Identification of TIAR as a Protein Binding to the Translational Regulatory AU-rich Element of Tumor Necrosis Factor α mRNA

Abstract

In monocyte/macrophages, the translation of tumor necrosis factor α (TNF-α) mRNA is tightly regulated. In unstimulated cells, translation of TNF-α mRNA is blocked. Upon stimulation with lipopolysaccharides, this repression is overcome, and the mRNA becomes efficiently translated. The key element in this regulation is the AU-rich element (ARE). We have previously reported the binding of two cytosolic protein complexes to the TNF-α mRNA ARE. One of these complexes (complex 1) forms with extracts of both unstimulated and lipopolysaccharide-stimulated macrophages and requires a large fragment of the ARE containing clustered AUUUA pentamers. The other complex (complex 2) is only detected after cell activation, binds to a minimal UUAUUUAUU nonamer, and is composed of a 55-kDa protein. Here, we report the identification of the RNA-binding protein TIAR as a protein involved in complex 1. The RNA sequence bound by TIAR and the cytoplasmic localization of this protein in macrophages argue for an involvement of TIAR in TNF mRNA posttranscriptional regulation. In monocyte/macrophages, the translation of tumor necrosis factor α (TNF-α) mRNA is tightly regulated. In unstimulated cells, translation of TNF-α mRNA is blocked. Upon stimulation with lipopolysaccharides, this repression is overcome, and the mRNA becomes efficiently translated. The key element in this regulation is the AU-rich element (ARE). We have previously reported the binding of two cytosolic protein complexes to the TNF-α mRNA ARE. One of these complexes (complex 1) forms with extracts of both unstimulated and lipopolysaccharide-stimulated macrophages and requires a large fragment of the ARE containing clustered AUUUA pentamers. The other complex (complex 2) is only detected after cell activation, binds to a minimal UUAUUUAUU nonamer, and is composed of a 55-kDa protein. Here, we report the identification of the RNA-binding protein TIAR as a protein involved in complex 1. The RNA sequence bound by TIAR and the cytoplasmic localization of this protein in macrophages argue for an involvement of TIAR in TNF mRNA posttranscriptional regulation. tumor necrosis factor lipopolysaccharides AU-rich element untranslated region electrophoretic shift assay. Tumor necrosis factor-α (TNF-α)1 is a cytokine predominantly produced by macrophages but also by lymphocytes, NK cells, astrocytes, and other cell types. The most powerful inducers of TNF-α production by macrophages are the lipopolysaccharides (LPS), which are membrane components released by Gram-negative bacteria in the course of infection (1Beutler B. Tumor Necrosis Factors: The Molecules and Their Emerging Role in Medicine. Raven Press, Ltd., New York1992: 485-513Google Scholar). It is now well established that the induction of TNF-α production upon stimulation of macrophages by LPS results from both an enhancement of TNF-α gene transcription and a translational derepression of the mRNA. In unstimulated macrophages, TNF-α mRNA translation is blocked. Upon stimulation with LPS, this repression is overcome, and TNF-α mRNA becomes efficiently translated (2Han J. Brown T. Beutler B. J. Exp. Med. 1990; 171: 465-475Crossref PubMed Scopus (431) Google Scholar). The key element involved in this regulation is the AU-rich element (ARE) located in the 3′-untranslated region (-UTR) of TNF-α mRNA (3Han J. Beutler B. Eur. Cytokine Netw. 1990; 1: 71-75PubMed Google Scholar). This 70-nucleotide-long sequence is composed of several repeats of the AUUUA pentamer. The physiological importance of TNF-α mRNA translational control is demonstrated by the fact that the expression of a TNF-α transgene lacking its 3′–UTR in mouse leads to severe inflammatory disorders (4Keffer J. Probert L. Cazlaris H. Georgopoulos S. Kaslaris E. Kioussis D. Kollias G. EMBO J. 1991; 10: 4025-4031Crossref PubMed Scopus (1350) Google Scholar). Similar AREs are found in the 3′–UTR of a growing number of mRNAs encoding cytokines, protooncogenes, or other transiently expressed proteins (5Caput D. Beutler B. Hartog K. Thayer R. Brown Shimer S. