Analysis of the LAMB3 gene in a junctional epidermolysis bullosa patient reveals exonic splicing and allele-specific nonsense-mediated mRNA decay
Analysis of the LAMB3 gene in a junctional epidermolysis bullosa patient reveals exonic splicing and allele-specific nonsense-mediated mRNA decay
复制标题
DOI：
10.1038/labinvest.3700164
复制
发表时间：
2004-10-01
影响因子：
5
通讯作者：
Bauer, JW
中科院分区：
文献类型：
--
作者：
Buchroithner, B;Klausegger, A;Bauer, JW
剪接是前信使RNA（mRNA）中内含子去除的过程，如何在人类细胞中以如此逼真的方式进行仍然不清楚，尽管主要通过体外实验提出了一些一般规则。这些规则目前正在重新定义的剪接机制的分析，在病人提出剪接缺陷。我们分析了材料的病人患有交界性大疱性表皮，遗传性起泡皮肤病。免疫组化分析和免疫电镜显示，患者皮肤真皮-表皮交界处层粘连蛋白-5蛋白缺失，半桥粒发育不良。随后的DNA分析显示LAMB 3基因中的突变R635 X和3009 C--> T为杂合性。后者不改变密码子翻译，但在外显子20中引入外显子剪接位点。有趣的是，这个外显子剪接位点，其剪接分数仅为68.6，优先使用的剪接体超过野生型剪接位点在外显子20-内含子20边界，其显示剪接分数为92.2。在RT-PCR分析中仍然可以检测到LAMB 3 mRNA，尽管异常剪接的mRNA导致在外显子21中的终止密码子，通常假定的3'边界的5'用于无义介导的mRNA衰变。这些结果描述了前mRNA剪接和RNA降解的拟议规则的例外。
How splicing, the process of intron removal in pre-messenger RNA (mRNA), is carried out with such fidelity in human cells is still not understood, although some general rules are being proposed mainly by in vitro experiments. These rules are currently being redefined by analysis of splicing mechanisms in patients presenting splicing defects. We analysed material of a patient suffering from junctional epidermolysis bullosa, a heritable blistering skin disease. Absence of laminin-5 protein together with hypoplastic hemidesmosomes at the dermo-epidermal junction in the patient's skin was shown by immunohistochemical analysis and immunoelectron microscopy. Subsequent DNA analysis revealed heterozygosity for the mutations R635X and 3009C --> T in the LAMB3 gene. The latter did not alter codon translation, but introduced an exonic splice site in exon 20. Interestingly, this exonic splice site, which presented a splice score of only 68.6, was preferentially used by the spliceosome over the wild-type splice site at the exon 20-intron 20 border, which showed a splice score of 92.2. LAMB3 mRNA was still detectable in RT-PCR analysis although the aberrantly spliced mRNA leads to a stop codon in exon 21, 5' of the commonly assumed 3' border for nonsense-mediated mRNA decay. These results describe an exception to the proposed rules of pre-mRNA splicing and RNA degradation.