Substrate specificity of human MCPIP1 endoribonuclease

(1)

Published:xxxxxxxx

1

SCientiFiCRepoRts| ( 2 0 1 8 ) 8:7381| DOI:10.1038/s41598- 018-25765-2

www.nature.com/scientificreports

OPE N

Received:29December2017 Accepted:27April2018

Substratespecificityofh umanMCPIP1endoribon uclease

MateuszWilamowski¹,AndrzejGorecki²,MartaDziedzicka- Wasylewska²&JolantaJura¹

MCPIP1,alsoknownasRegnase-

1,isaribonucleasecrucialforregulationofstabilityoftranscriptsrelatedtoinflammat oryprocesses.Here,wereportthatMCPIP1actsasanendonucleasebydegradingseve ralstem-loopRNAstructuresandsingle-

strandedRNAs.Ourstudiesrevealedcleavagesitespresentinthestem- loopsderivedfromthe3′untranslatedregionoftheinterleukin-

6transcript.Furthermore,MCPIP1inducedendonucleasecleavageattheloopmotifo fstem-loopstructures.Additionally,weobservedthatMCPIP1couldcleavesingle- strandedRNAfragments.However,

RNAsubstratesshorterthan6nucleotideswerenotfurtheraffectedbyMCPIP1nucl eolyticactivity.Inthisstudy,wealsodeterminedthedissociationconstantsoffull- lengthMCPIP1D141NanditsribonucleasedomainPIND141Nwithtwelveoligonucle otidessubstrates.Theequilibriumbindingconstants(Kd)forMCPIP1D141Nandthe RNAtargetswereapproximately10nM.Interestingly,

weobservedthatthepresenceofazincfingerinthePINdomainincreasestheaffinityofthi sproteinfragmentto25-nucleotide-longstem-

loopRNAbutnottoshorterones.Furthermore,sizeexclusionchromatographyoft heMCPIP1andPINproteinssuggestedthatMCPIP1undergoeshomooligomeriza tionduringinteractionwithRNAsubstrates.Ourresultsprovideinsightintothem echanismofMCPIP1substraterecognitionanditsaffinitytowardsvariousoligonu cleotides.

RibonucleasedegradationofmRNAisanessentialmechanismtocontrolthelevelofselectedtranscriptsincells.MCPIP 1(MonocyteChemoattractantProtein-1–InducedProtein1),alsoknownasRegnase1,regulatesRNAsta-

bilitythroughitsribonucleolyticactivity.RegulationofimmuneresponsesbyMCPIP1occursthroughthedirectdegrad ationoftranscriptsofmanycytokines,suchasIL-1β,IL-2,IL-6,IL-8,IL-12b,andIL-17^1–

7.MCPIP1wasdescribedasamodulatorofinflammatoryprocessesintheearlyphaseofinflammation⁵.MCPIP1alsoreg ulatesdifferentiation,tumorgrowthandangiogenesis^8–10.

TheenzymaticactivityofMCPIP1isduetothePINdomain(PilTN–terminus),whichpossessesribonucleo- lyticactivity^1,2.TheputativeMCPIP1activesiteconsistoffouraspartateresiduesthatareengagedincoordinationofasin glemagnesiumionlocalizedintheenzymecatalyticcleft¹¹.PINdomainsarecommonlypresentinvariouseukaryotican dprokaryoticnucleasesthatcleavedifferentclassesofRNAmolecules,includingmRNA,rRNA,tRNAandviralRNA s^12,13.OneofthosenucleasesistheDis3subunitoftheeukaryoticexosomecomplex,whichcontainsaPINdomainthatha sendonucleaseactivityagainstmRNA.Additionally,theC-

terminaldomainofDis3possessesprocessive3′to5′exonucleaseactivity¹⁴.PINdomainsarefrequentlypresentastheto xinagentofprokaryoticproteinsengagedintoxin-anti-

toxinsystems,includingtheVapBCsystemcontainingtheVapCPINribonuclease¹⁵.Recently,theCaenorhabditisele gansproteinREGE-1wasshowntocontainafunctionalnucleasePINdomain,indicatingclosehomologytoMCPIP1¹⁶. MCPIP1,whichisencodedbyZC3H12A,belongstotheMCPIPfamilycomprisingproductsofthegenesZC3H12A ,ZC3H12B,ZC3H12CandZC3H12D¹⁷.AspecificfeaturesharedbythisfamilyisasingleCCCHzincfinger(ZF)domai npositionedattheC-terminalregionofthePINribonucleasedomain.CCCH-

typeZFsarecharacteristicofproteinsinvolvedinRNAprocessing.SeveralrepresentativesofCCCHZFRNA- bindingpro-

(2)

2

teinsaretristetraproline(TTP),Roqu in1andRoquin2¹⁸.TheCCCHZFincr easestheefficiencyofRNAsubstrate cleavagecatalyzedbyMCPIP1^11,19. Additionally,thecrystalstructureoft hePINdomainrevealedthepositivel ychargedloopsequencethatislocate dnearthecatalyticcoreofMCPIP1.T hisloopmaymediatetheinteractionw ithnegativelychargedphosphategro upsofoligonucleotidebackbones¹¹. HomooligomerizationofMCPIP1o ccursthroughtheC-

terminaldomain,whichisenrichedin prolineresidues.Deletionofthisregi ondecreasedribonucleolyticactivit yofMCPIP1²⁰.Purifiedrecombinan tMCPIP1proteinwithamutationint henuclease

1DepartmentofGeneralBioch emistry,FacultyofBiochemist ry,BiophysicsandBiotechnol ogy,JagiellonianUniversity,K rakow,Poland.²Departmento fPhysicalBiochemistry,Facult yofBiochemistry,Biophysicsa ndBiotechnology,Jagiellonian University,Krakow,Poland.Co rrespondenceandrequestsfor materialsshouldbeaddressed toJ.J.

(email:jolanta.jura@uj.edu.pl)

(3)

www.nature.com/scientificre ports/

Name Sequence Length

RNAoligonucleotides

mIL-682–1063′FAM 5′-UGUUGUUCUC U A CGA AGAACUGACA-3′-FAM 25nt mIL-682–1065′FAM FAM-5′-UGUUGUUCUC U A CGA AGAACUGACA-3′ 25nt mIL-682–106R S FAM-5′-ACAGUCAAGAC U A CGA UCUUGUUGU−3′ 25nt mIL-682–106Y R FAM-5′-UGUUGUACAC U A CGA UGUACUGACA-3′ 25nt mIL-685–101shortstem FAM-5′-UGUUCUC U A CGA AGAAC-3′ 17nt hIL-682–99 FAM-5′-UGUUCUCU A UG GAGAACU-3′ 18nt consensusstem-loop FAM-5′-UGGAAAGU A U CUUUCCU-3′ 17nt

mIL-682–93 FAM-5′-UGUUGUUCUCUA-3' 12nt

mIL-682–88 FAM-5′-UGUUGUU-3' 7nt

poly-U FAM-5′-UUUUUUUUUUUU-3' 12nt

mIL-682–106int.ACA FAM-5′-UGUACAUCUC U A CGA AGAUGUUACA-3′ 25nt mIL-683–98ter.ACA FAM-5′-ACAGUUCUCU A UG GAGAACUGU−3′ 22nt

mIL-685–93ter.ACA FAM-5′-ACAUGUUCUCUA-3′ 12nt

mIL-61–45 FAM-5′-

UGCGUUAUGCCUAAGCAUAUCAGUUUGUGGA CAUUCCUCACUGUG-3′

45nt DNAoligonucleotides

mIL-682–93ssDNA FAM-5′-TGTTGTTCTCTA-3′ 12nt

mIL-682–93dsDNA ′-TGTTGTTCTCTA-3′

3′-ACAACAAGAGAT-5′ 12bp

FAM-5

Table1.NtsequencesoffluorescentlymodifiedoligonucleotidesusedfortheRNaseassaysandaffinitydeterminatio nassays.Ntsthatformloopfragmentsofstem-

loopstructuresareunderlined.Sequenceswithnumberedresiduesarepartofthe3′UTRoftranscriptsfrommouseorh umanIL-

6.Thesesequenceswerenumberedsuchthatthefirstntafterthestopcodonofthecodingsequenceismarkedas0.RS–

reversestemmodificationofmIL-682–106(alteredntsareinbold).YR–

purineandpyrimidineresiduemodificationofmIL-682–106( alteredntsareinbold).

catalyticsite(D141N)retainedtheabilitytorecognizeRNA,andformationofthenucleoproteincomplexwasobserved ingelshiftelectromobilityassays^19,21.

Thehalf-lifeoftranscriptsisprimarilymodulatedthroughRNA-bindingproteinsthatrecognizecis- regulatoryelements,suchasAU-richelements(AREs)orstem-loopstructures.MCPIP1recognizesstem- loopsinmRNAanddegradestranscriptsinanARE-

independentmanner^1,2,4.Analysesofsequencesobtainedfromhigh-

throughputsequencingofRNAisolatedbycrosslinkingimmunoprecipitation(HITS-CLIP)showedthatstem- loopsequencespreferablyrecognizedbyMCPIP1D141Ncontainpyrimidine-purine-

pyrimidine(YRY)loopmotifs⁵.TheseresultsindicatedthattheMCPIP1ribonucleaserecognizessequencespresentinc ertainstructuralmotifs.Interestingly,MCPIP1andRoquincooperateinposttranscriptionalgeneregulationbyproces singthesamesetoftargetmRNAs^5,22.TheYRYsequencemotifwasalsopreviouslyidentifiedintargetsrecognizedbyR oquin1,whichbindsstem-loopRNA²³.However,Roquin1itselfdoesnotpossessnucleaseactivity,andregula- tionoftranscriptsoccursthroughtherecruitmentoftheCCR4-NOTdeadenylasecomplex²⁴.

