The coupling of mRNA transcription and 3' end formation

描述(申请人提供)：在过去的十年中，人们已经清楚地看到，前mRNA的加工，包括5‘端封端、剪接、3’端多聚腺苷基化，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控转录在很大程度上是未知的。这一建议的前提是转录和加工因子的招募和交换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来协调，CTD是最大的RNAP II亚基上存在的一种独特结构。我们将集中讨论CPF加工因子的组成部分Ssu72 CTD Ser5-P磷酸酶在转录中的作用以及3‘端加工因子如何影响转录终止。这项研究涉及三个目标，第一个目标是确定Ssu72在转录周期中的作用位置，并确定与RNAP II相关的特定因素是否受Ser5-P去磷酸化的影响。染色质免疫沉淀(CHIP)将用于评估Ssu72如何影响RNAP II、转录因子和处理因子在体内转录基因中的分布。我们将使用体外转录系统来测试Ssu72是否促进了起始-延伸转换。我们的第二个目标是了解Ssu72是如何受到RNAP II转录机制和3‘端加工因子的调控的，这在拟议研究的两个目标中都有涉及。我们将确定与Ssu72基因相互作用的转录因子是否影响CTD Ser5-P水平和RNAP II Ser5-P在基因之间的分布。酵母遗传方法的强大组合将被用来识别与Ssu72相互作用的新因素。我们还将检验这一假设，即Ssu72与CPF以及3‘端加工复合体与CTD的相互作用对于将Ssu72活性定向到磷酸化的CTD是重要的。我们的第三个目标是确定切割/多聚腺苷酸化机制在Poly(A)依赖的终止中的作用。对于导致终止的分子事件有很大的争议，包括加工因子是否必须与RNAP II接触，新生转录本的切割是否重要，以及是否需要在切割位点下游降解转录本。我们将使用芯片分析，一种改进的转录运行分析，以及一种允许检查致命突变的策略来解决这些问题。拟议的实验建立在我们利用生物化学和遗传学成功研究转录和信使核糖核酸3‘末端处理的悠久历史基础上。我们预计，这些新的研究将促进我们对转录周期动态的洞察，并识别和澄清该周期早期和后期的关键检查点。此外，RNAP II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，我们预计从我们提议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.