Coordinate Regulation of GATA-3 and Th2 Cytokine Gene Expression by the RNA-Binding Protein HuR

Abstract

The posttranscriptional mechanisms whereby RNA-binding proteins (RBPs) regulate T cell differentiation remain unclear. RBPs can coordinately regulate the expression of functionally related genes via binding to shared regulatory sequences, such as the Adenylate-Uridylate-Rich Elements (ARE) present in mRNA’s 3′ untranslated region (UTR). The RBP HuR posttranscriptionally regulates IL-4, IL-13 and other Th2-cell-restricted transcripts. We hypothesized that the ARE-bearing GATA-3 gene, a critical regulator of Th2 polarization, is under HuR control as part of its coordinate posttranscriptional regulation of the Th2 program. We report that in parallel with stimulus-induced increase in GATA-3 mRNA and protein levels, GATA-3 mRNA half-life is increased after restimulation in the human T cell line Jurkat, in human memory and Th2 cells and in murine Th2-skewed cells. We demonstrate by immunoprecipitation of ribonucleoprotein complexes that HuR associates with the GATA-3 endogenous transcript in human T cells and found, using biotin pull-down assay, that HuR specifically interacts with its 3′UTR. Using both loss- and gain-of-function approaches in vitro and in animal models, we show that HuR is a critical mediator of stimulus-induced increase in GATA-3 mRNA and protein expression and that it positively influences GATA-3 mRNA turnover, in parallel with selective promotion of Th2 cytokine overexpression. These results suggest that HuR-driven posttranscriptional control plays a significant role in T cell development and effector function in both murine and human systems. A better understanding of HuR-mediated control of Th2 polarization may have utility in altering allergic airway inflammation in human asthmatic patients.

Posttranscriptional Gene Regulation of IL-17 by the RNA-Binding Protein HuR Is Required for Initiation of Experimental Autoimmune Encephalomyelitis

“…HuR has been reported to stabilize transcripts of multiple proinflammatory cytokines including, but not limited to, cyclooxygenase 2, TNF-a, vascular endothelial growth factor, and IL-13 (23)(24)(25). In this study, we report that HuR directly binds to IL-17 39 UTR mRNA and stabilizes its transcript, which in turn results in increases in both IL-17 mRNA and protein levels.…”

“…The RBP HuR positively regulates stability of many target mRNAs via binding AREs present in the 39 UTR, including inflammatory cytokines such as IL-4, IL-13, and TNF-a (23)(24)(25)(26). In this study, we demonstrated that IL-17 mRNA and protein levels in polarized Th17 cells from HuR conditional knockout (KO) mice are reduced compared with wild-type (WT) Th17 cells.…”

“…Briefly, Th17-polarized cells were lysed using polysomal lysis buffer (25,26). The lysates were precleared for 30 min at 4˚C by adding 30 mg IgG 1 (BD Bioscience) and 100 ml Protein-A Sepharose beads (50% slurry; Sigma-Aldrich) swollen in NT2 buffer (150 mM NaCl, 50 mM Tris [pH 7.4], 1 mM MgCl, and 0.05% Nonidet P-40) with 5% BSA.…”

“…RNA immunoprecipitation (RIP) was performed according to established protocol (25,26) with slight modification. Briefly, Th17-polarized cells were lysed using polysomal lysis buffer (25,26).…”

Regulation of CUG-binding Protein 1 (CUGBP1) Binding to Target Transcripts upon T Cell Activation

“…The molecular events leading to T cell activation must be tightly controlled to prevent the development of disease states, such as autoimmunity or malignancy (2)(3)(4), and it is becoming increasingly clear that posttranscriptional gene regulation at the level of mRNA degradation is critical for normal cellular activation, proliferation, and immune effector function (5,6). Indeed, over half of the gene expression changes in early T cell activation are a result of changes in mRNA half-life (7).…”