U6 snRNA expression prevents toxicity in TDP-43-knockdown cells

14 Pages·2017·1.63 MB·English

RESEARCHARTICLE
U6 snRNA expression prevents toxicity in TDP-
43-knockdown cells
MasaoYahara,AkiraKitamura,MasatakaKinjo*
LaboratoryofMolecularCellDynamics,FacultyofAdvancedLifeScience,HokkaidoUniversity,Sapporo,
Japan
*[email protected]
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Abstract
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a1111111111 Depletionofamyotrophiclateralsclerosis(ALS)-associatedtransactivationresponse(TAR)
a1111111111 RNA/DNA-bindingprotein43kDa(TDP-43)alterssplicingefficiencyofmultipletranscripts
andresultsinneuronalcelldeath.TDP-43depletioncanalsodisturbexpressionlevelsof
smallnuclearRNAs(snRNAs)asspliceosomalcomponents.Despitethisknowledge,the
relationshipbetweencelldeathandalterationofsnRNAexpressionduringTDP-43deple-
tionremainsunclear.Here,weknockeddownTDP-43inmurineneuroblastomaNeuro2A
OPENACCESS
cellsandfoundatimelagbetweenefficientTDP-43depletionandappearanceofcelldeath,
Citation:YaharaM,KitamuraA,KinjoM(2017)U6
snRNAexpressionpreventstoxicityinTDP-43- suggestingthatseveralmechanismsmediatebetweenthesetwoevents.TheamountofU6
knockdowncells.PLoSONE12(11):e0187813. snRNAwassignificantlydecreasedduringTDP-43depletionpriortoincreaseofcelldeath,
https://doi.org/10.1371/journal.pone.0187813
whereasthatofU1,U2,andU4snRNAswasnot.DownregulationofU6snRNAledtocell
Editor:EmanueleBuratti,InternationalCentrefor death,whereastransientexogenousexpressionofU6snRNAcounteractedtheeffectof
GeneticEngineeringandBiotechnology,ITALY
TDP-43knockdownoncelldeath,andslightlydecreasedthemis-splicingrateofDnajc5
Received:May31,2017 andSortilin1transcripts,whichareassistedbyTDP-43.Theseresultssuggestthatregula-
Accepted:October26,2017 tionoftheU6snRNAexpressionlevelbyTDP-43isakeyfactorintheincreaseincelldeath
uponTDP-43loss-of-function.
Published:November10,2017
Copyright:©2017Yaharaetal.Thisisanopen
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and Introduction
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. Transactivationresponse(TAR)RNA/DNA-bindingprotein43kDa(TDP-43)hasbeeniden-
tifiedasanamyotrophiclateralsclerosis(ALS)-associatedprotein.TDP-43ismainlylocalized
DataAvailabilityStatement:Allrelevantdataare
inthenucleusandshuttlesbetweenthenucleusandcytoplasmtomaintainseveralRNA-asso-
withinthepaperanditsSupportingInformation
files. ciatedfunctions(e.g.,localtranslation,translocation,splicing,andmicroRNAprocessing)[1].
However,inmotorneuronsfromALSpatients,TDP-43disappearsfromthenucleusand
Funding:A.K.wassupportedbyaJapanSociety
appearsincytoplasmicubiquitinatedinclusionbodies,alongwithcarboxyl-terminalfragments
forPromotionofScience(JSPS)Grant-in-Aidfor
ScientificResearch(C)(#26440090);byagrantfor (CTFs)ofTDP-43[2].TDP-43andTDP-43CTFsareaggregation-proneandexertcytotoxic-
DevelopmentofSystemsandTechnologiesfor ityinneuronalandnon-neuronalcelllines[3–5].Severalgroupsincludingoursreportedthat
AdvancedMeasurementandAnalysisfromthe RNAmaybeinvolvedintheaggregationprocessofTDP-43andTDP-43CTFs[6–9].There-
JapanAgencyforMedicalResearchand
fore,itisexpectedthattoxicgain-of-functionofRNA-involvedaggregationofTDP-43and
Development(AMED);byagrant-in-aidofThe
TDP-43CTFsmaybeimplicatedinneuronalcelldeath.
NakabayashiTrustforALSResearch(Tokyo,
Alternatively,sinceTDP-43knockoutinmurinemotorneuronscausesprogressivemotor
Japan);byagrant-in-aidofJapanAmyotrophic
LateralSclerosisAssociation(JALSA,Tokyo, neurondegeneration[10],loss-of-functionofTDP-43maybeinvolvedinALSpathogenesis.
