MCPIP1, alias Regnase-1 binds and cleaves mRNA of C/EBP/beta

(1)

PLOSONE|https://doi.o r g/10.13 7 1/journal.p o ne.0174 3 8 1 Marc h22,2017 1^/14

OPENACCESS

Citation:LipertB,WilamowskiM,GoreckiA,JuraJ(201 7)MCPIP1,aliasRegnase-

1bindsandcleavesmRNAofC/EBPβ.PLoSONE12(3):

e0174381.

https://doi.org/10.1371/journal.pone.0174381 Editor:YoonKiKim,KoreaUniversity,REPUBLICO FKOREA

Received:November11,2016 Accepted:March8,2017 Published:March22,2017

Copyright:©2017Lipertetal.Thisisanopenacces sarticledistributedunderthetermsoftheCreativeCo mmons Attribution License ,whichpermitsunrestrict eduse,distribution,andreproductioninanymedium ,providedtheoriginalauthorandsourcearecredited.

DataAvailabilityStatement:Allrelevantdataisst oredinFigshareatthefollowingDOI:https://dx.doi.

org/10.6084/m9.figshare.4569649.v1.

Funding:Thisstudywassupportedbytheresearch grantsfromtheNationalScienceCentre2013/11/B/

NZZ/00125and2015/17/B/NZ3/01051.

TheFacultyofBiochemistry,BiophysicsandBiotec hnologyofJagiellonianUniversityisapartneroftheL eadingNationalResearchCenter(KNOW)support edbytheMinistryofScienceandHigherEducation.

Competinginterests:Theauthorshavedeclaredth atnocompetinginterestsexist.

RESEARCHARTICLE

MCPIP1,aliasRegnase-

1bindsandcleavesmRNAofC/EBPβ

BarbaraLipert^1☯,MateuszWilamowski^1☯,AndrzejGorecki²,JolantaJura¹*

1JagiellonianUniversity,FacultyofBiochemistry,BiophysicsandBiotechnology,DepartmentofGeneralBioc hemistry,Krakow,Poland,2JagiellonianUniversity,FacultyofBiochemistry,BiophysicsandBiotechnology, DepartmentofPhysicalBiochemistry,Krakow,Poland

☯Theseauthorscontributedequallytothiswork.

*jolanta . jura@uj.edu.pl

Abstract

CCAAT/enhancer-

bindingproteinbeta(C/EBPβ)isatranscriptionfactorcontrollingabroadrangeofgenesessentialf orhomeostasis,includinggenesrelatedtoimmunefunctions,inflammation,metabolismandgro wth.Monocytechemoattractantprotein-1-inducedprotein1(MCPIP1)alsocalledasRegnase- 1isanRNaseandhasbeenshowntodecreasethesta-bilityofshort-

livedtranscriptscodingforinflammation-relatedproteins,includingIL-1β,IL-6,IL-2,IL-8,IL- 12b,IER-3,c-Rel.Wefoundpreviouslythatthehalf-lifeoftheC/EBPβtran-

scriptisregulatedbyMCPIP.Tounderstandthemechanismdrivingdown-

regulationofC/EBPβbyMCPIP1,weappliedaninvitrocleavageassay,followedbyaluciferase- reporterassayandRNAimmunoprecipitation(RIP).WedemonstratedthatMCPIP1recognizesr egionsofthe3’UTRofC/EBPβmRNAandpromotesitsdecaybyintroducingdirectendo-

nucleolyticcleavage.

Introduction

ModulationofmRNAstabilityhasbeenfoundtoberesponsiblefor40–

50%ofallchangesingeneexpression[1,2].Researchinthisfieldhasrevealedtheimportanceofcis- actingelementslocatedpredominantlyinthe3’untranslatedregionofanmRNA(3’UTR).Theseel ementsenabletheinteractionofmRNAtranscriptswithanmRNA-

decaycomplexwhereunspecificnucleasespromotedegradation.Sincethedegradationinvolve s5’-to-3’and/or3’-to-5’exonu-cleases,theirattackmustbeprecededbyahydrolysisofthe7- metylo-guaninecaponthe5’endand/orremovalofapoly(A)-

tailonthe3’endofanmRNA[3].However,anefficientwayforinstantaneousexpositionofbothmR NAendsfordegradationisanintramolecularcleavagebyendonucleases.Arecentlydiscovereden donucleasecapableofmRNAcleavageisknownasMCPIP1.Thisproteinisessentialforthedegrad ationofshort-livedtranscriptscodingforinflammation-relatedproteins,includingIL-1β,IL-6,IL- 2,IL-8,IL-12b,IER-3,c-Rel[4–

8].IthasbeenshownthatMCPIP1bindingofanmRNAdependsonaconservedstem-loopstruc- tureinthe3’UTR.TheribonucleolyticactivityofMCPIP1hasbeenattributedtoaPIN(PilTNtermin us)likedomain,wherefourAspresidues(D141,D225,D226,D244)inthecatalyticcenterdetermi neRNaseactivity[9].Interestingly,thePIN-likedomainsoftwoMCPIP1

(2)

MCPIP1bindsandcleavesmRNAofC/EBPβ

moleculeshavebeenshowntointeractwitheachotherandthisseemstobecriticalforMCPIP1acti vityinvitro[10].

Inthisstudy,weaimedtoanalyzehowMCPIP1regulatesthestabilityofmRNAcodingfor C/EBPβ(CCAAT/enhancer-

bindingproteinbeta).C/EBPβisabZIP(basicleucinzipper)transcriptionfactorandbelongstoala rgerfamilyofC/EBPproteins.C/EBP-

bindingmotifshavebeenfoundinthepromotersofvariousgenes,includinggenesencodingthein flamma-torycytokinesIL-6,IL-1β,TNF-α,IL-8andIL-12b[11–

16].OurpreviousworkhasshownthatMCPIP1decreasesthehalf-

lifeofmRNAcodingforC/EBPβinHepG2cells,andthatthiseffectrequiresthepresenceofPINdo main[17].Here,wedemonstratethatMCPIP1degradesmRNAcodingforC/EBPβ.Weindicater egionsofits3’UTRthatarerecognizedanddirectlycleavedbyMCPIP1invitro.Ourobservationsa refurtherconfirmedinHepG2cellsbyalucif-erase-

reporterassayandRNAimmunoprecipitation(RIP).

