Protein RNA Rearrangements in the Spliceosome

摘要 剪接体从前体信使rna中剔除内含子是几乎所有 人类的基因表达。这一过程受到高度调控，与基因的转录和 其他处理事件，如多聚腺苷化和核苷酸修饰。 剪接体识别化学事件的确切位置以及如何识别的机制 这些反应是被催化的，目前还不是很清楚。该项目的长期目标是了解 剪接体成分与作为底物的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，要素的多重重新安排和多重 分支机构现场的表彰活动。对这些事件的调查--这些事件不被理解 机械-将阐明核心组件之间的相互作用和重新安排，并可能作为 剪接体和其他RNP机器中重排的范例。这项提案的重点是 剪接体动力学影响剪接保真度的机制。 该项目的目标集中在剪接体和SnRNA的两个新发现的方面： (1)研究六磷酸肌醇(IP6)在剪接体功能中的作用。这个小分子 最近被冷冻-EM意外地确定为剪接复合体的一部分。没有生物化学或遗传学 到目前为止已经探索了它的重要性。我们利用IP6生物合成途径中的突变体来探索其贡献。 剪接以及肌醇多聚磷酸(IPP)异构体水平是否会影响剪接事件。(2)一洞 在我们目前对RNA生物学的理解中，SnRNA突变在疾病中的作用。有两个突变是 最近发现在U1SnRNA的5‘端，与胶质母细胞瘤(GBM)和其他癌症有关。我们会 使用我们的电子单链RNA基因组来查询TCGA全基因组数据集(WGS)并研究，在 特别是，我们在膀胱癌和膀胱癌类器官中发现的U2 SnRNA突变高度丰富 台词。

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.