Molecular Cell Article Widespread Regulation of Transl - PDF document

MolecularCellWidespreadRegulationofTranslationbyElongationPausinginHeatShockReutShalgi,JessicaA.Hurt,IrinaKrykbaeva,MikkoTaipale,SusanLindquist,andChristopherB.BurgeDepartmentofBiologyDepartmentofBiologicalEngineeringMassachusettsInstituteofTechnology,Cambridge,MA02142,USAWhiteheadInstituteforBiomedicalResearch,Cambridge,MA02142,USAHowardHughesMedicalInstitute*Correspondence:cburge@mit.eduhttp://dx.doi.org/10.1016/j.molcel.2012.11.028 MolecularCell,439Ð452,February7,20132013ElsevierInc. usedribosomefootprintproÞlingtogloballymapthelocationsofindividualribosomesalongmRNAs(Ingoliaetal.,2009etal.,2011),inconjunctionwithRNAsequencing(RNA-seq)toassessmRNAabundance.RibosomefootprintandRNA-seqlibrarieswerepreparedfromNIH3T3mouseÞbroblastsundernormalgrowthconditions(control,37C),andafter8hrofmildC,HS8M,chronicheatstress)or2hrofsevereheatstressC,HS2S,acutestress)(FiguresS1AÐS1Eavailableonline).NeitheroftheseconditionsinducedsigniÞcantcelldeath,andcellsappearedtofullyrecover24hrafterwithdrawalfromheatstress(FigureS1F).Changesinlevelsoftotalandribosome-associatedmRNAwereobservedformanygenesfollowingbothmildandsevereheatstress(FigureS1A).HSPsshowedasigniÞcantupregulationintheirtranslationlevelsunderbothconditions,asmeasuredbyfootprintRPKM(FiguresS1AnalyticalpolysomeproÞlingrevealedasubstantialdecreaseintheproportionofheavypolysomesandtheaccumulationofmonosomesinbothmildandsevereheatstress(Figure1 0 10 20 30 40 50 60 70 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 Distance (mm)OD (254 nm) Control HS8M HS2S 35 40 45 50 55 60 65 70 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 Distance (mm)OD (254 nm) 80 0 100 200 300 400 500 0 0.5 1 1.5 2 2.5 position along ORF (nt) normalized footprint density 100 0 100 200 300 400 500 0 2 4 6 8 10 12 position along ORF (nt)no. of footprint readsSerpine1 0.062 0.125 0.25 0.5 1 2 4 8 16 32 64 0 200 400 600 800 1000 1200 102 101 100 101 102 102 101 100 101 102 5’LR Control 5’LR HS8M 102 101 100 101 102 102 101 100 101 102 5’LR Control 5’LR HS2S MP40S 60S 80S 2 3 4 polysomes (5+)ABCDE Control HS8M HS2S Control 5’LR=1.29 HS8M 5’LR=1.07 HS2S 5’LR=4.20 HS8M mean=1.04 HS2S mean=3.08 r5’LRnumber of genes Figure1.HeatShockInducesaGlobalIncreaseinRibosomeOccupancyaround5EndsofOpenReadingFrames(A)PolysomeproÞlesofmouseÞbroblasts(3T3cells)undernormalgrowthconditions(blue)oraftermildheatshock(42for8hr,green)orseverebutnotlethalheatshock(44for2hr,red).ThepolysomeregionwasdeÞnedas5-somesandhigher.P:M(polysometomonosome)ratiois0.79incontrol,0.18inHS8M,and0.08inHS2S.ProÞleisrepresentativeofthreereplicateexperiments,whererelativeP:MratiosinHS8Mwere22.5%±0.9%ofcontrolvalues,andrelativeP:MratiosinHS2Swere7.2%±2.7%ofcontrol.(B)NormalizedfootprintdensityalongmRNAs(seeExperimentalProcedures(C)Rawfootprintcountperposition(smoothed)acrosstheSerpine1transcript(ENSMUST00000041388,5UTRandÞrst500ntofthetranscriptareshown).(D)Distributionofr5LRvaluesinHS8M(green)andHS2S(red).Populationmeansareindicated.(E)Changesin5LRvaluesafterheatshock.SeealsoFigureS1TableS1 MolecularCellTranslationElongationPausinginHeatShockMolecularCell,439Ð452,February7,20132013ElsevierInc. consistentwithpreviousobservationsandwithaglobalreduc-tionintranslation(McCormickandPenman,1969).Notably,lighterpolysomefractionsrepresentingtwotofourribosomesactuallyincreasedsomewhatinsevereheatstress(Figure1discussedfurtherbelow.ThedistributionofribosomefootprintsequencereadsalongindividualmRNAs,orinaÔÔmetageneÕÕanalysisofreadsmappingtoallmRNAs,revealedamorecomplexpictureFigures1Band1C).Whilechangesinresponseto8hrofmildheatshockweregenerallymodest,dramaticchangesinrelativeribosomeoccupancyoccurredinresponseto2hrofsevereheatshockinfourdistinctregions.Theseincludedincreasesinthe5untranslatedregion(UTR)andtheÔÔinitiationregionÕÕ(readsindicatingpositioningoftheribosomalPsiteintheÞrst15basesoftheopenreadingframe[ORF]),andan1.7-foldincreaseinthenext180nucleotides(nt)aftertheinitiationregion,whichshiftedtoa1.7-folddecreaseintheremainderoftheORF(Figures1BandJ).Theoverallshapeofthisdistributionremainedunchangedwhethergene-levelnormalizationwasused(asinFigures1J,andS1K)orsimplyplottingrawreadcounts(FigureS1L).ManyindividualgenesthathadsufÞcientlyhighreadcoveragedisplayedadistri-butionsimilartothemetagenedistribution,asillustratedforSerpine1(Figure1C;additionalexamplesareshowninureS1M).Similarbiasesinrelativeribosomeoccupancyaftersevereheatshockwereobservedinabiologicalreplicateexper-imentdescribedbelow.ThesewholesalechangesinribosomeoccupancyalongmRNAsimplymajorshiftsintranslationalregulationinresponsetoheatshock.Theaccumulationofribo-somesinthe200ntaftertheinitiationsiterelativetotheremainderoftheORFwasparticularlynotablebecauseitimpliedextensivepostinitiationregulationoftranslation.Suchapossibilityhasbeennoted(BallingerandPardue,1983)butnotspeciÞcallycharacterizedpreviouslyincellularresponsestostress.Wethereforechosetofocusoureffortsinthisstudyoncharacterizingthisphenomenonindetailandexploringitspotentialcausesandconsequences.Toobjectivelymeasuretheextentofribosomeaccumulationinthe5endsofORFs,wedeÞnedthe5loadingratio(5ofageneastheratioofthefootprintreaddensitybetweenbases16and195(codons6Ð65)oftheORFtothedensityalongtheremainingdownstreampositionsintheORF.Thismeasure,whichisanalogoustomeasuresusedtoquantifytranscriptionalpausing(Rahletal.,2010),reßectstheextenttowhichORF-associatedribosomesarepreferentiallyaccumulatedordepletedatthe5endofanORF.ToassesstheeffectofheatstressontranslationofindividualmRNAs,wecalculatedtherelativeloadingratio,r5LR,deÞnedastheratioofthe5underheatstresstothatincontrolconditions.Followingmildheatstress,nosigniÞcantincreasein5LRwasobservedinthemetageneanalysisorformostindividualgenes:r5LRvalueswerecenteredaround1.0orslightlyabove(Figures1B,1D,and1E).Thedistributionof5LRvaluesincreaseddramaticallyfollowingsevereheatstress,withameanr5LRvalueabove3Figure1D).Theincreasein5LRinsevereheatstresswasapparentformostindividualmRNAsexamined(Figure1TableS1),indicatingaglobalchangeinthetranslationalTranslationElongationIsTransientlyPausedinSevereHeatStressMultipleregulatorymechanismsmightproducesuchaglobalincreasein5LR,including(1)transient(reversible)pausingoftranslationelongationaroundcodon65onmostofthegeneÕsmRNAs(resultinginaccumulationofribosomes5ofthepause),(2)irreversiblestallingoftranslationelongationaroundcodon65onasubsetofthegeneÕsmRNAs(alsoresultinginaccumulationofribosomesupstream),(3)accelerationoftranslationelonga-tionaftercodon65,or(4)prematureterminationoftranslationaroundcodon65onasubsetofageneÕsmessages.Todiscrim-inatebetweenthesepotentialexplanationsfortheincreasein5LRaftersevereheatshock,weperformedanalyticalpolysomeproÞlingafterinhibitionoftranslationinitiationwiththedrugHarringtonine,whichblocksinitiationaftersubunitjoiningbypreventingelongationduringtheÞrstroundofpeptidebondformation(Huang,1975Ingoliaetal.,2011).SincetreatmentwithHarringtonineinhibitsnewlyinitiatingribosomeswhileallowingpreviouslyengagedribosomestocontinuetranslatingthroughtermination,thisdrugenablesthefatesofelongatingribosomestobefollowedunderdifferentconditions.Asexpected,undercontrolconditions,treatmentwithHar-ringtonineresultedinadecreaseinheavypolysomesandanincreaseinmonosomesthatwasreadilyapparentafter1minandmorecompleteafter3min(Figure2A).Undersevereheatshockconditions,1minofHarringtoninetreatmenthadonlyaminimaleffectonthepolysomeproÞle,but3mintreatmentresultedinadistinctreductioninheavypolysomes,suggestingthatmostribosomesarenotirreversiblystalled(Figures2AandA).Anaccelerationoftranslationaftercodon65wouldpredictmorerapidratherthanslowercollapseofpolysomesinheatshockconditions,andprematureterminationwouldalsonotbeexpectedtoslowthecollapseofpolysomes.Theslowerkineticsofthiscollapsearethereforemostconsistentwiththemodelinwhichribosomesaretransientlypausedduringtransla-tionelongationinheatshockconditions.ToconÞrmthatthereducedcollapserateofpolysomesunderheatstressafterHarringtoninetreatmentwasduetoalteredelongationrates,weusedthetranslationalinhibitorpuromycin,whichcausesdissociationofribosomesthatareactivelytrans-lating.Treatmentwithpuromycinyieldedasimilarreductioninpolysomesinheat-stressedcellsasundercontrolconditionsFigureS2B),indicatingthatundersevereheatstress,asincon-trolconditions,mostribosomesaretranslatingandareneitherirreversiblystallednorprematurelyterminating.Together,thesedatasupportthattheslowedkineticsofpolysomecollapseresultfromalteredelongationrates.Ribosomepausingwascenteredaroundcodon65basedonthemetageneanalysis,butsuchanaveragedrepresentationcouldhidevariabilitybetweengenes.Tocharacterizethisphenomenoninmoredepth,wecalculatedtheinferredlocationandstatisticalsigniÞcanceofshiftsinribosomefootprintdensityalongindividualgenesusingamodiÞedKolmogorov-Smirnov(KS)test.WereasonedthatiftranslationofanmRNAispausedinheatshockedcells,ribosomesshouldaccumulateupstreamofthepause,sothedensityoffootprintreadsshouldbesigniÞ-cantlyhigherbetweentheAUGandthelocationofthepauserelativetotheremainderoftheORF;thiscanbedeterminedby MolecularCellTranslationElongationPausinginHeatShockMolecularCell,439Ð452,February7,20132013ElsevierInc. theKStest(SupplementalExperimentalProcedures).Forexample,thistestidentiÞedahighlysigniÞcantshiftinfootprintdensityatnucleotide189(codon63)oftheVimentinORF,aposi-tionwecalltheÔÔKSlocation,ÕÕsuggestingthatribosomespauseinthisvicinityandaccumulateupstream(Figure2Genome-wideKSanalysisindicatedthat75%ofthetrans-latedmRNAshadasigniÞcantelongationpauseundersevereheatshockconditionsatafalsediscoveryrate(FDR)cutoffof5%.ThelocationdetectedbytheKSanalysisbeyondwhichfootprintdensitybegantoplateaumostlyoccurredbetween 0 10 20 30 40 50 60 70 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 Distance (mm)OD (254 nm) Control Control+Har 1min Control+Har 3min 0 10 20 30 40 50 60 70 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 Distance (mm)OD (254 nm) HS2S HS2S+Har 1min HS2S+Har 3min 35 40 45 50 55 60 65 70 0.01 0.04 0.07 0.1 Distance (mm)OD (254 nm) 35 40 45 50 55 60 65 70 0.01 0.04 0.07 0.1 Distance (mm)OD (254 nm) 0 300 600 900 1200 0 2 4 6 8 10 12 14 16 18 20 position along ORF (nt)no. of footprint readsVimentin Control 5’LR=0.90 HS8M 5’LR=1.09 HS2S 5’LR=4.95 91 128 181 256 362 512 724 1024 1448 2048 2896 4096 0 200 400 600 800 1000 1200 KS location (nt from AUG) no. of significant genes HS8M (358 sig at FDR 5%) HS2S (3856 sig at FDR 5%) 0 300 600 900 1200 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 position along ORF (nt)cumulative fraction of footprints Control HS2S 200 0 200 400 600 800 1000 0 2 4 6 8 10 Atf4 uORF1uORF2MPACBDAUGMP189Control 5’LR=1.36HS8M 5’LR=1.48HS2S 5’LR=1.59position along ORF (nt)no. of footprint reads Figure2.RibosomesArePausedafterTranslatingabout65AminoAcidsinSevereHeatShock(A)PolysomeproÞlesofHarringtonine-treatedcontrol(leftgraph,Control)andsevereheatshock(rightgraph,HS2S)-treatedcells.Heatshockedorcontrolcellswereharvestedaftertreatmentwith0.1mMHarringtoninefor1min(darkblueandpurplelinesforcontrolandHS2S,respectively)or3min(cyanandpinklinesforcontrolandHS2S,respectively)orwithoutanytreatment(blueandredlines).P:Mratiosare0.8incontrol,0.33incontrol+Har1min,0.07incontrol+Har3min,0.10inHS2S,0.09inHS2S+Har1min,and0.05inHS2S+Har3min.Areplicateexperimentshowedasimilarpolysomecollapse(FigureS2(B)IllustrationoftheKSlocationandpvaluecalculationforVimentin(ENSMUST00000028062).CumulativeplotsofreadsalongtheORF(bottompanel)areshownforcontrol(blue)andHS2S(red)(seetheExperimentalProcedures(C)DistributionoftheKSlocationsforalluniquetranslatedgeneswhichwerefoundtobesigniÞcantlypaused(atanFDRof5%)inHS8Mrelativetocontrol(green)andinHS2Srelativetocontrol(red).ThenumberofsigniÞcantgenesisindicated,outof5,125genes.(D)Rawfootprintcounts(smoothed)alongthetranscript(ENSMUST00000109605)showanescapefromtheglobalpause.uORFpositionsaremarked.SeealsoFigureS2TableS2 MolecularCellTranslationElongationPausinginHeatShockMolecularCell,439Ð452,February7,20132013ElsevierInc. 197and466ntfromtheinitiationcodon(25and75Figures2CandCandTableS1),withamedianestimatedpositionof263nt.Thus,individualgeneanalysisindicatedanelongationpauseintheÞrstfewhundredbasesforthemajorityofmousemRNAsinresponsetosevereheatshock,aphenom-enonwecallÔÔ5ribosomepausing.ÕÕAribosomepausedintheÞrstcoupleofhundredbasesofanORFmightbeexpectedtocauseaccumulationofafewribosomesupstream,likelyrunningasalightpolysome,consistentwiththeobservedincreaseinthelightpolysomefractionseeninsevereheatshock(Figure1Whileouranalysessuggestedthatthevastmajorityofgenesexperience5ribosomepausinginsevereheatshock,theincreasein5LRwasnotuniversal.OnegenesetofinterestwasthesetoftranscriptsthatwerenotsigniÞcantlypaused(basedontheKStest).Functionalenrichmentanalysisofthissetdetectedenrichmentfortranscriptionalregulators(GOtermÔÔtranscriptionfactoractivity,ÕÕp=3.5,FDR1%),whichmightcontributetothetranscriptionalresponsetoheatshock.WeusedamorestringentsetofcriteriatodeÞneÔÔescapersÕÕasmRNAswhose5LRdidnotsigniÞcantlychange(ordecreased)aftersevereheatshock(SupplementalExperimental).WedidnotobserveHSPstobeamongtheescapers,suggestingthattheupregulationoftranslationofHSPsnotedabove(FiguresS1GÐS1I)hasothercauses,e.g.,increasedmRNAlevelsand/orincreasedtranslationinitiation.However,thesecriteriadididentify187mRNAsthatescapedtranslationalpausing,includingseveraltranscriptionfactorsTableS2).Interestingly,thetranscriptionfactorgeneswerebothamongtheescapers,withr5LRvaluesof1.18and1.05,respectively,farbelowthemeanvalueofaroundFigures2DandD).ATF5istranslationallyinducedafteravarietyofstresses,includingheatshock(Zhouetal.,2008ThehomologousATF4factorisamajorregulatorofoxidative,endoplasmicreticulum(ER),andaminoacidstarvationstressHardingetal.,2000)buthasnotbeenpreviouslyconnectedtotheheatshockresponse.BothofthesefactorsareknowntobetranslationallyupregulatedunderstressconditionsviaamechanisminvolvingtranslationofupstreamORFs(uORFs)Hardingetal.,2000Zhouetal.,2008).Ourobservationssuggestthatescapefromtheglobalelongationandinitiationblocksareusedtoregulatedistinctsubsetsofgenes.RibosomeAssociationofHsp70ChaperonesIsAlteredduringHeatShockWenextsoughttoexplorepotentialmechanismsunderlyingthegeneralphenomenonof5ribosomepausing.Thenear-universalscopeandrelativelyspeciÞclocationofthepausesuggestedtheinvolvementofageneralaspectoftranslationelongation,whichmightbedependentmoreonthenascentpeptidethanongene-speciÞcproperties.NascentpeptidesareboundbyavarietyoffactorssuchasSRP(discussedbelow)andHsp70chaperonesKrameretal.,2009),whoseregulationisalsoahallmarkoftheheatshockresponse.ThecytosolicHsp70(70kilodalton[kda]heatshockprotein)chaperonesHSC70andHSP70ÑtheconstitutiveandinducibleformsofcytosolicHsp70,respec-tivelyÑareamongthemajorchaperonesinthecellthatmediateproteinfolding.Duringtranslation,theseproteinsassociatewithribosomesandinteractwithnascentchainsastheyemergefromtheribosomeexittunnel(Beckmannetal.,1990Nelsonetal.,).ThisfunctionofHsp70familyproteinshasbeenshowntobeimportantforcellgrowthbothinyeastandinmammalsJaiswaletal.,2011Nelsonetal.,1992).Heatstressplacesasubstantialburdenonchaperonesasaresultofthemisfoldingofexistingcytosolicproteins,and,inresponse,cellsupregulatestress-speciÞchomologsofthesechaperones,includingHSP70(encodedbythegenesinmice),whichsupplementsthelevelsoftheconstitutiveHSC70,encodedbyToassesstheassociationofHsp70chaperoneswiththeribo-someafterheatshock,wefractionatedpolysomegradientsfromcontrolorheat-shockedcellsandmeasuredtheamountofHSC70/HSP70thatcomigratedwithpolysomesusingapan-Hsp70antibody.Wedetectedaconsistent2-to4-foldreductionintheassociationofHSC70/HSP70withmonosomesandpoly-somesinsevereheatshock(Figures3Aand3B).RelativelylittlecomigrationofHSC70/HSP70wasobservedinthepolysomalportionofthegradientfollowingdissociationofribosomesbyEDTAtreatment,supportingthespeciÞcityofthedetectedasso-ciation(FigureS3A).SigniÞcantdepletionofHSC70/HSP70wasdetectedinseveralfractionsrangingfrommonosomesto5-somes(Figure3B).mRNAswithribosomespausedattypicallocations(around200nt)areexpectedtomigratemostlyaslighterpolysomesormonosomes,dependingonwhetheraddi-tionalribosomesaccumulateupstreamofthepause.ReducedassociationofHsp70chaperoneswithtranslatingribosomescouldaffecttheribosomeinvariousways,includingtheexpo-sureofnascentchains.SigniÞcantlyPausedmRNAsHaveFeaturesAssociatedwithHsp70InteractionHSC70/HSP70bindnascentchainsgenerallybuthavehigherafÞnityforhydrophobicpeptides,andhydrophobicpeptidesareexpectedtobemorereliantoninteractionwithHSC70/HSP70forproperfolding(Hartletal.,2011).TheÞrst20Ð25resi-duesofproteinstendtobemorehydrophobicthantherestoftheprotein,particularlyforproteinscontainingasignalpeptide(Heijne,1981),butalsotoalesserextentforotherproteinsFigures3CandC).WefoundthatgeneswithsigniÞcant5ribosomepausingencodedmorehydrophobicNterminithandidnonsigniÞcantlypausedgenes(Figure3C,ttestpvalue=).HydrophobicityoftheNterminusalsodifferedbetweenmRNAsgroupedbytheirKSlocation.ThosewithKSlocationsbetweencodons44Ð171wereassociatedwithhigherhydrophobicityoftheNterminusthanthe(smaller)setsofmessageswithmoredistallyormoreproximallylocatedpausesites(FigureS3E),suggestingthataminoterminalhydropho-bicitycontributestopausinginthisregion.Hsp70isknowntohaveaparticularlyhighafÞnityforstretchesofÞvehydrophobicresiduesßankedbyfourpositivelychargedaminoacidsoneachside(digeretal.,1997).AnalysisofthebindingsitecontentofproteinNterminiwiththescoringsystemproposedbyRudigerandcolleaguesrevealedthatsigniÞcantlypausedmRNAshadbetteraverageHsp70bindingsitescores,reßectinggreaterpropensityforHsp70binding(Figure3D),suggestingthatthesepeptideswouldbemoredependentonHsp70binding.Consis-tently,theNterminiofnonsigniÞcantlypausedmRNAsencoded MolecularCellTranslationElongationPausinginHeatShockMolecularCell,439Ð452,February7,20132013ElsevierInc. 10 20 30 40 50 60 70 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4 3.9 position along protein (aa)Hsp70 binding site score (Rudiger) all mRNAs (4805) sig paused mRNAs (3645) non sig mRNAs (1160) 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Control HS2S CHS2SControlHS2S CHS2S 7525 40/60m1m22some3some4-55-77+heavyunboundOD (254 nm) m1 m2 2some 3some 4-5 5-7 7+ heavy RPL10A * m1 m2 2some 3some 4-5 5-7 7+ ControlHS2S PM 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 all mRNAs (4805) sig paused mRNAs (3645) non sig mRNAs (1160) background 10203040506070 Figure3.AssociationofHSC70/HSP70withPolysomesIsAlteredafterHeatShock(A)WesternblotofHSC70/HSP70(withpan-HSP70antibody,seetheExperimentalProceduresfordetails)proteinlevels(middlepanel)acrossapolysomegradientincontrolandsevereheatshockcells(HS2S,toppanel).RPL10Awasusedtoassessribosomeabundanceineachfraction(middlepanel).Levelsof(legendcontinuedonnextpage) MolecularCellTranslationElongationPausinginHeatShockMolecularCell,439Ð452,February7,20132013ElsevierInc. signiÞcantlyfewerstrongHsp70bindingsites(FigureS3mRNAsthatencodedsignalpeptidesexhibitedasimilardegreeof5ribosomeaccumulationasmRNAsthatdidnot(FigureS3anddifferencesinhydrophobicitybetweenpausedandnon-pausedmRNAswereobservedformRNAsencodingproteinswithandwithoutsignalpeptides(FigureS3F),suggestingthatthisregulationoccursindependentlyofthesecretorypathway.BoththelocationofthishydrophobicstretchattheNterminus,andthehigherhydrophobicityofsigniÞcantlypausedmRNAsareconsistentwithpausingbeinginßuencedbynascentpeptides,aroundwheretheymightbeexpectedtoÞrstrequireinteractionwithchaperones.ModulationofHsp70ChaperoneActivityRegulatesTranslationElongationPausingTotestthepotentialroleofHsp70chaperonesinheatshock-inducedelongationpausing,weÞrstanalyzedthermotolerance.Chaperoneexpressionisinducedbymildheatshock(KelleyandSchlesinger,1978Lindquist,1980FigureS3B),explain-ingwhymildheatshocktreatmentpriortosevereshockhasprotectiveeffectsoncellsinthephenomenonknownasthermo-tolerance(GernerandSchneider,1975Henleetal.,1978).Ther-motoleranceisdependentonproteinsynthesisandenablesenhancedtranslationundersevereheatshockconditions(quist,1980PetersenandMitchell,1981).Toassesshowprein-ductionofchaperonesinßuencesribosomedensitypatterns,weperformedathermotoleranceexperiment,pretreatingcellswith8hrofmildheatshockpriorto2hrofsevereheatshock,alongwithabiologicalreplicateofthecontrol,mild,andseveretreat-ments.Pretreatmentofcellswithmildheatshockresultedinpartialrescueofthepolysomecollapsethatoccurredduringsevereheatshock(Figure4A),consistentwiththeexpectedrelieffromtranslationalinhibition.ReplicateribosomefootprintdatayieldedmetageneproÞlesundercontrol,mild,andsevereheatshockconditions(Figure4B)similartothoseobservedinure1.Remarkably,pretreatmentwithmildheatshockalmostcompletelyrescuedthe5ribosomepausingobservedafter2hrofsevereheatshock(Figures4BandA).Theseobserva-tionssuggestthat5ribosomalpausingisregulatableandisreducedbyfactorsinducedbymildheatstresssuchasTomorespeciÞcallylookattheroleofHSC7/HSP70chaper-onesinthephenomenon,weexploredwhetherinhibitionofHsp70activityundercontrolconditionswouldaffecttranslationelongation.Totestthispossibility,wesubjectedcellstoashorttreatmentofasmall-moleculedrug,VER-155008,thatinhibitstheATPaseactivityofbothconstitutiveandinducibleHsp70proteins,anactivitythatisrequiredfortheirchaperonefunctionMasseyetal.,2010).TreatmentwiththeHsp70inhibitorforashortperiodledtoareproduciblereductioninheavypolysomesandanaccumulationofmonosomes(Figure4supportingthedirectinvolvementofHsp70proteinsinthecontroloftranslationinmammaliansystems.FootprintproÞlingrevealedapronouncedaccumulationofribosomesnearthe5endsofmRNAsincellstreatedwiththedrugfor3hrintheabsenceofstress(Figure4AnalysisofindividualmRNAsrevealedthat2,500mRNAsweresigniÞcantlypausedaftertreatmentwithHsp70inhibitor,themajorityofwhichwerealsosigniÞcantlypausedundersevereheatshock(overlapof2072,p=10)withasimilarbutsomewhatbroaderdistributionofinferredKSlocationsFigureS4C).Similartowhatweobservedundersevereheatshock(Figure3C),mRNAsthatweresigniÞcantlypausedafterHsp70inhibitortreatmentencodedsigniÞcantlymorehydro-phobicNterminithannonpausedmRNAs(p=5FigureS4F).TheinferredKSlocationswereinthevicinityofthoseseenundersevereheatshockfor60%ofthemRNAs,comparingthesamemRNAsinbothexperiments(DandS4E),whilethemetageneanalysisshowedaccumula-tionsomewhat5tothatobservedinheatshock.DifferencesbetweentheeffectsofinhibitortreatmentandsevereheatshockonfootprintproÞlesmayreßectincompleteinhibitionofHsp70activityorfunctionsofHsp70sthatdonotrequireATPhydrolysis.Similartothetrendsobservedinsevereheatstress,moreproximalKSlocationscorrelatedwithgreaterN-terminalhydrophobicity,whilemoredistalKSlocationswereassociatedwithreducedN-terminalhydrophobicityFigureS4ToverifythatHsp70chaperonesdirectlyaffectribosomeaccumulationandtoexplorethisphenomenoninanotherspecies,weoverexpressedtheinducibleHSP70()inhuman293Tcells.WeÞrstsubjected293Tcellsto2hrofsevereheatstress.Aftersevereheatshock,293Tcellsshowedasimilarribosomeaccumulationtothatobservedinmouse3T3cells,withasimilarpauselocationataround200ntintotheORFbasedonmetageneanalysis(Figure4E),ofsomewhatsmaller comigratingHSC70/HSP70proteinandRPL10AproteinweremeasuredwithImageJsoftware.RibosomalassociationofHSC70/HSP70wasquantiÞedbynormalizingtheamountofHSC70/HSP70proteinbytheamountofRPL10Aproteinineachfraction([HSC70/HSP70]/[RPL10A])forbothcontrol(bluebars)andheatshock(redbars)conditions.P:Mratiois1.9incontroland0.18inHS2S.ProÞleisrepresentativeofsixexperiments,whererelativeP:MratiosofHS2Swere8.5%±2.9%ofcontrol.(B)QuantiÞcationandsigniÞcanceofthereductioninHSC70/HSP70ribosomeassociationafterheatshock.Meanandstandarderroroftheratio(log)ofHSC70/HSP70association([HSC70/HSP70]signal/[RPL10A]signal),inHS2Stocontrolconditions,ineachfraction,overÞvebiologicalreplicateexperimentsisshown.FractionsinwhichHSC70/HSP70and/orRPL10Asignalwasundetectablewereexcludedfromanalysis(seetheExperimentalProceduresfordetails).Signif-icanceofreductioninHSC70/HSP70ribosomalassociationisgivenbyttest.*p0.05,**p0.01.(C)Hydrophobicityscores(seetheSupplementalExperimentalProcedures)oftheÞrst70aminoacidsencodedbyallhighlytranslatedmRNAs(inblack),ofsigniÞcantlypausedmRNAs(inmagenta)andofnonsigniÞcantlypausedmRNAs(inorange).ThebackgroundrepresentingtheoverallaverageKyte-DoolittlehydrophobicityofallhighlytranslatedmRNAs(graydashedline)isshownforreference.ThettestpvalueforthedifferenceinthemeanhydrophobicityvaluesoftheÞrst25aminoacidsbetweenallsigniÞcantlypausedmRNAsandnonsigniÞcantmRNAswas3.8(D)Hsp70bindingsitescores(SupplementalExperimentalProcedures)wereplottedasin(C).ThettestpvalueforthedifferenceinthemeanHsp70scoresoftheÞrst25aminoacidsbetweenallsigniÞcantlypausedmRNAsandnonsigniÞcantmRNAswas5.3SeealsoFigureS3 MolecularCellTranslationElongationPausinginHeatShockMolecularCell,439Ð452,February7,20132013ElsevierInc. magnitudethanthatseenin3T3cells.Thisobservationextendsthephenomenonofheatstress-induced5ribosomalaccumula-tiontoasecondmammalianspecies.EctopicexpressionofHsp70sisgenerallyinhibitorytocellgrowth(Federetal.,1992),butwewereabletoachievemoderatelevelsofoverexpressionofHSP70,about1.5-foldin 0 10 20 30 40 50 60 70 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 Distance (mm)OD (254 nm) 30 35 40 45 50 55 60 65 70 0 0.01 0.02 0.03 0.04 0.05 0.06 Distance (mm)OD (254 nm) 80 0 100 200 300 400 500 0 0.5 1 1.5 2 2.5 3 3.5 position along ORF (nt) Normalized footprint density Control HS8M HS2S HS8M2S 0 10 20 30 40 50 60 70 0.04 0.06 0.08 0.1 0.12 0.14 0.16 Distance (mm)OD (254 nm) Control Hsp70 inhibitor 80 0 100 200 300 400 500 0 0.5 1 1.5 2 2.5 3 3.5 position along ORF (nt) Normalized footprint density 80 0 100 200 300 400 500 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 position along ORF (nt) Normalized footprint density 30 35 40 45 50 55 60 65 70 0.04 0.05 0.06 0.07 0.08 Distance (mm)OD (254 nm)ABDCEMPMPControlHsp70 inhibitorHS2S Control HS8M HS2S HS8M2S 293T GFP293T GFP+HS2S293T Hspa1a293T Hspa1a+HS2S Figure4.ModulationofHsp70ChaperoneActivityRegulatesTranslationElongationPausing(A)PolysomeproÞlesofathermotoleranceexperiment.3T3cellsweretreatedwithmild(green),severe(red),ormildpriortosevere(HS8M2S,magenta)heatshock,orleftuntreated(blue).P:Mratiosare1.4incontrol,0.33inHS8M,0.1inHS2S,and0.2inHS8M2S.ProÞleisrepresentativeoftworeplicateexperiments,whererelativeP:Mratioswere22.4%±1.3%ofControlinHS8M,5.8%±1.7%ofcontrolinHS2S,and12.2%±2.7%ofcontrolinHS8M2S.(B)NormalizedfootprintdensityalongmRNAsforabiologicalreplicateexperimentandforcellspretreatedwithmildheatshock(8hrat42C)priortosevereheatshock(magenta).(C)PolysomeproÞlesofcellstreatedwiththeHsp70inhibitorVER-155008(Masseyetal.,2010)ata20Mconcentration(Hsp70inhibitor,cyanline)orDMSO(control,blueline)for3hr.P:Mratiosare1.52incontroland0.7inHsp70inhibitor.ProÞleisrepresentativeofthreereplicateexperiments,whererelativeP:MratiosforHsp70inhibitorwere46.5%±3.8%ofcontrol.(D)NormalizedfootprintdensityalongmRNAsfor3T3cellsafter3hrofHsp70inhibitor(VER-155008)treatment(cyan).Controlandsevereheatshockplots(dottedlines)areidenticaltothosein(B).