Materialsandmethods Luciferaseassay

Fragmentsofthe3’UTRC/EBPβwereamplifiedwithPCRusingaspecificprimers(Table1)toampli fyC/EBPβsequence(accessionnumber:NM_005194.3).ThelastnucleotideoftheSTOPcodonofC/

EBPβmRNAinthispaperwasnumbered“0”.Amplified3’UTRC/EBPβfragmentswereclonedtop mir-

GLOplasmid(FJ376737;Promega)atXhoIandSalIrestrictionsitesandsequenced.ThePCRprod uctofMCPIP1(accessionnumber:NM_025079)generatedbyPCRwith5’atggatccgagt ctgagctatgagtgg3’and5’ggaattccctcactggggtgctgg3’p rimerswasinsertedintothepc-

DNA3.1vector(Invitrogen)atBamHIandEcoRIrestrictionsites.HepG2cellswereco- transfectedwithluciferasereporterplasmidpmir-

GLOcontainingthewhole3’UTRofC/EBPβoritsfragmentsandexpressionvectorpc- DNA3.1codingforawild-typeMCPIP1oritsmutantMCPIP1-D141N.Cellswereco-trans- fectedwithanemptypmir-

GLOandpcDNA3.1vectorsservedasanexternalreference.After24hofincubation,cellswerelys edandsupernatantsweresubjectedforanalysisperformedwiththeDual-

LuciferaseReporterAssaySystem(E1910;Promega).

Proteinexpressionandpurification

FulllengthhumanMCPIP1(WT)andMCPIP1(D141N)recombinantproteinswerepro- ducedaccordingtoprocedurespreviouslydescribed[18].Twoadditionalconstructsexpress- ingPINdomain:MCPIP1PINandMCPIP1PIN-D141N,spanning131–

327residues,weregeneratedbyPCRwiththefollowingprimers:5’atggatccgatctg cgcccggtggttatc3’and5’tactcgagttagctcggacgttccggg tggaa3’,thenclonedtoBamHIandXhoIrestrictionsiteinmodifiedpET-

21aexpressionvectorcontainingaHis-tagattheN-

Table1.Primersusedforamplificationof3’UTRC/EBPβmRNA.

3’UTRofC/EBPβmRNA Forwardprimer5’-3’ Reverseprimer5’-3’

-60-624 ttactcgagctgcggaacttgttcaagcag attgtcgactttttttactgcccccaaaaggc

15–624 attctcgagcgtccccctgccggcc attgtcgactttttttactgcccccaaaaggc

15–270 attctcgagcgtccccctgccggcc attgtcgacggcagagggagaagcagaga

268–450 attctcgagtctctgcttctccctctgcc attgtcgaccatcaacagcaacaagcc

(3)

MCPIP1bindsandcleavesmRNAofC/EBPβ

432–624 attctcgagggcttgttgctgttgatg attgtcgactttttttactgcccccaaaaggc

PrimerssequencedesignwasdoneonthebasisofcDNA(NM_005194.3)sequence.

https://d o i.org/10.1371/ j ournal.pon e .0174381.t001

(4)

terminus.MCPIP1PINandMCPIP1PIN-D141NwerepurifiedbyNi^2+affinitychromatogra- phy,followedbysizeexclusionchromatography.

InvitromRNAssynthesisandpurification

HumanC/EBPβtranscriptswereamplifiedbyPCRonthebasisoftheC/EBPβsequence:NM_005 194.3.PCRproductswereinsertedintoapcDNA3.0vector(Invitrogen)atHindIIIandEcoRIrestric tionsites.Toobtain3’UTRC/EBPβmRNA(-3–19)synthetizedoligonucleo-

tides5’ttaaagctttagcgcggcccccgcgcgcgtcgaattctta3’and5’taa gaattcgacgcgcgcgggggccgcgctaaagctttaa3’weredenaturedthenann ealedtoachievedsDNAtemplate,followedbyphosphorylationwithT4polynucleotidekinase.Ne xt,thetem-

platewasligatedtopcDNA3.0vectoratthebluntendDraIrestrictionsite.C/EBPβtranscriptfragme ntsweresynthetizedusingaT7RNApolymerasekit(TranscriptAidT7highYieldTranscriptionKit;

ThermoFisher)accordingtothemanufacturer’sprocedures.PriormRNAsynthesistemplateswe relinearizedattheEcoRVsite.Synthetizedtranscriptswerepurifiedusingaphenol-

chloroformextractionprocedure[19].Next,C/EBPβtranscriptswereprecipi- tatedovernightinabsoluteethanolat-

20˚C.Afterprecipitation,transcriptsweredissolvedinRNAsefreewater.RNAconcentrationwas determinedusingaNanoDrop1000spectropho-

tometer(ThermoScientific).Purifiedtranscriptswerestoredat-

80˚C.Beforeconductingexperiments,transcriptswerethawedandthendenaturedat90˚Cfor5mi n.

mRNAscleavageassay

PurifiedrecombinantMCPIP1orMCPIP1PINwasincubatedwithselected3’UTRC/EBPβtranscripts .Cleavagereactionswereconductedat37˚Cinabuffer(50mMTris,150mMNaCl,2.5mMMgCl2,2.5m MDTT,0.5mMEDTA,0.025mMZnCl2,pH8.3).Sampleswerepreparedat20μlinmolarratio:1pmolR NAto5pmolMCPIP1.Sampleswerecollectedatselectedtimepointsandfrozentostopthereaction.Cl eavedtranscriptswerevisualizedthrough4%denaturingpolyacrylamidegelelectrophoresisinTBEb uffer.Beforeloadingonthegel,samplesweremixedwithRNAGelLoadingDye(#R0641ThermoFishe r)anddena-

turedfor15minutesat70˚C.Electrophoresiswasperformedat80V,thenthegelwasstainedwithSimpl eSafe(EuRX)andvisualizedusingaChemiDocMPimagingsystem(BioRad).

