The molecular basis of coupling between poly(A)-tail length ...

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These mRNAs were made by in vitro transcription from DNA templates that encoded the mRNA body followed by the poly(A) tail as well as the ... Sharethisarticle Doi Copytoclipboard Citethisarticle KehuiXiang DavidPBartel (2021) Themolecularbasisofcouplingbetweenpoly(A)-taillengthandtranslationalefficiency eLife10:e66493. https://doi.org/10.7554/eLife.66493 Copytoclipboard DownloadBibTeX Download.RIS Article Figuresanddata Abstract eLifedigest Introduction Results Discussion Materialsandmethods Dataavailability References Decisionletter Authorresponse Articleandauthorinformation Metrics Inanimaloocytesandearlyembryos,mRNApoly(A)-taillengthstronglyinfluencestranslationalefficiency(TE),butlaterindevelopmentthiscouplingbetweentaillengthandTEdisappears.Here,weelucidatehowthiscouplingisfirstestablishedandwhyitdisappears.Overexpressingcytoplasmicpoly(A)-bindingprotein(PABPC)inXenopusoocytesspecificallyimprovedtranslationofshort-tailedmRNAs,therebydiminishingcouplingbetweentaillengthandTE.Thus,strongcouplingrequireslimitingPABPC,implyingthatincoupledsystemslonger-tailmRNAsbettercompeteforlimitingPABPC.InadditiontoexpressingexcessPABPC,post-embryonicmammaliancelllineshadtwootherpropertiesthatpreventedstrongcoupling:terminal-uridylation-dependentdestabilizationofmRNAslackingboundPABPC,andaregulatoryregimewhereinPABPCcontributesminimallytoTE.Thus,theseresultsrevealedthreefundamentalmechanisticrequirementsforcouplinganddefinedthecontext-dependentfunctionsforPABPC,whichpromotesTEbutnotmRNAstabilityincoupledsystemsandmRNAstabilitybutnotTEinuncoupledsystems. Cellsaremicroscopicbiologicalfactoriesthatareconstantlycreatingnewproteins.Todoso,acellmustfirstconvertitsmastergeneticblueprint,theDNA,intostrandsofmessengerRNAormRNA.Thesestrandsaresubsequentlytranslatedtomakeproteins.Cellshavetwowaystoadjustthenumberofproteinstheygeneratesotheydonotproducetoomanyortoofew:bychanginghowmanymRNAmoleculesareavailablefortranslation,andbyregulatinghowefficientlytheytranslatethesemRNAmoleculesintoproteins. Inanimals,bothunfertilizedeggsandearly-stageembryoslacktheabilitytocreateordestroymRNAs,andconsequentlycannotadjustthenumberofmRNAmoleculesavailablefortranslation.ThesecellscanthereforeonlyregulatehowefficientlyeachmRNAistranslated.Theydothisbychangingthelengthoftheso-calledpoly(A)tailattheendofeachmRNAmolecule,whichismadeupofalongstretchofrepeatingadenosinenucleotides.ThemRNAswithlongerpoly(A)tailsaretranslatedmoreefficientlythanthosewithshorterpoly(A)tails.However,thisdifferencedisappearsinolderembryos,whenbothlongandshortpoly(A)tailsaretranslatedwithequalefficiency,anditislargelyunknownwhy. Tofindoutmore,XiangandBartelstudiedfrogeggs,anddiscoveredthatartificiallyraisinglevelsofaproteinthatbindspoly(A)tails,alsoknownasPABPC,improvedthetranslationofshort-tailedmRNAstocreateasituationinwhichbothshort-andlong-tailedmRNAsweretranslatedwithnear-equalefficiency.Thissuggestedthatshort-andlong-tailedmRNAscompeteforlimitedamountsofthetranslation-enhancingPABPC,andthatlong-tailedmRNAsarebetteratitthanshort-tailedmRNAs.FurtherinvestigationrevealedthateggsalsohadtoestablishtherightconditionsforPABPCtoenhancetranslationandhadtoprotectmRNAsnotassociatedwithPABPCfrombeingdestroyedbeforetheycouldbetranslated. Overall,XiangandBartelfoundthatineggsandearlyembryos,PABPCandpoly(A)tailsenhancedthetranslationofmRNAsbutdidnotinfluencetheirstability,whereaslaterindevelopment,theyenhancedmRNAstabilitybutnottranslation. Thisresearchprovidesnewinsightsintohowproteinproductioniscontrolledatdifferentstagesofanimaldevelopment,fromunfertilizedeggstoolderembryos.Understandinghowthisprocessisregulatedduringnormaldevelopmentiscrucialforgaininginsightsintohowitcanbecomedysfunctionalandcausedisease.Thesefindingsmaythereforehaveimportantimplicationsforresearchintoareassuchasinfertility,reproductivemedicineandraregeneticdiseases. MosteukaryoticmRNAsarepolyadenylatedattheir3′endsinaprocessassociatedwithtranscriptionaltermination.Inthenucleus,thesepoly(A)tailscanfacilitatemRNAnucleocytoplasmicexport(KühnandWahle,2004),whereasinthecytoplasm,theyserveasmoleculartimersformRNAdecay,withtheirlengthsbecomingprogressivelyshorterbydeadenylation,whicheventuallyleadstomRNAde-cappingandturnover(ChenandShyu,2011;Eisenetal.,2020;GoldstrohmandWickens,2008). Thelengthofapoly(A)tailcanalsoinfluencemRNAtranslationalefficiency(TE).Pioneeringstudiesinmaturingoocytesandearlyembryosshowthatlengtheningofpoly(A)tailsthroughcytoplasmicpolyadenylationiscriticalforregulatinggeneexpressionduringtheseearlystagesofanimaldevelopment(Richter,1999;Sallésetal.,1994;Sheetsetal.,1995).Resultsfromtheseandothersingle-genestudiesinoocytesandearlyembryoshadledtothenotionthatthelengthofapoly(A)tailgenerallycorrelateswithTE(Eckmannetal.,2011;Weilletal.,2012).Morerecenttranscriptome-widestudiesconfirmastrongglobalrelationshipbetweentaillengthandTEinoocytesandearlyembryos(Eichhornetal.,2016;Limetal.,2016;Subtelnyetal.,2014).However,infish,frogs,andflies,thiscorrelationdiminishesnearthetimeofgastrulation,andcouplingbetweenpoly(A)-taillengthandTEisessentiallynonexistentinpost-embryonicsystems(Eichhornetal.,2016;J.-E.Parketal.,2016;Subtelnyetal.,2014).Thus,theseglobalanalysesrevealadevelopmentaltransitioninhowtranslationisregulated(Subtelnyetal.,2014),whichcloselyfollowsthelong-knownmaternal-to-zygotictransitionintranscriptionalcontrol.Theexistenceofthistransitionintranslationalcontrolbringstotheforemechanisticquestionsastohowcouplingbetweenpoly(A)-taillengthandTEisestablishedinoocytesandearlyembryosandwhythiscouplingdisappearslaterindevelopment. Cytoplasmicpoly(A)-bindingproteins(PABPCs)arehighlyconservedRNA-bindingproteinsineukaryotes(Mangusetal.,2003).AlthoughSaccharomycescerevisiaehasonlyonePABPC(Pab1p),mostanimalscontainmultipleparalogsthathavespatiallyandtemporallyvariedexpressionpatterns(Smithetal.,2014;Wigingtonetal.,2014).PABPCshavehighaffinitytopoly(A)sequencesinvitro(Kd ~5nMforA25)andrequireatleast12Asforefficientbinding(KühnandWahle,2004).BindingofPABPCstomRNApoly(A)tailscanenhancetranslation,butthemechanismofthisenhancementisunclear.OnemodelpositsthatthemRNAformsaclosed-loopstructuremediatedbytheassociationoftheeukaryotictranslationinitiationfactoreIF4G(ascaffoldingprotein)withbothPABPCandthecap-bindingproteineIF4E(Hinnebusch,2014;ThompsonandGilbert,2017;Wellsetal.,1998).ThisassociationisproposedtostabilizetheinteractionbetweeneIF4EandthemRNA5′capandfacilitaterecruitmentand/orrecyclingofribosomestoincreasetranslationinitiation(Kahvejianetal.,2001).However,despitedirectvisualizationofloop-likeassembliesbothwithinsomecellsandinaninvitroreconstitutedsystem(Christensenetal.,1987;Wellsetal.,1998),resultsofseveralstudieshavequestionedtheuniversalityofthismodelamongdifferentmRNAsandbiologicalsystems(Adivarahanetal.,2018;Amranietal.,2008;Costelloetal.,2015;Risslandetal.,2017;ThompsonandGilbert,2017). PABPCscanalsoinfluencemRNAstability,asshowninyeast.GeneticablationofyeastPab1pislethalandcauseslengtheningofsteady-statepoly(A)-taillengths(SachsandDavis,1989),whichisattributedtopre-maturemRNAdecappingandcompromiseddeadenylation(CaponigroandParker,1995).BothyeastandmammalianPABPCscaninteractwithtwomRNAdeadenylationcomplexesPAN2-PAN3andCCR4-NOT,andeitherpromoteorinhibittheiractivitiesinvitro(Schäferetal.,2019;Uchidaetal.,2004;Websteretal.,2018;Yietal.,2018).BecausemRNAdecayiscoupledtodeadenylation(DeckerandParker,1993;Eisenetal.,2020),thedeadenylation-stimulatoryeffectsofPABPCwouldacceleratethedemiseofboundmRNAs,whichcontraststootherstudiessuggestingPABPCprotectsmRNAsfromdegradationincellextracts(Bernsteinetal.,1989;Wangetal.,1999).ThedichotomousandpotentiallyconflictingfunctionsofmetazoanPABPCexaminedinvitroraisethequestionoftheextentstowhichPABPCmightinfluencemRNApoly(A)-taillengthandstabilityinmetazoancells. PABPCsaregenerallythoughttocoatmRNApoly(A)tailsinthecytoplasm(KühnandWahle,2004;Mangusetal.,2003).However,thestoichiometrybetweenPABPCandpoly(A)sitesmightvaryindifferentbiologicalcontexts(Cossonetal.,2002;Voeltzetal.,2001),anditisunclearwhetherthispotentiallyvariablestoichiometrymightimpactgeneregulationincells.Moreover,thepossibilitythatPABPCmightinfluenceproteinsynthesisbyaffectingeithermRNAstabilityorTEcancomplicateanalysisofitsmolecularfunctionsindifferentbiologicalsystems,leavingitsmechanisticrolespoorlyunderstood. Here,weuncovermechanisticrequirementsforcouplingbetweenpoly(A)-taillengthandTEobservedinoocytesandearlyembryos,showingthatthiscouplingandthesubsequentuncouplingobservedlaterindevelopmentrelyonacontext-dependentswitchinthefunctionofPABPCs. Toassaytheinfluenceofpoly(A)-taillengthonTE,weusedaninvitrotranslationextractmadefromstageVIXenopuslaevisoocytes,wherecytoplasmicpolyadenylationleadstotranslationalactivationofthec-mos,cdk2andsomecyclinmRNAs (RichterandLasko,2011).IntothisextractweaddedNanolucluciferase(Nluc)reportermRNAswitheitherashort(29nt)oralong(139nt)poly(A)tail(Figure1A).ThesemRNAsweremadebyinvitrotranscriptionfromDNAtemplatesthatencodedthemRNAbodyfollowedbythepoly(A)tailaswellasthehepatitisdeltavirus(HDV)self-cleavingribozyme,whichcleavedduringinvitrotranscriptiontogeneratenotonlyadefined3′endatthedesiredpoly(A)-taillengthbutalsoa2′−3′-cyclicphosphatedesignedtoinhibitundesiredlengtheningorshorteningofthetail(Avisetal.,2012). Figure1with2supplementsseeall Downloadasset Openasset PABPCoverexpressionuncouplespoly(A)-taillengthandTEinfrogoocytes. (A)SchematicofcappedT7transcriptswithtwodifferenttaillengths,whichwereusedasreportermRNAs.AdditionalsequencesbeyondtheHDVsequencearenotdepicted.(B)Theeffectoftaillengthonrelativeyieldsofinvitrotranslationofshort-andlong-tailedNlucreportermRNAs,ineitherfrogoocyteextract(left)orrabbitreticulocytelysate(right).Thenumberaboveeachbracketindicatesthefolddifferenceofthemeannormalizedluciferasesignal(errorbars,standarddeviationfromthreetechnicalreplicates).(C)TheeffectofpurifiedPABPC1onrelativeyieldsofinvitrotranslationofshort-andlong-tailedNlucreportermRNAsinfrogoocyteextract.PurifiedeGFPandPABPC1wereeachaddedasindicated.Otherwise,thispanelisasin(B).(D)Experimentalschemeforserial-injectionofmRNAsintofrogoocytes.(E)TheeffectofoverexpressingPAPBC1andaPABPC1M161AmutantonrelativetranslationofNlucreportermRNAsinfrogoocytes.DifferentialPABPC1expressionwasachievedbyinjectingthe indicatedamountofmRNAinthefirstinjection(errorbars,standarddeviationfromthreebiologicalreplicates).Otherwise,thispanelisasin(B).