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

• Published in: Nitric oxide 2013-09, Vol.33, p.6-17
• Main Authors: Casper, Ingrid, Nowag, Sebastian, Koch, Kathrin, Hubrich, Thomas, Bollmann, Franziska, Henke, Jenny, Schmitz, Katja, Kleinert, Hartmut, Pautz, Andrea
• Format: Article
• Language: English
• Subjects: 3' Untranslated Regions; Binding Sites; Cell Line, Tumor; Cytokines - biosynthesis; Cytokines - genetics; Cytokines - metabolism; Down-Regulation; Humans; iNOS; mRNA stability; Mutation; Nitric Oxide Synthase Type II - biosynthesis; Nitric Oxide Synthase Type II - genetics; Nitric Oxide Synthase Type II - metabolism; PABP; Poly(A)-Binding Proteins - genetics; Poly(A)-Binding Proteins - metabolism; RNA Processing, Post-Transcriptional; RNA, Messenger - chemistry; RNA, Messenger - genetics; RNA, Messenger - metabolism; siRNA; UTR
• ISSN: 1089-8603; 1089-8611
• DOI: 10.1016/j.niox.2013.05.002

•PABP binds to the 5′- and 3′-UTR of the human iNOS mRNA.•Downregulation of PABP resulted in reduction of cytokine induced iNOS expression.•PABP-downregulation did not change human iNOS promoter activity but reduced iNOS mRNA stability.•Mutation or deletion of the PABP binding site in the 5′-UTR reduced reportergene expression.•PABP post-transcriptionally regulates human iNOS expression in a specific manner. 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.