Mechanisms of Spliceosome Assembly and Splice Site Recognition

剪接体组装和剪接位点识别的机制

• 批准号: 8996582
• 负责人: Aaron Andrew Hoskins
• 金额: $ 28.47万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8996582
• 关键词: 3' Splice Site; 5' Splice Site; ATP Hydrolysis; ATP phosphohydrolase; Address; Alternative Splicing; Binding; Biochemical; Blindness; Cell Extracts; Cells; Complex; Coupled; Defect; Disease; Dysmyelopoietic Syndromes; Eukaryota; Event; Excision; Exons; Fluorescence; Foundations; Gene Expression; Goals; Hereditary Disease; Human; In Vitro; Introns; Investigation; Laboratories; Lead; Length; Ligation; Link; Location; Malignant Neoplasms; Measurement; Messenger RNA; Methods; Modeling; Molecular; Monitor; Muscular Atrophy; Mutation; Outcome; Outcome Study; Participant; Pathway interactions; Play; Process; Property; Proteins; RNA Sequences; RNA Splicing; Reaction; Recombinants; Recruitment Activity; Research; Role; Signal Transduction; Site; Small Nuclear RNA; Small Nuclear Ribonucleoproteins; Spliceosome Assembly Pathway; Spliceosomes; Staging; Structure; System; Testing; Transcript; Translations; U1 Small Nuclear Ribonucleoprotein; U2 Small Nuclear Ribonucleoprotein; Work; Yeasts; design; effective therapy; genetic information; innovation; innovative technologies; insight; interdisciplinary approach; mRNA Precursor; macromolecular assembly; novel; programs; public health relevance; reconstitution; research study; single molecule; tool; transmission process

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.

描述（由申请人提供）： 项目摘要 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，并且必须精确发生。精度取决于称为剪接体的大分子机器对 RNA 内剪接位点的准确识别。剪接体在蛋白质和小核核糖核蛋白 (snRNP) 成分的转录物中的特定位置组装。在人类中，大多数 RNA 都是选择性剪接的，这意味着剪接体可以整合多个调节信号来控制给定转录物的剪接命运。调节选择性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/Mud2 蛋白异二聚体识别。这形成所谓的承诺复合体 (CC)，然后招募 U2 形成预剪接体。前剪接体的形成被认为决定了许多转录本的选择性剪接命运，而人类 CC 和前剪接体成分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。该项目的最终目标是了解剪接体在 RNA 上组装的途径。虽然这些过程中参与者的身份是已知的，但他们的作用机制仍不清楚。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，我们重点关注剪接体 CC 的形成及其向剪接前体的转变。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。在目标 1 中，我们将纯化 CC 的成分并在体外重建其组装体。目标 1 的一个关键成果是用于研究 CC 形成的纯化、生化特征系统。这是我们生化重建剪接体组装的长期目标的必要步骤。在目标 2 中，我们使用纯化组分和酵母细胞提取物的新型组合来研究 CC 单分子的分解和预剪接体的形成。在目标 3 中，我们使用多种方法来研究剪接体前形成过程中 Prp5 ATP 酶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。