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: 10894365
• 负责人: Elizabeth C Duran
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 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 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10894365
• 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，转录的rna从前体信使rna(pre-mrna)加工成 成熟的RNA在一个被称为剪接的过程中。在这个RNA处理机制中，非编码区 前-信使核糖核酸被移除，侧翼区域由一个被称为 剪接体。剪接体并不存在预先组装成剪接活性构象的现象。相反，拼接站点 (SS)是通过五个小的核核糖核蛋白复合体的逐步组装而具体选择的 由一个小的核糖核酸和大量的相关蛋白质组成。这些剪接体组件 负责正确识别和并列未在 前信使核糖核酸。增加剪接位点选择的复杂性，&gt；90%-95%的人类前mRNAs是交替的 通过改变连接和移除多外显子的区域的配置进行拼接 含有基因。与剪接位点的替代使用相关的剪接错误在很大程度上涉及 Hutchinson-Gilford早衰症(替代5‘SS)等人类疾病的数量扩大 心肌病(替代3‘SS)、骨髓增生异常综合征(改变3’SS偏好)和早发性 帕金森病(神秘剪接部位的使用)。尽管几十年来一直在研究剪接机制， 控制剪接位点使用的机制还不完全清楚。为了填补这一知识空白，长期的- 候选人的学期目标是描述控制剪接位点选择和剪接的机制 涉及的因素。在这个项目中，我提议研究剪接过程中蛋白质驱动的rna重排。 利用单分子荧光显微镜方法通过三个特定的目标进行催化。在《目标1》中，我将 实现单分子Förster共振能量转移(SmFRET)方法来表征守恒的 Prp22解旋酶驱动的剪接体重排导致连接的mRNA从 剪接体催化核心的保守区，U5 SnRNA环1。Prp22变体将被用来拖延 外显子连接后，将小体剪接到表面固定的前-mRNA上，然后从 剪接体。Prp22驱动的连接的mRNA的位移将随后用荧光进行监测 记者分别安装在U5的SnRNA环1和RNA底物上。具体目标2和3提出了 一种人类特有的蛋白质FAM32A的研究假设稳定了5‘外显子之间的相互作用 和U5环1，以便于与3‘SS的连接。总之，这项工作将回答有关保护动物的问题 以及后生动物特有的机制，涉及前mRNA剪接催化的后期阶段。这个项目将 在一定程度上推进申请者在学术机构运营独立实验室的职业目标 这结合了她在机械酶学方面的研究生培训和她正在进行的RNA博士后培训 分子生物学和生物物理学表征复杂大分子的作用机制和组装 其适当功能对人类健康至关重要的机器。