Abstract
A prolonged activation of the immune system is one of the main causes of hyperproliferation of lymphocytes leading to defects in immune tolerance and autoimmune diseases. Fas ligand (FasL), a member of the TNF superfamily, plays a crucial role in controlling this excessive lymphoproliferation by inducing apoptosis in T cells leading to their rapid elimination. Here, we establish that posttranscriptional regulation is part of the molecular mechanisms that modulate FasL expression, and we show that in activated T cells FasL mRNA is stable. Our sequence analysis indicates that the FasL 3'-untranslated region (UTR) contains two AU-rich elements (AREs) that are similar in sequence and structure to those present in the 3'-UTR of TNFα mRNA. Through these AREs, the FasL mRNA forms a complex with the RNA-binding protein HuR both in vitro and ex vivo. Knocking down HuR in HEK 293 cells prevented the phorbol 12-myristate 13-acetate-induced expression of a GFP reporter construct fused to the FasL 3'-UTR. Collectively, our data demonstrate that the posttranscriptional regulation of FasL mRNA by HuR represents a novel mechanism that could play a key role in the maintenance and proper functioning of the immune system.
Highlights
Fas ligand (FasL) mRNA Has a Short Half-life in Activated T Cells—Previous reports have shown that FasL mRNA is rapidly induced in T lymphocytes upon T cell receptor engagement and mitogen stimulation [1]
To define whether this up-regulation was associated with a stabilization of the FasL mRNA, we first assessed its steady-state levels in T cells exposed to various activators
The rapid increase in FasL mRNA expression and the difference in its half-life between the two treatments (PHA and PMA) indicate that for this message to be properly expressed, stabilization mechanisms, similar to those reported for other TNF family members, are activated at least for a short period of time
Summary
The human FasL 3Ј-UTR was PCR-amplified, and a 5ЈBamHI site followed by stop codon and a 3Ј-HindIII site were introduced (for primer sequences see supplemental Materials and Methods). Transfections were performed in 10-cm dishes with 8 –16 g of plasmid DNA and Lipofectamine reagent (Invitrogen) according to the manufacturer’s instructions. Transfections of siRNA were performed with 60 nM duplexes (siHuR or siCtrl)/10-cm cell culture dish, using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer’s instructions [25]. 2 l of cDNA was PCR-amplified with HotStarTaq (Qiagen) using actin, FasL, or GFP cDNA-specific primers (see supplemental Materials and Methods). The RNA binding assay was performed with purified 300 ng of GST and GST-HuR as described previously [18] except that upon incubation the RNA-protein complex was not treated with RNase T1. The cDNA was PCR-amplified with primers described in supplemental Materials and Methods, using the Quantitect SYBR Green kit (Qiagen) in a Corbett Rotor Gene real time thermocycler. The Ct value was used to calculate the amount of the cDNA of interest by extrapolation from a standard curve
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