Posttranscriptional regulation of IL-13 in T cells: Role of the RNA-binding protein HuR
Posttranscriptional regulation of IL-13 in T cells: Role of the RNA-binding protein HuR
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DOI：
10.1016/j.jaci.2007.12.1166
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发表时间：
2008-04-01
影响因子：
14.2
通讯作者：
Stellato, Cristiana
中科院分区：
文献类型：
--
作者：
Casolaro, Vincenzo;Fang, Xi;Stellato, Cristiana
背景：IL-13是过敏反应中的关键细胞因子，其调节机制尚不清楚。目的：我们研究了HuR对IL-13的转录后调节作用，HuR是一种与mRNA 3 '非翻译区（UTRs）中富含腺苷酸-尿苷酸的元件相关的蛋白质，可促进mRNA的稳定性和翻译。将IL-13 3'UTR亚克隆到诱导型β-珠蛋白报告基因中，该报告基因在不存在或存在过表达HuR的情况下在H2细胞中瞬时表达。通过核糖核蛋白复合物的免疫沉淀和生物素下拉试验检测HuR与IL-13 mRNA的关联。结果：IL-13 mRNA半衰期在再刺激的T（H）2-skewed细胞中较基线值显著增加。β-珠蛋白mRNA的衰减在用含有IL-13 3'UTR的质粒转染的H2细胞中比在携带对照载体的那些细胞中显著更快。HuR过表达增加β-珠蛋白IL-13 3 '-UTR报告基因半衰期。通过免疫沉淀Jurkat细胞核糖核蛋白复合物与抗HuR产生显著富集的IL-13 mRNA。HuR与IL-13 3'UTR的结合通过跨越IL-13 3'UTR的生物素标记的RNA探针的下拉测定来确认。二维Western印迹分析显示刺激诱导的HuR翻译后修饰。结论：丝裂原诱导的IL-13表达与转录周转的改变、HuR磷酸化的改变及其与mRNA 3 'UTR的相关性有关。
Background: IL-13, a critical cytokine in allergy, is regulated by as-yet-elusive mechanisms. Objective: We investigated IL-13 posttranscriptional regulation by HuR, a protein associating with adenylate-uridylate-rich elements in the 3' untranslated regions (UTRs) of mRNA, promoting mRNA stability and translation.Methods: IL-13 mRNA decay was monitored in human T(H)2-skewed cells by using the transcriptional inhibitor actinomycin D. The IL-13 3'UTR was subcloned into an inducible beta-globin reporter transiently expressed in H2 cells in the absence or presence of overexpressed HuR. Association of HuR with IL-13 mRNA was detected by means of immunoprecipitation of ribonucleoprotein complexes and a biotin pull-down assay. The effects of HuR transient overexpression and silencing on IL-13 expression were investigated.Results: IL-13 mRNA half-life increased significantly in restimulated T(H)2-skewed cells compared with baseline values. Decay of beta-globin mRNA was significantly faster in H2 cells transfected with the IL-13 3'UTR-containing plasmid than in those carrying a control vector. HuR overexpression increased the beta-globin IL-13 3'-UTR reporter half-life. Significant enrichment of IL-13 mRNA was produced by means of immunoprecipitation of Jurkat cell ribonucleoprotein complexes with anti-HuR. HuR binding to the IL-13 3'UTR was confirmed by means of pull-down assay of biotin-labeled RNA probes spanning the IL-13 3'UTR. Two-dimensional Western blot analysis showed stimulus-induced posttranslational modification of HuR. In Jurkat cells mitogen-induced IL-13 mRNA was significantly affected by HuR overexpression and silencing.Conclusions: Mitogen-induced IL-13 expression involves changes in transcript turnover and a change in phosphorylation of HuR and its association with the mRNA 3'UTR.