Novel Tools to Investigate Local and Global RNA Conformations in 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(外显子)被分开，由 含有多达数万个核苷酸的内含子。去除内含子，即所谓的“剪接”，是一个关键 基因表达的一步，允许精致的微调调节，并通过选择性剪接， 使单个基因多样化为一种以上的蛋白质。剪接由剪接体执行，剪接体是多个- 其功能需要前信使RNA之间相互作用的兆达顿大分子复合体 (前信使核糖核酸)底物，五个小核糖核蛋白颗粒(SNRNP)，以及许多额外的 蛋白质因素。确定这些组件的角色和它们之间的相互作用对于 了解许多人类疾病的分子机制，在这些疾病中异常剪接是 牵连其中。 最近单分子显微镜在剪接体中的应用揭示了 剪接的分子机制。然而，SnRNAs和Pre-mRNA之间的相互作用 由于制备位点特异的荧光团的SnRNA的挑战，仍然很难进行探测 贴上了标签。此外，只能在特定的长度尺度上跟踪构象变化，受 使用的实验技术的灵敏度，这些技术通常基于Förster共振能量转移 (烦躁)。为了应对这些挑战，具体目标1将研究U5之间相互作用的重新安排 小核糖核酸和前信使核糖核酸解旋酶Prp22的作用。地点--特别是荧光团-- 标记的U5将通过使用短肽核酸齐聚物来阻止T7转录而制备 RNA聚合酶在所需的标记位置，这是一种通用的方法，可以避免其他方法的许多缺点 RNA标记方法。特定目标2提出了FRET滤波光谱(FFS)的新技术， 它将利用两个间隔很近的荧光团作为FRET供体，并使用额外的荧光团作为FRET供体 接受者。FFS将使用两个捐赠者之间的电子耦合来揭示他们的局部构象 它们与受体的距离的功能，并可以扩展到利用任何类型的荧光检测 光谱学作为读数。这项技术将应用于Cy3和Cy5标记的RNA来研究解离 Prp22对RNA双链的影响。特定目标3将目标1的标记方法与FFS相结合，利用FRET- 滤过性圆二色谱检测大鼠脑内局部前信使核糖核酸构象的变化 作为纯化的BACT中间体的分支点附近的腺苷通过剪接的第一步被追逐。 这项工作将回答有关局部和全球RNA之间的相关性的长期问题 剪接体中的构象，并涉及可推广到许多不同的新方法 生物系统。目标1和目标2的初步实验将在 申请人的研究导师，而目标2将完成，目标3将在#年启动和完成 申请人的独立实验室。 在授奖的指导阶段，申请者将在#年的密歇根大学工作 尼尔斯·沃尔特博士的实验室，他在培养成功的科学家方面有着很好的记录。申请人有 组建了一个咨询委员会，与沃尔特博士一起，将为她的研究和她的 过渡到独立的职业生涯。申请者的职业目标包括经营一家独立的实验室 在一家学术机构，她寻求将她在光谱学方面的研究生培训与正在进行的 核糖核酸分子生物学和生物物理学博士后培训。除了提供仪器之外 对于拟议的研究来说，密歇根大学主办了许多组织和活动， 将有助于申请者的培训和职业发展。这份建议书建立在申请人的所有 之前和正在进行的培训打开了了解剪接体功能的独特窗口。

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.