Targeted RNA-Seq profiling of splicing pattern in the DMD gene: exons are mostly constitutively spliced in human skeletal muscle
Targeted RNA-Seq profiling of splicing pattern in the DMD gene: exons are mostly constitutively spliced in human skeletal muscle
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DOI：
10.1038/srep39094
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发表时间：
2017-01-03
影响因子：
4.6
通讯作者：
Tuffery-Giraud, Sylvie
中科院分区：
文献类型：
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作者：
Bouge, Anne-Laure;Murauer, Eva;Tuffery-Giraud, Sylvie
我们分析了人类杜氏肌营养不良症（DMD） NB转录本在正常骨骼肌中的剪接模式。为了达到分析肌肉中这种低表达基因所需的覆盖深度，我们设计了一种靶向RNA-Seq程序，该程序结合了全长11.3 kb DMD cDNA序列的扩增和454测序技术。获得了高而均匀的cDNA序列覆盖率，从而可以对肌肉DMD转录物中的生理选择性剪接事件进行可靠的盘点。与之前的假设相反，我们证明了79个DMD外显子中的大多数是在骨骼肌中组成剪接的。仅确定了有限数量的12个备选剪接事件，所有剪接事件都存在于非常低的水平。这些包括先前已知的外显子跳跃事件，也包括新描述的假外显子内含物和替代的3'剪接位点，其中一个是在DMD基因中报道的第一个功能性NAGNAG剪接位点。这项研究提供了第一个基于rna - seq的骨骼肌DMD剪接模式参考，并报告了一个实验程序，非常适合检测这种低丰度转录物的条件特异性差异，这可能被证明对诊断、研究或基于rna的治疗应用有用。
We have analysed the splicing pattern of the human Duchenne Muscular Dystrophy (DMD) NB transcript in normal skeletal muscle. To achieve depth of coverage required for the analysis of this lowly expressed gene in muscle, we designed a targeted RNA-Seq procedure that combines amplification of the full-length 11.3 kb DMD cDNA sequence and 454 sequencing technology. A high and uniform coverage of the cDNA sequence was obtained that allowed to draw up a reliable inventory of the physiological alternative splicing events in the muscular DMD transcript. In contrast to previous assumptions, we evidenced that most of the 79 DMD exons are constitutively spliced in skeletal muscle. Only a limited number of 12 alternative splicing events were identified, all present at a very low level. These include previously known exon skipping events but also newly described pseudoexon inclusions and alternative 3' splice sites, of which one is the first functional NAGNAG splice site reported in the DMD gene. This study provides the first RNA-Seq-based reference of DMD splicing pattern in skeletal muscle and reports on an experimental procedure well suited to detect condition-specific differences in this low abundance transcript that may prove useful for diagnostic, research or RNA-based therapeutic applications.