Posttranscriptional regulation of IL-13 in T cells: role of the RNA-binding protein HuR.
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.
Casolaro
Vincenzo
V
Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Fang
Xi
X
Tancowny
Brian
B
Fan
Jinshui
J
Wu
Fan
F
Srikantan
Subramanya
S
Asaki
S Yukiko
SY
De Fanis
Umberto
U
Huang
Shau-Ku
SK
Gorospe
Myriam
M
Atasoy
Ulus X
UX
Stellato
Cristiana
C
D020413:3' Untranslated Regions / Q000235:genetics / Q000276:immunology; D000954:Antigens, Surface / Q000502:physiology*; D001483:Base Sequence; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D006801:Humans; D018793:Interleukin-13 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D008985:Monensin / Q000494:pharmacology; D012323:RNA Processing, Post-Transcriptional / Q000276:immunology*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018418:Th2 Cells / Q000276:immunology* / Q000378:metabolism*
D020413:3' Untranslated Regions; D000954:Antigens, Surface; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D018793:Interleukin-13; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D008985:Monensin; D013755:Tetradecanoylphorbol Acetate