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• W2024139218 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. 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. Fas and Fas ligand (FasL) 3The abbreviations used are: FasLFas ligandAREAU-rich elementFADDFas-associated protein with death domainHuRhuman antigen RIPimmunoprecipitationPHAphytohemagglutinin-MPMAphorbol 12-myristate 13-acetate. are a transmembrane receptor ligand pair of the TNF receptor and TNF family, primarily involved in maintaining the homeostasis of the immune system by eliminating antigen-activated lymphocytes which consequently limits the magnitude and duration of the immune response (1Strasser A. Jost P.J. Nagata S. Immunity. 2009; 30: 180-192Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). Fas/FasL mediates this effect by triggering an apoptotic response in these cells. This response involves recruiting the adaptor protein FADD to the intracellular tail of Fas via an interaction with a death domain. In turn, the FasL·Fas·FADD complex recruits procaspases 8 and 10 via homotypic death effector domain interactions leading to caspase cleavage and apoptosis (1Strasser A. Jost P.J. Nagata S. Immunity. 2009; 30: 180-192Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). The importance of Fas and FasL is evidenced by the fact that defects in their expression trigger excessive lymphoproliferation resulting in loss of immune tolerance and autoimmune diseases (1Strasser A. Jost P.J. Nagata S. Immunity. 2009; 30: 180-192Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 2Karray S. Kress C. Cuvellier S. Hue-Beauvais C. Damotte D. Babinet C. Lévi-Strauss M. J. Immunol. 2004; 172: 2118-2125Crossref PubMed Scopus (58) Google Scholar, 3Watanabe-Fukunaga R. Brannan C.I. Copeland N.G. Jenkins N.A. Nagata S. Nature. 1992; 356: 314-317Crossref PubMed Scopus (2696) Google Scholar, 4Senju S. Negishi I. Motoyama N. Wang F. Nakayama K. Nakayama K. Lucas P.J. Hatakeyama S. Zhang Q. Yonehara S. Loh D.Y. Int. Immunol. 1996; 8: 423-431Crossref PubMed Scopus (24) Google Scholar). Although the Fas receptor is constitutively expressed in most tissues, FasL is restricted to activated lymphocytes and sites of immune privilege (1Strasser A. Jost P.J. Nagata S. Immunity. 2009; 30: 180-192Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). This supports the idea that FasL but not Fas is the limiting factor in the Fas/FasL-induced signaling pathways. Fas ligand AU-rich element Fas-associated protein with death domain human antigen R immunoprecipitation phytohemagglutinin-M phorbol 12-myristate 13-acetate. The disruption of Fas/FasL signaling pathways by spontaneous mutations in mice or in human patients has been associated with diseases such as systematic lupus erythematosus or autoimmune lymphoproliferative syndrome (1Strasser A. Jost P.J. Nagata S. Immunity. 2009; 30: 180-192Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 3Watanabe-Fukunaga R. Brannan C.I. Copeland N.G. Jenkins N.A. Nagata S. Nature. 1992; 356: 314-317Crossref PubMed Scopus (2696) Google Scholar). Likewise, an increase in FasL-mediated apoptosis of normal, Fas-bearing, bystander cells causes certain immunopathologies such as hepatitis, which is linked to excessive T cell activation (1Strasser A. Jost P.J. Nagata S. Immunity. 2009; 30: 180-192Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). Hence, a tight regulation of FasL expression during T cell activation is critical to maintain the homeostasis and the proper functioning of the immune system. During the past decade, the majority of studies have focused on delineating the molecular mechanisms that modulate FasL expression at the transcriptional level. Several factors, such as NF-AT, NF-κB, and IRF-1, have been shown to activate the transcription of the fasl gene directly (5Chow W.A. Fang J.J. Yee J.K. J. Immunol. 