Yeast PUF3 control of mRNA expression

酵母 PUF3 控制 mRNA 表达

• 批准号: 7128339
• 负责人: CLYDE L DENIS
• 金额: $ 21.75万
• 依托单位: UNIVERSITY OF NEW HAMPSHIRE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7128339
• 关键词: RNA binding protein; Saccharomyces cerevisiae; binding sites; fungal genetics; fungal proteins; gene deletion mutation; genetic regulation; genetic translation; messenger RNA; posttranscriptional RNA processing; protein protein interaction; protein structure function; recombinant DNA; reporter genes; ribonucleoproteins; transcription factor

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.

描述（由申请人提供）：这项研究的重点是表征酵母菌蛋白在控制mRNA表达时的分子作用机理。 PUF蛋白是包含由6-8 pumilio重复序列组成的RNA结合基序的蛋白质家族的成员。酵母和较高的真核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。尽管已经提出了PUF作用的其他部位，但已证明对mRNA降苯甲基化过程的PUF控制已被证明是这两种效应的共同要求。在酵母中，每种PUF蛋白可以结合相对不同的mRNA，每组mRNA编码的蛋白质在功能上相关。这些观察结果表明，PUF在控制细胞中蛋白质集的表达中起重要的转录后作用。我们已经确定了PAB1-MRNP结构的几个特征，这些特征影响了苯基化过程，这些特征可能是PUF蛋白可以通过的位点。我们已经表明，酵母菌可以结合CCR4-not dearenylase复合物，这表明该复合物的PUF3保留可以帮助体内加速deadenylation。我们还观察到，PUF可以结合翻译起始因子，支持PUFS对翻译起始的作用，这是PUF在该提案中影响mRNA降解的一种手段，我们将在MRNA Deadenylation方面检验几种关于酵母菌PUFS蛋白作用机制的假设。使用由PUF控制的模型mRNA（COX17），我们将通过改变涉及翻译起始复合物，翻译终止因子和PAB1的MRNP结构的几种不同特征来确定PUFS蛋白是否通过改变MRNP结构的几种不同特征来加速deadenylation。此外，我们将测试通过募集CCR4-not dearenylase到mRNA的CCR4，该模型可以加速降苯甲基化。基于酵母菌的大蛋白质尺寸，它们可能会产生多种蛋白质接触并通过各种方法作用，以确保对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。本科生将参与该研究项目的几个方面，包括突变PUF和翻译起始因素，以便本地化相互联系的领域。通过研究如何控制蛋白质表达，该提案与公共卫生有关。调节何时及在多大程度上合成蛋白质的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。