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.

项目总结