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J Allergy Clin Immunol. 2008 Apr;121(4):853-9.e4. doi: 10.1016/j.jaci.2007.12.1166. Epub 2008 Feb 14.

Posttranscriptional regulation of IL-13 in T cells: role of the RNA-binding protein HuR.

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  • 1Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Abstract

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.

PMID:
18279945
[PubMed - indexed for MEDLINE]
PMCID:
PMC2666917
Free PMC Article

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