Nishida Atsushi, Minegishi Maki, Takeuchi Atsuko, Niba Emma Tabe Eko, Awano Hiroyuki, Lee Tomoko, Iijima Kazumoto, Takeshima Yasuhiro, Matsuo Masafumi
Department of Medical Rehabilitation, Faculty of Rehabilitation, Kobegakuin University, Kobe, Japan.
Department of Clinical Pharmacy, Kobe Pharmaceutical University, Kobe, Japan.
J Hum Genet. 2015 Jun;60(6):327-33. doi: 10.1038/jhg.2015.24. Epub 2015 Apr 2.
The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), comprises 79 exons that show multiple alternative splicing events. Intron retention, a type of alternative splicing, may control gene expression. We examined intron retention in dystrophin introns by reverse-transcription PCR from skeletal muscle, focusing on the nine shortest (all <1000 bp), because these are more likely to be retained. Only one, intron 40, was retained in mRNA; sequencing revealed insertion of a complete intron 40 (851 nt) between exons 40 and 41. The intron 40 retention product accounted for 1.2% of the total product but had a premature stop codon at the fifth intronic codon. Intron 40 retention was most strongly observed in the kidney (36.6%) and was not obtained from the fetal liver, lung, spleen or placenta. This indicated that intron retention is a tissue-specific event whose level varies among tissues. In two DMD patients, intron 40 retention was observed in one patient but not in the other. Examination of splicing regulatory factors revealed that intron 40 had the highest guanine-cytosine content of all examined introns in a 30-nt segment at its 3' end. Further studies are needed to clarify the biological role of intron 40-retained dystrophin mRNA.
在杜氏肌营养不良症（DMD）中发生突变的肌营养不良蛋白基因包含79个外显子，这些外显子会发生多种可变剪接事件。内含子保留作为一种可变剪接类型，可能会控制基因表达。我们通过对骨骼肌进行逆转录PCR来检测肌营养不良蛋白内含子中的内含子保留情况，重点关注9个最短的内含子（均<1000 bp），因为这些内含子更有可能被保留。在mRNA中仅保留了一个内含子，即内含子40；测序显示在第40外显子和第41外显子之间插入了完整的内含子40（851 nt）。内含子40保留产物占总产物的1.2%，但在第5个内含子密码子处有一个提前终止密码子。内含子40保留在肾脏中最为明显（36.6%），在胎儿肝脏、肺、脾脏或胎盘中未检测到。这表明内含子保留是一种组织特异性事件，其水平在不同组织间存在差异。在两名DMD患者中，其中一名患者检测到内含子40保留，而另一名患者未检测到。对剪接调节因子的检测显示，内含子40在其3'端30 nt片段中的鸟嘌呤-胞嘧啶含量在所有检测的内含子中最高。需要进一步研究以阐明内含子40保留的肌营养不良蛋白mRNA的生物学作用。
