Mechanisms of Spliceosome Assembly and Splice Site Recognition

描述（由申请人提供）： RNA剪接-内含子的去除和外显子的连接-是真核基因表达的重要步骤，必须精确地发生。精确度取决于被称为剪接体的大分子机器对RNA内剪接位点的准确识别。剪接体在转录本的特定位置由蛋白质和小核核糖核蛋白（snRNP）组分组装而成。在人类中，大多数RNA是选择性剪接的，这意味着剪接体可以掺入多种调控信号来控制给定转录物的剪接命运。调节可变剪接和剪接效率的许多关键步骤发生在剪接体组装的最早阶段。在酵母中的这些步骤中，5'剪接位点（SS）和分支位点（BS）首先分别被U1 snRNP和BBP/Mud 2蛋白异源二聚体识别。这形成了所谓的承诺复合体（CC），然后招募U2形成前剪接体。前剪接体形成被认为决定了许多转录物的选择性剪接命运，并且人CC和前剪接体组分中的缺陷与包括骨髓增生异常综合征（MDS）在内的遗传疾病有关。这个项目的最终目标是了解剪接体在RNA上组装的途径。虽然这些进程中的参与者的身份是已知的，但其作用机制仍不清楚。对这些事件的研究将有助于更好地理解这一基本过程，并为与剪接相关的疾病提供新的见解。在这里，我们专注于形成的剪接体CC和其过渡到前剪接体。在这些实验中，我们利用单分子荧光的独特功能作为我们的主要工具。目的一：纯化CC的组分，并在体外重组其组装体。目标1的一个关键成果是一个纯化的，生物化学特征的系统，用于研究CC的形成。这是我们生化重组剪接体组装的长期目标中的必要步骤。在目标2中，我们研究了拆卸的CC和形成前剪接体的单分子使用一种新的组合纯化的成分和酵母细胞提取物。在目的3中，我们使用多种方法来研究Prp 5 ATP酶在前剪接体形成期间的结合和构象动力学。这些实验将为剪接体组装及其调控方式提供急需的新见解。

DESCRIPTION (provided by applicant): PROJECT SUMMARY RNA splicing-the removal of introns and ligation of exons-is an essential step in eukaryotic gene expression and must occur precisely. Precision depends on accurate recognition of the splice sites within RNAs by a macromolecular machine called the spliceosome. Spliceosomes are assembled at particular locations in transcripts from protein and small nuclear ribonucleoprotein (snRNP) components. In humans, most RNAs are alternatively spliced meaning that the spliceosome can incorporate multiple regulatory signals to control the splicing fate of a given transcript. Many of the key steps in regulating alternative splicing and splicing efficiency occur in the earliest stages of spliceosome assembly. During these steps in yeast, the 5' splice site (SS) and the branchsite (BS) are first recognized by the U1 snRNP and the BBP/Mud2 protein heterodimer, respectively. This forms the so-called commitment complex (CC) that then recruits U2 to form the pre-spliceosome. Pre-spliceosome formation is believed to determine the alternative splicing fate of many transcripts and defects in human CC and pre-spliceosome components are linked to genetic diseases including myelodysplastic syndrome (MDS). The ultimate goals of this project are to understand the pathways by which spliceosomes assemble on RNAs. While the identities of the players in these processes are known, their mechanisms of action remain unclear. Investigation of these events will lead to a better understanding of this fundamental process as well as provide new insights into diseases linked to splicing. Here, we focus on formation of the spliceosomal CC and its transition into the pre-spliceosome. In these experiments, we exploit the unique capabilities of single molecule fluorescence as our primary tool. In Aim 1, we will purify the components of CC and reconstitute its assembly in vitro. A key outcome of Aim 1 is a purified, biochemically characterized system for studying CC formation. This is a necessary step in our long-term objective of biochemically reconstituting spliceosome assembly. In Aim 2, we study the disassembly of single molecules of CC and the formation of pre-spliceosomes using a novel combination of purified components and yeast cell extracts. In Aim 3, we use a variety of approaches to study the binding and conformational dynamics of the Prp5 ATPase during pre- spliceosome formation. Together these experiments will provide much needed new insights into spliceosome assembly and the ways in which it can be regulated.