PLOSONE|https://doi.org/10.1371/journal.pone.0187813 November10,2017 1/14
U6snRNApreventstoxicityofTDP-43depletion
Japan)forALSresearch;andbyagrantofThe TDP-43knockoutinmiceexhibitsearlyembryoniclethality[11–13].Moreover,TDP-43
AkiyamaLifeScienceFoundation(Sapporo, depletioninvariousmammalianculturedcellsandembryonicstemcellsresultsincelldeath
Japan).M.K.waspartiallysupportedbyagrant-in-
[14–17].TheseresultspointatanessentialroleofTDP-43incellsurvival;however,the
aidofNakataniFoundationforAdvancementof
detailedmechanismofcelldeathduringTDP-43loss-of-functionhasnotbeenelucidated.
MeasuringTechnologiesinBiomedical
TDP-43depletionbothinmurinebrainandmammalianculturedcellscauseswidespread
Engineering.
alterationsoftheRNA-splicingstatesuchaschangesinexoninclusion[18–21].Defectsin
Competinginterests:Theauthorsdeclareno
RNAsplicingareimplicatedincelldeathinmanyneurodegenerativediseasesincludingALS
conflictsofinterest.
[22,23].TheseresultsimplythatTDP-43loss-of-functionmaycausecelldeaththroughalter-
ationsoftheRNA-splicingstate.AnimportantmachineryduringRNAsplicingineukaryotes
isthespliceosome,composedofsmallnuclearRNAs(snRNAs)includingU1,U2,U4,U5,and
U6snRNA,inadditiontoarangeofsmallnuclearribonucleoproteins(snRNPs)[24].Reports
showthattheexpressionprofilesofsuchsnRNAsarealteredinTDP-43-knockeddowncul-
turedcellsandspinalcordfromALSpatients[25,26].Onestudyreportedthattheexpression
levelofU6snRNAwasdecreasedinthespinalcordofALSpatients,butthatofU1,U2,U4,
andU5snRNAwasnot[25].Conversely,anotherstudyreportedthattheexpressionlevelof
U6snRNAinthespinalcordofALSpatientswasnotdecreased,whereasthatofU1,U2,U4,
andU5snRNAwasincreased[26].Moreover,theamountofU6snRNAinTDP-43-knocked
downhumanneuroblastomaSH-SY5Ycellswasnotdecreasedineitherstudy[25,26].Thus,
TDP-43depletioncandisturbexpressionofsnRNAs;however,theexpressionprofileofindi-
vidualsnRNAsduringTDP-43depletionremainsunclear.
Therefore,tounveiltherelationshipbetweencelldeathandthealterationofsnRNAexpres-
sionduringTDP-43depletion,weinvestigatedwhethertheexpressionlevelofU6snRNAcan
bemodifiedusingTDP-43-knockeddownmurineneuroblastomaNeuro2Acells,whichshow
asignificantincreaseofcelldeathpost-TDP-43depletion.Finally,weinvestigatedwhethercell
deathduringTDP-43depletioncanbepreventedbyrestoringtheexpressionlevelsofU6
snRNA.
Materialandmethods
PlasmidDNAs
PlasmidDNAsencodingGFP(pmEGFP-N1)andpmEGFP-N1toexpressC-terminallyGFP-
taggedTDP-43(TDP-43-GFP)werepreparedasestablishedpreviously[6].SyntheticcDNAof
TDP-43thatisnotrecognizedbysiRNAagainstmurineTDP-43mRNAwassynthesizedby
ThermoFisherScientific(Waltham,MA).ThecDNAwassubclonedintopmEGFP-N1to
expressC-terminallyGFP-taggedTDP-43(T43-GFP)accordingtoapreviousstudy[6].Exog-
enousexpressionofU6snRNAwasdrivenbythehumanH1promoterintheplasmidpSuper-
ior.neo(Oligoengine,Seattle,WA).SyntheticU6snRNAoligonucleotidewassynthesizedby
ThermoFisherScientific(S1ATable)andsubclonedintopSuperior.neo(pU6).Emptyvector
(pEV)wasusedasanegativecontrol.
