Casolaro Vincenzo, Fang Xi, Tancowny Brian, Fan Jinshui, Wu Fan, Srikantan Subramanya, Asaki S Yukiko, De Fanis Umberto, Huang Shau-Ku, Gorospe Myriam, Atasoy Ulus X, Stellato Cristiana
Johns Hopkins University School of Medicine, Baltimore, MD, USA.
J Allergy Clin Immunol. 2008 Apr;121(4):853-9.e4. doi: 10.1016/j.jaci.2007.12.1166. Epub 2008 Feb 14.
IL-13, a critical cytokine in allergy, is regulated by as-yet-elusive mechanisms.
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.
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.
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.
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.
白细胞介素-13（IL-13）是过敏反应中的一种关键细胞因子，其调控机制尚不清楚。
我们研究了HuR对IL-13的转录后调控作用，HuR是一种与mRNA 3'非翻译区（UTR）富含腺苷酸-尿苷酸元件结合的蛋白质，可促进mRNA的稳定性和翻译。
使用转录抑制剂放线菌素D监测人Th2偏向性细胞中IL-13 mRNA的降解。将IL-13 3'UTR亚克隆到可诱导的β-珠蛋白报告基因中，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的结合。研究了HuR瞬时过表达和沉默对IL-13表达的影响。
与基线值相比，再刺激的Th2偏向性细胞中IL-13 mRNA半衰期显著延长。转染含IL-13 3'UTR质粒的H2细胞中β-珠蛋白mRNA的降解明显快于携带对照载体的细胞。HuR过表达增加了β-珠蛋白IL-13 3'UTR报告基因的半衰期。用抗HuR对Jurkat细胞核糖核蛋白复合物进行免疫沉淀，可显著富集IL-13 mRNA。通过对跨越IL-13 3'UTR的生物素标记RNA探针进行下拉试验，证实了HuR与IL-13 3'UTR的结合。二维蛋白质印迹分析显示刺激诱导的HuR翻译后修饰。在Jurkat细胞中，丝裂原诱导的IL-13 mRNA受到HuR过表达和沉默的显著影响。
丝裂原诱导的IL-13表达涉及转录本周转的变化以及HuR磷酸化的改变及其与mRNA 3'UTR的结合。
