Translational control of the interferon regulatory factor 2 mRNA by IRES element

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Translational control of the interferon regulatory factor 2 mRNA by IRES element
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  Nucleic Acids Research, 2007,  1–13 doi:10.1093/nar/gkm524 Translational control of the interferon regulatory factor 2 mRNA by IRES element Debojyoti Dhar, Swagata Roy and Saumitra Das* Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore-560012, India Received May 17, 2007; Revised and Accepted June 22, 2007  ABSTRACTTranslational control represents an important modeof regulation of gene expression under stressconditions. We have studied the translation of inter-feron regulatory factor 2 (IRF2) mRNA, a negativeregulator of transcription of interferon-stimulatedgenes and demonstrated the presence of internalribosome entry site (IRES) element in the 5 ’ UTRof IRF2 RNA. Various control experiments ruledout the contribution of leaky scanning, crypticpromoter activity or RNA splicing in the internalinitiation of IRF2 RNA. It seems IRF2-IRESfunction is not sensitive to eIF4G cleavage, sinceits activity was only marginally affected in presenceof Coxsackievirus 2A protease. Interferon  a  treat-ment did not affect the IRF2-IRES activity or theprotein level significantly. Also, in cells treatedwith tunicamycin [an agent causing endoplasmicreticulum (ER) stress], the IRF2-IRES activity andthe protein levels were unaffected, although thecap-dependent translation was severely impaired. Analysis of the cellular protein binding with theIRF2-IRES suggests certain cellular factors, whichmight influence its function under stress conditions.Interestingly, partial knockdown of PTB proteinsignificantly inhibited the IRF2-IRES function.Taken together, it appears that IRF2 gene expres-sion during stress condition is controlled by theIRES element, which in turn influences the cellularresponse.INTRODUCTION Interferon regulatory factors (IRFs) are DNA-bindingproteins that control interferon (IFN) gene expression.IRF1 has been shown to function as an activator of IFNand IFN-inducible genes, whereas IRF2 represses theaction of IRF1 (1). Since, interferon induction is followedby translational attenuation; it is plausible that thesynthesis of IRF2 protein, which is required to repressand regulate the IFN stimulated genes, is allowed tocontinue under such condition using an alternate mecha-nism of translation (2). In fact, the repressor of IFN- b promoter, NRF (NF- k B repressing factor) protein hasbeen shown to be translationally regulated to providesufficient level of NRF protein for the complete silencingof the IFN- b  genes (3).Initiation of translation is the rate-limiting step of protein synthesis and hence it is tightly regulated.Although the general mode of translation of cellularmRNAs involves cap-dependent translation initiation,a sizeable proportion of mRNAs was shown to beassociated with polyribosomes in poliovirus-infected cellsat a time when cap-dependent initiation is impaired (4).The most widespread mechanism of cap-independentmode of translation initiation is mediated by internalribosome entry sites (IRESs), which directly recruitsribosome bypassing the requirement for 5 0 cap structureand the cap-binding protein eIF4E (5,6). Many mRNAsthat contain IRESs encode proteins that play importantroles in cell growth, proliferation, differentiation andregulation of apoptosis (7–10). Stress conditions, such asstarvation of growth factors, heat shock, hypoxia andendoplasmic reticulum (ER) stress leads to down regula-tion of protein synthesis through phosphorylation of eIF2 a  (11). However, a number of cellular mRNAscontaining IRES elements such as vascular endothelialgrowth factor (VEGF) (12),  c-Myc  (13), cat-1 mRNA (14),NRF (3) and PITSLRE kinase (15) continue to betranslated under conditions when cap-dependent transla-tion is severely impaired. Similarly, inhibition of proteinsynthesis during apoptosis is accompanied by a caspase-dependent cleavage of initiation factor eIF4G (16).However, there is strong evidence that translation of death associated protein (DAP5) (17), X-chromosomelinked inhibitor of apoptosis protein (XIAP) (18),apoptotic protease activating factor (Apaf1) mRNAs ismaintained under these conditions and is driven by theirrespective IRES elements. This indicates that thesemRNAs containing IRES may probably have a reducedrequirement for the intact eIF4G, allowing the translation *To whom correspondence should be addressed. Tel: +91 80 293 2886; Fax: +91 80 360 2697; Email: sdas@mcbl.iisc.ernet.in   2007 The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.   Nucleic Acids Research Advance Access published August 13, 2007   b  y g u e  s  t   onF  e  b r  u a r  y2  5  ,2  0 1 4 h  t   t   p :  /   /  n a r  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   of mRNAs containing them to continue under stressconditions (19). This mode of initiation of translationprobably protects cells from hostile conditions or at leasthelp them to tide over transient stress conditions.Here, we have investigated the presence of IRESelement in the 5 0 untranslated region (UTR) of ‘interferonregulatory factor 2 0 or IRF2, which belongs to interferonregulatory factor family (1). Our results suggest that IRF25 0 UTR (177nt) contains an IRES element, which under-goes translation initiation in an eIF4G-independentmanner. Also, it seems that IFN- a  treatment does notinhibit the IRF2-IRES function to the extent observed incase of HCV or BiP IRES activity (GRP78). Analysis of the cellular protein binding with the IRF2-IRES showedspecific binding of certain cellular factors, which mightinfluence its function under stress conditions. In fact PTBprotein has been shown to specifically interact with theIRF2 5 0 UTR and partial knock down of PTB proteinresulted in significant decrease in IRF2-IRES activity.Additionally, we have studied the effect of ER stress onIRF2-IRES function. In cells treated with tunicamycin,the IRF2 protein level as well as the IRES function wasfound to be largely unaltered. These results suggest thatthe IRES element of the IRF2 mRNA allow translationinitiation under stress condition and may play a role in thecellular response. MATERIALS AND METHODS Plasmid constructs The cDNA corresponding to the 5 0 UTR of IRF2,was amplified from the RNA isolated from HeLa cellsand cloned in pCDNA 3.1 (+). The primers were usedaccording to the GenBank sequence NM_002199 andconfirmed by DNA sequencing (Gene Bank Acc. No. forIRF2 5 0 UTR, DQ409328). The construct pR  ENullF wasa kind gift from Dr Peter Sarnow (Stanford University).All the bicistronic constructs contain respective 5 0 UTRsequences (pRIRF2F, pRHAVF and pRBipF) clonedbetween Renilla luciferase (RLuc) and firefly luciferase(FLuc) genes, in pCDNA 3.1 in between HindIII andEcoRI sites. The eukaryotic promoter less bicistronicconstruct, the pGEMT-R-IRF-F, containing the IRF25 0 UTR and also the pR  EnullF bicistronic cassettewere cloned in pGEMT easy vector (Promega) under T7promoter. The T7pRCVB3F was cloned in the pBluescriptvector (Stratagene) under T7 promoter. The landscape of structure derived from inactive   EMCV IRES sequencewas cloned upstream of Rluc gene in the upstream hairpin(uphp) bicistronic plasmid (4). The Nsp bicistronicconstruct (pRNspF) contains 264nt from La ORF(120–204 amino acid encoding region) between Rluc andFluc (20). For constructing IRF2 monocistronic plasmid(pIRF2Fluc), IRF2-Fluc was digested with HindIII andApaI enzymes (NEB) from the plasmid pRIRF2F andligated in