Transfectionandcellpreparation
MurineneuroblastomaNeuro2AcellswerekindlyprovidedbyProf.KazuhiroNagataatKyoto
SangyoUniversity.Thestrainofthecellwasassameasthatinthepreviousstudies,andculture
conditionswerereportedpreviously[6,27].ForknockdownofTDP-43andU6snRNA,Neu-
ro2Acells(5.5×105)wereplatedina10cmcellculturedish(CORNING,Corning,NY).A
senseandantisenseTDP-43-targetingsiRNAduplex(T43-siRNA;5’-GUUAGAAAGAAGUGGA
AGATT-3’and5’-UCUUCCACUUCUUUCUAACTT-3’,respectively;synthesizedbyNippon
Gene,Tokyo,Japan),oranon-targetingsiRNA(NC-siRNA;#AM4611;ThermoFisherScien-
tific)asanegativecontrol,weretransfectedusing24μLLipofectamineRNAiMAXtransfection
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U6snRNApreventstoxicityofTDP-43depletion
reagent(ThermoFisherScientific)and160pmolofeachsiRNAinOpti-MEMImedium
(ThermoFisherScientific).AsenseandantisenseU6snRNA-targetingsiRNAduplex(U6-
siRNA;5’-GCUUCGGCAGCACAUAUACTT-3’and5’-GUAUAUGUGCUGCCGAAGCTT-3’,
respectively;alsosynthesizedbyNipponGene)wasmodifiedfromapreviousstudy[28].
ForexpressionofU6snRNAinTDP-43-knockeddowncells,Neuro2Acells(2.8×105)
wereplatedina10cmculturedish.Aftera20hincubation,cellsweretransfectedusing
12.8μLLipofectamine2000transfectionreagent(ThermoFisherScientific)andamixtureof
0.5μgpmEGFP-N1andeither4.5μgpU6orpEV.Thetransfectionmixwasincubatedwith
thecellsfor4h.Aftertheincubation,themediumwasreplacedandsiRNAwastransfectedfol-
lowingtheprotocoldescribedabove(S1AandS1BFig).CellviabilityassaysandWesternblot-
tingwereperformedusing3.5cmculturedishesandbyreducingthenumberofcellsand
amountsofplasmidDNAandreagentsto1/8scale,accordingly.
Westernblotting
RecoveryofcelllysatesandWesternblottingtodetecttheproteinamountofTDP-43andα-
tubulinwereperformedaspreviouslyreported[6].
PCRfornon-codingRNAsormRNAs
TotalRNAwasisolatedwithTRIzol(ThermoFisherScientific)andpurifiedusingPureLink
RNAMiniKit(ThermoFisherScientific)accordingtothemanufacturer’sinstructions.
ExtractsweretreatedwithDNaseI(TaKaRa,Shiga,Japan)beforeperformingadditional
assays.
Todetectnon-codingRNAs,totalRNA(750ng)wasusedforsynthesisoffirst-strand
cDNAusingMir-XmiRNAFirstStrandSynthesisKit(TaKaRa)accordingtothemanufactur-
er’sinstructions.Real-timequantitativePCR(qPCR)wasperformedusingSYBRPremixEx
Taq(TliRNaseHPlus;TaKaRa),ROXReferenceDyeII(TaKaRa),0.2μMforwardprimer,
0.2μMreverseprimer,and2μLtemplatecDNA(correspondingto15ngtotalRNA).Am-
plificationanddetectionwereperformedusingareal-timePCRsystem(Mx3005P;Agilent
Technologies,SantaClara,CA).ThePCRwasperformedinatwo-stepprotocolunderthefol-
lowingconditions:initialdenaturingat95˚Cfor30s,followedby35cyclesofdenaturingat
95˚Cfor10s,andannealing/extensionat60˚Cfor20s.
Todetectthesplicingstateoftranscripts,first-strandcomplementaryDNAsynthesiswas
performedusingatranscriptase(PrimeScript;TaKaRa)accordingtothemanufacturer’s
instructions.PCRwasperformedusingathermalcycler(BioerTechnology,Binjiang,China).
ThePCRwasperformedinathree-stepprotocolunderthefollowingconditions:initialdena-
turingat98˚Cfor30s,followedby21cyclesofdenaturingat98˚Cfor10s,annealingat60˚C
for30s,andextensionat72˚Cfor60s.PCRproductswereseparatedin5%polyacrylamide
gelanddetectedusingLAS4000mini(Fujifilm,Tokyo,Japan).PCRprimersaredescribedin
S1BTable.
Urea-polyacrylamidegelelectrophoresis(Urea-PAGE)forRNA
SamplesincludingtotalextractRNA(2.75μg)weremixedwithloadingbufferconsistingof
1.5×Tris-acetateEDTA(TAE),10Murea,10%(w/v)sucrose,0.05%(w/v)bromophenolblue,
and0.05%(w/v)xylenecyanol,incubatedat65˚Cfor15min,andkeptoniceuntilelectropho-
reticseparation.Sampleswereseparatedina12%polyacrylamidegelcontaining7%ureain
1×TAEbuffer.RNAswerestainedusingSYBRGold(ThermoFisherScientific).Fluorescent
intensitiesweredetectedusingLAS4000mini(Fujifilm)usinga460nmexcitationlightanda
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U6snRNApreventstoxicityofTDP-43depletion
long-passfilter(Y515;Fujifilm).QuantificationwasperformedusingImageJ1.47software
(NationalInstitutesofHealth,Bethesda,MD).