ElectrophoreticMobilityShiftAssay(EMSA)

PurifiedfulllengthMCPIP1-

D141NorproteinfragmentcontainingmutantformofPINdomain,MCPIP1PIN- D141Nwereincubatedwithselected3’UTRC/EBPβtranscripts.Reac-

tionswereperformedat37˚Cinabuffer(25mMTris,150mMNaCl,2.5mMMgCl2,2.5mMDTT,0.5mME DTA,0,025mMZnCl2,pH8.3).Samplesweremixedusingconstant2pmolamountofRNA(RNA:protei nmolarratiowereusedasfollows:1:0;1:2.5;1:12.5;1:25;1:37.5;1:50)andwereincubatedonicefor20 minutes.Subsequently,sampleswereloadedon0.8%agarosegelthatwaspre-

stainedwithSimpleSafe(EuRX)dye.ElectrophoresiswascarriedoutinTAEbufferat65V.

Cross-linkedRNAimmunoprecipitation

Humanhepatocellularlivercarcinomacellline(HepG2)waspurchasedfromATCC.Cellsweregr owninahumidifiedatmosphereat5%CO2inDMEMcontaining1g/lofglucose(Lonza,Switzerlan d)andsupplementedwith5%FBS(Lonza,Switzerland).Fortheexperi-

ments,cellsonpassages100–110wereseededonthepoly-L-lysine-

(5)

coatedcellcultureplates(BDFalcon,USA).PCRproductsofMCPIP1generatedby5’atgcgg ccgcagctatgagtg

(6)

gc3’and5’ttaggatccacaggcagcttactcactg3’wereinsertedintoa pFLAG- CMV-2vectoratNotIandBamHIrestrictionsites.CellsweretransfectedwithMCPIP1pFLAG- CMV-2expressionvectororemptypFLAG-CMV-2vector(E7033;SIGMA).Toper-

formcrosslinking,cellsontheplatesweretreatedwithformaldehyde(432173111;POCh,Poland)i nafinalconcentrationof1%for10minutes.Cellswerecollectedfromplatesandwashedtwiceusing coldPBSbufferwiththeadditionofaproteaseinhibitorscocktail(P8340,Sigma-

Aldrich).Cellswerecentrifugedat120RCFfor10minutesatroomtemperatureandlysedinabuffer containing50mMHEPES,400mMNaCl,1mMEDTA,1mMDTT,10%glycerol,0.5%TritonX- 100(pH7.5),supplementedwithanRNaseinhibitor(037–

1000;A&ABiotechnology)andproteaseinhibitorcocktail(P8340,Sigma- Aldrich).Thepelletwasre-sus-

pendedinthelysisbuffer,frozeninliquidnitrogenandthenthawedrapidlytopromotecelldisruption .SubsequentimmunoprecipitationofMCPIP1complexeswithRNAwasdoneinIPbuffer:50mMH EPES,150mMNaCl,1mMEDTA,2mMDTT,10%glycerol,0.5%Triton

X.100(pH7.5)supplementedwithanRNaseinhibitorandproteaseinhibitorcocktail.Sam- pleswereincubatedovernightwith50μlProteinAagarose(11719408001;Roche)and30μgmono clonalanti-

FLAGM2antibody1:1000(F1804;SIGMA).Next,sampleswerewashedfivetimesin1mlIPbuffer, followedbycentrifugationat300RCFfor2minutes.ForproteinaseKtreatment(V302B,Promega), 50μgofenzymewasaddedtosamples.Reverseofcross-

linkingwasdoneinRIPbuffer:50mMHEPES,100mMNaCl,5mMEDTA,10mMDTT,10%glycerol,1,5%SDS,0.5%TritonX-

100(pH7.5)supplementedwithRNaseinhibitor.SampleswereincubatedinRIPbufferat70˚Cfor 1hour,thensupernatantswerecollectedforfurtheranaly-

sis.AlloftheabovebuffersweremadeusingDEPCtreatedwater.Transcriptswereextractedusing aphenol-

chloroformprocedure[19].BeforeRNAprecipitationinisopropanol,VividVioletwasaddedtosam plesasaco-precipitatingfactor(E4502-01;EURx).Precipitationwasperformedat-

20˚Cfor2hours.PurifiedRNAwasreversetranscribedtocDNAusingrandomhexamerprimers(E 0101-

01;EURx)andMMLVreversetranscriptase(M1701;Promega).RealtimePCRanalysisofmRNA wasdoneusingReal-Time2xPCRMasterMix(2005–100;A&ABiotechnology)andtheEcoReal- TimePCRSystem(Illumina).Thefollowingprimerswereused:C/EBPβ5’agcgacgagta caagatccg3’and5’gctgctccaccttcttctgc3’,actin5’caagagatgg ccacggctgctt3’and5’caggtctttgcggatgtccacg3’,28S

RNA5’cacccactaatagggaacgtg3’and5’ctgacttagaggcgttca gtc3’.

Westernblotting

Forwesternblottinganalysisthefollowingantibodieswereused:monoclonalanti- FLAGM2antibody1:1000(F1804;SIGMA)andperoxidase-conjugatedgoatanti-

mouseantibody1:10000(554002;BDPharmingen).Immobilontransfermembrane(IPVH00010;Mi llipore)wasincubatedwithImmobilonWesternChemiluminescentHRPSubstrate(WBKLS0500;Mi llipore)andvisualizedusingtheChemiDocMPimagingsystem(BioRad).

Results

MCPIP1recognizes3’UTRofC/EBPβmRNA

Tospecifywhich3’UTRregionoftheC/EBPβmRNAisinvolvedintheMCPIP1-drivendown- regulation,wepreparedasetofreportervectorsharboringdifferentsectionsofthe3’UTRofC/E BPβmRNA(Fig1A )whichwerelinkedtoaluciferasecodingsequence(CDS).Constructswerein

(7)

troducedtoHepG2cellstogetherwithplasmidscodingforMCPIP1orMCPIP1lackingRNaseactiv ity(MCPIP1-

D141N).Luciferaseassayresultshowed,thatcellsexpressingluciferasecDNAwiththefull- length3’UTR(-60-624)exibitloweractivityof

(8)

Fig1.IdentificationofMCPIP1targetregionswithinthe3’UTRofC/EBPβmRNA.A.Schematicrepresentationofgeneticconstructsusedinlucifera seactivityassays.B.LuciferaseassayofHepG2cellstransfectedwithapmir-

