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: 9765599
• 负责人: Karla M Neugebauer
• 金额: $ 44.06万
• 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/9765599
• 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; Precursor RNA; 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; 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 在序列、结构和功能上与其前体 RNA 不同。巨大的 前体 mRNA 加工的调节能力受到转录的影响，因为 RNA 加工 机器以共转录方式起作用，可以直接接触 RNA 聚合酶 II (Pol II) 和/或染色质。在 反过来，前 mRNA 加工中的每个步骤 - 5' 末端加帽、剪接和 3' 末端切割 - 都与 Pol II 行为的变化，例如暂停。这笔赠款的重点是转录之间的协调， 酿酒酵母和粟酒裂殖酵母中的剪接和 3' 末端切割。我们在调查期间的发现 过去 3 年重新定义了我们对 mRNA 这三个方面之间相互影响的看法 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全球范围内延伸 Pol II 位置的函数，从而产生 有两个重大发现： (1) 我们已经证明剪接体的运行时间比之前预想的要快得多 作用时接近Pol II。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' 末端切割相协调。瞄准 1，我们测试了关于响应 RNA 序列、RNA 结构的剪接体组装时间的模型， 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 来自新生 mRNA 的催化剪接体，我们称之为剪接体驱逐，对于促进 mRNA 是必要的 在 Pol II 暂停位点成熟——并通过实验评估这个想法与其他模型的对比。 (2) 我们的方法之一，长读测序，确定新生 RNA 从 5' 到 3' 端的完整序列 （Pol II 位置），使我们能够追踪粟酒裂殖酵母中丰富的多内含子转录物的剪接 “实时”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“全有或全无”的方式进行拼接，其中一半以上被快速且完全地拼接。相比之下， 18% 的新生转录本完全未剪接，未能进行 3' 末端切割，并被 核外泌体。这些“死胡同”的转录本显示转录通读，最近已被 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 定义转录本命运的分子机制，包括剪接、3' 末端切割和降解。 我们新目标的影响将是定义决定动力学的内含子特征。 体内剪接体组装；识别转录和 mRNA 成熟中的协调转变；和 发现转录、剪接和 3' 末端切割如何与成功或失败相关 正常生长和细胞应激。