Mechanisms of Spliceosome Assembly and Regulation

项目总结/摘要 RNA剪接是人类基因表达的关键特征，也是基因扩增的主要贡献者。 通过选择性剪接获得信息。剪接是由一个大型的动态细胞机器进行的， 剪接体剪接体由小的核核糖核蛋白（snRNP）组成， 前体转录物（pre-mRNAs）以去除内含子并将外显子拼接在一起。这个过程必须发生 正是为了保存mRNA中携带的遗传信息。对拼接至关重要的是 RNA键切割和形成位点的鉴定[5 '和3'剪接位点（SS）和分支 地点（BS）]。许多不同的ATP酶有助于SS和BS识别的保真度以及携带 剪接体的广泛组成和构象重塑。最近，生物化学研究 通过对酵母和人类的几十种不同结构的测定， 通过冷冻电镜观察剪接体。尽管发生了这场结构性革命， 剪接反应本实验室的研究目标是阐明剪接体的机制 组装和调节的生化深度使用各种技术。我们经常用单分子 荧光显微镜去卷积的复杂和异质反应途径所采用的 拼接机械在最近的工作中，我们研究了5'SS和BS的识别，组装和 动力学的U6 snRNP，并开发了荧光标记，纯化和抑制RNA的方法 和RNP。我们未来五年的愿景是将从剪接体结构中获得的见解 单分子，生物化学，计算和遗传实验，以解决我们的突出差距， 关于Splicing这些差距包括RNP折叠和组装的基本原理， 调节剪接的机制，以及缺乏特异性和有效的化学抑制剂， 剪接体作为这一愿景的一部分，我们将使用多学科方法回答以下问题： 1)RNA和蛋白质如何共同折叠以组装U6 snRNP？ 2)剪接体在其活性位点形成过程中是如何重塑的？ 3)调节蛋白如何促进弱5'SS的剪接？ 4)我们如何用小分子抑制剂阻断剪接过程中的ATP酶依赖性转换？ 5)我们如何定量分析、比较和整合剪接体的冷冻电镜结构？

PROJECT SUMMARY/ABSTRACT RNA splicing is a key feature of human gene expression and a major contributor to expansion of genetic information by alternative splicing. Splicing is carried out by a large and dynamic cellular machine called the spliceosome. Spliceosomes are composed of small nuclear ribonucleoproteins (snRNPs) that assemble on precursor transcripts (pre-mRNAs) to remove introns and splice together exons. This process must occur precisely in order to preserve the genetic information carried in the mRNA. Critical for splicing is the correct identification of the sites of RNA bond cleavage and formation [the 5' and 3' splice sites (SS) and the branch site (BS)]. A number of different ATPases contribute to the fidelity of SS and BS recognition as well as carry out extensive compositional and conformational remodeling of the spliceosome. Recently, biochemical studies of splicing have been transformed by determination of dozens of different structures of yeast and human spliceosomes by cryo-EM. Despite this structural revolution, much remains unknown about central features of the splicing reaction. The goal of my laboratory’s research is to elucidate mechanisms of spliceosome assembly and regulation in biochemical depth using a variety of techniques. We often use single molecule fluorescence microscopy to deconvolute the complex and heterogeneous reaction pathways employed by the splicing machinery. In recent work, we have studied mechanisms of 5'SS and BS recognition, assembly and dynamics of the U6 snRNP, and developed methods for fluorescently-labeling, purifying, and inhibiting RNAs and RNPs. Our vision for the next five years is to merge the insights obtained from structures of spliceosomes with single molecule, biochemical, computational, and genetic experiments to address outstanding gaps in our knowledge of splicing. These gaps include fundamental principles of RNP folding and assembly, the mechanisms of regulated splicing, and the scarcity of specific and effective chemical inhibitors of the spliceosome. As part of this vision, we will answer the following questions using multi-disciplinary approaches: 1) How do RNA and protein co-fold to assemble the U6 snRNP? 2) How is the spliceosome remodeled during creation of its active site? 3) How do regulatory proteins promote splicing at weak 5'SS? 4) How can we block ATPase-dependent transitions during splicing with small molecule inhibitors? 5) How do we quantitatively analyze, compare, and integrate cryo-EM structures of spliceosomes?