10.1261/rna.1611309Access the most recent version at doi: published on - PDF document

10.1261/rna.1611309Access the most recent version at doi: published online July 27, 2009RNA Rui Zhou, Benjamin Czech, Julius Brennecke, et al.Loquacious isoform endo-siRNAs depends on a specificDrosophilaProcessing of   MaterialSupplemental http://rnajournal.cshlp.org/content/suppl/2009/07/23/rna.1611309.DC1.htmlPPublished online July 27, 2009 in advance of the print journal.Open AccessFreely available online through the RNA Open Access option. serviceEmail alerting click heretop right corner of the article or object identifier (DOIs) and date of initial publication. publication). Advance online articles are citable and establish publication priority; they are indexedappeared in the paper journal (edited, typeset versions may be posted when available prior to final http://rnajournal.cshlp.org/subscriptions go to: RNATo subscribe to Copyright © 2009 RNA Society Cold Spring Harbor Laboratory Press on August 21, 2009 - Published by rnajournal.cshlp.orgDownloaded from Processingofendo-siRNAsdependsonaspecificLoquaciousisoformRUIZHOU,BENJAMINCZECH,JULIUSBRENNECKE,RAVISACHIDANANDAM,JAMESA.WOHLSCHLEGEL,NORBERTPERRIMON,andGREGORYJ.HANNONHarvardMedicalSchool,DepartmentofGenetics,HowardHughesMedicalInstitute,Boston,Massachusetts02115,USAWatsonSchoolofBiologicalSciences,HowardHughesMedicalInstitute,ColdSpringHarborLaboratory,ColdSpringHarbor,NewYork11724,DepartmentofBiologicalChemistry,DavidGeffenSchoolofMedicine,UniversityofCaliforniaatLosAngeles,LosAngeles,California90095,DrosophilamelanogasterexpressesthreeclassesofsmallRNAs,whichareclassifiedaccordingtotheirmechanismsofbiogenesis.MicroRNAsare22–23nucleotides(nt),ubiquitouslyexpressedsmallRNAsthataresequentiallyprocessedfromhairpin-likeprecursorsbyDrosha/PashaandDcr-1/Loquaciouscomplexes.MicroRNAsusuallyassociatewithAGO1andregulatetheexpressionofprotein-codinggenes.Piwi-interactingRNAs(piRNAs)of24–28ntassociatewithPiwi-familyproteinsandcanarisefromsingle-strandedprecursors.piRNAsfunctionintransposonsilencingandaremainlyrestrictedtogonadaltissues.Endo-siRNAsarefoundinbothgermlineandsomatictissues.These21-ntRNAsareproducedbyadistinctDicer,Dcr-2,anddonotdependonDrosha/Pashacomplexes.TheypredominantlybindtoAGO2andtargetbothmobileelementsandprotein-codinggenes.Surprisingly,asubsetofendo-siRNAsstronglydependfortheirproductiononthedsRNA-bindingproteinLoquacious(Loqs),thoughtgenerallytobeapartnerforDcr-1andacofactorformiRNAbiogenesis.Endo-siRNAproductiondependsonaspecificLoqsisoform,Loqs-PD,whichisdistinctfromtheone,Loqs-PB,requiredfortheproductionofmicroRNAs.ParallelingtheirrolesinthebiogenesisofdistinctsmallRNAclasses,Loqs-PDandLoqs-PBbindtodifferentDicerproteins,withDcr-1/Loqs-PBcomplexesandDcr-2/Loqs-PDcomplexesdrivingmicroRNAandendo-siRNAbiogenesis,respectively.Drosophilamelanogaster;Dicer;double-strandedRNA-bindingproteins(dsRBPs);Loquacious;endo-siRNAprocessing;transposonsilencingINTRODUCTIONDrosophilamelanogasterexpressesawidevarietyofsmallRNAs,whichareclassifiedbasedontheirmechanismofbiogenesisandtheArgonauteproteinstowhichtheybind.MicroRNAs(miRNAs)areaclassofubiquitouslyexpressedsmallRNAs,typically,22–23nucleotides(nt)inlength.Theyarederivedfromendogenoustranscriptscapableofforminghairpin-likestructures,whicharesequentiallyprocessedbyDrosha/PashaandDcr-1/Loqscomplexes(Leeetal.2003,2004;Denlietal.2004;Forstemannetal.2005;Jiangetal.2005;Saitoetal.2005).Theypredomi-nantlyassociatewithArgonaute-1(AGO1)andregulatetheexpressionofprotein-codinggenes(Bartel2004;BushatiandCohen2007;Eulalioetal.2008).Piwi-interactingRNAs(piRNAs),typically,24–28ntinlength,associatewithPiwi-familyproteins.TheexpressionofpiRNAsismainlyrestrictedtogonadaltissues,wheretheyfunctioninsilencingofmobileelementsandrepeats(Aravinetal.2007;Brenneckeetal.2007;Gunawardaneetal.2007;KlattenhoffandTheurkauf2008).Recently,athirdclassofendogenoussmallRNAswasidentifiedinboththegerm-lineandthesomaofDrosophila:endogenoussmallinter-feringRNAs(endo-siRNAs)(Czechetal.2008;Ghildiyaletal.2008;Kawamuraetal.2008;Okamuraetal.2008).Endo-siRNAsarepredominantly21ntinlengthandarederivedeitherfromlongendogenoustranscriptscapableofformingextensivefold-backstructures,orareprocessed