HindIII, ApaI digested pCDNA 3.1-Fluc.Coxsackievirus 2A protease gene was amplified fromCVB3 cDNA (a generous gift from Nora Chapman,Nebraska) using the primers with BamH1 and EcoR1 sitesrespectively and cloned in pCDN3.1 His C (pCD2A pro ).The primers used are as follows: Cox(F):5 0 ATTAggATCCggCgCATTTggACAA3 0 ; Cox(R):5 0 ACgCgAATTCCTgTTCCATTgCATC 3 0 . Bicistronic plasmids pRHAVF andpRBipF were constructed as described earlier (21,22).The primers used for the amplification of IRF2 5 0 UTRare as follows: IRF2(F)-5 0 CggCAAgCTTTCTCCTTgTTTTgCT3 0 ;IRF2(R)- 5 0 ATATgAATTCggTgCCCTCTCAgTg3 0 . Cell linesand transfection Hela S3, Huh7 cells were maintained in DMEM(Invitrogen) with 10% fetal bovine serum (GIBCO,Invitrogen). Cells were transfected with various bicistronicplasmids and pSV40ß-gal using Tfx 20 reagent (Promega)and luciferase assay was performed using Dual luciferaseassay reagent (Promega). In experiments using eukaryoticpromoter-less bicistronic constructs, cells were infectedwith vaccinia virus expressing T7 RNA polymerase,VTF7.3 (generous gift from Dr B. Moss, NIH) (23)prior to transfection with bicistronic plasmids. Luciferaseassay was performed by dual luciferase assay reporterreagent (Promega) in a TD 20/20 luminometer (TurnerDesign, CA, USA). For the interferon experiment, Huh 7cells was transfected with the bicistronic plasmidspRIRF2F, pRHCVF, pRBiPF followed by treatment of 1000IU/ml of IFN–alpha 2b (Virchow Ltd). For the 2Aprotease experiment, co-transfection was performed usingpRCVB3F, pRIRF2F and pRHAVF bicistronic plasmidswith Coxsackie 2A pro plasmid (pCD2Apro) constructs.Luciferase assay was performed after 24h of transfection.For tunicamycin treatment, cells were incubated inpresence of 2.5 m g/ml of tunicamycin (Calbiochem) for14h. Co-transfection of siRNA with bicistronic plasmidwas performed in HeLa S3 cells growing in monolayerusing lipofectamine-2000 transfection reagent andoptiMEM-I prepared without addition of antibiotic(Invitrogen). Cells were seeded onto 35mm dishes oneday prior to transfection in similar manner. For eachtransfection, 100nM of pre-characterized siPTB(Dharmacon) and 1 m g of bicistronic DNA were dilutedwith optiMEM-I to a final volume of 100 m l. In a separatetube, 6 m l of lipofectamine-2000 was diluted with 94 m l of optiMEM-I to a final volume of 100 m l followed byincubation at room temperature for 5min. The contentsof the two tubes were mixed and incubated at roomtemperature for 20min. Subsequently, 800 m l of optiMEM-I was added to the transfection mixture,which was then layered onto cells. Six hours later,the medium was replaced with 2ml of DMEM(with antibiotic) and 10% FBS. Thirty-six hours post– transfection, the cells were washed, lysed with passive lysisbuffer and luciferase enzymes assayed in a similar way.For RNA transfections, capped bicistronic RNAswere synthesized  in vitro  from different constructs ( RIRF2F, RBipF, R  EnullF) using T7RNA polymerase(Ribomax kit, Promega). Ten microgram of the abovesynthesized RNAs were used to transfect HeLa cells usingLipofectamine 2000 and optiMEM-I (Invitrogen) asdescribed above. After 6h, medium was replaced with2ml of DMEM (with antibiotic) and luciferase assay was 2  Nucleic Acids Research, 2007    b  y g u e  s  t   onF  e  b r  u a r  y2  5  ,2  0 1 4 h  t   t   p :  /   /  n a r  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   performed by dual luciferase assay reporter reagent(Promega) after 8h incubation. Enzyme activity wasmeasured in a TD 20/20 luminometer (Turner Design,CA, USA). The transfection efficiency was normalized andthe relative luciferase activities were plotted. In vitro  transcription To make antisense FLuc probe RNA, pCD Luc DNA waslinearized with HindIII (NEB) and transcribed by SP6RNA polymerase (Promega) and 10 m Ci/ m