The coupling of mRNA transcription and 3' end formation

mRNA转录和3末端形成的耦合

• 批准号: 7534920
• 负责人: CLAIRE L MOORE
• 金额: $ 4.56万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7534920
• 关键词: Address; Affect; Attention; Back; Biochemistry; Cell Survival; Complex; Coupled; Coupling; Event; Funding; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Heterogeneous Nuclear RNA; Human; Human Biology; In Vitro; Lead; Link; Mediating; Medicine; Messenger RNA; Methods; Molecular; Mutation; Nuclear Export; Numbers; Organism; Peptide Signal Sequences; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Poly A; Polyadenylation; Process; Protein Dephosphorylation; Proteins; RNA Polymerase II; RNA Processing; RNA Splicing; RNA chemical synthesis; RNA polymerase II largest subunit; Recording of previous events; Recycling; Research; Research Personnel; Role; Run-On Assays; Site; Specificity; Staging; Structure; System; Terminal Repeat Sequences; Testing; Transcript; Transcription Factor 3; Transcription Process; Yeasts; chromatin immunoprecipitation; in vivo; insight; mRNA Precursor; melting; mutant; novel; promoter; research study; spleen exonuclease; transcription factor; transcription termination; yeast genetics

During the past decade, it has become clear that processing of pre-mRNA, including 5' capping, splicing, 3' end polyadenylation, and even nuclear export, is coupled with transcription by RNA polymerase II (RNAP II). However, the mechanisms linking transcription to processing and how processing regulates transcription are largely unknown. The premise of this proposal is that recruitment and exchange of transcription and processing factors is coordinated with the transcription cycle by phosphorylation and dephosphorylation of Ser2 and Ser5 of the carboxy-terminal repeat domain (CTD), a unique structure present on the largest RNAP II subunit. We will focus on the role of the Ssu72 CTD Ser5-P phosphatase, a component of the CPF processing factor, in trans- cription and how 3' end processing factors affect transcription termination. The research addresses 3 objectives The first objective is to determine where in the transcription cycle Ssu72 acts and to identify specificfactors whose associationwith RNAP II is affected by Ser5-P dephosphorylation. Chromatin immunoprecipitation (ChIP) will be used to evaluate how Ssu72 affects the distribution of RNAP II, transcription factors, and processing factors across transcribed genes in vivo. We will use an in vitro transcription system to test whether Ssu72 facilitates the initiation-elongation transition. Our second objective,which is addressed in two aims of the proposed research, is to understand how Ssu72 is regulated by the RNAP II transcription machinery and by 3' end processing factors. We will determine whether transcription factors that genetically interact with Ssu72 affect CTD Ser5-P levels and the distribution of RNAP II Ser5-P across genes. A powerful combination of yeast genetic methods will be used to identify novel factors that interact with Ssu72. We will also test the hypothesis that the interactions of Ssu72 with CPF, and of the 3' end processing complex with the CTD, are important to direct Ssu72 activity to the phosphorylated CTD. Our third objective is to determine the role of the cleavage/polyadenylation machinery in poly(A)-dependent termination. There is much debate over the molecular events that lead to termination, including whether the processing factors must contact RNAP II, whether cleavage of the nascent transcript is important, and whether degradation of the transcript downstream of the cleavage site is needed. We will use ChIP analysis, a modified transcription run-on assay, and a strategy that allows the examination of lethal mutations to address these issues. The proposed experiments build upon our long history of using biochemistry and genetics to successfully study transcription and mRNA 3' end processing. We anticipate that these new studies will advance our insight into the dynamics of the transcription cycle and identify and clarify critical checkpoints in the early and late phases of this cycle. Moreover, RNAP II transcription and RNA processing are highly conservedprocesses, orchestrated by phylogenetically similar proteins. Consequently, we expect that information gained from our proposed studies will be directly applicable to human biology and medicine.

在过去的十年中，已经清楚的是，前体mRNA的加工，包括5'加帽，剪接，3'端修饰， 末端多聚腺苷酸化，甚至核输出，与RNA聚合酶II（RNAP II）的转录偶联。 然而，将转录与加工联系起来的机制以及加工如何调节转录， 大部分未知。这个建议的前提是，招聘和交流的转录和处理 这些因子通过磷酸化和去磷酸化丝氨酸2和丝氨酸5与转录周期相协调， 羧基末端重复结构域（CTD），一种存在于最大RNAP II亚基上的独特结构。我们将 关注Ssu 72 CTD Ser 5-P磷酸酶（CPF加工因子的一种成分）在反式- 以及3'端加工因子如何影响转录终止。该研究涉及三个目标 第一个目标是确定Ssu 72在转录周期中的作用位置，并鉴定特异性因子 其与RNAP II的结合受到Ser 5-P去磷酸化的影响。染色质免疫沉淀 （ChIP）将用于评估Ssu 72如何影响RNAP II、转录因子和 体内转录基因的加工因子。我们将使用体外转录系统来测试 Ssu 72促进起始-伸长转变。我们的第二个目标是在《联合国宪章》的两个目标中提出的， 这项研究的目的是了解Ssu 72是如何被RNAP II转录机制和3'端调控的。 结束处理因素。我们将确定与Ssu 72基因相互作用的转录因子是否 影响CTD Ser 5-P水平和RNAP II Ser 5-P在基因间的分布。一种强大的酵母组合 遗传学方法将用于鉴定与Ssu 72相互作用的新因子。我们还将检验假设 Ssu 72与CPF的相互作用以及3'端加工复合物与CTD的相互作用对于 将Ssu 72活性直接作用于磷酸化的CTD。我们的第三个目标是确定 切割/多聚腺苷酸化机制中的poly（A）依赖性终止。关于这个问题存在很多争论 导致终止的分子事件，包括加工因子是否必须接触RNAP II， 新生转录物的切割是否重要，以及转录物下游的降解是否 需要切割位点。我们将使用ChIP分析，一种改良的转录运行分析，以及一种策略， 允许检测致命突变来解决这些问题。 拟议的实验建立在我们长期使用生物化学和遗传学成功地 研究转录和mRNA 3'末端加工。我们预计这些新的研究将促进我们的洞察力 转录周期的动态，并确定和澄清早期和晚期的关键检查点， 这个周期的各个阶段。此外，RNAP II转录和RNA加工是高度保守的过程， 由遗传相似的蛋白质组成。因此，我们希望从我们的 拟议的研究将直接适用于人类生物学和医学。