PDF(Pubmed)
Abstract：
Deposition of the exon junction complex (EJC) upstream of exon-exon junctions helps maintain transcriptome integrity by preventing spurious re-splicing events in already spliced mRNAs. Here we investigate the importance of EJC for the correct splicing of the 2.2-megabase-long human DMD pre-mRNA, which encodes dystrophin, an essential protein involved in cytoskeletal organization and cell signaling. Using targeted RNA-seq, we show that knock-down of the eIF4A3 and Y14 core components of EJC in a human muscle cell line causes an accumulation of mis-splicing events clustered towards the 3\' end of the DMD transcript (Dp427m). This deregulation is conserved in the short Dp71 isoform expressed ubiquitously except in adult skeletal muscle and is rescued with wild-type eIF4A3 and Y14 proteins but not with an EJC assembly-defective mutant eIF4A3. MLN51 protein and EJC-associated ASAP/PSAP complexes independently modulate the inclusion of the regulated exons 71 and 78. Our data confirm the protective role of EJC in maintaining splicing fidelity, which in the DMD gene is necessary to preserve the function of the critical C-terminal protein-protein interaction domain of dystrophin present in all tissue-specific isoforms. Given the role of the EJC in maintaining the integrity of dystrophin, we asked whether the EJC could also be involved in the regulation of a mechanism as complex as skeletal muscle differentiation. We found that eIF4A3 knockdown impairs myogenic differentiation by blocking myotube formation. Collectively, our data provide new insights into the functional roles of EJC in human skeletal muscle.
摘要：
外显子-外显子连接上游的外显子连接复合物（EJC）的沉积有助于通过防止已经剪接的mRNA中的虚假重新剪接事件来维持转录组完整性。在这里，我们研究了EJC对于2.2兆碱基长的人DMD前mRNA的正确剪接的重要性，编码肌营养不良蛋白，参与细胞骨架组织和细胞信号传导的必需蛋白。使用靶向RNA-seq，我们显示，在人肌肉细胞系中，EJC的eIF4A3和Y14核心成分的敲除会导致聚集在DMD转录本（Dp427m）3'末端的错误剪接事件的积累。这种失调在成年骨骼肌中普遍表达的短Dp71同种型中是保守的，并且用野生型eIF4A3和Y14蛋白而不是用EJC组装缺陷突变体eIF4A3拯救。MLN51蛋白和EJC相关的ASAP/PSAP复合物独立地调节受调节外显子71和78的包含。我们的数据证实了EJC在保持剪接保真度方面的保护作用，在DMD基因中，这对于保留存在于所有组织特异性同工型中的肌营养不良蛋白的关键C端蛋白-蛋白相互作用域的功能是必需的。鉴于EJC在维持肌营养不良蛋白完整性方面的作用，我们询问EJC是否也可能参与骨骼肌分化等复杂机制的调节.我们发现eIF4A3敲低通过阻断肌管形成来损害肌源性分化。总的来说，我们的数据为EJC在人类骨骼肌中的功能作用提供了新的见解。
