The role of HuR in the post-transcriptional regulation of interleukin-3 in T cells.

Human Interleukin-3 (IL-3) is a lymphokine member of a class of transiently expressed mRNAs harboring Adenosine/Uridine-Rich Elements (ARE) in their 3' untranslated regions (3'-UTRs). The regulatory effects of AREs are often mediated by specific ARE-binding proteins (ARE-BPs). In this report, we show that the human IL-3 3'-UTR plays a post-transcriptional regulation role in two human transformed cell lines. More specifically, we demonstrate that the hIL-3 3'-UTR represses the translation of a luciferase reporter both in HeLa and Jurkat T-cells. These results also revealed that the hIL-3 3'-UTR-mediated translational repression is exerted by an 83 nt region comprised mainly by AREs and some non-ARE sequences. Moreover, electrophoretic mobility shift assays (EMSAs) and UV-crosslinking analysis show that this hIL-3 ARE-rich region recruits five specific protein complexes, including the ARE-BPs HuR and TIA-1. HuR binding to this ARE-rich region appears to be spatially modulated during T-cell activation. Together, these results suggest that HuR recognizes the ARE-rich region and plays a role in the IL-3 3'-UTR-mediated post-transcriptional control in T-cells

The hIL-3 ARE-rich region is recognized by specific RNA-binding protein complexes in both HeLa and Jurkat T-cells.

<p>(A) Radiolabeled RNA probes corresponding to the hIL-3 3'-UTR UP, ARE and Down regions were incubated with (lanes 5–8) or without (lanes 1-4) cytoplasmic extracts from HeLa cells. Arrows indicate the obtained gel shifts. (B) To assess the specificity of the RBP complexes that recognize the IL-3 ARE-rich region in HeLa cells, an EMSA competition assay was performed using cold RNA competitors at increasing fold-excess (10¹–10⁴): Up (lanes 2–5), ARE (lanes 6–9) or Down (lanes 10–13). Non- competitor RNA (nc) was added in lane 1. The pGem7z multiple cloning site (80nt) was used as a negative control RNA (c-RNA). (C) EMSAs were also performed using cytoplasmic extracts from Jurkat cells. (D) EMSA reactions were treated with 15 μg of Proteinase K (PK).</p

T cell activation modulates HuR binding towards the hIL-3 ARE-rich region.

<p>(A) HuR EMSA supershift analysis was carried out with Jurkat cytoplasmic extracts activated at 0, 6, 12 and 24 hours. (B) Graphic representation of HuR supershift quantification during T cell activation. Values represent mean ± standard error of the mean (SEM) from two experiments. Fold changes were normalized to 0 hours of T cell-activation with PMA/Ionomycin.</p

RNA-binding proteins ranging from 34-88 kDa recognize the hIL-3 ARE-rich region.

<p>(A) IL-3 Up, ARE and Down radiolabeled RNAs were incubated with HeLa cytoplasmic protein extracts (lanes 2–4) and the RNA-protein interactions were UV cross-linked. Samples were subjected to RNase digestion and to SDS-10% polyacrylamide gel electrophoresis. (B) UV-cross linking assays were also performed with Jurkat cytoplasmic cell extracts. IL-3 Up, ARE and Down radiolabeled RNAs were incubated with Jurkat cytoplasmic protein extracts (lanes 6-8).</p

Knockdown of HuR increases the repression exerted by the IL-3 3'-UTR.

<p>Jurkat cells were co-transfected with the firefly reporter constructs (Luc and 3’UTR) and siRNA against HuR (siHuR) or control non-targeting siRNA (siCtrl). (A) Total protein extracts were used in Western blot analysis for HuR. β-actin was used as a loading control. (B) 48 hrs post-transfection, cells were harvested and luciferase activities were measured. Two-tailed <i>t</i>-tests were used for statistical analysis. The asterisk indicates a statistical significant (<i>P</i><0.05) result when compared to the siCtrl.</p

The hIL-3 ARE-rich region is recognized by HuR and TIA-1 ARE-BPs.

<p>(A) The ³²P-labeled IL-3 ARE-rich sequence was incubated with HeLa cytoplasmic protein extracts and TIA-1, TIAR, AUF-1 and HuR antibodies. Non-immune goat serum (IgG) antibody was used as a negative control (lane 7). IL-3 ARE incubated with the HeLa cytoplasmic extract without antibody addition was used as an additional control in the analysis (lane 1). (B) Jurkat cytoplasmic protein extracts were also used in the EMSA super shift assays.</p

T cell activation modulates HuR spatial protein concentration.

<p>(A-B) Cytoplasmic and total protein extraction of Jurkat cells activated with DMSO and PMA/Ionomycin (P/IO) during 0, 6, 12 and 24 hours were used to perform a HuR immunoblotting. (C) β-actin and hnRNP C1/C2 protein distribution was determined in Jurkat cytoplasmic and nuclear protein extracts. Fold changes were normalized to 0 hours of T cell-activation.</p