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: 10351379
• 负责人: Elizabeth C Duran
• 金额: $ 10万
• 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 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10351379
• 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; Event; Exons; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Gene Expression; Genes; Genetic; Goals; Health; Hela Cells; Human; Immobilization; Immunoglobulin Joining Region; Impairment; Institution; Introns; Investigation; Kinetics; Knowledge; Label; Laboratories; Lead; 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; 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; base; biophysical techniques; career; cyanine dye 5; early onset; fluorophore; helicase; human disease; human error; insight; mRNA Precursor; post-doctoral training; 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RNA的剪接过程。在这种RNA加工机制中， 前体mRNA被去除，侧翼区域由一个大的分子机器连接起来， 剪接体剪接体不存在预先组装成剪接活性构象。相反，剪接位点 (SS)是通过五个小的核核糖核蛋白复合物的逐步组装而特别选择的 由一个小的核RNA和大量的相关蛋白质组成。这些剪接体组装体 负责正确识别和并列剪接位点，这些剪接位点不是在细胞中明确编码的序列。 前mRNA。增加了剪接位点选择的复杂性，>90-95%的人前体mRNA可选择性地被剪接。 通过改变连接区域和从多外显子移除区域的构型来剪接 含有基因。与剪接位点的选择性使用相关的剪接错误涉及大量的 许多人类疾病，如Hutchinson-Gilford早衰综合征（替代5 'SS），扩张性 心肌病（替代3 'SS）、骨髓增生异常综合征（改变的3' SS偏好）和早发性 帕金森病（隐蔽剪接位点使用）。尽管几十年来研究了剪接机制的特征， 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，长期以来- 候选的术语目标是表征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，我建议研究剪接过程中蛋白质驱动的RNA重排 通过三个特定的目标，使用单分子荧光显微镜方法进行催化。在目标1中，我将 实施单分子Förster共振能量转移（smFRET）方法来表征保守的 由Prp 22解旋酶驱动的剪接体重排，导致连接的mRNA从 剪接体催化核心中的保守区，U 5 snRNA环1。Prp 22变体将用于失速 在外显子连接后但在从膜释放之前，将剪接体剪接到表面固定的前mRNA上。 剪接体Prp 22驱动的连接的mRNA的置换随后将使用荧光标记进行监测。 报告基因分别安装在U 5 snRNA环1和RNA底物上。具体目标2和3建议， 一种人类特异性蛋白质FAM 32A的研究，该蛋白质被假设为稳定5'外显子之间的相互作用。 和U 5环1，以便于连接到3' SS。总之，这项工作将回答有关保守的问题， 和后生动物特异性机制参与前体mRNA剪接催化的晚期阶段。该项目将 推进申请人在学术机构运行独立实验室的职业目标， 该研究所将她在机械酶学方面的研究生培训与她正在进行的RNA博士后培训相结合 分子生物学和生物物理学来表征复杂大分子的机制和组装 这些机器的正常功能对人类健康至关重要。