Yeast PUF3 control of mRNA expression

酵母 PUF3 控制 mRNA 表达

• 批准号: 7850034
• 负责人: CLYDE L DENIS
• 金额: $ 0.46万
• 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/7850034
• 关键词: Affect; Applications Grants; Binding; Biochemical Genetics; Cell Nucleus; Cells; Chromatin; Complex; Consensus Sequence; Cytoplasm; Development; Disease; Ensure; Eukaryota; Excision; Genetic Transcription; In Vitro; Mediating; Messenger RNA; Mitochondrial Proteins; Modeling; Molecular Mechanisms of Action; Mutate; Pattern; Peptide Initiation Factors; Play; Poly A; Process; Production; Protein Family; Proteins; Public Health; RNA; RNA Binding; Recombinant DNA; Recruitment Activity; Regulation; Research; Research Project Grants; Role; Saccharomyces cerevisiae; Set protein; Site; Structure; Students; Techniques; Testing; Translation Initiation; Translational Repression; Translations; Untranslated Regions; Yeasts; base; in vivo; mRNA Decay; mRNA Expression; mRNA Transcript Degradation; member; messenger ribonucleoprotein; programs; protein expression; research study; spindle pole body; termination factor; yeast protein

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）：本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 蛋白是含有由 6-8 个 pumilio 重复组成的 RNA 结合基序的蛋白家族的成员。酵母和高等真核 PUF 与 mRNA 3' UTR 序列的 RNA 结合已被证明对于翻译抑制和加速 mRNA 降解非常重要。尽管已经提出了 PUF 作用的其他位点，但 PUF 对 mRNA 去腺苷化过程的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，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 在控制细胞中蛋白质组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 结构的几个影响去腺苷化过程的特征，这些特征可能是 PUF 蛋白发挥作用的位点。我们已经证明酵母 PUFS 可以结合 CCR4-NOT 去腺苷酶复合物，这表明 PUF3 保留该复合物有助于加速体内去腺苷化。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。在本提案中，我们将测试有关酵母 PUFS 蛋白作用机制的几个假设mRNA 去腺苷酸化。使用 PUFS (COX17) 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 PAB1 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 CCR4-NOT 去腺苷酸招募到 mRNA 来加速去腺苷化的模型。由于酵母 PUF 的蛋白质尺寸较大，它们很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 表达的正确调节。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多个方面，包括突变 PUFS 和翻译起始因子，以便本地化用于相互联系的域。该提案通过研究蛋白质表达的控制方式与公共健康相关。调节蛋白质合成时间和程度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。

项目成果

CAF1 plays an important role in mRNA deadenylation separate from its contact to CCR4.

• DOI: 10.1093/nar/gkm196
• 发表时间: 2007
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Ohn T;Chiang YC;Lee DJ;Yao G;Zhang C;Denis CL
• 通讯作者: Denis CL

Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein.

• DOI: 10.1007/s00438-012-0709-5
• 发表时间: 2012-09
• 期刊: Molecular genetics and genomics : MGG
• 作者: Richardson R;Denis CL;Zhang C;Nielsen MEO;Chiang YC;Kierkegaard M;Wang X;Lee DJ;Andersen JS;Yao G
• 通讯作者: Yao G

The RRM1 domain of the poly(A)-binding protein from Saccharomyces cerevisiae is critical to control of mRNA deadenylation.

• DOI: 10.1007/s00438-013-0759-3
• 发表时间: 2013-09
• 期刊: Molecular genetics and genomics : MGG
• 作者: Zhang C;Lee DJ;Chiang YC;Richardson R;Park S;Wang X;Laue TM;Denis CL
• 通讯作者: Denis CL