Title: AUF1 Is a bcl-2 A + U-rich Element-binding Protein Involved in bcl-2 mRNA Destabilization during Apoptosis
Abstract: We previously identified a conserved A + U-rich element (ARE) in the 3′-untranslated region of bcl-2mRNA. We have also recently demonstrated that the bcl-2ARE interacts with a number of ARE-binding proteins (AUBPs) whose pattern changes during apoptosis in association with bcl-2mRNA half-life reduction. Here we show that the AUBP AUF1 bindsin vitro to bcl-2 mRNA. The results obtained in a yeast RNA three-hybrid system have demonstrated that the 1–257-amino acid portion of p37 AUF1 (conserved in all isoforms), containing the two RNA recognition motifs, also binds to thebcl-2 ARE in vivo. UVC irradiation-induced apoptosis results in an increase of AUF1. Inhibition of apoptosis by a general caspase inhibitor reduces this increase by 2–3-fold. These results indicate involvement of AUF1 in the ARE/AUBP-mediated modulation of bcl-2 mRNA decay during apoptosis. We previously identified a conserved A + U-rich element (ARE) in the 3′-untranslated region of bcl-2mRNA. We have also recently demonstrated that the bcl-2ARE interacts with a number of ARE-binding proteins (AUBPs) whose pattern changes during apoptosis in association with bcl-2mRNA half-life reduction. Here we show that the AUBP AUF1 bindsin vitro to bcl-2 mRNA. The results obtained in a yeast RNA three-hybrid system have demonstrated that the 1–257-amino acid portion of p37 AUF1 (conserved in all isoforms), containing the two RNA recognition motifs, also binds to thebcl-2 ARE in vivo. UVC irradiation-induced apoptosis results in an increase of AUF1. Inhibition of apoptosis by a general caspase inhibitor reduces this increase by 2–3-fold. These results indicate involvement of AUF1 in the ARE/AUBP-mediated modulation of bcl-2 mRNA decay during apoptosis. A + U-rich element ARE-binding protein three-hybrid system RNA electrophoretic mobility shift assay antibody untranslated region transketolase nucleotide(s) benzyloxycarbonylVal-Ala-Asp(OMe)-fluoromethylketone The bcl-2 gene encodes the multifunctional Bcl-2 protein known to be involved in cell growth, differentiation control, and prevention of apoptosis (1Tsujimoto Y. Shimizu S. FEBS Lett. 2000; 466: 6-10Crossref PubMed Scopus (635) Google Scholar). Down-regulation of bcl-2expression is a general response of the cell to apoptotic stimuli (2Suzuki A. Matsuzawa A. Igushi T. Oncogene. 1996; 13: 31-37PubMed Google Scholar,3Miyashita T. Krajewski S. Krajewska M. Wand H.-G. Lin H.-K. Hoffman B. Lieberman D. Reed J.C. Oncogene. 1994; 9: 1799-1805PubMed Google Scholar). The mechanisms by which Bcl-2 exerts a protective activity against apoptosis are still unclear, but the mechanism by whichbcl-2 expression is regulated has recently been partially elucidated. A large amount of evidence indicates that up- and down-regulation of bcl-2 expression is modulated both at transcriptional and posttranscriptional levels, the latter of which includes mRNA stability and protein activity control. Expression of the bcl-2 gene was known to be regulated transcriptionally by a negative regulatory element (4Young R.L. Korsmeyer S.J. Mol. Cell. Biol. 1993; 13: 3686-3697Crossref PubMed Scopus (182) Google Scholar). Two estrogen-responsive elements within the coding region involved in transcriptional regulation ofbcl-2 have also been characterized recently in a breast cancer cell line (5Perillo B. Sasso A. Abbondanza C. Palumbo G. Mol. Cell. Biol. 2000; 20: 2890-2901Crossref PubMed Scopus (291) Google Scholar). One of the posttranscriptional control mechanisms of bcl-2 expression has been described to be mediated by phosphorylation of Bcl-2 protein at different amino acid positions (6Huang Y. Sheikh M.S. Fornace Jr., A.J. Holbrook N.J. Oncogene. 1999; 18: 3431-3439Crossref PubMed Scopus (74) Google Scholar,7Breitschopf K. Haendeler J. Malchow P. Zeiher A.M. Dimmeler S. Mol. Cell. Biol. 2000; 20: 1886-1896Crossref PubMed Scopus (290) Google Scholar). Another posttranscriptional mechanism of bcl-2regulation has been identified in our laboratory. This mechanism modulates bcl-2 mRNA stability and involves a cis-acting A + U-rich element (ARE)1 located in the 3′-UTR of bcl-2 mRNA (8Schiavone N. Rosini P. Quattrone A. Donnini M. Lapucci A. Citti L. Bevilacqua A. Nicolin A. Capaccioli S. FASEB J. 2000; 14: 174-184Crossref PubMed Scopus (98) Google Scholar) that binds to a number of ARE-binding proteins (AUBPs) whose pattern undergoes modifications during apoptosis in association with bcl-2 mRNA decay (9Donnini M. Lapucci A. Papucci L. Witort E. Tempestini A. Brewer G. Bevilacqua A. Nicolin A. Capaccioli S. Schiavone N. Biochem. Biophys. Res. Commun. 