Jin Chongfei, Jiang Jin, Wang Wei, Yao Ke
Eye Center of the Second Affiliated Hospital, Medical College of Zhejiang University, Hangzhou, China.
Mol Vis. 2010 Nov 2;16:2253-8.
To investigate the consequence of a major intrinsic protein MIP splice-site mutation (c.607-1G>A) in a four-generation Chinese pedigree afflicted with autosomal dominant congenital cataracts (ADCC).
Both a mutated minigene with c.607-1G>A, and a wild-type minigene were constructed using the pTARGET mammalian expression vector. They were transiently transfected into HeLa cells and human lens epithelial cells, respectively. After 48 h incubation, RNA extraction and RT-PCR analysis were performed and PCR products were separated and confirmed by sequencing. Structural models of the wild-type and the mutant aquaporin 0 (AQP0) were generated and analyzed using SWISS-MODEL.
The G>A transition activated a cryptic acceptor splice site (c.965-966) in the 3' untranslated region (3' UTR), resulting in the absence of the coding region and most of the 3'UTR in exon 4 of the mature mRNA. Moreover, homology modeling of the mutant protein suggested that the sixth transmembrane helix and carboxyl terminus were replaced with the Leu-His-Ser tripeptide (AQP0-LHS).
The MIP splice-site mutation (c.607-1G>A) activates a cryptic acceptor splice site in the 3' UTR, which may result in substitution of the sixth transmembrane helix and carboxyl terminus for AQP0-LHS. To our knowledge, this is the first report of activation of a cryptic splice site in the 3' UTR in a human disease gene.
研究一个患常染色体显性遗传性先天性白内障（ADCC）的四代中国家系中主要内在蛋白MIP剪接位点突变（c.607-1G>A）的后果。
使用pTARGET哺乳动物表达载体构建携带c.607-1G>A的突变小基因和野生型小基因。它们分别被瞬时转染到HeLa细胞和人晶状体上皮细胞中。孵育48小时后，进行RNA提取和RT-PCR分析，PCR产物经分离并通过测序确认。使用SWISS-MODEL生成并分析野生型和突变型水通道蛋白0（AQP0）的结构模型。
G>A转换激活了3'非翻译区（3'UTR）中的一个隐蔽受体剪接位点（c.965-966），导致成熟mRNA的外显子4中编码区和大部分3'UTR缺失。此外，突变蛋白的同源建模表明，第六个跨膜螺旋和羧基末端被亮氨酸-组氨酸-丝氨酸三肽（AQP0-LHS）取代。
MIP剪接位点突变（c.607-1G>A）激活了3'UTR中的一个隐蔽受体剪接位点，这可能导致AQP0-LHS取代第六个跨膜螺旋和羧基末端。据我们所知，这是人类疾病基因中3'UTR隐蔽剪接位点激活的首次报道。
