Spliceosome Mechanism Dissected at the Single Molecule Level

单分子水平剖析剪接体机制

• 批准号: 8415518
• 负责人: NILS G WALTER
• 金额: $ 27.22万
• 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/8415518
• 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.

描述（由申请人提供）：在单分子级别上剖析的剪接体机制摘要：剪接体是一种多麦达尔顿RNA - 蛋白质复合物，在所有真核生物中催化了内含子的去除和外显子在Pre pre splin splin pre splining pre splin的结扎率的催化 mrnas。在人类中，所有前MRNA中有94％都经历替代剪接，这允许从单个基因通过细胞和组织特异性剪接事件的细胞和组织特异性网络的各种蛋白质同工型的动态表达。据估计，通过破坏剪接代码的所有导致人类疾病的突变的最多50％。由于具有独特的遗传和生化操纵工具的可用性，酿酒酵母的萌芽酵母菌已提供了一种中央模型系统，用于剖析真核前mRNA剪接的机制。尽管进行了25年的研究，但仍然对与酵母剪接体功能相关的组成和构象重排，时间和协调鲜为人知。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，这些分子已开始在拼接过程中剖析MRNA前构象变化。特别是，我们已经确定了一个小的，有效剪接的酵母前MRNA，其中可以将供体和受体荧光团放置在与5'和3'剪接位点相邻的外显子中，并使用它来表明剪接体接近热平衡。在这里，我们建议对这一进步进行跟进，并开始剖析单分子水平的剪接机理。在特定目标1中，我们将通过添加到我们的 工具集：（i）关闭照明与先进的隐藏马尔可夫建模和原位杂交相结合，以忠实地跟踪整个拼接过程中单个Pre-mRNA底物分子的构象动力学； （ii）耗尽融合方法，引入功能活性，荧光团标记为小核RNA（SNRNA）和剪接组的蛋白质成分，以进行巧合分析（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性蛋白质因子； （iv）一种优化的亲和力纯化技术，用于分离特定的剪接体 具有荧光团标记成分的复合物，用于聚焦探测。在特定的目标2中，我们将跟进我们的观察结果，即我们的内含子表现出重要的二级结构，其侧翼外显子比线性序列距离的预期要近得多。我们将测试以下假设：该二级结构通过引入系统损害的系统突变集，然后恢复预测的二级结构，并通过测试每个突变体进行剪接，从而具有功能影响。在特定的目标3中，我们将剖析DEXD/H盒解旋酶PRP2在准备活化的Bact中的机械作用。剪接剪接的第一步是通过将其重新排列到B*复合物中，并带有裸露的mRNA分支点。综上所述，这些进步将为资助期间的酵母菌剪接和研究人类的替代剪接的广泛机理研究铺平道路。