Implication of alternative splicing for expression of a variant NAD(P)H:quinone oxidoreductase-1 with a single nucleotide polymorphism at 465C>T.
Implication of alternative splicing for expression of a variant NAD(P)H:quinone oxidoreductase-1 with a single nucleotide polymorphism at 465C>T.
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DOI：
10.1097/00008571-200208000-00009
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发表时间：
2002-08
期刊：
影响因子：
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通讯作者：
S. Pan;Yusheng Han;P. Farabaugh;H. Xia
中科院分区：
文献类型：
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作者：
S. Pan;Yusheng Han;P. Farabaugh;H. Xia
人还原型烟酰胺腺嘌呤二核苷酸磷酸三种等位基因：苯醌氧化还原酶-1(NQO1)，野生型，609C>；T变异体，465C>；T变异体，分别命名为NQO1*1，NQO1*2，NQO1*3。以前，我们在HCT-116细胞的一个等位基因中发现了NQO1*3，在丝裂霉素C抗性亚系HCT-116R30A的两个等位基因中都发现了NQO1*3。HCT-116R30A的NQO1蛋白含量是HCT-116的5%。逆转录聚合酶链式反应显示，两种细胞株中都存在外显子4缺失的NQO1mRNA，表明存在选择性剪接。然而，NQO1在HCT-116R30A中表达降低的原因尚不清楚。目前的数据表明，在不进行RNA剪接的情况下，HCT-116R30A细胞能够表达来自转基因载体的NQO1蛋白。半定量聚合酶链式反应测得Hct-116和Hct-116R30A的全长与缺失外显子4的比例分别为：36和34：66。所有其他细胞株的比例为90：10，包括HT-29、NIH-125和NCI-H1688(纯合子NQO1*1)；MCF-7和HL-60(杂合子NQO1*1/*2)；以及MDA-MB231(纯合子NQO1*2/*2)。在具有NQO1*3的细胞中，内含子-4 5‘-剪接点的NQO1选择性剪接增加。465C&>T单核苷酸多态(SNP)破坏了5’-剪接点的共同序列，这是剪接体中U1小核RNA(U1 SnRNA)结合所必需的。通过用U1 SnRNA构建体转染HCT-116R30A细胞，这种有缺陷的RNA剪接被部分纠正，U1 SnRNA构建体含有碱基变化，以补偿465个SNP。NQO1蛋白和酶活性随着正确剪接的增加而增加。在HCT-116R30A细胞中，465SNP是导致NQO1蛋白表达降低和选择性剪接增加的主要原因。
Three alleles of the human reduced nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase-1 (NQO1) gene are known; wild-type, 609C>T variant, and 465C>T variant; designated as NQO1*1 and NQO1*2, and NQO1*3, respectively. Previously, we found NQO1*3 in one allele of HCT-116 cells, and in both alleles of the mitomycin C-resistant subline, HCT-116R30A. The NQO1 protein content of HCT-116R30A is 5% that of HCT-116. RT-PCR revealed an exon-4-deleted NQO1 mRNA in both cell lines indicating alternative splicing. However, the cause of the lower expression of NQO1 in HCT-116R30A is unknown. Current data show that HCT-116R30A cells are able to express NQO1 protein from transfected cDNA constructs when RNA splicing is omitted. The ratio of full-length to exon-4-deleted mRNA measured by semiquantitative PCR shows that HCT-116 and HCT-116R30A have ratios of 64 : 36 and 34 : 66, respectively. All other cell lines tested have a ratio of 90 : 10, including HT-29, NIH-125 and NCI-H1688 (homozygous NQO1*1); MCF-7 and HL-60 (heterozygous NQO1*1/*2); and MDA-MB231 (homozygous NQO1*2/*2). Alternative splicing of NQO1 at the 5'-splice site of intron-4 increased in cells with NQO1*3. The 465C>T single nucleotide polymorphism (SNP) disrupts the consensus sequence at the 5'-splice site, which is required for binding by U1 small nuclear RNA (U1 snRNA) in spliceosomes. This defective RNA splicing was partially corrected by transfecting HCT-116R30A cells with U1 snRNA constructs, containing base changes to compensate for the 465 SNP. NQO1 protein and enzymatic activity increased with corrected splicing. The 465 SNP was the major cause of increased alternative splicing and decreased expression of NQO1 protein in HCT-116R30A cells.