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

作者：Casper Ingrid; Nowag Sebastian; Koch Kathrin; Hubrich Thomas; Bollmann Franziska; Henke Jenny; Schmitz Katja; Kleinert Hartmut^*; Pautz Andrea

来源：Nitric Oxide-Biology and Chemistry, 2013, 33: 6-17.

DOI：10.1016/j.niox.2013.05.002

• iNOS
• PABP
• UTR
• mRNA stability
• siRNA

Affinity purification using the 3%26apos;-untranslated region (3%26apos;-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%26apos;-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 beta 2-microglobuline (beta 2M). Along with the mRNA also iNOS protein expression was reduced by siPABP-treatment, whereas in the same cells protein expression of STAT-1 alpha, NF-kappa B p65, or GAPDH was not altered. Reporter gene analyses showed no change of the inducibility of the human 16 kb 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 pm 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%26apos;-UTR and two different binding sites in the 3%26apos;-UTR of the human iNOS mRNA. Mutation or deletion of the binding site in the 5%26apos;-UTR but not in the 3%26apos;-UTR reduced luciferase expression in DLD-1 cells transfected with iNOS-5%26apos;-UTR or iNOS-3%26apos;-UTR luciferase reporter constructs. %26lt;br%26gt;In summary, our data demonstrate that PABP by binding to specific sequence elements in the 5%26apos;-UTR post-transcriptionally enhances human iNOS mRNA stability and thereby iNOS expression.

• 出版日期2013-9-1