2001; 287: 1063-1069Crossref PubMed Scopus (17) Google Scholar). AREs represent a class of cis-acting elements that modulate mRNA stability (10Chen C.Y. Shyu A.B. Trends Biochem. Sci. 1995; 20: 465-470Abstract Full Text PDF PubMed Scopus (1681) Google Scholar). They are present in a variety of mRNAs of genes required to be rapidly and finely modulated under particular conditions, such as response to growth factors, serum starvation, and apoptosis (8Schiavone N. Rosini P. Quattrone A. Donnini M. Lapucci A. Citti L. Bevilacqua A. Nicolin A. Capaccioli S. FASEB J. 2000; 14: 174-184Crossref PubMed Scopus (98) Google Scholar, 11Xu N. Loflin P. Chen C.Y. Shyu A.B. Nucleic Acids Res. 1998; 26: 558-565Crossref PubMed Scopus (136) Google Scholar, 12Rodriguez-Pascual F. Hausding M. Ihrig-Biedert I. Furneaux H. Levy A.P. Forstermann U. Kleinert H. J. Biol. Chem. 2000; 275: 26040-26049Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 13Madireddi M.T. Dent P. Fisher P.B. Oncogene. 2000; 19: 1362-1368Crossref PubMed Scopus (56) Google Scholar). GM-CSF and c-fos mRNAs were the first to be studied in detail (14Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3123) Google Scholar, 15You Y. Chen C.Y. Shyu A.B. Mol. Cell. Biol. 1992; 12: 2931-2940Crossref PubMed Google Scholar). Compared with the AREs of these genes (16Winstall E. Gamache M. Rayamond V. Mol. Cell. Biol. 1995; 15: 3796-3804Crossref PubMed Scopus (48) Google Scholar), the bcl-2 ARE possesses a moderate, constitutive, destabilizing activity that is dramatically enhanced upon application of apoptotic stimuli (8Schiavone N. Rosini P. Quattrone A. Donnini M. Lapucci A. Citti L. Bevilacqua A. Nicolin A. Capaccioli S. FASEB J. 2000; 14: 174-184Crossref PubMed Scopus (98) Google Scholar, 9Donnini M. Lapucci A. Papucci L. Witort E. Tempestini A. Brewer G. Bevilacqua A. Nicolin A. Capaccioli S. Schiavone N. Biochem. Biophys. Res. Commun. 2001; 287: 1063-1069Crossref PubMed Scopus (17) Google Scholar). The mechanism of action of ARE-mediated mRNA decay is under investigation by many laboratories. A number of AUBPs acting as trans-acting factors are known. In particular, some AUBPs belonging to the heterogeneous nuclear ribonucleoprotein family are involved in mRNA localization and stability control (17Dreyfuss G. Matunis M.J. Pinol-Roma S. Burd C.G. Annu. Rev. Biochem. 1993; 62: 289-321Crossref PubMed Scopus (1336) Google Scholar, 18Krecic A.M. Swanson M.S. Curr. Opin. Cell Biol. 1999; 11: 363-371Crossref PubMed Scopus (716) Google Scholar). Among them, AUF1 and the embryonic lethal abnormal vision-like protein HuR are able to enhance or inhibit mRNA degradation, respectively. For example, there is evidence for binding and stabilization of c-fos, plasminogen activator inhibitor 2, and vascular endothelial growth factor mRNAs by HuR (19Peng S.S. Xu C.Y. Chen N. Shyu A.B. EMBO J. 1988; 17: 3461-3470Crossref Scopus (656) Google Scholar, 20Maurer F. Tierney M. Medcalf R.L. Nucleic Acids Res. 1999; 27: 1664-1673Crossref PubMed Scopus (62) Google Scholar, 21Levy N.S. Chung S. Furneaux H. Levy A.P. J. Biol. Chem. 1998; 273: 6417-6423Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 22Fan X.C. Steits J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (747) Google Scholar, 23Blaxall B.C. Dwyer-Nield L.D. Bauer A.K. Bohlmeyer T.J. Malkinson A.M. Port J.D. Mol. Carcinog. 2000; 28: 76-83Crossref PubMed Scopus (104) Google Scholar). This protein has also been implicated in the increase of p53-induced p21waf1 mRNA after apoptotic stimuli (24Wang W. Furneaux H. Cheng H. Caldwell M.C. Hutter D. Liu Y. Holbrook N. Gorospe M. Mol. Cell. Biol. 2000; 20: 760-769Crossref PubMed Scopus (469) Google Scholar). AUF1 was first identified as an mRNA-binding protein with selective affinity for AREs located within mRNAs such as c-myc, c-fos, and GM-CSF (25Zhang W. Wagner B.J. Ehrenman K. Schaefer A.W. DeMaria C.T. Crater D. DeHaven K. Long L. Brewer G. Mol. Cell. Biol. 1993; 13: 7652-7665Crossref PubMed Scopus (497) Google Scholar, 26Brewer G. Mol. Cell. Biol. 1991; 11: 2460-2466Crossref PubMed Scopus (404) Google Scholar). Although its destabilizing function is well documented, some studies suggest that AUF1 may have a role in the stabilizing complex on the α-globin mRNA (27Kiledijian M. DeMaria C.T. Brewer G. Novick K. Mol. Cell. Biol. 1997; 11: 4870-4876Crossref Google Scholar) and, more recently, as a parathyroid hormone mRNA-binding protein that modulates mRNA stability (28Sela-Brown A. Silver J. Brewer G. Naveh-Many T. J. Biol. Chem. 2000; 275: 7424-7429Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). AUF1 is comprised of four isoforms of 37, 40, 42, and 45 kDa (25Zhang W. Wagner B.J. Ehrenman K. Schaefer A.W. DeMaria C.T. Crater D. DeHaven K. Long L. Brewer G. Mol. Cell. Biol. 1993; 13: 7652-7665Crossref PubMed Scopus (497) Google Scholar, 29Sheflin L.G. Spaulding S.W. Am. J. Physiol. Endocrinol. Metab. 