Protein RNA Rearrangements in the Spliceosome

剪接体中蛋白质 RNA 重排

基本信息

批准号：
8373631
负责人：
CHARLES C QUERY
金额：
$ 51.14万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-08-01 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8373631
关键词：
3&apos Splice Site ATP phosphohydrolase Active Sites Affect Alternative Splicing Binding Binding Sites Bypass Catalysis Catalytic Domain Chemicals Chromatin Code Commit DNA Sequence Rearrangement Data Defect Development Elements Event Excision Fission Yeast Gene Expression Genetic Screening Genetic Transcription Goals Human Individual Infection Introns Investigation Joining Exons Ligands Link Mammalian Cell Mediating Modeling Modification Nucleotides Pathway interactions Pattern Polyadenylation Positioning Attribute Predisposition Process Proteins RNA RNA Splicing Reaction Rest Role Saccharomyces cerevisiae Series Site Small Nuclear RNA Spliceosome Assembly Pathway Spliceosomes Staging Structure Substrate Interaction System Techniques Testing Time U2 Small Nuclear Ribonucleoprotein U2 small nuclear RNA Yeasts genome-wide helicase improved in vivo mRNA Precursor novel research study single molecule tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): 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. A better understanding of pre- mRNA splicing will be essential to further understand mechanisms that regulate splicing, that control patterns of alternative splicing, and that contribute to development, oncogenesis and retroviral infections. 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 altered spliceosomal dynamics impact splicing fidelity. Experiments will investigate contributions of the U1-U2 protein-interaction network and the ATPase Prp5 to intron definition and to the fidelity of spliceosome assembly. As the first ATP-dependent event, this step provides a unique commitment to spliceosome assembly and a simplified system for studying the action of a spliceosomal ATPase. Further experiments will focus on the binding site of the 3' splice site substrate for the second step of splicing, which will use an 'orthogonal spliceosome' system in yeast, and on the mechanism by which the second-step substrate replaces first-step product in the catalytic core. Finally, we are investigating the role of novel RNA-RNA interactions in the transition from the first-to-second step of splicing. Together, the models proposed in these aims will greatly improve our understanding of the dynamic range of RNA-RNA interactions at consecutive stages of splicing. PUBLIC HEALTH RELEVANCE: PUBLIC HEALTH RELEVANCE STATEMENT Excision of introns from pre-mRNA is critical in the pathway of gene expression. This excision occurs within spliceosomes and requires precise recognition - during both spliceosome assembly and splicing catalysis - of three sites (two splice sites and a branch site) to appropriately join exonic coding sequences. Mechanisms by which spliceosomes recognize the exact sites and catalyze the reactions are not well understood. Mutations in components of the U1-U2 snRNP interaction network during spliceosome assembly are implicated in myelodysplastic syndrome (MDS). Our studies will provide a better understanding of interactions and rearrangements between spliceosome components and the RNA ligands that are substrates for catalysis.

描述（由申请人提供）：剪接体从前体信使 RNA 中切除内含子是几乎所有人类基因表达的关键步骤。该过程受到高度调控，与基因转录和其他加工事件（例如多腺苷酸化和核苷酸修饰）紧密相关。更好地了解 mRNA 前体剪接对于进一步了解调节剪接、控制选择性剪接模式以及促进发育、肿瘤发生和逆转录病毒感染的机制至关重要。剪接体识别化学事件的确切位点的机制以及反应如何催化尚不清楚。该项目的长期目标是了解剪接体成分和作为催化反应底物的 RNA 配体之间的相互作用和重排。充足的证据表明，分支位点存在多种因子重排和多种识别事件。对这些事件的研究（机械上尚未理解）将阐明核心组件之间的相互作用和重排，并可作为剪接体和其他 RNP 机器中重排的范例。该提案重点关注改变剪接体动力学影响剪接保真度的机制。实验将研究 U1-U2 蛋白质相互作用网络和 ATPase Prp5 对内含子定义和剪接体组装保真度的贡献。作为第一个 ATP 依赖性事件，该步骤为剪接体组装提供了独特的承诺，并为研究剪接体 ATP 酶的作用提供了一个简化的系统。进一步的实验将集中于第二步剪接的3'剪接位点底物的结合位点，这将使用酵母中的“正交剪接体”系统，以及第二步底物取代第一步产物的机制在催化核心中。最后，我们正在研究新型 RNA-RNA 相互作用在剪接第一步到第二步转变中的作用。总之，这些目标中提出的模型将极大地提高我们对连续剪接阶段 RNA-RNA 相互作用的动态范围的理解。公共卫生相关性：公共卫生相关声明从前 mRNA 中切除内含子对于基因表达途径至关重要。这种切除发生在剪接体内，并且需要在剪接体组装和剪接催化过程中精确识别三个位点（两个剪接位点和一个分支位点）以适当地连接外显子编码序列。剪接体识别确切位点并催化反应的机制尚不清楚。剪接体组装过程中 U1-U2 snRNP 相互作用网络组件的突变与骨髓增生异常综合征 (MDS) 有关。我们的研究将更好地理解剪接体成分和催化底物 RNA 配体之间的相互作用和重排。