New function for the RNA helicase p68/DDX5 as a modifier of MBNL1 activity on expanded CUG repeats
New function for the RNA helicase p68/DDX5 as a modifier of MBNL1 activity on expanded CUG repeats
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DOI：
10.1093/nar/gkr1228
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发表时间：
2012-04-01
影响因子：
14.9
通讯作者：
Marie, Joelle
中科院分区：
文献类型：
--
作者：
Laurent, Francois-Xavier;Sureau, Alain;Marie, Joelle
强直性肌营养不良I型（DM1）是由肌营养不良性肌强直蛋白激酶（DMPK）基因的3' UTR中CTG三联体的异常扩增引起的，导致突变转录物在核RNA灶中聚集。扩增的突变体转录物促进MBNL1剪接因子的螯合，导致可变剪接事件的子集的误调节。在这项研究中，我们确定了DEAD盒RNA解旋酶p68（DDX5）在复合物组装到体外转录的CUG重复。我们发现，在表达含有扩增的CTG重复序列的DMPK基因的3'UTR的细胞中，p68与RNA灶共定位。我们发现p68增加了MBNL1与病理重复序列和心肌肌钙蛋白T（TNNT2）前mRNA内的茎环结构调控元件的结合，其剪接在DM 1中被错误调节。p68解旋酶核心的突变阻止了蛋白质对MBNL1结合的刺激作用和p68与CUG重复序列的共定位，这表明p68对RNA二级结构的重塑有利于MBNL1结合。我们还发现p68调控TNNT 2外显子5的能力依赖于MBNL1结合位点的完整性。我们建议，p68作为MBNL1活性剪接靶点和致病性RNA的修饰剂。
Myotonic Dystrophy type I (DM1) is caused by an abnormal expansion of CTG triplets in the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene, leading to the aggregation of the mutant transcript in nuclear RNA foci. The expanded mutant transcript promotes the sequestration of the MBNL1 splicing factor, resulting in the misregulation of a subset of alternative splicing events. In this study, we identify the DEAD-box RNA helicase p68 (DDX5) in complexes assembled onto in vitro-transcribed CUG repeats. We showed that p68 colocalized with RNA foci in cells expressing the 3'UTR of the DMPK gene containing expanded CTG repeats. We found that p68 increased MBNL1 binding onto pathological repeats and the stem-loop structure regulatory element within the cardiac Troponin T (TNNT2) pre-mRNA, splicing of which is misregulated in DM1. Mutations in the helicase core of p68 prevented both the stimulatory effect of the protein on MBNL1 binding and the colocalization of p68 with CUG repeats, suggesting that remodeling of RNA secondary structure by p68 facilitates MBNL1 binding. We also found that the competence of p68 for regulating TNNT2 exon 5 inclusion depended on the integrity of MBNL1 binding sites. We propose that p68 acts as a modifier of MBNL1 activity on splicing targets and pathogenic RNA.