Báo cáo khoa học: The 3¢-UTR of the mRNA coding for the major protein kinase C substrate MARCKS contains a novel CU-rich element interacting with the mRNA stabilizing factors HuD and HuR ppt

Eur J Biochem 270, 350–365 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03396.x The 3¢-UTR of the mRNA coding for the major protein kinase C substrate MARCKS contains a novel CU-rich element interacting with the mRNA stabilizing factors HuD and HuR Georg Wein1, Marek Rossler1, Roland Klug1 and Thomas Herget1,2 ă Laboratory of Molecular Neurobiology, Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg-University, Mainz, Germany; 2Axxima Pharmaceuticals AG, Martinsried, Germany The expression of the major protein kinase C substrate MARCKS (myristoylated alanine-rich C kinase substrate) is controlled by the stability of its mRNA While the MARCKS mRNA is long living in quiescent fibroblasts (t1/2 ¼ 14 h), its half-life time is drastically reduced (t1/2 ¼ h) in cells treated with phorbol esters to activate protein kinase C (PKC) or treated with growth factors In a first step to study the underlying mechanism we identified both a cis-element on the MARCKS mRNA and the corresponding trans-acting factors Fusing the complete 3¢-UTR or specific regions of the 3¢-UTR of the MARCKS gene to a luciferase reporter gene caused a drastic decrease in luciferase expression to as low as 5–10% of controls This down-regulation was a result of destabilization of the chimeric transcript as shown by RNA run-off and Northern blot-assays By RNase/EMSA and UV-cross-linking experiments, we identified a stretch of 52 nucleotides [(CUUU)11(U)8] in the 3¢-UTR of the MARCKS mRNA specifically recognized by two RNA-binding proteins, HuD and HuR These trans-acting factors are members of the ELAV gene family and bind the MARCKS CU-rich sequence with high affinity Overexpression of HuD and HuR in murine fibroblasts caused a striking stabilization of the endogenous MARCKS mRNA even under conditions when the MARCKS mRNA is normally actively degraded, i.e after treating cells with phorbol ester These data imply, that the identified CU-rich cis-element of the MARCKS 3¢-UTR is involved in conferring instability to mRNAs and that members of the ELAV gene family oppose this effect Based on its structural and functional properties, the (CUUU)11(U)8 sequence described here can be grouped into class III of AU-rich elements Expression of many genes that control cellular proliferation and differentiation is, at least in part, adjusted by regulation of the stability of their transcripts (reviewed in [1–3]) Such transcripts include proto-oncogenes such as c-myc, transcription factors, cytokines, lymphokines, growth factors and their receptors The decay rates of many of these transcripts are governed by a sequence determinant called the AU-rich element (ARE) This cis-acting element, which varies in length and sequence, is characterized by a high degree of uridylate and, sometimes, adenylate residues and often contains one or more AUUUA pentamers [4,5] mediating transcript instability Moreover, ARE-directed mRNA degradation is influenced by many exogenous factors, including phorbol esters, calcium ionophores, cytokines and transcription inhibitors, consistent with the possibility that AREs play a critical role in the regulation of gene expression during cell proliferation and differentiation [5–8] To date, three classes of AREs have been identified based on their presence, number of repeats of the pentamer AUUUA, and their subsequent effects on RNA decay [9] Many ARE-specific RNA-binding proteins have been identified; however, the molecular mechanism by which these proteins target mRNA for rapid degradation remains largely to be determined It is not yet clear whether AREs are the actual targets for ribonucleases and/or whether trans-acting factors lead to an increase in the rate of deadenylation, which often is the first step in ARE-directed mRNA decay (reviewed in [8,10,11]) A number of trans-acting factors interacting with AREs have been identified Among them are the ELAV-like proteins, also called Hu antigens, which are the mammalian orthologues of the elav (embryonic lethal abnormal vision) gene of Drosophila [11] Hu proteins are also known as autoimmune antigens in human paraneoplastic disorders [12] In each species of vertebrates, there are four different Hu proteins, which are produced from distinct genes [13] HuC, also referred to as ple21 or ElrC, and HuD, also referred to as ElrD, are expressed specifically in neurons, Correspondence to T Herget, Axxima Pharmaceuticals AG, Am Klopferspitz 19, 82152 Martinsried, Germany Fax: + 49 89 740 165 20, Tel.: + 49 89 740 165 30, E-mail: herget@axxima.com Abbreviations: ARE, AU-rich element; CDS, coding sequence; CstF64, cleavage stimulation factor of 64 kDa; ELAV, embryonic lethal abnormal vision; GAP-43, growth associated protein of 43 kDa; Luc, luciferase; MARCKS, myristoylated alanine-rich C kinase substrate; PDB, phorbol-12,13-dibutyrate; PKC, protein kinase C; RA, all-trans retinoic acid (Received 10 September 2002, revised November 2002, accepted 26 November 2002) Keywords: RNA stability; AU-rich elements; protein kinase C; MARCKS; Hu-proteins Ó FEBS 2003 Hu-proteins control MARCKS mRNA stability (Eur J Biochem 270) 351 whereas HuB, also referred to as Hel-N1 in humans, Mel-N1 in mice or ElrB, is expressed mainly in neurons, testes and ovaries Another Hu protein, HuR, also referred to as HuA or ElrA, is expressed in all tissues tested [14] All four members of the Hu family encode RNA-binding proteins containing three RNA-interacting domains of the RRM (RNA recognition motif) type [11] The first two domains recognize and specifically associate with a target motif, the ARE, whereas the third domain seems to bind the mRNA poly(A) tail [15] It is generally accepted that Hu proteins exert a stabilizing function on labile mRNAs [16,17] HuD/ HuR proteins are reported to travel between the nucleus and cytoplasm due to the presence of a nuclear-cytoplasmic shuttling signal [16,18–20] As Hu proteins are associated with mRNAs, this shuttling may represent a mechanism used to distribute bound messengers and determine the amounts of mRNA available for translation [20] We previously showed that the cellular level of the mRNA coding for the myristoylated alanine-rich C kinase substrate, called MARCKS or 80K, is cell cycle dependent In quiescent Swiss 3T3 and murine embryonic fibroblasts (MEF), levels of MARCKS mRNA are high However, upon stimulating cells with growth factors like PDGF [21] or activating protein kinase C (PKC) with phorbol esters [22], the MARCKS mRNA levels were drastically reduced within hours Nuclear run-off experiments showed that this down-regulation was not due to a shut-down of the promoter of the unique MARCKS gene, but to an enhanced degradation of the MARCKS mRNA [21,22] An inverse correlation between expression of MARCKS and progression through the cell cycle was also shown after subculturing Swiss 3T3 cells in fresh medium [23] Furthermore, overexpression of the MARCKS protein in fibroblasts caused slow growth rate, a low final cell density and enhanced susceptibility towards calmodulin antagonists [24] Additionally, the finding that many tumor cell lines have no or reduced MARCKS expression supports the hypothesis that MARCKS is a tumor and/or growth suppressor gene in some cell types [25–27] The precise physiological role of this widely distributed PKC substrate has not been convincingly established yet (reviewed in [28–30] Phosphorylation of MARCKS appears to be involved in controlling cell shape changes, possibly via regulating cytoskeleton-membrane linkage [31], and/or adjusting the level of free cellular calmodulin [24] The present study shows that the 3¢-UTR of the MARCKS transcript contains a sequence that confers mRNA instability when fused to a reporter gene In the 3¢-UTR of the MARCKS mRNA we identified an ARElike sequence that is specifically recognized by several proteins including HuD and HuR Overexpression of HuD induces stabilization of the MARCKS mRNA under conditions which otherwise cause its degradation Materials and methods Cell lines and culture conditions Stock cultures of Swiss 3T3 fibroblasts [32] were propagated as described previously [33] For experiments, · 105 cells were subcultered