2000; 278: E50-E57Crossref PubMed Google Scholar, 30Wagner B.J. DeMaria C.T. Sun Y. Brewer G. Genomics. 1998; 48: 195-202Crossref PubMed Scopus (238) Google Scholar). Although the role of each isoform has yet to be fully characterized, a direct correlation has been observed between each AUF1 isoform's binding affinity and its RNA-destabilizing activity toward different AREs, with isoforms p37 and p42 being the most effective (31De Maria C.T. Brewer G. J. Biol. Chem. 1996; 271: 12179-12184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Here, we demonstrate that AUF1 is a bcl-2 mRNA-binding protein and that potentially all its isoforms are able to form complexes with the bcl-2 ARE. At doses able to induce apoptosis, UVC irradiation induced an increase of cytoplasmic levels of the p45 AUF1 isoform, which was paralleled by enhancement of a bcl-2 mRNA-AUF1 complex and mediated by a mechanism that requires caspase activation. These results indicate that ARE-mediated bcl-2 mRNA down-regulation during apoptosis involves AUF1 and suggest different roles for its four isoforms. The schematic diagram of bcl-2 mRNA and the sequences of ARE segments used for either in vitrotranscription or cell transfection are shown in Fig. 1. A 396-bp segment encoding the human bcl-2 mRNA located in the 3′-UTR from nucleotide 752–1147, named bcl-2 ARE+ (GenBankTM accession number M14745; Ref. 32Cleary M.L. Smith S.D. Sklar J. Cell. 1986; 47: 19-28Abstract Full Text PDF PubMed Scopus (1076) Google Scholar), was obtained by PCR amplification using the plasmid pBS-SK-H-Bcl-2 (33Seto M. Jaeger U. Hockett R.D. Graninger W. Bennett S. Goldman P. Korsmeyer S.J. EMBO J. 1988; 7: 123-131Crossref PubMed Scopus (457) Google Scholar) as template, with 5′- AGTCAACATGCCTGC-3′ forward (FW1) and 5′- GTGATCCGGCCAACAAC-3′ reverse (RV2) primers. The bcl-2 ARE+ was cloned in the TA cloning site of the pCRII plasmid according to the TA Cloning Kit specifications (Invitrogen), yielding pCRII/bcl-2 ARE+, and used to synthesize the bcl-2 ARE+ riboprobe. A 289-bp segment of the human bcl-2 mRNA located in the 3′-UTR from nucleotide 752–1147, named bcl-2 ΔARE, was obtained by nested deletion of the cDNA from nucleotide 944–1050 (32Cleary M.L. Smith S.D. Sklar J. Cell. 1986; 47: 19-28Abstract Full Text PDF PubMed Scopus (1076) Google Scholar) using the plasmid pBS-SK-H-Bcl-2 (33Seto M. Jaeger U. Hockett R.D. Graninger W. Bennett S. Goldman P. Korsmeyer S.J. EMBO J. 1988; 7: 123-131Crossref PubMed Scopus (457) Google Scholar) as template. Two partially overlapping PCR products were synthesized for this purpose. The first PCR product was amplified with FW1, as described above, and 5′-TTCGACGTTTTGCCTGAAGACT-3′ reverse (RV1) primers. The second PCR product was amplified with 5′-CAAAACGTCGAACGACCACTAATTGCCAAGC -3′ (FW2) and RV2 primers. The 12 overlapping nucleotides are underlined in RV1 and FW2 primers. The segment bcl-2 ΔARE was cloned in plasmid pCRII as described above, yielding pCRII/bcl-2 ΔARE, and used for the synthesis of bcl-2 ΔARE riboprobe. A 107-bp segment of human bcl-2 mRNA located in the 3′-UTR from nucleotide 944–1050 (a short region with the highest evolutionary conservation containing AUUUA pentamers and UUAUUUAUU nonamer particularly rich in A + U motifs and therefore considered the ARE core), named bcl-2 ARE (32Cleary M.L. Smith S.D. Sklar J. Cell. 1986; 47: 19-28Abstract Full Text PDF PubMed Scopus (1076) Google Scholar), was obtained by PCR amplification using the plasmid pBS-SK-H-Bcl-2 (33Seto M. Jaeger U. Hockett R.D. Graninger W. Bennett S. Goldman P. Korsmeyer S.J. EMBO J. 1988; 7: 123-131Crossref PubMed Scopus (457) Google Scholar) as template, with 5′-TCAGCTATTTACTGCCAAAG-3′ forward and 5′-GATTTCCAAAGACAGGAG-3′ reverse primers. The bcl-2 ARE product was cloned in pCRII as described above, yielding pCRII/bcl-2 ARE to synthesize thebcl-2 ARE riboprobe. The remaining polylinkers of pCRII/bcl-2 ARE+, pCRII/bcl-2 ΔARE, and pCRII/bcl-2 ARE were removed by ApaI digestion and religation. Plasmids pBBB4 (obtained by Dr. Ann-Bin Shyu, University of Texas, Houston Health Science Center, Houston, TX) and pBBB-U1 have been described previously (8Schiavone N. Rosini P. Quattrone A. Donnini M. Lapucci A. Citti L. Bevilacqua A. Nicolin A. Capaccioli S. FASEB J. 2000; 14: 174-184Crossref PubMed Scopus (98) Google Scholar). Plasmid pBBB-U2 was obtained by cloning the BglII/BamHI segment of pCRII/bcl-2 ΔARE into the unique BglII site of plasmid pBBB4. Plasmids pBBB4, pBBB-U1, and pBBB-U2, used for RNase protection analysis