CYP3A5 mRNA degradation by nonsense-mediated mRNA decay
CYP3A5 mRNA degradation by nonsense-mediated mRNA decay
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DOI：
10.1124/mol.105.014225
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发表时间：
2005-09-01
影响因子：
3.6
通讯作者：
Cresteil, T
中科院分区：
文献类型：
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作者：
Busi, F;Cresteil, T
CYP 3A 5 *1等位基因携带者的总CYP 3A 5 mRNA水平显著高于CYP 3A 5 *3纯合子。大多数CYP 3A 5 *3 mRNA包括一个内含子序列（外显子3B），在外显子3和4之间含有提前终止密码子（PTC）。用两种模型研究CYP 3A 5 mRNA的降解：一种是由CYP 3A 5外显子和内含子3 ~ 6组成的CYP 3A 5小基因转染MCF 7细胞，另一种是内源性CYP 3A 5基因在HepG 2细胞中表达。3 '-非翻译区G. 31611 C>T突变对CYP 3A 5 mRNA的降解无影响。含有外显子3B的剪接变体比野生型（wt）CYP 3A 5 mRNA更不稳定。放线菌酮阻止核糖体对PTC的识别：在转染的MCF 7和HepG 2细胞中，放线菌酮减缓了含有外显子3B的剪接变体的降解，表明参与了无义介导的衰变（NMD）。当从假外显子3B中去除PTC或当UPF 1小干扰RNA用于削弱NMD机制时，剪接变体的衰变减少，证实NMD参与CYP 3A 5剪接变体的降解。诱导可能代表CYP 3A 5表达的变异性来源，并可能改变剪接变体的比例。研究了暴露于巴比妥类或类固醇后CYP 3A 5诱导的程度：与未治疗的新生儿相比，儿童人群中CYP 3A 4明显诱导。然而，在总CYP 3A 5 RNA、剪接变体RNA比例或蛋白水平中均未检测到影响。因此，在这些携带者中，诱导不太可能在CYP 3A 5 *3/*3携带者中开启表型CYP 3A 5表达。
The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g. 31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudo-exon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.