Yeast PUF3 control of mRNA expression

描述（由申请方提供）：本研究的重点是表征酵母PUF蛋白在mRNA表达控制中的分子作用机制。PUF蛋白是含有由6-8个pumilio重复组成的RNA结合基序的蛋白质家族的成员。酵母和高等真核生物PUF与mRNA 3' UTR序列的RNA结合已显示对翻译抑制和加速mRNA降解是重要的。已证明，PUF对mRNA去腺苷化过程的控制是这两种效应的共同要求，尽管已经提出了其他的PUF作用位点。在酵母中，每种PUF蛋白可以结合相对不同的mRNA，并且由每组mRNA编码的蛋白质在功能上是相关的。这些观察结果表明，PUFs在控制细胞中蛋白质组的表达中发挥重要的转录后作用。我们已经确定了PAB 1- mRNP结构的几个特征，这些特征影响去腺苷化过程，这些特征可能是PUF蛋白作用的位点。我们已经表明，酵母PUFS可以结合CCR 4-NOT去腺苷酶复合物，表明PUF 3保留该复合物可以帮助加速体内去腺苷化。我们还观察到，PUFS可以结合翻译起始因子，支持PUFS调节翻译起始的作用，作为PUFS影响mRNA降解的一种手段。在该提案中，我们将测试几种假设，关于酵母PUFS蛋白在mRNA去腺苷化方面的作用机制。使用由PUFS（COX 17）控制的模型mRNA，我们将确定PUFS蛋白是否通过改变涉及翻译起始复合物、翻译终止因子和PAB 1的mRNP结构的几个不同特征来加速去腺苷化。此外，我们将测试PUFS通过将CCR 4-NOT去腺苷酶募集到mRNA中来加速去腺苷化的模型。基于酵母PUFs的大蛋白质尺寸，它们可能会进行多种蛋白质接触，并通过各种方式发挥作用，以确保RNA表达的适当调节。这些实验将利用生物化学、遗传学和重组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。这项建议通过研究蛋白质表达是如何控制的，与公共卫生有关。对蛋白质合成时间和程度的调控因素的表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.