l of alpha  32 PUTP (NEN) as per manufacturer’s guidelines. The HCV5 0 UTR RNA probe was made from HCV-GFP DNA (22)linearized with EcoRI and was transcribed by T7 RNApolymerase. Similarly, the  32 P-labeled RNA probescorresponding to the 5 0 UTRs of IRF2 and HAV weremade from their respective plasmid DNAs after linearizingwith either NcoI or EcoRI and transcribed with either Sp6or T7 RNA polymerase, respectively. The non-specificRNA was made from linearized pGEMT as describedelsewhere (22). pRIRF2F, pRBipF, pR  EnullF bicis-tronic plasmids were linearized with Pme1 (NEB) and thecorresponding bicistronic RNAs were synthesized usingRibomax kit (Promega) following manufacturer’sprotocol. Northern blotting and RT-PCR Total RNA from the HeLa cells, transfected withpRIRF2F, pRCVB3F bicistronic plasmids were extractedusing TRIZOL (Sigma), followed by DNase I treatment.Firefly luciferase RNA (Promega) and above extractedRNAs were resolved on a 0.8% agarose–formaldehydegel, blotted on positively charged Nylon membrane(Millipore) and hybridized with a  32 P-labeled riboprobecorresponding to the FLuc gene. Total RNA from HeLacells transfected with pRIRF2F bicistronic plasmid wasextracted using TRIZOL (Sigma). Reverse transcriptionwas performed using AMV RT (Promega) followed byPCR with taq polymerase (Invitrogen). Western blot hybridization Huh7 and HeLa cells were harvested and the cell pelletwas resuspended in 1   RIPA buffer (10mM sodiumphosphate, pH 7.5, 150mM NaCl, 2mM EDTA,1% sodium deoxycholate, 1% NP-40, 0.1% SDS,0.1%  b ME, 1mM PMSF, 50mM sodium fluoride).Extracts were suspended with 5   SDS gel loading buffer(100mM Tris-Cl, 200mM DTT, 4% SDS, 0.2% bromo-phenol blue, 20% glycerol) and resolved on SDS–10%polyacrylamide gel, followed by electrotransfer of proteinsto nitrocellulose membranes. The expression of IRF2 andtubulin was analyzed using antibody specific to IRF2(a generous gift from Dr Angela Battistini of IstitutoSuperiore di Sanita, Rome, Italy) and anti rabbitsecondary antibody (SIGMA). For detecting tubulin,anti-tubulin antibody (SantaCruz Biotech) was usedfollowed by anti-mouse secondary antibody (SIGMA).TFIID was detected using anti-TFIID antibody (SantaCruz Biotech) and eIF4G was detected using anti-eIF4G antibody against N-terminal region (Santa CruzBiotech). The signal was detected by using enhancedchemiluminescence (ECL) detection kit (Amersham-Pharmacia). Similarly for detecting endogenous PTB,anti-PTB antibody was used (Calbiochem). UV cross-linking S10 extract was prepared from HeLa and Huh7 cells asdescribed before (21). [ a - 32 P] 5 0 UTR RNAs were allowedto form complex with S10 extracts as described earlier,followed by cross-linking with UV light. The unboundRNAs were digested with RNaseA treatment. Theprotein–RNA complexes were then resolved in a SDS– 10% polyacrylamide gel followed by phosphorimaginganalysis. Purification of recombinant PTB The expression of recombinant PTB from PET28a-PTB(a generous gift from Dr J.G. Patton) was inducedby 0.6mM IPTG in  Escherichia coli   (BL21 DE3) cellstransformed with the expression vector. His-taggedprotein was purified using Ni 2+ -nitrilotriacetic acidagarose (Qiagen) under non-denaturing conditions andeluted with 250mM imidazole. 