(E)NormalizedfootprintdensityalongmRNAsfor293TcellsoverexpressingGFPor,withorwithout2hrofsevereheatshock.SeealsoFigureS4 MolecularCellTranslationElongationPausinginHeatShockMolecularCell,439Ð452,February7,20132013ElsevierInc. excessoverHSC70/HSP70levelsobservedinnormalgrowthconditions(FigureS4H).Wenotethat,asiscommonlyobservedintransformedlines,293TcellsexpressbothHSC70andHSP70undercontrolconditions(FigureS4H).HSP70-overexpressingcellswerethensubjectedto2hrofsevereheatshock.Strikingly,thismoderateectopicexpressionofHsp70resultedinapartialrescueoftheelongationpausingfollowingsevereheatshock,asseeninFigure4E(seealsoFiguresS4IandS4J).Theobser-vationsthatHsp70inhibitortreatmentleadsto5pausingintheabsenceofstress,andthatmoderateoverexpres-sionofHsp70partiallyrelievesheatshockinducedelongationpausing,directlyimplicateHsp70chaperoneintheregulationoftranslationelongationduringsevereheatshock. TP53 RPS14 RPS6 RPL3 RPS3 RPS2 RPLP1 BAG2 DNAJA4 DNAJA3 BAG1 BAG3 BAG5 STUB1 HSF1 BAG4 RPL13A RPL18 DNAJC7 RPL12 RPL15 RPL23A RPL6 RPS18 RPL11 RPS16 RPL28 RPL4 RPS10 RPL10A RPSA RPL14 RPL13 RPLP0 RPS3A RPS19 RPS4Y1 RPL7L1 RPS17 RPL10L RPS11 HSC70 EIF2S3 EIF4H EIF3I RPS6KB2 EIF3M EIF2AK2 EIF2AK4 EIF2B2 EIF4E2 EIF4E3 EEF2 EEF1A2 EEF1G EEF1A1 EEF1D EIF2AK1 GSPT2 EIF4A2 GSPT1 EIF5 EIF3D EIF3K EIF4B EIF3C EIF2B4 EIF5B EIF3F EIF3E EIF3L DNAJB4 DNAJA2RibosomalProteinsCochaperonesElongation ReleaseInitiation Log2 Fold Change HS/Control (Hsp70/FLAG) 543210-1-2-3-4-5-6HSF1EEF1A1EIF4E2EIF2AK4EIF2B2RPS6KB2RPS3RPLP0RPL28RPL23ARPS2RPL18RPL22RPL4GSPT1GSPT2EIF3I EEF1A1RPS6KB2RPL23ARPL4 HSF1 C HS HSP70ABC C HS C HS C HS C HS DNAJB1 Figure5.TheHsp70Chaperone-Transla-tionMachineryInteractome(A)HSC70-translationalmachineryinteractomeinanetworkrepresentation,wasdrawnusingCytoscape.Proteinsarerepresentedbycircles,andthesizeofthecirclecorrespondstotheHSC70interactionscorelog([HSC70,lumines-cence]/[FLAGELISA]).Differentgroupsofinterestareindicated.(B)Hsp70interactomechangesupon2hrofsevereheatshockwastestedwithcoIP.BlotswerequantiÞedwithImageJandshownarelog2ofHsp70(interaction)normalizedtoFLAG(expres-sion),ofproteinsthatchangedmorethan1.5-fold.StripedbarsrepresentproteinswithincreasednormalizedinteractionandsigniÞcantdestabiliza-tion,deÞnedasasigniÞcantreductioninFLAGlevelsÑatleast3-foldinonereplicateandatleast2-foldintheother.DarkgraybarsrepresentincreasedinteractionswithHsp70withstableproteinlevels.LightgraybarsrepresentdecreasedinteractionswithoutsigniÞcantdestabilization.Meanandstandarddeviationoftwoexperimentsisshown.(C)CoIPWBdataforseveralproteins.SeealsoFigureS5TableS3,andTableS4,aswellastheSupplementalExperimentalProceduresTheHsp70-TranslationalMachineryInteractome:CharacterizationandStress-AssociatedChangesHsp70chaperonesareknowntobindnumerousproteins(Callonietal.,2012KampingaandCraig,2010).Here,itwasofparticularinteresttocharacterizeinteractionsbetweenHsp70sandthetranslationmachinery.Consistentwitharecentobservation(Jaiswaletal.,2011thattheribosomalcochaperoneDNAJC2(MPP11)doesnotinßuencetheribosomalassociationofHSC70(thepredominantHsp70proteinin3T3cellsinbothcontrolconditionsandafter2hrofsevereheatshock),knockdownofthisfactorin3T3cellsdidnotappreciablyaffectthedistri-butionofribosomesundernormalgrowthconditions(datanotToexplorepotentialwaysinwhichHsp70chaperonesmayinßuenceelongationpausing,wenextsoughttocharacterizedirectinteractionsbetweenHsp70sandthetranslationmachin-eryusingthehigh-throughputLUMIERwithBACONassay(LUMIERwithBaitcontrol,ExperimentalProceduresTaipaleetal.,2012).Usingthisassay,weexaminedtheinteractionof504clonesrepresentingabroadpanelof377translation-relatedgenes(TableS3)ina293TcelllinestablyexpressingRenillataggedHSC70.WefoundthatHSC70interactswithabouthalfofthesefactorstovaryingextents(213genes,abovethe75centileoftheestimatedbackgroundluminescence,Figure5 MolecularCellTranslationElongationPausinginHeatShockMolecularCell,439Ð452,February7,20132013ElsevierInc. andwith109ofthemstrongly(atthe99.9percentileofback-FiguresS5AandS5B).TheknownHsp70clientproteinsHSF1(Abravayaetal.,1992)andp53(Zyliczetal.,2001)wereamongthetopinteractors(ranked4and33intheirluminescencescore),aswereahandfulofcochaperoneswhichwereincludedaspositivecontrols.Wenextaskedwhethertheseinteractionschangedinresponsetostress.TheLUMIERassayisbasedRenillaluciferasetagging,andluciferaseactivityismarkedlyreducedafterheatshock(datanotshown).Thus,forapplicationofthismethod,weinsteadtreatedcellswithanotherproteotoxicstress,theproteasomeinhibitorbortezomib.Ofcourse,someaspectsofthecellularresponsestobortezomibandheatstressmaydiffer.ButtheresultinginteractiondatashowedaglobalreductionintheinteractionofHSC70withribo-somalproteins(FigureS5C),parallelingthereducedribosomalassociationofHsp70observedinsevereheatshock.Toexplorepotentialchangesintheinteractionsoftheendog-enousHsp70chaperoneswiththetranslationalmachineryfollowingheatstress,weusedalower-throughputcoimmuno-precipitation(coIP)assay.Thisassaywasappliedafter2hrofsevereheatshocktoeachofasetof48translation-relatedproteins,includingprimarilytranslationfactorsandribosomalproteins(andseveralpositivecontrols)representingaspectrumofbasalHSC70interactionscores(Figures5B,5C,andDandTableS4).Inthisassay,transfectedFLAGtaggedproteinswerepulleddownandproteinexpressionquantiÞedbyanti-FLAGantibody,whileinteractionwiththeendogenousHsp70swasmeasuredwithapan-Hsp70antibody.BecausetheFLAGtagswerelocatedattheCterminus,thisassaymeasuresinteractionwithmatureratherthannascentproteins.Interactionscoreswerethencalculatedastheratioof[Hsp70]/[FLAG],controllingforeffectsonexpression.HSF1,theheatshocktranscriptionfactor,hadreducedinteractionafterheatstress(Figures5and5C),consistentwithitsphosphorylation,trimerization,andconcomitantactivationofitstranscriptionalactivity(rkandSistonen,2010Heatshockisknowntocausemisfoldingofpre-existingproteins,whicharethendetectedbyHsp70andotherchaper-onesforrefolding.Ifthechaperonesareunabletorefoldthoseproteins,theyaretargetedfordegradation,thuspreventingaccumulationandaggregationofmisfoldedproteinsinthecellHartletal.,2011).WeclassiÞedeachofthetestedproteinsaccordingtothechangeinitsHsp70interactionscore(increasedordecreased),andproteinlevels(stableordestabilized,asmeasuredbyFLAG),afterheatshock.Severalproteinsshowedalargereductionintheiroverallproteinlevelswhiletheirinterac-tionwithHsp70chaperonesremainedunchangedorwaselevated,resultinginanincreaseintheirinteractionscores(ure5B).Thisgroupisexpectedtoconsistofproteinsthatbecomemisfoldedanddegradedasaresultofsevereheatshock,andhenceshowanincreaseinHsp70interaction.AmongthosearethereleasefactorGSPT2(eRF3component),andtheinitiationregulatorRPS6KB2.OnlytwoproteinsshowedaconsistentupregulationintheirinteractionwithHsp70withoutarespectivedecreaseinexpression:RPL22andRPL4(Figure5B).Interestingly,boththeseproteinsprotrudeintotheinterioroftheribosomeexittunnelandhavebeenshowntoplayaroleingene-speciÞcelongationstallinginbacteriaandeukaryotes(WilsonandDoudnaCate,2012Thelargestclassofchangesincludedproteinsthatwererela-tivelystableandshowedreducedHsp70interactions(Figure5Weexpectthisgrouptoreßectregulatoryinteractions.Hsp70wasfoundtointeractwithÞveelongationfactorsundernormalgrowthconditions(Figure5A),outofwhichEEF1A1wasfoundtohavereducedHsp70interactionsduringheatstress.SeveralribosomalproteinsalsoshowedthistypeofreducedinteractionwithHsp70afterheatshock,furthersupportingthereducedassociationofHsp70withribosomesduringheatstress.Inparticular,RPL23A,whichislocatedontheoutsideoftheexittunnel,wasdownregulatedinitsinteractionwithHsp70aftersevereheatshock(Figures5Band5C).ThissupportsthenotionthatHsp70chaperonesarepresentinproximitytotheexittunnelundernormalgrowthconditions,andtheirassociationisreducedundersevereheatshock.Inthisstudyweidentifyanunanticipatedaspectofthecellularresponsetoheatstress,inwhichribosomesaccumulateattheendsofopenreadingframesofmostmRNAs,apparentlyasaresultoftemporarypausingoftranslationelongation(Figure6Underheatstress,cellularprioritiesshiftfromgrowthtocytopro-tectivefunctions,includingpreventionofproteinmisfoldingandaggregation.ReducedHSC70/HSP70associationwithribo-somesandalteredinteractionswiththetranslationalmachinerywereobservedafter2hrofsevereheatstress.Thesechangesmayserveseveralfunctions,includingreversibleinhibitionoftranslationelongationthat,togetherwithinhibitionoftranslationinitiation,mayhelptoreducecellularproteinproductionandtheassociatedburdenonthecellularchaperonemachinery.Revers-ibilityoftheelongationpausebyinductionofchaperoneswouldallowcellstorapidlyaccelerateproteinsynthesisandgrowthoncetheyhaveeffectivelyadaptedtothestressassociatedwithproteinmisfolding,orafterareturntononstressconditions.Weobservedelongationpausinginheatstressinbothmouseandhumancells.TheseÞndings,togetherwithamuchearlierreportpointingtochangesinelongationrateswithheatshockcells(BallingerandPardue,1983),suggestthatthemechanismmaybeadeeplyconservedfeatureoftheresponsetoproteotoxicstress.OurdatashowthatthelevelsandactivityofHsp70simpactelongationpausing.Avarietyofpossiblemechanismsfortheseeffectscanbeimagined,includingalteredinteractionsofHsp70withelongationfactors,Hsp70-dependentregulationofribosomalproteinsintheexittunnel,oraberrantexposureofnascentpeptidesinheatstress.MultiplechaperonesareknowntointeractwithribosomesandwithnascentpeptidesinbothprokaryotesandeukaryotesKrameretal.,2009),anditispossiblethatadditionalribo-some-associatedchaperonesplayaroleintranslationelonga-tionundernormalorstressconditions.AnalyzingtheHsp70-translationalmachineryinteractome,wefoundthatHsp70interactedwithÞveelongationfactorsundernormalgrowthconditions,andthatitsinteractionwithEEF1A1wasreducedaftersevereheatstress.ThisraisesthepossibilitythatHsp70ÕsalteredinteractionwithEEF1A1inheatstressmight MolecularCellTranslationElongationPausinginHeatShockMolecularCell,439Ð452,February7,20132013ElsevierInc. inßuenceelongationrates,thoughwhetherandhowthiscouldcontributetoelongationpausingisunclear.AhandfulofinitiationfactorsalsohaddecreasedinteractionswithHsp70underheatstress,suggestingapossibleconnectionbetweenHsp70andregulationoftranslationinitiationunderheatshockbeyonditspreviouslyreportedrole.However,anylinktoinitia-tionfactorsislikelydistinctfromHsp70ÕsroleinelongationOuranalysisidentiÞedRPL4andRPL22,theonlytworibo-somalproteinsthatextendintothewalloftheribosomeexittunnel,formingaÔÔconstrictionÕÕ(WilsonandDoudnaCate,),astheonlytwostableproteinsintheHsp70-translationalmachineryinteractomethatshowedaconsistentincreaseintheiroverallextentofHsp70interactioninheatshock.Theexittunnelitselfwaslongconsideredapassiveregionintheribosome.Morerecently,evidencehasemergedthatstructuralchangesinvolvingRPL22andRPL4canoccurinsidetheexittunnel,leadingtoregulatoryeffectsonelongation(Berisioetal.,2003WilsonandDoudnaCate,2012).Forexample,thebacterialL22proteininteractswiththenascentpeptideofgawaandIto,2002)andtheeukaryoticRPL4proteininteractswiththenascentpeptideofauORFoftheCMVproteinBhushanetal.,2010),bothleadingtogreaterconstrictionoftheexittunnelandstallingofelongation.OnepossibilityisthatduringheatshockHsp70chaperonesregulatetheconformationoftheexittunnelviainteractionswithRPL22and/orRPL4.TheoverallassociationofHSC70/HSP70withribosomeswasdownregulatedafter2hrofsevereheatstress(Figures3Aand Normal AUG m7G Severe heat shock AUG exit tunnelm7G 120 nt 360 nt 120 nt 240 nt HSC70/HSP70 Modulation of Hsp70 levels/activity affects elongation pausingTOP HSC70/HSP70 Figure6.HeatShock-InducedTranslationElongationPausingIsModulatedbyHsp70Duringsevereheatshock,ribosomesoftenpauseat5endsofmRNAsaftertranslatingabout65aminoacids(Figures1),resultinginaccu-mulationofribosomesupstreamanddepletionofribosomesdownstreamofthispoint;aftersevereheatshock,HSC70/HSP70associationwiththeribosomeisreduced(Figures3Aand3B).AsHsp70proteinsinteractwithnascentpeptidechainsastheyemergefromtheexittunnel(mannetal.,1990Nelsonetal.,1992),nascentchainsmightbeexposed.MoresigniÞcantlypausedmRNAstendtoencodeNterminiwithstrongerHsp70requiringcharacteristic(Cand3D).ModulationofHsp70levels,byther-motoleranceoroverexpression,rescuesheatshock-inducedelongationpausing,whileinhibitionofHsp70activityleadstopausingintheabsenceofstress(Figure4).Hsp70chaperonesinteractwithvarioustranslationfactorsandribosomalproteins,andthisinteractionisregulatedafterheatshockFigure5).HSC70/HSP70mayaffectribosomedynamicsviaitsinteractionwithelongationfactors,regulationofribosomalproteinsintheexittunnel,orthroughexposureofnascentchains.3B),andconsistentreductionintheinter-actionwithseveralribosomalproteinswasshownbycoIP(Figures5Band5C).AnumberofribosomalproteinshavepreviouslybeenshowntoefÞcientlyincorporateintoribosomeswhenC-termi-nallytagged,e.g.,RPL22(Sanzetal.,2009),RPL23A(etal.,2002Rossetal.,2007),RPL18,andRPL16(etal.,2009),suggestingthatourassayoftendetectedinteractionwithproteinsincorporatedintoribosomes.TheextentofpausingcorrelatedwithhydrophobicityoftheNterminiandwithpres-enceofHsp70bindingmotifs.Hsp70proteinshaveevolvedtorecognizeexposedhydrophobicpatches,particularlywhenßankedbybasicresidues,asasignofamisfoldedoraggre-gatedprotein.Intheabsenceofribosome-associatedHsp70,nascentpeptideswithstrongerHsp70bindingmotifsmightbemoredependentonHsp70andthushaveagreatertendencytomisfoldoraggregate,potentiallyimpactingtheefÞciencyoftranslation.MoredistantKSlocationswereassociatedwithreducedN-terminalhydrophobicity(Figures3C,3D,andconsistentwithribosomesbeingabletotranslatefurtherdownstreambeforepausing.Theseobservationssuggestthatexposureofnascentpeptidesmightimpactelongation.Finally,RPL23A(inyeast),whichservesasthedockingsitefornascentchainbindingaccessoryfactors(Krameretal.,2009interactedstronglywithHsp70undernormalgrowthconditions,andshowedreducedinteractionsunderheatstress(Figures5and5C).ThisobservationsuggeststhatHsp70ispresentinthevicinityoftheexittunnelundernormalgrowthconditions,butmuchlesssoduringheatstress.IndeedHsp70hasbeenproposedinthepasttoplayaroleinaidinginthepassageofnascentpeptidesthroughtheexittunnel(Nelsonetal.,1992 