Comprehensive Identification of mRNA-Binding Proteins of Leishmania donovani by Interactome ...

16 Pages·2016·1.39 MB·English

RESEARCHARTICLE
Comprehensive Identification of mRNA-
Binding Proteins of Leishmania donovani by
Interactome Capture
DevkiNandan1,SnehaA.Thomas1,AnneNguyen1,Kyung-MeeMoon2,LeonardJ.Foster2,
NeilE.Reiner1,3*
1 DepartmentsofMedicine,UniversityofBritishColumbia,Vancouver,BC,Canada,2 UniversityofBritish
Columbia,CentreforHigh-ThroughputBiologyandDepartmentofBiochemistry&MolecularBiology,
Vancouver,BC,Canada,3 DepartmentofMicrobiologyandImmunology,UniversityofBritishColumbia,
a1111111111
Vancouver,BC,Canada
a1111111111
a1111111111 *[email protected]
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Abstract
Leishmaniaareunicellulareukaryotesresponsibleforleishmaniasisinhumans.Likeother
trypanosomatids,leishmaniaregulateproteincodinggeneexpressionalmostexclusivelyat
OPENACCESS
thepost-transcriptionallevelwiththehelpofRNAbindingproteins(RBPs).Duetothepres-
Citation:NandanD,ThomasSA,NguyenA,Moon
enceofpolycystronictranscriptionunits,leishmaniadonotregulateRNApolymeraseII-
K-M,FosterLJ,ReinerNE(2017)Comprehensive
IdentificationofmRNA-BindingProteinsof dependenttranscriptioninitiation.Recentevidencesuggeststhatthemaincontrolpointsin
LeishmaniadonovanibyInteractomeCapture. geneexpressionaremRNAdegradationandtranslation.Protein-RNAinteractionsare
PLoSONE12(1):e0170068.doi:10.1371/journal.
involvedineveryaspectofRNAbiology,suchasmRNAsplicing,polyadenylation,localiza-
pone.0170068
tion,degradation,andtranslation.Adetailedpictureoftheseinteractionswouldlikelyprove
Editor:ThomasPreiss,JohnCurtinSchoolof
tobehighlyinformativeinunderstandingleishmaniabiologyandvirulence.Wedevelopeda
MedicalResearch,AUSTRALIA
strategyinvolvingcovalentUVcross-linkingofRBPstomRNAinvivo,followedbyinterac-
Received:August12,2016
tomecaptureusingoligo(dT)magneticbeadstodefinecomprehensivelythemRNAinter-
Accepted:December28,2016 actomeofgrowingL.donovaniamastigotes.Theproteinmassspectrometryanalysisof
Published:January30,2017 capturedproteinsidentified79mRNAinteractingproteinswhichwithstoodverystringent
washingconditions.Strikingly,wefoundthat49ofthesemRNAinteractingproteinshadno
Copyright:©2017Nandanetal.Thisisanopen
accessarticledistributedunderthetermsofthe orthologsorhomologsinthehumangenome.Consequently,thesemayrepresenthigh
CreativeCommonsAttributionLicense,which qualitycandidatesforselectivedrugtargetingleadingtonoveltherapeutics.Theseresults
permitsunrestricteduse,distribution,and
showthatthisunbiased,systematicstrategyhasthepromisetobeapplicabletostudythe
reproductioninanymedium,providedtheoriginal
mRNAinteractomeduringvariousbiologicalsettingssuchasmetabolicchanges,stress
authorandsourcearecredited.
(lowpHenvironment,oxidativestressandnutrientdeprivation)ordrugtreatment.
DataAvailabilityStatement:Themass
spectrometryproteomicsdatafilesavailablefrom
ProteomeXchangeConsortiumidentifier
PXD005601.
Introduction
Funding:ThisworkwasfundedbyGrantsMOP–
125879toNERand77688toLJFfromthe Regulationofgeneexpressionrequiresanarrayofcoordinatedmechanismsthatareusedby
CanadianInstitutesforHealthResearch.The
cellstomodulatetheproductionofspecificgeneproducts.Ineukaryoticcells,generegulation
fundingagencieshadnoroleindesign,collection,
startsattheleveloftranscriptioninitiation,followedbyregulationatthepost-transcriptional
analysis,orinterpretationofdata;neitherwerethey
level.NascentmRNAsaresubjecttoregulationviaRNAprocessing,transport,localization,
involvedinwritingthemanuscript;orinthe
decisiontosubmitthemanuscriptforpublication. translationanddegradation.Duringbiosynthesisandaftercompletionoftranscription,
PLOSONE|DOI:10.1371/journal.pone.0170068 January30,2017 1/16
IdentificationofLeishmaniaInVivomRNA-Interactome
CompetingInterests:Theauthorshavedeclared nascentmRNAsassociatewithRNAbindingproteinstoformmessengerribonucleoprotein
thatnocompetinginterestsexist. (mRNP)complexesthatregulatemostaspectsofmRNAmetabolismandfunction.Intheir
dynamicassociationwithmRNPcomplexes,arepertoireofRBPsplayscriticalrolesinregulat-
ingRNAprocessing,transport,localization,translationanddegradation.Infact,thereisevi-
dencefortheexistenceofanextensivenetworkofRBPs,whichthroughcombinatorialbinding
affectsthepost-transcriptionalfateofeverymRNAinthecell[1–4].Itisbecomingincreas-
inglyclearthatpost-transcriptionalprocessesplayequallyimportantrolesintheoutputof
geneproductswhencomparedtotranscriptionalcontrol.Notably,Leishmaniaisonegenus
thathasevolvedtobypasstheregulationofgeneexpressionatthetranscriptioninitiationstage
[5].
