Exon-skipping and mRNA decay in human liver tissue: molecular consequences of pathogenic bile salt export pump mutations
Exon-skipping and mRNA decay in human liver tissue: molecular consequences of pathogenic bile salt export pump mutations
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DOI：
10.1038/srep24827
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发表时间：
2016-04-26
影响因子：
4.6
通讯作者：
Kubitz, Ralf
中科院分区：
文献类型：
--
作者：
Droege, Carola;Schaal, Heiner;Kubitz, Ralf
胆盐输出泵BSEP介导胆汁形成。超过150个BSEP突变与进行性家族性肝内胆汁淤积2型（PFIC-2）相关，很少有特异性特征。我们检测了两名PFIC-2患者的肝脏组织，这些患者的剪接位点突变为C .150 + 3A > C和C .2783_2787dup5，导致移码带有过早终止密码子（儿童1）或p.R832C（儿童2）。用minigene系统和患者肝脏mRNA测序分析剪接。免疫荧光法检测蛋白表达。使用minigene， C .150 + 3A > C导致外显子3完全跳过。在儿童1的肝组织中，c.2783_2787dup5在DNA上存在，但在mRNA水平上不存在，提示c.2783_2787dup5存在时存在无义介导的mRNA衰变（NMD）。尽管如此，BSEP蛋白以及带或不带外显子3的mRNA都可以检测到，并且可以分配给C .150 + 3A > C等位基因。在儿童2的肝脏中也证实了C .150 + 3A > C的正确剪接转录本。总之，我们提供了(1)BSEP移码突变导致的有效NMD和(2)C. 150 + 3A > c导致的部分外显子跳变的证据。结果表明，外显子跳变的程度取决于基因组和细胞背景，并且剪接的调节可能具有治疗潜力。
The bile salt export pump BSEP mediates bile formation. Over 150 BSEP mutations are associated with progressive familial intrahepatic cholestasis type 2 (PFIC-2), with few characterised specifically. We examined liver tissues from two PFIC-2 patients compound heterozygous for the splice-site mutation c.150 + 3A > C and either c.2783_2787dup5 resulting in a frameshift with a premature termination codon (child 1) or p.R832C (child 2). Splicing was analysed with a minigene system and mRNA sequencing from patients' livers. Protein expression was shown by immunofluorescence. Using the minigene, c.150 + 3A > C causes complete skipping of exon 3. In liver tissue of child 1, c.2783_2787dup5 was found on DNA but not on mRNA level, implying nonsense-mediated mRNA decay (NMD) when c.2783_2787dup5 is present. Still, BSEP protein as well as mRNA with and without exon 3 were detectable and can be assigned to the c.150 + 3A > C allele. Correctly spliced transcripts despite c.150 + 3A > C were also confirmed in liver of child 2. In conclusion, we provide evidence (1) for effective NMD due to a BSEP frameshift mutation and (2) partial exon-skipping due to c.150 + 3A > C. The results illustrate that the extent of exon-skipping depends on the genomic and cellular context and that regulation of splicing may have therapeutic potential.