RNA-binding protein HuR and the members of the miR-200 family play an unconventional role in the regulation of c-Jun mRNA.pdf

RNA-binding protein HuR and the members of the miR-200family play an unconventional role in the regulationof c-Jun mRNAGIORGIA DEL VECCHIO,1,4FRANCESCA DE VITO,1,4SITA J.SAUNDERS,2ADELE RISI,1CECILIA MANNIRONI,3IRENE BOZZONI,1and CARLO PRESUTTI11Dipartimento di Biologia e Biotecnologie,Universit“Sapienza,”00185 Rome,Italy2Bioinformatics Group,Department of Computer Science,Albert-Ludwigs-University Freiburg,79110 Freiburg,Germany3IBPMCNR,00185 Rome,ItalyABSTRACTPost-transcriptional generegulation is afundamental step forcoordinating cellular response inavarietyof processes.RNA-bindingproteins(RBPs)and microRNAs(miRNAs)are the most important factors responsible for this regulation.Here we report thatdifferent components of the miR-200 family are involved in c-Jun mRNA regulation with the opposite effect.While miR-200binhibits c-Jun protein production,miR-200a tends to increase the JUN amount through a stabilization of its mRNA.This actionis dependent on the presence of the RBP HuR that binds the 3UTR of c-Jun mRNA in a region including the mir-200a bindingsite.The position of the binding site is fundamental;by mutating this site,we demonstrate that the effect is not micro-RNAspecific.These results indicate that miR-200a triggers a microRNA-mediated stabilization of c-Jun mRNA,promoting thebinding of HuR with c-Jun mRNA.This is the first example of a positive regulation exerted by a microRNA on an importantoncogene in proliferating cells.Keywords:miRNA;HuR;mRNA stability;c-JunINTRODUCTIONPost-transcriptional gene regulation is crucial to maintain afine and coordinated production of proteins necessary tothecellsinavarietyofprocesseslikedevelopment,homeosta-sis,and disease.To achievethis result,post-transcriptional gene expressionis controlled at multiple levels:pre-mRNA splicing and mat-uration,mRNA stability in the nucleus,mRNA transport,editing,mRNA stability in the cytoplasm,storage,and trans-lation(Bousquet-Antonelli et al.2000;Mitchell and Toller-vey 2000;Orphanides and Reinberg 2000).mRNA turnoverandtranslationareverywell-suitedstepsofcontroltoaccom-plish quick changes in the pattern of expressed proteins fol-lowing environmental perturbations.The most importantcellular factors that modulate RNA stability and translationare noncoding RNAs,in particular microRNAs(miRNAs),and turnover and translation regulatory RNA-binding pro-teins(TTR-RBPs).These factors associate with specific ciselements in mRNAs to perform their activity.miRNAs areshort noncoding RNAs that strongly influence gene expres-sion;according to the most recent data,2588 mature humanmiRNAs have been identified and sequenced(Kozomara andGriffiths-Jones 2014).They are produced from long primarytranscripts synthesized by RNA Pol II(pri-miRNA)andprocessed in the nucleus by“microprocessor,”a complexof factors containing Drosha and DiGeorge Critical Region8(DGCR8)ribonucleases to generate precursor miRNA(pre-miRNA).After its translocation to the cytoplasm by Expor-tin5,the pre-miRNA is cleaved by another ribonuclease,Di-cer,toformaduplexRNAof22nucleotides(nt).Onestrandis then loaded into miRNA-loaded RNA-induced silencingcomplex(miRISC),which comprises,among others,Argo-naute proteins(for review,see Ha and Kim 2014).The acti-vated RISC can target specific mRNAs containing,generallyat the 3untranslated region,sequences complementary tomiRNA.mRNA and miRNA form a partial hybrid character-izedbythepresenceofa“seed”region(nucleotides27ofthemiRNA)perfectly paired between the two.The interaction ofmiRISC with an mRNA usually inhibits its translation andthis effect is often accompanied by a decrease in the stability4These authors