Leishmaniaarethecausativeagentsofleishmaniasesinhumans.Transmittedbysandflies,
theseinfectionsareresponsibleforseveremorbidityandmortality,worldwide.Todate,there
arenoeffectivevaccinesagainsttheseparasites.Moreover,currentlyavailabledrugshavesig-
nificanttoxicitiesandovertime,theseorganismsareknowntodevelopdrugresistance.Leish-
maniahaveadigeneticlife-cyclealternatingbetweentheirmammalianhosts(amastigote
stage)andtheirsandflyvectors(promastigotestage).Adaptationtodistinctenvironments
requiresactivationofspecificgenenetworks.Asdescribedabove,mosteukaryotesactivate
genenetworksbytranscriptionalmechanismssingularly,orinparallelwithpost-transcrip-
tionalregulatorymechanisms.However,inleishmaniaandotherrelatedtrypanosomatids,
theseresponsesappeartobeaccomplishedmainlybypost-transcriptionalmechanisms[5].In
thissetting,RNA-bindingproteins(RBPs)areemergingaskeyregulatoryfactorsthataidin
post-transcriptionalgeneregulation.Theshiftfromtranscriptionaltopost-transcriptional
controlintheseorganismsappearstobeduetoananomalousarrangementoftrypanosomatid
proteincodinggenes[6].Inadditiontothisspecializedarrangement,thegeneslackcanonical
promoters[7].Transcriptionalcontrolofgeneexpressionposesachallengeintheseorganisms
asRNApolymeraseIItranscribesallproteinencodinggenesatcomparablerates[8].Never-
theless,thefinaloutputofmaturemRNAsandproteinscanvarytremendouslyevenbetween
adjacentgenes.Onepost-transcriptionalregulatorymechanismusedbyleishmaniatocontrol
geneexpressioninvolvestheprocessingofpolycistronicpre-mRNAsintomaturemRNAs.
ThisRNAprocessingtoolresultsinthetrans-splicingofacappedspliced-leaderexonatthe
5’-endandapolyadenylationatthe3’-end[9,10].
TheassociationofRBPswiththe3’-UTRsofmRNAsusuallyleadstochangesinstability,
translation,orlocalizationoftargetmRNAs,andthismechanismseemstobeoperativeintry-
panosomatids[11].Overthepasttwodecades,researchershaveidentifiedandcharacterized
numerousRBPsintrypanosomatids,particularlyTrypanosomacruziandTrypanosomabrucei.
Recentadvancementsinsequencingandannotationofthetrypanosomatidgenomeshaveled
tovariouspredictionsofnovelRBPs[12,13].Thesepredictionswerebasedonfindingclassical
mRNAstructuralmotifsthatserveasligandsfordifferentRBPs[14].Differenttranscriptscar-
ryingthesamesignaturemotifsarelikelytoberegulatedinsimilarways[15].RBPsinteract
withmRNAmotifsviauniquefunctionaldomainssuchasRNA-RecognitionMotifs(RRM),
ZincFingerdomains(ZF)andPumiliodomains(PUM);andthesemotifshavebeenfoundin
theRBPsoftrypanosomatids[16].Forexample,insilicoscreeningofthegenomesofTrypano-
somabrucei,TrypanosomacruziandLeishmaniamajoridentifieduniquezincfingerproteins
whichareindicativeofRNAbinding[17].Inaddition,computationalanalysisandbioinfor-
maticshavebeenusedtopredictandcharacterizeRBPsoftrypanosomes[15].Invitroap-
proacheshavebeenusedtopulldownproteinsboundtospecifictarget3’-UTRtranscripts
or,conversely,topulldownRNAtranscriptsboundtoknownRNA-bindingproteins[18].
