Molecular mechanism of mRNA 3'-end formation in yeast

酵母中mRNA 3端形成的分子机制

• 批准号: 7988814
• 负责人: CLAIRE L MOORE
• 金额: $ 12.14万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-17 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7988814
• 关键词: Address; Animal Model; Binding Proteins; Biochemical; Cell Nucleus; Cells; Chromosome Segregation; Cleaved cell; Color; Commit; Complex; Coupled; Cytoplasm; DNA Repair; DNA Sequence Rearrangement; Eukaryota; Event; Figs - dietary; Gene Expression; Genes; Genetic Transcription; Genome; Hand; Length; Link; Messenger RNA; Modification; Molecular; Mutation; Nature; Oranges; Paper; Phosphorylation; Poly A; Poly(A) Tail; Poly(A)-Binding Proteins; Polyadenylation; Polymerase; Polynucleotide Adenylyltransferase; Positioning Attribute; Process; Progress Reports; Proteins; RNA; Regulation; Research; Role; Saccharomyces cerevisiae; Scaffolding Protein; Site; Strigiformes; System; Tail; Tissues; Touch sensation; Transcript; Translating; Work; Yeasts; insight; mRNA Cleavage and Polyadenylation Factors; mRNA Export; mRNA Precursor; mutant; polyadenylated messenger RNA; protein expression

DESCRIPTION (provided by applicant): Project summary: Synthesis of mRNA in eukaryotes, and its utilization in the cytoplasm, requires modification at the RNA's 3' end by addition of a poly(A) tail. This process also serves as a point at which the cell can regulate the type and amount of mRNA derived from a particular gene. Even though mRNA 3' end formation occurs in an unexpectedly large complex, most, if not all, of the subunits of this machinery have been identified in the yeast S. cerevisiae. However, little is known about how these 21 proteins cooperate with each other to insure processing that is accurate as well as coupled in a timely fashion to other events in mRNA synthesis and packaging. With the 3'-end processing components in hand, and activities for several of these factors newly defined, a unique opportunity now exists to rigorously address the mechanism by which this essential and universal step in gene expression occurs and is regulated. The central hypothesis of this proposal is that definable rearrangements of protein partners occur within the complex as it evaluates the authenticity of the processing site, commits to cleavage at the poly(A) site, reorganizes to position the poly(A) polymerase for tail synthesis, and releases the final RNA product. The objective is to understand how such reorganizations drive the cycle of mRNA 3' end processing. The research focuses on four events that are likely to be critical transition points in this cycle yet whose underlying mechanisms are not understood. These include the initiation of cleavage and the role of the Ssuy2 protein in this step, the transition from cleavage to poly(A) addition and its regulation by phosphorylation of the Ptai scaffold protein, the control of poly(A) polymerase activity by interactions at its amino-terminus, and the release of processing factors following tail synthesis. The studies will use yeast as the model organism because of the ease of introducing tags and mutations into the genome, the availability of numerous 3' end processing mutants, the ease of purifying processing factors for biochemical studies, and the high degree of conservation of the 3' end processing machinery across eukaryotes. Relevance: Without poly(A) tails, mRNA is targeted for degradation in the nucleus, it does not get out of the nucleus very well, and it is not translated efficiently or turned over at the appropriate rate in the cytoplasm. Maturation of mRNA 3' ends is functionally linked to other essential processes such as transcription, mRNA export, chromosome segregation, DNA repair, and tissue-specific protein expression. Mistakes in polyadenylation can impact on all of these processes. The proposed research should significantly advance our insight into the dynamics of this essential step in mRNA synthesis and identify points at which the constitutive process is likely to be regulated.

