Analysis of the transcriptional and post-transcriptional regulation of human iNOS expression

• Applicants: Professor Dr. Hartmut Kleinert; Professorin Dr. Andrea Pautz
• Subject Area: Pharmacology ; Biochemistry ; Immunology
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 273680753

• Final Report Year: 2019

Final Report Abstract

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. Sequence database analyses and qRT-PCR analyses showed an upregulation of human iNOS mRNA expression in human iPSC or ESC induced for neuronal differentiation (using different induction regimen). 5’-RACE analysis showed that the iNOS mRNA expressed in the differentiating iPSC lacks exon 1and 2 of the iNOS gene and contains an alternative neuron-specific exon 1 corresponding to the intron 2 sequence of the human iNOS gene. The functionality and the importance of this neuronal iNOS has to be established.

Publications

• (2019) Regulation of human inducible nitric oxide synthase expression by an upstream open reading frame. Nitric Oxide, 88, 50-60
  Gather, F., Schmitz, K., Koch, K., Vogt, L.M., Pautz, A. and Kleinert, H.
  
• (2019) The KH-type splicing regulatory protein (KSRP) regulates type III interferon expression post-transcriptionally. Biochem J, 476, 333-352
  Schmidtke, L., Schrick, K., Saurin, S., Kafer, R., Gather, F., Weinmann-Menke, J., Kleinert, H. and Pautz, A.