Theseauthorscontributedequallytothiswork.Presentaddresses:IMBA–InstituteofMolecularBiotechnology,Dr.Bohr-Gasse3,1030Vienna,Austria;DepartmentofGeneticsandGenomicSciences,MountSinaiSchoolofMedicine,1425MadisonAvenue,NewYork,NY10029,USA.Reprintrequeststo:GregoryJ.Hannon,WatsonSchoolofBiologicalSciences,HowardHughesMedicalInstitute,ColdSpringHarborLabora-tory,ColdSpringHarbor,NY11724,USA;e-mail:hannon@cshl.edu;fax:(516)367-8874.Articlepublishedonlineaheadofprint.Articleandpublicationdateareathttp://www.rnajournal.org/cgi/doi/10.1261/rna.1611309.(2009),15:00–00.PublishedbyColdSpringHarborLaboratoryPress.Copyright2009RNASociety. Cold Spring Harbor Laboratory Press on August 21, 2009 - Published by rnajournal.cshlp.org Downloaded from fromdouble-strandedregionsformedbyintermolecularhybridizationofconvergentlytranscribedmRNAs.Endo-siRNAsusuallyjoinArgonaute-2(AGO2)andfunctionintheregulationofgeneexpressionandtransposonsilencing.Thebiogenesisofendo-siRNAsandmiRNAsdependsonanumberofproteincomplexescontainingRNAprocessingenzymesandtheirdsRNA-bindingprotein(dsRBPs)part-ners.InthecaseofmiRNAprocessing,thenucleartypeIIIribonucleaseDroshaassociateswiththedsRBP,Pasha,andprocessesprimarymiRNAtranscriptstopre-miRNAs(Leeetal.2003;Denlietal.2004;Gregoryetal.2004).Inasecondstep,thecytoplasmicDicerenzymeDcr-1,assistedbythedsRBPLoquacious(Loqs/R3D1),furtherprocessespre-miRNAstomaturemiRNAs(Forstemannetal.2005;Jiangetal.2005;Saitoetal.2005).Incontrast,processingoflongdsRNAprecursorsintosiRNAduplexesdependsonasecondDrosophilaDicerprotein,Dcr-2(Leeetal.2004).ThecanonicalDcr-2partnerR2D2seemsnottoberequiredfortheproductionofsiRNAs.Instead,itwasfoundtoimpacttheloadingofsiRNAduplexesintotheRNA-inducedsilencingcomplex(RISC)andproperguidestrandselection(Liuetal.2003;Tomarietal.2004).Ingeneral,itisbelievedthatthedsRBPscontributetothesubstratespecificityoftheirpartnerRNAprocessingenzymes.ThedsRNAbindingproteinLoquaciouswasidentifiedinDrosophilaasacomponentofacomplexthatalsocontainsthetypeIIIRNaseDicer-1(Dcr-1).GeneticexperimentssuggestedthatLoqswasrequiredforefficientmiRNAbiogenesis(Forstemannetal.2005;Jiangetal.2005;Saitoetal.2005;Liuetal.2007).LossofmainlyimpactedthefinalstepofmiRNAprocessingasindicatedbytheac-cumulationofpre-miRNAs,whichareformedbyDrosha/Pashacomplexes.MutationsinalsoreducedlevelsofasubsetofmaturemiRNAs,consistentwiththeimpactsoftheselesionsonviabilityandfertility(Forstemannetal.2005;Jiangetal.2005;Saitoetal.2005;Liuetal.2007;Parketal.2007;Yeetal.2007).Recently,itwasfoundthatlossofstronglyreducedlevelsofendogenoussiRNAs(endo-siRNAs)derivedfromstructuredlociinbothS2cellsandflies(Czechetal.2008;Okamuraetal.2008).Drosophila,thealternativesplicingofloqstranscriptswasreportedtoproducethreedistinctisoforms:loqs-RA,and(Forstemannetal.2005;Jiangetal.2005).Thesearetranslatedintothreeproteinisoforms,Loqs-PA,PB,andPC.istheisoformpredominantlyexpressedinovaries,whereasistheprincipalisoformfoundinmales.ThethirdmRNAisoform,,wasdetectedonlyinS2cells(Forstemannetal.2005).WhileLoqs-PBwassufficienttorescuethemiRNAprocessingdefectsofflies,Loqs-PAwasincapableofrestoringpropermiRNAprocessing(Parketal.2007),indicatingthattheseLoqsisoformshaddistinctfunctionsduringdevelopment.HereweexaminedtherolesofindividualLoqsisoformsindifferentsmallRNApathwaysandcharacterizedtheactivityofanovelLoqsisoform,Loqs-PD.WeshowthatcoordinateddepletionofallLoqsisoformsinculturedcellsaffectsthebiogenesisofbothmiRNAsandendo-siRNAs,whereascellssinglydepletedofLoqs-PBorLoqs-PDshowanimpactonlyonthemiRNAorontheendo-siRNApathway,respectively.Whilethere-expressionofLoqs-PDrestoredendo-siRNAlevelsinculturedcellsthathadbeendepletedofallLoqsisoforms,Loqs-PDwasincapableofrescuingmiRNAprocessingdefects.Moreover,weshowthatLoqs-PDpreferentiallyinteractswithDcr-2,theenzymeresponsiblefortheprocessingofallendo-siRNAspecies.Consideredtogether,ourstudiesdemonstratethatasingleLoquaciousisoform,Loqs-PD,isnecessaryandsufficientforthebiogenesisofseveraltypesofendogenoussiRNAs.RESULTSANDDISCUSSIONUsingquantitativeproteomicsofLoqsimmunoprecipitatesfromfliesandDrosophilaS2cells,weidentifiedphysicalinteractionsofLoqswithbothDcr-1andDcr-2(Czechetal.2008).InordertoidentifytheLoqsisoformsinvolvedintheseinteractions,weanalyzedourproteomicsdatafromimmunoprecipitates,preparedusinganantibodyspecifictotheNterminusofendogenousLoqsproteins,forisoform-specificpeptides.WefoundnosignificantpeptideevidenceforLoqs-PCinculturedcellsandflies,whereasbothisoformsPAandPBwerepresent.Inaddition,wedetectedpeptidescorrespondingtoanas-yet-uncharacterizedformofLoquacious.ThesecouldbeassignedtoanovelisoformidentifiedbyForstemannandcolleaguesandtermedLoqs-PD(Fig.1A,1B;JVHartig,SEsslinger,RBottcher,KSaito,andForstemannK,unpubl.).TocharacterizetherolesofindividualLoqsisoformsinsmallRNAbiogenesis,weexaminedisoform-specificeffectsonmiRNAandendo-siRNAprocessing.WedepletedvariouscombinationsofLoqsproteinsusingisoform-specificdsRNAsinDrosophilaS2cells(Fig.1A).TreatmentofS2cellswithdsRNAstargetingloqs-RA,orsinglyorincombinationledtoareductioninsteady-statelevelsofthecorrespondingtranscripts,asmeasuredbyRT-PCR(datanotshown).Inaddition,WesternblottingconfirmedthedepletionofspecificLoqsisoformsbythecorrespondingdsRNAs(Fig.1C).Notably,whileLoqs-PCandPDcouldnotbeefficientlyresolvedbyPAGE,cellstreatedwithdsRNAsspecificallytargetingthePDisoformwereeffectivelydepletedoftheLoqsproteinspeciesmigratingatthepositionofPC/PD(Fig.1C,lane11).ThisindicatedthattheLoqs-PCisoformwasnotdetectablyexpressedandconfirmedourfindingsfromthequantitativeproteomicanalysisofLoqsimmunoprecipitates.Weexaminedtheimpactofisoform-specificknock-downsonthebiogenesisofaprevalentendo-siRNA(Fig.)andamiRNA(Fig.1D,miR-bantam).Deple-tionofallisoformsupontreatmentwithdsRNAstargeting Zhouetal.RNA,Vol.15,No.10 Cold Spring Harbor Laboratory Press on August 21, 2009 - Published by rnajournal.cshlp.org Downloaded from FIGURE1.(Legendonnextpage) Cold Spring Harbor Laboratory Press on August 21, 2009 - Published by rnajournal.cshlp.org Downloaded from common5-UTRorORFsequencesresultedintheac-cumulationofthemiR-bantamprecursorandinastrongreductionofesi-2.1levels(Fig.1D,lanes6–8),whereasdepletionofisoformsPA,PB,andPCbydsRNAstargetingisoform-specific3-UTRsonlyaffectedmiRNAprocessing(Fig.1D,lane12).WeobservedmiRNAprocessingde-fectsinallcellstreatedwithdsRNAsthatcotargetedLoqs-PB(Fig.1D,lanes6–9,12–14),whereasthebiogenesisofendo-siRNAwasaffectedonlyifcellsweretreatedwithdsRNAsthattargetLoqs-PD,eithersinglyortogetherwithotherisoforms(Fig.1D,lanes6–11).Notably,depletionofLoqs-PDalonecausedastrongreductioninlevels,whilemiRNAprocessingwasunaffected(Fig.1D,lane11).Tovalidatethesefindings,wedepletedallLoqsisoformsinS2cellsusingdsRNAtargetingshared5-UTRsequencesandtestedwhetherthesubsequentintroductionofRNAi-resistantORFsdirectingexpressionofindividualLoqsisoformswascapableofrescuingdefectsinendo-siRNAormiRNAprocessing.Incontrolcells,depletionofallLoqsisoformsledtoasignificantreductioninlevelsof(Fig.1E,lanes3,4),whereasdsRNAstargeting-or-UTRsortargetingORFscausedamoderateaccumula-tionofthemiR-bantamprecursor(Fig.1E,lanes3–5).TheexpressionofLoqs-PA,Loqs-PC,orR2D2failedtorescueanyobservedbiogenesisdefect(Fig.1E,lanes6–10,16–20,26–30).Incontrast,there-expressionofLoqs-PBeffectivelyrescuedmiRNA-processingdefects(Fig.1E,cf.miR-bantamlevelsinlanes14,15andlane13).How-ever,Loqs-PBfailedtorestorenormalendo-siRNAlevels(Fig.1E,lane14).There-expressionofLoqs-PDrestoredlevelsofesi-2.1inLoqs-depletedcells(Fig.1E,lane24)butwasincapableofrescuingmiRNA-processingdefects(Fig.1E,cf.lanes24,25andlane23).OurobservationthattheexpressionofR2D2wasincapableofrescuinganysmallRNA-processingdefectcausedbydepletionofLoqsstronglysuggeststhatR2D2andallLoqsisoformscannotfunctioninaredundantmanner.Consideredtogether,thesedataindicatethatonlytheLoqs-PBisoformisrequiredforthebiogenesisofmiRNAsandsuggestthatonlyLoqs-PDisessentialforendo-siRNAproduction.Tosupportresultsemergingfromcellculture,wealsoexaminedtherolesofLoqsisoformsinvivo.Fliesexpress-ingtheLoqs-PBisoformunderthecontrolofitsendoge-nousregulatoryelementscouldrescueboththepre-miRNA-processingdefectsandthepronouncedphenotypes-nullanimals(Parketal.2007).Strikingly,homozy-gousmutantfliescarryingaLoqs-PBtransgenedidnotregainnormallevelsoftheendo-siRNA,,butdidexpressnormalamountsof(SupplementalFig.S1A).Similarly,theintroductionofLoqs-PBalsorestorednormallevelsinhomozygousmutantflies,whereasendo-siRNAbiogenesisdefectswerenotaffected(Supple-mentalFig.S1B).TheexpressionofLoqs-PAinhomozygousmutantflieswasincapableofrestoringeithermiRNAorendo-siRNAprocessing(SupplementalFig.S1B).Theseresultsareconsistentwiththeobservationfromcell-basedstudiesshowingthatneitherLoqs-PAnorPBisrequiredfortheendo-siRNApathway.WeconcludethatLoqs-PBisrequiredformiRNAbiogenesisinmultiplecelltypes,whereasLoqs-PDsupportsendo-siRNAbio-genesis.Loqs-PCseemsneithertobeexpressedatsignifi-cantlevelsnortoimpactsmallRNAbiogenesis.ThefunctionofLoqs-PA,whichisexpressedbothinculturedcellsandinanimals,remainselusive.GiventheunexpectedrequirementforLoqsinthebiogenesisofendogenoussiRNAs(endo-siRNAs)(Czechetal.2008;Okamuraetal.2008)andthephysicalinteractionbetweenLoqsandbothDcr-1andDcr-2(Czechetal.2008),wesoughttoinvestigatewhetherindividualLoqsisoformsmightshowspecificityforeitherDicerprotein.ImmunoprecipitationofFlag-taggedDcr-2fol-lowedbyimmunoblottingwithanantibodyspecifictotheN-terminusofendogenousLoqs,andthusrecognizingallknownLoqsisoforms,revealedastrongsignalcorrespond-ingtothemolecularweightofLoqs-PC/PD(Fig.2A,lane2).SincethelevelsofendogenousLoqs-PCinS2cellsarenegligible,thepredominantDcr-2-interactingendogenousLoqsisoformappearstobeLoqs-PD.Next,weexaminedtheinteractionsbetweenvariousLoqsisoformsandDcr-2byexpressingFlag-taggedDcr-2togetherwithT7-taggedLoqsisoformsinS2cells.Cellextractsweresubjectedtoanti-Flagimmunoprecipitation,andcoimmunoprecipi-tatedproteinsweredetectedusingananti-T7antibody.AllfourtaggedLoqsisoformsandthepositivecontrolT7-R2D2wereabletointeractwithDcr-2(Fig.2B,lanes7–11), FIGURE1.AspecificLoqsisoform,Loqs-PD,isrequiredforthebiogenesisofendo-siRNAsderivedfromstructuredloci.()ShownisaschemeoftheannotatedgenomicstructureofthefourisoformsofLoquacious.Theregionstargetedbyisoform-specificdsRNAsareindicated.(Thinlines)Introns;(thinboxes)UTRs;(thickboxes)ORFs.()AschemeshowingdomainstructuresandmolecularweightsoftheLoqsisoforms.(Smallboxes)dsRNA-bindingdomains.()Westernblotsshowingsteady-stateproteinlevelsofLoqsisoformsupontreatmentofS2cellswiththeindicateddsRNAs.-Tubulinservedasaloadingcontrol.ToincreasethelengthofdsRNAstofacilitatedsRNAuptakebyS2cells,somedsRNAswerefusedwithacarriersequence(indicatedby‘‘-Z’’).()Northernblotsprobinglevelsofanendo-siRNAderivedfromastructuredlocus,,andthemicroRNA(pre-miRNAindicatedbytheasterisk)inS2cellstreatedwithdsRNAagainsttheindicatedgenes(asin).Asaloadingcontrol,themembranewasstrippedandre-probedfor2SrRNA.()ExpressionofLoqs-PDissufficienttorescueendo-siRNAprocessingdefects,whileonlyLoqs-PBrestorespropermiRNAbiogenesis.ControlcellsexpressingtheTAPepitopealoneorthoseexpressingvariousTAP-taggedproteinsweretreatedtwicewithvariousdsRNAs(asindicatedonofthepanel)foratotaldurationof8d.Theexpressionofthetransgeneswascontrolledbythebasalactivityofthepromoter.TotalRNAswerepreparedandsubjectedtoNorthernblottingusingprobesagainstmiR-bantam,and2SrRNA. Zhouetal.RNA,Vol.15,No.10 Cold Spring Harbor Laboratory Press on August 21, 2009 - Published by rnajournal.cshlp.org Downloaded from