in 90-mm dishes (Falcon) with 10 mL Dulbecco’s modified Eagle’s medium (DMEM) supplemen- ted with 12.5% fetal bovine serum (Life-Technologies, Eggenstein, Germany) and incubated in a humidified atmosphere of 10% CO2 and 90% air at 37 °C Cells were rendered quiescent by incubating under these conditions for 8–10 days before use Swiss 3T3 cells were used for stable transfection with a chimeric luciferase-MARCKS construct and for transient transfection with the cDNAs coding for human HuD and HuR The mouse embryonic carcinoma cell line PCC7-Mz1 is a subclone of the PCC7-S-AzaR1 (clone 1009) cell line Its properties and culture conditions have been described in detail elsewhere [34,35] The stem cells were maintained in tissue culture dishes in DMEM supplemented with 12.5% fetal bovine serum (PAA Laboratories, Colbe, Germany) at ă 37 C in 90% humidified air/10% CO2 Expression of chimeric luciferase pcDNA3-luc-MARCKS 3¢-UTR (pDK1) was constructed as follows: pGEMÒ (Promega, Mannheim, Germany) containing the luciferase gene was digested with StuI and HindIII, and the luc-fragment was cloned into pcDNA3 (Invitrogen, Karlsruhe, Germany) via a filled-in EcoRI site and a HindIII site downstream of the CMV promoter, resulting in the plasmid pLuc The MARCKS 3¢-UTR fragment was excised with NotI from pBS-DC1 and ligated into NotI digested pLuc downstream of the luciferase coding sequence The construction of 3¢ deletions of pDK1 was performed as described for pBS-DC1 deletion clones (see Preparation of RNA transcripts) except with digestion of pDK1 by ApaI and XhoI The resulting vectors were sequenced and named according to the lengths of the MARCKS sequence contained (pDK2– pDK10) Swiss 3T3 cells (4.8 · 104) were stably transfected with 1.5 lg of pDK1 (MARCKS 3¢-UTR: 1287 bp), pDK2 (999 bp), pDK8 (252 bp) or pLuc(–) plasmid DNA using Lipofectamine plus reagent (Life Technologies, Karlsruhe, Germany) according to the manufacturer’s instructions Two days after transfection, selection for stably transfected cells began by incubation with geneticin (750 lgỈmL)1) (G418; Sigma, Steinheim, Germany) containing medium which was changed every days After weeks, resistant colonies were isolated by trypsinization within a glass cylinder (ring cloning) and cloned by the limited dilution technique Clones were analyzed when cultures reached confluence The luciferase activities of the established cell lines were quantified with the luciferase reporter gene assay (Roche, Mannheim, Germany) according to the manufacturer’s instructions using the Lumat LB 9501 luminometer (Berthold, Wildbad, Germany) The relative light units (RLU) measured over s were normalized to the amount of protein concentration of each clone The luciferase mRNAs were detected by Northern blotting as described in the following paragraph To monitor the transcriptional activity of the transfected pLuc and pDK1 constructs, Swiss 3T3 cells nuclear run-off analyses were performed as described previously [22] with the following modifications The cell lysate prepared from quiescent cells was washed twice in NP-40 lysis buffer (0.5% Ó FEBS 2003 352 G Wein et al (Eur J Biochem 270) NP-40, 10 mM NaCl, mM MgCl2 and 10 mM Tris/HCl pH 7.4) prior resuspending the nuclear pellet in glycerol storage buffer (40% glycerol, mM MgCl2, 0.1 mM EDTA and 50 mM Tris/HCl pH 8.3) and snap freezing in 100 lL aliquots in liquid nitrogen The run-off transcription assay was performed using [a-32P]UTP as described previously [36] followed by purification of the 32P-labeled RNA using the RNeasy Total RNA kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions The incorporation of [a-32P]UTP into newly synthesized RNA was determined by Cerenkov counting, and equal amounts of radioactivity ( 106 c.p.m.ỈmL)1) were incubated with filter-immobilized plasmids as described for Northern blotting The target plasmids (5 lg each) used were pBluescript KSÒ, p809.1 [22], pc-myc, pTH82 containing a 1.6-kb cDNA fragment from the coding region of mouse cytochrome c oxidase subunit [35] and pGEMÒ with the luciferase gene Before binding to membrane (Hybond N; Amersham-Pharmacia, Freiburg, Germany) plasmids were linearized by digestion with either StuI (pGem) or EcoRI, denatured by incubation with 0.2 M NaOH for at room temperature, 10 at 65 °C and on ice, and neutralized by addition of · NaCl/Cit/1.4 M Tris/HCl pH 3.5 After hybridization, the filters were washed three times with · NaCl/Cit for at room temperature followed by incubation with · NaCl/Cit supplemented with 10 lgỈmL)1 RNase A at 37 °C for additional 30 Subsequently, the membranes were washed twice with · NaCl/Cit, 0.2% SDS for 20 at 50 °C, and then twice with 0.2 · NaCl/Cit, 0.2% SDS at 65 °C for 20 prior to audioradiography with Kodak X-OMAT AR X-ray film Northern blot analysis Cultures were washed twice with ice-cold NaCl/Pi and cellular RNA was extracted using the RNeasy Total RNA kit (Qiagen) Five micrograms of total RNA per lane were separated in 1.2% (w/v) agarose/2.2 M formaldehyde gels and transferred onto HybondTM-N nylon membrane (Amersham-Pharmacia) by capillary blot with 10 · NaCl/Cit Prehybridization was performed in · NaCl/Cit containing · Denhardt’s solution, 0.5% (w/v) SDS, 250 lgỈmL)1 denatured salmon sperm DNA and 50% (v/v) formamide at 42 °C for h For MARCKS detection, the EcoRI insert of clone p809.1 [22] containing 1.2 kb of the MARCKS cDNA was gel-purified and labeled by random-priming in the presence of [a-32P]dCTP [37] The luciferase probe was a radioactively labeled 1.7 kb HindIII/NotI fragment of the pLuc plasmid The 32P-labeled probes were added to freshly prepared prehybridization solution at 106 c.p.m.ỈmL)1 and incubated with the membranes overnight at 42 °C Filters were washed three times with · NaCl/Cit, 0.2% (w/v) SDS at 42 °C for 15 each, followed by three times with 0.2 · NaCl/Cit, 0.2% (w/v) SDS at 60 °C for 15 each The blots were exposed overnight at )80 °C to Kodak X-OMAT AR films with intensifier screens Western blot analysis For Western blotting 20 lg protein per lane were separated by electrophoresis in 10 and 12.5% (w/v) SDS-polyacryl- amide gels and transferred onto poly(vinylidene difluoride) membranes (PVDF; Immobilon P, Millipore, Eschborn, Germany) by semidry blotting The membranes were blocked with NaCl/Pi/Triton [0.1% (w/v) Triton X-100 in NaCl/Pi, pH 7.2], supplemented with 5% (w/v) low fat milk powder, for h at room temperature and then incubated overnight at °C with the antibody The Hu antiserum was diluted : 1000 in NaCl/Pi/Triton containing 1% (w/v) low fat milk powder, the anti-CstF64 Ig (murine mAb 3A7, kindly provided by I Mattaj, EMBL Heidelberg) was diluted : 100 After five washes with the NaCl/Pi/Triton, the membranes were incubated with horseradish peroxidase-conjugated goat-anti rabbit Ig for Hu detection and with horseradish peroxidase-conjugated rabbit-anti mouse Ig for CstF64 detection, respectively, for 1–2 h at room temperature Both secondary antibodies (Dako, Hamburg, Germany) were diluted : 2000 in NaCl/Pi/Triton with 1% (w/v) low fat milk powder Membranes were washed five times in NaCl/Pi/Triton and bound antibodies were visualized by enhanced chemiluminescence (ECL) detection system using Fuji medical X-ray films Preparation of RNA transcripts To clone the complete MARCKS 3¢-UTR sequence by PCR we used oligonucleotides as DNA primers whose sequences were deduced from the murine MARCKS cDNA [38] The PCC7-MzN1 NM 1149–cDNA library [39] was utilized as template and the 1287 bp PCR fragment was cloned into the EcoRI and HindIII site of pBluescript (Stratagene, Amsterdam, Netherlands) Sequence comparison of the resulting plasmid pBS-MARCKS 3¢-UTR (pBSDC1) revealed identity with the published MARCKS sequence [22,38] pBluescript plasmids containing 3¢ truncated MARCKS 3¢-UTR sequences were constructed by exonucleolytic digestion [40,41] Following digestion with KpnI and XhoI pBS-MARCKS 3¢-UTR was treated with exonuclease III (100 lg)1 DNA) at 37 °C for various periods of time (1.5–15 min) The overhanging 5¢- and 3¢-ends were blunted by incubation with nuclease S1 (20 U) for 30 at 30 °C Finally, the reaction was stopped by adding EDTA (final concentration 60 mM) and Tris/HCl pH 8.0 (final concentration 0.3 M) The DNA fragments were gel-purified, autoligated, and transformed in E coli C600 cells The precise 