Department of Pharmacology, University Medical Center of the Johannes Gutenberg University Mainz, Obere Zahlbacher Str. 67, 55101, Mainz, Germany.
Department of Pharmacology, University Medical Center of the Johannes Gutenberg University Mainz, Obere Zahlbacher Str. 67, 55101, Mainz, Germany.
Nitric Oxide. 2019 Jul 1;88:50-60. doi: 10.1016/j.niox.2019.04.008. Epub 2019 Apr 18.
The human inducible nitric oxide synthase (iNOS) gene contains an upstream open reading frame (uORF) in its 5'-untranslated region (5'-UTR) implying a translational regulation of iNOS expression. Transfection experiments in human DLD-1 cells revealed that the uORF although translatable seems not to inhibit the translation start at the bona fide ATG. Our data clearly show that human iNOS translation is cap-dependent and that the 5'-UTR of the iNOS mRNA contains no internal ribosome entry site. Translation of the bona fide coding sequence is most likely mediated by a leaky scanning mechanism. The 5'-UTR is encoded by exon 1 and exon 2 of the iNOS gene with the uORF stop codon located in front of the first intron indicating an involvement of the nonsense mediated RNA decay (NMD) in iNOS regulation. SiRNA-mediated down-regulation of Upf1 resulted in enhanced endogenous cytokine iNOS expression in human DLD-1 cells. Transfection of constructs containing iNOS exon 1, intron 1 and exon 2 in front of a luciferase gene showed a clear effect of the mutation of the uORF-ATG on luciferase reportergene expression. Our data indicate that the uORF in the 5'-UTR sequence of human iNOS gene reduces its expression via the NMD mechanism.
人类诱导型一氧化氮合酶（iNOS）基因在其 5'-非翻译区（5'-UTR）中含有一个上游开放阅读框（uORF），暗示 iNOS 表达的翻译调控。在人 DLD-1 细胞中的转染实验表明，虽然 uORF 可翻译，但似乎不会抑制真实 ATG 处的翻译起始。我们的数据清楚地表明，人类 iNOS 翻译是依赖帽的，并且 iNOS mRNA 的 5'-UTR 不含内部核糖体进入位点。真实编码序列的翻译最有可能通过渗漏扫描机制介导。5'-UTR 由 iNOS 基因的外显子 1 和外显子 2 编码，uORF 终止密码子位于第一个内含子之前，表明无义介导的 RNA 降解（NMD）参与 iNOS 调节。siRNA 介导的 Upf1 下调导致人 DLD-1 细胞中内源性细胞因子 iNOS 表达增强。转染包含 iNOS 外显子 1、内含子 1 和外显子 2 在前的荧光素酶基因的构建体显示，uORF-ATG 的突变对荧光素酶报告基因表达有明显影响。我们的数据表明，人类 iNOS 基因 5'-UTR 序列中的 uORF 通过 NMD 机制降低其表达。