35 S Protein labeling and immunoprecipitation HeLa cells were treated with either interferon  a  ortunicamycin for the different time periods as mentionedin the text followed by starvation of the cells for 45min inMEM-medium lacking methionine (SIGMA). Cells werewashed and incubated with 100 m Ci of   35 S-methionine(trans-label, BARC) for 45min at 37 8 C. Cells wereharvested and the pellet was resuspended in 2   IP buffer(2% triton X-100 and 0.1% NP40 in TBS) and kept in icefor 1h. The supernatant was collected and proteinestimation was performed using Bradford’s reagent(BIORAD). Equal amount of protein was resolved ina SDS–10%PAGE and analyzed by autoradiography. ForIRF2 immunoprecipitation, 150 m g of untreated/treatedcell extracts were incubated with IRF2 antibody (SantaCruz Biotech) overnight at 4 8 C. The immunocomplexwas separated by protein A-sepharose beads (SIGMA)for 2h at 4 8 C on a rocker. The beads were washed threetimes with 1   IP buffer and the bound proteins wereanalyzed by SDS–10% PAGE followed by detection byautoradiogram. RESULTS 5 ’ UTR of IRF2 iscapable of mediating internal initiationoftranslation To determine whether IRF2 5 0 UTR can mediate cap-independent internal initiation of translation to providebasal level of protein under stress; we have investigatedthe presence of an IRES element in the 5 0 UTR of IRF2RNA. For this purpose, IRF2 5 0 UTR was amplified byRT-PCR from total RNA isolated from HeLa cells. Thenucleotide sequence of the IRF2 5 0 UTR has been shownin Figure 1A. Zuker’s MFOLD algorithm predicteda stable secondary structure with a minimal free energyof    41.7kcal/mol (Figure 1B) (24). Similar secondary Nucleic Acids Research, 2007   3   b  y g u e  s  t   onF  e  b r  u a r  y2  5  ,2  0 1 4 h  t   t   p :  /   /  n a r  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   structure was predicted by MFOLD for the BiP IRES(data not shown). It would be interesting to investigatewhether IRF2 5 0 UTR contains the ‘Y’-type stem-loopstructure that has been suggested as the characteristicfeature of certain cellular IRESs (25).To investigate the presence of IRES element in the5 0 UTR of IRF2, the amplified IRF2 5 0 UTR wassubsequently cloned in a bicistronic construct in betweentwo reporter genes. The upstream reporter (Renillaluciferase) in this bicistronic RNA is translated by cap-dependent mode, whereas the downstream reporter(Firefly luciferase) will be translated if the intergenicregion contained a functional IRES element. The bicis-tronic plasmid, pRIRF2F was transiently transfected intoHeLa cells followed by luciferase assay. The resultsshowed appreciable amount of firefly luciferase (Fluc)translation mediated by the IRF2 5 0 UTR. The Flucactivity was found to be around 12.5-fold highercompared to the negative control bicistronic plasmid,pR  EnullF. Interestingly, the bicistronic plasmidpRBipF, containing Bip IRES as positive control (26),showed   17-fold increase in the Fluc activity comparedto null bicistronic plasmid control (Figure 1D and E).However, cap-dependent translation of the renilla plt22jpg by 0.stewart and M. Zukerc 2005 Washington University B  CA IRF2-5  ′ UTR  UCUCCUUGUUUUGCUUUCG  A  UCUGG  A  CUGUUCUC  A  GGC  AA  GCCGGGG  A  GU  AA  CUC pR  ∆ ENullF  pR ∆ ENullFpR ∆ ENullF pRIRF2F  pRIRF2FpRIRF2F pRIRF2F pRIRF2F pR  D EIRF2F pR  D EIRF2F    IRF2 IRF2 IRF2 5  ′ UTR 5  ′ UTR 5  ′ UTR p  D ERIRF2F p  D ERIRF2F     P  e  r  c  e  n   t   l  u  x   i   f  e  r  a  s  e  a  c   t   i  v   i   t  y   P  e  r  c  e  n   t   l  u  x   i   f  e  r  a  s  e  a  c   t   i  v   i   t  y R Luc R Luc R Luc  F GED F Luc F Luc F Luc  800250200150100500110064126001200012000 R Luc  RLucFLucConstruct F Luc pRBipF  pRBipFpRBipF2000180016001400120010008006004002000 UUU  A  GUUUUGCUCCUGCG  A  UU  A  UUC  AA  CUG  A  CGGGCUUUC  A  UUUCC  A  UUUC  A  C  A  C  A  CCCU  A  GC  AA  C  A  CUU  A  U  A  CCUUGCGG  AA  UUGU  A  UUGGU  A  GCGUG  AAAAA  GC  A  C  A  CUG  A  G  A  GGGC  A  CC   IRF2 Bip null  Figure 1.  