Pettersson Olof J, Aagaard Lars, Andrejeva Diana, Thomsen Rune, Jensen Thomas G, Damgaard Christian K
Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 4, Building 1240, DK-8000 Aarhus C, Denmark.
Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers Allé 3, building 1131, DK-8000 Aarhus C, Denmark.
Nucleic Acids Res. 2014 Jun;42(11):7186-200. doi: 10.1093/nar/gku352. Epub 2014 May 3.
Myotonic dystrophy type 1 (DM1) is caused by CUG triplet expansions in the 3' UTR of dystrophia myotonica protein kinase (DMPK) messenger ribonucleic acid (mRNA). The etiology of this multi-systemic disease involves pre-mRNA splicing defects elicited by the ability of the CUG-expanded mRNA to 'sponge' splicing factors of the muscleblind family. Although nuclear aggregation of CUG-containing mRNPs in distinct foci is a hallmark of DM1, the mechanisms of their homeostasis have not been completely elucidated. Here we show that a DEAD-box helicase, DDX6, interacts with CUG triplet-repeat mRNA in primary fibroblasts from DM1 patients and with CUG-RNA in vitro. DDX6 overexpression relieves DM1 mis-splicing, and causes a significant reduction in nuclear DMPK-mRNA foci. Conversely, knockdown of endogenous DDX6 leads to a significant increase in DMPK-mRNA foci count and to increased sequestration of MBNL1 in the nucleus. While the level of CUG-expanded mRNA is unaffected by increased DDX6 expression, the mRNA re-localizes to the cytoplasm and its interaction partner MBNL1 becomes dispersed and also partially re-localized to the cytoplasm. Finally, we show that DDX6 unwinds CUG-repeat duplexes in vitro in an adenosinetriphosphate-dependent manner, suggesting that DDX6 can remodel and release nuclear DMPK messenger ribonucleoprotein foci, leading to normalization of pathogenic alternative splicing events.
1型强直性肌营养不良症（DM1）是由肌强直性营养不良蛋白激酶（DMPK）信使核糖核酸（mRNA）的3'非翻译区（UTR）中的CUG三联体扩增引起的。这种多系统疾病的病因涉及由CUG扩增的mRNA “吸附” 肌肉盲家族剪接因子的能力引发的前体mRNA剪接缺陷。尽管含有CUG的mRNA颗粒在不同病灶中的核聚集是DM1的一个标志，但其稳态机制尚未完全阐明。在这里，我们表明，一种DEAD盒解旋酶DDX6在DM1患者的原代成纤维细胞中与CUG三联体重复mRNA相互作用，并在体外与CUG-RNA相互作用。DDX6的过表达减轻了DM1的错误剪接，并导致核DMPK-mRNA病灶显著减少。相反，内源性DDX6的敲低导致DMPK-mRNA病灶数量显著增加，并导致MBNL1在细胞核中的隔离增加。虽然CUG扩增的mRNA水平不受DDX6表达增加的影响，但mRNA重新定位于细胞质，其相互作用伙伴MBNL1变得分散，也部分重新定位于细胞质。最后，我们表明DDX6在体外以三磷酸腺苷依赖的方式解开CUG重复双链体，这表明DDX6可以重塑和释放核DMPK信使核糖核蛋白病灶，从而导致致病性可变剪接事件正常化。
