Cross-regulation between transcription and pre-mRNA splicing

转录和前 mRNA 剪接之间的交叉调节

• 批准号: 10170363
• 负责人: Karla M Neugebauer
• 金额: $ 43.47万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10170363
• 关键词: Alternative Splicing; Behavior; Binding; Cellular Stress; Chemicals; Chromatin; Competence; DNA; DNA Polymerase II; DNA-Directed RNA Polymerase; Data; Development; Disease; Ensure; Excision; Exons; Failure; Fission Yeast; Gene Expression; Gene Proteins; Genes; Genetic Transcription; Goals; Grant; Growth; Health; Heat-Shock Response; Homing; Human; Individual; Introns; Kinetics; Length; Link; Malignant Neoplasms; Measures; Messenger RNA; Methods; Modeling; Molecular; Nuclear; Osmoregulation; Output; Phosphorylation; Polyadenylation; Positioning Attribute; Proteins; Proteome; RNA; RNA Degradation; RNA Precursors; RNA Processing; RNA Sequences; RNA Splicing; Reaction; Regulation; Regulator Genes; Role; Saccharomyces cerevisiae; Saccharomycetales; Site; Spliceosome Assembly Pathway; Spliceosomes; Stress; Structure; Symptoms; Testing; Time; Trans-Activators; Transcript; Transcription Elongation; Transcriptional Regulation; U1 Small Nuclear Ribonucleoprotein; U2 Small Nuclear Ribonucleoprotein; Variant; Virus Diseases; cleavage factor; exosome; gene repression; genomic locus; human disease; in vivo; insight; mRNA Export; mRNA Precursor; messenger ribonucleoprotein; nervous system disorder; novel; protein expression; recruit; response; single molecule; stem; success; transcriptome sequencing

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.

摘要/项目摘要 细胞RNA的序列，结构和功能与其前体RNA不同。巨大 前MRNA处理的调节能力受转录的影响，因为RNA处理 机械通过共转录起作用，可以直接接触RNA聚合酶II（POL II）和/或染色质。在 转弯，前MRNA处理中的每个步骤 - 5'端封，剪接和3'端裂解 - 与 Pol II行为的变化，例如暂停。该赠款重点是转录之间的协调 酿酒酵母和schizosacchomyces pombe的剪接和3'端裂解。我们在 过去3年重新定义了我们如何看待mRNA这三个方面之间的串扰 成熟。我们开发了两种完整的单分子RNA-seq方法，它们直接测量 剪接反应的进展是在全球范围内伸长pol II的位置的函数，导致 在两个主要发现中： （1）我们已经证明，剪接体的运行时间比以前所欣赏的时间快得多，并且 当它起作用时，它接近pol II。剪接和转录之间的短滞后引发了有关 在Gene Termini上，剪接如何与转录，MRNP成熟和3'端裂解协调。目标 1，我们测试了有关RNA序列，RNA结构，RNA结构的剪接体组装时间的模型 以及跨性别因素的作用。在AIM 2中，我们研究了一个新的假设 - 去除后 我们称为剪接体驱逐的新生mRNA的催化剪接体是促进MRNP的必要条件 Pol II暂停的地点的成熟 - 并对其他模型对此思想进行了实验评估。 （2）我们的方法之一是长读测序，确定了新生RNA的完整序列从5'端到3' （Pol II位置），使我们能够跟踪S. Pombe和 在“实时”中确定共转录内含子去除的顺序。值得注意的是，大多数新生的成绩单 以“全或无”的方式进行剪接，以至于一半以上是迅速且完全剪接的。相比之下， 18％的新生成绩单完全没有填充，未能进行3'末端裂解，并被脱落 核外泌体。这些“死胡同”成绩单显示了转录读取，最近已经 在对压力，癌症和病毒感染的细胞反应中实施。在AIM 3中，我们建议确定 针对剪接，3'末端裂解和降解的转录本命运的分子机制。 我们新目标的影响是定义内含子功能的曲目，这些功能决定了动力学的动力学 体内剪接组件；确定转录和MRNP成熟中的协调转变；和 发现转录，剪接和3'末端裂解如何在上下文中取得成功或失败 正常生长和细胞应激。