whilenointeractionwasdetectedforthenegativecontrolT7-Ran(Fig.2B,lane12).Therefore,Dcr-2seemscapableofinteractingwithallisoformsofLoqsiftheyareover-expressed.Interestingly,wealsofoundthatLoqsisoformsarecapableofforminghomo-andheterodimers(orevenoligomers)witheachother,asFlag-taggedLoqsisoformsPA,PB,andPCarecapableofpullingdownT7-taggedLoqs-PAincoimmunoprecipitationassays(SupplementalFig.S3;datanotshown).ThiscouldaccountfortheapparentlackofspecificityintheinteractionbetweenDcr-2withindividualLoqsisoformsuponoverexpression.WealsoprobedtheinteractionbetweenLoqsisoformsandDcr-1.Wedetectedrobustinteractionbetweenendog-enousDcr-1andTAP-Loqs-PBandaweakinteractionbetweenDcr-1andTAP-Loqs-PA(Fig.2C,lanes1–4).Incontrast,noendogenousDcr-1wasdetectedintheTAP-Loqs-PC,TAP-Loqs-PD,orTAP-R2D2complexes.Totestwhethertheobservedbindingresultedfromprotein–proteininteractionorfromLoqsandDicerpro-teinsbindingindependentlytothesameRNAsubstrates,wetreatedimmunoprecipitateswithRNase.Thishadnosignificanteffectontheobservedinteractions(Fig.2C;datanotshown).Overexpressionofneitherapri-miRNA(pri-miR-bantam),noraprecursoroftheendo-siRNAcluster,alteredpatternsofdifferentialaffinity(datanotshown),indicatingthatourexperimentsdetectedRNA-independentprotein–proteininteractions.Therefore,weconcludethattwoLoqsisoforms,Loqs-PBandtoalesserextentLoqs-PA,arecapableofinteractingwithDcr-1,aresultconsistentwithfindingsreportedbyForstemannetal.(2005).Incontrast,Dcr-2predominantlyinteractswithendogenousLoqs-PD,whileotherisoformsinteractwithDcr-2toasignificantextentonlywhenoverexpressed.Consideringtherestrictionofloqs-PCtranscriptstoS2cellsrstemannetal.2005)andthenegligibleproteinlevelsofLoqs-PC,weconcludethatendogenousLoqs-PDisthepredominantisoformthatinteractswithDcr-2.InordertoinvestigatetheeffectsofLoqsdepletiononsmallRNAprofilesinmoredetail,wepreparedsmallRNAlibrariesfromS2cellstreatedwithloqs-ORFdsRNAsandfromcontrolknockdowncells,including,anduntreatedcells(‘‘mock’’).Theseweredeep-sequencedusingtheIlluminaplatform.Normalizedcloningcountswereplottedbyreadlengthtocreatesizeprofiles(Fig.3A).TheindicatedsmallRNAcategorieswereisolatedfromthetotallibrarybioinformatically(Czechetal.2008).MaturemicroRNAspopulatedabroadpeakcenteredaround22–23nt,andtheseweredecreasedmostpromi-nentlyintheknockdown.MicroRNAsappearedtobeslightlyincreasedinloqs-ORFdcr-2knockdownsascomparedto(Fig.3A),probablybecauseofanartifactoflibrarynormalizationduetothestrongimpactonendo-siRNAlevels(Figs.1D,3B).Endo-siRNAsmappingtooverlappingtranscripts(exonicantisense)werestronglyreducedinloqs-ORFknockdowns(Fig.3A).Amoderatereductionwasalsoobservedforendo-siRNAsderivedfromtheklarsichtlocusuponLoqsdepletion,whileknockdownshadmoreprominentimpacts.Finally,knockdownsofcausedasubstantial(50%)reductioninendo-siRNAscorrespondingtorepeatandtransposonsequences,whilethelevelsofthesesmallRNAsremainedunchangedinallotherknockdownstested(Fig.WevalidatedresultsfromsmallRNAsequencingbyNorthernblotting.Wesawdecreasedlevelsofthreeindependentendo-siRNAsderivedfromstructuredlociesi-1.2esi-2.1)inloqs-ORFknockdowns,whileknockdownofhadnosimilarimpact(Fig.3B).ConsistentwiththesesmallRNAsequencingandNorthernblottingresults,depletionofallLoqsisoforms, FIGURE2.InteractionofLoqsisoformswithDicerproteins.()Flag-taggedDcr-2coimmunoprecipitatesendogenousLoqs-PD.Flag-Dcr-2immunoprecipitatesweresubjectedtoimmunoblottingusingantibodiesagainsttheNterminusofendogenousLoqsandagainsttheFlagepitope.)Flag-Dcr-2wasexpressedinS2cellstogetherwithT7-taggedR2D2orvariousLoqsisoforms.T7-Ranservedasacontrol.Flag-Dcr-2wasimmunoprecipitatedusingananti-Flagantibody,andtheinteractionwithcoexpressedproteinswasexaminedbyWesternblottingusingananti-T7antibody.()CoimmunoprecipitationofendogenousDcr-1withTAP-taggedLoqsisoforms.TAP-taggedproteinswereexpressedinS2cells,immunoprecipitatedusingantibodiesagainsttheTAPepitope,andsubjectedtoimmunoblottingusingantibodiesagainstendogenousDcr-1andtheTAPtag,respectively.RNasetreatment(asindicatedonofthepanel)revealedthatthestronginteractionbetweenDcr-1andLoqs-PBaswellastheweakinteractionbetweenDcr-1andLoqs-PAareRNA-independent. DistinctfunctionsofLoquaciousisoforms Cold Spring Harbor Laboratory Press on August 21, 2009 - Published by rnajournal.cshlp.org Downloaded from butnotdepletionofLoqs-BCorLoqs-C,causedade-repressionofaRenillaluciferasesensorforesi-2.1(Sup-plementalFig.S2).WealsonotedareductionofklarsichtsiRNAsandsequencesderivedfromthetransposonDM1731loqs-ORFknockdown,butnotuponLoqs-BCdepletion(Fig.3B).ProbingthesamemembraneforthreemiRNAsrevealedaslightreductioninmaturemiRNAlevelsandaslightbutdetectableaccumulationofpre-miRNAsindcr-1loqs-ORF,andknockdowncells(Fig.3B).Consideredtogether,theseresultssupporta FIGURE3.Loqsisinvolvedinthebiogenesisofseveralclassesofendo-siRNAs.