Molecular Recognition during pre-mRNA Splicing

mRNA 前体剪接过程中的分子识别

• 批准号: 7652230
• 负责人: CLARA KIELKOPF
• 金额: $ 37.11万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7652230
• 关键词: 3' Splice Site; ATP Hydrolysis; ATP phosphohydrolase; Address; Adenosine; Adopted; Affinity; Architecture; Arginine; Binding; Cell Differentiation process; Collaborations; Complex; Consensus; Consensus Sequence; Development; Dimensions; Ensure; Eukaryota; Excision; Figs - dietary; Fluorescence; Gene Expression; Goals; Health; Hereditary Disease; Human; Human Genetics; Introns; Investigation; Laboratories; Lead; Malignant Neoplasms; Maps; Metabolic Diseases; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Myotonic Dystrophy; Neurologic; Phosphorylation; Phosphorylation Site; Positioning Attribute; Process; Proteins; RNA; RNA Splicing; Reaction; Recruitment Activity; Resolution; Roentgen Rays; Role; SF1; Serine; Side; Signal Transduction; Site; Site-Directed Mutagenesis; Small Nuclear RNA; Solutions; Source; Spliceosome Assembly Pathway; Spliceosomes; Staging; Structure; Surface; Testing; Thermodynamics; Transcript; U2 Small Nuclear Ribonucleoprotein; Variant; Water; Work; X-Ray Crystallography; Yeasts; acrosome stabilizing factor; analog; base; human disease; interest; leukemia; mRNA Precursor; macromolecular assembly; molecular recognition; novel; protein protein interaction; public health relevance; synchrotron radiation; three dimensional structure

DESCRIPTION (provided by applicant): Splicing of pre-mRNAs provides a major source of transcript diversity for cell differentiation and development. It is known that this process requires a splicing machine (spliceosome) composed of ~100 proteins and 5 small nuclear (sn)RNAs. The early stages of spliceosome assembly on pre-mRNA splice sites are key regulated steps that often go awry in human genetic diseases and cancers. Yet, exactly how the spliceosome selects and excises the correct splice sites from amidst thousands of competing pre-mRNA sequences remains poorly understood at the molecular level. Hence, the overall goal of this proposal is to understand the sequential three- dimensional interactions that guide 3' splice site selection in the early stages of spliceosome activation. A complex of the essential splicing factors U2AF and SF1 recognizes pre-mRNA sequences adjacent the 3' splice site, and in turn stabilizes association of the core spliceosome. Pre-mRNA contacts by an arginine-serine (RS) region of U2AF, and the U2AF-associated ATPase UAP56, are required to accomplish this task. Specific aims of this proposal address the following central questions concerning the critical early stages of pre-mRNA splicing: (1) By what means does U2AF recognize diverse splice sites? (2) By what means does SF1 enhance splice site recognition by U2AF? (3) By what means does U2AF recruit UAP56, and in turn, what is the action of UAP56 on U2AF at the splice site? (4) By what means does an RS region promote association of spliceosomal snRNAs with the pre-mRNA? We have already made significant advances towards these aims by (i) determining three-dimensional structures of U2AF bound to splice sites, (ii) evaluating thermodynamic and structural contributions of SF1 domains to U2AF binding, and (iii) characterizing RNA interactions by RS domains. These studies will significantly advance our understanding of this fundamental step of gene expression. PUBLIC HEALTH RELEVANCE: Errors in pre-mRNA splicing contribute to major human diseases, including cancers, leukemias, myotonic dystrophies, neurological and metabolic disorders. The investigation of normal splice site recognition to be gained by the proposed work would serve as a basis for understanding, and in the long term developing treatments against, harmful splice variants of human disease.

描述（由申请人提供）：前MRNA的剪接为细胞分化和发育提供了主要来源。众所周知，此过程需要一台由约100种蛋白质和5个小核（SN）RNA组成的剪接机（剪接）。剪接体组装在前MRNA剪接位点上的早期阶段是关键的调节步骤，通常在人类遗传疾病和癌症中出现问题。然而，剪接体如何从成千上万的竞争前MRNA序列中选择和切除正确的剪接位点在分子水平上仍然很少了解。因此，该提案的总体目标是了解在剪接体激活的早期阶段指导3'剪接位点选择的顺序三维相互作用。基本剪接因子U2AF和SF1的复合物认识到3'剪接位点附近的前mRNA序列，进而稳定了核心剪接体的关联。 U2AF的精氨酸 - 丝氨酸（RS）区域以及与U2AF相关的ATPase UAP56的前MRNA接触才能完成此任务。该提案的具体目的解决了以下有关MRNA剪接的关键早期阶段的中心问题：（1）U2AF认识到不同的剪接站点是什么方式？ （2）SF1通过什么方式增强U2AF的剪接站点识别？ （3）U2AF招募UAP56的方式，而UAP56在U2AF在剪接网站上的作用是什么？ （4）RS区域促进剪接SNRNA与前MRNA的关系是什么？我们已经通过（i）确定与剪接位点结合的U2AF的三维结构，（ii）评估SF1域对U2AF结合的热力学和结构贡献，以及（iii）通过RS域表征RNA相互作用。这些研究将大大提高我们对基因表达的基本步骤的理解。公共卫生相关性：前MRNA剪接的错误会导致主要的人类疾病，包括癌症，白血病，肌发育不良，神经系统疾病和代谢性疾病。对拟议工作所获得的正常剪接部位识别的调查将是理解的基础，并在长期开发针对人类疾病的有害剪接变异的治疗方法中。