Understanding the mechanism of pre-mRNA splicing

了解前体 mRNA 剪接的机制

• 批准号: 10387298
• 负责人: Francesca Danielle De Bortoli
• 金额: $ 6.76万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10387298
• 关键词: 3' Splice Site; 5' Splice Site; ATP phosphohydrolase; Address; Alternative Splicing; Area; Automobile Driving; Binding; Biochemical; Biochemistry; Biological Assay; Complex; Cryoelectron Microscopy; Development; Disease; Elements; Event; Exons; Frameshift Mutation; Genetic; Genetic Diseases; Growth; Human; Human Genetics; In Vitro; Introns; Investigation; Lead; Liquid substance; Malignant Neoplasms; Mass Spectrum Analysis; Modeling; Molecular Conformation; N-terminal; Onset of illness; Pattern; Point Mutation; Process; Proteins; RNA; RNA Helicase; RNA Splicing; Regulation; Research; Site; Spliceosomes; Structure; Substrate Specificity; System; Testing; Therapeutic; Time; Transcript; U1 Small Nuclear Ribonucleoprotein; U2 Small Nuclear Ribonucleoprotein; Untranslated RNA; Yeasts; density; flexibility; helicase; in vitro activity; in vivo; insight; mRNA Precursor; novel; recruit

PROJECT SUMMARY Splicing is an essential step in pre-mRNA processing during which introns are excised, and exons are ligated. This is catalyzed by the spliceosome which recognizes splice sites (ss) and assembles on its pre-mRNA substrate in a stepwise manner. The spliceosome undergoes multiple rearrangements, largely driven by DExD/H-box helicases, during the splicing cycle. Splice site recognition and structural rearrangement of the spliceosome need to be carried out with high fidelity. Errors in splicing contribute to around 30% of human genetic disorders and the development of many other diseases including cancer. In spite of decades of research, the mechanism of initial splice site recognition is still largely a mystery. There are currently two models for ss recognition, namely intron and exon definition. In the former model, the spliceosome initially recognizes and assembles across introns which is then excised, ligating the flanking exons. In the latter model, the spliceosome first assembles around an exon and is remodeled into a cross- intron complex before splicing out the intron. It is unclear whether complexes assembled across an exon or an intron are the same or different from each other, and how exactly exon definition remodeling occurs. Our lab recently determined the yeast E-complex structure. The structure suggest that the exact same spliceosome can assemble on an exon and carry out exon definition without the need of additional components or structural rearrangements. To provide the first experimental evidence for this hypothesis, we will solve the structure of an early spliceosomal complex assembled on both an intron and an exon. Results from this project will significantly advance our understanding of initial intron/exon recognition by the spliceosome. DExD/H-box helicases are important in driving spliceosome transitions and all four DEAH-box spliceosomal helicases (Prp2, Prp16, Prp22 and Prp43) involved in late splicing share intriguing structural similarities. In spite of extensive biochemical and structural analyses of these helicases, it is unclear what structural element in DEAH-box helicases is responsible for the helicase activity. It is also unclear how they are specifically recruited to the spliceosome and regulated to perform their dedicated action at the right time and place. I aim to address these questions using Prp22 as a model with mostly biochemical approaches. These results may provide important insights into the mechanism and regulation of all four DEAH-box spliceosomal helicases.

项目概要 剪接是前 mRNA 加工中的一个重要步骤，在此过程中内含子被切除，外显子被连接。 这是由剪接体催化的，剪接体识别剪接位点 (ss) 并在其前 mRNA 上组装 基质以逐步的方式进行。剪接体经历多次重排，很大程度上是由 DExD/H-box 解旋酶，在剪接周期中。剪接位点识别和结构重排 剪接体需要以高保真度进行。剪接错误导致人类约 30% 遗传性疾病和包括癌症在内的许多其他疾病的发展。 尽管经过了数十年的研究，初始剪接位点识别的机制在很大程度上仍然是个谜。那里 目前ss识别有两种模型，即内含子和外显子定义。在前一个模型中， 剪接体最初识别内含子并组装，然后切除内含子，连接侧翼 外显子。在后一种模型中，剪接体首先围绕外显子组装，并被重塑为交叉结构。 剪接内含子之前的内含子复合物。目前尚不清楚复合物是否跨外显子或跨外显子组装 内含子彼此相同或不同，以及外显子定义重构究竟如何发生。我们的实验室 最近确定了酵母E复合物的结构。该结构表明完全相同的剪接体 可以在外显子上组装并进行外显子定义，而不需要额外的组件或结构 重新安排。为了为这一假设提供第一个实验证据，我们将解决一个 早期剪接体复合物在内含子和外显子上组装。该项目的结果将 显着增进了我们对剪接体初始内含子/外显子识别的理解。 DExD/H 盒解旋酶对于驱动剪接体转变非常重要，并且所有四种 DEAH 盒剪接体 参与后期剪接的解旋酶（Prp2、Prp16、Prp22 和 Prp43）具有有趣的结构相似性。在 尽管对这些解旋酶进行了广泛的生化和结构分析，但尚不清楚是什么结构元件 DEAH 盒解旋酶中负责解旋酶活性。也不清楚它们具体是如何 被招募到剪接体中并受到调节以在正确的时间和地点执行其专门的行动。我的目标 使用 Prp22 作为模型并主要采用生化方法来解决这些问题。这些结果可能 为所有四种 DEAH 盒剪接体解旋酶的机制和调节提供了重要的见解。