2000; 164: 3512-3518Crossref PubMed Scopus (80) Google Scholar, 6Hsu S.C. Gavrilin M.A. Lee H.H. Wu C.C. Han S.H. Lai M.Z. Eur. J. Immunol. 1999; 29: 2948-2956Crossref PubMed Google Scholar, 7Li-Weber M. Laur O. Krammer P.H. Eur. J. Immunol. 1999; 29: 3017-3027Crossref PubMed Scopus (64) Google Scholar). It is well established for other TNF family members such as TNFα, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and several interleukins, that although transcription is tightly regulated, the amount of mRNA produced does not correlate with the protein expression levels (8Beutler B. Krochin N. Milsark I.W. Luedke C. Cerami A. Science. 1986; 232: 977-980Crossref PubMed Scopus (1009) Google Scholar). In many cases, although the steady-state levels of these messages remain unchanged, the protein levels increase significantly in response to extracellular stimuli (9Jacob C.O. Lee S.K. Strassmann G. J. Immunol. 1996; 156: 3043-3050PubMed Google Scholar, 10Jacobs D.B. Mandelin 2nd, A.M. Giordano T. Xue I. Malter J.S. Singh L.D. Snyder A.K. Singh S.P. Life Sci. 1996; 58: 2083-2089Crossref PubMed Scopus (5) Google Scholar). This increase is because the expression of these mRNAs is also regulated posttranscriptionally at the level of subcellular localization, mRNA turnover, and translational efficiency. These posttranscriptional effects are mediated mainly by AU-rich elements (AREs) in the 3′-untranslated regions (3′-UTRs) of these and other messages (11Antic D. Keene J.D. J. Cell Sci. 1998; 111: 183-197Crossref PubMed Google Scholar, 12Barreau C. Paillard L. Osborne H.B. Nucleic Acids Res. 2005; 33: 7138-7150Crossref PubMed Scopus (761) Google Scholar, 13Asirvatham A.J. Magner W.J. Tomasi T.B. Cytokine. 2009; 45: 58-69Crossref PubMed Scopus (127) Google Scholar). AREs are known to regulate a variety of transiently expressed cytokines during T cell activation. These include IFNγ, GM-CSF, CD83, TNFα, and CD40L (14Tobler A. Miller C.W. Norman A.W. Koeffler H.P. J. Clin. Invest. 1988; 81: 1819-1823Crossref PubMed Scopus (51) Google Scholar, 15Chemnitz J. Pieper D. Grüttner C. Hauber J. Eur. J. Immunol. 2009; 39: 267-279Crossref PubMed Scopus (16) Google Scholar, 16Ford G.S. Barnhart B. Shone S. Covey L.R. J. Immunol. 1999; 162: 4037-4044PubMed Google Scholar). This regulation is due to the stabilization of these messages by a mechanism that involves their association with ARE-binding proteins such as HuR (12Barreau C. Paillard L. Osborne H.B. Nucleic Acids Res. 2005; 33: 7138-7150Crossref PubMed Scopus (761) Google Scholar, 17Sakai K. Kitagawa Y. Saiki M. Saiki S. Hirose G. Mol. Immunol. 2003; 39: 879-883Crossref PubMed Scopus (13) Google Scholar, 18Di Marco S. Hel Z. Lachance C. Furneaux H. Radzioch D. Nucleic Acids Res. 2001; 29: 863-871Crossref PubMed Google Scholar). HuR belongs to the ELAV (embryonic lethal abnormal vision) family of RNA-binding proteins that contains three other members, HuB, HuC, and HuD (19Keene J.D. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 5-7Crossref PubMed Scopus (257) Google Scholar). Of the four ELAV family members, only HuR is ubiquitously expressed, and it is particularly well expressed in primary and secondary lymphoid tissues such as the thymus, spleen, and the gut (20Atasoy U. Watson J. Patel D. Keene J.D. J. Cell Sci. 1998; 111: 3145-3156Crossref PubMed Google Scholar, 21Mukherjee N. Lager P.J. Friedersdorf M.B. Thompson M.A. Keene J.D. Mol. Syst. Biol. 2009; 5: 288Crossref PubMed Scopus (75) Google Scholar). Recently, it has been shown that in a tissue-specific knock-out mice, disrupting the hur gene in T lymphocytes causes a severe defect in their maturation (22Papadaki O. Milatos S. Grammenoudi S. Mukherjee N. Keene J.D. Kontoyiannis D.L. J. Immunol. 