Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy
Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy
复制标题
DOI：
10.1242/jcs.02404
复制
发表时间：
2005-07-01
影响因子：
4
通讯作者：
Cooper, TA
中科院分区：
文献类型：
--
作者：
Ho, TH;Savkur, RS;Cooper, TA
强直性肌营养不良I型（DM 1）是由肌营养不良性肌强直蛋白激酶（DMPK）基因中的非编码CTG重复扩增引起的RNA介导的疾病。在突变DMPK的转录本中扩增的CUG重复序列形成核灶，其募集肌盲样（MBNL）蛋白，一种选择性剪接因子家族。尽管突变体DMPK和MBNL蛋白的转录物在核RNA焦点中积累，但尚不清楚焦点形成是否是剪接误调节所必需的。在这里，我们使用共转染策略，以表明CUG和CAG重复序列形成RNA焦点，共定位与绿色荧光蛋白（GFP）-MBNL 1和内源性MBNL 1。然而，只有CUG重复序列改变了两种测试的前mRNA，心肌肌钙蛋白T（cTNT）和胰岛素受体（IR）的剪接。使用FRAP，我们证明了GFP-MBNL 1在CUG和CAG病灶中具有相似的恢复半衰期和固定分子的分数，表明GFP-MBNL 1被CUG和CAG重复序列结合。我们还发现DM 1成纤维细胞中GFP-MBNL 1的不动部分和内源性CUG RNA灶中类似的快速交换。因此，RNA焦点的形成和MBNL 1调节的剪接的破坏是可分离的事件。
Myotonic dystrophy type I (DM1), which is caused by a non-coding CTG-repeat expansion in the dystrophia myotonica-protein kinase (DMPK) gene, is an RNA-mediated disease. Expanded CUG repeats in transcripts of mutant DMPK form nuclear foci that recruit muscleblind-like (MBNL) proteins, a family of alternative splicing factors. Although transcripts of mutant DMPK and MBNL proteins accumulate in nuclear RNA foci, it is not clear whether foci formation is required for splicing misregulation. Here, we use a co-transfection strategy to show that both CUG and CAG repeats form RNA foci that colocalize with green fluorescent protein (GFP)-MBNL1 and endogenous MBNL1. However, only CUG repeats alter splicing of the two tested pre-mRNAs, cardiac troponin T (cTNT) and insulin receptor (IR). Using FRAP, we demonstrate that GFP-MBNL1 in CUG and CAG foci have similar half-times of recovery and fractions of immobile molecules, suggesting that GFP-MBNL1 is bound by both CUG and CAG repeats. We also find an immobile fraction of GFP-MBNL1 in DM1 fibroblasts and a similar rapid exchange in endogenous CUG RNA foci. Therefore, formation of RNA foci and disruption of MBNL1-regulated splicing are separable events.