()LengthprofilesforsmallRNAsisolatedfromS2cellstreatedwiththeindicateddsRNAsareshown.KnownmiRNAsanddistinctclassesofendo-siRNAs(asindicated)weresplitcomputationally,andnormalizedcloningcountswereplottedovertheirlength.()NorthernblotsshowlevelsofmiRNAs(pre-miRNAsindicatedbyasterisks),endo-siRNAsderivedfromstructuredloci,theLTRtransposon,andtheS2cell-specificlocusinS2cellstreatedwiththeindicateddsRNAs.2SrRNAservedasacontrolforequalloading. Zhouetal.RNA,Vol.15,No.10 Cold Spring Harbor Laboratory Press on August 21, 2009 - Published by rnajournal.cshlp.org Downloaded from dependenceofvariouscategoriesofendo-siRNAsontheLoqs-PDisoform.Giventheeffectofloqs-ORFknockdownsonrepeatendo-siRNAs,weprobedthepotentialimpactofmutationsontransposonsilencing.Allendo-siRNAsthatcorrespondtorepeatsandthatwere21ntinlengthwereextractedbioinformatically,andheatmapsshowingtheirrelativeabundancewerecreatedfortheindicatedlibraries(Fig.4A).Knockdownofcausedareductionofendo-siRNAsequencesforthemajorityoftransposableelementsascomparedtothenumberofreadsinthelibrary,whileuntreatedcells(‘‘mock’’),downs,showedonlyminor,ifany,effects.DepletionofallLoqsisoformsreducedlevelsofrepeatendo-siRNAs,whichatleastinpartcorrelatedwithdcr-2depletion.Interest-ingly,wenotedthatknockdownofshowedaweakimpactonthoserepetitiveelementsthatdidnotdependonLoqsdepletion.PlottingtheabundanceofrepeatsiRNAsinLoqsorDcr-2-depletedcellsagainstthoseinthecontrollibraryshowedsimilarpatterns,withsomesiRNAsappearingunaffectedandothersshowingdrasticreductionloqs-ORFknockdowns(Fig.4B,C).Plottingthenormalizedreadnumberofendo-siRNAsmatchingtothetransposableelementDM1731,whichshowsthehighestlevelsoftransposon-derivedendo-siRNAsinS2cells,for,andloqs-ORFlibrariesalsoshowscleardependenceon(Fig.4D).Interestingly,depletionofDcr-2andLoqsdoesnotshowidenticalimpactsonthepatternsofindividualsequencesoverthisrepresentativetransposon(Fig.4D).Thiscouldindicatemultipleoverlappingbiogenesispathwaysordifferentialaffinitiesofonemachineryfordifferentprocessingsites.PreviousstudiesindicatedthatDcr-1preferentiallybindsLoqs-PB(Forstemannetal.2005;Yeetal.2007),andthatDcr-2iscapableofinteractingwithLoqsproteins,althoughisoformswerenotexaminedinthatstudy(Czechetal.2008).HerewehavecharacterizedtheroleofanovelLoqsisoform,Loqs-PD,inthebiogenesisofvariouscategoriesofendo-siRNAs.WepresentevidencethatLoqs-PD,inadditiontothecanonicalpartnerR2D2(Liuetal.2003),isthepredominantinteractingpartnerforDcr-2inS2cells.OurgeneticstudiesclearlydemonstratethedifferentialrequirementforLoqs-PDandLoqs-PBinthebiogenesisofendo-siRNAsandmiRNAs,respectively.Thus,itislikelythatbesidestheintrinsicsubstratespecificityoftheindi-vidualDicerproteins,theidentityofDicerinteractingcofactorsisalsocrucialforthebiogenesisofdifferentclassesofsmallRNAs.AlthoughsmalldsRNA-bindingproteinsareclearlyimportantforsmallRNAproductionandactascofactorsforDicerandDroshaenzymes,theirprecisebiochemicalrolesremainunclear.OurstudiesaddcomplexitytothelandscapeoftherolesofdsRBPsinsmallRNAbiogenesisandraisefundamentalquestionsaboutwhysomanydistinctformsoftheseproteinsarerequiredforthesepathwaystooperate.MATERIALSANDMETHODSDNAconstructs500-base-pairDNAfragmentencompassingthesequencewasamplifiedbyPCRandclonedintopRmHa-3usingBamHI/EcoRIsitestogeneratepRmHa-3-Bantam.ApairofoligoscontainingthreeimperfectbindingsitesforwereannealedandclonedintopRmHa-3-(Zhouetal.2008)usingSalItogeneratethesensorconstructsfor-Bantamsensor).Togenerateepitope-taggedexpressionconstructs,DNAfragmentsencodingepitope-taggedproteinswereamplifiedbyPCRandclonedintopRmHa-3usingthefollowingrestrictionenzymes(SacI/KpnIforFlag-Dcr-2;EcoRI/BamHIforallT7-taggedLoqsisoforms;KpnI/BamHIforT7-R2D2;EcoRI/BamHIforT7-Ran).ForTAP-taggedvectors,apairofoligoswasannealedandclonedintopMK33-NTAP(Veraksaetal.2005)usingXhoIandBamHIsitestogeneratepMK33-RZ-NTAP.DNAfragmentsencodingthecorrespondingproteinswereeitheramplifiedbyPCRandclonedintopMK33-RZ-NTAPusingthefollowingrestrictionenzymes(SacI/KpnIforLoqs-PA,PC,andPD),orsubclonedfromtheFlag-taggedpRmHa-3constructsusingthefollowingrestrictionenzymes(KpnI/BamHIforR2D2;EcoRIblunt/BamHI-treatedcDNAfragmentclonedintoSacIblunt/BamHI-digestedvectorforLoqs-PBandRan).DNAoligonucleotidesequencesarelistedinSupplementalTableS1.ImmunoprecipitationandimmunoblottingCellsweretransfectedwithexpressionconstructsforepitope-taggedproteins,inducedwith500MCuSO2daftertrans-fection,andharvestedanother24hlater.ImmunoprecipitationandRNasetreatmentwereperformedaspreviouslydescribed(Czechetal.2008;Zhouetal.2008).TheimmunoprecipitatedsampleswereresolvedbySDS-PAGE,transferredtonitrocellulosemembranes,andprobedwithantibodiesagainsttheT7epitope(Novagen),theFlag-epitope(Sigma),theTAP-tag(OpenBiosys-tems),orrabbitantibodiesagainstDcr-1orLoqs.-Tubulinwasdetectedusingananti--tubulinantibodyraisedinmouse(Sigma).CellcultureanddsRNAtreatmentS2-NPcellsweremaintainedinSchneider’smedium(Invitrogen)supplementedwith10%FBSand1%pen-strep(Invitrogen).FordsRNAtreatment,S2-NPcellsweresoakedin1.5mLofserum-freeSchneider’smediumcontaining10gofdsRNAsin6-wellplates,and3mLofserum-containingmediumwasadded45minlater.After4dofinitialdsRNAtreatment,cellsweretreatedwithdsRNAsforasecondroundandharvestedanother4dlater.SequencesoftheprimersforgenerationthedsRNAsarelistedinSupplementalTableS1.RT-PCRTomeasurelevelsofvarioustranscriptsupondsRNAtreatment,totalRNAfromS2cellswasextractedusingTrizol(Invitrogen).RNAwastreatedwithRQ1DNase(Promega)accordingtothemanufacturer’sinstructions.cDNAwassynthe-sizedbyreversetranscriptionusingQuantiscriptreversetran-scriptase(QIAGEN)andamixtureofoligo-dTandrandom DistinctfunctionsofLoquaciousisoforms Cold Spring Harbor Laboratory Press on August 21, 2009 - Published by rnajournal.cshlp.org Downloaded from hexamerprimers.cDNAwasamplifiedusingprimersthatamplifyallthreeisoforms(productsvarybysize).ThePCRproductswereseparatedbyagarosegelelectrophoresis.Analysiswascarriedoutwithtwobiologicalreplicatespersample.ThesequencesoftheDNAoligonucleotidesusedinthisstudyarelistedinSupplemen-talTableS1. FIGURE4.DepletionofLoqsinS2cellsresultsinreducedlevelsofendo-siRNAsderivedfromrepeatsandtransposableelements.()Heatmapsingrayscaleshowcloningfrequenciesof21-nt-sizesiRNAsmatchingindividualtransposonsinS2cellstreatedwithdsRNA.Foldchangesofcloningcountsrelativetothoseinthelibraryareshownincolor(red-greenscale)inlogscale.Notethatonlytransposonspassingacloningcountthresholdof5inasinglelibraryand500inalllibrariestogetherareshown.()AllsiRNAsmatchingtothetransposonsindicatedinwithmorethan50readsinthelibraryareplottedagainstthelibraries,respectively.()All21-ntendo-siRNAsinthe,andlibrariesthatmatchtothetransposableelementareplottedoverthetransposonsequencewiththeirnormalizedcloningcountsonthe Zhouetal.RNA,Vol.15,No.10 Cold Spring Harbor Laboratory Press on August 21, 2009 - Published by rnajournal.cshlp.org Downloaded from NorthernblottingTotalRNAwasisolatedusingTrizol(Invitrogen).Twentymicro-gramsoftotalRNAfromS2cellsor40goftotalRNAfromflieswasseparatedon15%denaturingpolyacrylamidegelsandtransferredontoHybond-Nmembranes(AmershamBioscien-ces)in0.5TBE.RNAwasUVcross-linkedtothemembraneandpre-hybridizedinULTRAhyb-Oligobuffer(Ambion)for1h.DNAprobes(sequencesarelistedinSupplementalTableS1)complementarytotheindicatedendo-siRNAs,miRNAs,and2SrRNAwere5radio-labeledandaddedtothehybridizationbuffer(hybridizationovernightat30C).Membraneswerewashedfourtosixtimesin1SSCwith0.1%SDSat30CandexposedtoPhosphorImagerscreensfor12–72h.Probeswerestrippedbyboilingthemembranetwicein0.2SSCcontaining0.1%SDSinamicrowave.ReporterassaysTransfectionswereperformedin384-wellplateformat.Foreachwell,atotalof100ngofplasmidDNAwastransfected.Tosensoractivity,100