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1670-1674Crossref PubMed Scopus (1218) Google Scholar). These sequences have also been shown to regulate mRNA stability (6Chen C.Y. Shyu A.B. Trends Biochem. Sci. 1995; 20: 465-470Abstract Full Text PDF PubMed Scopus (1689) Google Scholar). In former studies, we reported that TNF-α mRNA ARE can form two complexes with proteins present in cytosolic macrophage extracts. One of these complexes (complex 1) forms with extracts of both unstimulated and LPS-stimulated macrophages and requires a large fragment of the ARE containing clustered AUUUA pentamers. The other complex (complex 2) is only detected after cell activation, binds to a minimal UUAUUUAUU nonamer, and is composed of a 55-kDa protein (7Gueydan C. Houzet L. Marchant A. Sels A. Huez G. Kruys V. Mol. Med. 1996; 2: 479-488Crossref PubMed Google Scholar, 8Lewis T. Gueydan C. Huez G. Toulmé J.J. Kruys V. J. Biol. Chem. 1998; 273: 13781-13786Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). To identify the proteins involved in both complexes, we designed a cloning strategy based on the differential screening of a macrophage cDNA expression library with TNF-α mRNA 3′–UTR riboprobes containing or not the ARE. By this method, we isolated the cDNA encoding the short 40-kDa isoform of the RNA-binding protein TIAR. We show that TIAR specifically binds TNF-α ARE and corresponds to the protein involved in the formation of complex 1. Moreover, analysis of TIAR subcellular localization by immunostaining reveals that TIAR is mainly found in the cytoplasm of murine macrophages. Enzymes used in this study were purchased from Boehringer Mannheim and Life Technologies Inc. LPS (Escherichia coli strain 0.127.B8), diethyl pyrocarbonate, and anti-actin antibody were obtained from Sigma. Isopropyl-1-thio-b-d-galactopyranoside and oligonucleotides were purchased from Life Technologies Inc. Lysozyme was purchased from Appligene Oncor. DNase I and polyC were purchased from Amersham Pharmacia Biotech. Goat anti-TIAR polyclonal antibody directed against a C-terminal peptide of TIAR and rabbit anti-NF-κB antibody were purchased from Santa Cruz. The goat IgG control antibody was purchased from Rockland (Gilbertsville, Pa). Mouse actin cDNA cloned in the pBluescript SK(−) phagemid was purchased from Stratagene. RAW 264.7 mouse macrophages were maintained as described previously (7Gueydan C. Houzet L. Marchant A. Sels A. Huez G. Kruys V. Mol. Med. 1996; 2: 479-488Crossref PubMed Google Scholar). LPS was added at a final concentration of 10 ng/ml for 2 h in all experiments. A RAW 264.7 mouse macrophage cDNA library was prepared according to a previously described method (5Caput D. Beutler B. Hartog K. Thayer R. Brown Shimer S. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1670-1674Crossref PubMed Scopus (1218) Google Scholar) and was inserted into the pUC-19 vector within thePstI and BamHI restriction sites. The library or the pGEX3X-37CR plasmid-expressing AUF1 (generously provided by Dr. Gary Brewer, Bowman Gray School of Medicine, Wake Forest University, Winston Salem, North Carolina) or the pGEX5X-1-actin plasmid-expressing actin (a gift from V. Dilbeck, University of Brussels) were electroporated into MC1061 bacteria and plated to obtain 10,000 colonies/dish (dish diameter: 13.5 cm). After overnight incubation, 1 replicate/dish was performed on nitrocellulose filters (Schleicher & Schuell). These “master” replicates were used to perform secondary (2 to 5) replicates. The master and the secondary replicates were then placed in dishes containing ampicillin (100 μg/ml) or ampicillin (100 μg/ml) and isopropyl-1-thio-b-d-galactopyranoside (1 mm), respectively, and incubated overnight at 37 °C. The master replicates were subsequently stored at 4 °C. The secondary replicates were hung up in a sealed tube containing a 1-cm layer of pure chloroform for 35 min. Filters were then transferred into plastic bags (6 filters/bag maximum) and soaked in 300 ml of autoclaved washing buffer (50 mm Tris, pH 8, 150 mm NaCl, 5 mm MgCl2, 1/1000 (v/v) diethyl pyrocarbonate ) containing lysozyme (1 mg/l) and DNase I (1 mg/l) for 2 h at room temperature with light shaking. The washing buffer was then replaced by 300 ml of fresh washing buffer, and the incubation was prolonged for 4 additional h. The washing buffer was replaced for a second time, and the filters were soaked overnight. The filters were then rinsed three times for 5, 30, and 60 min, respectively, in 300 ml of binding buffer (40 mm Tris, pH 8, 4 mm EDTA, 200 mm NaCl, 3.6 mm 2-mercaptoethanol, 1/1000 (v/v) diethyl pyrocarbonate) at room temperature with light shaking. For the binding with the RNA probe, each membrane was transferred into a plastic bag containing 35 ml of binding buffer and incubated for 10 min at room temperature. Heparin (0.55 mg/ml) was added, and a subsequent incubation of 10 min was performed. PolyC (10 μg/ml) was then