of mRNA stability, contain the rabbit β-globin gene transcriptionally driven by the serum-inducible c-fos promoter (pBBB4) with inserted the bcl-2ARE (pBBB-U1) or bcl-2 ΔARE (pBBB-U2). The Jurkat T-cell leukemia line (clone E61; European Collection of Animal Cell Cultures) was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mm glutamine, 50 IU/ml penicillin, and 50 μg/ml streptomycin in a humidified atmosphere, 5% CO2, at 37 °C. The two mouse fibroblast NIH 3T3 polyclonal cell lines transfected with plasmids pBBB4 or pBBB-U1 have been described previously (8Schiavone N. Rosini P. Quattrone A. Donnini M. Lapucci A. Citti L. Bevilacqua A. Nicolin A. Capaccioli S. FASEB J. 2000; 14: 174-184Crossref PubMed Scopus (98) Google Scholar). The third NIH 3T3 polyclonal cell line was obtained upon transfection with plasmid pBBB-U2 described above. All NIH 3T3 cell lines were maintained in Dulbecco's modified Eagle's medium supplemented as described above for RPMI 1640 medium. Reporter β-globin mRNA stability was determined by the serum-inducible transcriptional pulse system reported previously (8Schiavone N. Rosini P. Quattrone A. Donnini M. Lapucci A. Citti L. Bevilacqua A. Nicolin A. Capaccioli S. FASEB J. 2000; 14: 174-184Crossref PubMed Scopus (98) Google Scholar). The plasmids pCRII/bcl-2ARE+, pCRII/bcl-2 ΔARE, and pCRII/bcl-2 ARE were linearized with SmaI and used for in vitrorun-off transcription from the T7 promoter using an RNA labeling Kit (Amersham Biosciences) in the presence of [α-32P]UTP (800 Ci/mmol; Amersham Biosciences) to obtain three radiolabeledbcl-2 ARE riboprobes (Fig. 1). The isoforms of AUF1 were prepared from 1 μg of each of four different pcDNA-AUF1 plasmids (p45, p42, p40, and p37). Templates p45 and p42 were prepared byNotI digestion, whereas templates of p37 and p40 were prepared by ApaI digestion. The cDNAs were in vitro-transcribed/translated in the presence or absence of [35S]methionine (Amersham Biosciences) with the TnT-coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions. Cells (107) induced to apoptosis by irradiation with UVC (15 J/m2, 254 nm), with or without a 2-h pretreatment with 100 μm Z-VAD-fmk (Bachem AG), were collected at various time points, washed with ice-cold phosphate-buffered saline, and lysed in 100 μl of lysis buffer (10 mm HEPES, pH 7.9, 40 mm KCl, 3 mmMgCl2, 1 mm dithiothreitol, 5% glycerol, 0.2% Nonidet P-40, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride) for 10 min on ice. Nuclei were pelleted by centrifugation at 14,000 rpm for 30 s in a microcentrifuge, and extracts were processed immediately or stored at −70 °C. REMSAs were performed by incubating the radiolabeledbcl-2 ARE+, bcl-2 ΔARE, and bcl-2ARE riboprobes (5 × 105 cpm) with cytoplasmic protein extracts (30 μg, determined by BCA reagent; Pierce) or with in vitrosynthesized AUF1 isoforms in a reaction mixture (20 μl) containing 10 mm Tris, pH 7.5, 0.1 m potassium acetate, 5 mm magnesium acetate, 2 mmdithiothreitol, 15 units of RNasin (Promega), and 50 μg of heparin for 20 min at room temperature, followed by digestion for 20 min at room temperature with 5 units of RNase T1 (La Roche Ltd.), which cuts RNA downstream to guanosine residues in a number of fragments. Samples were separated on a native polyacrylamide gel (6% polyacrylamide:bisacrylamide, 60:1). In REMSA/supershift experiments, RNase T1 was added after incubation of samples with 50 μg/ml polyclonal rabbit anti-AUF1 antibody (Ab) or with nonspecific control anti-transketolase (TK) polyclonal Ab, a kind gift of Prof. F. Paoletti (University of Florence), for 20 min at room temperature, electrophoresed as described above, and exposed to Hyperfilm MP (Amersham Biosciences). Cytoplasmic proteins (50 μg, prepared as described above) were separated by 12.5% SDS-PAGE, electroblotted (Miniprotean apparatus; Bio-Rad) onto Hybond-C membrane (Amersham Biosciences), and detected with ECL Western blotting analysis system (Amersham Biosciences) with 300 ng/ml anti-AUF1 Ab. Each isoform was identified on the basis of molecular mass markers (Bio-Rad). Aliquots of cytoplasmic proteins (50 μg, prepared as described above) were incubated with the 32P-labeled bcl-2 ARE riboprobe (109 cpm/μg, 5 × 105 cpm) in the presence of 0.5 mg/ml heparan sulfate and 2 μg of tRNA in a microplate (total volume, 10 μl) at room temperature for 10 min. RNA-protein complexes were cross-linked on ice by exposure to UVC for 5 min with 3000 μW/cm2 in a Stratalinker 1800 (Stratagene). Samples were incubated with RNase A (1 μl, 1 mg/ml) for 30 min at 37 °C to digest unbound RNA. Proteins were immunoprecipitated by a 1-h incubation with polyclonal anti-AUF1 rabbit Ab (10 μg/ml), followed by overnight incubation with protein A-agarose (Sigma) at 4 °C. The immunoprecipitates were separated by 12.5% SDS-PAGE, autoradiographed, and analyzed by a Storm PhosphorImager (Molecular Dynamics). The molecular mass of each complex was evaluated on the basis of the in vitro-synthesized radiolabeled isoforms used as external standards and molecular mass markers (Bio-Rad). The yeast strain L40-coat and plasmids pIIIA/MS2-1, pIIIA/IRE-MS2, pAD-IRP1, and pACT2 (34Kraemer B. Zhang B. Sen Gupta D. Fields S. Wickens M. Methods Enzymol. 