However,alloftheseapproachesarelimitedandcannotprovideacomprehensivepictureof
RBPsintheorganismsofinterest.IdentificationoftherepertoireoftrypanosomatidmRNA
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IdentificationofLeishmaniaInVivomRNA-Interactome
interactingproteinsisessentialfororganizingourcurrentmolecularandgeneticunderstand-
ingofgeneexpressionandinparticular,post-transcriptionalgeneregulation.Accordingly,it
waspertinenttodevelopanalternativestrategyforcharacterizingRBPsinthesepathogenic
protozoa.
Recently,aspartofanefforttoidentifymammalianinvivomRNA-boundproteomes,three
independentstudiesusedacombinationofUVRNA-proteincross-linkinginvivofollowed
byPoly(A)-RNApulldowns[19–21].Thiscomprehensiveapproachistermed‘‘interactome
capture”,andthesestudiesgeneratedanatlasofmammalianmRNA-bindingproteinsin
vivo.Recently,usingagenome-widetetheringscreen,inwhichrandomproteinfragments
wereartificiallyboundtoareportermRNAasbait,over300proteinswereidentifiedaspos-
siblemRNA-fateregulatorsbypost-transcriptionalmechanismsinT.brucei[22].Sinceran-
domproteinfragmentswereartificiallyboundtoareportermRNA,theresultsobtained
maynotbetrueforfull-lengthproteins.Subsequently,thetetheringscreenwasextendedby
thesamegroupusingasmall-scalelibraryoffull-lengthopenreadingframesandthis
resultedinacatalogueofabout100proteinsthatmayregulateT.bruceimRNA-fate[23].
ThissamestudyalsoreportedanmRNA-boundproteomeconsistingof155high-confi-
denceRBPsfrombloodstreamformsofT.brucei.However,tothebestofourknowledgeno
effortshavebeenreportedtoidentifycomprehensivelytherepertoireofRBPsinleishmania.
WeusedLeishmaniadonovani,initsamastigotestage,asaparadigmforthisstudy.We
developedtheinteractomebycombiningUVcross-linkingandoligo(dT)capturetopull
downproteinsboundtomRNAsinviableL.donovaniamastigotes.Weshowthatthein
vivoleishmaniamRNAinteractomeconsistsofatleast79proteins,49ofwhichshowno
significanthomologytoidentifiedhumanRBPsinthedatabaseandwediscussresulting
insightsintotheRNAbiologyofleishmania.
MaterialsandMethods
Parasitecultures
Leishmaniapromastigoteculture. L.donovaniSudanstrainS2promastigoteswererou-
tinelyculturedinM199(Sigma-Aldrich)with10%heatinactivatedfetalbovineserum(FBS,
Gibco),20mMHEPES(Sigma-Aldrich),6μg/mLhemin(Sigma-Aldrich),10μg/mLfolicacid
(Sigma-Aldrich),2mML-glutamine(Stemcell),100U/mLpenicillin/streptomycin(Sigma-
Aldrich)and100μMadenosine(Sigma-Aldrich)at26˚C.Every3daystheparasitesweresub-
cultured1:10infreshmediumandwerekeptincultureforamaximumof20–25passages.In
ordertomaintainvirulenceandinfectivity,freshparasiteswereroutinelyobtainedbypurifica-
tionofamastigotesfromspleensof6–8weekinfectedSyrianGoldenhamstersfollowedbyin
vitrotransformationintopromastigotesbyculturingfor5–7daysat26˚Cinpromastigote
medium.PriorapprovalforanimalexperimentswasobtainedfromtheAnimalCareCommit-
teeofUniversityofBritishColumbia(approval#A14-00218)
Leishmaniaaxenicamastigoteculture. Invitrocultureofamastigoteswascarriedoutas
follows:latelogarithmicphaseculturesofL.donovanipromastigotesweretransferredfrom
promastigotemediumat26˚CandpH7.4toRPMI1640mediumsupplementedwith10%
fetalbovineserumandincubatedat37˚C(5%CO )for16–18h.Subsequently,cellswere
2
collectedbycentrifugation(1200×gatroomtemperaturefor10min)andresuspendedin
amastigotemedium(RPMI1640with25%fetalbovineserum,100μMadenosineandpHwas
titratedtopH5.5with1MMES)at37˚Cin5%CO environment.Threedaysafterinitiation
2
oftransformation,cellsweredilutedinfreshamastigotemediumandmaintainedbychanging
mediaevery4thday.
