Biochemical analyses of muscleblind complexes in myotonic dystrophy

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。

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.