Post-transcriptional regulation of the human inducible nitric oxide synthase (iNOS) expression by the cytosolic poly(A)-binding protein (PABP)

Abstract

Affinity purification using the 3′-untranslated region (3′-UTR) of the human inducible nitric oxide synthase (iNOS) mRNA identified the cytosolic poly(A)-binding protein (PABP) as a protein interacting with the human iNOS 3′-UTR. Downregulation of PABP expression by RNA interference resulted in a marked reduction of cytokine-induced iNOS mRNA expression without changes in the expression of mRNAs coding for the major subunit of the RNA polymerase II (Pol 2A) or β2-microglobuline (β2M). Along with the mRNA also iNOS protein expression was reduced by siPABP-treatment, whereas in the same cells protein expression of STAT-1α, NF-κB p65, or GAPDH was not altered. Reporter gene analyses showed no change of the inducibility of the human 16kb iNOS promoter in siPABP cells. In contrast, the siPABP-mediated decline of iNOS expression correlated with a reduction in the stability of the iNOS mRNA. As the stability of the Pol 2A and β2M mRNA was not changed, siPABP-treatment seems to have a specific effect on iNOS mRNA decay. UV-crosslinking experiments revealed that PABP interacts with one binding site in the 5′-UTR and two different binding sites in the 3′-UTR of the human iNOS mRNA. Mutation or deletion of the binding site in the 5′-UTR but not in the 3′-UTR reduced luciferase expression in DLD-1 cells transfected with iNOS-5′-UTR or iNOS-3′-UTR luciferase reporter constructs.In summary, our data demonstrate that PABP by binding to specific sequence elements in the 5′-UTR post-transcriptionally enhances human iNOS mRNA stability and thereby iNOS expression.