描述（由申请人提供）： 项目摘要：真核生物中 mRNA 的合成及其在细胞质中的利用，需要通过添加聚 (A) 尾在 RNA 的 3' 末端进行修饰。这个过程也是细胞调节源自特定基因的 mRNA 类型和数量的一个点。尽管 mRNA 3' 末端的形成发生在一个意想不到的大复合体中，但该机制的大多数（如果不是全部）亚基已在酿酒酵母中得到鉴定。然而，人们对这 21 种蛋白质如何相互协作以确保加工准确以及及时与 mRNA 合成和包装中的其他事件耦合的情况知之甚少。有了 3' 端加工组件，以及新定义的其中几个因子的活性，现在存在一个独特的机会来严格解决基因表达中这一重要且普遍的步骤发生和调节的机制。该提案的中心假设是，当复合物评估加工位点的真实性、致力于在 Poly(A) 位点进行切割、重组以定位 Poly(A) 聚合酶以进行尾部合成并释放最终 RNA 产物时，复合物内会发生可定义的蛋白质伴侣重排。目的是了解此类重组如何驱动 mRNA 3' 末端加工循环。该研究重点关注可能成为这一周期中关键转变点的四个事件，但其潜在机制尚不清楚。这些包括切割的起始和 Ssuy2 蛋白在此步骤中的作用、从切割到 Poly(A) 添加的转变及其通过 Ptai 支架蛋白磷酸化的调节、通过其氨基末端的相互作用控制 Poly(A) 聚合酶活性，以及​​尾部合成后加工因子的释放。这些研究将使用酵母作为模型生物，因为在基因组中引入标签和突变很容易，大量3'端加工突变体的可用性，生化研究中纯化加工因子的容易性，以及真核生物中3'端加工机制的高度保守性。相关性：如果没有 Poly(A) 尾，mRNA 就会在细胞核中被靶向降解，它不能很好地脱离细胞核，并且不能有效翻译或在细胞质中以适当的速率周转。 mRNA 3' 末端的成熟在功能上与其他重要过程相关，例如转录、mRNA 输出、染色体分离、DNA 修复和组织特异性蛋白质表达。聚腺苷酸化中的错误可能会影响所有这些过程。拟议的研究将显着推进我们对 mRNA 合成这一重要步骤的动态的了解，并确定组成过程可能受到调节的点。

项目成果

The role of the Brr5/Ysh1 C-terminal domain and its homolog Syc1 in mRNA 3'-end processing in Saccharomyces cerevisiae.

Brr5/Ysh1 C 末端结构域及其同源 Syc1 在酿酒酵母 mRNA 3 末端加工中的作用。

• DOI: 10.1261/rna.2267606
• 发表时间: 2006
• 期刊: RNA (New York, N.Y.)
• 作者: Zhelkovsky,Alexander;Tacahashi,Yoko;Nasser,Tommy;He,Xiaoyuan;Sterzer,Ulrike;Jensen,TorbenHeick;Domdey,Horst;Moore,Claire
• 通讯作者: Moore,Claire

DNA damage induces targeted, genome-wide variation of poly(A) sites in budding yeast.

• DOI: 10.1101/gr.144964.112
• 发表时间: 2013-10
• 期刊: Genome research
• 影响因子: 7
• 作者: Graber JH;Nazeer FI;Yeh PC;Kuehner JN;Borikar S;Hoskinson D;Moore CL
• 通讯作者: Moore CL

Structure-function relationships in the Saccharomyces cerevisiae poly(A) polymerase. Identification of a novel RNA binding site and a domain that interacts with specificity factor(s).

酿酒酵母聚腺苷酸聚合酶的结构-功能关系。

• DOI: 10.1074/jbc.270.44.26715
• 发表时间: 1995
• 期刊: The Journal of biological chemistry
• 作者: Zhelkovsky,AM;Kessler,MM;Moore,CL
• 通讯作者: Moore,CL

Cleavage factor II of Saccharomyces cerevisiae contains homologues to subunits of the mammalian Cleavage/ polyadenylation specificity factor and exhibits sequence-specific, ATP-dependent interaction with precursor RNA.

酿酒酵母的裂解因子 II 含有与哺乳动物裂解/聚腺苷酸化特异性因子亚基的同源物，并与前体 RNA 表现出序列特异性、ATP 依赖性相互作用。

• DOI: 10.1074/jbc.272.16.10831
• 发表时间: 1997
• 期刊: The Journal of biological chemistry
• 作者: Zhao,J;Kessler,MM;Moore,CL
• 通讯作者: Moore,CL

Transcription termination downstream of the Saccharomyces cerevisiae FBP1 [changed from FPB1] poly(A) site does not depend on efficient 3'end processing.

酿酒酵母 FBP1 [从 FPB1 更改] Poly(A) 位点下游的转录终止不依赖于有效的 3 端加工。

• 发表时间: 1998
• 期刊: RNA (New York, N.Y.)
• 作者: Aranda,A;Pérez-Ortín,JE;Moore,C;delOlmo,ML
• 通讯作者: delOlmo,ML