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IdentificationofLeishmaniaInVivomRNA-Interactome
UV-crosslinkingofleishmaniaamastigotes
Dividingamastigotes(10x109)werewashedoncewithcoldphosphatebufferedsaline(PBS)
andresuspendedincoldPBStoaconcentrationof1.7x109perml.Thecellsuspension(6ml)
wasdispersedintoa100mmx20mmcellcultureplatewithoutlid,placedonicepack(imme-
diatelybeforecross-linking),andUV-irradiated3times(1mineachwith2minsgaponice)
with0.15Jcm−2at254nmusingUltra-LUMelectronicultravioletcrosslinkeratadistance
of15cmfromtheUVsource.AfterUVirradiation,cellswerecollectedbycentrifugation.
Preparationofparasiteproteinextracts
Pelletsfromnonirradiatedcontrolandirradiatedamastigoteswerelysedseparatelyin10ml
oficecoldstringentlysisbuffer(20mMTris-HCl(pH7.5),500mMLiCl,0.5%lithiumdode-
cylsulfate,1mMEDTAand5mMDTT)containing1mMphenylmethylsulfonylfluoride,
10μgofaprotinin/ml,10μgofleupeptin/ml,and2μgofpepstatinA/ml.Pelletswereresus-
pendedbypipettingupanddown.Thesuspendedsampleswerethenpassedatleastthree
timesthroughasyringewithanarrowneedleoruntilthehomogenatebecameclear.The
lysateswereincubatedfor10minat4˚C.
Oligo(dT)capture
300μlofOligo(dT) magneticbeads(NewEnglandBiolabs)persample,previouslyequili-
25
bratedinthreevolumesoflysisbuffer,wasaddedtothelysateandincubatedthemfor1hat
4˚Cwithgentleend-over-endrotation.Thetubeswereplacedonamagnetat4˚Cfor5mins
tocompletelycapturethemagneticbeads.Thebeadswereresuspendedin5mlofice-cold
lysisbufferandwashedfor5minsat4˚Cbygentleend-over-endrotation,andthenpelleted
withthehelpofamagnet.Recoveredbeadswereresuspendedin5mlofice-coldwashbuffer1
((20mMTris-HCl(pH7.5),500mMLiCl,0.1%LiDS,1mMEDTAand5mMDTT)and
incubatedfor5minsat4˚Cbygentleend-over-endrotationandbeadswerepelletedwiththe
helpofamagnet.5mlofice-coldwashbuffer2(20mMTris-HCl(pH7.5),500mMLiCland
1mMEDTA)wasaddedtopelletedbeadswhichwereresuspendedandincubatedfor5mins
at4˚Cbygentleend-over-endrotationandbeadswerepelletedusingamagnet.Afinalwash
wasgiventopelletedbeadswithlowsaltbuffer(20mMTris-HCl(pH7.5),200mMLiCland1
mMEDTA).ElutionofthemRNA-proteincomplexeswasperformedbyincubatingwashed
beadswith250μl/mlnosaltelutionbuffer(20mMTris-HCl(pH7.5)and1mMEDTA)for3
minsat55˚C.RNAconcentrationwasdeterminedusingaNanodropdevice.
RNAanalysis
TheRNAqualitywasassessedbyBioanalyzerchips(Agilent)usingbuffersandinstructions
providedbythemanufacturer.
RNAdigestionandproteinconcentration
TorecovermRNAboundproteins,mRNA-proteincomplexeswereincubatedwithRNAseA
(Fermentas)for1hat37˚C.TheRNAse-treatedeluatewasthenconcentratedbytransfertoa
2-mlAmiconUltra103-kDacutoffdeviceandtoppedupwithbuffer4(20mMTris-HCl
(pH7.5)and50mMNaCl).
Preparationsofsamplesformassspectrometryanalysis
Resultingproteinsolutionswerereducedandalkylatedasper[24].anddigestedwith1μgof
trypsin(Promega)at37˚Covernight.Peptidesampleswerepurifiedbysolidphaseextraction
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IdentificationofLeishmaniaInVivomRNA-Interactome
onC-18STopAndGoExtraction(STAGE)tips[25],andeachtreatmentwaschemically
labeledbyreductivedimethylationusingformaldehydeisotopologues[26].Thelabelledpep-
tidesweremixedandpurifiedagainbyC-18STAGEtips.
HPLCandmassspectrometeryanslysis
Finalpeptideswereloadedontoquadrupole–timeofflightmassspectrometer(ImpactII;Bru-
kerDaltonics)on-linecoupledtoanEasynanoLC1000HPLC(ThermoFisherScientific)
usinga40-50cmlonganalyticalcolumnpackedwith1.9μm-diameterReprosil-PurC-18-AQ
beads(Dr.Maisch,www.Dr-Maisch.com),fixedonanin-houseconstructedcolumnheaterset
at50˚C.BufferAconsistedof0.1%aqueousformicacid,andbufferBconsistedof0.1%formic
acidand80%acetonitrileinwater.SampleswereresuspendedinbufferAandloadedwith
thesamebuffer.Sampleswereanalyzedover90minpeptideseparationandthecolumnwas
washedwith100%BufferBfor15min,andre-equilibratedwithBufferA.Theanalysiswas
performedat0.25μl/minflowrateandtheImpactIIwassettoacquireinadata-dependent
auto-MS/MSmodewithinactivefocusfragmentingthe20mostabundantions(oneatthe
timeat18Hzrate)aftereachfull-rangescanfromm/z200Thtom/z2000Th(at5Hzrate).
