Regulation of human inducible nitric oxide synthase expression by an upstream open reading frame

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.