GLOreportervectorcarryingarelevantfragmentofthe3’UTRofC/EBPβmRNAlinkedtoacodingsequenceoffireflyluciferaseandpcDNA3.1expressio nvectorcodingforwildtypeMCPIP1orMCPIP1withabolishedRNaseactivity(MCPIP1-

D141N).EmptypcDNA3.1vectorservedasacontrol.Allvalueswerenormalizedtothesameexternalreferencei.e.valuemeasuredforcellsco- transfectedwithemptyluciferase-

expressingvectorandanemptypcDNA3.1expressionvector.Graphshowsmean±SDn=3;*p<0.05,**p<0.02***p<0.001,ns—

notsignificant(Student-ttest).

https:// d oi.org/10.137 1 /journal.pone . 0174381.g001

luciferaseinthepresenceofMCPIP1,butnotMCPIP1-

D141N(Fig1B ).Amongst3’UTRfragments,theinhibitoryeffectofMCPIP1wasobservedtohaves imilaractivityfortheregionsspanningnucleotides15to624and15to270aswellasforashortregio nneartheSTOPcodon(-3to19nt).MutantformofMCPIP1(MCPIP1-D141N)hadnoeffectonlucif- eraseactivity,thus,weconcludedthattheseregionsareimportantintheregulationofC/EBPβmRN AstabilitycontrolledbytheactiveformofMCPIP1.Onlydistantregionsofthe3’UTRofC/EBPβmR NA(268to624nt)remainedresistanttoMCPIP1activity.Interest-

ingly,acomparisonbetweenreferencecells(transfectedwithanemptyplasmids)revealedthatthe3’

UTRofC/EBPβmRNAstronglyinfluencedluciferaseactivity,alsoirrespectivelyofMCPIP1.Thi simpliesthatthe3’UTRofC/EBPβmRNAcontainsotherMCPIP1-inde-

pendentregulatoryelements.Thestrongestinhibitionofluciferaseactivitywasobservedforthefull- lengthofthe3’UTRofC/EBPβmRNA.Theregulatorymotifsarelocatedinthefirst(15–

270)andlast(432–

624)partofthe3’UTRandtheireffectonmRNAprocessingseemstobeadditive.

(9)

MCPIP1bindsthe3’UTRofC/EBPβmRNA

AtthispointitwasnotclearifthelossofluciferaseactivityresultsfromdirectorindirecteffectsofMCP IP1onmRNAprocessing.Forexample,MCPIP1mightdegradethetranscriptcodingfortheC/EB PβmRNAregulator.Thus,inthenextstepofourstudywecheckedifMCPIP1caninteractdirectlywi ththe3’UTRofC/EBPβmRNA.Toachievethisobjectiveweperformedelectrophoreticmobilityshi ftassays(EMSA).Additionally,toidentifythefunc-

tionalregionweanalyzedasetoffragmentsofthe3’UTRofC/EBPβmRNA.RNAwasincu- batedwithenzymaticallyinactivemutantofrecombinantMCPIP1(MCPIP1-

D141N)topreventdegradation.WefoundthatMCPIP1-

D141Nefficientlybindsallanalyzed3’UTRfrag-ments,includingthedistantregion(ranged268–

450nt)(Fig 2A ).

Weobservedthatanincreasedamountofproteinleadstotheformationofheaviercom- plexes.WeconcludedthattheseareoligomericformsofMCPIP1andthusahomo-

oligomerisafunctionalformofMCPIP1.Weanalyzedtheinteractionbetweenthe3’UTRfragmen tsofC/EBPβmRNAandthePINdomainofMCPIP1harboringthepointmutation,D141N.We

Fig2.MCPIP1-

D141NwithcompletelyabolishedRNaseactivityretainsinteractionabilityagainstthe3’UTRofC/EBPβmRNA.Resultsofelectrophoreticmobilityshifta ssaysperformedwithincreasingamounts(0–200pmol)ofrecombinantMCPIP1-D141NA.orPINdomainalone,MCPIP1PIN-

D141NB.incubatedwith2pmolofinvitro–transcribedthe3’UTRfragmentsofC/EBPβmRNA.

(10)

didnotobservetheoligomerizationofrecombinantPINdomainwithmutationofonecon- servedaminoacidresidue:MCPIP1PIN-

D141N,evenatthehighestquantityoftheRNA(200pmol).ThisclearlyshowsthatMCPIP1needs otherdomainstocreateoligomersandthat

thePINdomainaloneisnotsufficienttogeneratesuchcomplexes.Furthermore,MCPIP1PIN- D141Ndomainshowedweakpotentialtobindanyoftheanalyzedthe3’UTRfragmentsofC/EBPβ mRNA(Fig2B).ThisindicatesthatbindingofatargetRNAbyMCPIP1requiresthepresenceofoth erMCPIP1domains.Moreextensiveanalysisisneededtoelucidatethemechanismofsubstratepr ocessingbyMCPIP1.

TofurtherconfirmaninteractionbetweenMCPIP1andC/EBPβmRNA,weperformedaRIPass ay,weperformedaRIPassay.ToobtainthehighestpossibleamountofisolatedRNA,weoverexpre ssedtheenzymaticallyinactiveFLAG-MCPIP1-D141Nmutant.ThepresenceofMCPIP1-

D141Nwasverifiedbywesternblot(Fig 3A ).ResultsoftheRIPanalysisshowedthattheFLAG- MCPIP1-

D141NprecipitatewassignificantlyenrichedbyaC/EBPβtranscript(Fig3B).Ontheotherhand,tr anscriptcodingfor28SRNAwasonlyslightlyincreasedinFlag-MCPIP-

D141Nprecipitatedsampleincomparisontocontrol(actintranscript).Thisdemon- stratesaninvivointeractionbetweenMCPIP1andthe3’UTRofC/EBPβmRNA.

