Spliceosome Mechanism Dissected at the Single Molecule Level

单分子水平剖析剪接体机制

• 批准号: 8260192
• 负责人: NILS G WALTER
• 金额: $ 29.63万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8260192
• 关键词: 3' Splice Site; Address; Affect; Affinity; Affinity Chromatography; Alternative Splicing; Animal Model; Binding; Biochemical; Biological; Biological Assay; Biological Models; Biological Process; Boxing; Catalysis; Cells; Characteristics; Chemicals; Code; Complement; Complex; DNA Sequence Rearrangement; Data; Detection; Disease; Disputes; Dissociation; Equilibrium; Eukaryota; Event; Excision; Exhibits; Exons; Funding; Genes; Genetic; Human; In Situ Hybridization; In Vitro; Individual; Introns; Kinetics; Knowledge; Label; Lead; Ligation; Lighting; Molecular Conformation; Mutation; Names; Pattern; Peptide Signal Sequences; Process; Protein Isoforms; Proteins; Proteome; RNA; RNA Helicase; RNA Splicing; Role; Saccharomyces cerevisiae; Saccharomycetales; Signal Transduction; Site; Small Nuclear RNA; Spliceosome Assembly Pathway; Spliceosomes; Structure; Techniques; Testing; Time; Tissues; Transcript; U1 Small Nuclear Ribonucleoprotein; Yeasts; fluorophore; follow-up; helicase; human disease; mRNA Precursor; markov model; mutant; protein complex; single molecule; single-molecule FRET; tool

DESCRIPTION (provided by applicant): Spliceosome mechanism dissected at the single molecule level ABSTRACT: The spliceosome is a multi-megadalton RNA-protein complex that catalyzes in all eukaryotes the removal of introns and the ligation of exons during splicing of pre mRNAs. In humans, 94% of all pre-mRNAs undergo alternative splicing, which allows for the dynamic expression of various protein isoforms from a single gene through cell- and tissue-specific networks of regulated splicing events. It is estimated that up to 50% of all mutations leading to human disease act through disrupting the splicing code. Due to the availability of unique genetic and biochemical manipulation tools, the budding yeast Saccharomyces cerevisiae has long provided a central model system for dissecting the mechanism of eukaryotic pre-mRNA splicing. Despite 25 years of study, however, there is still little known about the compositional and conformational rearrangements, timing, and coordination associated with yeast spliceosome function. To address this challenge, we recently have developed single molecule fluorescence resonance energy transfer (smFRET) assays that have begun to dissect pre-mRNA conformational changes during splicing. In particular, we have identified a small, efficiently spliced yeast pre-mRNA, in which donor and acceptor fluorophores could be placed in the exons adjacent to the 5' and 3' splice sites, and have used it to show that the spliceosome operates close to thermal equilibrium. Here, we propose to follow up on this advance and begin to dissect the mechanism of splicing at the single molecule level. In Specific Aim 1, we will test the hypothesis that specific sets of conformational fluctuations lead to splicing, by adding to our tool set: (i) shuttered illumination combined with advanced hidden Markov modeling and in situ hybridization to faithfully track the conformational dynamics of single pre- mRNA substrate molecules over the entire time course of splicing; (ii) depletion-complementation approaches to introduce functionally active, fluorophore labeled small nuclear RNA (snRNA) and protein components of the spliceosome for coincidence analysis (CIA); (iii) covalent, small-tag fluorophore labeling approaches to non-invasively mark functional protein factors of the spliceosome; and (iv) an optimized affinity purification technique to isolate specific spliceosomal complexes with fluorophore labeled components for focused probing. In Specific Aim 2, we will follow up on our observation that our intron exhibits significant secondary structure, placing its flanking exons much closer than expected from their linear sequence distance. We will test the hypothesis that this secondary structure has a functional impact by introducing a systematic set of mutations that first impair, and then restore the predicted secondary structure, and by testing each mutant for splicing. In Specific Aim 3, we will dissect the mechanistic role of DExD/H-box helicase Prp2 in preparing the activated Bact. spliceosome for the first step of splicing by rearranging it into the B* complex with exposed pre- mRNA branch point. Taken together, these advances will pave the way for, over the funding period, extensive mechanistic studies of yeast splicing and for studying alternative splicing in humans in the longer term. PUBLIC HEALTH RELEVANCE: The spliceosome is the multi-megadalton RNA-protein complex present in all eukaryotes that catalyzes the removal of introns from pre-mRNAs. As a finely tuned process of great complexity and critical importance to the diversification of the proteome, it is thought that up to 50% of all mutations connected to human disease act through disrupting splicing. In this project, splicing in a model organism, baker's yeast, will be mechanistically dissected at an unprecedented single molecule level with the prospect of paving the way for studying human splicing diseases.

