Nitric oxide-p38 MAPK signaling stabilizes mRNA through AU-rich element-dependent and -independent mechanisms.
Regulation of mRNA stability by p38 MAPK has been linked to adenosine-uridine-rich elements (AURE) within the 3'-untranslated region (3'UTR) of mRNA. Using microarrays, we previously found that AURE-containing mRNA is over-represented among transcripts up-regulated by NO(*), an activator of p38 MAPK. Here, we investigated NO(*)-induced mRNA stabilization of specific AURE-containing genes to determine the sequence specificity and protein-binding interactions associated with this effect. IL-8, TNF-alpha, and p21/Waf1 3'UTRs were inserted into a luciferase (LUC) reporter gene system and found to decrease LUC activity and mRNA half-life in transfected THP-1 cells. The inhibitory effect of these 3'UTRs on LUC expression inversely correlated with the number of AUUUA motifs. Sequence truncation of the IL-8 3'UTR revealed that two segments, one with AURE sites and another without, contributed to mRNA destabilization. NO(*) activation of p38 MAPK increased LUC activity and mRNA half-life for reporter constructs that contained either of these IL-8 3'UTR segments. AURE-dependent and -independent NO(*) effects were blocked by p38 MAPK inhibition, and AURE-dependent effects were also blocked by site-directed mutagenesis of AUUUA sites. Two proteins, HuR and heterogeneous nuclear ribonucleoprotein A0, were identified, which bound to the AURE-containing region of exogenous and endogenous IL-8 mRNA in a NO(*)-p38 MAPK-dependent manner. These results demonstrate that NO(*)-p38 MAPK signaling can stabilize mRNA via AURE-dependent and -independent mechanisms.
Wang
Shuibang
S
Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.
Zhang
Jianhua
J
Zhang
Yi
Y
Kern
Steven
S
Danner
Robert L
RL
D020413:3' Untranslated Regions / Q000235:genetics*; D000241:Adenosine / Q000502:physiology; D002460:Cell Line; D017931:DNA Primers; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D009000:Monocytes / Q000502:physiology*; D009569:Nitric Oxide / Q000502:physiology*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014529:Uridine / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases / Q000502:physiology*
D020413:3' Untranslated Regions; D017931:DNA Primers; D012333:RNA, Messenger; D009569:Nitric Oxide; D008156:Luciferases; D048051:p38 Mitogen-Activated Protein Kinases; D000241:Adenosine; D014529:Uridine