MCPIP1directlycleavesthe3’UTRofC/EBPβtranscript

ToclarifywhetherMCPIP1bindingoftheC/EBPβmRNA3’UTRresultsindegradationofCEBPβtr anscriptweperformedacleavageassay,whererecombinantMCPIP1wasmixedandincubatedinvitr owiththe3’UTRfragmentsofC/EBPβmRNAfor0.5,1,4and8hours.Weobservedthatallanalyze d3’UTRfragmentsofC/EBPβmRNAunderwentMCPIP1-

mediateddegradation(Fig 4A ).However,thedegradationrateofthedistant3’UTRfragmentofC/

EBPβmRNA(268–450)wassignificantlyslowerthanthatofthefull-

length3’UTRofC/EBPβmRNA(-60-624).Moreover,theregionlocatedbetweenbetween- 3to19ntofthe3’UTRofC/EBPβmRNAwasalsoconfirmedasanimportantinmRNAdegradation assaywithrecom-binantwildtypeMCPIP1butnotwithitsmutantform(Fig4B).

Thepatternofbandsdetectedafter0.5hcleavageofthefull-length3’UTR(-60-

624)clearlyshowsthatthereareseveralspecificsitesofendonucleolyticcleavagewithinthe3’UT RofC/EBPβmRNA.Forthelongerreactiontimes,weobservedfragmentationofthe3’UTRthat

Fig3.MCPIP1bindsC/EBPβtranscriptinvivo.HepG2cellsweretransfectedwithanemptyplasmidorplasmidc odingforenzymaticallyinactiveMCPIP1-D141NwithlinkedFlagtagatN-

terminus.Celllysateswereimmunoprecipitatedwithanti-

FlagantibodiesfollowedbyRNAextraction.A.WesternblotshowingexpressionofaMCPIP1andtubulinasaloadin gcontrol.B.IsolatedRNAwasreversetranscribedandanalyzedbythereal-

timePCRusingprimersspecifictoC/EBPβor28SRNAwithactinspecificprimersasaninternalreference.Thesear erepresentativeresultsoftwoindependentexperiments.

(11)

https:// d oi.org/10.1371/ j ournal.po n e.0174381.g 0 03

(12)

Fig4.MCPIP1directlycleavesthe3’UTRofC/EBPβmRNA.A.Cleavageassayofthe3’UTRfragmentsofC/EBPβmRNAcombinedwithreco mbinantMCPIP1(residues1–599)orMCPIP1PIN(residues134–327).B.Cleavageassayofthesynthetized3’UTRofC/EBPβmRNA(-3- 19)stemloopstructurecombinedwithrecombinantMCPIP1-WTorMCPIP1-D141Nasacontrol.

https://d o i.org/10.1371/ j ournal.pon e .0174381.g0 0 4

(13)

mostlikelyresultsfromlessspecificinteractionsbetweenMCPIP1andtheRNA.Thisstudyreve aledthattherearenumeroussitesofinteractioninthe3’UTRofC/EBPβmRNA,whichmightdiff erintheiraffinityforMCPIP1.

SinceithasbeenshownthatthecatalyticdomainofMCPIP1iscapableofdegradingRNA,wepe rformedanadditionalcleavageassayforaMCPIP1PINdomain(Fig 4A ).Weobservedthatitwasc apableofdegradingRNAandthefinalproductsdisplayedthesamepatternasobservedforwholep rotein,althoughtheyappearedafterlongerreactiontimes.Thisdemon-

stratesthattheisolatedPINdomainofMCPIP1(residues134–327)exhibitspecificityfortran- scriptdegradationbutithassignificantlylowercatalyticactivityagainstthe3’UTRofC/EBPβmRN Athanthefull-lengthMCPIP1protein.

Identificationofpotentialhairpinsinthe3’UTRofC/EBPβmRNA

AllmRNAidentifiedsofarasatargetsofMCPIP1containastem-

loopinthe3’UTR[5,7,8].IthasbeenproventhatsubstratebindingbyMCPIP1requiresthepresenc eofastem-

loopwithinanRNA[7].ToinvestigatethepossibilityofMCPIP1involvementinC/EBPβmRNAstab ility,wesearchedforhairpinsinthe3’UTR.UsingtheEnsembledatabaseandClustalW[20,21]we aligned3’UTRsequencesofC/EBPβmRNAfrom8mammalianspecies.Thehighlyevolutionarily conservedregionshavebeenfurtheranalyzedforthepresenceofpotentialsec-

ondarystructuresusingthemfoldwebserver[22]

(Fig5).Weidentifiedmanysiteswithinthe3’UTRofC/EBPβmRNAthatfoldintohairpinswithahigh probability.Theseregionswere

Fig5.HighlyconservedregionsofC/EBPβmRNAmightfoldintohairpins.ClustalOmegaalignmentoftheC/EBPβmRNA3’UTRofeightmammalianspecie srevealedregionsofhighconservationstatus.Withinthese,asetofpotentialhairpinsismarkedinyellowandgreen.

(14)

spreadwithinthe3’UTRandwereappearedtobehighlyconserved.However,furtherfunc- tionalstudiesareessentialtoshowwhichstem-

loopsfromthe3’UTRofC/EBPβmRNAareessentialfordegradationtriggeredbyMCPIP1.

Discussion

C/EBPβbelongstoafamilyofleucine-

zippertranscriptionsfactors.AccordingtotheHumanProteinAtlas,C/EBPβisaubiquitousprotein withthehighestexpressionlevelsinintestine,lungs,skin,adiposetissue,breast,skeletalmuscleandl iver[23].SinceC/EBPβactivatesabroadrangeofgenesessentialforhomeostasis,includinggenes relatedtoimmunefunction,metabolism,andgrowth,theregulationofC/EBPβactivitymustbeacc urate.IthasbeenshownthatC/EBPβactivityiscontrolledontranscriptionalandpost-

translationallevels.How-ever,littleisknownaboutthepost-

transcriptionalcontrolofC/EBPβexpression.TheHuRproteinhasbeenshowntobindtheC/EBPβ transcriptandenhanceitsstability[24,25],butaccordingtoourknowledgenodestabilizingagentfo rtheC/EBPβtranscripthasbeendescribedsofar.

MCPIP1isanendonucleaseinvolvedinmRNAdecaybyselectivecleavageofmRNAswithintheir 3’UTR.WefoundpreviouslythatMCPIP1overexpressionledtoadecreaseinmouseC/ebpβmRNAa ndproteinlevels.Furthermore,experimentsdoneonHepG2cellswithactinomycinDconfirmedthat MCPIP1reduceshalf-lifeofhumanC/EBPβtranscript[17].Additionally,Yangandco-

workersshowedthatMCPIP1overexpressionreducestheamountoftheCREB[26],whichisatranscr iptionfactorpromotingC/EBPβgeneexpression.Here,weinvestigatedtheinfluenceofMCPIP1onth estabilityoftheC/EBPβtranscript.