(F)TheeffectofPAPBC1overexpressionontranslationofreportermRNAswithdifferent3′-endstructuresinfrogoocytes.ShownarerawluciferaseyieldsfromNlucreportersthathaveeitherashortpoly(A)tail,alongpoly(A)tail,ahistonemRNA3′-endstem-loop,oraMalat1triple-helix3′-endinoocytesoverexpressingeithereGFPorPABPC1(errorbars,standarddeviationsfromthreebiologicalreplicates).pvaluesarefromone-sidedt-tests(n.s.,notsignificant).Foroverexpression,2.4fmolmRNAwasinjectedperoocyte. Figure1—sourcedata1 SourcedataforluciferasevaluesshowninFigure1andFigure1—figuresupplement2. https://cdn.elifesciences.org/articles/66493/elife-66493-fig1-data1-v1.xlsx Downloadelife-66493-fig1-data1-v1.xlsx Whenaddedtothefrogoocyteextractstogetherwithafireflyluciferase(Fluc)mRNA,usedtonormalizeforoveralltranslationactivity,thelong-tailedreporterwastranslatedsubstantiallybetterthanwastheshort-tailedreporter(Figure1B).Incontrast,thesamereportermRNAsweretranslatednearlyequallywellinrabbitreticulocytelysate,apost-embryonicdifferentiatedsystemforwhichnocouplingbetweentaillengthandTEwasexpected(Subtelnyetal.,2014; Figure1B).SimilarresultswereobservedforananalogouspairofRenillaluciferasereportermRNAs(Figure1—figuresupplement2A).Inboththeoocyteandreticulocytesystems,thereportermRNAswerestablewithnodetectablechangestotheirtaillengths(Figure1—figuresupplement1A–C).Thus,thelargedifferenceinluciferasesignalobservedbetweentheshort-andlong-tailedreportersintheoocyteextractwasattributabletoadifferenceinTE.Theseresultsshowedthatthecausalrelationshipbetweenlongerpoly(A)-taillengthandgreaterTEobservedforsomematuration-specificmRNAsinfrogoocytes(Sheetsetal.,1995;Stebbins-BoazandRichter,1994)isnotuniquetothosemRNAs,andindicatedthatfrogoocyteextractsprovideasystemforprobingthemechanismthatcouplestaillengthtoTE. Whenconsideringthepotentialmechanismsforreadingouttaillengthandpromotingtranslation,aroleforPABPCseemedplausible.Forinstance,translationmightbesensitivetothenumberofPABPCmoleculesassociatedwithanmRNA.Inonemechanisticpossibility,PABPCmightbeinexcessoveritsbindingsiteswithintails,suchthattailsarecoatedwiththeprotein,asisgenerallythoughttooccur(KühnandWahle,2004;Mangusetal.,2003),inwhichcase,mRNAswithlongertailsmightbedetectedasthoseabletobindmorePABPCmolecules.Atanothermechanisticextreme,PABPCmightbelimiting,suchthatmRNAscompetewitheachotherforPABPCbinding,inwhichcase,thosewithlongpoly(A)-taillengthswouldcompetemoreeffectivelyandtherebypreferentiallybenefitfromanyenhancementinTEthatPABPCbindingconfers.Todistinguishbetweenthesepossibilities,weincreasedavailablePABPCinouroocyteextracts,reasoningthatifPABPCwerealreadycoatingthetails,addingmorewouldhavelittleeffect,whereasifPABPCwerelimiting,addingmorewoulddiminishthecompetitionforPABPCbindingandtherebyreducethedifferenceinTEobservedbetweenshort- andlong-tailedmRNAs.Accordingly,wepurifiedrecombinantXenopusPABPC1tonearhomogeneity(Figure1—figuresupplement1D)andexamineditsinfluencewhenaddedtotheinvitrotranslationextractderivedfromstageVIoocytes.AsmorePABPC1wasadded,translationoftheshort-tailedreporterincreased,withlittlechangeintranslationofthelong-tailedreporter,whereasaddingequivalentamountofeGFPhadlittleimpactontranslationofeitherreporter(Figure1C).Thisconcentration-dependentdiminutionofcouplingbetweentail-lengthandTEstronglysupportedthehypothesisthatlimitingPAPBCisrequiredforstrongcoupling. ToinvestigatewhetherthisrequirementoflimitingPABPCwasrestrictedtoourinvitroextractsorwhetheritalsoappliedtolivingoocytes,weperformedserial-injectionexperimentsinoocytes.StageVIfrogoocyteswerefirstinjectedwitheitherPABPC1mRNAoracontrol,andafterwaiting24hrtoallowPABPC1proteintoaccumulate(Figure1—figuresupplement1E),oocyteswereinjectedwiththereportermRNAsandassayedforluciferaseactivity(Figure1D).WhereasinjectingthecontrolmRNA,eGFP,hadnomoreinfluencethaninjectingwater,injectingPABPC1mRNAsignificantlyreducedtheextenttowhichpoly(A)-taillengthandTEwerecoupled(Figure1E).SimilarresultswereobservedforananalogouspairofRenillaluciferasereportermRNAsorwheninjectingePABmRNAratherthanPABPC1mRNA(Figure1—figuresupplement2B).Reporterpoly(A)-taillengthsdidnotchangeoverthecourseoftheexperiment(Figure1—figuresupplement1F),whichindicatedthattheincreasedrelativetranslationoftheshort-tailedreportermRNAwasnotduetoelongatedpoly(A)tails. IntroducingadditionalPABPCintofrogoocytesspecificallyimprovedtranslationoftheshort-tailedreporterwhilehavinglittleeffectontranslationofeitherthelong-tailedreporterorreportersforwhichtailswerereplacedwitheitherastem-loopfromthe3′endofahistonemRNA(Lingetal.,2002)oratriple-helixfromthe3′endoftheMalat1non-codingRNA(Wiluszetal.,2012; Figure1F).TheobservationthatmRNAsrequiredatailtobenefitfromaddedPABPCindicatedthattheeffectsofaddingPABPCweremediatedincisthroughtail-boundPABPCmolecules,andweredirectandnotsomesecondaryconsequenceofalteringtranslation.Moreover,theobservationthatPABPChadlittleeffectontranslationoflong-tailedmRNAssuggestedthatthesemRNAscompetedforthelimitingendogenousPABPCsoeffectivelythatbindingofadditionalPABPCimpartednodetectableadditionalbenefittotheirtranslation. IntroducingPABPC1(M161A),whichencodesaPABPC1mutantthatisunabletobindeIF4G(GroftandBurley,2002),alsodiminishedcouplingbutdidsobyrepressingtranslationofthelong-tailedreporter.Thereducedtranslationofthelong-tailedreporterwaspresumablyduetoadominant-negativeeffectofreplacingfunctionalendogenousPABPCmoleculeswithdefective ones.Theobservationthatthelong-tailedreporterwaspreferentiallyaffectedagreedwithourconclusionthatendogenousPABPCwaslimitingandpreferentiallybindingtolong-tailedmRNAs.TheideathattheM161AmutantwasunabletoenhancetranslationinfrogoocytesimpliedthattheabilityforPABPCtobindeIF4Gandformtheclosed-loopstructureisimportantforenhancingtranslationinthiscontext (Wakiyamaetal.,2000). Insummary,ourresultswithreportersinoocytesandoocyteextractsconfirmedboththepositiveeffectofPABPContranslationandthecausalrelationshipbetweenpoly(A)-taillengthandTEinthesesystems.Moreover,theseresultsrevealedthatstrongcouplingbetweenpoly(A)-taillengthandTErequireslimitingPABPC. ToexaminetheglobaleffectofincreasingPABPConthetranslationalregulatoryregimeactingintheoocyte,wemonitoredtherelationshipbetweentaillengthandTEforendogenousmRNAsoftheoocytes.Asexpectedfromresultsofsingle-geneexperimentsinfrogoocytes(Figure1E; Sheetsetal.,1995;Stebbins-BoazandRichter,1994)andthestrongcouplingbetweenpoly(A)-taillengthandTEobservedinbothfrogembryosandflyoocytes(Eichhornetal.,2016;Limetal.,2016;Subtelnyetal.,2014),wefoundthatpoly(A)-taillengthcorrelatedstronglywithTEinstageVIfrogoocytes(Figure2A).OverexpressingeitherPABPC1orePABintheseoocytessignificantlydiminishedthecoupling,withtheSpearmancorrelation(Rs)fortherelationshipbetweentaillengthandTEdroppingfrom0.62to0.36and0.38,respectively(Figure2A,bothp = 0,modifiedDunnandClark’sz-test[DiedenhofenandMusch,2015]).Incontrast,overexpressingeGFPhadnosignificantimpactonthecoupling(p = 0.11),whichindicatedthatthistranscriptome-wideeffectwasaresultofadditionalPABPCproteinratherthananon-specificeffectofaddingmoremRNA. Figure2with1supplementseeall Downloadasset Openasset IncreasedPABPCpromotestranslationofendogenousshort-tailedmRNAs,therebydiminishingcouplingbetweentaillengthandTE. (A)TheeffectofPABPConcouplingbetweentaillengthandTEinfrogoocytes.ShownistherelationshipbetweenTEandmedianpoly(A)-taillengthinoocytesinjectedwitheitherwaterormRNAsencodingeithereGFP,PABPC1,orePAB(injecting16fmolmRNAperoocyte).ResultsareshownformRNAsfromgeneswith ≥100poly(A)tags.Eachpoly(A)tagrepresentsapairofsequencingreadsthatidentifythemRNAanditspoly(A)taillength.RsistheSpearman’scorrelationcoefficient.(B)AglobalincreaseinTEobserveduponoverexpressingePAB.TEinePAB-overexpressingoocytesiscomparedwithTEineGFP-expressingoocytes.ExperimentwasasinA,exceptrRNAswerenotdepleted,andTEvaluesarenormalizedtothoseofmitochondrialmRNAs.Alsoshownareresultsfromaco-injectedshort-tailedNlucreporterandalong-tailedFlucreporter.(C)EffectofexpressingPABPC1onproteinsynthesisinoocytes.AttheleftisagelshowingtotalproteinafterinjectionofeitherwaterortheindicatedmRNA(injecting4fmolmRNAperoocyte),withorwithouttreatmentwithcycloheximide(CHX),asvisualizedbyCoomassiestaining.Attherightisthesamegelshowingproteinsynthesis,asvisualizedbyincorporationof35S-methionineand35S-cysteine.ProminentbandspresumablyrepresentPABPC1,eGFP,andanotfullydenaturedformofeGFP(asterisk)expressedfrominjectedmRNAs.(D)QuantificationoftheeffectofexpressingPABPC1onproteinsynthesisinoocytes,asmeasuredin(C)andtwoadditionalbiologicalreplicates.OnlyregionsabovethePABPC1band,andbetweenthePABPC1bandandthetopeGFPband,wereusedforquantification.Valueswerenormalizedtothatofthemeanvaluefromwater-injectedoocytes(errorbars,standarddeviation;pvalues,one-sidedt-tests;n.s.,notsignificant).(E)ThepreferentialeffectofoverexpressingePABontheTEofshort-tailedmRNAs.TEfoldchangesobservedbetweenePAB-overexpressingandeGFP-expressingoocytesareplottedasafunctionofmediantaillengthineGFP-expressingoocytes.TEandtail-lengthvalueswereobtainedfromdifferentbatchesofoocytes;resultsareshownformRNAsfromgeneswith ≥100poly(A)tags.TEvaluesarenormalizedtothoseofmitochondrialmRNAs.(F)EffectofoverexpressingePABonthedistributionofTEvaluesobservedinfrogoocytes.ShownistheTEdistributionobservedinePAB-overexpressingoocytesandthatobservedineGFP-expressingoocytes.Tail-lengthmeasurementsinthisfigurewereobtainedusingTAIL-seq. Figure2—sourcedata1 SourcedataforvaluesshowninFigure2D. https://cdn.elifesciences.org/articles/66493/elife-66493-fig2-data1-v1.xlsx Downloadelife-66493-fig2-data1-v1.xlsx AccompanyingthereducedcouplingobserveduponPABPCoverexpressionwasasignificantrelativeincreaseofTEforshort-tailedmRNAs,aneffectnotobservedineGFP-expressingoocytes(Figure2—figuresupplement1A).ThisTEincreasewasnotaccompaniedbycorrespondinglengtheningofpoly(A)tails(Figure2—figuresupplement1B),implyingthattail-lengthchangesdidnotcausethese relativeTEchanges.TomakecomparisonsofabsoluteTEchanges,werepeatedtheePAB-overexpressionexperimentbutomittedrRNAdepletionduringsequencinglibraryconstruction,therebyallowingustonormalizeTEusingmitochondrialmRNAs(Iwasakietal.,2016),whichwereotherwisedepletedbyIlluminaRibo-Zerokits(Figure2—figuresupplement1C).Inthisexperiment,wealsoinjectedoocyteswithashort-tailedNlucmRNAreporterandalong-tailedFlucmRNAreporterandmonitoredtheirabsoluteTEchangestogetherwiththoseofendogenousmRNAs.MostendogenousmRNAshadgreaterabsoluteTEinePAB-overexpressingoocytescomparedtoeGFP-expressingcontroloocytes(Figure2B).Thisresultwasconsistentwith35Smetabolic-labelingexperimentsshowingthatoverexpressionofPABPC1butnoteGFPsignificantlyincreasedglobalproteinsynthesisinoocytes(Figure2C–D).Moreover,themagnitudeoftheTEincreaseconferredbyePAB-overexpressionnegativelycorrelatedwithtaillength,whichshowedthattranslationofshort-tailedmRNAsimprovedsubstantiallymorethanthatoflong-tailedmRNAs(Figure2E),asobservedforourco-injectedreporters.Indeed,addingePABhadessentiallynooveralleffectonTEofendogenousmRNAswiththelongesttails(medianTEfoldchange = 1.06forthe54mRNAswithmediantaillengths > 80nt),asobservedforourlong-tailedreporters.ThepreferentialimprovementofTEsforshort-tailedmRNAsledtonotonlyanoverallshiftinTEbutalsonarrowingoftheTEdistribution(Figure2F)tomorecloselyresemblethedistributionsobservedincellsinwhichpoly(A)-taillengthandTEarenotcoupled(Subtelnyetal.,2014).TheseresultssupportedthehypothesisthatincreasingPABPCinoocytesincreasestheopportunityforshort-tailedmRNAstobindaPABPCmolecule,therebypromotingtranslation. Overall,theresultsofourglobalanalysesofmRNAsinfrogoocytesagreedwiththoseofreporterassays,therebyextendingtoendogenousmRNAssupportfortheconclusionthatlimitingPABPCplaysacriticalroleinconferringstrongcouplingbetweenpoly(A)-taillengthandTE. Ourglobalanalysisexaminingtherelationshipbetweenpoly(A)-taillengthandTEofendogenousmRNAsinoocytesdifferedfromourreporterassaysinthatthecomparisonwasmadebetweenmRNAsofdifferentgenes,whichcanbeconfoundedbyfeaturesotherthantaillengththatvarybetweenthesemRNAs.Toovercomethisissue,wedevelopedahigh-throughputmethodforcomparingeffectsondifferenttail-lengthisoformsfromeachgene.Thisapproachforintragenicanalyses,calledPAL-TRAP(Poly(A)tail-LengthprofilingfollowingTranslatingRibosomeAffinityPurification),resembledotherTRAPapproachesinthatribosomesweresparselytaggedsuchthattheirimmunoprecipitation(IP)preferentiallyisolatedmRNAisoformsassociatedwithmoreribosomes,whichwereinferredtobemorehighlytranslated(ChenandDickman,2017;Heimanetal.,2008).Inasysteminwhichpoly(A)-taillengthandTEwerecoupled,longer-tailmRNAswereexpectedtobeassociatedwithmoreribosomesandthereforeenrichedintheeluate(Figure3A),whereasinanuncoupledsystem,longer-tailmRNAswerenotexpectedtobeenrichedintheeluate. Figure3with1supplementseeall Downloadasset Openasset LimitingPABPCisrequiredforintrageniccouplingbetweenpoly(A)-taillengthandTE. (A)ThePAL-TRAPmethodformeasuringintrageniceffectsoftaillengthonTE.Ribosomesaresparselytagged(redstars)sothathighlytranslatedmRNAsaremorelikelytocontaintaggedribosomesandthusbeenrichedintheimmunoprecipitation(IP)eluate.TaillengthsofbothinputandeluatemRNAsweremeasuredandcomparedformRNAsofeachgene.Thedepictedenrichmentoflong-tailedisoformsintheeluateindicatesthatpoly(A)-taillengthandTEarecoupled,whereasnoenrichmentwouldindicateotherwise.(B)TheexperimentalschemeofPAL-TRAPinfrogoocytes.SeeFigure3—figuresupplement1B–CforresultsfrompulldownsusingtheeGFP-HAcontrol.(C)EffectofoverexpressingePABoncouplingbetweentaillengthandTE,asdetectedafterpoolingPAL-TRAPresultsformRNAsfromdifferentgenes.Plottedarecumulativedistributionsofpoly(A)-taillengthsinthePAL-TRAPinputandeluateobtainedafterexpressingeithereGFP(left)orePAB(right)inoocytes(injecting4fmolmRNAperoocyte).Medianvaluesareindicated(dashedlines)andlistedinparentheses.(D)TheeffectofoverexpressingePABonintrageniccouplingbetweentaillengthandTE.PlottedforeachmRNAisoformisthemedianpoly(A)-taillengthofmRNAsinthePAL-TRAPeluatecomparedtothatintheinput.ShownareresultsformRNAsfromoocyteseitherexpressingeGFPoroverexpressingePAB(leftandright,respectively).EachpointrepresentsanmRNAisoformwithaunique3′endrepresentedby ≥100poly(A)tagsinbothinputandeluate.Alsoindicatedareresultsfor(1)anRlucreportermRNApossessingavariable-lengthtail(reporter),whichwasco-injectedwithmRNAsexpressingeithereGFPorePAB,(2)anmRNAwithavariable-lengthtail,whichwasspikedintothelysateimmediatelybeforeIP(spike-in),and(3)syntheticRNAswithdefinedtaillengthsaddedtosamplespriortolibrarypreparation(tailstandards).Pointsforeightstandardswithlongertailsfelloutsidetheplotareas,asdidapointrepresentingthemRNA3′-endfromonegene(uqcrb.S)intheePABsample.(E)SummaryofdifferencesinmediantaillengthsobservedbetweentheeluateandtheinputofmRNAisoformsshownin(D).Boxandwhiskersindicatethe10th,25th,50th,75th,and90thpercentiles.(F)EffectofoverexpressingePABonintragenictail-lengthdistributionsinfrogoocytes.Shownaretail-lengthdistributionsofthereporterRluc(left),anendogenousoocytemRNAbtg4.S(middle),andthespike-inmRNANluc(right)ineGFP-expressing(top)orePAB-overexpressing(bottom)oocytes.Mediantail-lengthvaluesareindicated(verticallines)andlistedinparentheses.Tail-lengthmeasurementsinthisfigurewereobtainedusingPAL-seqv3. ToimplementPAL-TRAP,wefirstinjectedstageVIfrogoocyteswithanmRNAencodingC-terminalHA-taggedRPL3(ChenandDickman,2017)andallowedtimeforRPL3-HAproteinexpressionandincorporationintoribosomes(Figure3B).Asacontrol,HA-taggedeGFPwasexpressedinseparateoocytes.ToexaminetherequirementoflimitingPABPCforcouplingbetweenpoly(A)-taillengthandTE,wetheninjectedoocyteswithmRNAsthatexpressedeithereGFPorePAB.ConfirmingthatRPL3-HAwasincorporatedintofunctionalribosomes,HA-IPfromRPL3-HA-expressingoocytesenrichedforproteinsfrombothribosomalsubunits,cytoplasmicmRNAs,andePAB,whereasHA-IPfromeGFP-HA-expressingoocytesdidnot(Figure3—figuresupplement1A–C).IncontroloocytesexpressingeGFP,longer-tailmRNAswereenrichedintheeluatecomparedtotheinput(mediantaillengths41and36nt,respectively),whichreflectedthecouplingbetweentaillengthandTE(Figure3C).OverexpressionofePABreducedthisenrichmentforlonger-tailmRNAsintheeluatecomparedtotheinput(mediantaillengths37and35nt,respectively),asexpectediflimitingPABPCwasrequiredforthiscoupling(Figure3C).Wealsoanalyzedtheflowthroughfractionsfromthesepulldownexperiments.ForbotheGFP-andePAB-expressingoocytes,thecumulativedistributionofpoly(A)-taillengthsfromtheflowthroughwasnearlyidenticaltothatfromtheinput(Figure3—figuresupplement1D),asexpectedwhenconsideringthatonlyasmallfractionofinputwasdepletedbyHA-beads.Theseanalysesindicatedthatourmethodswereabletocapturesmalldifferencesintail-lengthdistributions. Whenanalyzing,foreachgene,mRNAswithdifferenttail-lengthisoforms,mRNAsfrommostgenes(84.4%)hadlongermedianpoly(A)-taillengthsintheeluatethanintheinput(Figure3D–E),implyingthatformRNAsfrommostgenes,long-tailedisoformsweretranslatedmoreefficientlythanshort-tailedones.Althoughthemedianofthemediantail-lengthdifferenceswasmoderate(+2.5nt),28.3%mRNAshada ≥ 5ntlongermediantaillengthintheeluate(Figure3E).Incontrast,wheninputandflowthroughwerecompared,themediantail-lengthdifferencescenteredon0nt,andmRNAsfromonly5.4%ofgeneshada ≥ 5ntlongermediantaillengthinoneofthesamples,asexpectedwhenconsideringthatonlyasmallfractionofinputwasdepletedbyHA-beads(Figure3—figuresupplement1E–F).OverexpressingePABreducedthenumberofgeneswithlong-tailedisoformsenrichedintheeluateandshiftedthedistributionofmediantail-lengthdifferencescloserto0nt(Figure3D–E),asexpectedifcouplingbetweenpoly(A)-taillengthandTEdiminished. InthesePAL-TRAPexperiments,amixtureofRlucreportermRNAmoleculeswithdifferentpoly(A)-taillengthswasco-injectedwithmRNAsthatexpressedeithereGFPorePAB.IneGFP-expressingoocytes,longer-tailRlucisoformswerehighlyenrichedintheeluatecomparedtotheinput,whereasinePAB-overexpressingoocytes,thisdifferencediminisheddramatically(Figure3F).Asexpected,NlucreportermRNA,whichwasaddedtothelysateasaspike-induringHApulldown,wasnotsignificantlyenrichedinlonger-tailspeciesintheeluate,regardlessofthetreatment,whichindicatedthatthechangesobservedforRlucmRNAreflectedchangesoccurringintheoocyte,priortolysis(Figure3F).AlthoughendogenousmRNAsfromsomegenes,suchasbtg4.S,alsounderwentlargechangesinmediantaildifferencesinresponsetoePAB-overexpression(Figure3F),changesweretypicallysmallerforendogenousmRNAs(Figure3E),whichwasatleastpartlyattributabletothenarrowerrangeofinitialtaillengthisoformsforendogenousmRNAscomparedtotheinjectedRlucmRNA. Takentogether,thePAL-TRAPresultsrevealedintrageniccouplingbetweenpoly(A)-taillengthandTEforendogenousmRNAsaswellasreportersinstageVIfrogoocytes.Moreover,thesePAL-TRAPanalysesshowedthatasobservedbothinourreporterassaysandinourglobalintergeniccomparisons,substantialcouplingbetweentaillengthandTErequireslimitingPABPC. HavingestablishedthenecessityoflimitingPABPCforcoupling,weinvestigatedifitcouldalsobesufficient,that is,whetherlimitingPABPCcouldconfercouplingbetweenpoly(A)-taillengthandTEincellsinwhichthesefeatureswerenormallyuncoupled.Tothisend,weknockeddownPABPCsinHeLacells.BasedonavailablemRNA-seqandmassspectrometryresults(Nagarajetal.,2011),PABPC1andPABPC4arethetwomajorPABPCparalogsinHeLacells,withPABPC1fourtimesmoreabundantthanPABPC4andthetwotogetheraccountingfor >95%ofallPABPCinHeLacells.ConsistentwiththeideathatPABPCisnotnormallylimitinginuncoupledsystems,PABPC1isestimatedtobepresentinthreefoldexcessoverthepoly(A)sitesinHeLacells(Görlachetal.,1994).ToreducePABPCtolimitinglevels,weusedsiRNAstoreduceeitherPABPC1orPABPC4aloneorbothPABPC1andPABPC4by >90%(Figure4—figuresupplement1A)andexaminedtherelationshipbetweenmediantaillengthsandTE.KnockingdownPABPC4alonehadlittleimpactonthecoupling(Figure4—figuresupplement1B),consistentwiththeinferencethatPABPC4constituteslessthan20%ofthetotalPABPCprotein.Althoughcorrelationbetweenmedianpoly(A)-taillengthandTEgraduallyincreasedasmorePABPCwasdepleted,itreachedanRsofonly0.18(p<10–19)indouble-knockdowncells(Figure4—figuresupplement1B),whichwasmuchweakerthanthatobservedinfrogoocytes(Figure2A)andfrogandfishearlyembryos(Subtelnyetal.,2014).MinorRsincreaseswereobservedinothermouseandhumanpost-embryoniccelllinesinwhichPABPCwasdepleted,butstrongcouplingbetweenpoly(A)-taillengthandTEwasnotestablished,withnoRsvaluesexceeding0.3(datanotshown).Thus,otherconditionsinadditiontolimitingPABPCmustalsobemettoconferstrongcouplingbetweenpoly(A)-lengthandTE. AstrikingconsequenceofdepletingPABPCinHeLacellswasasharpincreaseinmedianpoly(A)-taillengths,whichforHeLamRNAsincreasedanaverageof17and39ntinPABPC1-anddouble-knockdowncells,respectively(Figure4A).Substantialchangeswerealsoobservedinthedistributionsofglobalpoly(A)-taillength,whichshowedthatmRNAswithtailsrangingfrom10to50ntwere >2-folddepletedinthePABPC1-knockdowncellsandthatmRNAswithtailsrangingfrom10to135ntwere2-to20-folddepletedinthedouble-knockdowncells(Figure4B).SimilarresultswereobtainedinNIH3T3cells(Figure4—figuresupplement2A–B).Incontrast,lossofshort-tailedmRNAswasnotobservedformitochondria-encodedmRNAs,asexpectedforthesemRNAsthatneverencounterPABPC(Figure4A–B,Figure4—figuresupplement2A–B),andexaminationofinternalstandardsandreplicatesconfirmedthatthelossofshort-tailedcytoplasmicmRNAswasnotattributabletoinaccurateorvariablemeasurements(Figure4—figuresupplement2C–D).Moreover,knockingdowntheminorisoform(PABPC4)alonedidnothaveasimilareffect(Figure4—figuresupplement2E),suggestingthatthetail-lengthchangesobservedforcytoplasmicmRNAswereaconsequenceoflimitingPABPC. Figure4with3supplementsseeall Downloadasset Openasset PABPCdepletioncausesprematuredecayofshorter-tailcytoplasmicmRNAsinHeLacells. (A)TheeffectofPABPCknockdownonpoly(A)-taillength.Theplotscomparemedianpoly(A)-taillengthsineitherPABPC1-knockdowncells(left)orPABPC1andPABPC4double-knockdowncells(right)tothoseincontrolcells.ResultsareshownforcytoplasmicmRNAswith ≥100poly(A)tags(gray)andformitochondrialmRNAs(red),mergingdataforMT-ATP6andMT-ATP8andforMT-ND4andMT-ND4L,whicharebicistronicmitochondrialmRNAs.