Understanding the structure and function of U1 snRNP

了解 U1 snRNP 的结构和功能

• 批准号: 9751902
• 负责人: RUI ZHAO
• 金额: $ 45.17万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9751902
• 关键词: 3' Splice Site; 5' Splice Site; Alternative Splicing; Binding; Binding Sites; Biochemical; Biochemistry; Biological Models; C-terminal; Cells; Complex; Cryoelectron Microscopy; Disease; Elements; Essential Genes; Eukaryota; Event; Gene Expression; Genetic; Genetic Diseases; Human; Introns; Mammalian Cell; Mediating; Modeling; Molecular; Molecular Biology; Pathway interactions; Proteins; RNA; RNA Splicing; Reaction; Regulation; Resolution; Role; Saccharomyces cerevisiae; Sequence Homology; Site; Small Nuclear Ribonucleoproteins; Spliceosome Assembly Pathway; Spliceosomes; Structural Models; Structure; Testing; U1 Small Nuclear Ribonucleoprotein; U1 small nuclear RNA; Yeasts; analog; base; experience; human disease; human model; mRNA Precursor; paralogous gene; prevent; protein complex; recruit; scaffold; structural biology; transcriptome sequencing

Abstract Pre-mRNA splicing is essential for gene expression in all eukaryotes and errors in splicing cause genetic disorders and many other diseases. A thorough understanding of the molecular mechanisms of pre-mRNA splicing has the potential to provide useful approaches for human disease therapy. The splicing of introns is carried out through two transesterification reactions catalyzed by the spliceosome, a large RNA/protein complex composed of five snRNPs (U1, U2, U4, U5, U6) and many non-snRNP related protein factors. Despite the recent determination of multiple high resolution cryo EM structures of spliceosome complexes at later stages of the splicing pathway, there is a lack of structural and mechanistic understanding of the initial intron recognition and early spliceosome assembly events. U1 snRNP is critical for the initial recognition of 5' splice site (ss). Due to its important role in 5' ss recognition, U1 snRNP is a frequent target of the action of alternative splicing factors that either facilitate or prevent U1 snRNP from binding to 5' ss. Much of what we know today about the molecular mechanism and regulation of 5' ss recognition comes from genetic, biochemical, and structural studies of two commonly used model systems, S. cerevisiae (yeast) and human U1 snRNP. Intriguingly, the yeast U1 snRNP is much more complex than the human U1 snRNP. Yeast U1 snRNA contains a 3.5-fold larger RNA and seven additional proteins, most of which have human homologs that are weakly associated with U1 snRNP and are involved in alternative splicing. We have recently determined the cryoEM structure of yeast U1 snRNP to 3.6 Å resolution, generating many interesting hypotheses on how human alternative splicing factors recruit U1 snRNP and the unexpected role of human PrpF39 in alternative splicing. We will test these hypotheses using a combination of biochemistry and molecular biology approaches. We will also decipher the molecular mechanism of concerted recognition of all intron elements through a combination of structural biology and biochemical approaches. Results from this project will significantly advance our understanding of the molecular mechanism of intron definition and alternative splicing.

抽象的 前mRNA剪接对于所有真核生物中的基因表达至关重要，剪接中的误差会导致遗传 疾病和许多其他疾病。对前MRNA的分子机制有透彻的理解 剪接有可能为人类疾病疗法提供有用的方法。内含子的拼接是 通过剪接体催化的两种式式反应，一种大的RNA/蛋白 复合物由五个SNRNP（U1，U2，U4，U5，U6）和许多与SNRNP相关的蛋白质因子组成。尽管 稍后的剪接体复合物的多个高分辨率冷冻结构的最近确定 剪接途径的阶段，缺乏对初始内含子的结构和机械理解 认可和早期剪接组装事件。 U1 SNRNP对于5'剪接位点（SS）的初始识别至关重要。由于其在5'Ss识别中的重要作用， U1 SNRNP通常是促进或预防U1的替代剪接因子作用的目标 SNRNP从结合到5'Ss。我们今天对5'分子机制和调节的了解中的大部分内容 SS识别来自两个常用模型系统的遗传，生化和结构研究， S. cerevisiae（酵母）和人类U1 snrnp。有趣的是，酵母U1 SNRNP比 人U1 snrnp。酵母U1 snRNA含有3.5倍大的RNA和七个额外的蛋白质，大多数 具有与U1 SNRNP无关的人类同源物，并且参与了替代方案 剪接。我们最近确定了酵母U1 snrnp的冷冻结构至3.6Å分辨率，生成 关于人类替代剪接因素如何募集U1 SNRNP和意外的许多有趣的假设 人PRPF39在替代剪接中的作用。我们将使用组合来检验这些假设 生物化学和分子生物学方法。我们还将解密协调的分子机制 通过结构生物学和生化方法的结合来识别所有内含子元素。 该项目的结果将大大提高我们对内含子分子机制的理解 定义和替代剪接。