Afterapplyinganinvitrocleavageassayfollowedbyaluciferase-

reporterassayandRNAimmunoprecipitation(RIP),wedemonstratedthatMCPIP1canregulatelevel sofC/EBPβthroughadirectendonucleolyticcleavageofthe3’UTRoftheC/EBPβmRNA.Toidentifyth eregionofthe3’UTRthatmightbesucceptibleforthisdegradation,weanalysedMCPIP1-de-

pendentdegradationofforfourdifferentpartsofthe3’UTRspanning-3-19,15–270,268–450,432–

624nucleotides.Weobservedthatallanalysedregionsofthe3’UTRC/EBPβmRNAwerecleavedbyth efull-

lengthrecombinantMCPIP1withsimilarefficiency.However,aluciferaseassayofthedistantregionss panningthenucleotidesfrom268to624didnotconfirmtheirsusceptibilitytodegradation.Thismayres ultfromthelowsensitivityoftheluciferaseassay,sinceforbothtestedconstructsweobservedaslightde creaseinluciferaseactivity,althoughthisdecreasewasnotstatisticallysignificant.Nevertheless,thisd ataindicatesthatthe3’UTRofthehumanC/EBPβmRNAcontainsfewsitesofinteractionwiththeMCPI P1endonuclease.Analysisofevolutionalconservationfollowedbythepredictionofsecondarystructu resrevealedinthe3’UTRofC/EBPβthepresenceofmanypotentiallyrelevanthairpins.Twoofthese,lo catedinhumanC/EBPβ171–185and365–376nt,canbetargetedbyMCPIP1astheyexhibitaPy-Pu- Pypatternintheloopregions(TATandTGT,respectively)andeitheraGorAonthesecondpositioninth eloophasbeenshowntobecrucialforMCPIP1suppression[7].ItislikelythereareadditionalMCPIP1ta rgetsinthe3’UTRofC/EBPβmRNA.Weobservedthatregionspanningnucleotidesfrom-3-

19wasalsocleavedbyMCPIP1,bothinvitroandinvivo.Accordingtobioinformaticstoolsthisregionma yalsocreateahairpincon-tainingadifferentthanPy-Pu-

Pynucleotidepatternintheloop.However,Luandco-workersshowedthattargetsotherthanthePy- Pu-

PyloopsarealsosusceptiblefordegradationbyMCPIP1[27].TheimpactofthisparticularhairpinonC/

EBPβmRNAremainsuncertainasMinoandco-

workersshowedthatonlyadistancegreaterthan20ntbetweenthehairpinandstopcodonassuressup pressionofMCPIP1onIL-

63’UTR[7].Suchconditionswerecreatedinthegeneticconstructsweused,butwerenotpresentintheo riginalC/EBPβmRNA.

(15)

However,thishairpindoesnotexhibitevolutionaryconservation,asitwasnotpresentinC/EBPβmRNA ofanalysedrodents.Thesignificanceofstemloopstructuresdetectedwithbioin-

formaticstoolsinthe3’UTRofC/EBPβmRNAinthespecificdegradationtriggeredbyMCPIP1requiref urther,morecomplexstudies.

SinceithasrecentlybeenproposedthatcleavageofmRNAisexertedbyMCPIP1dimers[10],wetest edwhetherafragmentofMCPIP1thatisenzymaticallyactivebutunabletoformoligomerscandegradet he3’UTRofC/EBPβmRNAinvitro.WechoseaMCPIP1fragmentconsistingofresidues134–

327asbothC-terminalandN-

terminalfragmentshavebeendescribedasanimportantforMCPIP1dimerformation[10,28].Weobser vedsignificantlylowerefficiencyofRNAbidinganddegradationcapacityofanalysedMCPIP1fragment scom-paredtoawild-typeprotein.

Interestingly,arecentlyfoundinductorofMCPIP1expressionisC/EBPα[29].ConsideringthatC/EB PαandC/EBPβactcooperatively,onemightexpectthatexpressionofMCPIP1dependsonC/EBPβasw ell.Infavourofthishypothesisistheidentificationofanegativefeed-

backloopbetweenMCPIP1anditsotheractivator,NF-κB[30].

TherearefourC/EBPβtranscriptsresultingfromthealternativepositionsofthetranslationstart site.IsoformsLAP^*andLAPareconsideredtobetranscriptionalactivators,whileiso-

formLIPistheirnatural,dominant-

negativeinhibitorandthustranscriptionalrepressor[31].AlthoughmRNAscodingforC/EBPβisof ormsaredifferentinsize,their3’UTRshavethesamelength,thusareequallysusceptibletoMCPIP 1cleavage.ItislikelythattheratioofLAP^*/LIPorLAP/LIPisoformsismoreimportantthantheconce ntration.Fromthispointofview,MCPIP1mightberegardedasregulatorofC/EBPβactivity.Trans criptionallyactiveiso-formsofC/EBPβaregenerallyregardedasthekeyregulatorsofIL-

6signallingwhileotherMCPIP1targets,includingNF-κB,coordinatesignallingofIL-

1β.BothtranscriptionfactorsmustactsynergisticallytoactivategenescodingforIL-6,IL-8,andIL- 12[15,16].Interestingly,themRNAoftheseproteinsisalsotargetedbyMCPIP1[4,5,18].Moreov er,MCPIP1hasbeenproventoinhibitNF-κBactivityandcleavec-RelmRNA[30,32–34].

OurpreviousandpresentresultsindicatethatMCPIP1repressesexpressionofC/EBPβinmou seandhumans[17].Thisbringsnewinsightintounderstandingofregulatorynetworksandorchestr atedresponsetoIL-1andIL-

6cytokines.ThisalsodemonstratesvariousMCPIP1functionsintheprocessofsilencingofinflam matoryresponse.Down-regulationofC/EBPβ

byMCPIP1mightbealsopotentiallyrelevanttoclinicalapplications,sinceenhancedexpres- sionofC/EBPβexistsinthebackgroundofpathologicalconditionssuchas,cancerandmeta- bolicsyndrome[35–37].

