Human inducible nitric oxide synthase (iNOS) expression is regulated by nonsense mediated decay (NMD).

K. Schmitz, K. Koch, S. Hahn, J. Art, I. Forsch, A. Pautz, H. Kleinert. University Medical Center of the Johannes Gutenberg University Mainz, Department of Pharmacology, 55101, Germany

Nitric oxide (NO) generated by the inducible isoform of nitric oxide synthase (iNOS) is involved in complex immunomodulatory and antitumoral mechanisms and has been described to have multiple beneficial microbicidal, antiviral and antiparasital effects. However, dysfunctional induction of iNOS expression seems to be involved in the pathophysiology of many human diseases. Therefore iNOS has to be regulated very tightly.

Modulation of expression, on both the transcriptional and especially post-transcriptional level, is the major regulation mechanism for iNOS. In this report we describe the regulation of human iNOS expression by nonsense-mediated decay (NMD).

NMD is an important mechanism of RNA surveillance that eliminates aberrant transcripts coding for potentially hazardous proteins resulting from premature termination codons (PTCs) in the coding sequence. In addition NMD has been described to regulate the expression of genes containing upstream open reading frames (uORF) in the 5’-untranslated region (5’-UTR) of their mRNAs.

Analysis of the human iNOS gene sequence reveals the existence of a small uORF in exon 1. The stop codon of this uORF is located about 50 bp in front of the first intron of the human iNOS gene. Such a genomic structure indicates a putative involvement of the NMD in the regulation of gene expression.

We generated EGFP-expression constructs containing the human iNOS uORF sequence (without its stop codon) instead of the ATG start codon of EGFP. In transfection experiments with human DLD-1 cells a clear uORF-EGFP protein expression was observed. These data indicate that the uORF sequence of the human iNOS can be expressed.

To analyze the putative involvement of the uORF and NMD in human iNOS expression we created constructs containing the human iNOS gene sequence (exon 1, intron 1, exon 2 up to the start codon; Ex1_In1_Ex2_luc-constructs) with (wt) or without (mutORF) the start codon of the uORF in front of the luciferase reporter gene. In transfection experiments using human DLD-1 colon carcinoma cells we observed a twofold enhancement of luciferase activity in cell transfected with the mutORF constructs compared to wt transfected cells. This indicates an involvement of the uORF in the post-transcriptional regulation of human iNOS expression.

A central mediator of the NMD is the protein UPF1 (also called Rent1). Downregulation of UPF1 by siRNA in cytokine treated DLD-1 cells resulted in twofold enhanced human iNOS mRNA and protein expression. In addition, siRNA-mediated downregulation of UPF1 expression in DLD-1 cells stably transfected with the Ex1_In1_Ex2_luc-construct containing the human iNOS uORF sequence, also resulted in a twofold enhancement of the expression of the luciferase mRNA.

In summary our data clearly indicate that the uORF located in the 5’-UTR sequence of human iNOS gene reduce iNOS expression by activation the NMD mRNA decay mechanism.