Cellviabilityassay
Deadcellswerestainedby1.0μg/mLpropidiumiodide(PI;Sigma-Aldrich,St.Louis,MO)
andobservedusinganLSM510(CarlZeiss,Jena,Germany)andaPlan-Neofluar10×/0.3NA
objectiveasreportedpreviously[6,27].
Foralternativecellviabilityassaysbasedonmetabolism,at96haftersiRNAtransfection,
cells(0.5×105)in100μLcellculturemediumwerere-platedintoa96-wellplate(AsahiTech-
noglass,Shizuoka,Japan)andcultivatedfor20h.Thereafter,10μLWST-1mixture(Roche,
Basel,Switzerland)wasaddedtothecellculturemedium,sampleswereincubatedforanaddi-
tional4h,andthenthemediumwascollected.Absorbanceat490and690nmwasmeasured
usingaDU800spectrophotometer(BeckmanCoulter,Brea,CA).Absorbanceat490nmwas
subtractedfromthatat690nm.
Cross-linkingimmunoprecipitation(CLIP)assay
Neuro2Acellswerepassagedintofour10cmculturedishesataratioof1/5andtransfected
using16.0μLLipofectamine2000transfectionreagentand4.0μgpmEGFP-N1orTDP-
43-GFPexpressionplasmid(TDP-43-GFP).Afterincubationfor30h,theCLIPassaywasper-
formedaspreviouslyreported[29].Cellswerefixedinphosphate-bufferedsalinecontaining
3%formaldehydeandlysedbysonication.Immunoprecipitationwasperformedovernightat
4˚Cusinganti-GFPmonoclonalantibody-conjugatedagarosebeads(MBL,Nagoya,Japan).
Afterdissociationofcross-linkedcomplexesat70˚C,RNAwasextracted,reverse-transcribed,
andU6snRNAtranscriptswerequantifiedbyqPCRasdescribedinthe“PCRfornon-coding
RNAsormRNAs”sub-section.
Statistics
Student’sttestwasperformedtoevaluatestatisticalsignificance.
Results
TemporaldelaybetweenTDP-43depletionandcelldeath
WefirstsoughttoevaluatethetimelineofTDP-43depletionfollowingknockdown.Toknock
downTDP-43,siRNAtargetingTDP-43mRNA(T43-siRNA)ornon-codingsiRNA(NC-
siRNA)weretransientlytransfectedintoNeuro2Acells.TDP-43proteinlevelswereassessed
byWesternblotat72,96,and120haftertransfection.T43-siRNA-transfectedcellsshoweda
significantdecreaseinTDP-43levelscomparedwithNC-siRNA-transfectedcells.Inturn,
NC-siRNAtransfectiondidnothaveanysignificanteffectonthelevelsofTDP-43compared
withmock-transfectedcells.TheseresultsindicatethatT43-siRNA-mediatedknockdowncan
achieveanefficientreductionintheproteinlevelsofTDP-43(Fig1A).Moreover,theprotein
levelsofTDP-43remainedlow(<10%ofcontrol)untilatleast120haftersiRNAtransfection
(Fig1A).
Next,weanalyzedthetimelineofcelldeathafterTDP-43depletion.Quantificationofcell
deathshowedthattheproportionoflivecellsremainedstableat72and96haftertransfection
withsiRNAandsignificantlydecreasedat120haftersiRNAtransfection(Fig1B).Giventhat
theproteinlevelofTDP-43wassignificantlydecreasedat72and96haftertransfection(Fig
1A),whereastheproportionoflivecellsdidnotsignificantlydecreaseuntil120haftersiRNA
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U6snRNApreventstoxicityofTDP-43depletion
Fig1.TimecourseofcelldeathduringTDP-43depletion.(A)WesternblotanalysisofendogenousproteinexpressionofTDP-43and
α-tubulinduringsiRNA-mediatedTDP-43knockdown.TimeaftertransfectionofT43-siRNA,NC-siRNA(NC),ornosiRNAandtransfection
reagents(Mock)isindicated(72,96,and120h).α-tubulinwasusedtonormalizebandintensitiesofTDP-43.Barsinbottomgraphs
indicatethenormalizedamountofendogenousTDP-43(mean±SEM;n=3).SignificancebetweenindicatedpairswastestedbyStudent’s
ttest:*p<0.05,**p<0.01,and***p<0.001.(B)Theproportionoflivecellsat72,96,and120haftertransfectionofT43-siRNAandNC-
siRNAasanegativecontrol(circleandsquare,respectively;mean±SEM;n=3).SignificancebetweenT43-siRNA-andNC-siRNA-
transfectedcellswastestedbyStudent’sttest:*p<0.05.
