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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：
7735572
负责人：
LUCIO COMAI
金额：
$ 49.36万
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/7735572
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 Human In Vitro Individual Inherited Introns Laboratories Maintenance Mass Spectrum Analysis Mechanics Mediating Molecular Molecular Weight Mus Muscular Dystrophies Mutation Myoblasts Myotonic Dystrophy Names Non-Insulin-Dependent Diabetes Mellitus Normal Cell Nuclear Nuclear Extract Pancreatic ribonuclease Pathogenesis Pathology Patients Phenotype Play Principal Investigator Production Protein Kinase Proteins Protocols documentation RNA RNA Binding RNA Sequences RNA Splice Sites RNA Splicing Research Resistance Role Scheme Silver Staining Site Skeletal Muscle Spliceosome Assembly Pathway Testing in vivo mutant programs research study

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(申请人提供)：这个项目的广泛目标是在分子水平上分析受强直性肌营养不良1(DM1)患者细胞中病理性MBNL1巨型复合体形成控制的改变的RNA剪接的调节机制。DM1的遗传缺陷导致编码扩展的CUG束的突变RNA的产生。异常扩张的CUG束已被证明在细胞核内形成异常的巨型复合体，其中包含替代剪接因子MBNL1。有几条证据表明，这些高分子量复合体的形成涉及生理上重要的RNA子集的改变剪接，以及随后体内DM1病理的发展。为了确定MBNL1巨复合体的形成改变DM1剪接密码的机制，我们建议同时纯化正常的MBNL1复合体和在DM1成肌细胞中发育的异常的MBNL1巨复合体。在互补性实验中，将确定这些复合体在决定RNA剪接位点选择中的作用。本应用的目的是：1.正常MBNL1复合体的纯化及其在剪接体组装和RNA催化中的功能鉴定。2.DM1成肌细胞MBNL1巨复合体的纯化及体内形成巨复合体的机制研究3.阐明了MBNL1巨复合体的形成改变DM1成肌细胞剪接密码的机制。