(B)TheeffectofPABPCknockdownontheabundanceofmRNAswithdifferenttaillengths.Shownaretail-lengthdistributionsofallcytoplasmic(left)andmitochondrial(right)mRNApoly(A)tagsincontrol,PABPC1-knockdown,anddouble-knockdowncells.Foreachdistribution,theabundanceoftagswasnormalizedtothatofthespike-intail-lengthstandards.Duetodepletionoftail-lengthcallingatposition50,whichwasassociatedwithachangeinlaserintensityatthenextsequencingcycle,thevaluesatthistaillengthwerereplacedwiththeaverageofvaluesattaillengths49and51nt.(C)TheeffectofPABPCknockdownontheabundanceofmRNAswithdifferenttaillengths,comparingtail-lengthsmeasuredbysequencing(left)withthoseobservedonRNaseHnorthernblots(right).ResultsareshownforacytoplasmicmRNAGAPDH(top)andamitochondrialmRNAMT-CYB(bottom).Relativetagdensitywascalculatedbylog-transforminglineartagdensityusingnormalizedpoly(A)tagcounts.Mediantail-lengthvaluesareindicated(horizontallines)andlistedinparentheses.ForRNaseHnorthernblots,aDNAoligonucleotidecomplimentarytothe3′-UTRwasusedtodirectcleavageofthetargetmRNAbyRNaseH,leavinga35-ntfragmentofthe3′-UTRappendedtothepoly(A)tail,whichwasresolvedonadenaturinggelanddetectedbyaradiolabeledprobe.Taillengthsindicatedalongtheleftsideofeachgelareinferredfromlengthsofsizemarkers.(D)TheeffectofPABPCknockdownontheabundanceofmRNAswithdifferenttaillengths,extendingtheintragenicanalysistotail-lengthdistributionsfromthousandsofgenes.Heatmapscomparepoly(A)-taglevelsinPABPC1-knockdown(left)ordouble-knockdown(right)cellstothoseincontrolcells,afternormalizingtospike-intail-lengthstandards,asmeasuredusingtail-lengthsequencing.EachrowrepresentsmRNAsfromadifferentgene,androwsaresortedbasedonfoldchangeofmRNAabundancemeasuredusingRNA-seq.Onlygeneswith ≥100poly(A)tagsineachoftwosamplesbeingcomparedwereincludedintheanalyses(n = 5504).Columnsrepresentvaluesfrom5-nttail-lengthbinsrangingfrom0to244ntanda6-ntbinrangingfrom245to250nt.Tilecolorindicatesthefoldchangeofnormalizedtagcounts(key).(E)TheeffectofPABPCknockdownontheabundanceofmRNAswithdifferenttaillengths,reanalyzingdatafrom(D)toshowdistributionsofpoly(A)-tagchangesobservedatdifferenttail-lengths.Eachbox-whiskershowsthe10th,25th,50th,75th,and90thpercentileoffoldchangesinnormalizedpoly(A)-tagcountsobservedforeachtail-lengthbinof(D).Thecolorofeachboxindicatesthemedianvalue(key).Tail-lengthmeasurementsinthisfigurewereobtainedusingTAIL-seq. WealsousednorthernblotstoexamineeffectsofPABPCdepletionontail-lengthdistributionsofmRNAsfromindividualgenesandfoundthattheresultscorrespondedwelltothoseobservedbytail-lengthsequencing.Bothnorthernblotsandsequencingshowedstrongdepletionofshort-tailedisoformsforacytoplasmicmRNA(GAPDH)afterPABPCknockdownbutnosubstantialchangeinthetail-lengthdistributionofamitochondrialmRNA(MT-CYB)(Figure4C).Usingsequencingdatatoexaminetheintragenictail-lengthdistributionsofcytoplasmicmRNAfromeachofmorethanfivethousandothergenesrevealedfindingsresemblingthoseobservedforGAPDH(Figure4D–E).AfterknockingdownPABPC1,thereductioninshort-tailedmRNAswastypicallymostsevereformRNAisoformswithtaillengthsof ~25nt,andinthedoubleknockdown,thisdipat ~25ntbecamemorepronounced,withreductionsextendingtoallbutthelongest-tailisoforms(Figure4D–E).Indeed,formorethanhalfofthegenesexamined, ≥2-foldreductionsextendedtoisoformswithtailsaslongas135nt(Figure4D–E).Similarresultswereobservedwhenexaminingtail-lengthdistributionsintheNIH3T3dataset(Figure4—figuresupplement2B),andwhenexaminingindividualtail-lengthdistributionsformRNAsofeachofthetop-expressednucleargenesbutnotwhenexaminingthoseformRNAsofmitochondrialgenes(Figure4—figuresupplement2F–G). Theseresults,whichshowedthatmRNAswithshorttailswerepreferentiallydestabilizedwhenPABPCwasdepletedprovidedgeneticloss-of-functionevidencethatPABPCstabilizesmRNAsinmammaliancelllines.AlthoughgeneticevidencefortheroleofPABPCinmRNAstabilityhasbeenreportedinyeast(CaponigroandParker,1995;Colleretal.,1998),thisfunctionhadnotbeenestablishedinmammaliancells.AlthoughinprinciplethemRNAdestabilizationthatweobserveduponPABPC-depletionmighthavebeenindirect,twolinesofevidencesupporttheconclusionthatthisdestabilizationwasadirectconsequenceofthelossofPABPCbindingtopoly(A)tails.First,destabilizationpreferentiallyoccurredforshort-tailedmRNAs,whichwereexpectedtobetheleastsuccessfulatcompetingforbindingunderconditionsoflimitingPABPC.Second,destabilizationsharplydiminishedattaillengthsof10–15nt,whichcorrespondedtothe12ntpoly(A)sequencereportedtobetheminimallengththatcanbeboundbyPABPC1withhighaffinity(KühnandPieler,1996).Indeed,themodestlossobservedformRNAswithveryshortpoly(A)tails(Figure4D–E)suggestedthatevenincontrolcellsthathadabundantPABPC,mRNAswithtailsshorterthan12ntwerepoorlyboundbyPABPCs,andthusPABPCdepletiondidnotsubstantiallyinfluencetheirabundance. Arecentstudyobservedsimilarpoly(A)tail-lengthchangesinPABPC1-depletedcellsbutattributedthesechangestoimpaireddeadenylation(Yietal.,2018).BecausePABPCcanpromotedeadenylationinvitro(Schäferetal.,2019;Uchidaetal.,2004;Websteretal.,2018;Yietal.,2018),itisconceivablethatthelossofPABPCwouldslowdeadenylation,therebyincreasingmRNAmediantaillengths,asobservedinPab1-knockoutyeast(CaponigroandParker,1995).However,ouranalyses,whichhadthebenefitofquantitativetailstandardsthatenabledmeasurementofabsoluteabundancechanges,revealedlittleaddedaccumulationoflong-tailedisoformsinPABPC-depletedcells(Figure4B–E,Figure4—figuresupplements2B, F),indicatingthatadeadenylationdefectwasnotthemajorcausefortheperturbedtail-lengthdistributions.Moreover,wefoundthatmRNAhalf-lifevalues,asdeterminedbymetaboliclabeling,reducedsignificantlywhenPABPCwasknockeddown(Figure4—figuresupplement3A–C),whichconcurredwiththeconclusionthatPABPCknockdowndestabilizedshort-tailedmRNAisoformsandarguedagainstthepreviousassertionthatPABPCknockdownimpaireddeadenylation,inthatimpaireddeadenylationwouldhavelengthenedmRNAhalf-lives. Takentogether,theseresultsshowthatPABPCbindingstabilizesmRNAsofculturedmammaliancells;ifPABPCbecomeslimitinginthesecells,theshort-tailedmRNAsbecomedestabilized,presumablybecausetheycompetelesseffectivelyforPABPC.Mostimportantly,thedestabilizationofmRNAsthatcompetedpoorlyforPABPCbindinghelpsexplainwhylimitingPABPCwasinsufficienttocausestrongcouplinginmammaliancelllines,inthatstrongcouplingbetweentaillengthandTEwouldbedifficulttoestablishinaregulatoryregimeinwhichshort-tailedmRNAmoleculesthatlackPABPCbindingaredegradedratherthantranslatedlessefficiently.Thus,theseresultsidentifyasecondmechanisticrequirementforstrongcouplingbetweentaillengthandTE:InadditiontolimitingPABPC,strongcouplingrequiresmetabolicstabilityofthemRNAsthatcompetepoorlyforPABPCbinding. TheidentificationofthissecondrequirementforstrongcouplingbroughttotheforethequestionofwhymRNAsthatcompetedpoorlyforlimitingPABPCweredestabilized.Toexplorethepossiblemechanisms,wesearchedforperturbationsthatcouldrestorestabilityofshort-tailedmRNAsinHeLacellsundergoingPABPCknockdown,monitoringtail-lengthdistributionsofendogenousGAPDHusingnorthernblots.Asapositivecontrol,expressingansiRNA-resistantPABPC1restoredstabilityofshort-tailedspecies,asdidfrogePAB,whichfurtherillustratedfunctionalconservationofPABPCfromdifferentspeciesanddevelopmentalstages(Figure5A).Interestingly,aPABPC1variantwithsubstitutionsthatdisruptitsinteractionwitheIF4G(Chorghadeetal.,2017)alsorestoredthestabilityofshort-tailedspecies,implyingthattheclassicalclosedloopisnotnecessaryforPABPC1toprotectshort-tailedmRNAsfromdegradation.BecausePABPChasbeenimplicatedininhibitingmRNAterminaluridylationinvitro(Limetal.,2014)andincells(Yietal.,2018),andterminaluridylationhasbeenlinkedtomRNAdecay(Limetal.,2014;RisslandandNorbury,2009),weaskedifterminaluridylationcontributedtothelossofshort-tailedmRNAs.KnockingdownbothTUT4andTUT7inPABPC-depletedcellspartiallyrestoredshort-tailedGAPDHmRNAs(Figure5B).SimilarresultswereobservedinHCT116cells,inwhichwetaggedendogenousPABPC1withanauxin-inducibledegron(AID)andinduceddepletionbyaddingindole-3-aceticacid(IAA,aformofauxin)(Figure5C; Natsumeetal.,2016). Figure5with1supplementseeall Downloadasset Openasset DepletionofTUT4andTUT7attenuatesprematuredecayofmRNAcausedbyPABPCdepletion. (A)Rescueoflossofshorter-tailmRNAsinPABPC-depletedHeLacellsbyexpressingeitheransiRNA-resistanthumanPABPC1,ansiRNA-resistantPABPC1codingforamutantthatdoesnotbindeIF4G(4G_mut),oransiRNA-resistantfrogePAB.AtthetopleftisanRNaseHnorthernblotprobedforGAPDH,asinFigure4C.Atthetoprightaretheintensitylevelsquantifiedfromtheblot.AtthebottomisawesternblotprobedfortheindicatedproteinsortheFLAGtagappendedtotheCterminusofeachoftheexpressedPABPCproteins.(B)Partialrescueoflossofshorter-tailmRNAsinPABPC-depletedHeLacellsbyknockingdownTUT4andTUT7.AtthetopleftisanRNaseHnorthernblotprobedforGAPDH,asinFigure4C.Atthetoprightaretheintensitylevelsquantifiedfromtheblot.Atthebottomisawesternblotprobedfortheindicatedproteins.(C)Partialrescueoflossofshorter-tailmRNAsinPABPC-depletedHCT116cellsbyknockingdownTUT4andTUT7.EndogenousPABPC1wastaggedwithAID,andIAAwasaddedfor24hrtotargetthefusionproteinfordegradation.Otherwise,thispanelisasin(B).(D)TheeffectofPABPCknockdownandTUTknockdownonterminaluridylation.PlottedisthefractionofuridinesneartheterminiofcytoplasmicmRNAsinHeLacellstransfectedwiththeindicatedsiRNAs,asmeasuredusingtail-lengthsequencing.(E)TheeffectofPABPCknockdownandTUTknockdownonterminaluridylationoftailswithdifferentlengths.ShownisterminaluridylationfrequencyofcytoplasmicmRNAsasafunctionofpoly(A)-taillengthinHeLacellstransfectedwiththeindicatedsiRNAs,asmeasuredusingtail-lengthsequencing.Theinsetshowsahigher-resolutionviewoftheshort-tailregion.