描述（由申请人提供）：在单分子水平上剖析的剪接体机制 摘要：剪接体是一种多兆道尔顿的 RNA-蛋白质复合物，在所有真核生物中，在前体剪接过程中催化内含子的去除和外显子的连接。 mRNA。在人类中，94% 的前 mRNA 会经历选择性剪接，从而允许单个基因通过受调控剪接事件的细胞和组织特异性网络动态表达各种蛋白质亚型。据估计，导致人类疾病的所有突变中，高达 50% 是通过破坏剪接代码而起作用的。由于独特的遗传和生化操作工具的可用性，芽殖酵母酿酒酵母长期以来为剖析真核前体 mRNA 剪接机制提供了一个中心模型系统。然而，尽管经过 25 年的研究，人们对与酵母剪接体功能相关的组成和构象重排、时间安排和协调仍然知之甚少。为了应对这一挑战，我们最近开发了单分子荧光共振能量转移 (smFRET) 测定法，该测定法已开始剖析剪接过程中前 mRNA 构象的变化。特别是，我们已经鉴定了一种小的、有效剪接的酵母前 mRNA，其中供体和受体荧光团可以放置在邻近 5' 和 3' 剪接位点的外显子中，并用它来表明剪接体的运作密切相关。达到热平衡。在这里，我们建议跟进这一进展，并开始在单分子水平上剖析剪接机制。在具体目标 1 中，我们将通过添加到我们的 工具集：(i) 快门照明与先进的隐马尔可夫模型和原位杂交相结合，忠实地跟踪单个前 mRNA 底物分子在整个剪接过程中的构象动态； (ii) 耗竭-互补方法，引入功能活性、荧光团标记的小核 RNA (snRNA) 和剪接体的蛋白质成分，用于符合分析 (CIA)； (iii) 共价小标签荧光团标记方法，用于非侵入性标记剪接体的功能蛋白因子； (iv) 优化的亲和纯化技术以分离特定的剪接体 具有荧光团标记成分的复合物，用于聚焦探测。在具体目标 2 中，我们将继续观察我们的内含子表现出显着的二级结构，使其侧翼外显子的线性序列距离比预期的要近得多。我们将通过引入一组系统突变来测试这种二级结构具有功能影响的假设，这些突变首先会损害然后恢复预测的二级结构，并测试每个突变体的剪接。在具体目标 3 中，我们将剖析 DExD/H-box 解旋酶 Prp2 在制备激活的 Bact 中的机制作用。剪接体通过将其重新排列成具有暴露的前mRNA分支点的B*复合物来进行剪接的第一步。总而言之，这些进展将为在资助期内对酵母剪接进行广泛的机制研究以及从长远来看人类选择性剪接的研究铺平道路。 公共健康相关性：剪接体是所有真核生物中存在的多兆道尔顿 RNA-蛋白质复合物，可催化前体 mRNA 中内含子的去除。作为一个极其复杂且对蛋白质组多样化至关重要的精细调节过程，人们认为与人类疾病相关的所有突变中有高达 50% 通过破坏剪接起作用。在这个项目中，模型生物——面包酵母中的剪接将以前所未有的单分子水平进行机械解剖，有望为研究人类剪接疾病铺平道路。