2000; 328: 297-321Crossref PubMed Google Scholar) were gifts from Dr. M. Wickens (University of Wisconsin). The plasmid pRevR2 (35Putz U. Skehel P. Kuhl D. Nucleic Acids Res. 1996; 24: 4838-4840Crossref PubMed Scopus (73) Google Scholar) was a gift from Dr. U. Putz (University of Hamburg, Hamburg, Germany). The hybrid RNA vector pIIIA/MS2-B2ARE harbors the 107-nucleotide sequence of the bcl-2 ARE region. The sequence was PCR-amplified from pBS-SK-H-Bcl-2, with 5′-GACCCGGGTCAGCTATTTACTGCCAAAG-3′ forward and 5′-GACCCGGGGATTTCCAAAGACAGGAG-3′ reverse primers, subcloned in pCRII vector (Invitrogen), and inserted into the SmaI site of plasmid pIIIA/MS2-1. The resulting plasmid, pIIIA/MS2-bcl2, was transformed into L40-coat, and the chimeric RNA levels were assayed by Northern analysis (data not shown). To prevent transcription termination due to an RNA polymerase III termination signal in the poly-U stretch, a mutation at nucleotide 981 was introduced by PCR. The mispaired primers 5′-CATTTATTTgTTACATTATTAAG-3′ (forward) and 5′-CTTAATAATGTAAcAAATAAATG (reverse) were used to insert a single-base substitution (T→G, as indicated by lowercase letters), and the resulting segment was cloned into the pIIIA/MS2-1 plasmid as described previously. The AUF1 cDNA corresponding to the first 257 amino acids of p37 was amplified from pBAD/HISB-p37AUF1 (36Wilson G. Brewer G. Methods. 1999; 17: 74-83Crossref PubMed Scopus (55) Google Scholar) with the 5′-GAGGATCCGAATGTCGGAGGAGCAG-3′ forward and 5′-GACTCGAGTCTTCCTGCAAATCCTCC-3′ reverse primers to test the interaction between AUF1 and bcl-2 ARE. The PCR product was digested and inserted into the BamHI/XhoI sites of pACT2, in-frame with the GAL4 activation domain. In previous work (9Donnini M. Lapucci A. Papucci L. Witort E. Tempestini A. Brewer G. Bevilacqua A. Nicolin A. Capaccioli S. Schiavone N. Biochem. Biophys. Res. Commun. 2001; 287: 1063-1069Crossref PubMed Scopus (17) Google Scholar), we demonstrated that the mRNA-destabilizing ARE of bcl-2 binds specifically to cytoplasmic AUBPs, whose pattern undergoes modifications during apoptosis. The observation that proteins ranging from 30–50 kDa underwent the most noticeable increase led us to hypothesize that AUF1 could be a bcl-2 ARE-binding protein. To examine this possibility, REMSA supershift analysis of cytoplasmic protein complexes with bcl-2 ARE radiolabeled riboprobes (Fig.1) has been carried out using an anti-AUF1 Ab and a nonspecific control anti-TK Ab (Fig.2). When bcl-2 ARE+ orbcl-2 ARE riboprobes are used, the anti-AUF1 Ab (lane 3 of each panel), but not the nonspecific anti-TK Ab (lane 4 of each panel), is able to produce a supershifted complex, indicating AUF1 binding. By contrast, when the bcl-2 ΔARE riboprobe (in which the 107-nt ARE core was deleted) was used, supershift did not occur, indicating no AUF1 binding. Other REMSA experiments have been carried out with each AUF1 isoform (p37, p40, p42, and p45) synthesized in vitro. As shown in Fig.3, all AUF1 isoforms shiftbcl-2 ARE+ and bcl-2 ARE riboprobes but not thebcl-2 ΔARE riboprobe, indicating that binding of thebcl-2 mRNA to AUF1 requires the ARE core.Figure 3Binding of all AUF1 isoforms to thebcl-2 ARE. All AUF1 isoforms synthesized in vitro were assayed in REMSA experiments for RNA binding activity with the three ARE riboprobes. Left panel, thebcl-2 ARE+ riboprobe incubated with each AUF1 isoform.Center panel, the bcl-2 ΔARE riboprobe incubated with each AUF1 isoform. Right panel, thebcl-2 ARE riboprobe incubated with each AUF1 isoform.FR represents free RNA digested with RNase T1. p37, p40, p42, and p45 indicate each AUF1 isoform used in shift experiments. Complexes are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To evaluate the possibility that mRNA destabilizing activity and AUF1 binding of the bcl-2 ARE are two related events, analysis of mRNA half-life in cells expressing a reporter gene with or without the bcl-2 ARE was performed. For this purpose, three NIH 3T3 cell lines stably expressing a rabbit β-globin gene transcriptionally driven by the c-fos serum-inducible promoter with either bcl-2 ARE+ (pBBB-U1) or the ARE-deletedbcl-2 ΔARE (pBBB-U2) or without insert (pBBB4) were used. Following