Expanded CUG Repeats Dysregulate RNA Splicing by Altering the Stoichiometry of the Muscleblind 1 Complex
Expanded CUG Repeats Dysregulate RNA Splicing by Altering the Stoichiometry of the Muscleblind 1 Complex
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DOI：
10.1074/jbc.m111.255224
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发表时间：
2011-11-04
影响因子：
4.8
通讯作者：
Reddy, Sita
中科院分区：
文献类型：
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作者：
Paul, Sharan;Dansithong, Warunee;Reddy, Sita
为了了解剪接调节因子muscleblind 1（MBNL 1）在强直性肌营养不良I型（DM 1）中RNA剪接缺陷发展中的作用，我们从正常人成肌细胞中纯化了RNA非依赖性MBNL 1复合物，并检测了这些复合物在DM 1成肌细胞中的行为。识别MBNL 1变体（MBNL 1（CUG））的抗体可以在DM 1细胞核中形成的毒性CUG RNA灶中螯合，用于从正常成肌细胞中纯化MBNL 1（CUG）复合物。在正常成肌细胞中，MBNL 1（CUG）结合10种参与重塑核糖核蛋白复合物的蛋白质，包括hnRNP H、H2、H3、F、A2/B1、K、L、DDX 5、DDX 17和DHX 9。在这些蛋白质中，只有MBNL 1（CUG）与DM 1 CUG病灶（> 80%的病灶）广泛共定位，其配偶体存在于< 10%的病灶中。重要的是，MBNL 1（CUG）复合物的化学计量在DM 1成肌细胞中发生了改变，表明其9种伴侣蛋白的稳态水平增加。这些变化是概括的表达扩增CUG重复RNA在Cos 7细胞。MBNL 1（CUG）复合物的化学计量改变是由异常的蛋白质合成或稳定性引起的，与PKC α功能无关。在正常成肌细胞中模拟这些变化表明，hnRNP H、H2、H3、F和DDX 5水平的增加独立地使重叠RNA亚群中的剪接失调。因此，扩增的CUG重复序列的表达改变了MBNL 1（CUG）复合物的化学计量，以允许RNA加工缺陷的增强和扩增。
To understand the role of the splice regulator muscleblind 1 (MBNL1) in the development of RNA splice defects in myotonic dystrophy I (DM1), we purified RNA-independent MBNL1 complexes from normal human myoblasts and examined the behavior of these complexes in DM1 myoblasts. Antibodies recognizing MBNL1 variants (MBNL1(CUG)), which can sequester in the toxic CUG RNA foci that develop in DM1 nuclei, were used to purifyMBNL1(CUG) complexes from normal myoblasts. In normal myoblasts, MBNL1(CUG) bind 10 proteins involved in remodeling ribonucleoprotein complexes including hnRNP H, H2, H3, F, A2/B1, K, L, DDX5, DDX17, and DHX9. Of these proteins, only MBNL1(CUG) colocalizes extensively with DM1 CUG foci (> 80% of foci) with its partners being present in < 10% of foci. Importantly, the stoichiometry of MBNL1(CUG) complexes is altered in DM1 myoblasts, demonstrating an increase in the steady state levels of nine of its partner proteins. These changes are recapitulated by the expression of expanded CUG repeat RNA in Cos7 cells. Altered stoichiometry of MBNL1(CUG) complexes results from aberrant protein synthesis or stability and is unlinked to PKC alpha function. Modeling these changes in normal myoblasts demonstrates that increased levels of hnRNP H, H2, H3, F, and DDX5 independently dysregulate splicing in overlapping RNA subsets. Thus expression of expanded CUG repeats alters the stoichiometry of MBNL1(CUG) complexes to allow both the reinforcement and expansion of RNA processing defects.