Manytranscriptsthatweredeterminedinhigh-

throughputsequencinganalysisasatranscriptnegativelyregulatedbyMCPIP1donotpossesstheYRYmotifintheloo pstructureofstem-loops.Moreover,someofthesetranscriptswerealsovalidatedastargetsforMCPIP1-

induceddegradation.Itwasshownthatfragmentsderivedfrom3′UTRofthetranscriptscodingforinterleukin-2121–

140,BCL2L1andBIRC3deprivedofYRYmotifinthestemloopsarenottargetsforMCPIP1induceddegradation.4,9Int erestingly,MCPIP1haspotentialtorec-ognizethestem-

loopsequenceswithawiderangeofsizes.Forexample,thereportedconsensussequencefromHITS- CLIPanalysisis7nt-3nt-7nt(stem-loop-

stem)5.However,validatedasatargetforMCPIP1thestemloopfrom3′UTRtranscriptcodingformouseSTAT31739–

1765contains10nt-8nt-10nt(stem-loop-

stem)motif²⁵.Therefore,itispossiblethatMCPIP1recognizesloopsequenceswithvariousnucleotide(nt)contentandst ruc-

tures.Thus,inthisstudy,wefocusedondescribingthespecificityofMCPIP1substraterecognitionusingRNAcleavage assaysandaffinitydeterminationassays.

Resul

ts

DeterminationofsubstratespecificityforMCPIP1.WepurifiedrecombinanthumanMC PIP1WT

andMCPIP1D141N,whichwereexpressedinE.colicells.Thepurityoftheanalyzedproteinswasconfirmedby SDS-PAGEanalysis(SupplementaryFig.S1).TodefinetheMCPIP1nucleasesubstratespecificity,weperformed RNasecleavageassaysusing4typesofoligonucleotides:17-25-nt-longRNAformingstem-loopstructures(mIL-682–

106,mIL-685–101shortstem,hIL-682–99),7-12-nt-longsingle-strandedRNA(mIL-682–93,mIL-682–88),12-nt-longsingle-

(4)

www.nature.com/scientificre

ports/ strandedDNA(mIL-6_82–93ssDNA)and12-nt-longdouble-strandedDNA(mIL-6_82–

93dsDNA).Thesesequenceswerederivedfromthe3′untranslatedregion(UTR)oftheIL- 6transcript.Furthermore,wealsoana-lyzedconsensusstem-

loopsequencesthatwerepreviouslyidentifiedasMCPIP1targets⁵^andsingle-strandedpoly-

URNAsequences.DetailedinformationabouttheappliedoligonucleotidesequencesispresentedinTable1.

BecausetheactivityofMCPIP1isdependentonthepresenceofMg^2+orMn^2+metalions,alldegradationassayswerep erformedinbufferwiththedivalentcationMg²⁺.Additionally,todecreasenon-specificelectrostatic

(5)

Figure1.R NAfragmentsobtaineduponMCPIP1-catalyzedcleavage.(A)Sequencesandstructuresofthe 25-nt-longRNAstem-loopsderivedfromthemIL-682–1063′UTRfragmentaredepicted.

(B)DegradationassayofthemodifiedmIL-682–106stem-loop(RS–reversestemalteration).

(C)DegradationassayofthemodifiedmIL-682–106stem-loop(YR–pyrimidineandpurinealterations).

(D)DegradationassayofthemIL-682–106stem-

looplabeledatthe3′end.Theoligonucleotideswerelabeledatthe5′or3′endwithFAMdye.Thereactionproductswere resolvedon20%denaturingPAGEandvisualizedwithafluorescenceimagingsystem.Concentrationsofthelabeledol igonucleotidesandMCPIP1proteinwere7.5μMand2μM,respectively.

TheD141NmutationofaconservedaspartateofthePINdomaincatalyticcenteroftheMCPIP1decreaseditsribonu cleolyticactivity.(A–

D)WeremadebytheseparationofgelsthatareshownatSupplementaryFig.S2A,D.MajorsitesofMCPIP1- inducedcleavageareindicatedbytheoligonucleotidefragmentlength.

(E)DensitometricanalysisofthekineticsofRNAdegradationpresentedastheleveloftheremaininguncleavedoligon ucleotidesfromtheRNaseassay.Thegraphshowsthelevelofthestem-loopsequence:mIL-682–

106RSduringdegradation.(F)DensitometricanalysisoftheMCPIP1WTinduceddegradationofthemIL-682–

1065′FAMoligonucleotide.AnalysiswascarriedoutseparatelyforeachofdegradationproductsofthemIL-682–

1065′FAM.Oligonucleotidelevelswerenormalizedatthetime30min.

interactionsbetweenMCPIP1andnucleicacids,thecleavagestudieswereperformedatphysiologicalsaltcon- centration(150mMNaCl).TheobservedMCPIP1ribonucleaseactivityproductswerereproduciblyconsistentforprot einsobtainedfromdifferentbatches.

Ineachcase,theRNAcleavageassaywascarriedoutfor30,60,120,180and240minutes.Weinitiallyper- formedanRNaseassayofthemIL-682–106stem-

loopstructure.WeobservedthatMCPIP1WTinduceddegradationstartingfromthe3′endofthemIL-682–

1065′FAM,andMCPIP1_WTcleavedthe25^thsinglentasthefirstone.Then,the24^thntwascleaved(Fig.1A).Simultaneously, mIL-682–1065′FAMstem-

loopcleavageoccurredattheloopsite,betweentheC10andU11nts(Fig.1A).Intheconsequence,a10ntsingle- strandedRNAfragmentwasgeneratedfromthe5′endofthemIL-682–1065′FAMstem-

loopstructure.Next,additionalprocessivedegradationofthenascent10-nt- longssRNAwasobserved(Fig.1A).However,MCPIP1-

induceddegradationwasnotobservedforssRNAfragmentsconsistingof6nt(Fig.1A).

Next,toverifythestereospecificityofMCPIP1-inducedcleavage,wereversedthesequenceofthemIL-682–

106atthestemsiteofthisstem-loop.Surprisingly,afterreversingthestemsequences(mIL-682–106RSoligonucle- otide),weobservedasinglentproductinducedbyMCPIP1WTactivity,indicatingthatenzymatichydrolysisoccurredb etweenfirst(A)ntandthesecond(C)nt(Fig.1B).Thus,thereversestemsequence(mIL-682–

106RS)wascleavedbetweenthesamentsasthebasicmIL-682–106stem-

loop,inwhichMCPIPWTtriggeredenzymatichydrolysisbetweenA25andC24ntinthevicinityofthe3′endoftheoligonucl eotide(Fig.1A,B).Moreover,accu-mulationofthe7-nt-longdegradationproductofthemIL-682–

106R SoligonucleotideindicatedthatMCPIP1WTintroducedcleavagebetweentheA7andA8nts(Fig.1B).

WealsoexaminedwhethertheanalysisofoligonucleotidedegradationwasaffectedbypotentialE.colicon- taminantsremainingfromtheproteinpurificationprocedure.Therefore,weanalyzedoligonucleotidecleavageinduce dbyMCPIP1D141NwithasubstitutionoftheconservedaspartateatthecatalyticcenterofthePINdomain.Noribonuclease sactivityoftheMCPIP1D141NwasobservedforthemIL-682–106RSstem-loopoligonucleotide(Fig.1B,E).However,low- efficacynucleaseactivityofMCPIP1D141NwasobservedformIL-682–

1065′FAM,asshowninFig.1A.MCPIP1_D141Ninducedcleavageoccurredonlyat3′endofthisoligonucleotide.Thus,theD 141NmutationdoesnotcompletelyabolishinvitroenzymaticactivityofMCPIP1.

(6)

ThecharacteristicfeatureoftheunmodifiedmIL-6_82–106stem-

loopisahighpresenceofpyrimidineresiduesatthe5′siteofthestem.Therefore,toassesstheroleofthischaracteristicpatt ern,wemodifiedthestemsequencetoachievebalanceddistributionofthepurineandpyrimidineresiduesatthestemsite ofthisstem-loop.Nts6–9and16–19werechangedinmIL-6_82–

106YR(Table1andFig.1C).OurresultsshowedthatMCPIP1_WT-induced

(7)

Figure2.R NAfragmentsobtaineduponcleavagecatalyzedbyMCPIP1.MajorsitesofMCPIP1-

inducedcleavageareindicatedbytheoligonucleotidefragmentlength.(A)Sequencesandstructuresoftheshortstem- loopsaredepicted.Thesesequencesform17-18-nt-longstem-loops.(B)Degradationofthesingle-

strandedRNAs.Twelveand7-nt-longsingle-strandedRNAsequenceswerepartofthemIL-682–106stem-loop.Poly- UhomopolymerRNAcontains12uracilresidues.Theoligonucleotideswerelabeledatthe5′endwithFAMdye.Thereac tionproductswereresolvedon20%denaturingPAGEandvisualizedwithafluorescenceimagingsystem.Concentratio nsofthelabeledoligonucleotidesandMCPIP1proteinwere7.5μMand2μM,respectively.TheD141Nmutationofacon servedaspartateofthePINdomaincatalyticcenteroftheMCPIP1decreaseditsribonucleolyticactivity.

(A,B)WeremadebytheseparationofgelsthatareshownatSupplementaryFig.S2B–D.

