权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Novel Tools to Investigate Local and Global RNA Conformations in the Spliceosome

研究剪接体中局部和整体 RNA 构象的新工具

基本信息

批准号：
9814290
负责人：
Julia Reed Widom
金额：
$ 24.9万
依托单位：
UNIVERSITY OF OREGON
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-16 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9814290
关键词：
5&apos Splice Site Address Adenosine Adopted Advisory Committees Alternative Splicing Award Binding Biochemical Biological Biophysics Circular Dichroism Circular Dichroism Spectroscopy Code Complex Coupled Coupling DNA Defect Detection Disease Eukaryota Event Excision Exciton Exhibits Exons Fluorescence Fluorescence Resonance Energy Transfer Gene Expression Genes Genetic Diseases Genetic Transcription Goals Human Human Genetics Institution Introns Label Laboratories Length Light Macromolecular Complexes Measurement Measures Mentors Messenger RNA Methods Michigan Microscopy Modeling Molecular Molecular Biology Molecular Conformation Mutation Nucleic Acid Conformation Nucleotides Open Reading Frames Pathway interactions Peptide Nucleic Acids Phase Process Protein Biosynthesis Proteins Protocols documentation RNA RNA Conformation RNA Helicase RNA Splicing Regulation Reporting Research Role Running Sampling Scientist Signal Transduction Site Small Nuclear RNA Small Nuclear Ribonucleoproteins Spectrum Analysis Spliceosomes Structure System T7 RNA polymerase Techniques Testing Training U5 small nuclear RNA Universities Work analog base biological systems career career development chromophore cyanine dye 5 dimer experimental study fluorophore human disease instrumentation intein literature survey mRNA Precursor novel post-doctoral training prevent response single molecule therapy development tool

项目摘要

Project Summary In eukaryotes, the vast majority of genes have their protein-coding regions (exons) split up, separated by introns containing up to tens of thousands of nucleotides. The removal of introns, called “splicing”, is a critical step in gene expression that allows for exquisitely fine-tuned regulation and, through alternative splicing, diversifies a single gene into more than one protein. Splicing is executed by the spliceosome, a multi- megaDalton macromolecular complex whose function requires interactions between the pre-messenger RNA (pre-mRNA) substrate, five small nuclear ribonucleoprotein particles (snRNPs), and numerous additional protein factors. Determining the roles of and interactions between these components is of central importance to understanding the molecular mechanisms of the many human diseases in which aberrant splicing is implicated. The recent application of single-molecule microscopy to the spliceosome has shed much light on the molecular mechanism of splicing. However, the interactions between the snRNAs and the pre-mRNA have remained difficult to probe due to the challenge of preparing snRNAs that are site-specifically fluorophore- labeled. Furthermore, conformational changes can be tracked only on certain length scales, limited by the sensitivity of the experimental techniques used, which are often based on Förster resonance energy transfer (FRET). To address these challenges, Specific Aim 1 will study the rearrangement of interactions between U5 snRNA and the pre-mRNA in response to the action of RNA helicase Prp22. Site-specifically fluorophore- labeled U5 will be prepared through by using a short peptide nucleic acid oligomer to stall transcription by T7 RNA polymerase at the desired labeling site, a general approach that avoids many of the downsides of other RNA labeling methods. Specific Aim 2 proposes the novel technique of FRET-filtered spectroscopy (FFS), which will utilize two closely spaced fluorophores as a FRET donor, and an additional fluorophore as an acceptor. FFS will use electronic coupling between the two donors to reveal their local conformation as a function of their distance from the acceptor, and can be expanded to utilize any type of fluorescence-detected spectroscopy as a readout. This technique will be applied to Cy3- and Cy5-labeled RNA to study the unwinding of RNA duplexes by Prp22. Specific Aim 3 combines the labeling method of Aim 1 with FFS, utilizing FRET- filtered circular dichroism spectroscopy to determine the changes in local pre-mRNA conformation in the vicinity of the branchpoint adenosine as purified Bact intermediates are chased through the first step of splicing. This work will answer longstanding questions about the correlations between local and global RNA conformations in the spliceosome, and involves novel methods that can be generalized to many different biological systems. Aim 1 and the initial experiments for Aim 2 will be pursued in the laboratory of the applicant's research mentor, while Aim 2 will be completed and Aim 3 will be both initiated and completed in the applicant's independent laboratory. During the mentored phase of the award, the applicant will be working at the University of Michigan in the laboratory of Dr. Nils Walter, who has a strong record of training successful scientists. The applicant has assembled an advisory committee who, together with Dr. Walter, will provide guidance on her research and her transition into an independent career. The applicant's career goals involve running an independent laboratory at an academic institution, and she seeks to combine her graduate training in spectroscopy with her ongoing postdoctoral training in RNA molecular biology and biophysics. In addition to providing the instrumentation necessary for the proposed research, the University of Michigan hosts numerous organizations and events that will contribute to the applicant's training and career development. This proposal builds on all of the applicant's previous and ongoing training to open a unique window into the function of the spliceosome.

项目总结