Understanding the structure and function of U1 snRNP

摘要 前体mRNA剪接对于所有真核生物中的基因表达是必不可少的，并且剪接中的错误导致遗传性疾病。 疾病和许多其他疾病。对前mRNA分子机制的深入了解 剪接具有为人类疾病治疗提供有用方法的潜力。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、U 5、U6）和许多非snRNP相关蛋白因子组成的复合物。尽管 近年来剪接体复合物高分辨冷冻电镜结构测定 剪接途径的各个阶段，缺乏对初始内含子的结构和机制的理解 识别和早期剪接体组装事件。 U1 snRNP对5'剪接位点（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是选择性剪接因子作用的常见靶点，选择性剪接因子促进或阻止U1 snRNP与5' ss结合。我们今天所知道的大部分关于5'端的分子机制和调控 SS识别来自于两种常用模型系统的遗传、生物化学和结构研究， S.酿酒酵母（酵母）和人U1 snRNP。有趣的是，酵母U1 snRNP比酵母U1 snRNP复杂得多。 人U1 snRNP。酵母U1 snRNA含有3.5倍大的RNA和7种额外的蛋白质，其中大部分是 其具有与U1 snRNP弱相关的人类同源物，并参与替代性的 拼接我们最近确定了酵母U1 snRNP的cryoEM结构，分辨率为3.6 nm， 关于人类选择性剪接因子如何募集U1 snRNP的许多有趣的假设和意想不到的 人PrpF 39在可变剪接中的作用。我们将使用以下组合来测试这些假设： 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物化学方法的组合识别所有内含子元件。 本项目的研究结果将极大地促进我们对内含子分子机制的理解 定义和选择性剪接。

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.