serum addition to induce a pulse of transcription of each reporter gene, total RNA was extracted at various times, and β-globin mRNA levels were quantitated by RNase protection using glyceraldehyde-3-phosphate dehydrogenase as an internal standard (Fig.4). With respect to the β-globin transcript without insert (BBB4), the insertion of bcl-2ARE+ (BBB-U1), which binds AUF1, reduces the half-life of reporter mRNA by 6-fold (from >12 h to 2 h). The insertion ofbcl-2 ΔARE (BBB-U2), which does not bind AUF1, does not affect the half-life of β-globin mRNA. All these results clearly indicate that the 107 nt of the ARE ofbcl-2 (shared by bcl-2 ARE and bcl-2ARE+), but not its flanking regions (bcl-2 ΔARE), contain the binding site for AUF1 and are also required for mRNA destabilizing activity. Thus, we have used the bcl-2 ARE as a riboprobe in further experiments. We assumed that the smallest isoform, p37, was the prototype of AUF1 because its entire sequence, harboring two RNA recognition motifs, is contained in the other three isoforms. The yeast RNA THS was used to demonstrate that recombinant p37 binds to the ARE of bcl-2 in vivo (Fig. 5). The reporter gene activation of the host yeast was tested in parallel with the indicated positive and negative controls. The 1–257-amino acid segment of p37 containing the two RNA recognition motifs of AUF1 elicited the activation of both LacZ and HIS3 genes, clearly demonstrating that binding of AUF1 to the bcl-2 ARE also occurs in vivo (Fig. 5). The possibility that enhanced decay of bcl-2 mRNA during apoptosis (8Schiavone N. Rosini P. Quattrone A. Donnini M. Lapucci A. Citti L. Bevilacqua A. Nicolin A. Capaccioli S. FASEB J. 2000; 14: 174-184Crossref PubMed Scopus (98) Google Scholar, 9Donnini M. Lapucci A. Papucci L. Witort E. Tempestini A. Brewer G. Bevilacqua A. Nicolin A. Capaccioli S. Schiavone N. Biochem. Biophys. Res. Commun. 2001; 287: 1063-1069Crossref PubMed Scopus (17) Google Scholar) is associated with changes of AUF1 levels was evaluated. Jurkat cells were irradiated with UVC at a dose previously established to commit cells to apoptosis (data not shown). REMSA experiments demonstrated an increase of the supershifted AUF1-bcl-2 ARE complex at 8 h compared with no UVC (Fig. 6, comparelane 3 with lane 4). To establish the involvement of the AUF1 isoforms in the increase of supershifted complex, we carried out time course Western blot analyses and immunoprecipitation experiments of UV-cross-linked complexes with anti-AUF1 Ab (Fig.7, a and b, respectively). Each AUF1 isoform was identified by immunodetection and comparison with standard molecular mass markers. During the time course (0–10 h) after apoptotic stimulation (Fig. 7a), the cytoplasmic p45 isoform markedly increased within 2 h after UVC irradiation, peaking at 10 h. Pretreatment with Z-VAD-fmk attenuated this increase by 2–3-fold. The increase of p37 and p40/p42 isoforms after UVC irradiation was very low and was not affected by Z-VAD-fmk pretreatment. To determine whether the AUF1 variations observed during apoptosis are accompanied by an increase of some isoform-specific ARE binding activity, immunoprecipitation analyses of UV-cross-linked complexes were performed (Fig.7b). Standard molecular mass markers and labeled AUF1 isoforms translated in vitro and run in the same gel were used for comparison. During the time course, the AUF1 isoform-bcl-2 ARE complex with an apparent molecular mass of about 45 kDa underwent a marked increase, which paralleled the increase of p45 AUF1 (Fig. 7a) in Western analysis and, analogously, is attenuated by pretreatment with Z-VAD-fmk. Another complex with lower molecular mass (about 37 kDa) was detected as a very faint band. No other complexes have been detected within the molecular mass range of the AUF1 isoforms. The additional bands with higher molecular masses observed in the immunoprecipitate (indicated bydouble-headed arrows) may represent AUF1 isoform dimers or multimers stabilized by UV-cross-linking. From all described results, we conclude that AUF1 is a bcl-2 AUBP and that an isoform-specific mechanism is involved in ARE-mediated bcl-2mRNA decay during apoptosis. Furthermore, our results suggest that the p45 AUF1 isoform is the most likely candidate to play a pivotal role in this mechanism.Figure 7Effect of UVC irradiation on AUF1 levels andbcl-2 ARE/AUF1 binding. Western blots (a) and immunoprecipitations (b) were carried out with cytoplasmic extracts from Jurkat cells obtained at various time points after UVC irradiation that had been pretreated or not pretreated with Z-VAD-fmk, as indicated. In Western blot analysis, immunodetection and molecular mass markers run in the same gel were used to identify