的分子机制。PUF蛋白是一个包含6-8个短片段重复序列的RNA结合基序的蛋白家族的成员。酵母和更高的真核puf与mRNA 3' UTR序列的RNA结合已被证明对翻译抑制和加速mRNA降解很重要。PUF对mRNA死烯化过程的控制已被证明是这两种作用的共同要求，尽管PUF作用的其他位点已被提出。在酵母中，每个PUF蛋白可以结合相对不同的mrna，并且每组mrna编码的蛋白在功能上是相关的。这些观察结果表明，puf在控制细胞中一系列蛋白质的表达方面发挥着重要的转录后作用。我们已经确定了影响死基化过程的PAB1- mRNP结构的几个特征，这些特征可能是PUF蛋白起作用的位点。我们已经证明酵母PUFS可以结合CCR4-NOT deadenylase复合物，这表明PUF3的保留可以帮助体内加速deadenylase。我们还观察到PUFS可以结合翻译起始因子，支持PUFS调控翻译起始的作用，作为PUFS影响mRNA降解的一种手段。在本提案中，我们将测试关于酵母PUFS蛋白在mRNA死烯化方面的作用机制的几个假设。利用PUFS （COX17）控制的模型mRNA，我们将确定PUFS蛋白是否通过改变包括翻译起始复合体、翻译终止因子和PAB1在内的mRNP结构的几个不同特征来加速死蛋白化。此外，我们将测试PUFS通过将CCR4-NOT死烯化酶募集到mRNA上来加速死烯化的模型。基于酵母PUFs的大蛋白尺寸，它们很可能通过多种蛋白质接触并通过各种手段来确保RNA表达的适当调节。这些实验将利用生化、遗传和重组DNA技术。本科生将从事本研究项目的几个方面，包括突变PUFS和翻译起始因子，以定位用于相互联系的域。这一建议通过研究如何控制蛋白质表达与公众健康相关。调节蛋白质合成的时间和程度的因素的特征将阐明异常蛋白质产生导致特定疾病状态的过程。

DESCRIPTION (provided by applicant): The focus of this research is to characterize the molecular mechanism of action of the yeast PUF proteins in the control of mRNA expression. The PUF proteins are members of a family of proteins containing an RNA binding motif consisting of 6-8 pumilio repeats. RNA binding by yeast and higher eukaryotic PUFs to mRNA 3' UTR sequences has been shown to be important to translational repression and accelerated mRNA degradation. PUF control of the mRNA deadenylation process has been shown to be the common requirement for both of these effects, although additional sites of PUF action have been suggested. In yeast each PUF protein can bind relatively distinct mRNAs, and the proteins encoded by each group of mRNAs are functionally related. These observations suggest that the PUFs play important post-transcriptional roles in controlling the expression of sets of proteins in the cell. We have identified several features of the PAB1- mRNP structure which affect the deadenylation process and these features may be sites through which PUF proteins act. We have shown that yeast PUFS can bind the CCR4-NOT deadenylase complex, suggesting that PUF3 retention of this complex can help accelerate deadenylation in vivo. We have also observed that PUFS can bind translation initiation factors, supporting a role of PUFS regulation of translation initiation as a means by which PUFS affects mRNA degradation In this proposal we will test several hypotheses concerning the mechanism of action of the yeast PUFS protein in terms of mRNA deadenylation. Using model mRNA controlled by PUFS (COX17), we will determine if the PUFS protein accelerates deadenylation by altering several different features of the mRNP structure involving the translation initiation complex, the translation termination factors, and PAB1. In addition, we will test the model that PUFS accelerates deadenylation by recruiting the CCR4-NOT deadenylase to the mRNA. Based on the large protein sizes of yeast PUFs, it is likely that they make multiple protein contacts and act through various means to ensure proper regulation of RNA expression. These experiments will utilize biochemical, genetic, and recombinant DNA techniques. Undergraduate students will be engaged in several aspects of this research project including that of mutating PUFS and translation initiation factors in order to localize the domains used in contacting each other. This proposal is relevant to public health by studying how protein expression is controlled. The characterization of the factors that regulate when and to what extent proteins are synthesized will illuminate the processes by which aberrant protein production leads to particular disease states.