Department of Cellular & Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada.
Hum Mol Genet. 2010 Apr 1;19(7):1211-20. doi: 10.1093/hmg/ddp591. Epub 2010 Jan 6.
The molecular mechanisms regulating expression of utrophin A are of therapeutic interest since upregulating its expression at the sarcolemma can compensate for the lack of dystrophin in animal models of Duchenne Muscular Dystrophy (DMD). The 5'-UTR of utrophin A has been previously shown to drive cap-independent internal ribosome entry site (IRES)-mediated translation in response to muscle regeneration and glucocorticoid treatment. To determine whether the utrophin A IRES displays tissue specific activity, we generated transgenic mice harboring control (CMV/betaGAL/CAT) or utrophin A 5'-UTR (CMV/betaGAL/UtrA/CAT) bicistronic reporter transgenes. Examination of multiple tissues from two CMV/betaGAL/UtrA/CAT lines revealed that the utrophin A 5'-UTR drives cap-independent translation of the reporter gene exclusively in skeletal muscles and no other examined tissues. This expression pattern suggested that skeletal muscle-specific factors are involved in IRES-mediated translation of utrophin A. We performed RNA-affinity chromatography experiments combined with mass spectrometry to identify trans-factors that bind the utrophin A 5'-UTR and identified eukaryotic elongation factor 1A2 (eEF1A2). UV-crosslinking experiments confirmed the specificity of this interaction. Regions of the utrophin A 5'-UTR that bound eEF1A2 also mediated cap-independent translation in C2C12 muscle cells. Cultured cells lacking eEF1A2 had reduced IRES activity compared with cells overexpressing eEF1A2. Together, these results suggest an important role for eEF1A2 in driving cap-independent translation of utrophin A in skeletal muscle. The trans-factors and signaling pathways driving skeletal-muscle specific IRES-mediated translation of utrophin A could provide unique targets for developing pharmacological-based DMD therapies.
调节肌联蛋白 A 表达的分子机制具有治疗意义，因为在 Duchenne 肌营养不良症（DMD）的动物模型中，在肌膜上调其表达可以弥补肌营养不良蛋白的缺乏。肌联蛋白 A 的 5'-UTR 先前已被证明可以在肌肉再生和糖皮质激素治疗时驱动无帽依赖性内部核糖体进入位点（IRES）介导的翻译。为了确定肌联蛋白 A IRES 是否具有组织特异性活性，我们生成了携带对照（CMV/betaGAL/CAT）或肌联蛋白 A 5'-UTR（CMV/betaGAL/UtrA/CAT）双顺反子报告基因的转基因小鼠。对来自两条 CMV/betaGAL/UtrA/CAT 线的多种组织进行检查后发现，肌联蛋白 A 5'-UTR 仅在骨骼肌中驱动报告基因的无帽依赖性翻译，而在其他检查的组织中则没有。这种表达模式表明，骨骼肌特异性因子参与肌联蛋白 A 的 IRES 介导的翻译。我们进行了 RNA 亲和层析实验，并结合质谱分析来鉴定与肌联蛋白 A 5'-UTR 结合的反式因子，并鉴定出真核延伸因子 1A2（eEF1A2）。UV 交联实验证实了这种相互作用的特异性。与 eEF1A2 结合的肌联蛋白 A 5'-UTR 区域也介导了 C2C12 肌肉细胞中的无帽依赖性翻译。与过表达 eEF1A2 的细胞相比，缺乏 eEF1A2 的培养细胞的 IRES 活性降低。这些结果表明，eEF1A2 在驱动骨骼肌中肌联蛋白 A 的无帽依赖性翻译中起重要作用。驱动肌联蛋白 A 在骨骼肌中 IRES 介导的翻译的反式因子和信号通路可以为开发基于药理学的 DMD 治疗方法提供独特的靶标。