3¢ ends of the MARCKS 3¢-UTR deletion clones were determined by sequencing and the deletion clones named according to their lengths, i.e pBS-DC2 contained a 1097bp insert and pBS-DC10 a 75-bp insert The pBS-MARCKS 52 nt CU-element plasmid (pBSMARCKS 52 nt) was constructed by annealing the two synthetic oligonucleotides (sense: 5¢-CCC CGG GCC CGA ATT CCT TTC TTT CTT TCT TTC TTT CTT TCT TTC TTT CTT TCT TTC TTT TTT TTT TTC TCG AGC CCC-3¢; antisense: 5¢-GGG GCT CGA GAA AAA AAA AAA GAA AGA AAG AAA GAA AGA AAG AAA GAA AGA AAG AAA GAA AGG AAT TCG GGC CCG GGG-3¢) representing base pairs 1830–1881 of the MARCKS cDNA [38] and cloning the resulting doublestranded DNA into the SmaI site of pBluescript For in vitro transcription pBS-DC1 was linearized with HindIII and the deletion clones with PvuII The Ó FEBS 2003 Hu-proteins control MARCKS mRNA stability (Eur J Biochem 270) 353 pBS-MARCKS 52 nt plasmid was digested with BamHI when in vitro transcription (T3 polymerase) of RNA in sense orientation and with EcoRV when transcription (T7 polymerase) of antisense RNA was performed The same restriction sites and RNA polymerases were applied when the empty pBluescript (pBS) vector was used for production of negative control RNAs All templates were phenol/ chloroform extracted and ethanol precipitated before use The reaction mix contained mM MgCl2, mM spermidine, 10 mM dithiothreitol, 40 mM Tris/HCl pH 8.0, 0.75 mM each of ATP, GTP and CTP, 30 lM UTP, 50 lCi [a-32P]UTP (3000 CiỈmmol)1, ICN, Eschwege, Germany), 40 U RNasin (MBI-Fermentas, St Leon-Roth, Germany), lg of template DNA and 20 U of T7 or T3 polymerase (Roche, Mannheim, Germany) After h at 37 °C the reaction was terminated by digestion of template DNA with 40 U DNase I (Roche, Mannheim, Germany) for further 20 Following removal of unincorporated nucleotides via Sephadex G-75 (Amersham-Pharmacia, Freiburg, Germany) gel filtration, the RNA was phenol/chloroform extracted and ethanol precipitated The nonlabeled competitor transcripts were synthesized under the same conditions, except the concentration of all four ribonucleotides was 0.75 mM and the [a-32P]UTP was omitted Production of recombinant Hu proteins To acquire recombinant GST-HuD fusion protein we amplified a cDNA encoding residues 2–373 of HuD by PCR using clone kuniZAP-265114 as template and BamHI and SmaI sites containing primers (sense: 5¢-TAG CGG ATC CGA GCC TCA GGT GTC AAA TGG-3¢; antisense: 5¢-AAT GCC CGG GTC AGG ACT TGT GGG CTT TGT-3¢) The plasmid kuniZAP-265114, kindly provided by M Kock, BASF, Germany, bears the complete HuD coding sequence and 3¢-UTR The resulting PCR product (1139 bp) was cloned in frame via the BamHI and SmaI sites downstream of the GST (glutathionine S-transferase) gene into GEX-2T (Amersham-Pharmacia) and was called pGEX-HuD pGEX-HuR was constructed in a similar way and was a gift of H Kleinert, Mainz, Germany Both vectors (pGEX-HuD/pGEX-HuR) were transformed in E coli (XL-1 Blue, Stratagene) and the expressed GST-HuD/GST-HuR fusion proteins were purified exactly as described for GST-MARCKS [42] To produce His-tagged HuD fusion protein, the HuD PCR-fragment was digested with BamHI and SmaI as for GST-HuD and cloned into pQE30 (Qiagen) digested with the same enzymes, resulting in plasmid pQE30-HuD The His6-tagged HuD fusion protein was expressed in E coli (XL-1 Blue) and purified as described for His6-MARCKS [43] The protein concentrations of GST-HuD, GST-HuR and His6-HuD were determined by loading aliquots on 10% SDS/polyacrylamide gels and comparison with defined amounts of BSA standards after Coomassie Blue staining Gel retardation assay The interaction of recombinant Hu-proteins with RNA transcripts were analyzed by gel retardation assays described by Chung and coworkers [44] Approximately 3000 c.p.m (2.5 ng) of labeled RNA was incubated with protein in a buffer containing 150 mM NaCl, 0.25 mgỈmL)1 tRNA, 0.25 mgỈmL)1 BSA and 50 mM Tris pH 7.0 in a final volume of 20 lL The reaction mixture was incubated at 37 °C for 10 and then lL of a dye mixture [50% (v/-v) glycerol, 0.1% (w/v) bromophenol blue and 0.1% (w/v) xylene cyanol] were added Twenty-five percent of the sample (6 lL) were immediately loaded on a 0.8% (w/v) agarose gel in Tris/acetate/EDTA buffer (1 mM EDTA, 40 mM Tris acetate pH 7.0) and gel electrophoresis was carried out at 40 V for 2–3 h Finally, the gels were dried under vacuum and exposed to Kodak X-OMAT AR films at )80 °C Generation of rabbit anti-Hu serum The full length recombinant GST-HuD protein was used for immunization of a rabbit as described for MARCKS [42] and GAP-43 [45] The serum was affinity-purified using the recombinant His6-HuD fusion protein coupled to Affi-Gel 10 matrix (Bio-Rad, Munchen, Germany) as described ă [45,46] Preparation of nuclear and cytosolic cell extracts For total cytoplasmic extracts quiescent or PDB (phorbol12,13-dibutyrate) (Sigma) treated cells (5 h, 200 nM) were rinsed twice with ice-cold NaCl/Pi and scraped off the dish with a rubber policeman in 100 lL lysis buffer [20 mM KOAc, 50 mM MgCl2, mM dithiothreitol, 0.5% (v/v) Nonidet P-40, 30 mM Tris/HCl pH 7.1, 0.1 mM Na3VO4, 10 mM NaF, 20 mM 2-glycerophosphate, 400 nM okadaic acid, 100 lgỈmL)1 leupeptin, 100 lgỈmL)1 aprotinin, 10 mM benzamidine and mM phenylmetylsulfonyl fluoride] Following 10 of incubation on ice for complete lysis the homogenate was centrifuged for 10 at 10.000 r.p.m (centrifuge 5417R, Eppendorf, Hamburg, Germany) at °C and the supernatants were removed and stored at )20 °C For fractionation into nuclear and cytosolic extracts the cells were rinsed once with ice-cold NaCl/Pi, scraped off the dish in mL NaCl/Pi and centrifuged for at 2000 r.p.m (Megafuge 1.0R, Heraeus, Hanau, Germany) and °C The pellet was resuspended in 100 lL of buffer A (1.5 mM MgCl2, 10 mM KCl, mM dithiothreitol, 10 mM Hepes pH 7.9, 0.1 mM Na3VO4, 10 mM NaF, 20 mM 2-glycerophosphate, 400 nM okadaic acid, 100 lgỈmL)1 leupeptin, 100 lgỈmL)1 aprotinin, 10 mM benzamidine and mM phenylmethylsulfonyl fluoride) and swelled on ice for 15 The cells were homogenized by pressing through a narrow-gauge hypodermic needle and the extract was centrifuged for with 14 000 r.p.m at °C (Eppendorf centrifuge 5417R) The supernatant containing the cytosolic fraction was collected and kept on ice The nuclear pellet was resuspended in 100 lL of buffer C (1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.42 M NaCl, mM dithiothreitol, 20 mM Hepes pH 7.9, 0.1 mM Na3VO4, 10 mM NaF, 20 mM 2-glycerophosphate, 400 nM okadaic acid, 100 lgỈmL)1 leupeptin, 100 lgỈmL)1 aprotinin, 10 mM benzamidine and mM phenylmethylsulfonyl fluoride), extracted for 30 on ice and centrifuged with 14 000 r.p.m for 15 at °C in parallel with the Ó FEBS 2003 354 G Wein et al (Eur J Biochem 270) cytosolic fractions The supernatants were collected and stored at )80 °C The protein concentrations of all extracts were determined using the BCA reagent (Pierce, Bonn, Germany) Technologies, Karlsruhe, Germany) according to manufacturer’s instructions After 24 h, half of the cultures were treated with 200 nM PDB (5 h) Cells were harvested and analyzed by Northern blotting as described above RNase/EMSA and UV-crosslinking Results For RNase/EMSA analysis, between and 20 lg protein were incubated with 32P-labeled transcript (2.5 ng in vitro transcribed RNA,  3000 c.p.m.) in 30 lL of 50 mM KCl, 5% (v/v) glycerol, 0.1% (v/v) Nonidet P-40, mM MgCl2 mM dithiothreitol, 10 mM Tris/HCl pH 8.0 and 10 lg yeast tRNA for 20 at room temperature For competition studies the nonlabeled competitor RNAs were preincubated with the proteins for 10 before the 32 P-labeled transcript was added In supershift experiments NP-40 was omitted from the buffer and the proteins were preincubated with lL of the a-Hu and the a-CstF64 antibodies for 30 on ice To eliminate non-protein covered RNA sequences, 10 U of RNase T1 (Roche, Mannheim, Germany) were added and incubation continued for an additional 30 Samples were then subjected to electrophoresis (2 h at 200 V; °C) performed on a 5% (w/v) native polyacrylamide gel (37 : 1) using Tris/borate/ EDTA as electrophoresis buffer Gels were dried under vacuum and exposed to Kodak X-OMAT AR films at )80 °C for 1–3 days For UV crosslinking assays, 5–20 lg cell extract or different amounts of recombinant Hu-proteins were incubated with 2.5 ng