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(申请人提供)：这项研究的重点是表征酵母PUF蛋白在控制mRNA表达中的分子作用机制。PUF蛋白是含有6-8个Pumilio重复序列的RNA结合基序的蛋白质家族的成员。酵母和更高真核生物的PUF与mRNA3‘非编码区序列的结合对翻译抑制和加速mRNA降解是重要的。PUF对mRNA去烯化过程的控制已被证明是这两种作用的共同要求，尽管已经提出了PUF作用的其他部位。在酵母中，每个PUF蛋白可以结合相对不同的mRNAs，每组mRNAs编码的蛋白质在功能上是相关的。这些观察表明，PUF在控制细胞中一组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PAB1-mRNP结构的几个影响去烯化过程的特征，这些特征可能是PUF蛋白发挥作用的部位。我们已经证明酵母PUFS可以结合CCR4-非死烯基酶复合体，这表明PUF3保留该复合体可以帮助加速体内的死烯基化。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降解的一种方式。在这个建议中，我们将从mRNA去烯化的角度检验几种关于酵母PUFS蛋白作用机制的假说。利用PUFS(COX17)控制的模型mRNA，我们将确定PUFS蛋白是否通过改变mRNP结构的几个不同特征来加速死烯基化，这些特征涉及翻译起始复合体、翻译终止因子和PAB1。此外，我们还将测试PUFS通过将CCR4-非死烯基酶重新招募到mRNA来加速死烯基化的模型。基于酵母PUF的蛋白质大小，它们很可能通过多个蛋白质接触并通过各种方式发挥作用，以确保适当地调节RNA的表达。这些实验将利用生化、遗传和重组DNA技术。本科生将参与这一研究项目的几个方面，包括突变的PUF和翻译启动因素，以定位相互联系所使用的领域。这项建议通过研究蛋白质表达是如何控制的，与公众健康相关。对调节蛋白质合成的时间和程度的因素的表征将阐明蛋白质产生异常导致特定疾病状态的过程。