Spliceosome Mechanism Dissected at the Single Molecule Level

描述（由申请人提供）：在单分子水平上剖析剪接体机制摘要：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，它催化前体剪接过程中内含子的去除和外显子的连接。 mRNA。在人类中，94%的前体mRNA经历选择性剪接，这允许通过调节剪接事件的细胞和组织特异性网络从单个基因动态表达各种蛋白质亚型。据估计，高达50%的导致人类疾病的所有突变通过破坏剪接密码发挥作用。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提供了一个中央模型系统解剖真核前mRNA剪接的机制。尽管25年的研究，然而，仍然是知之甚少的组成和构象重排，时间和协调与酵母剪接体功能。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，已开始解剖前mRNA的构象变化在剪接。特别是，我们已经确定了一个小的，有效剪接的酵母前mRNA，其中供体和受体荧光团可以被放置在邻近5'和3'剪接位点的外显子，并已使用它来显示剪接体接近热平衡运行。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。在具体目标1中，我们将测试特定构象波动导致剪接的假设，通过添加我们的 工具集：（i）与先进的隐马尔可夫建模和原位杂交相结合的快门照明，以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，以引入功能活性的荧光团标记的小核RNA（snRNA）和剪接体的蛋白质组分，用于重合分析（CIA）;（iii）共价、小标签荧光团标记方法，以非侵入性地标记剪接体的功能蛋白因子;和（iv）优化的亲和纯化技术，以分离特异性剪接体 用于聚焦探测的荧光团标记组分的复合物。在具体目标2中，我们将继续我们的观察，即我们的内含子表现出显着的二级结构，将其侧翼外显子比预期的线性序列距离更近。我们将通过引入一组系统的突变，首先损害，然后恢复预测的二级结构，并通过测试每个突变体的剪接，来测试这种二级结构具有功能影响的假设。在具体目标3中，我们将剖析DExD/H-box解旋酶Prp 2在制备活化的Bact中的机制作用。剪接体通过将其重排成具有暴露的前mRNA分支点的B* 复合物用于剪接的第一步。总的来说，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和长期研究人类的选择性剪接铺平道路。

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.