radiolabeled transcript in the same buffer as described above for RNase/EMSA analysis After 20 samples were placed on ice and irradiated by 180 mJ UV light with a Stratalinker UV 1800 (Stratagene) Then the covalently linked RNA:protein complexes were treated with 10 lg RNase A (Roche) for 30 at room temperature Reaction was stopped by addition of 30 lL · SDS-sample buffer and heating for 10 at 95 °C The samples were loaded on a 12.5% (w/v) SDS-polyacrylamide gel and separated overnight with 50 V at room temperature Finally, the gel was dried and analyzed directly by autoradiography Transient expression of HuD and HuR For transient expression of HuD and HuR in Swiss 3T3 cultures, we employed the plasmids pTetoff and pTRE (expression vector with the tTA-regulated promoter) of the TetoffTM system (Clontech, Heidelberg, Germany) [47] For construction of pTRE-HuD the plasmid kuniZAP-265114 was digested with XhoI, filled-in with DNA polymerase I, and subsequently restricted with SacII The purified fragment (1524 bp) was ligated into SacII and XbaI-blunted sites of pTRE Finally, the authenticity of the obtained plasmid was verified by restriction site mapping and sequencing The cDNA of HuR was amplified using plasmid pZeoSV(–)HuR sense (kindly provided by A Levy, Technion, Institute of Technology, Haifa, Israel) as template The HuR-PCR fragment (1133 bp) was cloned using EcoRI (upstream) and XbaI (downstream) restriction sites into the pTRE vector Swiss 3T3 were plated the day before transfection at a density of 2.9 · 104 cellsỈcm)2 in 90-mm dishes Cells were transfected with either pTRE-HuD or pTRE-HuR in the presence of plasmid pTetoff with lipofectamine 2000 (Life MARCKS 3¢-UTR mediates mRNA instability of luciferase reporter gene We previously showed that elevated levels of MARCKS mRNA and protein in quiescent Swiss 3T3 cells drastically decreases when cultures are treated with activators of PKC (e.g PDB or growth factors) or when cells are plated at low density in fresh medium [21–23] This pronounced downregulation of MARCKS was caused by post-transcriptional mechanisms involving destabilization of the MARCKS mRNA [21,22] As the stability of most mRNAs has been shown to be regulated by sequences in their 3¢-UTR [3,6,48] we explored whether the MARCKS 3¢-UTR is involved in controlling mRNA stability We fused the complete MARCKS 3¢-UTR cDNA with a luciferase reporter gene cloned into the pcDNA3 vector and stably transfected Swiss 3T3 cells with this construct (pDK1) (Fig 1A) In the same way we transfected the constructs pDK2 and pDK8 containing truncated sequences of MARCKS 3¢-UTR or as a control the luciferase reporter gene without additional sequences (pLuc) into Swiss 3T3 cells (Fig 1A) Cell extracts of the established cell lines were prepared, luciferase activities measured, normalized to protein concentration of each clone and the average of each construct presented as RLU (Fig 1B) The activity of the reporter gene decreased dramatically with the length of the fused MARCKS 3¢-UTR sequences Swiss 3T3 cells transfected with pDK8 or pLuc bearing only limited or no MARCKS sequences (pDK8, pLuc) showed high luciferase activities (mean: pDK8, 2003 RLUặlg protein)1, n ẳ 6; pLuc, 2642 RLUặlg protein)1, n ẳ 7) Signicantly less luciferase activity was detected when pDK1 (complete MARCKS 3¢-UTR) or pDK2 (MARCKS 3¢-UTR missing only the last 288 nucleotides) were transfected (mean: pDK1, 53 RLUặlg protein)1, n ẳ 15; pDK2, 184 RLUặlg protein)1, n ẳ 21) To investigate whether the low luciferase activities were due to reduced mRNA levels we performed Northern blot analyses (Fig 1C) Total RNA of four randomly chosen clones transfected with the various luciferase constructs and, as control, mock (pcDNA3) transfected Swiss 3T3 cells was isolated and hybridized with a radioactively labeled luciferase cDNA probe The detected luciferase mRNA band migrated accordingly to the length of the fused MARCKS 3¢-UTR sequences (Fig 1A) In pDK1- and pDK2-transfected cells the luciferase mRNA was hardly evident, however, considerable signals were obtained in pDK8 and pLuc transfectants (Fig 1C) Thus, the amount of luciferase mRNA of each clone corresponded closely with the level of luciferase activity (Fig 1B) Discrepancies may be due to clonal selection of the transfected cell line investigated for RNA expression As expected, in mock transfected Swiss 3T3 cells (3T3) no luciferase-mRNA was detected For monitoring the rate of transcription in pLuc- and pDK1-transfected cells we performed nuclear run-off assays The nuclei of pLuc- and pDK1-transfected Ó FEBS 2003 Hu-proteins control MARCKS mRNA stability (Eur J Biochem 270) 355 Fig The MARCKS 3¢-UTR confers mRNA instability when fused to the luciferase reporter gene (A) The chimeric constructs consisting of luciferase coding sequence (CDS) and MARCKS 3¢-UTR were driven by a CMV-promoter and used for transfection of Swiss 3T3 fibroblasts Length of the MARCKS 3¢-UTR sequences [38] of pDK1: 1310–2597 bp (complete 3¢-UTR of MARCKS); pDK2: 1310– 2309 bp; pDK8: 1310–1562 bp The poly(A) signal of the bovine growth hormone was provided by the pcDNA3 vector (B) Luciferase activity of transfected Swiss 3T3 cells were measured and the average (± SEM) obtained with each construct is depicted The transfectants (1 · 105 cells) were seeded on 90-mm dishes and grown to confluence Cell extracts were prepared and luciferase activity measured using luminometer Lumat LB 9501 Signals within s were normalized to the protein concentration of each sample and expressed as relative light units (RLU) The lowest activity of luciferase was observed when the complete MARCKS 3¢-UTR was fused to the luciferase gene (pDK1) (C) The level of luciferase mRNA was determined by Northern blot analysis pDK1, pDK2, pDK8, pLuc and pcDNA3-transfected Swiss 3T3 cells were seeded on 90-mm-dishes (1 · 105 cells) After week total RNA was isolated and lg per lane loaded onto a 1.2% agarose/2.2% formaldehyde gel After electrophoresis and transfer on a nylon membrane, RNA was hybridized with a 32P-radiolabeled luciferase cDNA probe (1.7 kb HindIII/NotI fragment of pLuc) and exposed to X-ray film (Kodak AR) with intensifier screens at )80 °C for days The upper panel shows the autoradiography Ethidium bromide staining proved equal loading (lower panel) The positions of 28S and 18S rRNAs are indicated (D) Transcriptional activity of the luciferase gene in pLuc- and pDK1-transfected Swiss 3T3 cells were studied by nuclear run-off assays Plasmids containing inserts encoding for MARCKS (p809.1), luciferase (pGEM), c-myc (pc-myc), cytochrome c oxidase (pTH82) and the control vector pBluescript (KS+) (pBS), were hybridized with 32P-labeled run-off transcripts from nuclei isolated from confluent Swiss 3T3 cultures transfected with pLuc and pDK1 Equal amounts of radioactivity was used for hybridization All genes analyzed are transcribed to similar degrees in pDK1- and pLuctransfected cell lines There was no hybridization signal with the pBS vector DNA detectable Filters were exposed to X-ray film (Kodak AR) for days at )80 °C with intensifier screens Swiss 3T3 cells were isolated and after incubation with [a-32P]UTP the radioactively labeled transcripts were hybridized to filter-immobilized cDNAs of luciferase, MARCKS (coding region), c-myc and cytochrome c oxidase As negative control pBluescript vector DNA was spotted on the filter The CMV promoter activity driving the luciferase gene was similar in pDK1- and pLuc-transfectants (Fig 1D) Transcription rate of the control mRNAs (MARCKS, c-myc, cytochrome c oxidase) were similar in both cell lines Taken together, these data reflecting the transcriptional activity as well as the RNA- and protein levels demonstrate that MARCKS 3¢-UTR sequences of pDK1 and pDK2 destabilize the chimeric luciferase-MARCKS mRNA via post-transcriptional mechanisms and that the MARCKS 3¢-UTR contains a regulatory cis-element(s) mediating mRNA instability A 52 nt CU-rich element is recognized by Swiss 3T3 proteins To identify proteins binding to the MARCKS mRNA, we cloned the complete murine MARCKS 3¢-UTR into the pBluescript vector (pBS-DC1; Fig 2A), synthesized radioactively labeled MARCKS 3¢-UTR RNA by in vitro transcription and incubated the resulting transcript with cytoplasmic extract of quiescent Swiss 3T3 cells After RNaseT1 digestion and native PAGE the formation of two RNA:protein complexes (C1 and C2) were observed (Fig 2B, lane 4) Complexes were absent when the transcript was incubated with an unrelated protein (BSA, lane 1) To localize more precisely the site within the MARCKS 3¢-UTR that interacts with Swiss 3T3 proteins, we performed RNase/EMSA analyses with 10 3¢ truncated MARCKS 3¢-UTR transcripts (data not shown) Strong RNA:protein complexes were only observed with RNA containing sequences between nucleotides 1773 and 1950, i.e with RNA derived from pBSDC4 (lane 5) but not with RNA from pBS-DC5 (Fig 2B, lane 6) Because previous work showed the importance of U-rich sequences in mRNA stability we suspected that the highly CU-rich sequence of 52 nucleotides on the pBSDC4 RNA could be involved in protein interaction (Fig 2A) To examine protein-binding capacity we cloned the 52 nt sequence into pBluescript, synthesized radiolabeled RNA and incubated the transcript with cytoplasmic protein of Swiss 3T3 cultures As shown in Fig 3A (left panel) the formation of two RNA:protein complexes with identical mobility to those observed with the fulllength 3¢-UTR (C1, C2) were observed with the 52 nt RNA probe To monitor the specificity of protein binding to the CU-rich sequence we transcribed the pBSMARCKS-52 nt construct in the antisense orientation 356 G Wein et al (Eur J Biochem 270) Ó FEBS 2003 Fig Formation of two major complexes between Swiss 3T3 proteins and the 3¢-UTR of the MARCKS mRNA (A) The MARCKS 3¢-UTR, the stop codon UAA of the coding sequence (CDS) and the poly(A) sequence are depicted The box within the 3¢-UTR marked the identified CU-rich sequence interacting with Swiss 3T3 proteins (top) The fragments of the 3¢-UTR cloned into plasmids (pBS-DC1, pBS-DC4, pBS-DC5 and pBS-MARCKS-52nt) are schematically presented (sequence according to [38]) These constructs were used to synthesize truncated RNA segments of the MARCKS 3¢-UTR The 3¢ termini of pBS-DC4 and pBS-DC5 are marked with arrows and the 52-nucleotide CU-rich sequence is underlined (bottom) (B) Interaction between Swiss 3T3 proteins and the MARCKS 3¢-UTR RNA was monitored by RNase/EMSA analysis pBS-DC1 was digested with HindIII, pBSDC4 and pBS-DC5 with PvuII and used as templates for in vitro transcription in the presence of [a-32P]UTP and T7-RNA polymerase 2.5 ng of the radiolabeled RNAs were incubated for 20 at room temperature with lg bovine serum albumin (BSA) (lanes 1–3) and lg Swiss 3T3 cytoplasmic extract from quiescent cells (lanes 4–6) Following RNase T1 digestion for 30 at room temperature the samples were loaded on 5% native polyacrylamide gel After electrophoresis at °C the gel was dried and exposed to X-ray film (Kodak AR) with intensifier screens at )80 °C for day The positions of the two RNA:protein complexes (C1, C2) are indicated but not with 52 nt antisense RNA (lane 10–14) We therefore conclude that the 52 nt CU-rich sequence represents a cis-element interacting with proteins from Swiss 3T3 cells By screening the MARCKS 3¢-UTR for sequences related to the 52 nt element, we identified a series of 18 U residues (nucleotides 1585–1603) It is likely that this element binds, albeit more weakly, the same proteins as the 52 nt CU element and therefore is responsible for the faint formation of complexes C1 and C2 with construct pDC5 (Fig 2B, lane 6) We did not detect any protein binding sequences within the first 200 bp of the 3¢-UTR (nucleotides 1310–1409) of the MARCKS mRNA (data not shown) Detection of four proteins binding to the MARCKS 52 nt CU-rich RNA and incubated this RNA with extracts in the same way There was essentially no binding activity detected (Fig 3A, right panel) The sequence specificity of the RNA:protein complexes was further demonstrated by competition experiments (Fig 3B) showing the complex formation was only inhibited by the addition of increasing amounts of unlabeled 52 nt sense transcripts (lane 4–9) To identify proteins responsible for the formation of the two complexes identified in RNase/EMSA analyses, we performed high resolution UV-crosslinking assays (Fig 3C) Five micrograms of protein of cytoplasmic Swiss 3T3 extract were incubated with radiolabeled MARCKS 52 nt sense-RNA and subjected to UV light irradiation (180 mJ) The crosslinked samples were treated with RNase A and resolved by electrophoresis on a 12.5% (w/v) SDS-polyacrylamide gel Four strong bands resulting from proteins crosslinked with the radioactively labeled RNA were detected (Fig 3C, lane 1) The sizes of these proteins were 55, 40, 36 and 30 kDa Because a common feature of RNA-binding proteins is that they can also bind single-stranded DNA [79], we preincubated the 3T3 proteins with increasing amounts of 52 nt sense DNA-oligonucleotide Addition of 10 ng and more of the 52 nt sense DNA-oligo resulted in a strong competition with the radioactive RNA for proteins and consequently a decrease in detection of proteins crosslinked Ó FEBS 2003 Hu-proteins control MARCKS mRNA stability (Eur J Biochem 270) 357 with the radioactive CU-rich RNA (Fig 3C) A similar competition in UV crosslinking assays was observed with unlabeled 52 nt CU-rich RNA (data not shown) Using the corresponding antisense sequence of the 52 nt CU-rich sequence as RNA or DNA for competition did not result in loss of detection of the RNA:protein complexes (data not shown) Furthermore, using the 52 nt element in antisense orientation as probe for UV crosslinking experiments did not reveal the four RNA:protein complexes (Fig 3C, lane 5) Taken together, the four RNA-binding proteins identified (Fig 3C) interact specifically with the CU-rich sequence Binding of ELAV/Hu proteins to the MARCKS 52 nt CU-rich RNA One of the identified proteins binding to the MARCKS CU-rich element has an apparent molecular mass of about 36 kDa (Fig 3C) A possible candidate for this protein might be HuR, an ubiquitously expressed, 36-kDa member of the ELAV/Hu gene family [49] These RNA-binding proteins recognize U-rich sequences of RNAs coding for proteins regulating cell growth and differentiation [49,50] To determine if proteins of the ELAV/Hu family bind the MARCKS 52 nt RNA, we focused on two members known 358 G Wein et al (Eur J Biochem 270) Fig Swiss 3T3 proteins recognize the MARCKS 52 nt CU-rich RNA element with high sequence-specificity (A) pBS-MARCKS-52nt was linearized with BamHI (sense) or EcoRV (antisense) and in vitro transcribed with [a-32P]UTP using T3-RNA polymerase (sense) or with [a-32P]ATP and T7-RNA polymerase, respectively 2.5 ng of the radiolabeled RNAs were incubated for 20 at room temperature with reaction buffer alone (lanes and 4), with lg bovine serum albumin (BSA) (lanes and 5), with lg protein of total extract from Swiss 3T3 cells (lanes and 6) After RNase T1 digestion (lanes 2, 3, and 6) samples were resolved by 5% native PAGE The gel was dried and exposed to Kodak AR X-ray film with screens at )80 °C for one day Swiss 3T3 proteins bound the MARCKS 52 nt cis-element when transcribed in sense orientation (B) RNase/EMSA analysis of the complete MARCKS 3¢-UTR was performed in the presence of the indicated amounts of the CU-rich element transcribed in sense and antisense orientation The nonlabeled sense and antisense pBSMARCKS 52 nt transcripts were incubated with lg cytoplasmic extract from Swiss 3T3 cells for 10 at room temperature (sense: lanes 4–9, antisense: lanes 10–14) Then, 2.5 ng of the complete, 32 P-labeled MARCKS 3¢-UTR RNA was added and incubation prolonged for 20 at room temperature Controls were: reaction buffer (lane 1), BSA (lane 2) and Swiss 3T3 extract without competitor RNA (lane 3) Undigested probe (lane 1) and RNase T1 digested samples (lanes 2–14) were loaded on native 5% polyacrylamide gel followed by electrophoresis at °C The dried gel was exposed to X-ray film (Kodak AR) with screens at )80 °C for day Effective competition with the MARCKS 3¢-UTR for binding Swiss 3T3 proteins was only detectable by the sense transcript Note, that the amounts of the 52 nt antisense RNA was 10-fold higher (lanes 11–14) than of the respective competitor sense transcript (lanes 5–9) The positions of the two RNA:protein complexes (C1, C2) are indicated (C) Four RNA-binding proteins recognize the MARCKS 52 nt RNA In UV-crosslinking experiments with lg extract from Swiss 3T3 cells, proteins of about 30, 36, 40 and 55 