Protein RNA Rearrangements in the Spliceosome

摘要 剪接体从前体信使RNA中切除内含子是几乎所有基因组中的关键步骤。 人类基因表达。这一过程受到高度调控，与基因转录密切相关， 其他加工事件，如聚腺苷酸化和核苷酸修饰。 剪接体识别化学事件的确切位点的机制以及如何识别化学事件的确切位点的机制 催化的反应还没有很好地理解。这个项目的长期目标是了解 剪接体成分和作为底物的RNA配体之间的相互作用和重排 用于催化反应。充分的证据表明，多种因素和多重 在分支站点的识别事件。对这些事件的调查-不了解 机制-将阐明核心组件之间的相互作用和重排，并可能作为一个 在spliceosome和其他RNP机器重排的范例。该提案的重点是 剪接体动力学影响剪接保真度的机制。 该项目的目的集中在剪接体和snRNAs的两个新发现的方面： (1)我们将研究肌醇六磷酸（IP 6）在剪接体功能中的作用。这种小分子 最近被冷冻电镜意外地鉴定为剪接复合物的一部分。没有生物化学或遗传学 到目前为止，他已经探索了它的重要性。我们使用IP 6生物合成途径中的突变体来探索其贡献 以及多磷酸肌醇（IPP）同工型水平是否会影响剪接事件。(2)一个孔 在我们目前对RNA生物学的理解中，最重要的是snRNA突变在疾病中的作用。两个突变是 最近在U1 snRNA的5 '端描述，与胶质母细胞瘤（GBM）和其他癌症相关。我们将 使用我们的计算机snRNA基因组来询问TCGA全基因组数据集（WGS），并研究， 特别是我们发现在膀胱癌和膀胱癌类器官中高度富集的U2 snRNA突变， 线

ABSTRACT Excision of introns from precursor messenger RNA by the spliceosome is a critical step in almost all human gene expression. This process is highly regulated, integrally linked with the transcription of genes and other processing events, such as polyadenylation and nucleotide modification. The mechanism by which the spliceosome recognizes the exact sites for the chemical events and how the reactions are catalyzed are not well understood. The long-term goals of this project are to understand interactions and rearrangements between spliceosome components and the RNA ligands that are substrates for the catalytic reactions. Ample evidence argues for multiple rearrangements of factors and multiple recognition events at the branch site. Investigation of these events — which are not understood mechanistically — will elucidate interactions and rearrangements among core components and may serve as a paradigm for rearrangements in the spliceosome and in other RNP machines. This proposal focuses on mechanisms by which spliceosomal dynamics impact splicing fidelity. The Aims of this project focus on two newly-discovered aspects of the spliceosome and snRNAs: (1) We will investigate the role of inositol hexakisphosphate (IP6) in spliceosome function. This small molecule was recently identified unexpectedly by cryo-EM as part of splicing complexes. No biochemistry or genetics has so far explored its importance. We use mutants in the IP6 biosynthetic pathway to explore its contribution to splicing and whether inositol polyphosphate (IPP) isoform levels may influence splice events. (2) One hole in our current understanding of RNA biology is the role of snRNA mutations in disease. Two mutations were recently described in the 5'-end of U1 snRNA, associated with glioblastoma (GBM) and other cancers. We will use our in silico snRNA genome to interrogate TCGA whole genome datasets (WGS) and investigate, in particular, U2 snRNA mutations that we find highly enriched in bladder cancer and bladder cancer organoid lines.