Molecular Mechanisms Coupling Transcription and Splicing

项目摘要 前体信使RNA（pre-mRNA）加工是基因表达的一个重要方面， 同时通过RNA聚合酶II进行转录。前体mRNA加工的一个重要步骤是去除 非编码内含子和编码外显子通过剪接体连接在一起（前mRNA剪接）。的 剪接体在转录过程中在新生RNA上组装，剪接在内含子后不久完成 已经被转录了。因此，剪接和转录机制在空间和时间上是耦合的- 协同工作以确保细胞mRNA的及时和准确表达。突变或扰动 这两种过程都改变基因输出，并经常与人类疾病有关。值得注意的是， 协调剪接与转录的机制知之甚少。拟议的工作结合了 已建立的技术和新的方法来阐明这两个细胞过程如何相互调节。 纽介堡实验室最近开发了单分子内含子跟踪（SMIT）和其他基于RNAseq的 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，并决定如何共同 转录剪接可以影响基因输出。Specific Aim 2结合了SMIT和其他基于RNAseq的 Pol II突变背景的方法，以揭示C-末端结构域和翻译后 RNA聚合酶II对剪接的修饰。特别是，这一目标询问CTD如何有助于 在单个转录物中有效剪接多个内含子。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。具体 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剂和遗传工具，使剪接体保持与 新生的成绩单这些方法和目标将为发展以下方面的专门知识提供一个切入点： 转录、生物信息学、基于RNA-seq的方法和其他计算方法。此外该 拟议的工作将产生前所未有的分子洞察力之间的相互作用的基本过程 在基因表达和提供基础知识，将是至关重要的，在未来的研究如何剪接和 转录在疾病中改变。

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.