kDa were crosslinked to [a-32P]UTP labeled MARCKS CU-rich sense RNA Specificity of this interaction was demonstrated by preincubation of the Swiss 3T3 cytoplasmic extract in RNase/EMSA buffer for 10 with the indicated amounts of the respective sense DNA oligonucleotide (employed for cloning of pBS-MARCKS-52nt, see Materials and methods) prior addition of 2.5 ng 32P-labeled transcript After another 10 incubation at room temperature samples were subjected to UV-crosslinking (Stratalinker) and RNase A digestion The denatured samples (10 at 95 °C in SDS-sample buffer) were loaded on 12.5% SDS-polyacrylamide gel and visualized by exposure to X-ray film The corresponding antisense probe did not bind any proteins (lane 5) Position and size of protein markers are indicated on the right to regulate mRNA stability: the wide-spread HuR and the neuron-specific HuD [51] The cDNAs of human HuR and HuD were cloned into prokaryotic expression vectors and recombinant GST-HuD and HuR were expressed in E coli Purified GST-HuD and GST-HuR fusion proteins were incubated with radioactively labeled 52 nt sense transcripts and HuD:RNA or HuR:RNA complex formation assayed by gel retardation analysis [44] The 52 nt RNA was bound by both Hu-proteins very efficiently, and complex formation was easily detectable with HuD (0.8 nM) (Fig 4A) or HuR (4 nM) (Fig 4B), respectively Using the complete MARCKS 3¢-UTR as RNA probe complex formation with GST-HuD and GST-HuR could be observed in the same way (data not shown) In contrast, no specific interaction Ó FEBS 2003 was observed with control RNA (pBSD52ntCU-RNA) and GST-HuD (up to 800 nM) (Fig 4C) Furthermore, complex formation was not detected with GST alone and the 52 nt CU-rich RNA (Fig 4D) To explore whether the crosslinked protein of 36 kDa is endogenous HuR (Fig 3C), we performed an UV-crosslinking assay with cytoplasmic extract prepared from Swiss 3T3 cells and the radioactively labeled MARCKS 52 nt sense transcript as probe The 36-kDa complex of the RNA with an endogenous protein (Fig 4E, lane 1) was disrupted by adding increasing amounts of recombinant His6-HuD to the incubation mix (lanes 2–7) HuD has similar binding characteristics to HuR but is not expressed in murine fibroblasts [19] (see also Fig 6B) and is of different size Due to their different sizes, the cellular 36-kDa HuR protein can easily be discriminated from the recombinant His6-HuD protein (39 kDa) Addition of His6-HuD apparently displaced the endogenous HuR (p36) protein from the complex with the CU-rich element demonstrating that both proteins are competing for similar RNA sequences (lanes 2–7) Interestingly, adding His6-HuD (5 ng and more) efficiently prevented both binding of cellular HuR and of the unknown 55-kDa protein to the MARCKS 52 nt RNA probe arguing that more than one protein recognize the same site (Fig 4E, lanes 5–7) Analysis of HuR and CstF64 in Swiss 3T3 cells The data so far showed that HuR binds the CU-element efficiently in vitro and that addition of recombinant HuD to Swiss 3T3 extracts can compete with an endogenous RNA:protein complex probably containing HuR It is known that HuR shuttles between nucleus and cytoplasm and displays a cell-type specific subcellular distribution To study the identified RNA-binding proteins in more detail we compared nuclear and cytoplasmic fractions of Swiss 3T3 cells in their capacity to bind the CU-rich sequence with the expression pattern of HuR Firstly, we generated and affinity-purified a polyclonal antiserum directed against recombinant GST-HuD fusion protein This Hu antiserum recognized several members of the ELAV/Hu family due to their high degree of sequence identity [49,51,78] Using our Hu-specific serum and nuclear and cytoplasmic extracts from quiescent Swiss 3T3 cells for Western blot analysis resulted in one specific band of 36 kDa, consisting of the HuR protein (Fig 5A) In Swiss 3T3 cells HuR was mainly localized in the nucleus and only a minor portion resided in the cytoplasm (Fig 5A, left panel) as described previously for other cell types [16,20,51–53] To validate the quality of our fractionation procedure, we analyzed the samples for the known nuclear RNA-binding protein CstF64 that recognizes U-rich sequences and contributes to polyadenylation of mRNAs [54,55] The CstF64-specific monoclonal antibody 3A7 [55] was used for Western blot analysis CstF64 was not detected in the cytoplasmic fraction, but was exclusively in the nuclear fraction (Fig 5A, right panel) Using these fractionated extracts for UV crosslinking studies with the radiolabeled 52 nt CU-rich RNA revealed that the intensity of the p36 RNA:protein complex (Fig 5B) Ó FEBS 2003 Hu-proteins control MARCKS mRNA stability (Eur J Biochem 270) 359 Fig HuD and HuR bind the MARCKS 52 nt CU-rich RNA (A) For gel retardation assay, 2.5 ng ( 3000 c.p.m.) of 32P-labeled MARCKS 52 nt sense RNA was incubated without protein or with the indicated concentrations of HuD After 10 at 37 °C the reaction mix was resolved on a 0.8% (w/v) agarose gel The dried gel was exposed to X-ray film (Kodak AR) at )80 °C for days Complex formation was detectable with 0.8 nM of GST-HuD protein (B) Gel retardation assay was performed with the 52 nt sense CU-rich RNA and GST-HuR; a RNA:protein complex was formed with nM of HuR protein C: As negative control, GST-HuD gel retardation assays were done with labeled pBluescript RNA (pBSD52ntCU-RNA) containing the identical vector sequences as the 52 nt CU-RNA probe (A, B, D, E) but without the 52 nt CU-element No complex formation was detectable with this probe D: There was also no complex identified when the 52 nt CU-rich RNA was incubated with GST E: UV-crosslinking of Swiss 3T3 proteins with the CU-rich RNA was performed in the presence of increasing amounts of recombinant His6-HuD 20 lg of cytoplasmic extract of Swiss 3T3 cells was incubated with the radiolabeled MARCKS 52 nt CU-rich transcript (lane 1) Increasing amounts of recombinant His6-HuD was added to the mix, as indicated After UV-crosslinking (180 mJ) the samples were digested with Rnase A and separated by denaturing electrophoresis on a 12.5% SDSpolyacrylamide gel Crosslinked proteins were visualized by autoradiography using Kodak AR X-ray films Twenty-five nanograms recombinant His6-HuD efficiently blocked the interaction of Swiss 3T3 protein HuR and the RNA probe (crosslinked proteins indicated by arrows) The positions of protein markers are indicated on the right correlated precisely with the levels of HuR in these samples (Fig 5A) supporting the finding that the p36 protein identified (Figs 3C and 4E) is HuR RNase/EMSA analyses demonstrated that the 52 nt CU-rich RNA can form the complexes C1 and C2 with both the nuclear (Fig 5C, lane 1) and the cytoplasmic fraction (lane 4) To directly explore the involvement of HuR in forming a complex with the CU-rich sequence, we used the Hu-antiserum in combination with RNase/EMSA for supershift analysis The nuclear and cytoplasmic extracts of quiescent Swiss 3T3 cells were preincubated with the CstF64 antibody 3A7 or with the Hu-specific antiserum Following incubation with the labeled MARCKS 52 nt probe and RNase T1 digestion, the resulting complexes were resolved by electrophoresis on a native polyacrylamide gel The Hu-specific antiserum caused a supershift of the RNA:protein complexes with the nuclear fraction (Fig 5C, lane 2) which was also detectable with cytoplasmic extract, although much weaker (Fig 5C, lane 5) Thus, the Hu-supershift pattern (Fig 5C) matches accurately the Hu-Western Blot data (Fig 5A, left panel) and the UV-crosslinking results (Fig 5B) further supporting the finding that HuR is one protein binding the MARCKS 3¢-UTR in Swiss 3T3 cells In contrast to the Hu antiserum, no interaction of the CstF64 control antibody with the MARCKS RNA-binding proteins was observed revealing that CstF64 is not involved in formation of the RNA:protein complexes C1 and C2 (Fig 5B, lanes and 6) The addition of increasing amounts of the Hu-specific serum to nuclear Swiss 3T3 proteins resulted in detection of up to three supershift complexes in addition to the RNA:protein complexes C1 and C2 (Fig 5D) It is therefore likely, that more than one Hu-protein binds the 52 nt CU-rich RNA Alternatively, the multiple bands may be due to various post-translational modifications e.g HuR protein