TheisolationwindowforMS/MSwas2to3Thdependingonparentionmasstochargeratio
andthecollisionenergyrangedfrom23to65eVdependingonionmassandcharge.Parent
ionswerethenexcludedfromMS/MSforthenext0.4minandreconsiderediftheirintensity
increasedmorethan5times.SinglychargedionswereexcludedsinceinESImodepeptides
usuallycarrymultiplecharges.Strictactiveexclusionwasapplied.Massaccuracy:errorof
massmeasurementistypicallywithin5ppmandisnotallowedtoexceed10ppm.Thenano
ESIsourcewasoperatedat1700Vcapillaryvoltage,0.20Barnanobusterpressure,3L/min
dryinggasand150˚Cdryingtemperature.
Massspectrometrydataanalysis
AnalysisofMassSpectrometryDatawasperformedusingMaxQuant1.5.1.0.Thesearchwas
performedagainstadatabasecomprisedoftheproteinsequencesfromUniprot’sLeishmania
donovani(bpk282a1)pluscommoncontaminantsusingthefollowingparameters:peptide
massaccuracy10ppm;fragmentmassaccuracy0.05Da;trypsinenzymespecificity,fixed
modifications—carbamidomethyl,variablemodifications—methionineoxidationandN-ace-
tylprotein.Onlythosepeptidesexceedingtheindividuallycalculated99%confidencelimit
(asopposedtotheaveragelimitforthewholeexperiment)wereconsideredasaccurately
identified.
Dataavailability
ThemassspectrometryproteomicsdatahavebeendepositedtotheProteomeXchangeCon-
sortiumviathePRIDEpartnerrepositorywiththedatasetidentifierPXD005601[27]
ResultsandDiscussion
InvivocaptureofleishmaniaamastigotemRNAbindingproteins
TocharacterizeleishmaniaamastigoteinvivomRNAinteractingproteins,wedevelopedan
approachbasedonUVirradiationofliveamastigotes.UVirradiationgenerateshighlyreac-
tive,short-livedstatesofthenucleotidebaseswithinRNAs,resultingincovalentbondforma-
tiononlywithaminoacidsindirectcontact.InordertocovalentlycoupleRBPstomRNAsin
aphysiologicalinvivostate,dividingamastigoteswereirradiatedwithUVlightat254nm.
Infact,ithasbeenshownthatUVlightat254nmcross-linksthenaturallyphotoreactive
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IdentificationofLeishmaniaInVivomRNA-Interactome
nucleotidebases,especiallypyrimidines,withaminoacidssuchasPhe,Trp,Tyr,Cys,andLys
[28,29].Notably,thismethodofferedseveraladvantagesoverpreviousleishmaniaRBPidenti-
ficationmethods:(i)UVirradiationinducescovalentbondformationbetweennucleotide
baseswithinRNAsonlywithaminoacidsindirectcontact(zerodistance][30];however,UV
irradiationdoesnotpromoteprotein-proteincross-linking[30,31],(ii)UVlightwasdirectly
appliedtolivingamastigotes,thuscapturingphysiologicalinvivoRNA-proteininteractions,
and(iii)mRNApoly(A)tail-oligo(dT)hybridizationisverystableinhighsaltandionicdeter-
gentcontainingbuffersthusallowingefficientremovalofnon-specificinteractions.Inorder
toincreasetheefficiencyofUVcross-linking,approximately10x109leishmaniaamastigotes
wereusedasstartingmaterialforeachanalysis.AfterUVirradiationoflivingamastigotesat
254nm,cellswerelysedinbuffercontaininghighsaltandionicdetergent.Followingcelllysis,
RBPscovalentlyboundtopolyadenylatedRNAsinvivowerecapturedonoligo(dT)magnetic
beads.Asanegativecontrol,weincludednonirradiatedamastigotescelllysatesintheoligo
(dT)captureassay.Thisnucleicacidhybridizationbasedstrategyallowedwashingsofaffinity
beadsusinghighlystringentbiochemicalconditionsincluding500mMlithiumchlorideand
0.5%lithiumdodecylsulfate.Thisprotein-denaturingconditionduringthepurification
ensuredthestringentisolationofproteinsindirectcontactwithleishmaniamRNAsthrough
covalentbondsandallowedustominimizecontaminants.Followingstringentwashes,pro-
teinswerereleasedfromcomplexesbyRNasetreatmentandwereidentifiedusingMS(Fig1).