MolecularCellTranslationElongationPausinginHeatShockMolecularCell,439Ð452,February7,20132013ElsevierInc. Together,theaboveobservationssuggestthatexposednascentpeptidesemergingfromtheribosomemightbeinvolvedintheprocessofelongationpausing(Figure6Theprevalenceofelongationpausingintheheatstressresponsesofbothmouseandhumancellsmakeitanintriguingphenomenontoexploreinotherstressesandorganisms.Elon-gationpausingcouldfacilitatetranslationalrepressionundercertainconditions,suchasproteotoxicstresses,whileinductionofchaperonesmightcounteractstress-inducedelongationpausing.Forexample,elevatedlevelsofchaperonesareoftenobservedincancer(MosserandMorimoto,2004andLindquist,2005).Thisphenomenonmaythereforeberele-vanttoabroadspectrumofstressesanddiseases.EXPERIMENTALPROCEDURESHeatShockandDrugTreatmentConditionsMouseÞbroblast3T3cellswereplatedatlowdensityandthentreatedwithmild(8hrat42C)orsevereheatshock(2hrat44C)toinducechronicoracuteheatstressresponses,respectively(SupplementalExperimentalProcedures).Harringtoninetreatmentswereperformedat0.1mMharringto-ninefor3min,or1minof0.1mMharringtoninefollowedby2minof0.1mg/mlcycloheximide(CHX),whichwereaddedtothemedia.Puromycintreatmentwasdoneat1mg/mlfor3min.TreatmentwiththeHsp70inhibitorVER-155008(Masseyetal.,2010)(TocrisBioscience)wasperformedatMfor3hr.RibosomeFootprintProÞlingRibosomefootprintingwasperformedasdescribedby(Ingoliaetal.,)withmodiÞcationsdescribedintheSupplementalExperimentalProceduresTransfectionsTransfectionconditionsaredetailedintheSupplementalExperimentalProceduresMappingofFootprintReadsRibosomefootprintreadsweretrimmedfromthe3endfrom36basesto32bases,andmultipletrailingadenosine(A)baseswereremoved.Next,footprintreadsofsize22Ð32basesweremappedtothemousegenome(mm9)orthehumangenome(hg18)withBowtie(Langmeadetal.,).Readsmappinguniquelytoexonicpositionsweresubsequentlymap-pedtoEnsemblgeneandtranscriptannotationÞles(downloadedfromUCSC)usingcustompythonandPerlscripts(SupplementalExperimentalProceduresPolysomeProÞling3T3cellsweretreatedwith0.1mg/mlcycloheximide(CHX)for5min,thenlysed(SupplementalExperimentalProcedures).ProÞleswerenormalizedbyloadingofequalamountsofRNAODunitsofallsamplesinasingleexperiment.Tocomparebetweenexperiments,weusedrelativeP:Mratio,calculatedastheP:Mratiointhegiventreatmentdividedbythatinthecorre-spondingcontrolconditionandexpressedasapercentage.ProteinAssociationwithPolysomeFractionsAfterultracentrifugation,24equalvolumefractionswerecollectedandpooledasdescribedinFigure3A,toppanel,andprecipitatedbyadditionoficecoldacetoneandovernightincubationatC.SigniÞcanceofthereductioninribosomalassociationofHSC70/HSP70wascalculatedbyttestoverÞvereplicatesusingMATLAB.FurtherdetailsprovidedintheSupplementalExperimentalProceduresAntibodiesAntibodiesaredetailedintheSupplementalExperimentalProceduresFootprintDensityPlotsandTestsofPauseSigniÞcanceandAlluniquelymappingexonicfootprintreadsweremappedtoEnsemblmRNAtranscriptswiththeirgenomicannotationsintheensGenetablefromtheUCSCdatabase.Foreachtranscript,aproÞlewasgeneratedasavectorcontainingthenumberofreadsforwhichthe5endmapped12nt5eachpositionofthetranscript,andthennormalizedbydividingbythemeannumberofreadsperpositionalongtheÞrst450nucleotidesoftheCDS,simi-larlytoIngoliaetal.(2009).PresentedintheÞguresaretheaveragedproÞlesfortheÞltereduniquesetoftranscriptsineachcondition(seethementalExperimentalProceduresforfurtherdetailsonthisandonKSanalysis).Anelevationofribosomedensityatthe5endsofyeastgenes,whichwasgraduallydecreasingupto600basesoftheORF,hasbeenobservedinyeastundernormalgrowthconditionsintheÞrstfootprintingstudy(Ingoliaetal.,).ThiselevationhasbeenattributedtoeffectsoftransferRNAabundanceontranslationalefÞciency(Qianetal.,2012Tulleretal.,2010HydrophobicityProÞlesandHsp70BindingSiteScoringHydrophobicityproÞlesandHsp70bindingsitescoringaredescribedintheSupplementalExperimentalProceduresHsp70TranslationMachineryInteractomeLUMIERwithBACONassaywasdoneasinTaipaleetal.(2012),witha293Tcelllinestablyexpressing-taggedHSC70().CoIPwithendoge-nousHsp70chaperoneswasperformedasinTaipaleetal.(2012).FurtherdetailsareprovidedintheSupplementalExperimentalProceduresACCESSIONNUMBERSHigh-throughputsequencingdatahavebeensubmittedtoGEO(accessionnumberGSE32060).SUPPLEMENTALINFORMATIONSupplementalInformationincludesSupplementalExperimentalProcedures,ÞveÞgures,andfourtablesandcanbefoundwiththisarticleonlineathttp://dx.doi.org/10.1016/j.molcel.2012.11.028ACKNOWLEDGMENTSWethankWendyGilbertandmembersofherlabforhelpfuladviceandsuggestions,useofequipmentthroughoutthisstudy,andforcriticalreadingofthemanuscript.WethankEricWangforhelpwithmappingoffootprintdataandforusefuldiscussions,SandroSantagataandLukeWhitesellforhelp-fuladvice,andDanielTreacyforhelpwithfootprintlibrarypreparation.R.S.isanAwardeeoftheWeizmannInstituteofScience-NationalPostdoctoralAwardProgramforAdvancingWomeninScience.ThisworkwassupportedbyanEMBOlong-termfellowshipandtheMachiahfoundation(R.S.),NIGMSfellowshipnumberF32GM095060(J.A.H.),andgrantsfromtheNIH(C.B.B.).Received:March27,2012Revised:September27,2012Accepted:November30,2012Published:January3,2013Abravaya,K.,Myers,M.P.,Murphy,S.P.,andMorimoto,R.I.(1992).Thehumanheatshockproteinhsp70interactswithHSF,thetranscriptionfactorthatregulatesheatshockgeneexpression.GenesDev.,1153Ð1164.Akerfelt,M.,Morimoto,R.I.,andSistonen,L.(2010).Heatshockfactors:inte-gratorsofcellstress,developmentandlifespan.Nat.Rev.Mol.CellBiol.Ballinger,D.G.,andPardue,M.L.(1983).ThecontrolofproteinsynthesisduringheatshockinDrosophilacellsinvolvesalteredpolypeptideelongationrates.Cell,103Ð113. MolecularCellTranslationElongationPausinginHeatShockMolecularCell,439Ð452,February7,20132013ElsevierInc. Beckmann,R.P.,Mizzen,L.E.,andWelch,W.J.(1990).InteractionofHsp70withnewlysynthesizedproteins:implicationsforproteinfoldingandassembly.,850Ð854.Berisio,R.,Schluenzen,F.,Harms,J.,Bashan,A.,Auerbach,T.,Baram,D.,andYonath,A.(2003).Structuralinsightintotheroleoftheribosomaltunnelincellularregulation.Nat.Struct.Biol.,366Ð370.Bhushan,S.,Meyer,H.,Starosta,A.L.,Becker,T.,Mielke,T.,Berninghausen,O.,Sattler,M.,Wilson,D.N.,andBeckmann,R.(2010).Structuralbasisfortranslationalstallingbyhumancytomegalovirusandfungalarginineattenuatorpeptide.Mol.Cell,138Ð146.Biamonti,G.,andCaceres,J.F.(2009).CellularstressandRNAsplicing.TrendsBiochem.Sci.,146Ð153.rk,J.K.,andSistonen,L.(2010).Regulationofthemembersofthemamma-lianheatshockfactorfamily.FEBSJ.,4126Ð4139.Bouche,G.,Amalric,F.,Caizergues-Ferrer,M.,andZalta,J.P.(1979).EffectsofheatshockongeneexpressionandsubcellularproteindistributioninChinesehamsterovarycells.NucleicAcidsRes.,1739Ð1747.Calderwood,S.K.,Khaleque,M.A.,Sawyer,D.B.,andCiocca,D.R.(2006).Heatshockproteinsincancer:chaperonesoftumorigenesis.TrendsBiochem.Sci.,164Ð172.Calloni,G.,Chen,T.,Schermann,S.M.,Chang,H.C.,Genevaux,P.,Agostini,F.,Tartaglia,G.G.,Hayer-Hartl,M.,andHartl,F.U.