contributed equally to this work.Corresponding author:carlo.presuttiuniroma1.itArticle published online ahead of print.Article and publication date are athttp:/www.rnajournal.org/cgi/doi/10.1261/rna.057588.116.2016 Del Vecchio et al.This article is distributed exclusively by the RNASociety for the first 12 monthsafter the full-issuepublication date(see http:/rnajournal.cshlp.org/site/misc/terms.xhtml).After 12 months,it is availableundera CreativeCommonsLicense(Attribution-NonCommercial4.0Inter-national),as described at http:/creativecommons.org/licenses/by-nc/4.0/.1510RNA 22:15101521;Published by Cold Spring Harbor Laboratory Press for the RNA Societyof the mRNA.However,it has been reported that under spe-cific conditions,the activated RISC can also promote transla-tion(Vasudevan et al.2007).There are hundreds of RBPs in the human genome;therole of most of them is still poorly understood.However,itis now clear that RBPs,together with miRNAs and probablyother noncoding RNAs(ncRNAs),target mRNAs in an or-chestrated way to regulate their localization,stability,and fi-nally the amount of protein synthesized.The combination ofall these effects on mRNAs is known as“post-transcriptionalregulatory code”(Keene 2007).Indeed,functionally relatedgroups of mRNAs are tagged in their coding and noncodingregions early in their lives;in this way,their subsequent des-tinies are organized and coordinated at the various steps ofprocessing and expression.This complex network of interac-tion is beginning to be addressed in eukaryotic cells wherespecific techniques and procedures have been devised to ex-amine the coordinated changes in mRNAs expression.Among the RBPs,the highly conserved ELAV/HU familyconsists of four family members,including three that arepredominantly cytoplasmic and neurospecific(HuB/Hel-N1,Huc,andHuD)andonethatcanshuttlebetweennucleusand cytoplasm of all human cells,HuR/HuA/ELAVL1(HuRfrom now on)(Keene 1999;Hinman and Lou 2008).HuR isinvolved in the regulation of cell cycle,cell migration,tumor-igenesis and apoptosis;consequently the HuR expressionchangesinmanytypesofcancerslikebreast,ovary,colon,andbrain,and its increase is often associated with poor prognosis(Wang et al.2013).HuR is also implicated in gametogene-sis,cell differentiation,and stem/progenitor cell survival(Levadoux-Martin et al.2003;Ghosh et al.2009).In macro-phages,HuR is involved in the regulation of inflammatoryand angiogenic processes(Zhang et al.2012;Lu et al.2014)HuR binds to mRNAs containing AU-rich element(ARE)and U-rich element(URE)(Abdelmohsen and Gorospe2010).While these sequences are known to be destabilizingfor the RNAs that contain them,the binding of HuR andHuD increases the stability of the target mRNAs and some-times activates their translation(Jain et al.1997).Thus,HuR protein appears to be one of the few RBPs found to sta-bilize ARE-and URE-containing mRNAs under most condi-tions(Simone and Keene 2013).The exact mechanisms of stabilization havenot been eluci-dated,butthebindingofHuRtoatargetmRNAisbelievedtoblock the interaction of other RBPs capable of driving themRNA to sites of decay like processing bodies or to facilitatethe entering of the exosome.However,HuR also modulatesthetranslationofseveraltargetmRNAs.Insomecases,thisef-fectiscarriedoutthroughtheassociationofHuRwith5UTRinternal ribosome entrysite(IRES),while in someothers,theeffect is due to a competition between HuR and miRNAs.In this respect,there are some important studies that ana-lyze transcriptome-wide HuR-mRNA interactions using thePAR-CLIP technique.These results have been compared toanalogous known data about miRNAmRNA interactionsto elucidate the resultant regulation(Lebedeva et al.2011;Mukherjee et al.2011).Preliminary results and interpreta-tions of these data