https://doi.org/10.1371/journal.pone.0187813.g001
treatment(Fig1B),theseresultsindicatethatthereisatimelagbetweenTDP-43depletion
andtheconstitutionofcelldeathphenotype.
U6snRNAisdownregulatedinTDP-43-knockeddowncells
Next,wecheckedtheexpressionlevelofU6snRNAinTDP-43-knockeddownNeuro2Acells
at72hafterthetransfectionofsiRNAsusingqPCR.TheamountofU6snRNAinT43-siRNA-
transfectedcellswassignificantlydecreasedcomparedwiththatinNC-siRNA-transfectedcells
(S2Fig).Theamountof18SribosomalRNA(18SrRNA)and7SLRNA,ubiquitouslyexpressing
smallRNAstranscribedfromhousekeepinggenes,wasnotchangedbyTDP-43knockdown,
butthatofsmallnucleolarRNA202(snoRNA202)wasincreased,indicatingthat18SrRNA
and7SLRNAcanbeusedasaninternalcontrolduringTDP-43depletion(S2Fig).Thus,to
evaluatetheamountofU6snRNAinTDP-43-knockeddowncells,weused18SrRNAasan
internalcontrol.Asaresult,theexpressionlevelofU6snRNAinTDP-43-knockeddowncells
wasdecreasedtoapproximately50%ofthatinNC-siRNA-transfectedcells(Fig2A).
WenextelucidatedtheexpressionlevelsofotherUsnRNAsusingurea-polyacrylamidegel
electrophoresis(Urea-PAGE).ThemigrationpatternofU1,U2,U4,andU6snRNAwas
unequivocallyassignedusingtheirpredictedmolecularsizes(164,187,145,and107nucleo-
tides,respectively).Unfortunately,U5snRNAwasnotdistinguishedinthismigrationpattern
(Fig2B).ThedecreasedamountofU6snRNAduringTDP-43depletionwasconsistentwith
theresultsobtainedusingqPCR,whereastheamountofU1,U2,andU4snRNAwasnot
changed(Fig2C).TheseresultssuggestthatU6snRNAisselectivelydownregulatedduring
TDP-43depletioninNeuro2Acells.
Moreover,toinvestigatewhetherTDP-43canassociatewithU6snRNA,wequantifiedthe
amountofU6snRNAco-immunoprecipitatedwithTDP-43usingqPCR.TheamountofU6
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U6snRNApreventstoxicityofTDP-43depletion
Fig2.DownregulationofU6snRNAinTDP-43-knockeddownNeuro2Acells.(A–C)Quantificationoftheexpressionchangeof
smallRNAsduringTDP-43knockdown.SignificancewastestedbyStudent’sttest:*p<0.05.(A)RelativeexpressionofU6snRNAin
T43-siRNA-transfectedcellscomparedwiththatinNC-siRNA-transfectedcells.Theexpressionlevelof18SrRNAwasusedasan
internalcontrol(mean±SEM,n=3).(B)GelimageofUrea-PAGE.Leftbarindicatesmolecularsizeinnucleotides(nt)ofmarker.The
twodominantbandscorrespondto5.8Sand5SribosomalRNA(rRNA).(C)RelativeamountofU1,U2,U4,andU6snRNAsin
T43-siRNA-transfectedcellscomparedwiththatinNC-siRNA-transfectedcells(mean±SEM,n=3).(D)Quantificationofthebinding
ofU6snRNAtoTDP-43byCLIP.TheamountofU6snRNAinthesamplecontainingTDP-43-GFPwasnormalizedagainstthatinthe
samplecontainingGFPasanegativecontrolineachinputcelllysate(INPUT)orimmunoprecipitatedmixture(IP)(mean±SEM;n=3).
SignificanceindicatedinthegraphwastestedbyStudent’sttest:*p<0.05.
https://doi.org/10.1371/journal.pone.0187813.g002
snRNAdidnotdifferbetweenlysatesofTDP-43-GFP-expressingandGFP-expressingcells
(Fig2D;INPUT).However,thelevelofU6snRNAco-precipitatedwithTDP43-GFPwas
14.7-foldhigherthanthatco-precipitatedwithGFP(Fig2D;IP).Thus,TDP-43maydirectly
bindtoU6snRNAandtherebyregulateitsstability.