(2012).DnaKfunctionsasacentralhubintheE.colichaperonenetwork.CellRep,251Ð264.Duncan,R.,andHershey,J.W.(1984).Heatshock-inducedtranslationalalterationsinHeLacells.InitiationfactormodiÞcationsandtheinhibitionoftranslation.J.Biol.Chem.,11882Ð11889.Feder,J.H.,Rossi,J.M.,Solomon,J.,Solomon,N.,andLindquist,S.(1992).Theconsequencesofexpressinghsp70inDrosophilacellsatnormaltemper-atures.GenesDev.,1402Ð1413.Gerner,E.W.,andSchneider,M.J.(1975).InducedthermalresistanceinHeLacells.Nature,500Ð502.Gibson,G.(2008).TheenvironmentalcontributiontogeneexpressionproÞles.Nat.Rev.Genet.,575Ð581.Halbeisen,R.E.,Scherrer,T.,andGerber,A.P.(2009).AfÞnitypuriÞcationofribosomestoaccessthetranslatome.Methods,306Ð310.Harding,H.P.,Novoa,I.,Zhang,Y.,Zeng,H.,Wek,R.,Schapira,M.,andRon,D.(2000).Regulatedtranslationinitiationcontrolsstress-inducedgeneexpressioninmammaliancells.Mol.Cell,1099Ð1108.Hartl,F.U.,Bracher,A.,andHayer-Hartl,M.(2011).Molecularchaperonesinproteinfoldingandproteostasis.Nature,324Ð332.Henle,K.J.,Karamuz,J.E.,andLeeper,D.B.(1978).Inductionofthermotoler-anceinChinesehamsterovarycellsbyhigh(45degrees)orlow(40degrees)hyperthermia.CancerRes.,570Ð574.Holcik,M.,andSonenberg,N.(2005).Translationalcontrolinstressandapoptosis.Nat.Rev.Mol.CellBiol.,318Ð327.Huang,M.T.(1975).Harringtonine,aninhibitorofinitiationofproteinbiosynthesis.Mol.Pharmacol.,511Ð519.Inada,T.,Winstall,E.,Tarun,S.Z.,Jr.,Yates,J.R.,3rd,Schieltz,D.,andSachs,A.B.(2002).One-stepafÞnitypuriÞcationoftheyeastribosomeanditsassoci-atedproteinsandmRNAs.RNA,948Ð958.Ingolia,N.T.,Ghaemmaghami,S.,Newman,J.R.,andWeissman,J.S.(2009).Genome-wideanalysisinvivooftranslationwithnucleotideresolutionusingribosomeproÞling.Science,218Ð223.Ingolia,N.T.,Lareau,L.F.,andWeissman,J.S.(2011).RibosomeproÞlingofmouseembryonicstemcellsrevealsthecomplexityanddynamicsofmamma-lianproteomes.Cell,789Ð802.Jaiswal,H.,Conz,C.,Otto,H.,Wolße,T.,Fitzke,E.,Mayer,M.P.,andRospert,S.(2011).Thechaperonenetworkconnectedtohumanribosome-associatedcomplex.Mol.Cell.Biol.,1160Ð1173.Jarosz,D.F.,andLindquist,S.(2010).Hsp90andenvironmentalstresstrans-formtheadaptivevalueofnaturalgeneticvariation.Science,1820Ð1824.Kampinga,H.H.,andCraig,E.A.(2010).TheHSP70chaperonemachinery:JproteinsasdriversoffunctionalspeciÞcity.Nat.Rev.Mol.CellBiol.Kelley,P.M.,andSchlesinger,M.J.(1978).TheeffectofaminoacidanaloguesandheatshockongeneexpressioninchickenembryoÞbroblasts.Cell1277Ð1286.Kramer,G.,Boehringer,D.,Ban,N.,andBukau,B.(2009).Theribosomeasaplatformforco-translationalprocessing,foldingandtargetingofnewlysynthesizedproteins.Nat.Struct.Mol.Biol.,589Ð597.Langmead,B.,Trapnell,C.,Pop,M.,andSalzberg,S.L.(2009).Ultrafastandmemory-efÞcientalignmentofshortDNAsequencestothehumangenome.GenomeBiol.,R25.Lindquist,S.(1980).VaryingpatternsofproteinsynthesisinDrosophiladuringheatshock:implicationsforregulation.Dev.Biol.,463Ð479.Lindquist,S.(1981).Regulationofproteinsynthesisduringheatshock.Nature,311Ð314.Lindquist,S.(2009).Proteinfoldingsculptingevolutionarychange.ColdSpringHarb.Symp.Quant.Biol.,103Ð108.Massey,A.J.,Williamson,D.S.,Browne,H.,Murray,J.B.,Dokurno,P.,Shaw,T.,Macias,A.T.,Daniels,Z.,Geoffroy,S.,Dopson,M.,etal.(2010).Anovel,smallmoleculeinhibitorofHsc70/Hsp70potentiatesHsp90inhibitorinducedapoptosisinHCT116coloncarcinomacells.CancerChemother.Pharmacol.,535Ð545.McCormick,W.,andPenman,S.(1969).RegulationofproteinsynthesisinHeLacells:translationatelevatedtemperatures.J.Mol.Biol.,315Ð333.Miller,M.J.,Xuong,N.H.,andGeiduschek,E.P.(1979).AresponseofproteinsynthesistotemperatureshiftintheyeastSaccharomycescerevisiae.Proc.Natl.Acad.Sci.USA,5222Ð5225.Mosser,D.D.,andMorimoto,R.I.(2004).Molecularchaperonesandthestressofoncogenesis.Oncogene,2907Ð2918.Nakatogawa,H.,andIto,K.(2002).Theribosomalexittunnelfunctionsasadiscriminatinggate.Cell,629Ð636.Nelson,R.J.,Ziegelhoffer,T.,Nicolet,C.,Werner-Washburne,M.,andCraig,E.A.(1992).Thetranslationmachineryand70kdheatshockproteincooperateinproteinsynthesis.Cell,97Ð105.Petersen,N.S.,andMitchell,H.K.(1981).Recoveryofproteinsynthesisafterheatshock:priorheattreatmentaffectstheabilityofcellstotranslatemRNA.Proc.Natl.Acad.Sci.USA,1708Ð1711.Qian,W.,Yang,J.R.,Pearson,N.M.,Maclean,C.,andZhang,J.(2012).BalancedcodonusageoptimizeseukaryotictranslationalefÞciency.PLoS,e1002603.Rahl,P.B.,Lin,C.Y.,Seila,A.C.,Flynn,R.A.,McCuine,S.,Burge,C.B.,Sharp,P.A.,andYoung,R.A.(2010).c-Mycregulatestranscriptionalpauserelease.,432Ð445.Richter,K.,Haslbeck,M.,andBuchner,J.(2010).Theheatshockresponse:lifeonthevergeofdeath.Mol.Cell,253Ð266.Ross,C.L.,Patel,R.R.,Mendelson,T.C.,andWare,V.C.(2007).FunctionalconservationbetweenstructurallydiverseribosomalproteinsfromDrosophilamelanogasterandSaccharomycescerevisiae:ßyL23acansubstituteforyeastL25inribosomeassemblyandfunction.NucleicAcidsRes.,4503Ð4514.diger,S.,Germeroth,L.,Schneider-Mergener,J.,andBukau,B.(1997).SubstratespeciÞcityoftheDnaKchaperonedeterminedbyscreeningcellu-lose-boundpeptidelibraries.EMBOJ.,1501Ð1507.Sanz,E.,Yang,L.,Su,T.,Morris,D.R.,McKnight,G.S.,andAmieux,P.S.(2009).Cell-type-speciÞcisolationofribosome-associatedmRNAfromcomplextissues.Proc.Natl.Acad.Sci.USA,13939Ð13944.Sonenberg,N.,andHinnebusch,A.G.(2009).Regulationoftranslationinitia-tionineukaryotes:mechanismsandbiologicaltargets.Cell,731Ð745.Spriggs,K.A.,Bushell,M.,andWillis,A.E.(2010).Translationalregulationofgeneexpressionduringconditionsofcellstress.Mol.Cell,228Ð237. MolecularCellTranslationElongationPausinginHeatShockMolecularCell,439Ð452,February7,20132013ElsevierInc. Taipale,M.,Krykbaeva,I.,Koeva,M.,Kayatekin,C.,Westover,K.D.,Karras,G.I.,andLindquist,S.(2012).QuantitativeanalysisofHSP90-clientinterac-tionsrevealsprinciplesofsubstraterecognition.Cell,987Ð1001.Tuller,T.,Carmi,A.,Vestsigian,K.,Navon,S.,Dorfan,Y.,Zaborske,J.,Pan,T.,Dahan,O.,Furman,I.,andPilpel,Y.(2010).Anevolutionarilyconservedmech-anismforcontrollingtheefÞciencyofproteintranslation.Cell,344Ð354.vonHeijne,G.(1981).Onthehydrophobicnatureofsignalsequences.Eur.J.Biochem.,419Ð422.Vries,R.G.,Flynn,A.,Patel,J.C.,Wang,X.,Denton,R.M.,andProud,C.G.(1997).Heatshockincreasestheassociationofbindingprotein-1withinitiationfactor4E.J.Biol.Chem.,32779Ð32784.Whitesell,L.,andLindquist,S.L.(2005).HSP90andthechaperoningofcancer.Nat.Rev.Cancer,761Ð772.Wilson,D.N.,andDoudnaCate,J.H.(2012).Thestructureandfunctionoftheeukaryoticribosome.ColdSpringHarb.Perspect.Biol..PublishedonlineMay1,2012.http://dx.doi.org/10.1101/cshperspect.a011536.Zhou,D.,Palam,L.R.,Jiang,L.,Narasimhan,J.,Staschke,K.A.,andWek,R.C.(2008).PhosphorylationofeIF2directsATF5translationalcontrolinresponsetodiversestressconditions.J.Biol.Chem.,7064Ð7073.Zylicz,M.,King,F.W.,andWawrzynow,A.(2001).Hsp70interactionswiththep53tumoursuppressorprotein.EMBOJ.,4634Ð4638. MolecularCellTranslationElongationPausinginHeatShockMolecularCell,439Ð452,February7,20132013ElsevierInc.