IRF2 5 0 UTR sequence mediate internal initiation of translation. ( A ) Nucleotide sequence of interferon regulatory factor 2 (IRF2) 5 0 UTRRNA. ( B ) MFOLD predicted secondary structure of IRF2 5 0 UTR. ( C ) Schematic representation of the bicistronic plasmids used in transienttransfections is indicated. ( D ) Bicistronic plasmids (1 m g) of pR  EnullF or pRIRF2F or pRBipF were transiently transfected into HeLa cells.Twenty-four hours post-transfection, respective luciferase activities corresponding to Fluc (white bar) and RLuc (gray bar) were measured and shownseparately as fold increase compared with that from control (pR  EnullF) taken as 100%. Transfection efficiencies were normalized by co-transfecting with a  b -galactosidase plasmid. The data mean  SD from three independent experiments. ( E ) Average absolute values of RLuc andFLuc activities (in relative light units) of the above transient transfection experiments conducted in trplicate are presented in the table. ( F ) Schematicrepresentation of hairpin containing bicistronic plasmids used in transient transfections is represented. ( G ) Bicistronic plasmids (1 m g) containing deltaEMCV sequence upstream or downstream of RLuc as indicated, were transfected into HeLa cells. The FLuc (white bar) and RLuc (gray bar)activities from the delta EMCV containing plasmids are shown as fold increase or decrease with respect to the corresponding controls, taken as 100.The data mean  SD from three independent experiments. 4  Nucleic Acids Research, 2007    b  y g u e  s  t   onF  e  b r  u a r  y2  5  ,2  0 1 4 h  t   t   p :  /   /  n a r  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   luciferase (RLuc) was found to be similar in all the threeplasmids as expected. These results indicate thatthe 5 0 UTR of IRF2 might contain an IRES element, theactivity of which is comparable to that of representativecellular IRES. The cap-independent translation ofIRF2 isnotdueto scanningor ribosomal read-through In order to rule out scanning or ribosomal read-throughas the possible reason for the IRF2 5 0 UTR-mediatedtranslation of the firefly luciferase, we have used IRF2bicistronic construct p  ERIRF2F, containing region of highly stable secondary structure upstream of renillaluciferase to prevent ribosome loading (Figure 1F) (4).When the p  ERIRF2F plasmid was transfected intoHeLa cells, RLuc activity was found to be significantlyinhibited. However, no significant change in Fluc transla-tion was observed compared to values obtained with thecontrol IRF2 bicistronic construct pRIRF2F (Figure 1G).Similarly, when the plasmid pR  EIRF2F containing theinternal hairpin structure inserted downstream of Rluc(Figure 1G) was trasnsfected into HeLa cells, FLucactivity was not inhibited. Interestingly, FLuc activitywas found to be marginally higher compared to control,probably due to a change in IRF2-IRES RNA structure inthe context of the internal hairpin. However, Rluc activityremained unchanged as expected (Figure 1G). The resultssuggest that the translation of the downstream cistronFluc was not due to ribosomal read-through of the firstcistron. IRF2 5 0 UTR does nothave crypticpromoter or splice sites It is possible that in the cells transfected with IRF2bicistronic plasmid, small amount of monocistronic FLucRNA is generated from the bicistronic construct due tocryptic promoter activity of the IRF2 5 0 UTR sequence.In order to rule out this possibility, HeLa cells weretransfected with IRF2 bicistronic construct that wascloned in a vector, which lacked conventional eukaryoticpromoter but contained a T7 phage promoter (Figure 2A).Similarly, null and coxsackievirus B3 (CVB3) bicistronicplasmids were used respectively as negative and positivecontrols in the experiment. Results showed luciferaseactivity almost equal to background level in absence of any eukaryotic promoter in the transfected constructs.But when cells were transfected with recombinant vacciniavirus expressing T7 RNA polymerase gene (VTF7.3) priorto transfection of the construct, significant levels of Fluc and Rluc activity were detected from the sameIRF2 and CVB3 bicistronic constructs (Figure 2A). In factFluc activity mediated