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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

基本信息

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
项目类别：
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&apos Splice Site 5&apos 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

项目摘要

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.

项目概要在真核生物中，转录的 RNA 从前体信使 RNA (pre-mRNA) 加工成 RNA 的成熟过程称为剪接。在这种 RNA 加工机制中，前 mRNA 被去除，侧翼区域由一个称为“ 剪接体。剪接体不存在预先组装成剪接活性构象的情况。相反，剪接位点 (SS)是通过逐步组装五个小核核糖核蛋白复合物而专门选择的由小核RNA和大量相关蛋白组成。这些剪接体组装体负责正确识别和并置未明确序列编码的剪接位点前mRNA。增加剪接位点选择的复杂性，>90-95% 的人类前 mRNA 是交替的通过改变连接哪些区域以及从多外显子中移除哪些区域的配置来进行剪接含有基因。与剪接位点的替代使用相关的剪接错误涉及大量许多人类疾病，例如 Hutchinson-Gilford 早衰综合症（替代 5'SS），扩张心肌病（替代 3'SS）、骨髓增生异常综合征（改变 3'SS 偏好）和早发型帕金森病（隐秘剪接位点使用）。尽管对剪接机制进行了数十年的研究，控制剪接位点使用的机制尚不完全清楚。为了填补这一知识空白，长期候选人的术语目标是描述控制剪接位点选择和剪接的机制所涉及的因素。在这个项目中，我建议研究剪接过程中蛋白质驱动的 RNA 重排使用单分子荧光显微镜方法进行催化可实现三个特定目标。在目标1中，我会实施单分子福斯特共振能量转移 (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 从剪接体催化核心、U5 snRNA 环 1 中的保守区域。Prp22 变体将用于阻止在外显子连接后但在从前体释放之前将剪接体固定到表面固定的前体mRNA上剪接体。随后将使用荧光监测 Prp22 驱动的连接 mRNA 位移记者分别安装在U5 snRNA环1和RNA底物上。具体目标 2 和 3 提出对人类特异性蛋白质 FAM32A 的研究，假设该蛋白质可稳定 5' 外显子之间的相互作用和 U5 环 1，以便于连接到 3' SS。这项工作将共同回答有关保守的问题以及参与前体 mRNA 剪接催化后期的后生动物特异性机制。该项目将以某种方式推进申请人在学术机构运营独立实验室的职业目标将她的机械酶学研究生培训与正在进行的 RNA 博士后培训结合起来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其正常功能对人类健康至关重要的机器。