DownregulationofU6snRNAresultsincelldeath
ToinvestigatewhetherU6snRNAisinvolvedincelldeath,U6snRNAwasknockeddown
usingsiRNA.At72haftertransfectionofU6-siRNA,U6snRNAexpressioninNeuro2Acells
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U6snRNApreventstoxicityofTDP-43depletion
Fig3.ViabilityofU6snRNA-depletedcells.(A)QuantificationofU6snRNAexpressionat72haftertransfectionofU6-siRNA
andNC-siRNA.Theexpressionlevelof18SrRNAwasusedasaninternalcontrol(mean±SEM,n=3).Significancewastested
byStudent’sttest:*p<0.05.(B)Theproportionoflivecellsat72haftertransfectionofU6-siRNAandNC-siRNA(mean±SEM;
n=3).SignificancewastestedbyStudent’sttest:*p<0.05.
https://doi.org/10.1371/journal.pone.0187813.g003
wasdecreasedto72%comparedwiththatinNC-siRNA-transfectedcells(Fig3A).Thepro-
portionoflivecellswassignificantlydecreaseduponU6snRNAknockdown(Fig3B),indicat-
ingthatdownregulationofU6snRNAincreasescelldeath.Thus,downregulationofU6
snRNAduringTDP-43depletionmaycontributetocelldeath.Theproportionofdeadcells
washigheruponTDP-43knockdown(21%,Fig1B)thanuponU6snRNAknockdown(6.5%,
Fig3B).ThismaybeduetoadifferenceinthedegreetowhichU6snRNAwasdownregulated
(28%uponU6snRNAknockdownand53%uponTDP-43knockdown).
ExpressionofU6snRNApreventscelldeathduringTDP-43depletion
TodeterminewhetherrestorationofU6snRNAexpressionrescuescelldeathduringTDP-43
depletion,weexaminedcellviabilitywhenexogenousU6snRNAwastransientlyexpressed
usingaplasmidDNAencodingU6snRNA(pU6)inT43-siRNA-transfectedNeuro2Acells.
Transfectionwithanemptyvector(pEV)asanegativecontroldecreasedtheproportionof
livecellsduringTDP-43knockdownat120h(Fig4A,lanes1&3).Interestingly,thepropor-
tionoflivecellsduringTDP-43knockdownwassignificantlyrestoredbytransfectionofpU6
(Fig4A,lanes3&4).TransfectionofpU6inNC-siRNA-transfectedcellsdidnotchangethe
proportionoflivecells(Fig4A,lanes1&2).Anotherassaybasedoncellularmetabolismalso
demonstratedtherecoveryofcellviabilitybytransfectionofpU6duringTDP-43knockdown
(S3Fig).
Next,toconfirmwhethertransfectionofpU6effectivelyincreasedtheamountofU6
snRNA,weperformedaqPCRat76haftertransfectionwithpU6.TheamountofU6snRNA
inNC-siRNA-transfectedcellsshowedaslightincreasebytransfectionwithpU6,butthedif-
ferencewasnotsignificant(Fig4B,lanes1&2).ApossiblereasonfortheslightincreaseofU6
snRNAexpressionmaybeautoregulatorytranscriptionalrepressionduringover-expressionof
U6snRNA[16].However,inthecaseofTDP-43knockdown,theamountofU6snRNAwas
significantlyhigherinpU6-transfectedcellsthanincontrolpEV-transfectedcells(Fig4B,
lanes3&4).WethereforeconcludedthattransientrescueofU6snRNAexpressionduring
TDP-43depletioncanbeachieved.Consequently,restorationofU6snRNAexpressioneffi-
cientlysloweddowntheconstitutionofcelldeathphenotypeduringTDP-43depletion.
WeconfirmedthattheproportionoflivecellsamongT43-siRNA-transfectedcellswas
restoredbyexogenousexpressionofaTDP-43thatisresistanttoknockdownbyT43-siRNA.