(F)Partialrescueoflossofshorter-tailcytoplasmicmRNAsinPABPC-depletedHeLacellsbyknockingdownTUT4andTUT7,extendingtheanalysistoallpoly(A)tags.Plottedaretail-lengthdistributionsfromHeLacellstransfectedwiththeindicatedsiRNAs.Foreachdistribution,tagcountswerenormalizedtospike-intail-lengthstandards.Medianvaluesareindicated(dashedlines)andlistedinparentheses.Tail-lengthmeasurementswereobtainedusingPAL-seqv3. Toexaminetheglobalrescueofshort-tailedmRNAsandatthesametimemonitormRNAterminaluridylationlevels,wemodifiedourtail-lengthsequencingprotocolbyincludingintheadaptor-ligationstepasplintoligonucleotidedesignedtoaccommodatetailswitha3′terminalU(Eisenetal.,2020).KnockdownofPABPC1alonesignificantlyincreasedtheterminaluridylationlevelsacrossessentiallyalltail-lengthisoforms(Figure5D–E),consistentwithapreviousreport(Yietal.,2018).KnockdownofPABPC4inadditiontoPABPC1furtherincreaseduridylationofmRNAisoformswithlongertail lengths(Figure5E).KnockdownofTUT4andTUT7inPABPC-depletedcellsbroughtterminaluridylationofalltail-lengthisoformstobackgroundlevels(Figure5D–E)and,moreimportantly,preferentiallyrescuedshorter-tailisoforms,therebydecreasingmediantaillengths(Figure5F).Theseresultswereconsistentwiththoseofournorthernassays,andtogether,ourresultsindicatedthatinthesemammaliancells,limitingPABPCmakesshort-andmedium-tailedmRNAisoformsthatpoorlycompeteforPABPCbindingmoresusceptibletoterminaluridylation,therebyacceleratingtheirdecay. Havingfoundthatdestabilizationofshort-tailedmRNAsdampenedcouplingbetweenpoly(A)-taillengthandTEinPABPC-depletedmammaliancells,akeyquestionremainedregardinghowmRNATE,ifnotinfluencedstronglybypoly(A)-taillength,wasaffectedinthesecells.Toanswerthisquestion,weconductedglobalprofilingofHeLacells48hraftersiRNAtransfection,atimepointatwhichPABPC1andPABPC4knockdownsweresubstantialbutsecondaryeffectswerepresumablynotyettoosevere(Figure6—figuresupplement1A).WeagainimplementedRNA-seqandribosome-profilingprotocolsthatenabledabsoluteTEcomparisonbyusingmitochondrialmRNAsfornormalization(Rooijersetal.,2013).Surprisingly,near-completedepletionofPABPCshadnodetectableeffectonglobalmRNATEs(Figure6A–B).Althoughtheproteinsynthesisrateindouble-knockdowncellswasreducedto75.7%ofthatobservedincontrolcells,asmeasuredbyaveragingribosome-footprintchangesobservedforthe9697analyzedgenes(Figure6A–B),whichagreedwithresultsofaglobalpuromycin-basedtranslationassay(78.7%)(Figure6C),thisreducedproteinsynthesiswasfullyexplainedbythedecreaseinmRNAlevels,asindicatedbyadistributionofTEchangesthatcenterednearzero(Figure6A–B).Examinationofourribosome-footprintingdatarevealednoupregulationofotherPABPCparalogs,althoughtheTEofPABPC1mRNAincreased2.5-fold(Figure6A),consistentwithitsknownautoinhibitorytranslationalcontrol(BagandWu,1996).Resultsofpolysomeprofilingconfirmedthoseofoursequencing-basedmethods,inthatthereductionintranslationoutput,asmeasuredbytheheightofpolysomepeaksindouble-knockdowncells,wasattributabletoanoveralldecreaseofmRNAlevelsratherthantodecreasedTEsthatwouldotherwisecauseashiftofmRNAdistributionfromheavytolightfractions(Figure6—figuresupplement1B).Together,theseresultsindicatedthatPABPCdepletioninHeLacellshadnegligibleeffectonmRNATE. Figure6with2supplementsseeall Downloadasset Openasset DepletionofPABPCinmammaliancelllineshasminimaleffectonTE. (A)EffectofPABPCknockdownonmRNAabundance(left),ribosome-footprintabundance(middle)andTE(right)inHeLacells,comparingvaluesindouble-knockdowncellstothoseincontrolcells.Foreachgene,valuesformRNAandribosome-footprintreadsperkilobaseareplottedafternormalizingtomeasurementsformitochondrialmRNAs.(B)DistributionsoftheeffectsofPABPCdoubleknockdownonmRNAabundance,ribosome-footprintabundanceandTE.Eachbox-whiskershowsthe10th,25th,50th,75th,and90thpercentileofthefoldchangesobservedin(A).(C)EffectofPABPCknockdownonproteinsynthesisinHeLacells.Plottedarerelativelevelsofproteinsynthesismeasuredbypulsepuromycinincorporation48hraftertransfectionwiththeindicatedsiRNAs(Figure6—figuresupplement1A;errorbars,standarddeviationofthreebiologicalreplicates;pvalues,one-sidedt-tests;n.s.,notsignificant).(D)WesternblotshowingrapiddegradationofPABPC1-AIDfusionproteinafteraddingIAAtogenome-engineeredHCT116cells.PABPC1proteinlevelswerequantifiedbyaveragingsignalsforAIDandPABPC1,afternormalizingtothatforGAPDH.(E)EffectofPABPC1depletiononabundanceofmRNAswithdifferenttaillengths.Shownaretail-lengthdistributionsofallpoly(A)tagsobtainedfromcytoplasmicmRNAsofHCT116PABPC1-AIDcellsaftertreatmentwithIAAfortheindicatedtime.Foreachdistribution,theabundanceoftagswasnormalizedtothatofthespike-intail-lengthstandards.Medianvaluesareindicated(dashedlines)andlistedinparentheses.Duetodepletionof101-nttaillengths,whichwasassociatedwithachangeinlaserintensityatthenextsequencingcycle,thevaluesatthislengthwerereplacedwiththeaverageofvaluesattaillengths100and102nt.(F)SummaryoftheeffectsofrapidPABPC1depletion.Foreachgene,ateachtimepointafteraddingIAAtoHCT116PABPC1-AIDcells,valuesformRNAabundance,ribosome-footprintabundanceandTEwereeachcomparedtothevalueobservedincellsnottreatedwithIAA(Figure6—figuresupplement1C),andthedistributionoffoldchangesisplotted.Eachbox-whiskershowsthe10th,25th,50th,75th,and90thpercentile.Tail-lengthmeasurementswereobtainedusingPAL-seqv4. Figure6—sourcedata1 SourcedataforvaluesshowninFigure6CandFigure6D. https://cdn.elifesciences.org/articles/66493/elife-66493-fig6-data1-v1.xlsx Downloadelife-66493-fig6-data1-v1.xlsx TheslowdynamicsofsiRNA-mediatedknockdown,whichdictatedtherelativelylate48hrtimepointforourglobalmeasurements,mighthavepreventeddetectionofanyTEchangesthathappenedearlier,beforeanewsteadystatehadbeenreached.Toexaminethispossibility,wemonitoredthedynamicsoftail-length,mRNA-abundance,andtranslationchangessoonafterPABPCdepletion,usingtheHCT116PABPC1-AIDdegroncellline,inwhichPABPC1wasrapidlyandefficientlydepletedafteraddingIAA(85%within30min, >99%within1hr,Figure6D).BecausePABPC1istheprimaryPABPCisoforminHCT116cells,depletionofPABPC1alonecausedsubstantialdestabilizationofshorter-tailmRNAs1hrafterIAAaddition,andthisdestabilizationfurtherincreasedafter3hr(Figure6E).Accompanyingthelossofshorter-tailmRNAswasacorrespondingreductionofmRNAabundanceformostgenes(Figure6F,Figure6—figuresupplement1C).Importantly,ribosomefootprintsdeclinedinlockstepwithmRNAabundance,leadingtomedianTEchangesthatcenterednearzeroovertheentirecourseofPABPC1depletion(Figure6F,Figure6—figuresupplement1C).Thus,asPABPCbecamelimiting,mRNAsthatlostPABPChadnodetectablereductioninTEbeforetheyweredestabilized. ThesedatafromthePABPCdegronlineallowedustoexaminewhethersomecouplingbetweentaillengthandTEmighthaveoccurredverysoonafterPABPCdepletion—duringthetimewindowinwhichPABPChadbecomelimitingbutshort-tailedmRNAshadonlystartedtodegrade.However,nocouplingbetweentaillengthandTEwasdetectedoverthecourseofrapidPABPCdepletion(Figure6—figuresupplement2).Thus,inthiscontextinwhichtwoconditionsforstrongcouplingweresatisfied(i.e.PABPCwaslimitingandshort-tailedmRNAswerelargelyintact),nocouplingwasobserved,presumablybecausecouplingalsorequiresaregulatoryregimeinwhichPABPCenhancestranslation. TheseresultsshowthatincontrasttomRNAsoffrogoocytesandpresumablythoseofothercoupledsystems,mRNAsofHeLaandHCT116cellsdonotrequirePABPCforefficienttranslation,whichexplainswhypoly(A)-taillengthswerenotabletostronglyinfluenceTEafterwereducedPABPCofthesecellstolimitinglevels.Thus,theseresultsidentifyathirdmechanisticrequirementforcouplingbetweenpoly(A)-taillengthsandTE:couplingrequiresaregulatoryregimeinwhichPABPCaffectsmRNAtranslation. Wefindthatthreefundamentalmolecularconditionsmustbemetforcellstousepoly(A)-taillengthstoeffectivelyregulateTE.First,PABPCmustbelimitingcomparedtothenumberofpoly(A)sitesavailableforbinding.Underthiscondition,short-tailedmRNAisoformsthatpoorlycompeteforPABPCarelesslikelytohavePABPCboundtotheir3′ends(Figure7).TotheextentthatPABPCisnotbound,theseisoformslosethetranslation-activatingcapabilityofPABPCobservedincoupledsystems.CooperativebindingofadjacentPAPBCmolecules(Linetal.,2012;Schäferetal.,2019)wouldfurtherenhancepartitioningoflimitingPABPCawayfromshort-tailedmRNAsandontolonger-tailedmRNAs.WhenadditionalPABPCisintroducedintoacoupledsystem,short-tailedmRNAsbenefitmorefromPABPCbindingthanlong-tailedones,whicharemorelikelytoalreadypossessthenumberofPABPCmoleculesrequiredformoreefficienttranslation. Figure7 Downloadasset Openasset Modelforcouplingbetweenpoly(A)-taillengthandTE,andcontext-dependentrolesofPABPC. Seetextfordetails. TherequirementoflimitingPABPCforcouplingTEtopoly(A)-taillengthraisesthequestionofthestoichiometrybetweenPABPCanditssitesinthepoly(A)tailsandwhetherthisstoichiometrychangesduringtheembryonicswitchofgene-regulatoryregimes.OursequencingresultsindicatedthatthemRNAsofastageVIfrogoocytehave ~2.8×1011PABPCsites(~7×1010sitesper1µgtotalRNA, ~4µgtotalRNAperoocyte),whichconcurredwiththepreviousestimateof2 ~ 3×1011sitesperoocyte(Sagataetal.,1980).TheamountofPABPC(includingbothePABandPABPC1)infrogoocyteshasbeenestimatedateither1×1011(Cossonetal.,2002)or1.4×1011(Peuchenetal.,2017;Smitsetal.,2014)moleculesperoocyte.OurresultsshowingthatPABPCactivityislimitinginfrogoocytes,agreedwiththeseestimatesthatimplythatPABPClevelsaresufficienttobindnomorethanhalfoftheavailablePABPCsites.Poly(A)-siteoccupancywouldbeevenlowerifsomePABPCproteinsweresequesteredfromthemRNApoolorinaninactiveform,whichcouldbeconferredbyfactorsthateitherbindtoorpost-translationallymodifyPABPCtoaffectitsabilitytoeitherbindpoly(A)tailsorpromotetranslation(Brooketal.,2012;Khaleghpouretal.,2001).Untilstage15,developingfrogembryosmaintainthenumberofPABPCsitesatalevelresemblingthatofoocytes(Sagataetal.,1980).Incontrast,thetotalamountofPABPCmoleculesincreasessignificantly,nearlytriplingbystage12(Peshkinetal.,2019)—thestageatwhichthecouplingbetweentaillengthandTEstartstodisappear(Subtelnyetal.,2014).ThisincreasedPABPCwouldshiftthestoichiometrytowardPABPCbeinglesslimiting.Importantly,dysregulationofthistightlycontrolledstoichiometrynotonlydisruptsthenormalgene-regulatoryregimebutalsocancausesevereconsequencesduringoocytematurationandembryonicdevelopment(Gorgonietal.,2011;Wormingtonetal.,1996). Inouroverexpressionexperiment,weincreasedthelevelofPABPC1to >6timesitsendogenouslevel(Figure1—figuresupplement1E).WhenconsideringthattheratiobetweenendogenousPABPC1andePABisabout1:3(Wühretal.,2014),theestimatedincreaseinoverallPABPClevelwas >2.5-fold,whichmighthavebeensufficienttosaturatethepoly(A)sites.Despitethispotentialsaturationanddiminishedcouplingbetweenpoly(A)-taillengthandTE,couplingwasnotcompletelylost(Figures1E,2Aand3C–E).Totheextentthatpoly(A)sitesweresaturated,theresidualcouplingsuggeststhatoocytesmighthaveamechanismforcountingboundPABCmoleculesthatenablessomecouplingtopersistevenafterPABPCoverexpressionsaturatesthepoly(A)sites.OnewaytoachievethiscountingwouldbeforacriticalPABPC-interactingfactortoalsobelimitingsuchthatlong-tailedmRNAs,withtheirhighernumberofboundPABPCmoleculeswouldmoreeffectivelycompeteforbindingtothislimitingfactor.AtopcandidateforsuchafactoriseIF4G.Indeed,wheneIF4GwasoverexpressedtogetherwithPABPC1infrogoocytes,thecouplingbetweenpoly(A)-taillengthandTEwasfurtherreduced(Figure1—figuresupplement2C).TheideathatPABPCinteractionswithlimitingeIF4Gmightfavortranslationoflong-tailedmRNAsinoocytesalsohelpstoexplainwhyoverexpressingthePABPC1M161Amutantwasmostdetrimentaltotranslationfromthelong-tailedreporter(Figure1E). AnotherwaythatsomecouplingcouldpersistevenafteroverexpressingsaturatinglevelsofPABPCisthroughsequestrationofsomemRNAsawayfromthetranslationmachinery.Onelikelylocationforsuchsequestrationwouldbegermgranules,whichhavebeenimplicatedinregulatingmRNAtranslationinoocytesofdiverseanimalspecies(Voroninaetal.,2011).Thismechanismmightreinforcecouplingbetweenpoly(A)-taillengthandTE,perhapsbyselectivelysequesteringshort-tailedmRNAsfromtheactivetranslationpool. Thesecondconditionrequiredforstrongcouplingbetweenpoly(A)-taillengthandTEisthesurvivalofshort-tailedmRNAsunderconditionsinwhichPABPCislimiting(Figure7).Thisconditionwasnotmetinthepost-embryonicmammaliancelllinesweexamined.WhenPABPCwasdepletedintheseuncoupledsystems,manymRNAmolecules,particularlyshort-tailedonesthatpresumablycompetedpoorlyfortheremainingPABPC,weredegraded.ThepreferentiallossofmRNAsnotboundbyPABPCreducedtherangeoftaillengths,whichcorrespondinglyreducedtherangeofTEsthatcouldpotentiallybeimpartedbycouplingbetweentaillengthandTE.Thisreducedrangealsopresumablyreducedtheabilitytodetectcoupling,althoughsomeabilitywasexpectedtoberetained,asindicatedbyananalysisinwhichdatafromacoupledsystemwassampledtomatchthemorerestrictedtail-lengthdistributionofanuncoupledsystem(Subtelnyetal.,2014).Moreimportantly,thelossofmRNAsnotboundbyPABPCreducedthenumberofPABPC-bindingsites,therebyreducingtheextenttowhichthesesiteswereinexcessoverPABPCandthusreducingcouplingbetweentaillengthandTE. Inoocytesandearlyembryos,mRNAdecappingisuncoupledfromdeadenylation(Gillian-Danieletal.,1998),whichhelpsexplainwhyshort-tailedmRNAssurviveinthesesystemsdespiteourfindingthattheyhavelimitingPABPCactivity.Wesuggesttwononexclusivemechanisticexplanationsforthisunusualdecouplingofdecappingfromdeadenylation.First,oocytesandearlyembryoshaverelativelylowexpressionofdecappingenzymes(Maetal.,2013;Peshkinetal.,2019).Second,mRNAterminaluridylationactivityisverylowinfrogoocytes(Figure5—figuresupplement1; Changetal.,2018),anditremainslowthroughoutearlyembryonicdevelopmentandonlystartstoincreasedramaticallyafterzygoticgenomeactivation(Changetal.,2018).Thisdevelopmentaldelayofterminaluridylationmighthelpensurethesurvivalofshort-tailedmRNAswhenPABPCislimitingandtherebyenablestrongcouplingbetweenpoly(A)-taillengthandTEinoocytesandearlyembryos.Laterindevelopment,short-tailedmRNAsareprotectedfromterminaluridylationbysaturatingPABPC,whichhelpsexplainwhydeletionofTUT4andTUT7inmousesomaticcellshaslittleimpactonmRNAabundance(Morganetal.,2017). ThethirdconditionrequiredforcouplingbetweentaillengthandTEisthatPABPCmusthavetheabilitytoinfluenceTEofboundmRNAs(Figure7).Inthecoupledsystemoffrogoocytes,increasingPABPClevelssubstantiallyimprovedTEofnearlyallmRNAs(Figure2B–E).Incontrast,inuncoupledsystemssuchasHeLaandHCT116cells,severedepletionofPABPC,suchthatshort-andmedium-tailedmRNAsweremarkedlydestabilized,hadnoconsistentimpactonmRNATE(Figure6BandF).WesuspectthatthedifferentialeffectofPABPConTEobservedincoupledanduncoupledsystemsisrelatedtothedivergentlevelsofbasaltranslationinitiationobservedbetweenthesesystems.Indeed,theoveralltranslationmeasuredbypolysomeprofilesinoocytesandearlyembryosismuchlowerthanthatobservedineitherlaterdevelopmentalstages(Woodland,1974)orpost-embryonicmammaliancelllines(Figure3—figuresupplement1A,Figure6—figuresupplement1B),whichprovidestheopportunityforatranslation-activatingeffectofPABPCtobemoreprominentinthecoupledsystems. OurresultsshowingthatPABPCs,whileplayingacrucialroleinprotectingmRNAfromprematuredecay,haveminimalcontributiontotranslationinpost-embryonicmammaliancelllinesmightseemtocontradictthewell-acceptedfunctionofPABPCasatranslationalactivator.However,manypreviousstudiesthatestablishedtheroleforPABPCinpromotingtranslationwerecarriedoutinfrogoocytesorearlyembryonicsystems(Smithetal.,2014),wherewefoundPABPCtogloballyenhanceTE.Otherpreviousstudieswereconductedinvitro,withmixedresults:inreconstitutedsystems,PABPCisdispensablefortranslationinitiation(Mitchelletal.,2010),andinrabbitreticulocytelysates,PABPChasaminimaleffectontranslation(Hintonetal.,2007),whereasinsomeothercellextracts,PABPCactivatestranslation(Kahvejianetal.,2005;TarunandSachs,1995).Additionalexperimentswillberequiredtodeterminewhetherthisdiscrepancybetweenresultsweobtainedfromlivingcellsandthoseobtainedinsomepost-embryoniccellextractsareattributabletodifferencesbetweencelltypesortodifferencesbetweencellularcytoplasmandinvitroextracts—perhapsimpartedbydilutionoftranslationalcomponentsinextracts.Suchstudieswillneedtodifferentiatebetweenthetranslation-activationandmRNA-stabilizationactivitiesofPABPC.Inthemeantime,itishelpfultoknowthatPABPCstabilizesmRNAsofpost-embryonicmetazoancellsandthatthetwoactivitiesofPABPCcanbecontextdependent,suchthatinfrogoocytesPABPCstronglyactivatestranslationandhasnoeffectonmRNAstability,whereasinmammaliancelllinesitstabilizesmRNAsandhasnodetectableeffectonTE. ThedualpotentialofPABPCinstabilizingmRNAandpromotingtranslationbestowsPABPCwithdistinctandcontext-dependentrolesinregulatingproteinsynthesis.Inoocytesandearlyembryos,thelackofmRNAtranscriptionanddegradationleavesdifferentialTEastheprimaryoptionformodulatingproteinsynthesis.OurworkindicatesthatinthiscontextlimitingPABPCproteinsbindprimarilytomRNAswithlongerpoly(A)tailsandactivatetheirtranslation.Incontrast,inpost-embryonicmammaliancelllines,wheretranscription,mRNAdegradation,andtranslationareeachoperatingathighefficiency,PABPCsprotectmRNAsfrompre-maturedecay,enablingthemtocontributetheproperamountofproteinduringtheirlifetimes,butwithoutanyadditionalenhancementofTE.Thesecontext-dependentactivitiesarenotonlycrucialforunderstandinghowcouplingbetweentaillengthandTEisestablishedinoocytesandearlyembryosandwhyitislostlaterindevelopment,theyalsoprovidemechanisticinsightintotheeffectsofthemanyposttranscriptionalregulatoryphenomenathatalterpoly(A)-taillengths.Forexample,duringmiRNA-mediatedrepression,theArgonaute–miRNAcomplexbindstargetmRNAsandrecruitsfactorsthatdisplacePABPCfromthepoly(A)tailandacceleratetailshortening(Fabianetal.,2009;Giraldezetal.,2005;Morettietal.,2012;Risslandetal.,2017;Wuetal.,2006).Inearlyzebrafishembryos,theseeffectsareexpectedtodisadvantagetargetmRNAsatcompetingforlimitedPABPC,whichinthiscontextwouldreducetheirTEwithoutchangingtheirstability,therebyexplainingwhymiRNAsprimarilycausetranslationalrepressionintheseearlyembryos(Bazzinietal.,2012;Subtelnyetal.,2014).However,inlaterembryonicdevelopmentaswellasinpost-embryonicmammaliancells,displacementofPABPCandacceleratedtailshorteningwouldreducePABPCbindingtotargetmRNAs,whichinthiscontextwouldreducetheirstabilitywithoutchangingtheirTE,therebyexplainingwhymiRNAsprimarilycausemRNAdestabilizationinthesecells(Bazzinietal.,2012;Guoetal.,2010;Subtelnyetal.,2014). HowPABPCpromotestranslationremainsanenigma,althoughtheclosed-loopmodelhasofferedasoundmechanisticexplanation.TheinteractionbetweeneIF4GandPABPCiswellcharacterizedandprovidesaphysicallinkconnectingbothendsofthemRNA,andsomestudiesareabletocatchaglimpseofpossiblecircularstructuresofmRNAsinfixedtissues(Christensenetal.,1987)orinvitro(Wellsetal.,1998).However,recentsingle-moleculeimagingstudiesinmammaliancellsquestionthewidespreadexistenceofmRNAclosed-loopstructures(Adivarahanetal.,2018;Kochetal.,2020).OurfindingthatdepletionofPABPChadminimalimpactonTEinmammaliancelllinessupportsamodelinwhichpervasiveeIF4G–PABPC-associatedloopingofmRNAsisgenerallylackinginuncoupledsystems.ThisideaisconsistentwiththefindingthattheinteractionbetweeneIF4GandPABPCisdispensableinyeast(E.-H.Parketal.,2011)andHEK293cells(Adivarahanetal.,2018),bothofwhichareuncoupledsystems(Subtelnyetal.,2014).Incontrast,theeIF4G–PABPCinteractioniscriticalduringfrogoocytematuration(Wakiyamaetal.,2000),aprocessthatreliesoncouplingbetweenpoly(A)-taillengthandTE(RichterandLasko,2011),andourresultsshowedthatincreasingPABPChadaglobaleffectonupregulatingTEinfrogoocytes.Moreover,translationofalong-tailedreporterwassubstantiallyrepressedwhenthePABPCM161Amutantwasoverexpressedinfrogoocytes(Figure1E).Thus,theclosed-loopstructure,ifitexists,mightbemoreprevalentincoupledsystems,suchasoocytesorearlyembryos. OurexperimentsexaminedonlyafewsystemsthatinherentlypossessedeitherallornoneofthethreemolecularconditionsthatwefoundtoberequiredforstrongcouplingbetweentaillengthandTE,that is,limitingPABPCactivity,stabilizationofmRNAslackingaboundPABPC,andPABPC-sensitivetranslation.Whethertheseconditionsarebroadlyapplicabletomanyothersystemsremainstobeinvestigated.Forexample,somecellsmightfallinbetweenthetwoextremes,processingoneortwooftheseconditionsbutnotallthree.Theresultsofourexperimentsinwhichweremovedoneoftheconditionsfromfrogoocytesorimposedoneortwooftheconditionsinpost-embryonicmammaliancells,predictthatsuchcellsthatinherentlyfallbetweenthetwoextremeshaveminimalifanycoupling.Indeed,theconceptthatmultipleconditionsmustbemetbeforestrongcouplingcanbeestablishedhelpstoexplainwhycouplingbetweentaillengthandTEhasbeensoinfrequentlydetectedoutsidethegeneregulatoryregimeoperatinginoocytesandearlyembryos(Subtelnyetal.,2014). Keyresourcestable Reagenttype(species)orresourceDesignationSourceorreferenceIdentifiersAdditionalinformationCellline(human)HCT116PABPC1-AID(sC152-C16)ThisstudysC152-C16SeeMaterials and methodsfordetailsCellline(human)HCT116PABPC1-AID(sC278-C2)ThisstudysC278-C2SeeMaterials and methodsfordetailsCellline(human)HCT116OsTIRNatsumeetal.,2016Cellline(human)HeLaNametal.,2014Cellline(human)HeLaRPL3-3xHA(sC262-4)ThisstudysC262-4SeeMaterials and methodsfordetailsCellline(mouse)NIH3T3Eisenetal.,2020Cellline(zebrafish)ZF4ATCC(CRL-2050)AntibodyAnti-β-actin(Rabbitmonoclonal)CellSignallingTechnology(D6A8)1:1000dilutionAntibodyAnti-AID(Mousemonoclonal)MBLInternational(M214-3)1:1000dilutionAntibodyAnti-ePAB(Rabbitpolyclonal)Wilkieetal.,20051:2000dilutionAntibodyAnti-GFP(Mousemonoclonal)CellSignallingTechnology(2955)1:1000dilutionAntibodyAnti-FLAG(Mousemonoclonal)MilliporeSigma(F9291)1:1000dilutionAntibodyAnti-GAPDH(Mousemonoclonal)Proteintech(60004)1:1000dilutionAntibodyAnti-HA(Mousemonoclonal)CellSignallingTechnology(6E2)1:1000dilutionAntibodyAnti-PABPC1(Rabbitpolyclonal)CellSignallingTechnology(4992S)1:1000dilutionAntibodyAnti-PABPC4(Rabbitpolyclonal)NovusBiologicals(NB100-74594)1:1000dilutionAntibodyAnti-puromycin(Mousemonoclonal)MilliporeSigma(MABE343)1:1000dilutionAntibodyAnti-RPL3(Rabbitpolyclonal)Abcam(ab154882)1:1000dilutionAntibodyAnti-RPS15(Rabbitpolyclonal)Proteintech(14957–1-AP)1:1000dilutionAntibodyAnti-RPS3(Rabbitmonoclonal)CellSignallingTechnology(D50G7)1:1000dilutionAntibodyAnti-TUT4(Rabbitpolyclonal)ABclonal(A5972)1:1000dilutionAntibodyAnti-TUT7(Rabbitpolyclonal)Proteintech(25196–1-AP)1:1000dilutionAntibodyAnti-Vinculin(Mousemonoclonal)Proteintech(66305)1:1000dilutionAntibodyIRDye800CWGoatanti-RabbitIgG(H + L)LI-COR(926–32211)1:10,000dilutionAntibodyIRDye680RDGoatanti-MouseIgGLI-COR(926–68070)1:10,000dilutionSequencebasedreagentAlloligosSupplementaryfile1RecombinantDNAreagentAllplasmidsSupplementaryfile2OtherReferencesequencesSupplementaryfile3OtherMaskedmitochondrialpseudogenesSupplementaryfile4OthermRNA3′-endannotationsSupplementaryfile5 