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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：
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
项目类别：
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&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.

项目总结