The coupling of mRNA transcription and 3' end formation

描述（由申请人提供）：在过去的十年中，已经很清楚，前mrna的加工，包括5‘盖帽，剪接，3’端聚腺苷酸化，甚至核输出，都与RNA聚合酶II （RNAP II）的转录相结合。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在很大程度上是未知的。该建议的前提是转录和加工因子的募集和交换是通过羧基末端重复结构域（CTD）的Ser2和Ser5的磷酸化和去磷酸化与转录周期协调的，CTD是RNAP II最大亚基上的一个独特结构。我们将重点关注Ssu72 CTD Ser5-P磷酸酶（CPF加工因子的一个组成部分）在转录中的作用以及3'端加工因子如何影响转录终止。该研究有三个目标：第一个目标是确定Ssu72在转录周期中起作用的位置，并确定与RNAP II相关的特定因子受到Ser5-P去磷酸化的影响。染色质免疫沉淀（ChIP）将用于评估Ssu72在体内如何影响RNAP II、转录因子和加工因子在转录基因中的分布。我们将使用体外转录系统来测试Ssu72是否促进起始-延伸过渡。我们的第二个目标是了解Ssu72如何受到RNAP II转录机制和3'端加工因子的调节，这在拟议研究的两个目标中得到了解决。我们将确定与Ssu72基因相互作用的转录因子是否会影响CTD Ser5-P水平以及RNAP II Ser5-P在基因间的分布。酵母遗传方法的强大组合将用于识别与Ssu72相互作用的新因子。我们还将验证Ssu72与CPF的相互作用，以及3'端加工复合物与CTD的相互作用，对于指导Ssu72活性对磷酸化CTD的作用是重要的。我们的第三个目标是确定切割/聚腺苷酸化机制在聚(A)依赖性终止中的作用。关于导致终止的分子事件有很多争论，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，以及是否需要在切割位点的下游降解转录物。我们将使用ChIP分析，一种改良的转录运行试验，以及一种允许检查致命突变的策略来解决这些问题。提出的实验建立在我们使用生物化学和遗传学成功研究转录和mRNA 3'端加工的悠久历史之上。我们预计这些新研究将推进我们对转录周期动力学的了解，并确定和阐明该周期早期和晚期的关键检查点。此外，RNAP II的转录和RNA加工是高度保守的过程，由系统发育相似的蛋白质协调。因此，我们期望从我们提出的研究中获得的信息将直接适用于人类生物学和医学。

DESCRIPTION (provided by applicant): 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 specific factors whose association with 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 conserved processes, orchestrated by phylogenetically similar proteins. Consequently, we expect that information gained from our proposed studies will be directly applicable to human biology and medicine.