ExpressionofexogenousTDP-43taggedwithGFP(T43-GFP)inNC-siRNA-transfectedcells
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U6snRNApreventstoxicityofTDP-43depletion
Fig4.AmeliorationofcelldeathbytransientexpressionofU6snRNAinTDP-43-knockeddowncells.Significanceindicatedin
thegraphwastestedbyStudent’sttest:*p<0.05and**p<0.01.(A)Theproportionoflivecellsat120haftertransfectionofT43-
siRNAorNC-siRNAamongU6snRNA-expressingornon-expressingcells(mean±SEM;n=4).(B)QuantificationofexpressionofU6
snRNAusingqPCRat72haftertransfectionofU6snRNAexpressionplasmidDNAoremptyvector(mean±SEM;n=4).(C)The
proportionoflivecellsat120haftertransfectionofT43-siRNAorNC-siRNAamongTDP-43-expressingorcontrolcells(mean±SEM;
n=4).(D)ProteinabundanceofTDP-43andα-tubulinduringtransientexpressionofT43-GFPfor76h.Top:Westernblotting
usingananti-TDP-43oranti-α-tubulinantibody.Bands(i)and(ii)indicateT43-GFPandendogenousTDP-43,respectively.Bottom:
QuantificationofrelativeexpressionofTDP-43(mean±SEM;n=4).
https://doi.org/10.1371/journal.pone.0187813.g004
didnotaltertheproportionoflivecells(Fig4C,lanes1&2).ExpressionofT43-GFPin
T43-siRNA-transfectedcellsincreasedtheproportionoflivecells(Fig4C,lanes3&4).Inthis
case,expressionofT43-GFPwasconfirmedat72haftertransfection(Fig4D).Quantification
ofproteinlevelsindicatedthattransfectionofexogenousTDP-43resultedina33.2%±9.8%
recoveryofTDP-43proteinlevelscomparedwiththeendogenouslevels(Fig4D,lanes1&4).
The33.2%abundanceofTDP-43maybesufficienttopreventtocelldeath.
Inaddition,exogenousexpressionofU6snRNAinTDP-43-knockeddowncellsdidnotalter
theamountofendogenousTDP-43until120haftertransfectionofT43-siRNA(S4AandS4B
Fig).Therefore,U6snRNAmayhaveaprotectiveroleincelldeathduringTDP-43depletion.
ExogenousexpressionofU6snRNApartiallyamelioratesmis-splicing
duringTDP-43depletion
ToinvestigateaplausiblemechanismbywhichanincreaseintheU6snRNAlevelameliorates
theviabilityofTDP-43-knockeddowncells,weexaminedtheaberrantmRNA-splicingstate
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U6snRNApreventstoxicityofTDP-43depletion
Fig5.Changeintheexon-exclusionstateoftranscriptsduringexpressionofU6snRNAinTDP-43-knocked
downcells.(A)ImagesofmigratedbandsofsplicedformsofDnajc5,Sort1,andPoldip3transcripts.RPS18was
usedasaninternalloadingcontrol.Splicedformsoftranscriptsweredistinguishedusingsplicing-dependentpairsof
PCRprimers.(B)Relativeamountsoftheexon-excludedformsofDnajc5,Sort1,andPoldip3transcripts
(mean±SEM;n=5).SignificancewastestedbyStudent’sttest:*p<0.05,**p<0.01,and***p<0.001.Numbersin
eachbarshowmeanvalues.
https://doi.org/10.1371/journal.pone.0187813.g005
duringTDP-43depletion.Basedonpreviousreports[18–20,30],weselectedfourtranscripts
(Dnajc5,Sortilin1[Sort1],Poldip3,andMadd)asmis-splicingtargetsduringTDP-43deple-
tion.ThesplicingofthesetranscriptswasalteredinTDP-43-knockeddowncells(Fig5A,
lanes1&3andS5AFig,lanes1&3).
Next,weanalyzedwhetherexpressionofexogenousU6snRNAchangestheamountofthe
splicedformofthesetranscriptsinTDP-43-knockeddowncells.Theamountoftheexon-
excludedformofDnajc5andSort1transcriptswasslightlyincreasedbyexogenousexpression
ofU6snRNAinTDP-43-knockeddowncells(Fig5B,lanes3&4),butnotinNC-siRNA-
transfectedcells(Fig5B,lanes1&2),whereasthesplicingofPoldip3andMaddtranscripts
wasnotchangedbyexogenousexpressionofU6snRNAinTDP-43-knockeddowncells
(Fig5BandS5BFig).TheseresultssuggestthatthesplicingefficiencyofDnajc5andSort1
PLOSONE|https://doi.org/10.1371/journal.pone.0187813 November10,2017 9/14
U6snRNApreventstoxicityofTDP-43depletion
transcriptsinTDP-43-knockeddowncellscanbepartiallyrestoredbyexogenousexpression
ofU6snRNA.Moreover,itispossiblethatU6snRNAmayhaveaprotectiveroleagainstthe
alterationofsplicingefficiencyofaportionoftranscriptsduringTDP-43depletion.