added, and the membranes were incubated for an additional 30 min. Finally, 100 × 106 cpm of riboprobe was added per bag, and the bags were incubated overnight at room temperature with light shaking. The membranes were washed twice for 10 min in 100 ml of binding buffer at room temperature with light shaking, dried for 15 min on 3MM paper, and autoradiographed. The DNA constructs used in this study were previously described (7Gueydan C. Houzet L. Marchant A. Sels A. Huez G. Kruys V. Mol. Med. 1996; 2: 479-488Crossref PubMed Google Scholar). The riboprobes used for the screening procedure and electrophoretic shift assay (EMSA) were synthesized with the transcription kit purchased from Epicentre (Madison, WI) according to the following method. Briefly, to generate 100 × 106 cpm (approximately 1.5 ×108cpm/μg), 4 μl of SP6 transcription buffer 5×, 2 μl of 100 mm dithiothreitol, 1 μl of 10 mm ATP, 1 μl of 10 mm CTP, 1 μl of 10 mm GTP, 3 μl of 1 mm UTP, 80 μCi of [α-32P]UTP (800 Ci/mmol), 3 μl of linearized DNA (0.5 μg/μl), and 1 μl of SP6 RNA polymerase were mixed and incubated for 2 h at 37 °C. The transcription reaction was then treated with DNase for 15 min, brought to a volume of 50 μl with H2O, and extracted with phenol/chloroform. The riboprobe was then purified on a P10 minicolumn, and the volume was increased to 300 μl. The probes were stored at −70 °C. DNA sequencing was performed using the Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham Pharmacia Biotech). S100 macrophage extracts and EMSA were carried out exactly as described previously (7Gueydan C. Houzet L. Marchant A. Sels A. Huez G. Kruys V. Mol. Med. 1996; 2: 479-488Crossref PubMed Google Scholar). Supershifts with anti-TIAR or control antibodies were performed by incubating 15 μg of S100 macrophage extract with 0.2 μg of anti-TIAR antibodies or control antibodies for 25 min on ice in a total volume of 15 μl before the EMSA. The EMSA were electrophoresed on nondenaturing 3.5% polyacrylamide gels. 150 μg of S100 extract from uninduced RAW macrophages were incubated with 4 μg of anti-TIAR antibody or control IgG in the presence of 50 μl of protein G-agarose beads (Santa Cruz, Ca.) for 16 h on a rotating wheel at 4 °C. The samples were centrifuged for 30 s, and the supernatant was incubated with fresh antibodies and protein G beads for 24 h. After elimination of the beads by centrifugation, the protein concentration of the samples was determined by using a BCA kit (Pierce), and 15 μg of each samples were used in an EMSA as described previously. Cytoplasmic RNA was isolated from RAW 264.7 cells according to the method previously described. Northern blot analysis was performed as described in Sambrook et al.(9Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) using 10 μg of total RNA. The blot was hybridized with a TIAR cDNA probe synthesized with the Rediprime kit (Amersham Pharmacia Biotech). The same blot was subsequently hybridized with an actin probe to normalize the amount of RNA loaded on the gel. Protein extracts were prepared by recovering the cells in 0.25 m Tris, pH 7.8, and were lysed by three cycles of freezing and thawing. The protein concentration in the extracts was determined by the BCA protein assay (Bio-Rad). Fifteen μg of each extract were run on a 10% SDS-polyacrylamide gel, and the Western blot was performed as described elsewhere (10Nanbru C. Lafon I. Audigier S. Gensac M.C. Vagner S. Huez G. Prats A.C. J. Biol. Chem. 1997; 272: 32061-32066Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). TIAR protein and actin were immunodetected using anti-TIAR and anti-actin antibodies, respectively. Briefly, 105cells were centrifuged on slides, fixed with methanol for 5 min at −20 °C, and stored at room temperature until use. The slides were rinsed in phosphate-buffered saline, incubated overnight with the primary antibody (anti-TIAR, anti-NF-κB, control IgG) (10 μg/ml) at 4 °C in a humidified chamber, and rinsed again in phosphate-buffered saline. Slides were subsequently incubated for 1 h in phosphate-buffered saline in the presence of the secondary antibody coupled to peroxydase (20 μg/ml). Peroxydase activity was revealed by using the AEC (3-amino-9-ethyl-carbazol) method (Sigma). The slides were mounted in aquatex (Merck) and observed at 100× magnification. TNF-α ARE sequence was previously shown to form two different complexes with proteins of macrophage S100 extracts. These two complexes differ in their electrophoretic mobilities and in their recognition motifs within the ARE. Moreover, although the