Biochemical analyses of muscleblind complexes in myotonic dystrophy

描述（由申请人提供）：本项目的主要目的是在分子水平上分析强直性肌营养不良1（DM 1）患者细胞中病理性MBNL1巨型复合物形成所控制的RNA剪接改变的调控机制。DM1的遗传缺陷导致产生编码扩展的CUG束的突变RNA。已显示异常扩增的CUG束在细胞核内形成含有选择性剪接因子MBNL1的异常巨型复合物。一些证据表明，这些高分子量复合物的形成涉及改变剪接的一个子集的生理上重要的RNA，并在随后的发展DM 1病理在体内。为了确定MBNL1巨型复合物的形成改变DM1中剪接密码的机制，我们建议纯化在DM1成肌细胞中发育的正常MBNL1复合物和异常MBNL1巨型复合物。在补充实验中，这些复合物在决定RNA剪接位点选择中的作用将被定义。本申请的目的是：1.正常MBNL1复合物在剪接体组装和RNA催化中的纯化和功能表征。2.从DM1成肌细胞中纯化MBNL1巨型复合物并定义体内巨型复合物形成的机制。3.阐明MBNL1巨型复合物的形成改变DM1成肌细胞中剪接密码的机制。

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.