Totesttheefficiencyofoligo(dT)pull-downs,wedeterminedtheamountsofbeta-tubulinand
glyceraldehyde3-phosphatedehydrogenase(GAPDH)mRNAsinoligo(dt)pull-downmate-
rialusingRT-qPCRascomparedwithtotalRNA.Therewasafivetosixfoldenrichmentinβ-
tublinandGAPDHmRNAS,intheoligo(dT)pulldownsincomparisontothestartingmate-
rial(S1Fig).ThisconfirmedthatthemRNAswereefficientlycapturedbytheoligo(dT)affinity
beads.EnrichmentofmRNAsoverrRNAswasindependentlyconfirmedusingaBioanalyzer
Chip(S2Fig).DNAdidnotcopurifyasnoPCRamplificationofbeta-tubulinorGAPDH
occurredwhensampleswereRNasetreatedbeforetheoligo(dT)pull-downorwhenreverse
transcriptase(RT)wasomittedfromtheRTreactionduringcomplementaryDNA(cDNA)
preparation(datanotshown).
IdentificationofleishmaniamRNA-boundproteomebymass
spectrometry
Toidentifyproteinscross-linkedtomRNAsinUVirradiatedleishmania,weperformedoligo
(dT)purificationsfollowedbythereleaseofboundproteinsbyRNAsetreatment.Theisolated
proteinswereconcentratedusinganAmiconUltra103-kDadevice.Theconcentratedpro-
teinswereseparatedbySDS-PAGEfollowedbyin-geldigestionwithtrypsin.Peptideswere
analyzedbynanoflowhigh-performanceliquidchromatographyandonlinemassspectrome-
try.ByprocessingthedatawithMaxQuant1.5.1.0,weselectedproteinswhichwereidentified
inproteomicanalysesofthreebiologicalUVcross-linkedreplicates.Inparallel,non-cross-
linkedmRNAboundsampleswereanalyzedforspecificitycontrol.Toperformstatisticaldata
analysis,proteinenrichmentinUVirradiatedsamples,ascomparedtonon-irradiatedsam-
ples,wasassessedbyconsideringintensityvalues.Thesevalueswerederivedbysummingup
alltheextractedioncurrentofmass-to-chargevaluesovertimeassociatedwiththeprotein
usingMaxQuant[32,33]andtheresultsarepresentedinSupportingInformation(SI)S1
Table.Forstatisticalanalysisweusedone-tailed,pairedt-testwiththeconfidenceintervalof
90%.Thisanalysisresultedinalistof79proteinsfoundinthreeindependentcross-linked
samples(S1Table).p-valueswerecorrectedusingBenjamini-Hockbergprocedure[34].Itis
likelythatthecoverageofRBPsfortheamastigoteformofL.donovaniisnotcomplete.This
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IdentificationofLeishmaniaInVivomRNA-Interactome
Fig1.SchematicrepresentationofL.donovaniamastigotesinteractioncapture&massspectrometry
(MS)analysis.LiveL.donovaniamastigoteswereUVirradiatedtostabilizemRNA-proteininteractions.
Thesecomplexeswereisolatedbyoligo(dT)magneticbeadscaptureandstringentwashings.Boundprotein-
mRNAcomplexeswereelutedwithRNasetreatmentandidentifiedbyMS.
doi:10.1371/journal.pone.0170068.g001
protocolwillnotdetectRBPsthatare(i)notinteractingwithpoly(A)RNAs;(ii)notexpressed
inamastigoteformofL.donovani;(iii)notactiveundertheexperimentalconditions;or(iv)
notinsufficientquantityinthestartingmaterialusedfortheanalysis.Nevertheless,the
mRNA-boundproteomeoftheamastigoteformofL.donovaniiscomprisedofacomplexmix-
tureofproteinsenrichedinRNA-relatedactivities(S2Table).
OverviewofidentifiedmRNA-interactingproteins
Wefirstclassifiedtheidentifiedproteinsintofunctionalcategoriesbasedongeneannotation.
Asexpected,ribosomalproteins,RNAhelicases,translationfactors,andclassicalRNA-bind-
ingproteinsweremostfrequent,makingupcloseto70%oftheidentifiedproteins(S2Table,
Fig2A&2B).Thelownumbersofhighlyexpressedcellularproteinssuchasmetabolic
enzymessuggestedthattheoligo(dT)purificationwasspecific.