(C)DensitometricanalysisoftheleveloftheremaininguncleavedoligonucleotidesfromtheRNaseassayatdiffere nttimepoints.ThepanelshowsacomparisonofthekineticsofdegradationofthemIL-682–

106andashorterfragmentofthisstem-loop,whichismIL-685–101.

(D)Thepanelshowsthelevelofdegradationofthe25-nt-longstem-loopmIL-682–

106incomparisonwiththe12ntssRNAsequenceofthemIL-682–93. (E)ThegraphcomparesthedegradationofthessRNAmIL682–

93withMCPIP1WTorMCPIP1D141N,whichpossessesattenuatedRNaseactivity.

(F)DensitometricanalysisoftheMCPIP1WTinduceddegradationofthemIL-682–

93oligonucleotide.Theanalysiswascarriedoutfor6ntand10ntlongmIL-682–

93d egradationproductsrespectively,oligonucleotidelevelswerenormalizedatthetime30min.

degradationofthemIL-682–106Y Roccursatthesametimeattheloopsiteofthestem- loopstructureoratthe3′endofthestem-

loopstructure(Fig.1C).Furthermore,wedeterminedthatafterdestabilizationofthemIL-682–

106YRstemloopstructurethroughloopcleavageinducedbyMCPIP1WT,the10-nt-

longssRNAwasincreasedandsubsequentlyprocessivelydegraded(Fig.1C).Additionally,weobservedthatdegradati onofmIL-682–106YRstopsatthefragmentconsisting6nt,similartothedegradationoftheunmodifiedmIL-682–

1065′FAMoligonucleotide(Fig.1A,C).Alterationofpurinewithpyrimidines(mIL-682–

106YR)didnotchangethecleavagesitesinthestemloopstructure,anddegradationwastriggeredasinthecaseofmIL-682–

1065′FAM(Fig.1A,C).Thus,weconcludedthatMCPIP1WT-

inducedinvitrodegradationisnotdependentonstemsequenceofthestem-loop.

Toavoidnegativeresultsduetodiminishedcleavagesusceptibilityofsiteswherentsaremodifiedbyfluores- centlabeling,welabeledthemIL-682–106stem-loopstructureatthe5′endorat3′end(mIL-682–1065′FAM,mIL-682–

1063′FAM,respectively).ForthemIL-682–1063′FAMsequence,weobserveda1-nt-

longdegradationproduct;therefore,thefirstcleavageinducedbyMCPIP1WToccursbetweenC24a ndA25ntsasshowninF ig.1D.Thus,cleavagebetweenC24andA25wasobservedforboththemIL-682–1065′FAMandmIL-682–

1063′FAMsequences.However,forthe3′FAM-

labeledoligonucleotide,thedegradationwaslessefficient.ComparisonofFig.1A,Dsuggeststhatthepresenceoffluor escentdyeonacleavedntdoesnotsignificantlyaffecttheMCPIP1activity.

KineticsofoligonucleotidedegradationtriggeredbyMCPIP1dependsonm anyfactors.Inthenextstep,weexaminedwhetherthestem-

(8)

loopstructure,oligonucleotidelengthornucleotidesequenceaffectedMCPIP1nucleolyticefficiency.Toverifythei mpactofdifferentstem-loopstructuresandsequencesonMCPIP1-triggeredcleavage,weusedasetofshortstem- loopsconsistingof17or18nts(mIL-6_85–101,hIL-6_82–99,consensusstem-loop)

(Table1).WeobservedthatMCPIP1_WTinducedcleavageoftwontsfromthe3′endoftheseoligonucleotidesandalsoacc umulationofbandsthatare10,9and7ntlong(Fig.2A).Thesefindingsindicated

(9)

thatMCPIP1WTintroducesendonucleolyticcleavageintheloopregionofthoseshortstem-loops(mIL-685–101,hIL-682–

99,consensusstem-loop).InitialMCPIP1WT-

inducedenzymatichydrolysisoccurssimultaneouslyforfourphosphodiesterbondsbetween9–

12ntattheloopmotifofthemIL-685–101sequence(Fig.2A).WedeterminedthatthepatternofloopcleavageofthemIL-685–

101stem-loopisdifferentthanthatformIL-682–

1065′FAM,whichwascutbetweentheC10andU11nts(Fig.1A).Therefore,thestemlengthofthestem- loopaffectsthecleavagesitesrecognizedbyMCPIP1.

Toverifytheinfluenceofsizeofhigh-

orderRNAbackbonestructuresontheoligonucleotidecleavagerates,weperformedkineticanalysis.Thekineticsofoli gonucleotidedegradationareshownasthelevelofuncleavedoligonucleotidesobtainedfromdensitometricanalysisof theresultsfromoligonucleotidedegradationassays.InthesubsequenttimepointsoftheRNaseassay,uncleavedmIL- 685–101oligonucleotidelevelsweresignifi-cantlydecreasedcomparedtouncleavedlevelsofthemIL-682–

106oligonucleotides(Fig.2C).WeobservedthatMCPIP1WT-triggeredcleavageofthemIL-685–101stem- loopwasrelativelyfasterthanthatofthehIL-682–99stem-

loop,whichpossesseslongerstems(Fig.2A).TheseresultsshowedthatthekineticsofdegradationofRNAstem- loopstructurescontainingshortstemsisfasterthanthatofstem-loopspossessinglongerstems.Wecon- cludedthatunwindingofshorterstemsfromthestem-

loopstructuresresultsinamoreefficientdegradation(Figs1Aand2A,C).Todeterminetheimportanceofloopfragment sinMCPIP1-triggeredstem-loopcleavage,wecomparedstem-

loopsthatcontaina3,4or6ntlongloopmotif.However,wedidnotobservemajordifferencesinMCPIP1- induceddegradationoftheseoligonucleotides(Fig.2A).

Subsequently,weassessedwhetherMCPIP1WTdegradesunstructuredssRNA.AfterMCPIP1WT- triggereddestabilizationofstem-

loopstructures,asubsequentcleavageoccurredinthenascentssRNA.Thus,weexamined12-nt- longssRNAoligonucleotidesfromthemIL-682–93and7-nt-longmIL-682–

88ssRNA(Fig.2B).UsingtheRNAfoldingsoftwaremFOLD²⁶,weconfirmedthatthemIL-682–93andmIL-682–

88sequencesdidnotshowbasepairinginteractionsatroomtemperature;thus,theydonotfoldintostablesecondarystruc tures.WenoticedthatfortheunstructuredssRNA,therateofMCPIP1WT-

induceddegradationwasincreasedcomparedtocleavageofthestem- loopsequences(Fig.2D).DegradationofeithermIL-682–93ormIL-682–

88indicatedthatssRNAsshorterthan6ribonucleotideswerenotefficientlycleavedbyMCPIP1WT(Fig.2B).Thelevelsof shortenedoligonucleotidesformedasaresultoftheMCPIP1WTinducedcleavageofthemIL-682–

1065′FAMindicatedhighincreaseofthe6ntlongtruncatedoligonucleotide(Fig.1F).Furthermore,weobserved11- foldincreaseofthelevelof6ntproductoftheMCPIP1WTinducedcleavageofthemIL-682–

93(Fig.2F).Moreover,therewasmarginalcatalyticactivityofMCPIP1D141NforssRNA,whichpresentedascleavageoftw ontsfromthe3′endofthemIL-682–93oligonucle-

otide(Fig.2B,E).ToverifythesequencespecificityofssRNAcleavage,weperformeddegradationassaysusingpoly- Usequences.However,itappearedthatMCPIP1WTprocessivelycleavedthepoly-

Uhomopolymer,andtheoligonucleotidedegradationstoppedwhenthefragmentconsistedof6nt(Fig.2B).Theseresult sindicatedthatMCPIP1WTcleavesunstructuredssRNAinasequence-independentmanner.

WenextinvestigatedwhetherMCPIP1exhibitsRNAsubstratespecificity.Therefore,inRNasecleavageassays,we usedsingle-strandedanddouble-

strandedDNA(ssDNAanddsDNA)asasubstrate.TheseDNAsequencesweresimilartoRNAsequencesconsistingof 12ntspresentinthemIL-682–

93oligonucleotide.WeobservedthatMCPIP1WTcleavesbothssDNAanddsDNA(Fig.3A).Degradationofthesesequen cesoccurredfromthe3′end;however,thekineticsoftheseprocesseswaslowercomparedwiththecleavageofmIL-682–

93ssRNA(Figs2B,Eand3A,B).DegradationofthemIL-682–

93ssDNAhadapproximatelyequalefficiencyusingeitherMCPIP1WTorMCPIP1D141N(Fig.3A,B).Therefore,theaspartate 141residueofMCPIP1iscrucialforRNAcleavagebutnotforDNAprocessing(Figs1A,B,2Band3A,B).

WeshowedthatMCPIP1D141NdoesnotpossessactivityagainstmIL-682–

106RS(Fig.1B).However,wehaveobservedthatMCPIP1D141Npossesseslownucleaseactivityinsomeoftheinvestigate dsystems(Figs1A,2Band3A).Therefore,tofurtherconfirmationthatpresentedRNAcleavageassayisnotaffectedby contamina-tionsfromE.coliextractweusedanothercontrolwhichisMCPIP1438–

599proteindeprivedofPINnucleasedomain.ApplyingMCPIP1438–

599toRNaseassaywedidnotobservedegradationofinvestigatedoligonucleotides(SupplementaryFig.S2E).Thus,ourr esultsarenotaffectedbycontaminationsandweconcludethatsinglemuta-

tionD141NofMCPIP1isnotsufficienttocompletelyabolishinvitroMCPIP1nucleaseactivity.Allidentifiedcleavage sitesobservedindegradationassaysarelistedinSupplementaryTableS1.Toverifythesequencespeci-

ficityofMCPIP1-

triggereddegradationofRNA,wepresentedtheidentifiedsitesofcleavageasaconsensuslogo(SupplementaryFig.S3).