by IRF2-IRES was found to bearound 15 times that of the null construct. As expected,the viral IRES (CVB3) showed much higher efficiencycompared to cellular IRES activity under this condition.The result rules out the possibility of cryptic promoteractivity in the IRF2 5 0 UTR.To rule out the possibility, that IRF2 5 0 UTR mightcontain splice sites which generate monocistronic FlucRNA  in vivo , northern blot hybridization assay wasperformed. For this purpose, total RNA was extractedfrom the cells transfected with IRF2 bicistronic plasmidand probed with a  32 P-labeled riboprobe complimentaryto Fluc gene. As a positive control, RNA extracted fromcells transfected with a similar bicistronic plasmid contain-ing Coxsackievirus B3 (CVB3) IRES was included inthe assay. In our northern blot assay, we failed to detectany smaller RNA products derived from either of thetransfected bicistronic plasmids (Figure 2B).We have also performed RT-PCR analysis fromthe total RNA extracted from cells transfected with theIRF2 bicistronic plasmid using different sets of primers asshown in Figure 2C, which showed the presence of intactbicistronic RNA  in vivo . Additionally, to investigate thepresence of shorter monocistronic RNA (if any), we haveperformed RT-PCR analysis of total RNA as above withdifferent dilutions of input RNA and primer sets asindicated above the panel in Figure 2D. In this experimentprimer set P5/P4 would amplify the region of Fluc geneonly, whereas the primer set P3/P4 would amplifythe full-length IRF2 5 0 UTR along with the Fluc gene.In the event of cryptic splice sites within IRF2 5 0 UTR inthe bicistronic construct, the ratio of the amplifiedproducts generated by using P5/P4 primers would besignificantly more than that of P3/P4 product. However,in our assay we did not find significant differences in theamplified products (Figure 2D).To further validate the IRF2-IRES activity, we haveperformed RNA transfection experiment. Capped bicis-tronic RNAs, synthesized  in vitro  from different plasmidconstructs (R  EnullF RIRF2F, RBipF, RCVB3F) weretransiently transfected into HeLa cells and the relativeluciferase activities were measured 8h post-transfection.The Fluc activity mediated by IRF2-IRES was foundto be 6-fold higher than that obtained for the negativecontrol pR  EnullF (Figure 2E). Similar fold increase inFluc activity was observed for Bip IRES, used as positivecontrol for cellular IRES (Figure 2E). Interestingly,the Fluc activity mediated by the viral IRES (CVB3)was found to be much higher than the cellular IRESs.However, the results are consistent with recent reports,which suggest that cellular IRESs are not as active as viralIRESs when RNA transfections are performed, possiblybecause cellular IRESs require ‘nuclear history’ for theiroptimum activity (27,28). Cellular protein bindingwith theIRF2 5 0 UTR To further characterize the IRES activity of IRF2 RNA,we have studied the requirement of canonical andnon-canonical initiation factors. Picornavirus 2A proteasehas been shown to cleave eIF4G and shut down cap-dependent translation of cellular mRNAs (29–31).Although viral IRES elements are not sensitive to eIF4Gcleavage, hepatitis A virus IRES has been shown torequire intact eIF4G for efficient translation (32).To investigate whether the IRF2-IRES needs intacteIF4G for its efficient function, we have used theCoxsackievirus B3-2A protease encoding plasmid CVB3-2A pro . This plasmid was co-transfected with IRF2bicistronic plasmid  pRIRF2F   in HeLa cells. In addition,bicistronic plasmids containing the IRES element of either Nucleic Acids Research, 2007   5   b  y g u e  s  t   onF  e  b r  u a r  y2  5  ,2  0 1 4 h  t   t   p :  /   /  n a r  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om 
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