complex of low electrophoretic mobility (complex 1) can be detected with S100 extracts from both unstimulated and LPS-stimulated macrophages, the other complex (complex 2) is formed only upon LPS stimulation (7Gueydan C. Houzet L. Marchant A. Sels A. Huez G. Kruys V. Mol. Med. 1996; 2: 479-488Crossref PubMed Google Scholar, 8Lewis T. Gueydan C. Huez G. Toulmé J.J. Kruys V. J. Biol. Chem. 1998; 273: 13781-13786Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). To clone the cDNAs encoding the proteins involved in these complexes, we developed an expression library screening method. We set up this method with a plasmid encoding AUF1, which has been shown to bind to AREs derived from several mRNAs (11DeMaria C.T. Brewer G. J. Biol. Chem. 1996; 271: 12179-12184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). As negative control, we used a plasmid encoding actin. Bacteria expressing either AUF1 or actin were plated and replicated to perform a binding with TNF-α mRNA 3′-UTR riboprobes containing or not containing the ARE (Fig.1 and “Experimental Procedures”). Although TNF 3′-UTR riboprobe bound to the replicate of bacteria encoding AUF1, no signal could be detected with bacteria expressing actin. The binding of TNF 3′-UTR riboprobe to AUF1-expressing colonies involves the ARE, because no signal was detectable with the TNF 3′-UTRΔAU riboprobe (Fig. 2).Figure 2Validation of the screening method with AUF-1 and actin expression vectors. E. coli strain MC1061 was transformed either with AUF-1 or actin-expression vectors and plated at 10,000 colonies/dish, replicated on nitrocellulose membranes, and incubated with the 3′TNF or 3′TNFΔAU RNA probes as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download We then the RAW 264.7 macrophage cDNA library in the same to identify encoding proteins specifically binding TNF ARE. In a detected by the binding of TNF 3′-UTR riboprobe were isolated and at replicates of the were prepared as described previously and to a second differential screening with 3′TNF and 3′TNFΔAU As in of the detected by the screening specifically bound to 3′TNF probe in the secondary differential The cDNA of colonies detected with the 3′TNF probe were and all were encoding the 40-kDa isoform of the RNA-binding protein TIAR. To TIAR is involved in of the two complexes that can form with TNF ARE, we the binding of TIAR to the probe (Fig. which has been previously shown to form complex 2 and not complex 1 8Lewis T. Gueydan C. Huez G. Toulmé J.J. Kruys V. J. Biol. Chem. 1998; 273: 13781-13786Abstract Full Text Full Text PDF PubMed Scopus (41) Google and As in the riboprobe binds to that TIAR not be involved in complex We performed with TNF 3′-UTR riboprobe and macrophage extracts in the presence of anti-TIAR or control IgG A that the of anti-TIAR antibody in the EMSA the electrophoretic mobility of complex 1 in with a control the other complex 2 is not by anti-TIAR that this complex not TIAR. To the involvement of TIAR in complex we performed with macrophage extract of TIAR by with anti-TIAR antibody library screening A in complex 1 was observed as with the obtained the was performed with a control IgG (Fig. 5 In we by Western blot analysis that of the extract by anti-TIAR antibody with an of TIAR present in the protein not of TIAR as a of complex 1. of complex 1 by anti-TIAR EMSA was performed with cytosolic extract from or RAW cells and the 3′TNF probe in the presence of anti-TIAR antibody or control The is of EMSA was performed with macrophage extract with either an anti-TIAR antibody or a control IgG library screening The is of two Large Image Figure ViewerDownload Hi-res image Download been previously described as a expressed gene at at the mRNA Moreover, can be expressed into two of and and from of the mRNA S. 1996; PubMed Scopus Google Scholar). an additional in the of the three RNA recognition of its in a of the RNA recognition this peptide has been to be for the protein RNA binding J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). We the expression of in unstimulated and LPS-stimulated macrophages at the RNA and protein The Northern blot analysis of TIAR mRNA that TIAR mRNA is expressed as a in both unstimulated and LPS-stimulated RAW cells (Fig. The of the not the of the mRNA encoding the two the protein we observed that the 40-kDa is expressed the As at the RNA TIAR proteins are expressed in unstimulated and LPS-stimulated macrophages which is a protein to has been described to DNA in A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and to be mainly in the upon