Forlogotypepreparation,weusedthesequencelogogeneratorsoftwareWebLogo²⁷.Wefiguredoutthatcleavagesites lackingGntsintheimmediatevicinityofthecutsitewerepreferableforMCPIP1-

inducedcleavage(SupplementaryFig.S3).

ObservedatFig.1oligonucleotidescleavagepatternsandresultspresentedatSupplementaryFig.S3revealedpossib leMCPIP1sequencespecificitywithin5′-ACA-

3′motif.Toconfirmthisobservationwepreparedthreeadditionaloligonucleotidesthatcontain5′-ACA- 3′modifications(Table1).Theinternalmodificationofstemsequenceto5′-ACA-3′wasintroducedtothemIL-682–

106(SupplementaryFig.S2F).WeobservedthatMCPIP1WTinducedcleavageofthemIL-682–

106int.ACAoccursatloopsitebetweentheC10andU11nts,then,additionalcleavageswerespottedbetweenA4-C5- A6nts(SupplementaryFig.S2F).However,incorporationofterminal5′-ACA-3′tothehIL-683–

(10)

98sequencerevealedthatforthisoligonucleotideMCPIP1_WTinducedcleavagetakesplacebetweensequencesU5-U6- C7(SupplementaryFig.S2F).ForsinglestrandedRNAadditionofterminal5′-ACA-3′tothemIL6_85–

93showedthatMCPIP1_WTinduceshydrolysisofbondbetweenC2andA3ntsofthemIL6_85–

93ter.ACA(SupplementaryFig.S2F).Nevertheless,processive3′to5′cleavageofsinglestrandedRNAishighlyefficientco mparedtoendonucleasecleavage(SupplementaryFig.S2F).

TofurtherconfirmationofourobservationaboutinvitrononspecificcleavageofRNAoligonucleotidesbyMCPIP 1_WTweperformedadditionalexperiments.WecheckedwhetherMCPIP1_WTmightcleavethetemplatewhichwereprevi ouslyreportedatinvivostudiesasnotdegradedbyMCPIP1nucleaseactivity.Thefragmentcomprising1–

81ntfromthemIL-63′UTRisnotregulatedthroughMCPIP1activityincellsstudies⁵.Thedistal

(11)

Figure3.DNAfragmentsobtaineduponcleavagecatalyzedbyMCPIP1.

(A)TheDNAsequencesarebasedonthemIL-682–

106sequence;oneoligonucleotideisssDNA,andthesecondisdsDNA.Theoligonucleotideswerelabeledatthe5′endwi thFAMdye.Thereactionproductswereresolvedon20%denaturingPAGEandvisualizedwithafluorescenceimaging system.ConcentrationsofthelabeledoligonucleotidesandMCPIP1proteinwere7.5μMand2μM,respectively.TheD 141NmutationofaconservedaspartateofthePINdomaincatalyticcenteroftheMCPIP1decreaseditsribonucleolytica ctivity.(A)WasmadebytheseparationofgelsthatareshownatSupplementaryFig.S2C,D.

(B)DensitometricanalysisoftheleveloftheremaininguncleavedssDNAanddsDNAoligonucleotidesfromtheMCP IP1nucleaseactivityassayatdifferenttimepoints.

partofthemIL-63′UTRcontainsthemIL-682–1063′UTRstemloopwhichistheputativeelementresponsibleforIL- 6transcriptsdestabilizationthroughMCPIP1nucleaseactivity.Duetolimitationsofsynthesismethodologyweused45 ntslongsequencefromthemIL-61–453′UTR(Table1andSupplementaryFig.S2F).UsingRNAfold-

ingsoftwaremFOLD^26weshowedthatthemIL-61–45oligonucleotidepossiblyformstwostem- loopsstructuresasshownatSupplementaryFig.S2F.DegradationofthemIL-61–

453′UTRclearlyindicatesendonucleaseactivityofMCPIP1WT.ThefragmentofthemIL-61–45tendstoformtwostem- loopsecondarystructures,thusobservedcleavageinducedbyMCPIP1WTshouldbeintroducedatloopsiteofthesesteml oops.Indeedweobservedendo-nucleasecleavageofthemIL-61–

45atbothloopsites(SupplementaryFig.S2F).However,duetoobtainedlowelectrophoresisresolutionwecouldnotpre ciselydescribetheexactnucleotidesbetweenwhichcleavagetakesplace(SupplementaryFig.S2F).

DissociationconstantsoftheMCPIP1complexwitholigonucleotides.Ourresu ltsfromoligo-

nucleotidedegradationassaysdidnotrevealastrongstructuralorsequencepreferenceofinvitroRNAcleavagebyreco mbinantMCPIP1_WT.However,wedeterminedthatsingle-strandedRNAor17-nt-longstem-

loopswerecleavedwithafasterratethan25-nt-

longstemloops.Forthatreason,weinvestigatedwhethertherewereanydifferencesinMCPIP1_D141Naffinityfortheteste doligonucleotides.WeusedFAM-

labeledoligonucleotidestodevelopamethodfordeterminationoftheMCPIP1_D141Naffinitytooligonucleotides.Previo usly,weusedelec-

trophoreticmobilityshiftassays(EMSAs)toshowthatMCPIP1_D141Nhasthepotentialtoformstablecomplexeswith3′U TRfragmentsoftheC/EBPβtranscriptandobtainedcomplexespossessingtwodistinctquaternarystructures²¹.Obser vedshiftsatEMSAassayindicatedthatthemarginalnucleaseactivityofMCPIP1_D141Ndidnotrepressformationofthenuc leoproteincomplex.EstimatedbindingaffinitiesofthecomplexesofMCPIP1_D141NwithRNAbasedonourresultspublis hedpreviouslybyLipertetal.werebetween640–

(12)

1580nM(SupplementaryTableS2)²¹.TheobtainedKdvariesfrompreviouslyusedthe3′UTRsequencefragmentsofth eC/EBPβtran-

script.However,inourpreviousEMSAexperiments,wewerenotabletodeterminetheequilibriumdissociationconstan tsoftheachievedcomplexes.

(13)

Figure4.(A)DomaincharacterizationofMCPIP1:UBA_43–89(Ubiquitin-associateddomain);PRR_100–126and_458–

536(Proline-richregion);PIN133–270(PilTN-terminusnucleasedomain);ZF305–325( z inc- fingermotif);disorderedregion_326–457;CTD545–598( C -

terminalconserveddomain).DepictedfragmentsofMCPIP1,PIN-ZFandPINthatwereusedinpresentedstudies.

(B)AffinityoftheMCPIP1interactionwitholigonucleotidesformingRNAstem-loopstructures:mIL-682–

1065′FAMandsingle-strandedRNAoligonucleotidesrepresentedbymIL-6_82–

93.Theanalyzedproteinswerethecatalyticmutatedforms:MCPIP1D141NanditsPIN-

ZFD141NandPIND141Nfragments.TheribonucleasePIND141Ndomainwasstudiedwithoutorwiththezincfingermotif attheC-terminalregion.Graphsillustratetheinteractionofselectedproteins(MCPIP1D141N,PIN-

ZFD141N,andPIND141N)witholigonucleotides.Functionswerefittedtothefluorescenceintensitydatapointsusing thesequentialbindingmodelN+P+PNP +PNPP(P–proteinN–

oligonucleotide).Thedepictederrorsbarsarestandarddeviations,n=3.

(C)Controlsoftheaffinitydeterminationassay.MCPIP1D141NinapresenceofthefreeFAMlabelandunlabeledhIL- 681–98RNAoligonucleotide.

Herein,wedeterminedtheapparentequilibriumdissociationconstantsofthehumanMCPIP1D141Ncom- plexeswithdifferenttypesofoligonucleotides:stem-

loopRNA,ssRNA,ssDNAanddsDNA(Fig.4B,SupplementaryFig.S4andTable2).Theslopesofdose-

responsecurveswereverysteepforproteinconcentrationvaluesbetween10nMand100nM.Amplitudesofthefluoresce ncesignalswerechangedapproximately2timesdependingonthesequence.Fluorescencepolarizationassaywhichisc

(14)

ommonlyusedforaffinitydeterminationmightbeaffectedbyhighfluorescenceintensitychangesobservedinourmeas urements.Thus,wedecidedto

(15)

MCPIP1D141N

1–599aa Kd(nM)

PIN- ZFD141N134–

327aa Kd(nM)

PIND141N13 4–297aa Kd(nM) Stem-loopRNAs

mIL-682–1065′FAM 6.5±2.1â 6.6±3.1^b 24.1±7.3âb mIL-682–1063′FAM 8.6±2.6â 4.8±1.8^b 21.8±6.3âb mIL-682–106R S 9.8±2.3â 15.1±7.3^b 39.3±8.6âb mIL-682–106Y R 9.5±5.2â 14.4±9.6 25.1±7.0â mIL-685–

101shortstem 9.2±4.0â 20±10 20.7±7.2â hIL-682–99 4.1±3.6â 13.2±6.8 15.1±4.5â consensusstem-loop 3.8±2.8â 9.1±3.2^b 36±17âb ssRNA

mIL-682–93 19±11â 34±18 41±12â mIL-682–88 14.5±8.1â 32±20 40±12â

poly-U 14.8±7.2^a 43±23 39±11^a

mIL-682–93ssDNA 18.3±8.2 36±20 21.7±6.6 mIL-682–93dsDNA 118±49 80±30 56±17

Table2.CalculatedequilibriumdissociationconstantsofMCPIP1complexeswithselectedoligonucleotides.Thean alyzedproteinsweremutatedformsofMCPIP1anditsribonucleasedomain(MCPIP1_D141N,PIN-