Acknowledgments

ThisstudywassupportedbyresearchgrantsfromtheNationalScienceCentre2013/11/B/NZZ/00125 and2015/17/B/NZ3/01051.TheFacultyofBiochemistry,BiophysicsandBiotech-

nologyofJagiellonianUniversityisapartneroftheLeadingNationalResearchCenter(KNOW)support edbytheMinistryofScienceandHigherEducation.WewouldliketothankDrAnetaKaszaforprovidinga backboneofpGL3vector.

AuthorContributions

Concep tualization:MWBLAGJJ.Formalan alysis:MWBL.Fundingacquisition:

JJ.

(16)

Investigation:MWBLAGJJ.Met hodology:MWBL.Projectadmi nistration:JJ.Resources:JJ.

Supervision:JJ.

Validation:MWBLAG.Visualization:

MWBLAGJJ.Writing–

originaldraft:MWBLJJ.

Writing–review&editing:MWBLAGJJ.

References

1. CheadleC,FanJ,Cho-

ChungYS,WernerT,RayJ,DoL,etal.StabilityregulationofmRNAandthecontrolofgeneexpression.Ann NYAcadSci.2005.pp.196–204.

2. FanJ,YangX,WangW,WoodWH,BeckerKG,GorospeM.Globalanalysisofstress- regulatedmRNAturnoverbyusingcDNAarrays.ProcNatlAcadSciUSA.2002;99:10611–

10616.https://doi. org/10.1073 / pnas.16221 2 39 9 PMID :12149460

3. SchoenbergDR,MaquatLE.RegulationofcytoplasmicmRNAdecay.NatRevGenet.2012;13:246–

259.https://d o i.org/10.103 8 /nrg316 0 PMID :22392217

4. MizgalskaD,WegrzynP,MurzynK,KaszaA,KojA,JuraJ,etal.Interleukin-1-inducibleMCPIPpro- teinhasstructuralandfunctionalpropertiesofRNaseandparticipatesindegradationofIL-

1βmRNA.FEBSJ.2009;276:7386–7399.https:// d oi.org/1 0 .1111/j.174 2 -4658.2009 . 07452. x PMID:

19909337

5. MatsushitaK,TakeuchiO,StandleyDM,KumagaiY,KawagoeT,MiyakeT,etal.Zc3h12aisanRNasee ssentialforcontrollingimmuneresponsesbyregulatingmRNAdecay.Nature.2009;458:1185–

1190.https: / /doi.org/10.1 0 38/natu r e0792 4 PMID :19322177

6. LiM,CaoW,LiuH,ZhangW,LiuX,CaiZ,etal.MCPIP1down-regulatesIL-2expressionthroughanARE- independentpathway.PLoSOne.2012;7.

7. MinoT,MurakawaY,FukaoA,VandenbonA,WesselsHH,OriD,etal.Regnase-

1androquinregulateacommonelementininflammatorymRNAsbyspatiotemporallydistinctmechanisms.

Cell.2015;161:1058–1073.https: / /doi.org/10.1 0 16/j.cell. 2 015.04. 0 2 9 PMID :26000482 8. KochanJ,WawroM,KaszaA.IF-

combinedsmRNAFISHrevealsinteractionofMCPIP1proteinwithIER3mRNA.BiolOpen.2016;5:889–

898.https://doi.o r g/10.12 4 2/bio.01801 0 PMID :27256408

9. XuJ,PengW,SunY,WangX,XuY,LiX,etal.StructuralstudyofMCPIP1N-

terminalconserveddomainrevealsaPIN-likeRNase.NucleicAcidsRes.2012;40:6957–

6965.https://doi.o r g/10.1093/ nar/gks35 9 PMID:22561375

10. YokogawaM,TsushimaT,NodaNN,KumetaH,EnokizonoY,YamashitaK,etal.Structuralbasisforthereg ulationofenzymaticactivityofRegnase-1bydomain-

domaininteractions.SciRep.2016;6:22324.https:/ / doi.org/10.1 0 38/srep 2 232 4 PMID :26927947 11. AkiraS,IsshikiH,SugitaT,TanabeO,KinoshitaS,NishioY,etal.AnuclearfactorforIL-6expression(NF-

IL6)isamemberofaC/EBPfamily.EMBOJ.1990;9:1897–1906.PMID:2112087

12. WedelA,SulskiG,Ziegler-HeitbrockHW.CCAAT/enhancerbindingproteinisinvolvedintheexpres- sionofthetumournecrosisfactorgeneinhumanmonocytes.Cytokine.1996;8:335–341.PMID:8726660 13. PopeRM,LeutzA,NessSA.C/EBPbetaregulationofthetumornecrosisfactoralphagene.JClinInvest.

1994;94:1449–1455.https://doi . org/10.1172/ J CI11748 2 PMID :7929820

14. ShirakawaF,SaitoK,BonaguraCA,GalsonDL,FentonMJ,WebbAC,etal.Thehumanprointerleukin1betag enerequiresDNAsequencesbothproximalanddistaltothetranscriptionstartsitefortissue-

specificinduction.MolCellBiol.1993;13:1332–1344.PMID:8441379

15. MatsusakaT,FujikawaK,NishioY,MukaidatN,MatsushimatK,KishimototT,etal.Transcriptionfac- torsNF-IL6andNF-KBsynergisticallyactivatetranscriptionoftheinflammatorycytokines,interleukin6

(17)

andinterleukin8(C/EBP/protein-proteininteraction/acute-phasereaction).Immunology.1993;90:

10193–10197.

16. PlevySE,GemberlingJH,HsuS,DornerAJ,SmaleST.Multiplecontrolelementsmediateactivationofthemu rineandhumaninterleukin12p40promoters:evidenceoffunctionalsynergybetweenC/EBPandRelprotein s.MolCellBiol.1997;17:4572–4588.PMID:92347 1 5

17. LipertB,WegrzynP,SellH,EckelJ,WiniarskiM,BudzynskiA,etal.Monocytechemoattractantpro-tein- inducedprotein1impairsadipogenesisin3T3-L1cells.BiochimBiophysActa—

MolCellRes.2014;1843:780–788.

