Molecular Mechanisms Coupling Transcription and Splicing

转录和剪接耦合的分子机制

• 批准号: 10369291
• 负责人: Tucker Joe Carrocci
• 金额: $ 1.71万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10369291
• 关键词: Affect; Alternative Splicing; Amino Acid Substitution; Biogenesis; Bioinformatics; C-terminal; Cell Nucleus; Cell physiology; Cells; Chromatin; Code; Coupled; Coupling; DNA Polymerase II; DNA-Directed RNA Polymerase; Data; Disease; Dominant-Negative Mutation; Elements; Ensure; Event; Excision; Exons; Fission Yeast; Future; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; High-Throughput Nucleotide Sequencing; Introns; Kinetics; Knowledge; Lead; Ligation; Malignant Neoplasms; Measures; Messenger RNA; Methods; Modeling; Modification; Molecular; Monitor; Mutation; Outcome; Output; Polymerase; Population; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; RNA; RNA Polymerase II; RNA Processing; RNA Sequences; RNA Splicing; Reaction; Role; Saccharomyces cerevisiae; Saccharomycetales; Site; Spliced Genes; Spliceosomes; System; Techniques; Time; Transcript; Untranslated RNA; Variant; Work; Yeasts; base; chemical genetics; design; developmental disease; experimental study; gene product; genome-wide analysis; human disease; in vivo; inhibitor/antagonist; insight; mRNA Precursor; man; mutant; next generation; novel strategies; rate of change; single molecule; tool; transcriptome; transcriptome sequencing

PROJECT SUMMARY Precursor messenger RNA (pre-mRNA) processing is an essential aspect of gene expression that occurs concurrently with transcription by RNA polymerase II. One essential step in pre-mRNA processing is the removal of non-coding introns and ligation of coding exons together (pre-mRNA splicing) by the spliceosome. The spliceosome assembles on the nascent RNA during transcription, and splicing is completed soon after the intron has been transcribed. Thus, the splicing and transcription machineries are spatially and temporally coupled – working in concert to ensure timely and accurate expression of cellular mRNAs. Mutations or perturbations of either process change gene output and are frequently associated with human disease. Remarkably, the mechanisms coordinating splicing with transcription are poorly understood. The proposed work combines established techniques and novel approaches to elucidate how the two cellular processes regulate one another. The Neugebauer lab recently developed single-molecule intron tracking (SMIT) and other RNAseq-based approaches to measure the in vivo kinetics of splicing relative to transcription. Specific Aim 1 investigates the contribution of intron sequence and other RNA features to the splicing reaction and determines how co- transcriptional splicing can influence gene output. Specific Aim 2 combines SMIT and other RNAseq-based approaches with Pol II mutant backgrounds to reveal the impact of the C-terminal domain and post-translational modifications of RNA polymerase II on splicing. In particular, this aim asks how the CTD contributes to the efficient splicing of multiple introns in a single transcript. These experiments will generate new mechanistic insights into how RNA polymerase interacts with the spliceosome to promote efficient RNA splicing. Specific Aim 3 investigates the influence of the splicing machinery on transcriptional dynamics and polymerase pausing using potent splicing inhibitors and genetic tools that cause the spliceosome to remain associated with the nascent transcript. These approaches and aims will provide an entry point for developing expertise in transcription, bioinformatics, RNA-seq based methods and other computational approaches. In addition, the proposed work will generate unprecedented molecular insight into the cross-talk between essential processes in gene expression and provide fundamental knowledge that will be vital in future studies on how splicing and transcription are altered in disease.

项目概要 前体信使 RNA (pre-mRNA) 加工是基因表达发生的一个重要方面 与 RNA 聚合酶 II 的转录同时进行。前 mRNA 加工中的一个重要步骤是去除 非编码内含子的连接和编码外显子通过剪接体连接在一起（前 mRNA 剪接）。这 转录过程中剪接体组装在新生RNA上，在内含子出现后不久剪接完成 已被转录。因此，剪接和转录机器在空间和时间上是耦合的—— 协同工作以确保细胞 mRNA 的及时、准确表达。突变或扰动 这两个过程都会改变基因输出，并且经常与人类疾病相关。值得注意的是， 人们对剪接与转录的协调机制知之甚少。拟议的工作结合了 已建立的技术和新方法来阐明这两种细胞过程如何相互调节。 Neugebauer 实验室最近开发了单分子内含子追踪 (SMIT) 和其他基于 RNAseq 的技术 测量剪接相对于转录的体内动力学的方法。具体目标 1 调查 内含子序列和其他 RNA 特征对剪接反应的贡献，并确定如何共同 转录剪接可以影响基因输出。具体目标 2 结合了 SMIT 和其他基于 RNAseq 的技术 使用 Pol II 突变体背景的方法揭示 C 末端结构域和翻译后的影响 RNA 聚合酶 II 在剪接上的修饰。特别是，该目标询问 CTD 如何为 单个转录本中多个内含子的有效剪接。这些实验将产生新的机制 深入了解 RNA 聚合酶如何与剪接体相互作用以促进有效的 RNA 剪接。具体的 目标 3 研究剪接机制对转录动力学和聚合酶暂停的影响 使用有效的剪接抑制剂和遗传工具，使剪接体保持与 新生的转录本。这些方法和目标将为发展专业知识提供切入点 转录、生物信息学、基于 RNA-seq 的方法和其他计算方法。此外， 拟议的工作将对基本过程之间的串扰产生前所未有的分子洞察力 基因表达方面的研究，并提供基础知识，这对于未来关于剪接和剪接如何进行的研究至关重要。 转录在疾病中发生改变。