C2c2结构

ArticleTwoDistantCatalyticSitesAreResponsibleforC2c2RNaseActivitiesGraphicalAbstractHighlightsdCrystalstructureofLeptotrichiashahiiC2c2-crRNAbinarycomplexandapoformdAbulge-containingcrRNAstemisessentialforC2c2RNaseactivitiesdHelical-1domainhasthecatalyticsiteforpre-crRNAprocessingdPromiscuousRNAcleavageduetodistancebetweenHEPNcatalyticsiteandcrRNAguideAuthorsLiangLiu,XueyanLi,JiuyuWang,...,JiazhiLi,GangSheng,[e-mail]alysisofC2c2revealsthatitsdualRNasecatalyticsiteslocateindependentlyintwophysicallydistantdomains,explainingitspromiscuouscleavageoftargetRNAandspecificprocessingofpre-CRISPRRNA.DataResources5WTJ5WTKLiuetal.,2017,Cell168,121–134January12,2017ª2017ElsevierInc.*1KeyLaboratoryofRNABiology,CASCenterforExcellenceinBiomacromolecules,InstituteofBiophysics,ChineseAcademyofSciences,Beijing100101,China2UniversityofChineseAcademyofSciences,Beijing100049,China3CollaborativeInnovationCenterofGeneticsandDevelopment,Shanghai200438,China4LeadContact*Correspondence:[e-mail]—oneforcuttingitsRNAtargetandtheotherforprocessingtheCRISPRRNA(crRNA).Here,wereportthestructuresofLep-totrichiashahiiC2c2initscrRNA-freeandcrRNA-boundstates.WhileC2c2hasabilobedstructurereminiscentofallotherClass2effectors,italsoex-hibitsdifferentstructuralcharacteristics.ItcontainstheREClobewithaHelical-1domainandtheNUClobewithtwoHEPNdomains.ThetwoRNasecata-lyticpocketsresponsibleforcleavingpre-crRNAandtargetRNAareindependentlylocatedonHeli-cal-1andHEPNdomains,respectively.crRNAbind-inginducessignificantconformationalchangesthatarelikelytostabilizecrRNAbindingandfacilitatetargetRNArecognition.ThesestructuresprovideimportantinsightsintothemolecularmechanismofdualRNaseactivitiesofC2c2andestablishaframe-workforitsfutureengineeringasaRNAeditingtool.INTRODUCTIONBacteriaandarchaeaareprotectedagainstinvadingnucleicacidsfromphagesandplasmidsbecauseofCRISPR(clusteredregularlyinterspacedshortpalindromicrepeats)-Cas(CRISPR-associatedproteins)systems,whichareRNA-guidedprokary-oticadaptiveimmunesystem(Barrangouetal.,2007;Barran-gouandMarraffini,2014;Marraffini,2015;vanderOostetal.,2014).CRISPR-Cassystemsarefoundinnearlyhalfofallbac-teriastudiedsofar,aswellasinthemajorityofarchaea.TheCRISPR-Cassystemdefendsagainstforeignnucleicacidsoriginatingfromvirusesandplasmidsinathree-stepprocess(vanderOostetal.,2014).ThefirstadaptationstepinvolvestheinsertionofafragmentoftheinvadingDNAintotheCRISPRlocus,thusgeneratingaspacerthatcapturesthepreviouslyinvadingnucleicacid.ThesecondstepinvolvescrRNAbiogenesis,wherebymaturecrRNAsaregeneratedfromalongpre-crRNAtranscript.Thispre-crRNAcontainsmulti-repeatsequencesflankingthespacers,whichareprocessedintoindividualshortmaturecrRNAconsistingofasinglespacer.Thefinalstepisinterference.ThecrRNAassembleswithCasproteinstoformtheeffectorcomplex,inwhichthecrRNAactsasaguidetorecognizethetargetDNA/RNAsequencebasedonthecomplementarityandCasproteinstoactasnucleasestocleavethetarget,thusdegradingtheinvadingforeignnucleicacids.Class2CRISPR-Cassystems,characterizedbyasingle-componenteffector,comprisetypeII,typeV,andtypeVIsys-tems.Cas9isadual-RNAguidedendonucleaseoftypeIICRISPR-Cassystems(Makarovaetal.,2015).Cas9cleavesthetargetdsDNAbyitsRuvCandHNHnucleasedomains(Ga-siunasetal.,2012;Jineketal.,2012).TypeVCRISPR-Cassys-temsuseasingleRuvCdomain-containingeffector,includingCpf1,C2c1,andC2c3,tocleavethetargetDNA(Shmakovetal.,2015).Cpf1isasingle-RNA-guidedDNAendonucleaseoftypeV-ACRISPR-Cassystem.C2c1isadual-RNA-guidedDNAendonucleaseoftypeV-BCRISPR-Cassystem.BothCpf1andC2c1cleavedouble-strandedtargetDNAthroughtheirRuvCdomainandaputativenucleasedomain,generatingaproductwithstaggeredends(Zetscheetal.,2015;Shmakovetal.,2015).AlloftheseClass2CRISPR-CasimmunesystemstargetDNAsubstrates.C2c2isanewlyidentifiedClass2typeVICRISPR-Cassystemendonuclease.SequenceanalysisrevealedthatC2c2lacksanidentifiableDNasecatalyticsite;instead,twoHEPNdomainscontaininghighlyconservedR-X-Hmotifwereidentified(Shma-kovetal.,2015).FurtherfunctionalstudiesrevealedthatC2c2isaRNA-guidedRNA-targetingCRISPReffector(Abudayyehetal.,2016).RecentbiochemicalanalysisfoundthatC2c2pos-sessesasecondRNaseactivity,whichisresponsibleforcrRNAmaturation(East-Seletskyetal.,2016).ThesetwoRNaseactiv-itiesofC2c2aremechanisticallydistinctfromeachother.WhilethesestudieshaveprovidedimportantbiochemicalinsightsofC2c2,thestructuralbasisofthedualRNaseactivitiesremainsunknown.TounderstandhowC2c2recognizesitscrRNAandtargetRNAandtoelucidatethemolecularmechanismofpre-crRNAprocessing,aswellascrRNA-guidedsinglestrandedRNAcleav-age,wedeterminedthecrystalstructureofLeptotrichiashahiiC2c2(LshC2c2)incomplexwithacrRNA,togetherwiththeCell168,