ZF_D141N,andPIN_D141N).K_dvaluesweredeterminedusingDynaFit4softwarewiththeimplementedmodelofseque ntialbindingoftwoproteinstoasingleoligonucleotidewithasingledissociationconstant.Errorsareshownasstan darddeviations,n=3.Statisticalsignificance(Pvalue<0.05)betweenselectedgroupsisshownbythefollowinginde xes:^aand^bforcomparisonoftheMCPIP1_D141NwithPIN_D141NandPIN-

ZF_D141NwithPIN_D141Ngroups,respectively.DifferencesobservedbetweentheMCPIP1_D141NandPIN- ZF_D141Ngroupsarenotstatisticallysignificant.

analyzeonlyfluorescenceintensitysignalwhichalsogivesusbetterresidualsofobtainedfits.Thetwo- phasecourseoffluorescenceintensitychanges,observedinallinvestigatedcases,promptedustomodeltheaffinit ydatawithacomplexdoubleequilibriumbindingequationwhereN+P+PNP+PNPP(N–oligonucleotide,P –protein)

(Fig.4BandSupplementaryFig.S4).Theseresultsshowedthattwoproteinmoleculessequentiallybindtoasingle oligonucleotide.Thebestmodeldescribingourdatawasasequentialbindingmodelwithequalequilibrium- bindingdissociationconstants,Kd1=Kd2.WealsoanalyzedthemodelcharacterizedbyKd1≠Kd2,whichwasrejecte dbecauseitinconsiderablyimprovedtheresidualdistribution,however,thestandarddeviationsofthecalculated KdwerehigherthanthoseforthemodelwithKd1=Kd2.Athirdanalyzedmodelwithasingleequilibriumconstant,N +P+PNPP,wasrejectedbecauseithadthehighestresidualsofcurvesthatwerefittedtothemeasureddatapoints.T hefinalselectedmodel(sequentialbindinganalysis,Kd1=Kd2)reflectsthemeas-

ureddatawell,andtheobtainedKdareshowninTable2.Thismodelwascharacterizedbytheloweststandarddeviat ionoftheobtaineddissociationconstantvaluesandlowresidualsofthefittedcurves.

Weobservedthatforthesetofinvestigatedoligonucleotidescomprisingstem-

loopstructures,ssRNA,andssDNA,wedidnotfindmajordifferencesbetweendissociationconstantsofthecomplexes withMCPIP1D141N(Table2).Therefore,MCPIP1D141Ncanefficientlybinddiverseoligonucleotidesequences.Minordi fferencesintheMCPIP1D141Naffinitytostem-loopstructuresorsingle-

strandedoligonucleotidessuggestthatthenucleicaciddouble-

strandedhelicalstructureisnotnecessarytointeractwithMCPIP1.Additionally,weobservedthatMCPIP1hasloweraf finitytodsDNAcomparingtootherinvestigatednts(Table2).Weshowedthattheaffinityoffull-

lengthMCPIP1D141Ntooligonucleotidesissignificantlyhigherthanthatforfragmentsofthisproteinrep- resentedonlybythenucleasedomain(PIND141N)

(Fig.4A,B,SupplementaryFig.S4andTable2).Moreover,wenoticedthatthezincfingerdomainincreasedtheaffinityo fthePIND141Nsubunitto25-nt-

longoligonucleotidesbutnottoshorteroligonucleotides(Fig.4A,B,SupplementaryFig.S4andTable2).

BindingassaysusingfreeFAMdyedidnotshowedsignificantchangesoffluorescenceintensityatinvesti- gatedsystems(Fig.4C).MCPIP1D141NandbufferconditiondidnotaffectfluorescenceemissionofthefreeFAMlabel.Th eunlabeledhIL-681–

98RNAoligonucleotidedidnotaffectfluorescenceemissionoffreeFAMlabelinthepresenceofMCPIP1D141N(Fig.4C).Th us,weassumethatdescribedinteractionsaretheeffectoftheassemblyoftheMCPIPD141Ncomplexwitholigonucleotides .TheshapeoffluorescencespectraoftheFAMlabeledoligonu-

cleotideswereconsistentforallexaminedMCPIPD141Nconcentrations(SupplementaryFig.S4A).Fluorescenceintensi tyoftheFAMlabeledoligonucleotideswerechangedduetoMCPIP1D141Nnucleoproteincomplexforma-

tionwhichaffectedFAMfluorescenceprobe(SupplementaryFig.S4A).

ObserveddissociationconstantsforMCPIP1D141NcomplexeswithmIL-682–

1065′FAMweresubstantiallyweakerforEMSAsystemthaninfluorescencebasedassay(Table2,SupplementaryFig.S5 andSupplementaryTableS2).Wesupposethatdifferencesindissociationsconstantsaretheresultsofthecomplexbindi ngkineticsofMCPIP1interactionwithRNAthatpossiblyischaracterizedbyrelativelyfastkoffrates.Incaseofhighkoffth eEMSAasanon-

(16)

equilibriummethodwillgivehigherdissociationconstantscomparedtoequilibriumtechniques.TheEMSAshiftformI L-6_82–93ssRNAandmIL-6_82–93ssDNAwereobservableatarelativelylowconcentrationofMCPIP_D141N(400nM) (SupplementaryFig.S5A)although,athigherconcentrationoftheMCPIP1_D141Ntheoligonucleotideswerenotcomplet elyboundedinnucleoproteincomplex.Therefore,wedidn’tcalculatetheKd

(17)

Figure5.HomooligomerizationoftheMCPIP1protein.

(A)SizeexclusionchromatographyresultsofMCPIP1.Chromatographywasperformedinabuffercomprisedof25 mMTris,pH7.9,300mMNaCl,10%

(w/v)glycerol,1mMDTT,and0.5mMEDTA.Additionally,forresultsshownasadottedline,thebufferwasenrichedi n1.6Murea.

(B)AmultipleGaussianpeakfitwasperformedtomodeltheobtainedelutionprofileofMCPIP1WT.Fittedpeaksillus tratedtetrameric,dimericandmonomericfractionsoftheMCPIP1WT.

(C)Calibrationcurveofthegelfiltrationcolumn.Greenpointsindicatetheapparentmolecularweightoftheinvestig atedproteinscalculatedusingthecalibrationcurve.Themolecularweightsoftheseproteinsareasfollows:MCPIP1:

65.7kDa;PIN-ZF:24.7kDa;PIN:21.1kDa.

(D)PercentagesoftheMCPIP1WTtetrameric,dimeric,andmonomericfractionswerecalculatedbasedontheareaoft hesizeexclusionchromatographypeaks.

(E)NativePAGEresultsofMCPIP1WTsampleinbufferscontaining50mMTris-HCl,pH8.3,150mMNaCl,10%

(w/v)glycerol,2.5mMMgCl2,1mMDTT,0.5mMEDTAand0.05mMZnCl2.Additionalbufferconditionchangeswere anincreasedconcentrationofNaClto500mMandadditionofureato1600mM.

fromthatresults.EMSAresultsmightsuggestthattherearedifferencesinquaternarystructuresofthecomplexbetween MCPIP1D141Nanddifferenttypesofsubstrates(SupplementaryFig.S5A).

HomooligomerizationofMCPIP1.Interestingly,thetwo-

phasecourseoffluorescenceintensitychangeswasobservedintheobtainedaffinityassaygraphsduringoligonucleot ide-

bindingprocesses(Fig.4B,SupplementaryFig.S4).Thus,twoproteinmoleculessequentiallybindtoasingleRNAmol ecule.Accordingtootherstudies,PINdomainsuperfamilyproteinsarefrequentlydescribedasoligomers:dimersortet ramers²⁸.Therefore,toobtainprecisedataoftheMCPIP1proteinoligomerizationstate,weanalyzedproteinsizeexclusi onchromatographyresults.Inbothcases,singleGaussianpeakswereobserved,indicatingthemonodispersityofthean alyzedproteinfragments.Analysisofproteinsizebasedontheretentionvolume(Fig.5A,C)indicatesthatboththePIN andPIN-

ZFdomainswereinamonomericstate.ThemousePINdomainwaspreviouslysuggestedtobeadimer¹⁹.Incontrast,for full-

lengthMCPIP1_WTandMCPIP1_D141N,weobservedwideelutionpeaksthatshiftedinfavorofpossibleoligomericforms(

Fig.5A).Toassesstheseelutionprofiles,weperformedmultipleGaussianpeakfitanalyses(Fig.5B).Comparingtheobt ainedmaximaoffittedpeakswiththecolumncalibrationcurve,weobservedthatthecalculatedmolecularmassesofthef ractionscorrespondedtotetrameric,dimericandmonomericformsofMCPIP1_WT(Fig.5C,D).Therefore,thefull- lengthMCPIP1_WTorMCPIP1_D141Nismostfrequentlypresentasadimerinnativeconditions,however,tetramericandmo nomericfractionswerealsopresentinsolution(Fig.5A–

D).Buffersupplementationwith1.6Murearesultedinnarrowingpeaksinsizeexclusionchromatography,indicatingt headditionalincreaseoftheMCPIP1dimersfractioninthesam-ple(Fig.5A–

D).NativePAGEelectrophoresisofMCPIP1revealedtwoheterogeneousbandsforMCPIP1_WT(Fig.5E).Theseresults confirmedthatMCPIP1homooligomerizationoccursinnativeconditions.Equilibriumbetweenthedimericandtetram ericstatesofMCPIP1couldbeinfluencedbychangingthebuffercomposition.AfteradditionofureatothenativePAGEb

(18)

uffer,theequilibriumwasshiftedinfavoroftheMCPIP1tetramericfraction(Fig.5D).Ureaasachaotropicagentalterna teshydrogenbondingbetweenwatermoleculesandpro-

teins,andthereforeaffecthydrophobicinteractions²⁹.Thesmallconcentrationofureaorothercommonlyuseddenatura tionagent:guanidinehydrochlorideincreasesstabilityofsomeproteinsandmightincreasereactionsrates³⁰.Ureaconc entration(1.6M)usedinourexperimenthasamuchlowerconcentrationthantypicallyisused

(19)

1 0

Figure6.(A)IdentificationofMCPIP1-triggeredcleavagesitesinthemIL-6_82–106stem- loopRNAstructure.NtsequencesandstructurescreatedduringMCPIP1-

inducedcleavageareillustrated.MappingofthecleavagesitesbasedontheRNaseassayresults,intermediatesandth emostsignificantsubsequentdegradationproductsarepresented.