AllDNAplasmidswereassembledbyrestriction-freecloningunlessexplainedotherwise(Ungeretal.,2010).Site-directedmutagenesiswasalsocarriedoutwiththismethod.Forplasmidsusedformammaliancelltransfection,humanPABPC1(fromHeLacellcDNA)andX.laevisePAB(fromoocytecDNA)codingsequenceswereclonedintopcDNA5/FRT/TO(ThermoFisher,V652020).ForsiRNA-resistanthumanPABPC1,silentmutationswereintroducedatD107,K108,S109,I110,D111,N112,V131,C132,D249,E250,N252,andG253.AdditionalsubstitutionsweremadeatI110L,D111E,D117E,A121G,G139A,Y140F,T147S,andR166KtodisrupttheinteractionbetweenPABPC1andeIF4G(Chorghadeetal.,2017).ForsiRNA-resistantX.laevisePAB,silentmutationswereintroducedatC128,K129,V130,V131,T249,E250,andN252.SequencesofoligosusedformutagenesisareprovidedinSupplementaryfile1.AlistofplasmidsusedinthisstudyisprovidedinSupplementaryfile2.PlasmidsandtheirsequenceinformationwillbeavailableatAddgene. PlasmidsforpreparingDNAtemplatesforinvitrotranscriptionwereassembledusingthepGEM-11Zf(+)(Promega)backbone,insertingtheappropriatesequencesegmentsaftertheT7promoter.HDVribozymesequencewasobtainedfromtheplasmidp2RZ(Avisetal.,2012).X.laevisPABPC1(pabpc1.S),ePAB(pabpc1l.L),andRPL3(rpl3.L)codingsequenceswereamplifiedfromcDNAgeneratedfromX.laevisoocytes.Renilla(Rluc)andfireflyluciferase(Fluc)codingsequenceswereobtainedfrompIS2andpIS0,respectively(Farhetal.,2005).NanoLuc(Nluc)codingsequencewasobtainedfrompNL1.1.TK(Promega).X.laevisβ-globin5′-and3′-UTRsequenceswereobtainedfrompT7TS(Addgene#17091).MouseMalat13′sequencewasobtainedfromtheComp.25mutantplasmid(Wiluszetal.,2012).RlucandFlucreporterscontained5′-and3′-UTRsequencesinheritedfromthepGEM-11Zf(+)backbone,whereasNlucreportershadtheX.laevisβ-globin5′-and3′-UTRsequences.Fragmentscontainingvariablepoly(A)lengthswereputindesiredplasmidsafterallotherDNAfragmentswereassembled,alsousingrestriction-freecloning,exceptthatC3040Hcompetentcells(NEB)wereusedtoamplifytheassembledplasmids.BecauselonghomopolymerstendtobecomeshorterorgetlostwhenplasmidsarepropagatedinE. coli,individualcloneswereselectedandcheckedbyPCRandSangersequencingtoconfirmthedesiredlengthofeachpoly(A)region.TheseplasmidpreparationswerethenusedtogeneratetemplatesforinvitrotranscriptionwithoutfurtherpropagationinE. coli. ForgeneratingDNAtemplatesforinvitrotranscription,PCRreactionswerecarriedoutusingKAPAHiFiHotStartPCRKit,withacommon5′primerupstreamoftheT7promoteranddifferent3′primers.FormakingRNAsendingindefinedlengthsofpoly(A)sequence,aprimer300 ~ 600ntdownstreamoftheHDVcleavagesitewasusedtofacilitateseparatingthe5′cleavageproductfromthe3′cleavageproductandtheuncleavedtranscript.FormakingRNAsnotendingindefinedlengthsofpoly(A)sequence,aprimerpairingtotheendofthedesired3′UTRsequencewasused.AllDNAtemplatesforinvitrotranscriptionwerepurifiedonagarosegelsusingtheQIAquickGelExtractionKit(QIAGEN).SequencesofoligosusedforgeneratingDNAtemplatesareprovidedinSupplementaryfile1. InvitrotranscriptionwascarriedoutwithT7RNApolymerasepurifiedinhouseandusedat3.2ng/µlfinalconcentrationinastandard50µlreactioncontaining40mMTrispH8.0,21mMMgCl2,2mMSpermidine(Sigma),1mMdithiothreitol(DTT,GoldBio),5mMNTP(bufferedATP,UTP,CTP,GTPmix,ThermoFisher),0.1unitsyeastinorganicpyrophosphatase(NEB),40unitsSUPERase•In(ThermoFisher),and1µgDNAtemplate.Afterincubationat37°Cfor2hr,2unitsofTURBODNase(ThermoFisher)wereadded,followedbyanother20minincubationat37°C.ForconstructswiththeHDVribozyme,thermalcyclingwasperformedtoenhanceHDVcleavage(65°Cfor90sand37°Cfor5min,threecycles,50µlofreactionpertube).Beforegelloading,2µl0.5MEDTApH8.0and50µl2xRNAGelLoadingDye(ThermoFisher,R0641)wereaddedtoallinvitrotranscriptionreactions,regardlessofwhethertheHDVcleavagestepwasperformedornot.Afterincubationat65°Cfor5min,RNAswereseparatedon5%acrylamidedenaturinggels(NationalDiagnostics,EC-829).DesiredRNAbandswereidentifiedbyUV-shadowing,excised,maceratedandelutedin300mMNaClat23°Covernightonarotator.ThegelpieceswereremovedusingSpin-Xcolumns(Corning8160),andRNAswereprecipitatedwithisopropanolandresuspendedinwaterfordownstreamreactions. CappingofRNAswascarriedoutwiththeVacciniaCappingSystem(NEB,M2080S)followingthemanufacturer’sprotocol.RNAswerethenpurifiedbyphenol/chloroformextractionandethanolprecipitation.Water-resuspendedRNAswereappliedtoMicroBio-Spincolumns(Bio-Rad,7326250)fordesalting.AllRNAswerecheckedforintegritybyvisualizingonformaldehyde-agarosedenaturinggelsbeforebeingstoredat–80°C. X.laevisovarieswereobtainedfromNasco(LM00935).Ovarieswerebrokendownto2–3cmpieceswithtweezersandincubatedincalcium-omittedOR-2buffer(82.5mMNaCl,2.5mMKCl,1mMMgCl2,1mMNa2HPO4,5mMHEPES,pH7.5)with0.2%(w/v)collagenaseA(Roche,11088793001)onarockerat23°Cfor ~3hr,atwhichpointmostoocytesweredefolliculated.Theoocyteswerethenwashedextensivelywithcalcium-omittedOR-2bufferfollowedbycompleteOR-2buffer(with1mMCaCl2).StageVandVIoocyteswereseparatedfromtherestwitha ~ 0.8mmdiametermeshsieve.Forinjectionexperiments,healthystageVIoocyteswerehand-pickedandincubatedincompleteOR-2bufferat18°Covernight(>16hr)forrecoverybeforeinjection. Whenpreparingoocyteextracts,thebulkstageVandVIoocytes(500–1,000)werewashedthreetimeswithampleoocyteextractionbuffer(10mMHEPESpH7.5,10mMsodiumacetate,1mMmagnesiumacetateand2mMDTT).Afterremovingallexcessbuffer,oocyteswerecentrifugedat20,000gfor20min.Themiddlelayercontainingtheextractwascollectedwithapipetteandtransferredtoanewtube.Thecollectedextractwascentrifugedat20,000gfor10min,andthemiddlelayerwasagaincollected,avoidingthetoplipidlayerandthebottominsolublelayerasmuchaspossible.Theextractwaspassedthrougha0.45µmfilter,thenaliquotedandstoredat–80°C.TheconcentrationoftheoocyteextractwasmeasuredusingtheBradfordassay(ThermoFisher,23236)andwasusuallyat35–45mg/ml. Five hundredµlrabbitreticulocytelysateobtainedfromPromega(L4151,untreated)wassupplementedwith1µl12.5mMHemin(Sigma,51280),25µl1MHEPESpH7.5,and2.5µl10mg/mlyeasttRNA(Sigma,10109495001).Invitrotranslationreactionswerecarriedoutwith5 µlofeithersupplementedrabbitreticulocytelysateoroocyteextractintotalvolumeof20µlcontaining10µMaminoacidmix(Promega,L4461),0.5unitsSUPERase•In,10mMcreatinephosphate(Roche10621714001),200µg/mlcreatinekinase(Roche,10127566001),20mMHEPESpH7.5,75mMpotassiumacetate,1.5mMmagnesiumacetate,and1fmol/µlRlucorNlucreporterRNAwithindicatedpoly(A)tails.WhenusingRlucreporters,2.5fmol/µlFlucRNAwitha120ntpoly(A)regionfollowedbyamutantmouseMalat13′endwasincludedineachreactionforuseasanormalizationcontrol.WhenusingNlucreporters,2.5fmol/µlFlucRNAwithahistonemRNA3′-endstem-loopwasincludedineachreactionforuseasanormalizationcontrol.Whenexaminingtheeffectsofaddingpurifiedproteins,thereactionalsoincluded2µloftheindicatedamountofeitherX.laevisPABPC1,eGFP,orbufferG(20mMTrispH7.5,150mMNaCl,5%glycerol,5mMDTT). X.laevisPABPC1(pabpc1.S)codingsequencewasamplifiedfromcDNAgeneratedfromX.laevisoocytes.eGFPwasobtainedfromtheplasmidpCS2+-eGFP(Chenetal.,2017).AllcodingsequenceswereclonedintopET28a(Novagen)vectorbyrestriction-freecloning.TherecombinantproteincarriedahexahistidinetagatitsCterminus.TheplasmidwastransformedintoE.coliBL21(DE3)Starcells.Aftergrowingcellsat37°Ctoanopticaldensity(OD600)of0.6,expressionofrecombinantproteinwasinducedwith0.5mMisopropylβ-D-thiogalactoside(GoldBio).ForpurificationofPABPC1,thecellscontinuedtogrowat18°Cfor16hr,afterwhichtheywerecollectedbycentrifugation,resuspendedin10volumesoflysisbuffer(20mMTrispH7.5,2MNaCl,5%(v/v)glyceroland5mM2-mercaptoethanol)andlysedbysonication.Lysatewasclearedbycentrifugationat25,000gfor30minandincubatedwithNi-NTAagarose(Qiagen,30210)at4°Cfor1hr(0.5mlresinper50mlsupernatant).Theresinwaswashedwith20resinvolumesofbufferH(20mMTrispH7.5,2MNaCl,5%glycerol,5mM2-mercaptoethanoland10mMimidazolepH7.5)andthenwith20resinvolumesofbufferL(20mMTrispH7.5,150mMNaCl,5%glycerol,5mM2-mercaptoethanoland20mMimidazolepH7.5).ProteinswereelutedwithbufferE(20mMTrispH7.5,150mMNaCl,5%glycerol,5mM2-mercaptoethanoland200mMimidazolepH7.5).Theeluatewasdiluted1:9withbufferCL(20mMTrispH7.5,25mMNaCl,5%glycerol,5mMDTT)andloadedontoaMonoScolumn(GEHealthcare).BoundproteinswereelutedbylinearNaClgradientwithbufferCLandbufferCH(20mMTrispH7.5,500mMNaCl,5%glycerol,5mMDTT).Fractionsfromthedesiredpeakwerepooled,concentratedwithanAmiconfilterunit(Millipore,UFC805024)andappliedtoaSuperdex200column(GEHealthcare)inbufferG(20mMTrispH7.5,150mMNaCl,5%glycerol,5mMDTT).Fractionsfromthedesiredpeakwerepooled,concentratedwithanAmiconfilterunit,flash-frozeninliquidnitrogenandstoredat−80°C.eGFPproteinwaspurifiedsimilarly,exceptallbuffershad150mMNaCl,andthecation-exchangestepwasomitted. HealthyX.laevisstageVIoocytesthathadrecoveredfromdefolliculationwereselectedforinjection.WhenexaminingtheeffectsofexpressingadditionalPABPC,eitherwaterortheindicatedamountofcappedmRNA(0.25–2pmol/µl)codingforeithereGFP,frogPABPC1,frogPABPC1(M161A),frogePAB,orhumaneIF4Gwasinjectedintooocytesinavolumeof2–8nlperoocyte(PLI-100PlusPico-Injector,HarvardApparatus)at23°C.InjectedoocyteswereincubatedincompleteOR-2bufferat23°Cfor20hr,afterwhichtheywereeitherlysedformakingsequencinglibrariesorco-injectedwithanFlucreportermRNAwithahistonemRNA3′-endstem-loop(20fmol/µl,usedfornormalization),andeitheranRlucreportermRNAwithindicatedpoly(A)-taillength(10fmol/µl)oranNlucreportermRNAwithindicatedpoly(A)-taillength(10fmol/µl)inatotalvolumeof2nlperoocyte,exceptinFigure1F,whereonly2nlNlucreportermRNAs(10fmol/µl)withindicated3′endswereinjected.TheseoocyteswereincubatedincompleteOR-2bufferat23°Cfor6hrbeforelysisfordualluciferaseassays. ForPAL-TRAP,oocyteswereinjectedwithmRNA(1pmol/µl)codingforeitherX.laevisRPL3-HAoreGFP-HAatavolumeof4nlperoocyte.AfterincubationincompleteOR-2bufferat23°Cfor1day,theseoocyteswereinjectedagainwithamixtureofmRNAcodingforeithereGFPorePAB(1pmol/µl)andapopulationofRlucreportermRNAswithdifferentpoly(A)-taillengths(0,30,63,98,and120nt,equimolarratio;combinedconcentration,166.5fmol/µl)inavolumeof4nlperoocyte.AfterincubationincompleteOR-2bufferat23°Cforanotherday,oocyteswerecollectedandlysedforPAL-TRAPanalysis. Five to 