each AUF1 isoform. In immunoprecipitation experiments, the extracts were cross-linked with the bcl-2 ARE riboprobe before incubation with the anti-AUF1 Ab. The molecular mass of the complexes was evaluated on the basis of labeled, in vitro-synthesized AUF1 isoforms and molecular mass markers run in the same gel.Double-headed arrows in b indicate other complexes with higher molecular masses than the 45-kDa complex. Quantitations of Western blots and immunoprecipitations are shown to the right in each panel.View Large Image Figure ViewerDownload Hi-res image Download (PPT) bcl-2 is one of the most studied apoptosis-related genes and is also implicated in cell cycle progression and cell differentiation. The expression of bcl-2 can be finely tuned by a variety of environmental or endogenous stimuli and regulated at both transcriptional (5Perillo B. Sasso A. Abbondanza C. Palumbo G. Mol. Cell. Biol. 2000; 20: 2890-2901Crossref PubMed Scopus (291) Google Scholar, 33Seto M. Jaeger U. Hockett R.D. Graninger W. Bennett S. Goldman P. Korsmeyer S.J. EMBO J. 1988; 7: 123-131Crossref PubMed Scopus (457) Google Scholar) and posttranscriptional levels (8Schiavone N. Rosini P. Quattrone A. Donnini M. Lapucci A. Citti L. Bevilacqua A. Nicolin A. Capaccioli S. FASEB J. 2000; 14: 174-184Crossref PubMed Scopus (98) Google Scholar). The diverse modes of regulation of bcl-2 expression probably reflect different requirements for down- or up-regulation in different physiological conditions or in pathological processes. We described a posttranscriptional level of regulation ofbcl-2 expression that is mediated by an ARE in the 3′-UTR ofbcl-2 mRNA. Under normal conditions, this element has a moderate destabilizing activity toward the bcl-2 transcript. This activity increases following apoptotic stimuli, leading to enhanced bcl-2 mRNA degradation (8Schiavone N. Rosini P. Quattrone A. Donnini M. Lapucci A. Citti L. Bevilacqua A. Nicolin A. Capaccioli S. FASEB J. 2000; 14: 174-184Crossref PubMed Scopus (98) Google Scholar). Thebcl-2 mRNA ARE binds to a number of AUBPs whose electrophoretic pattern changes after induction of apoptosis (9Donnini M. Lapucci A. Papucci L. Witort E. Tempestini A. Brewer G. Bevilacqua A. Nicolin A. Capaccioli S. Schiavone N. Biochem. Biophys. Res. Commun. 2001; 287: 1063-1069Crossref PubMed Scopus (17) Google Scholar). A number of AUBPs that modulated mRNA stability have been described for other ARE-containing mRNAs such as VEGF (37Claffey K.P. Shih S.C. Mullen A. Dziennis S. Cusick J.L. Abrams K.R. Lee S.W. Detmar M. Mol. Biol. Cell. 1998; 9: 469-481Crossref PubMed Scopus (159) Google Scholar, 38King P.H. Nucleic Acids Res. 2000; 28: E20Crossref PubMed Scopus (46) Google Scholar),GM-CSF (39Bickel M. Iwai Y. Pluznik D.H. Cohen R.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10001-10005Crossref PubMed Scopus (48) Google Scholar, 40Nakamaki T. Imamura J. Brewer G. Tsuruoka N. Koeffler H.P. J. Cell. Physiol. 1995; 165: 484-492Crossref PubMed Scopus (48) Google Scholar), and c-fos (15You Y. Chen C.Y. Shyu A.B. Mol. Cell. Biol. 1992; 12: 2931-2940Crossref PubMed Google Scholar, 41Gillis P. Malter J.S. J. Biol. Chem. 1991; 266: 3172-3177Abstract Full Text PDF PubMed Google Scholar, 42Chung S. Jiang L. Cheng S. Furneaux H. J. Biol. Chem. 1996; 271: 11518-11524Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). We first demonstrated that AUF1 is a bcl-2 AUBP involved inbcl-2 down-regulation during apoptosis. AUF1 is a heterogeneous nuclear ribonucleoprotein protein implicated in mRNA stability regulation, and it shuttles dynamically between the nucleus and cytoplasm (43Arao Y. Kuriyama R. Kayama F. Kato S. Arch. Biochem. Biophys. 2000; 380: 228-236Crossref PubMed Scopus (60) Google Scholar). Although the four isoforms of AUF1, namely, p45, p42, p40, and p37 (29Sheflin L.G. Spaulding S.W. Am. J. Physiol. Endocrinol. Metab. 2000; 278: E50-E57Crossref PubMed Google Scholar, 30Wagner B.J. DeMaria C.T. Sun Y. Brewer G. Genomics. 1998; 48: 195-202Crossref PubMed Scopus (238) Google Scholar), are present in both the nucleus and cytoplasm of Jurkat cells (data not shown), all our experiments have been carried out using cytoplasmic extracts because the cytoplasm is the compartment of mRNA degradation. The binding of AUF1 to the ARE of bcl-2 was demonstrated in vitro by means of supershift REMSA experiments. The cytoplasmic extracts were incubated simultaneously with bcl-2 ARE radiolabeled riboprobes and either anti-AUF1 or anti-TK Ab. In these experiments, only the specific anti-AUF1 Ab was able to supershift the bcl-2 ARE-protein complex. All AUF1 isoforms (p37, p40, p42, and p45) have the potential to bind to the bcl-2 mRNA, as indicated in vitro by results