ngofpRmHa-3-sensor(Czechetal.2008)and2ngofpRmHa-3-Firefly-long(Zhouetal.2008)(asacontrolfortransfectionefficiency)weretransfected.Tomeasuretheactivityofendogenous2ngofpRmHa-3-Firefly-long,50ngofpRmHa-3-Bantamsensor,and50ngofpRmHa-3(servingascarrierDNA)weretransfected.TheactivityofoverexpressedmiR-bantammeasuredbytransfecting2ngofpRmHa-3-Firefly-long,80ngofpRmHa-3-Renilla-Bantamsensor,and20ngofpRmHa-3-Bantam.Foreachwell,DNAwasmixedwith0.8LofEnhancerin15LofEC(QIAGEN)andincubatedfor5minatroomtemperature.Then0.35LofEffectenereagentwasadded,andthemixturewasimmediatelydispensedintoeachwellcontainingngofdsRNA.Afterincubationfor10minatroomtemperature,LofS2-NPcells(10cells/mL)weredispensedintothewell.Cellswereinducedwith200MCuSO144hpost-transfection,andluciferaseassayswereperformed24hlaterusingDualGloreagents(Promega).Foreachwell,thereporteractivity,referredtoasrelativeluciferaseunits(RLU),wascalculatedastheratioofRenillaluciferasetofireflyluciferase.TocalculatetheeffectofdsRNAtreatmentontheactivityofspecificsensors,thedatapointswerefirstnormalizedagainstcorrespondingdatapointswherepRmHa-3-Renillawastransfected(servingasno-sitecontrol),thennormalizedagainstsamplestransfectedwithdsRNAagainstLacZ.SmallRNAlibrariesSmallRNAsfromtotalRNAwereclonedasdescribed(Brenneckeetal.2007)(detailedprotocolavailableuponrequest).ThefollowingsmallRNAlibrariesfromtotalRNAwerepreparedforthisstudy:19–24ntfromuntreatedS2cells(‘‘mock’’);19–24ntfromS2cellstreatedwithdsRNAagainstDcr-1;19–24ntfromS2cellstreatedwithdsRNAagainstDcr-2;19–24ntfromS2cellstreatedwithdsRNAagainstLoqs-ORF;19–24ntfromS2cellstreatedwithdsRNAagainstR2D2;and19–24ntfromS2cellstreatedwithdsRNAagainstLacZ.LibrariesweresequencedusingtheIlluminasequencingplatform.SmallRNAsequencesweredepositedintheGeneExpressionOmnibus(www.ncbi.nlm.nih.gov/geo/)underaccessionnumberBioinformaticsanalysisofsmallRNAlibrariesSmallRNAlibrarieswereanalyzedasdescribed(Czechetal.2008).SmallRNAsequenceswerematchedtotherelease5genomeandgenomesofCvirus,Flockhousevirus,andCricketparalysisvirus.Onlyreadsperfectlymatchingtheflygenomeormatchingtoviralgenomeswithuptothreemismatcheswereusedforfurtheranalysis.ForannotationsweusedFlyBaseforprotein-codinggenes,UCSCfornon-codingRNAsandtransposons/repeats,andthemostrecentmiRNAcatalog(Rubyetal.2007;Starketal.2007).FlystocksfliesandfliesexpressingLoqs-PAandLoqs-PBisoformswereakindgiftofQinghuaLiu(UniversityofTexasSouthwesternMedicalCenter)(Parketal.2007).ThehypomorphicflieswereobtainedfromBloomington(stock#18371).AGO2flieswereakindgiftofHaruhikoSiomi(KeikoUniversitySchoolofMedicine)(Okamuraetal.2004),dcr-2flieswereakindgiftofRichardCarthew(NorthwesternUniversity)(Leeetal.2004),andr2d2flieswereakindgiftofDeanSmith(UniversityofTexasSouthwesternMedicalCenter)(Liuetal.2003).Fliesweredouble-balanced[double-balancerstock(CyOTM6C),agiftfromPhilZamore(UniversityofMassachusettsMedicalCenter)],thenhomozygousandheterozygotesflieswerecollected.Stock#2057fromBloomington(Celerasequencingstrain)wasusedaswildtype.SUPPLEMENTALMATERIALSupplementalmaterialcanbefoundathttp://www.rnajournal.org.ACKNOWLEDGMENTSWethankJ.V.HartigandK.Forstemannandcolleaguesforcommunicatingdatapriortopublication.WealsothankQ.Liu,R.Carthew,H.Siomi,D.Smith,andP.D.Zamoreforreagents.WearegratefultomembersoftheHannonandPerrimonlaboratoriesforhelpfuldiscussion,andtoM.RooksandD.McCombieforhelpwithdeepsequencing.R.Z.isaSpecialFellowoftheLeukemiaandLymphomaSociety.B.C.issupportedbyaPhDfellowshipfromtheBoehringerIngelheimFonds.ThisworkwassupportedinpartbygrantsfromtheNIHtoN.P.andG.J.H.,andagiftfromK.W.Davis(toG.J.H.).N.P.andG.J.HareinvestigatorsoftheHowardHughesMedicalInstitute.ReceivedFebruary19,2009;acceptedJune30,2009.REFERENCESAravinAA,HannonGJ,BrenneckeJ.2007.ThePiwi–piRNApathwayprovidesanadaptivedefenseinthetransposonarmsrace.BartelDP.2004.MicroRNAs:Genomics,biogenesis,mechanism,andBrenneckeJ,AravinAA,StarkA,DusM,KellisM,SachidanandamR,HannonGJ.2007.DiscretesmallRNA-generatinglociasmaster 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