InadditiontoknownmRNA-interactingproteins,weidentified19proteins(S2Table),
thathavenotpreviouslybeenannotatedasRNAbinding.Interestingly,2outof19seemto
haveRNAbindingmotifs(ProteinID:E9BH26has2RNA-RecognitionMotifs(RRMs)and
proteinID:E9B8L4hastryptophan-asparticacid(WD)repeatsregions(S2Table).Thesefind-
ingssuggestthattheapproachweusedhadthecapabilitytoidentifyRNA-interactingproteins
thatusedistinctorhighlydivergentRNA-bindingdomains.
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IdentificationofLeishmaniaInVivomRNA-Interactome
Fig2. AandB.GeneOntologyenrichmentanalysisofL.donovanimRNAinteractingproteins.
doi:10.1371/journal.pone.0170068.g002
SomeoftheRBPsweidentifiedareinvolvedinproteintranslation.Elongationfactor
1-alpha(S2Table)isanabundantmultifunctionalprotein.Inadditiontoitscanonicalrolein
translation,ithasalsobeeninvolvedinothercellularprocessessuchasnucleartransport,cel-
lularorganization,proteinqualitycontrol,co-translationaldegradation,mitochondrialtRNA
import[35,36]andmacrophageSHP-1activation[37].Elongationfactor-2(S2Table)is
involvedintranslocationoftheribosomealongthemRNAstrandduringtranslation[38,39].
TheLeishmaniagenomeencodesthreepoly(A)-bindingproteinsandwewereabletoiden-
tifyallthree(ProteinID:E9BHB2,EBSU0andE9BT55)(S2Table).Wealsoidentifiedthree
proteins(ProteinID:E9BE15,E9BM42andE9BTJ1)thatareinvolvedinenergymetabolism.
Thesewereunexpectedresults,sincetheseproteinsdonotpossessknownRNAbinding
domainsintheirstructures.However,inyeastandotherorganisms,metabolicmultifunctional
enzymescapableofbindingtoRNAsequenceshavebeenpreviouslyreported[40,41].Indeed,
anincreasingnumberofmultifunctionalenzymesarebeingdescribedinparasiticprotozoa
[42].Weidentifiedfivehelicases(ProteinID:E9B8A4,E9B909,E9BNE6,E9BRR9andE9BS1)
(S2Table)intheproteomeofleishmaniamRNAinteractingproteins.DEAD-boxRNAheli-
casesmediateconformationalchangeinRNAcontainingcomplexesandthesechangesare
requiredtounwindRNAduplexesordisruptRNA-proteininteractions[43].Twoproteins
-alphatubulinandbetatubulin-boundtomRNAswhichhavenoRNA-relatedgeneontology
annotation.However,interestinglythisunexpectedinteractionhasbeenshowninrecently
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IdentificationofLeishmaniaInVivomRNA-Interactome
publishedplantmRNAinteractome[44].Moreover,participationofthecytoskeletoninthe
spatialorganizationandregulationoftranslationhasrecentlybeensuggested[45].Inaddition,
wealsofoundthreemolecularchaperones(HSP-70,HSP-83-1andmitochondrialHSP-60)
(S2Table).RNA-bindingrolesarenotunprecedentedforHSPsasmammalianHSP70isable
tobindtothe3’UTRoflabilemRNAs[46].Inaddition,itisknownthathumanHSP-70binds
toandstabilizesendogenousAU-richelement-containingmRNAs[47].Moreover,HSP90is
involvedinRNAinterferenceinhumanandinyeast[48].ItisalsopossibletheseHSPsare
interactingnonspecificallyduetotheirstickynature.
ItwasofinteresttoaskwhetheranyoftheleishmaniaRBPsthatweidentifiedhadprevi-
ouslybeenimplicatedinleishmaniapathogenesis.Infact,wefoundthatthreeoftheRBPs
(A0A0R4J963):Elongationfactor1-alpha[37],E9BTN1:Universalminicirclesequencebind-
ingprotein[49]andE9BTJ1:Fructose-bisphosphatealdolase[50]areconsideredtopotentially
contributetothepathogenesisofleishmaniainfection(S2Table).
mRNAbindingdomainsofleishmaniaRBPs
TogaininsightsintomodesofinteractionofRBPswithmRNAs,theRBPswerecoveredwere
analyzedforthepresenceofassociatedstructureslikefunctionaldomains(S2Table).Forthis
weperformedPfamandInter-Prodomainenrichmentanalyses[14]whichshowedthatmost
ofthesignificantlyenricheddomainswerevariousRNA-interactionmotifs(Fig3).Inmost
eukaryotes,manyRBPsinteractwithmRNAsviamodularRNAbindingdomains(RBDs),
includingtheRNArecognitionmotif(RRM),heterogeneousnuclearRNPK-homology
domain(KH),zincfingers(Znf)andothers.[14].Thesemotifshaveinformedinsilicoalgo-
rithmstoidentifyotherproteinsharboringsimilarsignaturesasputativenovelRBPs[51].