2009; 182: 6779-6788Crossref PubMed Scopus (77) Google Scholar). Indeed, although these mice have a wild type thymic microenvironment, the HuR−/− thymocytes of these mice are unable to undergo positive selection, negative selection, and thymic egress (22Papadaki O. Milatos S. Grammenoudi S. Mukherjee N. Keene J.D. Kontoyiannis D.L. J. Immunol. 2009; 182: 6779-6788Crossref PubMed Scopus (77) Google Scholar). The defect in positive selection is attributed to an alteration in the T cell receptor signaling pathway. Likewise, the defect in thymic egress can be explained by defects in chemokine signaling required in this process such as the TNF receptor family members including Fas (22Papadaki O. Milatos S. Grammenoudi S. Mukherjee N. Keene J.D. Kontoyiannis D.L. J. Immunol. 2009; 182: 6779-6788Crossref PubMed Scopus (77) Google Scholar). The observations described above and the fact that FasL belongs to the TNFα family of cytokines raised the possibility that the expression of FasL could depend on posttranscriptional events involving ARE-binding proteins such as HuR. In this study we addressed this question and showed that the 3′-UTR of FasL mRNA contains AREs strikingly similar in structure to those of TNFα. Our data demonstrate that via these AREs, FasL mRNA associates with HuR and that this association is absolutely required for its expression. We also discuss the functional relevance of posttranscriptional regulation of FasL and its impact on T lymphocyte maturation. The human FasL 3′-UTR was PCR-amplified, and a 5′-BamHI site followed by stop codon and a 3′-HindIII site were introduced (for primer sequences see supplemental Materials and Methods). This PCR fragment was inserted into a pEGFP-C2 vector (Clontech) between the BglII and HindIII sites downstream of GFP. The GST and GST-HuR constructs were previously described (23Brennan C.M. Gallouzi I.E. Steitz J.A. J. Cell Biol. 2000; 151: 1-14Crossref PubMed Scopus (312) Google Scholar). All plasmids were prepared using the plasmid maxiprep kit (Qiagen) according to the manufacturer's instructions. The fusion proteins were purified as described in (23Brennan C.M. Gallouzi I.E. Steitz J.A. J. Cell Biol. 2000; 151: 1-14Crossref PubMed Scopus (312) Google Scholar, 24Frangioni J.V. Neel B.G. Anal. Biochem. 1993; 210: 179-187Crossref PubMed Scopus (825) Google Scholar) with the following modifications. The proteins were eluted from the glutathione-agarose beads with three applications of 500 μl of glutathione elution buffer (10 mm for the first elution, and 20 mm for the second and third elutions). Proteins were then dialyzed overnight against phosphate-buffered saline at 4 °C. The 20 mm glutathione eluates were the most pure (as determined by SDS-PAGE) and were used in all experiments. Jurkat cells (E6 clone) (American Type Culture Collection (ATCC)) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen) augmented with 10% FBS (Invitrogen). Jurkat cells were stimulated with PHA (Sigma) at 1 μg/ml or at 50 ng/ml for the times indicated. RNA stability curves were generated by treatment of cells with actinomycin D (Sigma) at 5 μg/ml. Transfections were performed in 12-well plates using 1 μg of plasmid DNA and TransPass RV (New England Biolabs) according to the manufacturer's instructions. HEK 293 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (Multicell) supplemented with 10% FBS (Sigma). HEK 293 cells were stimulated with 50 ng/ml PMA for the times indicated. Transfections were performed in 10-cm2 dishes with 8–16 μg 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-cm2 cell culture dish, using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions (25van der Giessen K. Di Marco S. Clair E. Gallouzi I.E. J. Biol. Chem. 2003; 278: 47119-47128Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). HEK 293 cells were transfected at 20% confluence then retransfected with siRNA 24 h later. Knockdown was assessed 52 h after the first transfection. Immunoprecipitation and RNA preparation were performed as previously described (26Tenenbaum S.A. Lager P.J. Carson C.C. Keene J.D. Methods. 