(B)VisualizationofthestoichiometryoftheMCPIP1interactionwithstem-

loops.Schematiccartoonrepresentationoftheternarycomplexmodel.Thesizeexclusionchromatographyresul tsshowedthatPINandPIN-ZFweremonomericandsuggestthatfull-

lengthMCPIP1mostfrequentlyoccursasadimerinnativecondition.StoichiometryoftheMCPIP1-

RNAinteractionwasbasedonthesizeexclusionchromatographyresultsandtheresultsfromaffinitydeterminati onassayswherethesequentialbindingmodelwereused.Thus,forfull-

lengthMCPIP1,weproposedasequentialbindingmodel:oligo+MCPIP1Dimer+MCPIP1dimeroligo- MCPIP1_dimer+MCPIP1dimeroligo-

MCPIP1_tetramer.Thepresenteddissociationsconstantsofthecomplexeswereestimatedbasedontheaffinitydetermin ationassaysshowninTable2.

fordenaturationofproteins,however,theconcentrationofthisosmolyteissufficienttosignificantlychangethecontent ofbulkwater²⁹.Ureacansubstantiallyinfluencethepolypeptidessolvation,increasingproteinsolventaccessiblearea whichmightconsequentlyleadtoconformationchangesofMCPIP1andprobablyitsmightaffectoligomerization³⁰.Al though,thisindirectmechanismappearstobethemostlikelyinourcase,therearereportsindicatingapossiblealternativ emechanism,inwhichtheureamoleculesdirectlyinteractswithproteinmole-culesinadivalentmanner³¹.

ThetwodistinctquaternarystructureofMCPIP1D141NnucleoproteincomplexeswerealsoobservedinEMSA,u singlongUTRsfragmentsaswellassinglestemloopofthemIL-6_82–

106,asshownbyLipertatal.inSupplementaryFig.5²¹.Together,thesizeexclusionchromatographyandnativePAGEre sultsindicatethatthebothdimericandtetramericformsofMCPIP1homooligomerswerefoundintheinvestigatedcondi tions.

Discuss

ion

PreviousstudiesindicatedthatMCPIP1isaselectiveribonucleasethatcleavestranslationallyactivemRNAatthe 3′UTR⁵.TodeterminehowMCPIP1recognizestheuniquemoleculartargetsthatwerereportedinbiologicalsystems,we appliedinvitroanalysisusingrecombinantMCPIP1.WeidentifiedMCPIP1asanendoribonucleasethatdegradesdivers esetsofRNAstem-

loopstructures.Collectively,ourdatadidnotindicateastrongstructuralorsequencepreferenceduringinvitrocleavage oftheRNAstem-

loops,asallinvestigatedsequenceswereaffectedbyMCPIP1.However,ourresultsrevealedthatunstructuredsingle- strandedRNAishighlypronetocleavagebyMCPIP1WT.WeobservedthatMCPIP1WT-induceddegradationofthemIL- 682–106stem-

loopstartsfromthe3′endofthesequence.Simultaneously,cleavageoccursattheloopsiteofthestemloop.Asaconseque nceofloopcleavage,thestem-

loopstructureisdestabilized,andssRNAfragmentsaregenerated,whicharefurtherprocessivelydegradedinthenextste

(20)

1 0

p(Fig.6A).Surprisingly,weobservedthat6-nt-longssRNAwasnotrapidlycleavedbyMCPIP1_WT.Apossibleexplana- tionforthisprocessmightbethatthe6ntRNAsubstrateistooshorttoreachthenucleasesite.Wehypothesizedthattheregi onofMCPIP1thatiscrucialforRNAbindingmustbeproximaltothecatalyticcleftinthePINdomainsincethe7-nt- longmIL-6_82–

88substratewasstillboundwithhighaffinitytothePIN_D141Ndomain.Wehypothesizedthatthepositivelychargedregionth atispresentinthestructureoftheMCPIP1PINdomainisessentialintheRNArecognition

(21)

11

processandprotectsboundfragmentsofshortssRNAfromfurthercleavage.Preferentialcleavageofoligoribonucle- otidestriggeredbyMCPIP1wasobservedforsequenceslackingaGntatpositions−1and+1ofthecleavagesite,however ,thisobservationmightbeaffectedbythelowcomplexityoftheanalyzedsequences.

Degradationof3′FAM-

labeledoligonucleotidesindicatedthatintroductionofthefluorescentlabeldidnotdisabletherecognitionofthecleava gesitesbyMCPIP1WT.However,for3′FAM-

labeledoligonucleotides,weobservedadecreaseinthenucleolyticefficiencyofMCPIP1WT.Moreover,weobservedt hatMCPIP1cleavedpoly-

UssRNAoligonucleotidesinaprocessivemanner.Thesefindingsmaysuggest3′to5′exonucleaseactivityofMCPIP1a gainstssRNA.Additionally,previousreportsrevealedthattheMCPIP1PINdomainshareshighstructuralhomology withtheT5D155′-exonuclease^11,32.Nevertheless,thesuccessiveexonucleasedegradationofsingle-

strandedRNAbyMCPIP1isnotrelevantinvivoduetothelowrateofobserved3′to5′exonucleasecleavageactivity.Mor eover,invitroendonucleaseactivityofrecombinantMCPIP1hadastrongbackgroundasshownintheresultsofcleavag eoftheloopsitesoftheinvestigatedstem-

loops.Additionally,previousresultshavealsoshowndegradationoflongertranscripts,suchasIL-6,IL-

8orCEBPβ,whichindicatedthatpreferablesitesofendonucleolyticcleavagearepresentinthesetranscripts^5,6,21.Theen donucleaseactivityofrecombinantMCPIP1wasalsoconfirmedfrominvitrodegradationofcircularRNAfragments⁵.

WehypothesizedthatthemarginalinvitroactivityobservedhereofeitherMCPIP1WTorMCPIP1D141NtowardsD NAmoleculesisirrelevantinvivo,sinceMCPIP1hasaprimarycytoplasmiclocalizationandshouldbeconsideredasarib onuclease.Incontrast,theEndoVnucleaseefficientlycleavesbothRNAandDNAsubstrates³³.Therearealsoevidences ofnuclearlocalizationoftheMCPIP1forwhichessentialisnuclearlocalizationsignal(RKKP)thatispresentinaminoa cidsequenceoftheMCPIP1³⁴.However,observedhereDNAcleavagewasinefficientthusweestimateDNAseactivity ofMCPIP1asbiologicallyinsignificant.

RibonucleasespossessingPINdomainsusuallylackstrongsequencespecificityininvitrostudieswithrecom- binantproteins¹³.However,proteinengineeringcanmodifythespecificityoftheseRNases.Oneexampleistheengineer edPIN-PUFnucleasethatpossessesahighsequencespecificityofRNAdegradation³⁵.Mostlikely,mod-

ificationofthePINdomainfromMCPIP1willenhanceitsspecificityandwillbebeneficialforthedevelopmentofahighl ysequence-specificmoleculartool.

Tothebestofourknowledge,theequilibriumdissociationconstantsofthecomplexofMCPIP1witholigo-

nucleotideshavenotbeenpreviouslydescribed.Toinvestigateoligonucleotides,wedeterminedtheKdvaluesofthecom plexwithMCPIP1D141N,PIN-

ZFD141NandPIND141N.ThedissociationconstantstudiesrevealedahighaffinityofMCPIP1D141Ntooligonucleotides,ho wever,theydidnotshowamajordifferenceinaffinityparam-

etersusingdifferentoligonucleotides.WeobservedthattheaffinityofMCPIP1D141NanditsfragmentstowardsssRNA,s sDNAanddsDNAsubstratesislowerthanthatforoligonucleotidesformingstemloops.Moreover,wedidnotobservesi gnificantdifferencesbetweentheaffinityofMCPIP1D141NorPIN-

ZFD141Ntotheinvestigatedoligonucleotides.Therefore,wehypothesizedthatthePIN-

ZFfragmentiscrucialformaintainingthecomplexwitholigonucleotides.Wealsoobservedthatthezinc- fingerdomainsignificantlyincreasedtheaffinityofthePIND141Ndomainto25-nt-

longoligonucleotides.Interestingly,forshorteroligonucleotides,wedidnotobservesignificantdifferencesintheaffini tyforPIND141NorPIN-

ZFD141N.Wehypothesizedthatthezincfingerdoesnotreachshortsubstrates,whichwerelocalizedinproximitytothecat alyticpocketofMCPIP1.Azincfingerteth-eredinthevicinityofthePINcatalyticdomainmightenhancethere- associationofthesubstrateandfacilitatesubsequentcleavage.Incontrast,previousdatasuggestedthatlongRNAfrag mentsderivedfromtheC/EBPβ3′UTRmRNAinteractwithfull-lengthMCPIPD141NbutnotwithPIN-

ZFD141N21.Wehypothesizedthatourprevi-ousresultsmightbeaffectedbythenon- equilibriumconditionsoftheEMSAmethod.ItisalsopossiblethatformaintainingofPIN-

ZFD141NinteractionwithlongmRNAfragments,additionaldomainsofMCPIP1arecrucialforthestabilityoftheternary complex.