phosphorylation [56,57] In summary, the MARCKS 52 nt CU-rich element represents an effective target sequence for ELAV/Huproteins One of the proteins of Swiss 3T3 cells binding the MARCKS CU-rich element was identified as the ELAV related protein HuR, which is predominately nuclear localized CstF64, a further nuclear RNA-binding protein with sequence specificity for U-residues is not involved in binding the MARCKS 52 nt CU-rich RNA element in Swiss 3T3 cells 360 G Wein et al (Eur J Biochem 270) Ó FEBS 2003 Fig HuR is expressed in the nucleus and binds the 52 nt CU-rich RNA (A) Quiescent Swiss 3T3 cells were harvested and extracts separated into nuclear and cytoplasmic fraction by centrifugation (see Materials and methods) Twenty micrograms of each fraction were loaded per lane on a 10% SDS-polyacrylamide gel, separated and transferred to PVDF membrane Hu-proteins was detected with the affinity-purified Hu-specific polyclonal antiserum (left panel) For CstF64 detection we applied the 3A7 monoclonal antibody (right panel) Bound antibody was detected by enhanced chemiluminescence HuR was predominantly localized in the nucleus, while CstF64 was exclusively localized in the nucleus (B) For UV-crosslinking experiments, 20 lg protein of the same extracts as used in Fig 5A were incubated with the 32P-labelled 52 nt CU-rich RNA probe and crosslinked by 180 mJ UV light (Stratalinker) The RNase A digested samples were run on a denaturating, 12.5% SDS-polyacrylamide gel and the dried gel was exposed to X-ray film (Kodak AR) with screens at )80 °C for days The band corresponding to the HuR protein is marked by an arrow The positions of protein markers in A and B are indicated on the right (C) Contribution of HuR in formation of the RNA:protein complexes was revealed by supershift analysis Ten micrograms of nuclear and cytoplasmic extracts of Swiss 3T3 cells were preincubated with either the Hu-specific antiserum (4 lL) (lanes and 5), the CstF64-specific antibodies (4 lL) (lanes and 6) or without antiserum (lanes and 4) for 30 on ice prior addition of 2.5 ng 32P-labeled MARCKS 52 nt CU-rich transcript The RNase T1 digested samples were loaded on a 4% native polyacrylamide gel and separated at °C The dried gel was exposed to X-ray film (Kodak AR) at )80 °C for one day Only the Hu-specific antiserum caused a supershift of the RNA:protein complexes (D) Supershift analysis with nuclear Swiss 3T3 extract was performed in the presence of increasing amounts of the Hu-specific antiserum Ten micrograms of the nuclear Swiss 3T3 extract (Fig 5A–C) were preincubated with 1, 2.5, and 10 lL of the Hu-specific antiserum (lanes 2–5) on ice for 30 prior addition of 2.5 ng 32P-labeled MARCKS 52 nt CU-rich transcript The formation of supershifts were analyzed as described in Fig 5C Up to three shifted complexes were observed in addition to complexes C1 and C2 PKC activation causes down-regulation of MARCKS mRNA in fibroblast but not in neural cells We recently observed that the MARCKS protein became phosphorylated in neural PCC7-Mz1 cells upon activation of PKC [58]; however, the protein was not down-regulated as demonstrated for murine fibroblasts [22] Northern Blot analyses showed that both cell lines expressed the 2.6 kb mature MARCKS mRNA and more weakly the 4.3 kb precursor RNA containing an intron of 1.7 kb RNA isolated from PCC7-Mz1 cultures treated with solvent or with PDB (200 nM) for h verified that the 2.6 kb MARCKS mRNA was not down-regulated in PCC7-Mz1 cells by PDB treatment as was the case in Swiss 3T3 cells analyzed in parallel (Fig 6A, upper panel) This difference in regulation of MARCKS mRNA expression was not Ó FEBS 2003 Hu-proteins control MARCKS mRNA stability (Eur J Biochem 270) 361 Fig Down-regulation of MARCKS mRNA in Swiss 3T3 fibroblasts but not in neural PCC7-Mz1 cells upon phorbol ester treatment (A) PCC7-Mz1 cells were seeded at a density of 1.75 · 104 cm)2 and the following day treated with PDB (200 nM, h) Swiss 3T3 cells (1 · 105 cells) were seeded on 90-mm cell culture dishes After week, when cultures reached confluence, cells were treated with 200 nM PDB for h For Northern blotting, total RNA was isolated and lg RNA separated per lane on a 1.2% (w/v) agarose/2.2% (v/v) formaldehyde gel After transfer on a nylon membrane by capillary blot the RNA was hybridized with a 32P-radiolabelled MARCKS cDNA probe (p809.1) [22], washed and exposed to X-ray film (Kodak AR) with intensifier screens at )80 °C for days (upper panel) PDB treatment caused down-regulation of MARCKS mRNA in murine fibroblasts (Swiss 3T3) but not in neural precursor cells (PCC7-Mz1) Ethidium bromide staining shows equal loading of the gel and integrity of the RNA (lower panel) The positions of 18S and 28S rRNA are indicated (B) Western blot analysis with the affinity-purified Hu-specific polyclonal antiserum was performed The following samples were loaded and separated in a 10% (w/v) SDS-polyacrylamide gel: 10 lg protein of untreated (–PDB) and PDB-treated (+PDB) (200 nM PDB, h) PCC7-Mz1 stem cells, 20 lg protein from extracts of untreated (–PDB) or PDB-treated (200 nM PDB, h) (+PDB) Swiss 3T3 cells, as indicated After gel electrophoresis, proteins were transferred onto PVDFmembrane Hu-proteins were detected with the Hu-specific antiserum and visualized by enhanced chemiluminescence Because of the high degree of sequence identity between members of the ELAV/Hu-family (about 70%) [78], the Hu-antiserum recognizes several members of this family In Swiss 3T3 cells, only one band of 36 kDa was recognized, corresponding to HuR In neural PCC7-Mz1 cells, additional Hu-proteins were detected demonstrating the expression of several members (e.g HuC, HuD and Hel-N1) of the ELAV/Hu gene family based on differences in the MARCKS mRNA sequences because cloning of the MARCKS cDNAs from both cell lines revealed complete identity (data not shown) Thus, diverse trans factors expressed in both cell lines seemed to be responsible for the different regulation of MARCKS mRNA stability (Fig 6A) Having shown that ELAV/Hu-proteins bind the CU-rich sequence of the MARCKS 3¢-UTR, we investigated whether there is a difference in expression of these proteins in neural PCC7-Mz1 cells and Swiss 3T3 fibroblasts Only the HuR protein of 36 kDa was detected in Swiss 3T3 extracts (20 lg) by Western blot analysis using the Hu-specific antiserum (Fig 6B) In PCC7-Mz1 cells (10 lg extract was loaded) levels of HuR were about fivefold higher than in Swiss 3T3 cells Furthermore, in extracts of neural PCC7-Mz1 cells additional bands of approximately 38–42 kDa were detectable, corresponding to neuronal members of ELAV/Hu-family (Hel-N1, HuC and HuD) [59] Staining of all bands could efficiently be prevented by preincubation of the Hu-antiserum with recombinant His6-HuD fusion protein showing the specificity of the detected bands (data not shown) Performing RNase/EMSA experiments with the CU-rich sequence and PCC7-Mz1 extracts (data not shown) revealed a third complex in addition to the two described complexes C1 and C2 (Figs 2B and 3A) Furthermore, in UV-crosslinking analysis with PCC7-Mz1 extracts (data not shown) a complex of about 39 kDa, not detected with Swiss 3T3 extracts (Fig 4E, lane 1), became evident, which was of the same size as the complex between His6-HuD and the 52 nt CU-rich RNA (Fig 4E, lanes 2–7) Based on these results, it was tempting to speculate that the high expression of members of the ELAV/Hu family is responsible for the MARCKS mRNA stability in PCC7Mz1 cells Role of ELAV/Hu in controlling stability of MARCKS mRNA We have demonstrated that ELAV/Hu proteins have a high affinity to the identified 52 nt CU-rich cis-element within the 3¢-UTR of the MARCKS mRNA Furthermore, we could show that PCC7-Mz1 cells express neuronal specific members of the ELAV/Hu family in addition to high levels of HuR (Fig 6B) To elucidate whether the neuronal HuD has a direct effect on controlling MARCKS mRNA stability, we cloned the cDNA coding for HuD into the eukaryotic expression vector pTRE The promoter activity is controlled by the tetracycline-controlled transactivator (tTA) This construct (pTRE-HuD) was transiently transfected into Swiss 3T3 cells together with the pTetoff plasmid coding for the tTA-protein Under these conditions the PDB initiated down-regulation of the MARCKS mRNA was completely