2002; 26: 191-198Crossref PubMed Scopus (234) Google Scholar) with several modifications. Briefly, cell extracts were prepared from PHA-stimulated Jurkat cells or PMA-stimulated HEK 293 cells. mRNP lysate (400 μl) was precleared with 8 μl of protein G-Sepharose, and the precleared lysate was subsequently divided for each immunoprecipitation (IP). IPs were performed using antibodies to HuR (3A2) and IgG1 isotype control antibody (Sigma). Specific messages associated with HuR were defined using RT-PCR. RNA was isolated from the immunoprecipitated mRNP complexes using the ChargeSwitch RNA isolation kit (Invitrogen) scaled to 20% of the manufacturer's instructions. Purified RNA was eluted in 30 μl of water, and 4 μl was reverse-transcribed using the Sensiscript reverse transcription kit (Qiagen) according to the manufacturer's protocol in 20 μl of final volume. Subsequently, 2 μl of cDNA was PCR-amplified with HotStarTaq (Qiagen) using actin, FasL, or GFP cDNA-specific primers (see supplemental Materials and Methods). The sequences of the primers, as well as the PCR conditions, are described in the supplemental Materials and Methods. The FasL RNA probes were produced by in vitro transcription as described previously (27Gallouzi I.E. Parker F. Chebli K. Maurier F. Labourier E. Barlat I. Capony J.P. Tocque B. Tazi J. Mol. Cell. Biol. 1998; 18: 3956-3965Crossref PubMed Scopus (167) Google Scholar). Regions of FasL 3′-UTR were PCR-amplified with sense primers containing the T7 promoter in 5′ (see supplemental Materials and Methods for primer sequences). The purified PCR fragment was used as a template for transcription using [32P]UTP with T7 RNA polymerase (Promega) according to the manufacturer's instructions. The RNA binding assay was performed with purified 300 ng of GST and GST-HuR as described previously (18Di Marco S. Hel Z. Lachance C. Furneaux H. Radzioch D. Nucleic Acids Res. 2001; 29: 863-871Crossref PubMed Google Scholar) except that upon incubation the RNA-protein complex was not treated with RNase T1. Northern blot analysis was performed as described (28Wojciechowski W. DeSanctis J. Skamene E. Radzioch D. J. Immunol. 1999; 163: 2688-2696PubMed Google Scholar). 15 μg of RNA was used and was isolated with the RNeasy Plus extraction kit (Qiagen) according to the manufacturer's instructions. After transferring to a Hybond-N membrane (Amersham Biosciences) and UV cross-linking, the blot was hybridized with GFP or 18 S rRNA probes prepared with [32P]dCTP by random priming with Ready-to-go DNA labeling beads (GE Healthcare) according to the manufacturer's instructions. PCR-amplified fragments of GFP and 18 S rRNA were used to generate labeled probes. After hybridization, the membranes were washed and subsequently exposed on BioMax films (Kodak). Cells were counted by using a hemocytometer and adjusted to a concentration of 1 × 106 cells/ml. 0.5 ml of cells was washed three times in 1.5 ml of PBS and then resuspended in 0.5 ml of PBS with 2% FBS. Acquisition of data was done on a FACScan (Becton Dickinson), and the analysis of these data was performed with FlowJo software. Total cell extracts were prepared as described (27Gallouzi I.E. Parker F. Chebli K. Maurier F. Labourier E. Barlat I. Capony J.P. Tocque B. Tazi J. Mol. Cell. Biol. 1998; 18: 3956-3965Crossref PubMed Scopus (167) Google Scholar). Western blotting was performed as described previously (23Brennan C.M. Gallouzi I.E. Steitz J.A. J. Cell Biol. 2000; 151: 1-14Crossref PubMed Scopus (312) Google Scholar). The blots were probed with antibodies to HuR (3A2), tubulin (Developmental Studies Hybridoma Bank), and GFP (JL8; Clontech). Immunofluorescence was performed as described previously (29Fan X.C. Steitz J.A. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 15293-15298Crossref PubMed Scopus (395) Google Scholar). Anti-HuR (3A2) was used at a 1:1500 dilution in 1% goat serum/PBS. HuR was detected using a 1:500 diluted Alexa Fluor 488-conjugated goat anti-mouse IgG polyclonal antibody. DAPI staining 1:20,000 was performed after secondary antibody. A Zeiss Axiovision 3.1 microscope was used to observe the cells with a 63 × oil objective, and an Axiocam HR (Zeiss) digital camera was used for immunofluorescence photography. RNA was isolated for quantitative RT-PCR by using the ChargeSwitch RNA isolation kit (Invitrogen) scaled to 20% of the manufacturer's instructions. RNA was quantitated using the Ribogreen kit (Molecular Probes), and 150 ng was reverse transcribed using the Sensiscript reverse transcription kit (Qiagen). Subsequently, 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. Previous reports have shown that FasL mRNA is rapidly induced in T lymphocytes upon T cell receptor engagement and mitogen stimulation (1Strasser A. Jost P.J. Nagata S. Immunity. 2009; 30: 180-192Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). 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. Jurkat T cells were treated with either PHA, a lectin that nonspecifically aggregates cell surface receptors or PMA, a PKC agonist (1Strasser A. Jost P.J. Nagata S. Immunity. 2009; 30: 180-192Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 30Chien E.J. Hsieh D.J. Wang J.E. J. Cell. Biochem. 2001; 81: 604-612Crossref PubMed Scopus (10) Google Scholar). We observed a rapid increase in the level of FasL mRNA at 3 h of PHA treatment. In contrast, PMA treatment had a smaller effect on the steady-state levels of FasL mRNA (Fig. 1A). Interestingly, levels of FasL mRNA return to baseline within 6–12 h of PHA stimulus, indicating that FasL is transiently expressed in response to lectins similarly to other cytokines. In the absence of PHA or PMA, however, the FasL mRNA was hard to detect (Fig. 1A). Next, we determined the half-life of FasL mRNA under these conditions. We performed actinomycin D pulse-chase experiments (31Dormoy-Raclet V. Ménard I. Clair E. Kurban G. Mazroui R. Di Marco S. von Roretz C. Pause A. Gallouzi I.E. Mol. Cell. Biol. 2007; 27: 5365-5380Crossref PubMed Scopus (89) Google Scholar) and determined the half-life of the FasL mRNA using quantitative RT-PCR analysis. Jurkat cells were treated with PHA or PMA for 3 h to induce maximal FasL mRNA expression and then treated with 5 μg/ml actinomycin D for different periods of time. We observed that whereas upon PHA treatment the half-life of FasL mRNA in Jurkat cells was slightly over 60 min, upon PMA treatment, the FasL mRNA half-life was >4 h (Fig. 1B). Of note, because in untreated Jurkat cells the FasL mRNA is difficult to detect, it was not possible to assess its half-life under these conditions. Therefore, 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. The stabilization of cytokine mRNAs is usually mediated by U-rich elements such as AREs located in their 3′-UTR (12Barreau C. Paillard L. Osborne H.B. Nucleic Acids Res. 2005; 33: 7138-7150Crossref PubMed Scopus (761) Google Scholar, 32Jacobson A. Peltz S.W. Annu. Rev. Biochem. 1996; 65: 693-739Crossref PubMed Scopus (575) Google Scholar). Our initial analysis of the primary sequence of FasL 3′-UTR indicated that it contains the typical AUUUA consensus sequence as well as a 300nt U-rich region (supplemental Fig. 1). The mFold prediction software for mRNA folding showed that both TNFα and FasL mRNAs could fold and form a highly similar secondary structure (Fig. 2A). This prediction method has highlighted the existence in the extreme 3′-end of the FasL 3′-UTR of two conserved U-rich sequences that are similar in structure and localization to the TNFα ARE-1 and ARE-2 (Fig. 2B) (33Fialcowitz E.J. Brewer B.Y. Keenan B.P. Wilson G.M. J. Biol. Chem. 2005; 