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.

项目总结 前体信使核糖核酸(前信使核糖核酸)的加工是基因表达的一个重要方面 与RNA聚合酶II的转录同时进行。前mRNA处理的一个基本步骤是去除 剪接体将非编码内含子和编码外显子连接在一起(前mRNA剪接)。这个 剪接体在转录过程中在新生的rna上组装，内含子之后不久就完成了剪接。 已经被转录了。因此，剪接和转录机器在空间和时间上耦合- 协同工作，确保细胞mRNAs的及时和准确表达。突变或扰动 这两个过程都会改变基因输出，并经常与人类疾病有关。值得注意的是， 协调剪接和转录的机制还知之甚少。拟议的工作结合了 已经建立的技术和新的方法来阐明这两个细胞过程是如何相互调节的。 纽格鲍尔实验室最近开发了单分子内含子追踪(SMIT)和其他基于RNAseq的 测量体内剪接相对于转录的动力学的方法。特定目标1调查 内含子序列和其他RNA特征对剪接反应的贡献，并决定如何协同- 转录剪接可以影响基因输出。特定目标2结合了SMIT和其他基于RNAseq的 用POL II突变背景揭示C-末端结构域和翻译后影响的方法 RNA聚合酶II对剪接的修饰。具体而言，这一目标是问CTD如何为 在单个转录本中高效地拼接多个内含子。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。特定的 目的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物和基因工具使剪接体保持与 新的成绩单。这些方法和目标将为开发以下领域的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， 拟议的工作将产生对基本过程之间的相互作用的前所未有的分子洞察力 并提供基础知识，这些基础知识将在未来关于剪接和 在疾病中转录会发生改变。