TIAR has been shown to be to the cytoplasm Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). we TIAR as a of complex which is formed upon incubation of TNF ARE with macrophage S100 cytosolic we determined TIAR subcellular localization in macrophages by an immunostaining This that to TIAR is predominantly found in the cytoplasm of RAW macrophages (Fig. This cytoplasmic localization of TIAR with its to form complex 1 from S100 cytosolic It is now well established that AREs a in the regulation of several transiently expressed These have been in two according to the number and the of AUUUA I AREs to three of the AUUUA in a of these I AREs are found in the 3′-UTR of and mRNAs and mRNA AREs are by the presence of clustered and are found in the 3′-UTR of cytokine mRNAs C.Y. Shyu A.B. Mol. Biol. 1997; PubMed Scopus Google Scholar). on the these AREs mRNA translational proteins have been reported to bind AREs in Cytoplasmic of RNA. New and E. 1998; PubMed Google Scholar). based on their and sequence of these to to the proteins involved in the complexes formed with TNF ARE that we previously described. The expression library screening method with RNA probes has been used to clone cDNAs of proteins specifically binding to RNA different of RNA binding J. PubMed Scopus Google Scholar, K. Mol. Biol. 1996; PubMed Scopus Google Scholar). we developed a but strategy based on the screening of colonies transformed with This method to the identification of as a protein specifically binding TNF ARE. Moreover, we have by that TIAR corresponds to the TNF protein present in macrophage cytosolic extract previously described as complex 1. EMSA performed in the presence of anti-TIAR antibody the electrophoretic mobility of complex 1. be that the to complex 1 is not and that the amount of anti-TIAR antibody not the not This from a of anti-TIAR the could to complex with complex 1. The of complex 1 by anti-TIAR antibody with the that complex 1 upon of TIAR from macrophage extract that this complex either or both TIAR The screening of of the RAW 264.7 cDNA library to the of and not of that is the isoform involved in complex 1. the expression of in macrophages could also the of the isoform by the screening We have shown by an immunostaining analysis that TIAR is cytoplasmic in murine macrophages. This with the identification of TIAR as a protein binding TNF ARE present in S100 cytosolic TIAR has been previously cloned on the of its to a protein mainly expressed in and shown to be to DNA in H. 1991; Full Text PDF PubMed Scopus Google Scholar). et Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) have also shown that TIAR is mainly located in the of cells and to the cytoplasm of these cells this the observed in the localization of TIAR in and murine macrophages is not TIAR localization be cell or TIAR subcellular could be with different of this protein. we that a in the by the antibodies used to determined TIAR localization could for the the antibody used to study TIAR in cells binds an located in the second RNA recognition of TIAR and a in Western This protein is not detected in Western blot performed with RAW 264.7 cell extract and an anti-TIAR antibody directed against a C-terminal peptide of TIAR and “Experimental Procedures”). Although TIAR was as a RNA-binding protein J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google this study the RNA sequence TIAR. We have previously reported that a of AUUUA is the minimal sequence within TNF ARE for complex 1 We have also shown that complex 1 can form with TNF and factor AREs and not with I ARE. TIAR a of complex 1 can be as a binding protein. TNF mRNA is both and in macrophages (2Han J. Brown T. Beutler B. J. Exp. Med. 1990; 171: 465-475Crossref PubMed Scopus (431) Google Scholar, Beutler B. Huez G. 1991; PubMed Scopus Google Scholar). a has been reported to TNF mRNA in macrophage by with TNF mRNA ARE E. 1998; PubMed Google but the proteins involved in the translational control of TNF mRNA are not As TIAR is located in the cytoplasm of macrophages and binds to TNF ARE of LPS of the cells, could TNF translational of macrophages with LPS leads to the of the translational and is by the binding of a 55-kDa protein on TNF ARE, which could be for this T. Gueydan C. Huez G. Toulmé J.J. Kruys V. J. Biol. Chem. 1998; 273: 13781-13786Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The identification of the 55-kDa protein with of TIAR in macrophage into the of cytokine mRNA translational control by We for T. and B. for their with the immunostaining for in T. and D. for