SizeexclusionchromatographyresultsindicatethatMCPIP1existsinequilibriumbetweenthedimericandtetramer icstate,weproposedastoichiometricmodeloftheMCPIP1-

RNAinteraction.TwomoleculesoftheMCPIP1dimerinteractwithasinglestem- loopstructure(Fig.6B).Thus,forthefull-lengthMCPIP1,wepro-

posedasequentialbindingmodel:oligo+MCPIP1_dimer+MCPIP1dimeroligo-MCPIP1dimer+MCPIP1dimeroligo- MCPIP1_tetramer.Wehypothesizedthatthebindingofoligonucleotidesubstratesinduceshomooligomeriza- tionoftheMCPIP1.TetramericoligomerizationofthePINdomainwaspreviouslyshownforNob1pandPAE2 754,wherethePINdomainsoftheseproteinsformaringstructurewithacentralholethatiswideenoughtoaccomm odatessRNAorssDNAbutnotdouble-

strandedoligonucleotides^36,37.Nevertheless,furthervalidationofthismodelofMCPIP1- RNAcomplexstoichiometryshouldbeperformed.Ourmodelisbasedonsizeexclu-

sionchromatographyresultsanddoublebindingequilibriumobservedintheaffinitycurves.Resolvingthequa- ternarystructureofMCPIP1withRNAoligonucleotidesiscrucialforunderstandingitsdetailedmechanismofR NAregulation.Todate,thereisnoresolvedholoenzymestructureofanyofthePINdomains;thus,furtherstudieso fMCPIP1complexesarehighlyinteresting.

Weobservedincreasedratesofdegradationofhairpinswithshortstems,whichareconsistentwithresultspublished byMinoandcoworkersfromHITS-CLIPanalyseswheresmallstem-loopsthatcontaineda3-nt-or4-nt-

longloopmotifwerepredominantlyidentifiedincomplexeswithMCPIP1_D141N⁵.Recentstudieshaverevealedtheimp ortanceofUPF1forcytoplasmicmRNAdecaycatalyzedbyMCPIP1⁵.UPF1isanRNAhel-

icasethatparticipatesindegradationofmRNAswithprematureterminationcodonsthatarecrucialfornonsense- mediatedmRNAdecay(NMD)³⁸.SMG6,oneofthekeyproteinsforNMD,alsocontainsaPINdomainattheC-

(22)

12

terminusthatisresponsibleforribonucleolyticactivityandmRNAturnover³⁹.ForinvitroRNAsubstratecleavageinduc edbyrecombinantSMG6,thepresenceofadditionalproteinswithhelicaseactivityisnotneces-

sary.WeshowedthatMCPIP1aloneissufficienttounwindanddegradesubstrateswithstem-

loopsecondarystructuresinvitro.Nevertheless,UPF1mightenhanceunwindingofMCPIP1substratesasanRNAhel icasesinceweobservedthatdegradationofmorestable(withlowGibbsfreeenergy)stem-loopswaslessefficient.

(23)

InteractionwithUPF1andotherproteinsmayincreasetherateofdegradationofselectedRNAtargetsandbroadenther ecognitionpotentialoftheMCPIP1complex.

OurbiochemicalstudiesrevealednumerouscleavagesitesintroducedbyrecombinantMCPIP1intheinves- tigatedsequences.WefoundthatMCPIP1inducedendonucleasecleavageintheloopmotifofstem-loopstructures.Wehypothesizedthatthepresenceofstrongandnon-sequence-

specificinteractionswithRNAwouldenableMCPIP1toefficientlysearchforthestem- loopelementsintranscripts,andidentificationofastem-

loopwouldresultinendonucleolyticcleavageandtranscriptdestabilization.Nevertheless,MCPIP1hasbeenidentified asselectiveribonucleasethatcleavestranslationallyactivemRNAsatthe3′UTR.Thisraisesthepossibilitythataddition alproteinsthatareelementsofaternarycomplexconsistingoftranscriptsandMCPIP1mightdeterminethefinalMCPIP 1specificity.

Metho

ds

Cloningandproteinpurification.ThehumanZC3H12AgenethatencodesMCPIP1wasoptimizedf orefficientexpressioninE.colistrainsandorderedasasyntheticgenefromGenScript(USA).Thecloning,expres- sionandpurificationofthefull-

lengthMCPIP1WTproteinanditsmutantformMCPIP1D141Nwerepreviouslydescribed⁶.Theproceduresforpurification oftheN-terminusHis6-taggedproteinsPIN-ZFWTandPIN-ZFD141N(134–

327residues)weredescribedpreviously²¹.ThesamemethodswereusedtopurifyPINWTandPIND141N(134–

297residues),whichwerealsotaggedwithHis6attheN-terminus.Briefly,E.coliBL21-CodonPlus-

RILculturesweregrownat37°CinLBmediumuntilreachinganOD600of0.5.Proteinexpressionwasinducedwithadditio nof0.5mMIPTG.Allproteinswereexpressedfor3hoursat37°C.Full-lengthMCPIPproteinswerepurifiedusingion- exchangechromatography(TMAE)indenaturingconditions.PINWT,PIND141N,PIN-ZFWTandPIN-

ZFD141NwerepurifiedusingNi-NTAaffinitychromatographyindenaturingconditions.Finally,allpro-

teinsweredialyzedandpurifiedusingagelfiltrationSuperdex200prepgrade10/300(GEHealthcare)columninabuffer comprisedof25mMTris,pH7.9,300mMNaCl,10%

(w/v)glycerol,1mMDTT,and0.5mMEDTA.ChromatographywasperformedusinganÄktaFPLCpurificationsystem(

AmershamPharmacia).

Fluorescent-

labelednucleicacidsequences.TheoligonucleotidesequenceslistedinTable1werepurchasedfr omSigma-Aldrich.Theseoligonucleotideswerefluorescentlylabeledusing6-carboxyfluorescein(6-

FAM).The5′endslabelingofoligonucleotidesweremadebyattaching6-

FAMtophosphategroupofthe5′terminalnucleotides.ThemIL-682–1063′FAMlabelingwasdonebycoupling6- FAMtophosphategroupofthe3′terminalnucleotide.Labeledandpurifiedwithhigh-

performanceliquidchromatographyoligonucleotideswerepurchasedfromSigma-Aldrich.Thedouble- strandedmIL-682–93dsDNAwaspreparedbymixingmIL-682–

93ssDNAandthecomplementaryoligonucleotideina1:1.2ratio.Subsequently,fordsDNA,oligonucleotideswereanne aledbyheatingto95°Cfor5minutesandcooledatroomtemperature.AnalysisofRNAsecondarystructureoftheinvesti gatedoligonucleotideswasperformedusingtheViennaRNAwebserver⁴⁰.

RNaseassays.InvitrocleavageassaysofFAM-labeledoligonucleotideswereperformedinbuffercontain- ing25mMTris-HCl,pH7.9,150mMNaCl,10%(w/v)glycerol,2.5mMMgCl2,1mMDTT,0.5mMEDTAand 1.5 mMZnCl2.LabeledoligonucleotidesandMCPIP1proteinconcentrationswere7.5µMand2µM,respectively.Sampleswereincubatedat37°C,andreactionsatdifferenttimepointswerestoppedbyfreezingindryice.Afteraddi tionoftwofoldexcessofconcentratedloadingdyeconsistingof95%(w/v)formamide,0.5mMEDTA,0.025%

(w/v)xylenecyanol,and0.025%

(w/v)bromophenolblue,reactionsproductsweredenaturedat95°Cfor1minute.AnalkalinehydrolysisRNAladderfor eacholigonucleotidewasgeneratedthroughdenaturationat95°Cfor25minutesinalkalinebuffercontaining50mMsodi umbicarbonate,pH9.5,and1mMEDTA.SampleswereresolvedindenaturinggelelectrophoresisinTBE(Tris/borate/

EDTA)buffer.Denaturinggelscontained20%polyacrylamideand7.5Murea.Fluorescencesignalsweredetectedusin gChemiDocgelimagingdevicewithImageLab5.2software(BioRadLaboratories).Signalacquisitiontimes0.5secwer ethesameforeachofthegels.

