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.

项目摘要 剪接是切除内含子并连接外显子的MRNA加工中的重要步骤。 这是由识别剪接位点（SS）并在其前MRNA上组装的剪接体催化的 以逐步方式底物。剪接体经历了多个重排，主要由 在剪接周期内，DEXD/H-box解旋酶。剪接站点识别和结构重排 剪接需要高保真进行。剪接的错误导致大约30％的人 遗传疾病和包括癌症在内的许多其他疾病的发展。 尽管进行了数十年的研究，但初始剪接站点识别的机制仍然很大程度上是一个谜。那里 目前是SS识别的两个模型，即内含子和外显子的定义。在以前的模型中 剪接体最初识别并组装跨内含子，然后切除，并连接侧翼 外显子。在后一种模型中，剪接体首先在外显子周围组装，并重塑为横架 插入内含子之前，内含子复合物。目前尚不清楚复合物是在外显子上组装还是 内含子彼此相同或不同，以及外显子定义如何重塑。我们的实验室 最近确定了酵母电子复合结构。结构表明完全相同的剪接体 可以在外显子上组装并执行外显子的定义，而无需其他组件或结构 重排。为了提供该假设的第一个实验证据，我们将解决 早期的剪接体复合物在内含子和外显子上均组装。这个项目的结果将 显着提高了我们对剪接体识别初始内含子/外显子识别的理解。 DEXD/H-box解旋酶在驱动剪接体过渡和所有四个DEAH-BOX剪接体中很重要 参与晚期剪接的解旋酶（PRP2，PRP16，PRP22和PRP43）具有有趣的结构相似性。在 这些解旋酶进行了广泛的生化和结构分析，目前尚不清楚哪种结构元素 在DEAH-box中，解旋酶负责解旋酶活性。还不清楚他们是如何专门的 被招募到剪接体中，并受到监管，以在正确的时间和地点执行其专门的行动。我瞄准 使用PRP22作为具有生化方法的模型来解决这些问题。这些结果可能 对所有四个DEAH-box剪接体解旋酶的机制和调节提供重要的见解。