Cross-regulation between transcription and pre-mRNA splicing

摘要/项目摘要 细胞RNA在序列、结构和功能上与其前体RNA不同。的巨大 前mRNA加工的调节能力受转录的影响，因为RNA加工 机器共转录地起作用，并且可以直接接触RNA聚合酶II（Pol II）和/或染色质。在 反过来，前体mRNA加工中的每一步- 5'端加帽，剪接和3'端切割-都与 Pol II行为的变化，例如暂停。这项资助的重点是转录， 在酿酒酵母和粟酒裂殖酵母中的剪接和3'末端切割。我们的发现在 在过去的3年里，我们重新定义了我们如何看待mRNA这三个方面之间的相互作用， 成熟我们开发了两种互补的单分子RNA-seq方法，可以直接测量 剪接反应的进展作为在全球范围内延长Pol II的位置的函数， 两个重大发现 (1)我们已经表明，剪接体的运作时间比以前认识到的要快得多， 在它行动的时候接近波尔二号剪接和转录之间的短暂滞后引发了一些有趣的问题， 剪接如何与转录、mRNP成熟和基因末端的3'末端切割协调。在Aim中 1，我们测试了关于剪接体组装对RNA序列，RNA结构， 以及反式作用因子的作用。在目标2中，我们研究了一个新的假设-去除后- 从新生mRNA中催化剪接体，我们称之为剪接体驱逐，是促进mRNP所必需的 成熟的网站Pol II暂停-和实验评估这个想法对其他模型。 (2)我们的方法之一，长读段测序，确定从5'到3'端的新生RNA的全序列 (Pol II位置），使我们能够跟踪剪接丰富的多内含子转录在S。粟酒和 在“真实的时间”中确定共转录内含子去除的顺序。值得注意的是，大多数新生的转录本 以“全部或全部没有”的方式拼接，使得超过一半被快速且完全地拼接。与此相反， 18%的新生转录物完全未剪接，未能进行3'端切割，并被降解。 核外泌体这些“死端”转录本显示转录通读，这是最近被 与细胞对压力、癌症和病毒感染的反应有关。在目标3中，我们建议确定 关于剪接、3'末端切割和降解，定义转录物命运的分子机制。 我们的新目标的影响将是定义内含子功能的库，这些功能决定了基因的动力学。 体内剪接体组装;鉴定转录和mRNP成熟中的协调转换;和 发现转录，剪接和3'端切割是如何连接成功或失败的背景下， 正常生长和细胞压力。

Abstract/Project Summary Cellular RNAs differ in sequence, structure, and function from their precursor RNAs. The enormous regulatory power of pre-mRNA processing is influenced by transcription, because the RNA processing machinery acts co-transcriptionally and can contact RNA polymerase II (Pol II) and/or chromatin directly. In turn, each step in pre-mRNA processing – 5' end capping, splicing, and 3' end cleavage – is associated with changes in Pol II behavior, such as pausing. This grant focuses on the coordination between transcription, splicing, and 3' end cleavage in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Our findings during the past 3 years have redefined how we think about the cross-talk between these three aspects of mRNA maturation. We developed two complementary single molecule RNA-seq methods that directly measure the progression of the splicing reaction as a function of the position of elongating Pol II on a global scale, resulting in two major discoveries: (1) We have shown that the spliceosome operates on a much faster time scale than previously appreciated and is close to Pol II when it acts. The short lag between splicing and transcription raises intriguing questions about how splicing is coordinated with transcription, mRNP maturation, and 3' end cleavage at gene termini. In Aim 1, we test models regarding the timing of spliceosome assembly in response to RNA sequence, RNA structure, and the action of trans-acting factors. In Aim 2, we investigate a novel hypothesis – that removal of the post- catalytic spliceosome from nascent mRNA, which we call spliceosome eviction, is necessary to promote mRNP maturation at sites of Pol II pausing – and experimentally evaluate this idea against other models. (2) One of our methods, long read sequencing, determines the full sequence of nascent RNA from 5' to 3' end (Pol II position), enabling us to track the splicing of abundant multi-intron transcripts in S. pombe and determine the order of co-transcriptional intron removal in “real time”. Remarkably, most nascent transcripts were spliced in an “all or none” fashion, such that more than half were rapidly and fully spliced. In contrast, 18% of nascent transcripts were totally unspliced, failed to undergo 3' end cleavage, and were degraded by the nuclear exosome. These “dead-end” transcripts display transcriptional readthrough, which has recently been implicated in cellular responses to stress, cancer, and viral infection. In Aim 3 we propose to identify the molecular mechanisms that define transcript fate with regard to splicing, 3' end cleavage, and degradation. The impact of our new aims will be to define the repertoire of intron features that determine the kinetics of spliceosome assembly in vivo; identify coordinated transitions in transcription and mRNP maturation; and discover how transcription, splicing and 3' end cleavage are linked for success or failure in the context of normal growth and cellular stress.