Rbfox1 downregulation and altered calpain 3 splicing by FRG1 in a mouse model of Facioscapulohumeral muscular dystrophy (FSHD).
Rbfox1 downregulation and altered calpain 3 splicing by FRG1 in a mouse model of Facioscapulohumeral muscular dystrophy (FSHD).
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DOI：
10.1371/journal.pgen.1003186
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发表时间：
2013
期刊：
影响因子：
4.5
通讯作者：
Gabellini D
中科院分区：
文献类型：
--
作者：
Pistoni M;Shiue L;Cline MS;Bortolanza S;Neguembor MV;Xynos A;Ares M Jr;Gabellini D
面肩肱型肌营养不良症（FSHD）是一种常见的肌肉疾病，其分子发病机制仍不清楚。小鼠、青蛙和蠕虫中 FSHD 区域基因 1 (FRG1) 的过度表达会扰乱肌肉发育并导致 FSHD 样表型。 FRG1 与剪接有关，我们通过在 FRG1 小鼠中进行全基因组分析来了解剪接如何参与 FSHD。我们发现剪接扰动与不同肌肉对 FRG1 过度表达和疾病进展的反应平行。有趣的是，Rbfox 剪接因子家族的结合位点在 FRG1 影响的剪接事件子集中过多。 Rbfox1 敲低、过表达和 RNA-IP 证实这些是 Rbfox1 的直接靶点。我们发现 FRG1 与 Rbfox1 RNA 相关并降低其稳定性。与此一致的是，在过度表达 FRG1 的小鼠和细胞以及 FSHD 患者中，Rbfox1 表达下调。受影响的基因包括 Calpain 3，它在肢带型肌营养不良症（一种表型与 FSHD 相似的疾病）中发生突变。在 FRG1 小鼠和 FSHD 患者中，缺乏外显子 6 (Capn3 E6–) 的钙蛋白酶 3 亚型增加。最后，Rbfox1 敲低和 Capn3 E6 过度表达抑制肌肉分化。总的来说，我们的结果表明 FSHD 发病机制的一个组成部分可能是由 FRG1 过度表达引起的，降低了 Rbfox1 水平，并通过剪接失调导致改变的钙蛋白酶 3 蛋白的异常表达。选择性剪接是人类细胞复杂性的一个主要因素，其破坏可能导致多种人类疾病。 FSHD 是最重要的肌肉疾病之一。虽然该疾病中肌肉分化缺陷已被广泛报道，但其分子机制在很大程度上尚不清楚。我们发现选择性剪接因子 Rbfox1 的表达是 FRG1 的直接靶标，并且其表达在 FSHD 小鼠模型和 FSHD 患者的肌肉中下降。此外，编码参与肌肉分化的蛋白酶的 Calpain 3 的选择性剪接受 Rbfox1 调节，并且在 FSHD 小鼠模型和 FSHD 患者的肌肉中发生改变。有趣的是，我们发现 Rbfox1 是肌肉分化所必需的，并且这种活性可能是由 Calpain 3 选择性剪接介导的。因此，我们的结果表明，Rbfox1 表达降低和 Calpain 3 剪接异常导致 FSHD 患者的肌肉分化缺陷。
Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (FRG1) in mice, frogs, and worms perturbs muscle development and causes FSHD–like phenotypes. FRG1 has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in FRG1 mice. We find that splicing perturbations parallel the responses of different muscles to FRG1 over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of FRG1-affected splicing events. Rbfox1 knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the Rbfox1 RNA and decreases its stability. Consistent with this, Rbfox1 expression is down-regulated in mice and cells over-expressing FRG1 as well as in FSHD patients. Among the genes affected is Calpain 3, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In FRG1 mice and FSHD patients, the Calpain 3 isoform lacking exon 6 (Capn3 E6–) is increased. Finally, Rbfox1 knockdown and over-expression of Capn3 E6- inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of FRG1, reducing Rbfox1 levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing. Alternative splicing is a major contributor to the complexity of human cells, and its disruption can lead to a wide range of human disorders. FSHD is one of the most important muscle diseases. While muscle differentiation defects have been widely reported in the disease, the molecular mechanisms responsible are largely unknown. We found that expression of the alternative splicing factor Rbfox1 is a direct FRG1 target, and its expression decreased in the muscles of a mouse model of FSHD and FSHD patients. Moreover, alternative splicing of Calpain 3, encoding for a protease involved in muscle differentiation, is regulated by Rbfox1 and is altered in the muscles of the mouse model of FSHD and FSHD patients. Interestingly, we found that Rbfox1 is required for muscle differentiation and that this activity is likely mediated by Calpain 3 alternative splicing. Hence, our results suggest that decreased expression of Rbfox1 and aberrant Calpain 3 splicing contribute to the muscle differentiation defects of FSHD patients.
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