Discussion
WeshowherethatTDP-43knockdowndownregulatestheamountofU6snRNAbutnotof
U1,U2,andU4snRNAsinNeuro2Acells.Twopossiblemechanismscouldexplainthisdown-
regulation:TDP-43couldbeinvolvedin(i)transcriptionalpromotionviaRNApolymeraseIII
and/or(ii)stabilityofU6snRNA.Inthefirstcase,U6snRNAistranscribedbyRNApolymer-
aseIII,whileU1,U2,andU4snRNAsaretranscribedbyRNApolymeraseII[24].However,
while7SLRNAisalsotranscribedbyRNApolymeraseIII[31],ourresultsshowthat7SLRNA
levelswerenotdecreasedduringTDP-43depletion(S2Fig).Therefore,transcriptionalpromo-
tionviaRNApolymeraseIIImaynotcontributetothedecreaseofU6snRNAlevelsduring
TDP-43depletion.Inthesecondcase,theU6snRNAsequencedoesnotcontaintypicalUG
repeats,whichTDP-43preferablyrecognizes;however,CLIPshowedthatalowamountof
U6snRNAbindstoTDP-43[19].WeconfirmedthatU6snRNAco-precipitatedwithTDP-
43-GFP(Fig2D),demonstratingthatTDP-43canassociatewithU6snRNA.Inaddition,
proteomicanalysisrevealedthatTDP-43interactswithSART1andPRPF3,twocomponents
ofU6snRNA-containingcomplexes[32].ThesefindingssuggestthatthedepletionofTDP-43
coulddestabilizethecomplex,releasingfreeU6snRNA,whichislikelydegraded.Therefore,
TDP-43maycontrolthestabilityofU6snRNAbydirectlymaintainingtheRNA-proteincom-
plexandnotviaanintermediateprocess(e.g.,anincreasedlevelofribonucleaseforU6
snRNAduringTDP-43depletion).However,itisunknownwhetherTDP-43bindstoU6
snRNAdirectlyorviaotherproteins,andthisshouldbeclarifiedinafurtherstudy.
AlthoughapreviousstudyusinghumanSH-SY5YcellsreportedthattheamountofU6
snRNAwasnotdecreasedbyTDP-43knockdown[25,26],weshowthatU6snRNAinTDP-
43-knockeddownmurineNeuro2Acellsisdownregulated(Fig2).Thisdiscrepancyislikely
duetodifferencesinthespeciesand/orcelllines,sincetheexpressionprofileofUsnRNAs
aftersiRNA-mediatedknockdowninsurvivalmotorneuronsprotein(SMN)differsbetween
humanandmurinecelllines[33].However,weshowthatexpressionofexogenousU6snRNA
canefficientlyrescuecelldeathduringTDP-43depletion(Fig4).Theseresultssuggestthat
downregulationofU6snRNAisinvolvedinthedeathofTDP-43-knockeddownmurineNeu-
ro2Acells.
Inaddition,ourresultsshowthatexpressionofU6snRNAinTDP-43-knockeddowncells
slightlyimprovedthemis-splicingefficiencyofDnajc5andSort1transcripts.Althoughthe
directeffectofU6snRNAonthealternativesplicingoftranscriptsintheTDP-43-knockdown
conditionhasnotbeenclarifiedyet,weexpectthatrestorationofU6snRNAlevelsduring
TDP-43depletionmightmaintainthealternativesplicingefficiencyofthesetranscripts.On
theotherhand,TDP-43loss-of-functioninhibitsvesiculartrafficking,includingendosomal
traffickingandautophagosome-lysosomefusion[15,34].Thesedefectsmaycontributetocell
deathbyalteringthetrophicstate.RestorationofU6snRNAexpressionfollowingTDP-43
depletionmaysuppresscelldeathbyregulatingtheexpressionofgenesimportantfortrophic
signaling.Futurestudiesshouldexplorethedetailedmechanismsbywhichdownregulation
ofU6snRNAexpressionaffectsalternativesplicingandincreasescelldeathuponTDP-43
depletion.
Althoughourresultsareconfinedtothecellularlevel,areportshowedadecreaseintheU6
snRNAlevelsinspinalcordfromALSpatients[26].Therefore,weexpectthatdownregulation
ofU6snRNAmightbeinvolvedinloss-of-functionofTDP-43inALSpathophysiologyand
PLOSONE|https://doi.org/10.1371/journal.pone.0187813 November10,2017 10/14