Wang Shuibang, Zhang Jianhua, Zhang Yi, Kern Steven, Danner Robert L
Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.
J Leukoc Biol. 2008 Apr;83(4):982-90. doi: 10.1189/jlb.0907641. Epub 2008 Jan 24.
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.
p38丝裂原活化蛋白激酶（p38 MAPK）对信使核糖核酸（mRNA）稳定性的调控与mRNA 3'非翻译区（3'UTR）内富含腺苷-尿苷的元件（AURE）有关。我们之前利用微阵列发现，在由p38 MAPK激活剂一氧化氮（NO*）上调的转录本中，含有AURE的mRNA占比过高。在此，我们研究了NO诱导的特定含AURE基因的mRNA稳定性，以确定与这种效应相关的序列特异性和蛋白质结合相互作用。将白细胞介素-8（IL-8）、肿瘤坏死因子-α（TNF-α）和p21/Waf1的3'UTR插入荧光素酶（LUC）报告基因系统，发现它们在转染的单核细胞增多性李斯特菌（THP-1）细胞中会降低LUC活性和mRNA半衰期。这些3'UTR对LUC表达的抑制作用与AUUUA基序的数量呈负相关。IL-8 3'UTR的序列截短显示，两个片段，一个带有AURE位点，另一个没有，都导致了mRNA的不稳定。p38 MAPK的NO激活增加了含有这两个IL-8 3'UTR片段之一的报告构建体的LUC活性和mRNA半衰期。p38 MAPK抑制可阻断AURE依赖性和非依赖性的NO效应，对AUUUA位点进行定点诱变也可阻断AURE依赖性效应。鉴定出两种蛋白质，即HuR和不均一核核糖核蛋白A0，它们以NO-p38 MAPK依赖性方式与外源性和内源性IL-8 mRNA的含AURE区域结合。这些结果表明，NO*-p38 MAPK信号传导可通过AURE依赖性和非依赖性机制稳定mRNA。