seem to indicate that when microRNAand HuR binding sites overlap or are in close proximity,the transcripts are preferentially regulated by HuR,whereaswhen the sites are nonoverlapping,the transcripts could bemainly regulated by miRNAs.However,these conclusionsare very limited and strongly related to specific mRNAsexamined.Although HuR,as stated before,tends to increase stability/translation of the target mRNAs,there are a few examples ofHuRs repressive effect on some mRNAs.In these cases HuRcould cooperate with microRNAs to repress target mRNAsthrough destabilization and/or inhibition of translation(Kullmann et al.2002;Yeh et al.2008).So the combined ac-tion of HuR and miRNAs on target mRNAs seems to be verycomplicated and sometimes variable on different mRNAs.In this article,we present the complex regulation ac-complished by miR-200 family microRNAs on the c-JunmRNAandtheinvolvementofHuRinthisprocess.JUNpro-teinisthemaincomponentoftranscriptionfactorAP1thatisan essential regulator of many different cellular processes.JUN is often deregulated in tumors and its coding gene,c-Jun,is considered one of the most important proto-onco-genes of the cell(Lopez-Bergami et al.2010).We show thatc-Jun mRNA is regulated in an opposite way by miR-200bwith respect to miR-200a:while the first has a classic inhib-itory effect on the production of JUN protein,the latter ap-pears to enhance the stability of c-Jun mRNA and toincrease the protein amount.To our knowledge this is oneof the very few examples in which a microRNA is able toup-regulate both the production of the protein as well asthe stability of the target mRNA.The opposite effect ofmiR-200a and miR-200b on the expression of such an im-portant protein like JUN raises interesting questions aboutthe coordinated control that miR-200 family members cancarry out on the same mRNA when expressed differently(Chu et al.2015)and the importance of this action in tumordevelopment and progression.RESULTSmiR-200 family affects the expression of c-Junproto-oncogene in an opposite wayJUN is the main component of the dimeric AP-1 transcrip-tion factor;this complex is involved in almost all areas ofeukaryotic cell behavior from cell proliferation and differen-tiation to stress response and apoptosis.Indeed,AP1 is acti-vated in response to several extracellular signals,fromcytokines and growth factors to stress and inflammation.Because of its central role in the cells,AP-1 is often involvedin tumor progression and malignant transformation duringwhich AP1 proteins are often deregulated by oncogenic sig-nals(Lopez-Bergami et al.2010).HuR and miRNA interplaywww.rnajournal.org1511Despite the fact that c-Jun is a central player in a variety ofbiological processes,little is known about its post-transcrip-tional regulation;then according to three independent targetprediction algorithms(TargetSCAN,www.targetscan.org;miRanda,www.microrna.org and PicTar pictar.mdc-berlin.de),we found that c-Jun mRNA has predicted seed matchesin its 3untranslated region(UTR),one for miR-200a/141and one for miR-200b/200c/429(Fig.1A).In ourexperimentsweutilized miR-200a and miR-200basrepresentative for the two miR-200 family functional groups.To determine whether miR-200s could target the 3UTR ofc-Jun mRNA,we cotransfected a reporter construct in whichFIGURE 1.(A)Schematic of the c-Jun 3UTR with the location of the predicted miR-200a/14 and miR-200b/c/429 target sites.(B)Schematic of theLuciferaseconstructswithc-Jun3UTRWT,mutantsAorB(leftpanel)andalignmentofc-Jun3UTRWTormutated(MUT-BorMUT-A)withseedregion of miR-200b or miR-200a(right panel);the mutated nucleotides are underlined.