However,numerousnoncanonicalRBDshavealsobeenreported[52–55],reflectinglimita-
tionsinthescopeofcomputationalpredictions.Analysisoftheleishmaniainteractome
showedthatabouthalfofthemRNAinteractingproteomecontainedknownRBDs.This
includedseveralclassicalandnonclassicalRBDs(Fig3).TheRNA-RecognitionMotif(RRM)
isoneofthemostcommonlyfoundproteindomainsinnature.Ouranalysisidentifiedeleven
RRMcontainingproteins(S2Table,Fig3).RRMsaremostofteninvolvedinsequence-specific
interactionswithsingle-strandedRNAs.ProteinsthatcontainRRMsareinvolvedinallpath-
waysoftheRNAcycle,fromsplicingtomRNAturnover.RRMsareusuallypresentinmultiple
copieswithinaproteinandthismultiplicityenhancesligandspecificity[14].
AnothercommonlyrecognizedRNAbindingdomainisthezincfingerdomain.This
domainwasfirstdescribedasaDNA-bindingdomainandwassubsequentlyfoundinproteins
thatinteractwithRNA[56].Wefoundseveninouranalysis,fourcontainingCCHCtypeand
threecontainingC3H1-typeZincfingermotifs(S2Table,Fig3).Besidesthecommonlyrecog-
nizedRNA-bindingdomains,wealsofoundPUMandhnRNPK-homology(KH)RNAbind-
ingdomains(Fig3).ThebestcharacterizedfunctionofthePUMcontainingPumilioprotein
isasapost-transcriptionalrepressor[57].WefoundtwoPUMcontainingproteinsinouranal-
ysis(S2Table,Fig3).hnRNPK-homologydomaincontainingproteinsareassociatedwith
multiplegeneexpressionregulatorymechanisms,suchastranscriptionalregulation,andtrans-
lationalcontrol[58].WefoundonlyonehnRNPK-homologydomaincontainingproteinin
theleishmaniainteractome(S2Table,Fig3).
ComparisonwithpreviouslyknownRBPsofleishmanial
ToestimatethedepthofthemRNA-boundproteomewecapturedinouroligo(dT)precipita-
tions,wecomparedthenumberofidentifiedproteinswithpreviouslyknownRBPsofleish-
mania.Forthispurpose,webeganbyexaminingthegenomeofL.donovaniforpotentialRBPs.
PLOSONE|DOI:10.1371/journal.pone.0170068 January30,2017 9/16
IdentificationofLeishmaniaInVivomRNA-Interactome
Fig3.GlobulardomainsinL.donovanimRNAinteractome.Numberofproteinsharboring(A)classicaland(B)non-classicalRNAbinding
motifsinL.donovanimRNAinteractingproteome.(C)ConsolidatednumberofRNAbindingmotifsin79identifiedproteins,including32unknown
RNAbindingdomains(RBDunknown).
doi:10.1371/journal.pone.0170068.g003
Recently,thewholegenomeofL.donovanistrainbpk282A1hasbeensequencedandannotated.
SearchingthisgenomeforpredictedproteinscontainingRNAbindingdomainsfromthecom-
pletepredictedproteomeofL.donovaniresultedin67proteins.Interestingly,outofthese67
predictedRBPs,13werepartoftheL.donovaniinteractomereportedhere(S3Table)providing
experimentalsupportfortheirinsilicoidentificationasputativeRNAbindingproteins.
Recently,L.braziliensisHSP70mRNAsinteractingproteinshavebeenreported[18].We
alsocomparedourlistofleishmaniaRBPswithRNA-bindingdomaincontainingproteins
observedinthisinvitrostudytoidentifyproteinsthatinteractwithL.braziliensisHSP70
mRNAs[18].Inthisstudythe5’UTRandthetwotypesof3’UTRsfromL.braziliensisHSP70
geneswereusedasbaitinpulldownassaysusingtotalproteinparasiteextracts.Thebound
proteinswereseparatedbytwo-dimensionalgelelectrophoresisandidentifiedbyMS.This
studyresultedintheidentificationof52differentproteinsbasedontheirinteractionwithL.
braziliensisHSP70-mRNAs[18].Outofthislistof52proteins,12wererepresentedinourin
vivoproteomeofL.donovaniamastigotemRNAinteractingproteins(S3Table).Thediffer-
encesincompositionofourmRNAinteractingproteomeandtheHSP70studycouldbedue
toinvivoversusinvitromRNAinteractions,differentleishmaniaspeciesordifferentwashing
PLOSONE|DOI:10.1371/journal.pone.0170068 January30,2017 10/16