Protein-driven dynamics of pre-mRNA splicing catalysis through single molecule microscopy

通过单分子显微镜观察蛋白质驱动的前 mRNA 剪接催化动力学

• 批准号: 10548142
• 负责人: Elizabeth C Duran
• 金额: $ 7.29万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10548142
• 关键词: 3' Splice Site; 5' Splice Site; ATP Hydrolysis; Active Sites; Address; Alternative Splicing; Biochemical; Biophysics; C-terminal; Cardiomyopathies; Catalysis; Catalytic Domain; Cell physiology; Charge; Code; Complex; DNA Sequence Alteration; Data; Development; Dilated Cardiomyopathy; Disease; Docking; Dysmyelopoietic Syndromes; Environment; Enzymatic Biochemistry; Esterification; Event; Exons; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Health; Hela Cells; Human; Immobilization; Immunoglobulin Joining Region; Impairment; Institution; Introns; Investigation; Kinetics; Knowledge; Label; Laboratories; Ligation; Macromolecular Complexes; Malignant Neoplasms; Mediating; Messenger RNA; Methods; Microscopy; Molecular; Molecular Biology; Molecular Chaperones; Molecular Conformation; Molecular Machines; Monitor; Mutate; Mutation; Nuclear; Nuclear Extract; Nucleotides; Organism; Parkinson Disease; Process; Progeria; Proteins; RNA; RNA Processing; RNA Splicing; RNA, Messenger, Splicing; Rationalization; Reaction; Reporter; Reporting; Research; Resolution; Role; Running; Site; Small Nuclear RNA; Specificity; Spectrum Analysis; Spliceosome Assembly Pathway; Spliceosomes; Structure; Substrate Interaction; Substrate Specificity; Surface; Syndrome; Testing; Training; Transcription Process; U5 small nuclear RNA; UBE2D2 gene; Untranslated RNA; Variant; Work; Yeasts; biophysical techniques; career; cyanine dye 5; early onset; fluorophore; helicase; human disease; insight; mRNA Precursor; post-doctoral training; posttranscriptional; preference; protein complex; real time monitoring; recruit; single molecule; therapy development

Project Summary In eukaryotic organisms, transcribed RNA is processed from precursor messenger RNA (pre-mRNA) into mature RNA in a process known as splicing. During this RNA processing mechanism, the non-coding regions of pre-mRNA are removed, and the flanking regions are joined by a large molecular machine known as the spliceosome. Spliceosomes do not exist pre-assembled into splicing active conformations. Instead, splice sites (SS) are specifically chosen through the stepwise assembly of five small nuclear ribonuclear protein complexes consisting of a small nuclear RNA and a large number of associated proteins. These spliceosome assemblies are charged with correctly identifying and juxtaposing splice sites that are not explicitly sequence encoded in the pre-mRNA. Adding to the complexity of splice site selection, >90-95% of human pre-mRNAs are alternatively spliced by varying the configuration of which regions are joined and which are removed from multi-exon containing genes. Splicing errors associated with alternative usage of splice sites are implicated in a large number of human diseases such as Hutchinson-Gilford progeria syndrome (alternative 5'SS), dilated cardiomyopathies (alternative 3'SS), Myelodysplastic syndromes (altered 3'SS preference) and early-onset Parkinson Disease (cryptic splice site usage). Despite decades of research to characterize splicing mechanisms, the mechanisms that control splice site usage are incompletely understood. To fill this knowledge gap, the long- term goal of the candidate is to characterize the mechanisms that control splice site selection and the splicing factors involved. In this project, I propose to investigate protein-driven RNA rearrangements during splicing catalysis using single-molecule fluorescence microscopy methods through three specific aims. In aim 1, I will implement a single molecule Förster resonance energy transfer (smFRET) approach to characterize a conserved spliceosome rearrangement driven by the Prp22 helicase that leads to displacement of ligated mRNA from a conserved region in the spliceosome catalytic core, U5 snRNA loop 1. A Prp22 variant will be used to stall spliceosomes onto a surface immobilized pre-mRNA just after exon ligation but prior to release from the spliceosome. Prp22-driven displacement of the ligated mRNA will subsequently be monitored using fluorescent reporters installed on U5 snRNA loop 1 and the RNA substrate, respectively. Specific Aims 2 and 3 propose the investigation of a human-specific protein, FAM32A, hypothesized to stabilize the interaction between the 5' exon and U5 loop 1 in order to facilitate ligation to the 3' SS. Together, this work will answer questions about conserved and metazoan-specific mechanisms involved in the late stages of pre-mRNA splicing catalysis. This project will advance the applicant's career goal of running an independent laboratory at an academic institution in a way that combines her graduate training in mechanistic enzymology with her ongoing postdoctoral training in RNA molecular biology and biophysics to characterize the mechanisms and assembly of complex macromolecular machines whose proper functions are vital to human health.

项目摘要 在真核生物中，将转录的RNA从前体信使RNA（前MRNA）处理到 在称为剪接的过程中成熟的RNA。在此RNA处理机制中，非编码区域 去除前mRNA，侧翼区域被称为 剪接体。剪接体不存在于剪接活性构象中。相反，剪接站点 （SS）是通过逐步组装的五个小核核糖核蛋白复合物来选择的 由小的核RNA和大量相关蛋白组成。这些剪接组件 负责正确识别和并置的剪接位点，这些剪接位点未明确编码在 前mRNA。增加了剪接站点选择的复杂性，> 90-95％的人类Pre MRNA替代 通过改变连接的区域的配置并从多exon中删除的配置来剪接 包含基因。与剪接站点的替代用法相关的剪接错误暗示在一个大的 人类疾病的数量，例如Hutchinson-Gilford Progeria综合征（替代5'S），扩张 心肌病（替代3'S），骨髓增生综合征（改变3's偏好）和早发性 帕金森氏病（隐秘的剪接现场使用）。尽管研究了数十年来表征剪接机制，但 控制剪接站点使用的机制尚不完全理解。为了填补这一知识差距，长期 候选人的术语目标是表征控制剪接站点选择和剪接的机制 涉及的因素。在这个项目中，我建议在剪接过程中研究蛋白质驱动的RNA重排 通过三个特定目的使用单分子荧光显微镜方法进行催化。在AIM 1中，我会 实施单个分子förster共振能量传递（SMFRET）方法来表征保守的 由PRP22解旋酶驱动的剪接体重排，导致连接的mRNA位移 剪接体催化核心中的保守区域，U5 snRNA环1。 外显子连接后，剪接到固定的前MRNA的表面，但在从释放之前 剪接体。随后将使用荧光监测带有连接mRNA的PRP22驱动的位移 记者分别安装在U5 snRNA循环1和RNA底物上。具体目的2和3提案 对人类特异性蛋白（FAM32A）的研究，假设是为了稳定5'外显子之间的相互作用 和U5循环1，以促进与3'SS结扎。一起，这项工作将回答有关配置的问题 以及参与前MRNA剪接催化的晚期涉及的特异性机制。这个项目将 促进申请人以某种方式在学术机构运营独立实验室的职业目标 这将她在机械酶学领域的研究生培训与她正在进行的RNA的博士后培训相结合 分子生物学和生物物理学以表征复合大分子的机制和组装 适当功能对人类健康至关重要的机器。