280: 22406-22417Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). These observations suggest that similarly to TNFα (18Di Marco S. Hel Z. Lachance C. Furneaux H. Radzioch D. Nucleic Acids Res. 2001; 29: 863-871Crossref PubMed Google Scholar), the expression of FasL mRNA could involve the association of these U-rich sequences (AREs 1 or 2) with the HuR protein. To test this possibility, we first assessed whether HuR can interact with the FasL mRNA in Jurkat T cells. To ensure the expression of FasL mRNA and reduce FasL-induced apoptosis, Jurkat cells were treated for only 2 h with PHA. These cells were then used to perform IP experiments with the anti-HuR antibody (34Gallouzi I.E. Brennan C.M. Stenberg M.G. Swanson M.S. Eversole A. Maizels N. Steitz J.A. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 3073-3078Crossref PubMed Scopus (272) Google Scholar) followed by RT-PCR analysis. Prior to the IP experiments, the cells were either exposed or not to UV irradiation for 4 min as described (34Gallouzi I.E. Brennan C.M. Stenberg M.G. Swanson M.S. Eversole A. Maizels N. Steitz J.A. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 3073-3078Crossref PubMed Scopus (272) Google Scholar). We observed that in PHA-treated Jurkat cells, with or without UV treatment, the FasL mRNA specifically associated with HuR compared with an isotype-matched control antibody (Fig. 3A). To map the HuR binding sites in the FasL 3′-UTR and to prove that the interaction between HuR and FasL mRNA is direct, the FasL 3′-UTR was divided into six 150-nucleotide regions that were used as probes for RNA electromobility shift assay (Fig. 3B). We observed that all regions other than region 2 (R2) bind purified GST-HuR (Fig. 3C). However, regions 5 and 6, which are particularly U-rich (supplemental Fig. 2A), show a better binding to HuR (Fig. 3C, compare lanes 15 and 18 with lanes 3, 6, 9, and 12). We also showed that this interaction is competed away by an excess of the same unlabeled probes (Fig. 3D) but not with an excess of unlabeled R2 probe (supplemental Fig. 3).FIGURE 3Fas ligand mRNA associates with HuR via U-rich sequences in the 3′-UTR. A, Jurkat cells stimulated with 1 μg/ml PHA for 2 h were either exposed to UV for 4 min (34Gallouzi I.E. Brennan C.M. Stenberg M.G. Swanson M.S. Eversole A. Maizels N. Steitz J.A. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 3073-3078Crossref PubMed Scopus (272) Google Scholar) or not. These cells were then used to prepare total cell extracts. HuR was immunoprecipitated from Jurkats with the anti-HuR (3A2) antibody (lanes 2 and 4). A mouse IgG1 antibody was used as an isotype-matched specificity control (lanes 1 and 3). IP was followed by RT-PCR for FasL (upper) and the β-actin mRNA (lower) that was used as a positive control. Representative blots of two independent experiments are shown. B, diagram indicates the location and length of probes for the gel shift assay. C, HuR associates with regions R1 (lane 3), R3 (lane 9), R4 (lane 12), R5 (lane 15), and R6 (lane 18) of the FasL 3′-UTR. Gel shift binding assays were performed by incubating 300 ng of purified GST or GST-HuR protein with radiolabeled probes (see B). Radiolabeled probe·GST·HuR complexes (HuR-C) are indicated with an asterisk. D, increasing concentrations of unlabeled probes for the regions R5 and R6 were incubated with GST-HuR in the presence of radiolabeled probes from the same regions. HuR-Cs are indicated by asterisks. GST was incubated with radiolabeled R2 (lane 1), R5 (lane 4), and R6 (lane 12) probes as negative controls. These gel shifts were performed with 300 ng of purified GST or GST-HuR and with 0.01 × (lanes 6 and 14), 0.1× (lanes 7 and 15), 1 × (lanes 8 and 16), 10× (lanes 9 and 17), and 100× (lanes 10 and 18) amounts of unlabeled probe. C and D, representative gel shift blots of two independent experiments