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。巨大的 前mRNA加工的调节能力受到转录的影响，因为RNA加工 机械参与共转录，可以直接接触RNA聚合酶II(POL II)和/或染色质。在……里面 反过来，mRNA前加工的每个步骤--5‘端封端、剪接和3’端切割--都与 Pol II行为的变化，如暂停。这笔赠款的重点是协调转录， 酿酒酵母和裂殖酵母的剪接和3‘端裂解。我们在调查期间的发现 过去3年重新定义了我们如何看待这三个方面的信使核糖核酸 成熟。我们发展了两种互补性的单分子rna-seq方法，可以直接测量 剪接反应作为延长POL II在全球范围内的位置的函数的进展，结果 在两个主要发现中： (1)我们已经证明，剪接体以比以前认识到的更快的时间尺度工作，并且 当它行动时接近波尔二号。剪接和转录之间的短暂延迟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‘端切割相协调。在AIM 1，我们测试了剪接体组装对RNA序列、RNA结构、 以及反式作用因子的作用。在目标2中，我们研究了一个新假设--去掉柱子-- 从新生的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。 在Pol II暂停的部位成熟--并对这一想法与其他模型进行实验评估。 (2)我们的方法之一，长读测序，确定了新生RNA从5‘端到3’端的全序列 (POL II位置)，使我们能够追踪丰富的多内含子在S.pombe和S.pombe中的剪接 “实时”确定共转录内含子移除的顺序。值得注意的是，大多数新生的成绩单 以“要么全有要么全不”的方式拼接，因此超过一半的片段被快速和完全拼接。相比之下， 18%的新生转录本完全没有剪接，没有经历3‘端切割，并被 核外切体。这些“死胡同”的成绩单显示了转录通读，这是最近 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 在剪接、3‘端切割和降解方面决定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特征的谱系，这些特征决定了 体内剪接体组装；识别转录和mRNP成熟过程中的协调转变；以及 了解转录、剪接和3‘端切割是如何联系在一起决定成败的 正常生长和细胞应激。

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.