A CTG repeat-selective chemical screen identifies microtubule inhibitors as selective modulators of toxic CUG RNA levels
A CTG repeat-selective chemical screen identifies microtubule inhibitors as selective modulators of toxic CUG RNA levels
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DOI：
10.1073/pnas.1901893116
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发表时间：
2019-10-15
影响因子：
11.1
通讯作者：
Berglund, J. Andrew
中科院分区：
文献类型：
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作者：
Reddy, Kaalak;Jenquin, Jana R.;Berglund, J. Andrew
DMPK基因中的CTG重复扩增是强直性肌营养不良1型（DM1）的致病突变。扩增的CTG重复序列的转录产生毒性功能获得性CUG RNA，导致疾病症状。以选择性方式靶向毒性CUG RNA的产生或稳定性的筛选平台具有提供新的生物学和治疗见解的潜力。生成了稳定表达来自DMPK的毒性r（CUG）480和类似r（CUG）0对照的DM1 HeLa细胞模型，并用于测量r（CUG）480相对于r（CUG）0的比率度量水平。该DM1 HeLa模型概括了DM1的致病特征，包括CUG核糖核病灶和肌盲（MBNL）选择性剪接因子的前体mRNA靶点的错误剪接。使用该细胞系重复选择性筛选导致意外地鉴定出多种微管抑制剂作为选择性降低r（CUG）480水平并部分挽救MBNL依赖性错剪接的命中物。这些结果通过在DM 1小鼠和原代患者细胞模型中使用食品和药物管理局批准的临床微管抑制剂秋水仙碱进行验证。发现作用机制涉及CTG扩增的选择性减少转录，我们假设涉及LINC（核骨架和细胞骨架的接头）复合物。微管抑制剂作为毒性CUG RNA的选择性调节剂的意外鉴定为这种形式的肌营养不良症开辟了研究方向，并可能揭示CTG重复扩增的生物学并提供治疗途径。这种方法有可能确定超过30个微卫星扩增疾病的含有扩增重复序列的基因表达的调节剂。
A CTG repeat expansion in the DMPK gene is the causative mutation of myotonic dystrophy type 1 (DM1). Transcription of the expanded CTG repeat produces toxic gain-of-function CUG RNA, leading to disease symptoms. A screening platform that targets production or stability of the toxic CUG RNA in a selective manner has the potential to provide new biological and therapeutic insights. A DM1 HeLa cell model was generated that stably expresses a toxic r(CUG)480 and an analogous r(CUG)0 control from DMPK and was used to measure the ratio-metric level of r(CUG)480 versus r(CUG) 0. This DM1 HeLa model recapitulates pathogenic hallmarks of DM1, including CUG ribonuclear foci and missplicing of premRNA targets of the muscleblind (MBNL) alternative splicing factors. Repeat-selective screening using this cell line led to the unexpected identification of multiple microtubule inhibitors as hits that selectively reduce r(CUG)480 levels and partially rescue MBNL-dependent missplicing. These results were validated by using the Food and Drug Administration-approved clinical microtubule inhibitor colchicine in DM1 mouse and primary patient cell models. The mechanism of action was found to involve selective reduced transcription of the CTG expansion that we hypothesize to involve the LINC (linker of nucleoskeleton and cytoskeleton) complex. The unanticipated identification of microtubule inhibitors as selective modulators of toxic CUG RNA opens research directions for this form of muscular dystrophy and may shed light on the biology of CTG repeat expansion and inform therapeutic avenues. This approach has the potential to identify modulators of expanded repeat-containing gene expression for over 30 microsatellite expansion disorders.