are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next, we mapped regions 5 and 6 more precisely, to determine the number of HuR binding sites in these regions. Regions 5 and 6 of the FasL 3′-UTR were further subdivided into six 50-nucleotide subregions (Fig. 4A). RNA electromobility shift assay experiments as described above showed strong interactions between HuR and some of the subregions, notably subregions 5.2, 5.3, 6.1, 6.2, and 6.3 (Fig. 4B). Therefore, our results indicate that there are at least four distinct U-rich HuR binding sites in regions 5 and 6 in addition to the weaker binding sites in regions 1, 3, and 4 (Fig. 3C). Furthermore, because the binding of HuR to these fragments can be competed by the same unlabeled probes (Fig. 4C), these interactions seem to be specific. Together, these observations argue that HuR binds directly to the 3′-UTR of FasL in an ARE-dependent manner. The data described above suggest that ARE sequences could collaborate with HuR to ensure the rapid expression of FasL mRNA during T cell activation. Hence, we assessed whether the expression of FasL mRNA depends on HuR in cells treated with activators such as PMA or PHA. Ideally, we would have liked to test this possibility in the context of the full-length FasL message by following its expression in the presence or absence of HuR. However, expressing the full-length FasL mRNA caused massive cell death in different cell lines, including Jurkat cells regardless of stimulus (data not shown). Therefore, we used GFP reporter constructs in which we fused the FasL 3′-UTR to the GFP coding sequence. The entire FasL 3′-UTR was included in the reporter construct because any or all of the HuR binding sites described in FIGURE 3, FIGURE 4 could mediate regulatory effects. Surprisingly, flow cytometry experiments showed that the level of expressed GFP was reduced by >55% in untreated Jurkat cells transfected with GFP-FasL 3′-UTR compared with GFP alone (Fig. 5A). This is probably due to other RNA-binding proteins which, in the absence of any stimulus, bind the FasL 3′-UTR in trans and promote its rapid decay. To eliminate the possibility that poor transfection efficiency in Jurkat cells led to the evaluation of a selected population, we transfected HEK 293 cells with the same constructs and obtained similar results by flow cytometry (Fig. 5B). In agreement with GFP protein levels, Northern blot analysis showed that in the absence of any treatment, the amount of GFP-FasL 3′-UTR mRNA in HEK 293 cells was significantly decreased compared with the GFP control (Fig. 5, C and D). These experiments suggest that in the absence of extracellular stimulus, the FasL 3′-UTR mediates the rapid decay of the GFP reporter mRNA. Due to the effects of PMA or PHA treatment on FasL expression, we investigated whether these actions were recapitulated on the expression of the GFP-conjugated FasL 3′-UTR. The GFP-FasL 3′-UTR or GFP plasmids were transfected into HEK 293 cells which were then treated or not with PMA. It is well established that HEK 293 cells activate the PKC pathway in response to PMA but not to PHA (35Böl G.F. Hülster A. Pfeuffer T. Biochim. Biophys. Acta. 1997; 1358: 307-313Crossref PubMed Scopus (40) Google Scholar). Additionally, our stability experiments presented in Fig. 1B clearly showed that PMA has a much stro" @default.
• W2024139218 created "2016-06-24" @default.
• W2024139218 creator A5004154228 @default.
• W2024139218 creator A5021378048 @default.
• W2024139218 creator A5071885510 @default.
• W2024139218 creator A5077905452 @default.
• W2024139218 creator A5084338307 @default.
• W2024139218 date "2010-10-01" @default.
• W2024139218 modified "2023-09-29" @default.
• W2024139218 title "FasL Expression in Activated T Lymphocytes Involves HuR-mediated Stabilization" @default.
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