Tristetraprolin Mediates Interferon-γ mRNA Decay

Darlisha A. Williams,Julius R. SternJohn,Bernd Rattenbacher,Heidi H. Hau,Paul R. Bohjanen,Rachel L. Ogilvie,Perry J. Blackshear,Irina A. Vlasova

doi:10.1074/jbc.m901229200

Abstract

Tristetraprolin (TTP) regulates expression at the level of mRNA decay of several cytokines, including the T cell-specific cytokine, interleukin-2. We performed experiments to determine whether another T cell-specific cytokine, interferon-gamma (IFN-gamma), is also regulated by TTP and found that T cell receptor-activated T cells from TTP knock-out mice overproduced IFN-gamma mRNA and protein compared with activated T cells from wild-type mice. The half-life of IFN-gamma mRNA was 23 min in anti-CD3-stimulated T cells from wild-type mice, whereas it was 51 min in anti-CD3-stimulated T cells from TTP knock-out mice, suggesting that the overexpression of IFN-gamma mRNA in TTP knock-out mice was due to stabilization of IFN-gamma mRNA. Insertion of a 70-nucleotide AU-rich sequence from the murine IFN-gamma 3'-untranslated region, which contained a high affinity binding site for TTP, into the 3'-untranslated region of a beta-globin reporter transcript conferred TTP-dependent destabilization on the beta-globin transcript. Together these results suggest that TTP binds to a functional AU-rich element in the 3'-untranslated region of IFN-gamma mRNA and mediates rapid degradation of the IFN-gamma transcript. Thus, TTP plays an important role in turning off IFN-gamma expression at the appropriate time during an immune response.

Highlights

Interferon-␥ (IFN-␥),2 a critical T cell-derived cytokine, plays a key role in immune responses by inducing differentiation, activation, and proliferation of T cells, B cells, natural killer cells, and macrophages [1]
Many of the labile transcripts expressed in T cells contain AU-rich elements (AREs), conserved sequence elements found in their 3Ј-untranslated region (UTR) that function as mediators of mRNA decay [19]
The results obtained in this study demonstrate that IFN-␥ mRNA expression in T cells is regulated by TTP.Tag plasmid (TTP)

Summary

EXPERIMENTAL PROCEDURES

Purification and Stimulation of Primary Murine and Human T Cells—Generation of homozygous TTP knock-out mice has been described previously [28]. Measurement of Murine IFN-␥ mRNA by Real Time Polymerase Chain Reaction—Total cellular RNA was isolated from purified T cells from wild-type and TTP knock-out mice using TRIzol reagent (Invitrogen) according to the manufacturer’s directions. To assay IFN-␥ mRNA decay rates, purified T cells from wild-type or TTP homozygous knock-out mice were stimulated with anti-CD3 antibodies for 6 h and treated with 10 ␮g/ml of actinomycin D, and total RNA was harvested after 0, 15, 30, and 45 min. IFN-␥ mRNA was expressed at a very low level in unstimulated T cells from both wild-type and TTP knock-out mice (medium). IFN-␥ protein production was not detected in T cells from wild-type or TTP knock-out mice following stimulation with medium alone. IFN-␥ mRNA decay half-lives were calculated based on a model of first order decay to be 23 min (95% confidence interval, 21–26 min) in T cells

RESULTS

Findings

DISCUSSION