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：
9164146
负责人：
Julia Reed Widom
金额：
$ 9万
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：
2016
资助国家：
美国
起止时间：
2016-09-16 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9164146
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 Electronics Energy Transfer Eukaryota Event Excision Exciton Exhibits Exons Fluorescence Fluorescence Resonance Energy Transfer Gene Expression Genes Genetic Transcription Goals Hereditary Disease Human Human Genetics Institution Introns Joining Exons 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 fluorophore human disease instrumentation intein literature survey mRNA Precursor novel post-doctoral training prevent research study 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.

项目摘要在真核生物中，绝大多数基因的蛋白质编码区域（外显子）分裂，被分开含有多达数万个核动脉的介绍。删除介绍称为“拼接”，是一个关键一步一步，允许精确调节调节，并通过替代剪接，将单个基因多样化为多种蛋白质。剪接是由剪接执行的 Megadalton大分子复合物的功能需要在预选前RNA之间进行相互作用（前MRNA）底物，五个小核核糖核蛋白颗粒（SNRNP），还有许多额外的蛋白质因子。确定这些组件之间的作用和相互作用对于了解许多人类疾病的分子机制在其中暗示。单分子显微镜在剪接体中的最新应用已大大阐明了剪接的分子机制。但是，snRNA和前mRNA之间的相互作用具有由于制备SNRNA的挑战，这些snRNA是特定于位点的荧光团 - 标记。此外，只能在某些长度尺度上跟踪构象变化，受到限制所使用的实验技术的敏感性通常基于Förster共振能量传递（烦恼）。为了应对这些挑战，具体目标1将研究U5之间相互作用的重排 SnRNA和前MRNA响应RNA解旋酶PRP22的作用。特定于现场的荧光团标记为U5将通过使用短肽核酸低聚物来制备T7的转录所需标记位点的RNA聚合酶，一种避免其他弊端的一般方法 RNA标记方法。特定目标2提案提出的新技术滤光光谱法（FFS），它将利用两个紧密间隔的荧光团作为粮食供体，以及一个额外的荧光团作为一个受体。 FFS将使用两个捐赠者之间的电子耦合来揭示其局部构象作为一个它们与受体的距离的功能，可以扩展以利用任何类型的荧光检测光谱作为读数。该技术将应用于CY3和CY5标记的RNA来研究放松 PRP22的RNA双链体。特定的AIM 3使用FRET-将AIM 1的标签方法与FFS结合在一起过滤的圆二色性光谱法确定局部前MRNA构象的变化分支点腺苷附近作为纯化的BACT中间体通过剪接的第一步挑战。这项工作将回答有关本地和全球RNA之间相关性的长期问题剪接体中的构象，涉及可以推广到许多不同的新方法生物系统。 AIM 1和AIM 2的最初实验将在实验室中进行申请人的研究心理，而AIM 2将完成，AIM 3将启动和完成申请人的独立实验室。在该奖项的修订阶段，申请人将在密歇根大学工作尼尔斯·沃尔特（Nils Walter）博士的实验室，他在培训成功的科学家方面有很强的记录。该应用程序具有组建了一个咨询委员会，该委员会与沃尔特博士一起为她的研究提供指导过渡到独立职业。申请人的职业目标涉及运营独立实验室在一家学术机构，她试图将光谱学研究生培训与她的持续培训相结合 RNA分子生物学和生物物理学的博士后培训。除了提供仪器拟议的研究必要的，密歇根大学举办了许多组织和活动将有助于申请人的培训和职业发展。该提案建立在所有申请人的基础上以前和正在进行的培训，可以打开一个独特的窗口，以了解剪接体的功能。