18. DoboszE,WilamowskiM,LechM,BugaraB,JuraJ,PotempaJ,etal.MCPIP-1,aliasRegnase-1,con- trolsepithelialinflammationbyposttranscriptionalregulationofIL-

8production.JInnateImmun.2016;8:564–578.https://doi.o r g/10.1159 / 00044803 8 PMID :27513529 19. ChomczynskiP,SacchiN.Thesingle-stepmethodofRNAisolationbyacidguanidiniumthiocyanate-

phenol-chloroformextraction:twenty-somethingyearson.NatProtoc.2006;1:581–

585.https://doi. org/10.1038 / nprot.2006.8 3 PMID :17406 2 85

20. HerreroJ,MuffatoM,BealK,FitzgeraldS,GordonL,PignatelliM,etal.Ensemblcomparativegeno- micsresources.Database.2016;pii:bav096.

21. LiW,CowleyA,UludagM,GurT,McWilliamH,SquizzatoS,etal.TheEMBL-

EBIbioinformaticswebandprogrammatictoolsframework.NucleicAcidsRes.2015;43:580–584.

22. ZukerM.Mfoldwebserverfornucleicacidfoldingandhybridizationprediction.NucleicAcidsRes.

2003;31:3406–3415.PMID:12824 3 37

23. UhlenM,FagerbergL,HallstromBM,LindskogC,OksvoldP,MardinogluA,etal.Proteomics.Tissue- basedmapofthehumanproteome.Science.2015;347:1260419.https://doi.o r g/10.1126 / science. 126041 9

PMID :25613900

24. JonesH,CarverM,PekalaPH.HuRbindstoasinglesiteontheC/EBP-betamRNAof3T3-L1adipo- cytes.BiochemBiophResCo.2007;355:217–220.

25. BergaletJ,FawalM,LopezC,DesjobertC,LamantL,DelsolG,etal.HuR- mediatedcontrolofC/EBPβmRNAstabilityandtranslationinALK-

positiveanaplasticlargecelllymphomas.MolCancerRes.2011;9:485–496.https://doi.o r g/10.1158 / 1541- 7786.M C R-10-035 1 PMID :21343335

26. YangL,ChaoJ,KookYH,GaoY,YaoH,BuchSJ.InvolvementofmiR-9/MCPIP1axisinPDGF-BB- mediatedneurogenesisinneuronalprogenitorcells.CellDeathDis.2013;4:e960.https://doi . org/10. 1038/

cd

d is.2013.4 8 6 PMID :24336080

27. LuW,NingH,GuL,PengH,WangQ,HouR,etal.MCPIP1selectivelydestabilizestranscriptsassoci- atedwithanantiapoptoticgeneexpressionprograminbreastcancercellsthatcanelicitcompletetumorregres sion.CancerRes.2016;76:1429–1440.https://doi.o r g/10.1158 / 0008-5472.CA N -15-1115

PMID:26833 1 20

28. SuzukiHI,AraseM,MatsuyamaH,ChoiYL,UenoT,ManoH,etal.MCPIP1ribonucleaseantagonizesdicera ndterminatesmicroRNAbiogenesisthroughprecursormicroRNAdegradation.MolCell.2011;44:424–

436.https://doi.o r g/10.1016 / j.molcel.20 1 1.09.01 2 PMID :22055188

29. MakkiMS,HaqqiTM.Histonedeacetylaseinhibitorvorinostat(SAHA,MK0683)perturbmiR-9- MCPIP1axistoblockIL-1beta-inducedIL-

6expressioninhumanOAchondrocytes.ConnectTissueRes.2016;

30. SkalniakL,MizgalskaD,ZarebskiA,WyrzykowskaP,KojA,JuraJ.RegulatoryfeedbackloopbetweenNF- kBandMCP-1-inducedprotein1RNase.FEBSJ.2009;276:5892–5905.https://doi.o r g/10.1111/j.

1742-4658. 2 009.07 2 73. x PMID :19747262 31. DescombesP,SchiblerU.Aliver-

enrichedtranscriptionalactivatorprotein,LAP,andatranscriptionalinhibitoryprotein,LIP,aretranslatedfr omthesammRNA.Cell.1991;67:569–579.PMID:1934061

32. LiangJ,SaadY,LeiT,WangJ,QiD,YangQ,etal.MCP-inducedprotein1deubiquitinatesTRAFpro- teinsandnegativelyregulatesJNKandNF-kappaBsignaling.JExpMed.2010;207:2959–

2973.https://doi.o r g/10.10 8 4/jem.20 0 9264 1 PMID :21115689

33. NiuJ,WangK,GrahamS,AzferA,KolattukudyPE.MCP-1-inducedproteinattenuatesendotoxin- inducedmyocardialdysfunctionbysuppressingcardiacNF-

κBactivationviainhibitionofIκBkinaseactivation.JMolCellCardiol.2011;51:177–

186.https://doi.o r g/10.10 1 6/j.yjmcc. 2 011.04.01 8 PMID: 21616078

34. UehataT,IwasakiH,VandenbonA,MatsushitaK,Hernandez-CuellarE,KuniyoshiK,etal.Malt1- inducedcleavageofregnase-

1inCD4(+)helperTcellsregulatesimmuneactivation.Cell.2013;153:1036–

1049.https: / /doi.org/10.1 0 16/j.cell. 2 013.04. 0 3 4 PMID :23706741

35. EatonEM,HanlonM,BundyL,SealyL.CharacterizationofC/EBPbetaisoformsinnormalversusneo- plasticmammaryepithelialcells.JCellPhysiol.2001;189:91–

105.https://doi.o r g/10.10 0 2/jcp.1139 PMID:11573 2 08

(18)

36. BundyLM,SealyL.CCAAT/enhancerbindingproteinbeta(C/EBPbeta)-

2transformsnormalmammaryepithelialcellsandinducesepithelialtomesenchymaltransitioninculture.On cogene.2003;22:869–883.https://d o i.org/10.103 8 /sj.onc.120 6 21 6 PMID :12584567

37. MillwardCA,HeaneyJD,SinasacDS,ChuEC,BedermanIR,GilgeDA,etal.Micewithadeletioninthegenef orCCAAT/enhancer-bindingproteinbetaareprotectedagainstdiet-inducedobesity.Diabe-

tes.2007;56:161–167.https://doi.o r g/10.2337 / db06-031 0 PMID :17192478