obtained in REMSA experiments carried out with each single in vitro-synthesized isoform. Using p37, considered as the AUF1 prototype, in the yeast RNA THS we demonstrated that the AUF1 RNA binding motifs common to all four isoforms are also able to bind to the bcl-2 ARE in vivo. Deletion of the evolutionary conserved ARE segment particularly rich in AU motifs (and therefore considered as the ARE core) not only abolishes the ability of the bcl-2 ARE to bind AUF1 but also abolishes its mRNA destabilizing activity. This indicates that the deleted segment harbors the binding site for AUF1 and that AUF1 is required as a trans-acting factor for functional activity of the bcl-2ARE. The implication of AUF1 as a trans-acting factor involved in ARE-mediated, bcl-2 mRNA degradation during apoptosis was further demonstrated by results obtained in supershift REMSA experiments carried out after UVC irradiation of cultured cells. Furthermore, we found that the level of the p45 isoform, as evaluated by Western blot, increased markedly until 10 h after UVC irradiation. In the same extracts, the level of one AUF1-bcl-2 ARE complex paralleled the increased level of p45 AUF1, and its molecular mass was also about 45 kDa. This complex was also the only one to undergo a clear decrease in Z-VAD-fmk-treated cells compared with untreated cells. This decrease also parallels the decrease in the 45-kDa isoform observed by Western blot of extracts from cells pretreated with Z-VAD-fmk with respect to untreated cells. Whereas these data are suggestive, the specific AUF1 isoform(s) involved in bcl-2 ARE binding and mRNA degradation during apoptosis could not be precisely identified on the basis of its molecular mass. Nevertheless, our results indicate that at least one AUF1 isoform is a candidate to play a pivotal role in this regulatory mechanism. Arao et al. (43Arao Y. Kuriyama R. Kayama F. Kato S. Arch. Biochem. Biophys. 2000; 380: 228-236Crossref PubMed Scopus (60) Google Scholar) found that p45 and p42 are predominantly nuclear proteins and that, consequently, the observed increase, if actually attributable to one of these isoforms, could be explained by nuclear to cytoplasm shuttling. This possibility is supported by our previous observation that the apoptotic stimulus by cycloeximide (50 μm), utilized to block protein synthesis, resulted in an analogous increase in bcl-2 ARE-bound, cytoplasmic AUBPs (9Donnini M. Lapucci A. Papucci L. Witort E. Tempestini A. Brewer G. Bevilacqua A. Nicolin A. Capaccioli S. Schiavone N. Biochem. Biophys. Res. Commun. 2001; 287: 1063-1069Crossref PubMed Scopus (17) Google Scholar). In our model, the relative balance of the AUF1 isoforms seems to determine the fate of the bcl-2 mRNA in response to apoptotic stimuli. We speculate that this balance could be affected in response to other types of endogenous and environmental stimuli. Results obtained from Western blot as well as immunoprecipitation assays suggest that binding of AUF1 isoforms to bcl-2mRNA in cellular extracts could also depend on the other amino acid sequence motifs by which the isoforms differ. We also speculate that the scarce signal of the relatively low molecular mass complex (about 37 kDa), the absence of other complexes in the range of the AUF1 molecular masses, and the presence of higher molecular mass complexes in immunoprecipitates could result from the AUF1 isoform ability to multimerize as reported by Wilson et al. (44Wilson G.M. Lu Y. Sun H. Brewer G. J. Biol. Chem. 1999; 274: 33374-33381Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Our results also strengthen the hypothesis that the various isoforms of AUF1 may reflect the need of cells to differentially modulate ARE-containing mRNAs during apoptosis. Furthermore, mRNA turnover of ARE-containing genes has already been demonstrated to be a finely tuned process. For example, Wang et al. (24Wang W. Furneaux H. Cheng H. Caldwell M.C. Hutter D. Liu Y. Holbrook N. Gorospe M. Mol. Cell. Biol. 2000; 20: 760-769Crossref PubMed Scopus (469) Google Scholar) demonstrated that the ARE-containing mRNA ofp21waf1, a p53-inducible gene responsible for cell cycle inhibition upon UVC irradiation, was stabilized by the AUBP HuR. This would account for the arrest of cell growth that occurs at the first stages of apoptosis. Other conditions, such as hemin-induced erythroid differentiation, are able to impair ARE activity by sequestration of AUF1 into a complex of proteins (45Loflin P. Chen C.Y. Shyu A.B. Genes Dev. 1999; 13: 1884-1897Crossref PubMed Scopus (263) Google Scholar). We thank Drs. A.-B. Shyu, M. Wickens, and U. Putz for plasmids, Prof. F. Paoletti for transketolase polyclonal antibody, and Marco Cutrì for technical support.