Spliceosome Mechanism Dissected at the Single Molecule Level

描述(申请人提供)：在单分子水平上剖析的剪接体机制摘要：剪接体是一个多兆吨的RNA-蛋白质复合体，在所有真核生物中催化在剪接过程中去除内含子和连接外显子。 MRNAs。在人类中，94%的前mRNAs经历了选择性剪接，这允许单个基因通过调节剪接事件的细胞和组织特异性网络动态表达各种蛋白质异构体。据估计，导致人类疾病的所有突变中，高达50%是通过扰乱剪接密码来起作用的。由于独特的遗传和生化操作工具的可获得性，萌芽酵母酿酒酵母长期以来一直为剖析真核细胞前mRNA剪接机制提供了一个中心模型系统。然而，尽管经过了25年的研究，与酵母剪接体功能相关的组成和构象重排、时间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)分析方法，该方法已经开始分析剪接过程中前mRNA的构象变化。特别是，我们已经确定了一个小的，高效剪接的酵母Pre-mRNA，其中供体和受体荧光团可以放置在5‘和3’剪接点附近的外显子中，并用它来表明剪接体的操作接近热平衡。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的机制。在特定的目标1中，我们将测试特定的构象波动集合导致剪接的假设，方法是添加 工具集：(I)闭合照明与先进的隐马尔可夫模型和原位杂交相结合，在剪接的整个时间过程中忠实地跟踪单个前mRNA底物分子的构象动力学；(Ii)耗尽-互补方法引入功能活跃的荧光团标记的小核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非侵入性地标记剪接体的功能蛋白因子；以及(Iv)优化的亲和纯化技术以分离特定的剪接体 与荧光团标记的组件的络合物，用于聚焦探测。在具体目标2中，我们将继续观察到我们的内含子显示出显著的二级结构，将其侧翼外显子从其线性序列距离放置得比预期的要近得多。我们将通过引入一组系统的突变来检验这种二级结构具有功能影响的假设，这些突变首先损害，然后恢复预测的二级结构，并测试每个突变体的剪接。在特定的目标3中，我们将剖析DExD/H-box解旋酶Prp2在制备活化的BaT中的机制作用。剪接体用于剪接的第一步，通过将其重新排列成具有暴露的前mRNA分支点的B*复合体。综上所述，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长期研究人类的替代剪接铺平道路。

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.