Biochemical analyses of muscleblind complexes in myotonic dystrophy

强直性肌营养不良肌盲复合体的生化分析

基本信息

批准号：
7352997
负责人：
LUCIO COMAI
金额：
$ 47.97万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-02-01 至 2012-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7352997
关键词：
19q 3&apos Untranslated Regions Adult Affinity Chromatography Alternative Splicing Antibodies Antibody Affinity Behavior Binding Biochemical Catalysis Cell Nucleus Cells Chromosomes Co-Immunoprecipitations Code Complex Data Defect Development Exons Fractionation Gel Chromatography Genes Heterogeneous-Nuclear Ribonucleoprotein Group F-H Human In Vitro Individual Inherited Introns Laboratories Localized Maintenance Mass Spectrum Analysis Measures Mechanics Mediating Molecular Molecular Weight Mus Muscular Dystrophies Mutation Myoblasts Myotonic Dystrophy Normal Cell Nuclear Nuclear Extract Pancreatic ribonuclease Pathogenesis Pathology Patients Phenotype Play Production Protein Kinase Proteins Protocols documentation RNA RNA Binding RNA Recognition Motif RNA Sequences RNA Splice Sites RNA Splicing Resistance Role Scheme Silver Staining Site Skeletal Muscle Spliceosome Assembly Pathway Testing in vitro Assay in vivo insight mutant novel research study restoration size stoichiometry

项目摘要

DESCRIPTION (provided by applicant): The broad objective of this project is to analyze at the molecular level the regulatory mechanisms of altered RNA splicing that are controlled by the formation of pathological MBNL1 mega-complexes in myotonic dystrophy 1 (DM1) patient cells. The genetic defect in DM1 results in the production of mutant RNAs encoding expanded CUG tracts. Abnormally expanded CUG tracts have been shown to form aberrant mega-complexes that contain the alternative splice factor, MBNL1, within the nucleus. Several lines of evidence implicate the formation of these high molecular weight complexes in altered splicing of a subset of physiologically important RNAs and in the subsequent development of DM1 pathology in vivo. To determine the mechanism whereby formation of the MBNL1 mega-complexes alters the splice code in DM1 we propose to purify both normal MBNL1 complexes and the aberrant MBNL1 mega-complexes that develop in DM1 myoblasts. In complementary experiments the role of these complexes in dictating RNA splice site choice will be defined. The Aims of this application are: 1. Purification and functional characterization of normal MBNL1 complexes in spliceosome assembly and RNA catalysis. 2. Purification of MBNL1 mega-complexes from DM1 myoblasts and definition of the mechanics of mega-complex formation in vivo. 3. Elucidation of the mechanisms by which formation of MBNL1 mega-complexes alters the splice code in DM1 myoblasts.

描述（由申请人提供）：该项目的广泛目的是在分子级分析由病理MBNL1 MEGA巨型复合物在肌动型1（DM1）患者细胞中形成的改变RNA剪接的调节机制。 DM1中的遗传缺陷导致编码扩展的CUG区域的突变RNA产生。已证明异常扩展的CUG道会形成异常的巨型复合物，这些大复合物在核内包含替代剪接因子MBNL1。几条证据表明，这些高分子量复合物的形成在改变了生理上重要的RNA子集的剪接以及随后在体内DM1病理学的发展中。为了确定MBNL1巨型复合物的形成的机制，改变了DM1中的剪接代码，我们建议净化正常的MBNL1复合物和在DM1成肌细胞中发展的异常MBNL1 MEGA-complexes。在互补实验中，将定义这些复合物在决定RNA剪接位点选择中的作用。该应用的目的是：1。剪接体组装和RNA催化中正常MBNL1复合物的纯化和功能表征。 2。从DM1成肌细胞中纯化MBNL1巨型复合物，以及体内巨型复合形成力学的定义。 3。阐明MBNL1巨型复合物的形成的机制会改变DM1成肌细胞中的剪接代码。