Affinitydeterminationassays.TheconcentrationofFAM-

labeledoligonucleotideswas2nMinasystemwithMCPIP1D141Nand20nMina systemcontainingPIND141NorPIN- ZF_D141Nproteins.FreeFAMlabel(6-Carboxyfluorescein,C0662Sigma-

Aldrich)wasusedasacontrolofaffinitydeterminationassay.UnlabeledandHPLCpurifiedhIL-681–

98RNAoligonucleotidewaspurchasedfromSigma-

Aldrich.Proteinconcentrationsweredeterminedbymeasuringtheabsorbanceat280nmusingaNanoDrop2000spectr ophotometer(ThermoScientific).Proteinsabsorptioncoefficientswerecalculatedonthebasisofaminoacidsequence .Sampleswerepreparedusingthetwofoldserialdilutionmethod;thus,ineachsample,theconcentrationofproteingrad

uallychanged.Thereactionbufferfordetectionofsamplefluorescencecontained25mMTris- HClpH7.9,150mMNaCl,5%

(w/v)glycerol,2.5mMMgCl2,1mMDTT,0.5mMEDTAand0.05mMZnCl2.Fluorescencesignalswerecollectedusi ngtheFluoroLogFL3–

12spectrofluorometer(HoribaJobinYvon).Excitationandemissionwavelengthswere495nmand514nm,respectively

(24)

.Measurementsoffluorescencewasperformedat25°Cusingatemperaturecontrolledcuvetteholder.Thedimensions ofthequartzcuvettewere3 ×3mm(Hellma).Dissociationconstants(Kd)weredeterminedusingDynaFitsoftware(ver sion4.07.111,BioKin)⁴¹.Determinationofthebindingmodelwasbasedonresidualdistributionoffittedcurvesandstan darddeviationofdeterminateddissociationconstants.Forcalculationofthedissociationconstants,asequentialbindi

ngmodelwasused:N+P+PNP+PNPP(N–oligonucleotide,P–

protein),whereKd1andKd2wereequaldissociationconstants.Additionally,twobindingmodelsweretested.Thefirston ewascharacterizedbyK_d1≠K_d2,andthesecondonewassimplifiedtothesingleequationN+PNP.Thegrapherrorsrepre sentstandarddeviationsfrom3independentexperiments.Forstatisticalanalysisofdifferencesbetweencalculateddiss

ociationconstants

(25)

foroligonucleotidecomplexeswithMCPIPD141N,PINZFD141NandPIND141None- wayANOVAfollowedbyTukey’smultiplecomparisontestwasused.

Gelfiltrationassays.AnalyticalsizeexclusionchromatographywasperformedusingaSuperdex200Inc rease10/300GLcolumn(GEHealthcare)thatwascalibratedwiththefollowingproteinstandards:myoglo-bin,α- chymotrypsinogen,β-

lactoglobulin,ovalbumin,bovineserumalbumin,apoferritinandthyroglobulin.TheapparentmolecularweightofM CPIP1proteinswasdeterminedbasedonthecolumncalibrationcurve.Fordeterminationofhomooligomerizationofth eanalyzedsamples,multipleGaussianpeakfitswereperformedforchromatogramdatausingOriginPro2017software(

OriginLab).

Nativepolyacrylamidegelelectrophoresis.Proteinsamplesfornativeelectrophoresiswerep reparedwiththeadditionoftwofoldexcessofconcentratedloadingdyethatcomprised62.5mMTris-

HCl,pH6.8,25%glycerol,and1%(w/v)bromophenolblue.Thegelscontained300mMTris- HCl,pH8.8,and6%polyacrylamide(concentrationsofacrylamide/bis-

acrylamidewere30%/1%w/v).Electrophoresiswasperformedat80Vusingrunningbuffercontaining25mMTrisand1 92mMglycine.GelswerestainedwithCoomassieBrilliantBlueG-250solution.

Dataavailability.Thedatasetsanalyzedduringthecurrentstudyareavailablefromthecorrespondingautho ronreasonablerequest.

References

1. Mizgalska,D.etal.Interleukin-1-inducibleMCPIPproteinhasstructuralandfunctionalpropertiesofRNaseandparticipatesin degradationofIL-1βmRNA.FEBSJ.276,7386–7399(2009).

2. Matsushita,K.etal.Zc3h12aisanRNaseessentialforcontrollingimmuneresponsesbyregulatingmRNAdecay.Nature458,1185–

1190(2009).

3. Iwasaki,H.etal.TheIκBkinasecomplexregulatesthestabilityofcytokine-encodingmRNAinducedbyTLR–IL- 1Rbycontrollingdegradationofregnase-1.Nat.Immunol.12,1167–1175(2011).

4. Li,M.etal.MCPIP1down-regulatesIL-2expressionthroughanARE-independentpathway.PLoSOne7,e49841(2012).

5. Mino,T.etal.Regnase-1androquinregulateacommonelementininflammatorymRNAsbyspatiotemporallydistinctmechanisms.

Cell161,1058–1073(2015).

6. Dobosz,E.etal.MCPIP-1,AliasRegnase-1,controlsepithelialinflammationbyposttranscriptionalregulationofIL-8production.

J.InnateImmun.8,,564–578(2016).

7. Monin,L.etal.MCPIP1/Regnase-1restrictsIL-17A-andIL-17C-dependentskininflammation.J.Immunol.198,767–775(2017).

8. Lipert,B.etal.Monocytechemoattractantprotein-inducedprotein1impairsadipogenesisin3T3-L1cells.Biochim.Biophys.Acta- Mol.CellRes.1843,780–788(2014).

9. Lu,W.etal.MCPIP1selectivelydestabilizestranscriptsassociatedwithanantiapoptoticgeneexpressionprograminbreastcancercellstha tcanelicitcompletetumorregression.CancerRes.76,1429–40(2016).

10. Marona,P.etal.MCPIP1downregulationinclearcellrenalcellcarcinomapromotesvascularizationandmetastaticprogression.

CancerRes.77,4905–4920(2017).

11. Xu,J.etal.StructuralstudyofMCPIP1N-terminalconserveddomainrevealsaPIN-likeRNase.NucleicAcidsRes.40,6957–6965(2012).

12. Senissar,M.,Manav,M.C.&Brodersen,D.E.StructuralconservationofthePINdomainactivesiteacrossalldomainsoflife.

ProteinSci.26,1474–1492(2017).

13. Matelska,D.,Steczkiewicz,K.&Ginalski,K.ComprehensiveclassificationofthePINdomain-likesuperfamily.NucleicAcidsRes.

45,6995–7020(2017).

14. Lebreton,A.,Tomecki,R.,Dziembowski,A.&Seraphin,B.EndonucleolyticRNAcleavagebyaeukaryoticexosome.Nature456,993–

996(2008).

15. Winther,K.S.,Brodersen,D.E.,Brown,A.K.&Gerdes,K.VapC20ofMycobacteriumtuberculosiscleavesthesarcin- ricinloopof23SrRNA.Nat.Commun.4,2796(2013).

16. Habacher,C.etal.Ribonuclease-mediatedcontrolofbodyfat.Dev.Cell39,359–369(2016).

17. Liang,J.etal.AnovelCCCH-zincfingerproteinfamilyregulatesproinflammatoryactivationofmacrophages.J.Biol.Chem.283,6337–

6346(2008).

18. Fu,M.&Blackshear,P.J.RNA-bindingproteinsinimmuneregulation:afocusonCCCHzincfingerproteins.Nat.Rev.Immunol.

17,130–143(2017).

19. Yokogawa,M.etal.StructuralbasisfortheregulationofenzymaticactivityofRegnase-1bydomain-domaininteractions.Sci.Rep.

6,22324(2016).

20. Suzuki,H.I.etal.MCPIP1ribonucleaseantagonizesdicerandterminatesmicroRNAbiogenesisthroughprecursormicroRNAdegradati on.Mol.Cell44,424–436(2011).

21. Lipert,B.,Wilamowski,M.,Gorecki,A.&Jura,J.MCPIP1,aliasRegnase- 1bindsandcleavesmRNAofC/EBPβ.PLoSOne12,e0174381(2017).

22. Jeltsch,K.M.etal.Cleavageofroquinandregnase-

1bytheparacaspaseMALT1releasestheircooperativelyrepressedtargetstopromoteTH17differentiation.Nat.Immunol.15,1079–

1089(2014).

23. Leppek,K.etal.RoquinpromotesconstitutivemRNAdecayviaaconservedclassofstem-looprecognitionmotifs.Cell153,869–81(2013).

24. Sgromo,A.etal.ACAF40-bindingmotiffacilitatesrecruitmentoftheCCR4- NOTcomplextomRNAstargetedbyDrosophilaRoquin.Nat.Commun.8,14307(2017).

25. Masuda,K.etal.Arid5aregulatesnaiveCD4⁺T

cellfatethroughselectivestabilizationofStat3mRNA.J.Exp.Med.213,605–619(2016).

26. Zuker,M.Mfoldwebserverfornucleicacidfoldingandhybridizationprediction.NucleicAcidsRes.31,3406–3415(2003).

27. Crooks,G.E.,Hon,G.,Chandonia,J.-M.&Brenner,S.E.WebLogo:asequencelogogenerator.GenomeRes.14,1188–1190(2004).

28. Arcus,V.L.,McKenzie,J.L.,Robson,J.&Cook,G.M.ThePIN-domainribonucleasesandtheprokaryoticVapBCtoxin- antitoxinarray.ProteinEng.Des.Sel.24,33–40(2011).

29. Salvi,G.,DeLosRios,P.&Vendruscolo,M.Effectiveinteractionsbetweenchaotropicagentsandproteins.ProteinsStruct.Funct.

Bioinforma.61,492–499(2005).

30. Bhuyan,A.K.Proteinstabilizationbyureaandguanidinehydrochloride.Biochemistry41,13386–94(2002).

31. Zhang,Y.&Cremer,P.S.ChemistryofHofmeisterAnionsandOsmolytes.Annu.Rev.Phys.Chem.61,63–83(2010).

32. Ceska,T.A.,Sayers,J.R.,Stier,G.&Suck,D.Ahelicalarchallowingsingle-strandedDNAtothreadthroughT55′-exonuclease.

Nature382,90–93(1996).