Understanding the structure and function of U1 snRNP

了解 U1 snRNP 的结构和功能

• 批准号: 10200085
• 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 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10200085
• 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; 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.

摘要 在所有真核生物中，前mRNA剪接是基因表达所必需的，剪接错误导致基因 疾病和许多其他疾病。深入了解前信使核糖核酸的分子机制 剪接有可能为人类疾病治疗提供有用的方法。内含子的剪接是 通过剪接体催化的两个酯交换反应进行，剪接体是一种大的RNA/蛋白质 由5个SnRNP(U1、U2、U4、U5、U6)和许多非SnRNP相关蛋白因子组成的复合体。尽管 剪接体复合体多个高分辨低温电子显微镜结构的最新测定 在剪接途径的各个阶段，对初始内含子缺乏结构和机制的理解 识别和早期剪接体组装事件。 U1 SnRNP对5‘剪接位点的初始识别至关重要。由于它在5‘s认知中的重要作用， U1 SNRNP是促进或阻止U1的选择性剪接因子作用的常见靶点 SnRNP从结合到5‘s。我们今天所知道的关于5‘的分子机制和调控的大部分 SS的识别来自对两个常用模型系统的遗传、生化和结构研究， 酿酒酵母和人U1单链核糖核酸酶。耐人寻味的是，酵母U1的SnRNP比 人U1单链RNP。酵母U1 SnRNA含有3.5倍大的RNA和7种额外的蛋白质，大多数 它们具有与U1 SnRNP弱相关的人类同源物，并参与替代 拼接。我们最近已经确定了酵母U1SnRNP的冷冻EM结构，分辨率为3.6？，产生了 关于人类选择性剪接因子如何招募U1 SnRNP和意想不到的 人类PrpF39在选择性剪接中的作用。我们将使用以下组合来检验这些假设 生物化学和分子生物学方法。我们还将破译协同作用的分子机制 通过结构生物学和生化方法的结合识别所有内含子元件。 该项目的结果将极大地促进我们对内含子分子机制的理解。 定义和替代拼接。