(C)Luciferase assay with HEK 293T cotransfected with re-porter construct(c-Jun 3UTR WT,c-Jun 3UTR MUT-A,c-Jun 3UTR MUT-B)and a microRNA overexpressing plasmid(miR-200a,miR-200b,miR-342)revealed an antithetical effect of miR-200a and miR-200b on c-Jun 3UTR.The mean values of the corresponding empty vector wereset to one.(D)Luciferase assay on HEK 293T,cotransfected with reporter construct c-Jun 3UTR MUT-A and microRNA overexpressing plasmids(miR-200a,miR-200b,miR-342,miR-200a-comp-mut),indicated that,in the presence of the compensatory mutation in the seed sequence of miR-200a,the inductive effect of the miRNA on the c-Jun 3UTR is recovered.The mean value of the corresponding empty vector was set to one.Datarepresent the mean SD and asterisks()indicate statistically significant modulations with respect to empty vector according to paired Studentstest.()P0.05;()P0.01.Del Vecchio et al.1512RNA,Vol.22,No.10the human c-Jun 3UTR WT was fused to Renilla Luciferase(RLuc)and a miR-200a or miR-200b expression plasmidinto the HEK 293T cell line;these cells express miR-200s ata very low level.The Rluc activity was strongly repressed incells transfected with the miR-200b overexpressing plasmid(Fig.1C;Bracken et al.2014;Jadhav et al.2014),as expectedfrom a targeting microRNA.Surprisingly,the overexpres-sion of miR-200a increased the RLuc activity.When bothmiRNAs were overexpressed together the luciferase activitydid not change compared to the control.In order to verifythattheobservedeffectcouldbeascribed tothedirectpairingbetween the microRNAs and the c-Jun 3UTR,we generatedtwo different mutants,one for the miR-200b binding site,named mutant B(MUT-B),and one for the miR-200a bind-ing site,named mutant A(MUT-A).The two mutants wereobtained by the replacement of three nucleotides of c-Jun3UTR from the second to the fourth nucleotide of miR-200 seeds,with 5-GUC-3(Fig.1B).The MUT-B 3UTRwas completely resistant to the suppressing activity of miR-200b while the inductive effect of miR-200a was preserved(Fig.1C).The opposite happened with the MUT-A 3UTR;using this construct the inductive effect of miR-200a waslost but we were still able to observe the suppressive actionof miR-200b(Fig.1C).We also generated an overexpressingplasmid carrying the sequence for miR-200a pre-microRNAwith a mutation in the seed sequence.We changed three nu-cleotides in the seed sequence(ACA GAC)to allow thebinding of this mutated miR-200a,named the miR-200acompensatorymutant(miR-200a-comp-mut),totheMUT-A 3UTR.With this mutation we restored the pairingbetweenthemicroRNAandthe mRNA 3UTR andrecoveredthe inductive effect of the miRNA(Fig.1D).To investigate the effect of miR-200a and miR-200b over-expression also on endogenous c-Jun,we performed a quan-titative real-time PCR(qRT-PCR)to measure the expressionlevel of c-Jun mRNA(Fig.2A)and a Western blot assayfor the protein(Fig.2B).We noticed that in the presenceof miR-200b we obtained a reduction in both mRNA andthe protein of c-Jun,compared to the cells transfected withthe empty vector.The opposite happened when wetransfect-ed the HEK 293T cells with miR-200a overexpressing plas-mid;the level of both c-Jun mRNA and JUN protein wereincreased.Sinceachangeinthelevelofc-JunmRNAcanbecausedbyeither an increase in the transcription level or an alteration ofmRNA stability,we further investigated the variationsobserved.Actinomycin D,an inhibitor of RNA polymeraseII,was added to the cell culture media 24 h after transfectionof HEK 293T cells with miR-200a overexpressing plasmid orthe empty vector control.qRT-PCR performed on totalmRNA,after 2,4,and 8 h by the addition of actinomycinD,showed substantial differences in c-Jun mRNA stability(Fig.2C).Also in this condition,the overexpression of miR-200a increased the c-Jun mRNA half-life up to 8 h with re-spect to the cells transfected with the empty vector wherethe c-Jun mRNA half-life was around 3 h(Fig.2C),confirm-