blocked (Fig 7, lane 4) A similar stabilization of the MARCKS mRNA after PDB treatment was also observed when HuR was transiently overexpressed in Swiss 3T3 cells (lane 6) The level of the MARCKS mRNA was even elevated in nontreated HuD and HuR-transfected cells (Fig 7, lanes and 5) These data clearly demonstrate that the MARCKS mRNA can be stabilized by members of the ELAV/Hu gene family Its high expression in PCC7-Mz1 cells seems to 362 G Wein et al (Eur J Biochem 270) Fig Transient overexpression of HuD and HuR stabilizes the MARCKS mRNA Swiss 3T3 cells were plated (3.5 · 105 cells per 90-mm dish) and transfected the following day with the plasmid pTetoff together with the empty pTRE vector (lanes 1, 2) or together with pTRE-HuD (lanes 3, 4) or together with pTRE-HuR (lanes 5, 6) by lipofection After one day cultures remained untreated (–PDB) or were treated with 200 nM PDB for h (+PDB) Total RNA was isolated and lg loaded on a 1.2% (w/v) formaldehyde/agarose gel for Northern blot analysis as described in legend to Fig 6A The degradation of the MARCKS mRNA by activation of PKC by PDB treatment (lane 2) was completely blocked by overexpression of HuD (lane 4) and HuR (lane 6) (upper panel) Staining of the gel with ethidium bromide revealed equal loading of the gel and integrity of the RNA (lower panel) The positions of the 28S and 18S rRNAs are indicated on the left be responsible for constitutive stabilization of the MARCKS mRNA Discussion The regulation of mRNA decay is a major control point in gene expression In the present study we show that: (a) a region containing an element of 52 nucleotides [(CUUU)11U8] of the 3¢-UTR of the MARCKS mRNA confers RNA instability when merged with a reporter gene; (b) this CU-rich sequence of 52 nucleotides of the MARCKS 3¢-UTR is specifically bound by members of the ELAV family (HuD, HuR); (c) upon activation of PKC, stability of the MARCKS mRNA is drastically reduced in murine fibroblasts, but not in neural stem cells expressing high levels of Hu-proteins; and (d) overexpression of HuD and HuR in fibroblasts caused a drastic stabilization of the MARCKS mRNA even when PKC was activated The identified CU-rich sequence of the 3¢-UTR of the MARCKS mRNA belongs to the AU-rich elements (ARE) previously found in the 3¢-UTRs of some short-living messengers, such as those for cytokines and lymphokines AREs can be divided into three classes based on their structural and functional properties Class I and II AREs are characterized by the presence of the pentanucleotide AUUUA, which is absent in class III AREs One to three copies of the pentanucleotide AUUUA are either distributed over the entire 3¢-UTR, e.g c-fos (class I) or three or more AREs are located in tandem, e.g TNFa (class II) [9,10] The mRNAs with class III AREs (e.g c-jun) not contain the Ó FEBS 2003 AUUUA pentanucleotide but only U-rich segments [60] Because the CU-rich element of the MARCKS 3¢-UTR causes RNA instability but does not contain the characteristic AUUUA element, we identified herewith a novel ARE motif belonging to the AREs of class III So far, 15 different proteins have been shown by UV-crosslinking and gel-shift assays to recognize AU- and U-rich RNA sequences: AUBF, AU-A (HuR) [61], AU-B, AU-C, Hel-N1, hnRNP D (AUF1), hnRNP A1, hnRNP C, AUH, GAPDH, hnRNP A0, HuD, tristetraprolin and TIAR However, only three of these proteins, hnRNP D and HuR and tristetraprolin seem to influence stability of ARE-containing mRNAs in vivo (reviewed in [51,62]) It is likely that HuR is involved in controlling the stability of the MARCKS mRNA Our data show that HuR binds with high affinity to the MARCKS ARE Furthermore, both elevated levels of endogenous Hu proteins as in neural PCC7-Mz1 cells and overexpression of HuD/HuR in fibroblasts dramatically stabilize the MARCKS mRNA Thus, the MARCKS mRNA is an additional example that Hu-protein overexpression stabilizes ARE-containing mRNAs [16,17,52,53,63] Presently we can only speculate on the mechanism by which ELAV/Hu proteins control the MARCKS mRNA stability A working model hypothesizes that the CU-rich element confers instability by recruiting RNases to the MARCKS mRNA, and that binding of Hu-proteins prevents this effect Therefore, one might envisage that the affinity of HuR to the MARCKS mRNA in Swiss 3T3 cells is controlled by protein kinases, as was recently found for several AREs [56,57] Our preliminary data show no difference in the capability of the 52 nt CU-rich element to form the complexes C1 and C2 with extracts of untreated and PDB-treated fibroblasts This might be due to the fact that in vitro binding of the 52 nt CU-element by ELAV proteins may not reflect the physiological situation, i.e treatment with PDB This interaction might be highly regulated intracellularly for this ARE in a cell type- and physiological state-dependent manner as has recently been shown for several AREs [80] Thus, four mammalian ligands, three of them known to be protein phosphatase 2A inhibitors, were recently identified, which modulate HuR’s ability to bind its target mRNAs in vivo [64] Other cis elements of the MARCKS mRNA, outside of the 3¢-UTR, and their corresponding trans factors may additionally contribute to control binding of ELAV proteins and consequently mRNA stability Recent studies show that HuR binds ARE-containing mRNAs in the nucleus and transports them as RNPs to the cytosol [16,22,65,75] It is likely that HuR together with other factors is involved in nucleo-cytoplasmic shuttling of the MARCKS mRNA An alternative explanation for the described stabilization of the MARCKS mRNA observed in 3T3 cells overexpressing HuR and HuD (Fig 7) is that these proteins are active in decay, but when overexpressed they sequester other factors needed for RNA degradation Antisense RNA- or RNAi-experiments to ablate HuR expression will help to elucidate this question Perturbations in the 3¢-UTR-mediated regulation was shown to cause loss of control over one or more genes Several disorders, such as carcinoma, inflammation, mytotonic dystrophy, a-thalassemia (reviewed in [66]) and Morbus Alzheimer [3,67] are caused by mutations in the Ó FEBS 2003 Hu-proteins control MARCKS mRNA stability (Eur J Biochem 270) 363 3¢-UTR sequences or in the 3¢-UTR-binding regulatory proteins Neoplastic transformation has been shown to stabilize ARE-containing mRNAs [68] and has been associated with the activation of c-Jun N-terminal kinase (JNK) [69] Similarly, activation of MAP kinase-activated protein kinase has been associated with the stabilization of ARE-containing mRNA in HeLa cells [70] Because MARCKS emerges as a growth and tumor suppressor gene [23,25], which is down-regulated in many transformed cell types, it will be of interest to investigate the reasons for its low expression in malignant cells It is possible that mutations in its 3¢-UTR prevent binding of factors like HuD/HuR which normally inhibit degradation of the transcript Alternatively, the MARCKS gene might be transcriptionally down-regulated as recently shown for immortalized rat hippocampal cells [71] Elucidating the exact mechanism by which ELAV proteins confer stability to the MARCKS mRNA will help to identify other genes following the same mechanism of regulation In search for such potential tumor suppressor genes, the sequence of the identified 52 nt cis-element will be useful However, RNA-binding proteins recognize both the primary sequence and the secondary structure [48,72–74] In this context it is interesting to mention that the computer programs MFOLD and MPLOT (German Cancer Research Institute Heidelberg, Germany) [76,77] predicted an extremely stable secondary structure for the MARCKS 3Â-UTR (DE ẳ )412.2 kJ at 22 °C) and that the 52 nt CU-sequence forms two helical and one stem-loop structure (data not shown) More than 70% of all bases of the 52 nt CU-rich element form double helical structures This robust conformation is maintained at 22 °C as well as 37 °C and may be necessary for the function of the CU-element, e.g for protein binding Therefore, the secondary structure of the MARCKS 3¢-UTR has to be taken into account when searching for genes regulated in the same fashion It is now becoming apparent that the combination of functionally and structurally distinct sequence motifs, 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