EXON SKIPPING DURING SPLICING OF DYSTROPHIN MESSENGER-RNA PRECURSOR DUE TO AN INTRAEXON DELETION IN THE DYSTROPHIN GENE OF DUCHENNE MUSCULAR-DYSTROPHY KOBE
EXON SKIPPING DURING SPLICING OF DYSTROPHIN MESSENGER-RNA PRECURSOR DUE TO AN INTRAEXON DELETION IN THE DYSTROPHIN GENE OF DUCHENNE MUSCULAR-DYSTROPHY KOBE
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DOI：
10.1172/jci115244
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发表时间：
1991-06-01
影响因子：
15.9
通讯作者：
NAKAMURA, H
中科院分区：
文献类型：
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作者：
MATSUO, M;MASUMURA, T;NAKAMURA, H
最近的分子研究表明，在患有杜氏肌营养不良症（DMD）的科比患者中，由于外显子88 bp中的52 bp缺失，肌营养不良蛋白基因的外显子19的大小减小到36 dp。 外显子19的5'和3'剪接位点处的共有序列未改变（Matsuo，M.，等，1990年。 生物化学、生物物理通信资源170：963-967）。 为了进一步阐明缺陷的分子本质，我们通过凝胶电泳和聚合酶链反应扩增的cDNA测序，研究了DMD科比患者跨外显子18、19和20连接处的胞质肌营养不良蛋白mRNA的一级结构。 使用随机引物逆转录编码肌营养不良蛋白的mRNA，然后在体外酶促扩增cDNA。 靶向片段小于基因组DNA分析的预期。 通过对扩增产物进行测序，我们发现外显子18直接连接到外显子20，因此外显子19完全缺失，这表明该外显子在抗肌萎缩蛋白mRNA前体的加工过程中被跳过。 扩增产物中的所有其他碱基均未改变。 因此，数据强烈表明，内部外显子缺失产生异常剪接的mRNA，其中外显子18的序列连接到外显子20的序列。 我们认为该缺失是DMD科比等位基因异常加工的原因。 这一发现对功能性剪接位点的决定因素具有重要意义。
Recent molecular studies have shown that in a patient with Duchenne muscular dystrophy (DMD) Kobe, the size of exon 19 of the dystrophin gene was reduced to 36 dp due to the deletion of 52 bp out of 88 bp of the exon. The consensus sequences at the 5' and 3' splice sites of exon 19 were unaltered (Matsuo, M., et al. 1990. Biochem. Biophys. Res. Commun. 170:963-967). To further elucidate the molecular nature of the defect, we examined the primary structure of cytoplasmic dystrophin mRNA of the DMD Kobe patient across the junctions of exons 18, 19, and 20 by gel electrophoresis and sequencing of polymerase chain reaction-amplified cDNA. The mRNA coding for dystrophin was reverse transcribed using random primers, and the cDNA was then enzymatically amplified in vitro. The targeted fragment was smaller than expected from the genomic DNA analysis. By sequencing of the amplified product, we found that exon 18 was joined directly to exon 20, so that exon 19 was completely absent, suggesting that this exon was skipped during processing of the dystrophin mRNA precursor. All other bases in the amplified product were unaltered. Therefore, the data strongly suggest that the internal exon deletion generates an abnormally spliced mRNA in which the sequence of exon 18 is joined to the sequence of exon 20. We propose that the deletion is responsible for abnormal processing of the DMD Kobe allele. This finding has important implications regarding the determinants of a functional splice site.