10oocyteswerelysedbyvigorousshakingandpipettinginPassiveLysisBuffer(Promega,E1980),using20µlperoocyte.Lysateswereclearedbycentrifugationat5000gfor5minat4°C,afterwhich20µlofeachsupernatantwastransferredtoa96-wellmicroplateandequilibratedtoroomtemperature.LuciferaseassayswerecarriedoutwithDual-LuciferaseReporterAssaySystem(Promega,E1980)inaVeritasMicroplateLuminometeraccordingtothemanufacturer’sprotocol,regardlessofwhetherNlucorRlucwasusedasthereporter. TotalRNA(1–10µg)wasseparatedonanagarose-formaldehydegel(MansourandPestov,2013)andtransferredtoanylonmembraneusingtheWhatmanNytranSuPerCharge(SPC)TurboBlottersystem(Sigma,WHA10416300).Afterovernighttransfer,RNAwascrosslinkedtothemembraneusingaUVStratalinker2400atwavelength254nmforatotalof1200µJ.ForprobingRlucand18SRNAs,membraneswerepre-incubatedwithULTRAhybUltrasensitiveHybridizationBuffer(ThermoFisher,AM8670)at68°Cunderrotationfor1hrandthenhybridizedunderthesameconditionsovernightwithDNAprobes,whichwerebody-labeledwith[α-32P]dCTP(PerkinElmer)usingaRandomPrimerDNALabelingKit(TaKaRa,6045)accordingtothemanufacturer’sprotocol.ThetemplatesforthelabelingreactionswereDNAfragmentsgeneratedbyPCRwithgene-specificprimers.Afterprobehybridization,membraneswerewashedtwotimes(5mineach)withalow-stringencybuffer(2XSSCand0.1%SDS)at68°Cwithrotation,andtwotimes(15mineach)withhigh-stringencybuffer(0.1XSSCand0.1%SDS)at68°Cwithrotation.ForprobingendogenousmRNAs,membraneswerehybridizedtoradiolabeledgene-specificDNAoligonucleotideprobesinULTRAhyb-Oligobuffer(ThermoFisher,AM8663)overnightat42°C.TheDNAprobeswerelabeledwithT4PNK(NEB,M0201S)and[γ-32P]ATP(PerkinElmer).Followinghybridization,membraneswerewashedthreetimes(20mineach)withawashbuffer(2XSSCand0.5%SDS)at42°Cwithrotation.TheblotswerethenanalyzedusingaTyphoonFLA7000phosphor-imager(GEHealthcareLifeSciences).BeforeprobingforasecondmRNAonthesameblot,theblotswerestrippedthreetimes(20mineach)inaboilingstrippingbuffer(0.04%SDS)withgentleshakingandthencheckedforanyresidualradioactivitybyextendedphosphorimaging.SequencesofoligosusedfornorthernblotsareprovidedinSupplementaryfile1. TotalRNA(1–10µg)wasmixedwithaDNAoligo(100pmol,Supplementaryfile1)complimentarytoasegmentofthe3′-UTRinwaterinavolumeof15µl.Insomeexperiments,twoDNAoligos,eachcomplementarytoadifferentmRNA,wereaddedtogetherinonereaction.AfterincubatingtheRNA–DNAmixat85°Cfor5min,thetemperaturewasgraduallyloweredto42°C(0.1°Cpers),andthen2µl10xHybridaseBuffer(500mMHEPESpH7.5,1MNaCl,100mMMgCl2),0.5µlHybridase(Lucigen,H39500,fiveunits/µl)and2.5µlwaterwereaddedtothemix.Afterincubationat42°Cfor20min,nucleicacidswereextractedwithphenol/chloroform,precipitatedwithethanol,resuspendedin1xRNAGelLoadingDye(ThermoFisher,R0641),andresolvedonan8%acrylamidedenaturinggel(NationalDiagnostics,EC-829).RNAwastransferredontoHybond-NXmembranes(GEHealthcare,RPN303T)usingaTrans-BlotSDSemi-DryTransferCell(Bio-Rad).Membraneswereincubatedat60°Cfor1hrwithEDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride),ThermoFisher,22981dilutedin0.17M1-methylimidazolepH8.0tochemicallycrosslink5′phosphatestothemembrane.BlotswerehybridizedtoradiolabeledDNAoligonucleotideprobesinULTRAhyb-Oligobuffer(ThermoFisher,AM8663)overnightat42°C.TheDNAprobeswerecomplimentarytothe3′-UTRregionsimmediatelyadjacenttothepoly(A)tailsandwerelabeledwithT4PNK(NEB,M0201S)and[γ-32P]ATP(PerkinElmer).Followinghybridization,membraneswerewashedthreetimes(20mineach)withawashbuffer(2XSSCand0.5%SDS)at42°Cwithrotation.TheblotswerethenanalyzedusingaTyphoonFLA7000phosphor-imager.BeforeprobingforasecondRNAonthesameblot,theblotswerestrippedasdescribedforconventionalnorthernblots.SequencesofoligosusedforRNaseHnorthernblotsareprovidedinSupplementaryfile1. SamplesforwesternblotswereboiledinNuPAGELDSSampleBuffer(ThermoFisher,NP0007),resolvedonNuPAGE4–12%Bis-Trisproteingels(ThermoFisher,NP0323BOX),andtransferredto0.2µmPVDFmembranes(ThermoFisher,LC2002)withaMiniGelTank(ThermoFisher,A25977),accordingtothemanufacturer’sprotocol.Membraneswerethenblockedwith5%(w/v)non-fatdrymilkinTBSbuffer(20mMTrispH7.5,150mMNaCl)for30min.Primaryantibodiesweredilutedat1:1000inTBSTbuffer(20mMTrispH7.5,150mMNaCl,0.1%(v/v)Tween20)andincubatedwiththeblotat4°Covernight.Secondaryantibodiesweredilutedat1:10,000inTBSTbufferandincubatedwiththeblotat23°Cfor1hr.BlotswereanalyzedusinganOdysseyClxmachine(LI-COR)andtheImageStudiosoftware(LI-COR).Fortotalproteindetection,Revert700TotalProteinStainKits(LI-COR,926–11010)wereusedbeforeincubationwithprimaryantibodies,accordingtothemanufacturer’sprotocol.TotalproteinlevelswerequantifiedwiththeImageQuantTLsoftware(GEHealthcareLifeSciences).IndividualproteinlevelswerequantifiedwiththeImageStudiosoftware(LI-COR,v5.2.5). Groupsoffiveoocyteswereincubatedwith0.5mCi/mlEasyTagEXPRESS35Sproteinlabelingmix(PerkinElmer,NEG772007MC)in100µlcompleteOR-2bufferat23°Cfor1hr.Foracontrol,agroupofoocyteswaspretreatedwith100µg/mlcycloheximidein100µlcompleteOR-2bufferfor5minbeforeaddingthelabelingmix.Afterlabeling,bufferswereremovedandoocyteswerewashedwith1mlcompleteOR-2buffer.Afterremovingallresidualwashbuffer,100µlicecoldlysisbuffer(20mMHEPESpH7.5,100mMKCl,5mMMgCl2,1%(v/v)TritonX-100,1xHaltproteinaseinhibitorcocktail(ThermoFisher,78429),2mMDTT)wasaddedtoeachgroup,andoocyteswerelysedbyvigorousshakingandpipetting.Lysateswereclearedbycentrifugingat5,000gat4°Cfor5min,andsupernatantswereboiledinNuPAGELDSSampleBufferandresolvedbySDS-PAGE.GelsweredriedandanalyzedusingtheTyphoonFLA7000phosphor-imagerandtheImageQuantTLsoftware(GEHealthcareLifeSciences). Injectedoocytes(~100)werewashedoncewithcompleteOR-2bufferandthreetimeswith1mlice-coldbufferRL(20mMHEPESpH7.5,100mMKCl,5mMMgCl2,1%(v/v)TritonX-100,100µg/mlcycloheximide,20units/mlSUPERase•In,cOmpleteproteaseinhibitorcocktail[Sigma,11836170001,1tabletper10mlbuffer]).Afterremovingallwashbuffer,oocyteswerelysedinbufferRL(10µl/oocyte)byvigorousshakingandpipetting.Lysateswereclearedbycentrifugationat5000gat4°Cfor10min.Aportionofeachsupernatant(5%)wassavedastheinputforwesternanalysisandRNAisolation.Theremainingsupernatantwasincubatedwithanti-HAmagneticbeads(ThermoFisher,88837)using5µlslurryper100µlsupernatant.ApopulationofNlucRNAswithvariedpoly(A)-taillengths(29,63,and139nt,equimolarratio)werespikedinat5ngper100µlsupernatant,andthebeadmixturewasincubatedat4°Cfor1hrwithend-to-endrotation.Beadswerethenimmobilizedusingamagneticstand,andthesupernatantwasremoved,withaportion(5%)savedastheflowthroughforwesternblotandRNAisolation.Beadswerewashedfourtimeswith1mlbufferRLandresuspendedin110µlbufferRL,10µlofwhichwastakenforwesternblotandtheremainderwasusedforRNAisolation.RNAwasisolatedwith10volumesofTriReagent(ThermoFisher,AM9738)accordingtothemanufacturer’sprotocol.OneµgpurifiedRNAwasincubatedat37°Cfor30minwith10unitsofT4PNK(NEB,M0201S)inPNKbuffercontaining20unitsSUPERase•Inina25µlreactiontoremove2′−3′-cyclicphosphatesofreporterRNAs.TwoPAL-seqv3librariesweremadefromeachinputandeluatesample,andaftermonitoringreproducibility,datafromeachpairofreplicatesweremergedduringanalyses,whereasonlyonelibrarywasmadefromtheeachflowthroughsample. Lysatepreparationandcentrifugationwereperformedthesameasforribosomeprofiling,exceptthatnonucleasewasaddedpriortocentrifugation.RNAwaspurifiedusing3volumesofTrizolLS(ThermoFisher,10296010)accordingtomanufacturer’sprotocolandproteinswereextractedbychloroform-methanolprecipitation. Allmammaliancellswereculturedat37°Cwith5%CO2.HeLacellswereobtainedfromtheBartellab(Nametal.,2014)andculturedinDMEM(VWR,45000–304)with10%FBS(TaKaRa,631106).HeLaRPL3-3xHA(sC262-4)cellsandtheirparentalcells,aFlp-InT-RexHeLacelllinethathadanOsTIR1geneobtainedfromAndrewHolland,wereculturedinDMEMwith10%FBS.HCT116OsTIR1cells,obtainedfromtheKanemakilab(Natsumeetal.,2016),andtheirderivativeHCT116PABPC1-AID(sC152-C16)cellswereculturedinMcCoy’s5Amedia(ThermoFisher,16600082)supplementedwith10%FBS,2mML-glutamine(ThermoFisher,25030081).HCT116PABPC1-AID(sC278-C2)cellswereculturedasHCT116OsTIR1cellsbutsupplementedwith600µg/mlG418(ThermoFisher,10131027).NIH3T3cellswereobtainedfromtheBartellab(Eisenetal.,2020)andculturedinDMEMwith10%BCS(Sigma,12133C).ZF4cellswereobtainedfromATCC(CRL-2050)andculturedinDMEMF-12media(ATCC,30–2066)and10%FBS,at28°Cwith5%CO2.Mycoplasmatestingwasperformedandnocontaminationwasobserved. ForsiRNAtransfection,cellswereplatedat2×106cellsper10cmplate,culturedfor12hr,andtransfectedwith15pmoltotalsiRNAsand45µlLipofectamineRNAiMAX(ThermoFisher,13778500)in1mlOpti-MEMmedia(ThermoFisher,31985062)accordingtomanufacturer’sprotocol.Cellsweresplit1:4ontonewplates24hraftertransfectionandthencollectedforanalysis48hr(Figure4—figuresupplement2A-B,Figure4—figuresupplement3,Figures5–6,Figure6—figuresupplement1)or72hr(Figure4,Figure4—figuresupplement1andFigure4—figuresupplement2C–G)aftertransfection.ForrescueofPABPC-knockdown,cellswereplatedat0.6×106cellsperwellin6-wellplates,andtransfectedwith2.5pmoltotalsiRNAsand7.5µlLipofectamineRNAiMAXin250µlOpti-MEMmedia.After24hr,cellsweretransfectedwith1µgDNAand3µlFuGENEHD(Promega,E2311)in150µlOpti-MEMmediaaccordingtomanufacturer’sprotocol,andcollectedforanalysis40hrafterDNAtransfection.ForotherDNAplasmidtransfections,cellswereplatedat0.6×106cellsperwellin6-wellplates,culturedfor24hr,transfectedwith1µgDNAand3µlFuGENEHD(Promega,E2311)in150µlOpti-MEMmediaaccordingtomanufacturer’sprotocol.siRNAswereallpurchasedfromDharmacon,includingsiControl(D-001810-10-05),humansiPABPC1(L-019598-00-0005),humansiPABPC4(L-011528-01-0005),mousesiPabpc1(L-060385-00-0005),humansiTUT4(L-021797-01-0005),andhumansiTUT7(L-026009-00-0005).PlasmidsandtheirsequenceinformationwillbeavailableatAddgene. Assayswereperformedasdescribed(Schmidtetal.,2009).Puromycin(ThermoFisher,A1113802)wasaddedtocellculturemediaatfinalconcentration1µg/ml.Cellswereincubatedat37°Cfor10minbeforeharvest.Foracontrol,cycloheximidewasaddedtothemediaatfinalconcentration100µg/ml5minbeforetheadditionofpuromycin. AIDwasintroducedattheCterminusofPABPC1usingCas9-mediatedgenomeengineeringinHCT116OsTIRcells.The5′and3′homologyarms(~500nt)flankingthestopcodonofPABPC1wereamplifiedfromgenomicDNAofHCT116OsTIR1cellsandclonedintoadonorplasmid(kindlyprovidedbyIainCheeseman).TheAIDcodingsequencefollowedbytheT2ApeptideandmCherry,whosesequenceswereobtainedfrompMK1221(Addgene,#84220),wasinsertedimmediatelybeforethePABPC1stopcodon.ThePAMregiontargetedbyCas9onthedonorplasmidwasmutated.TheCas9guideRNAwasclonedintopX330-BFP,asdescribed(McKinleyandCheeseman,2014).BoththedonorandtheCas9plasmidswereco-transfectedintoHCT116OsTIRcellsat0.5µgeachinasix-wellformat.After72hr,singlecellswithstrongmCherrysignalweresortedwithflowcytometryinto96-wellplates.CloneswereexpandedandgenotypedbyPCRandwesternblot.OnlyclonesthatwerehomozygousforAIDintegrationwereretained.Oneclonalline(sC152-C16)wasusedforresultsshowninFigure5C.SixhraftersiRNAtransfection,1µg/mldoxycyclinewasaddedtoinduceOsTIR1.Afteranother18hr,indole-3-aceticacid(IAA,GoldBio)wasdissolvedinethanolandaddedtocellsataconcentrationof0.5mM.Twenty-fourhrafterIAAaddition,cellswereharvestedforanalysis.SequencesofoligosusedformakingthedonorandtheCas9plasmidsareprovidedinSupplementaryfile1. IAA-induceddepletionofPABPC1inthePABPC1-AIDcellline(sC152-C16)wasrelativelyslowandgenerallyrequiredmorethan12hrfor90%depletion.Toachievefasterdepletiondynamics,morecopiesofOsTIR1wereintroduced.OsTIR1codingsequenceswereobtainedfrompBabePuroosTIR1-9Myc(Addgene#80074)andsubclonedintoPiggyBac-dCas9-Tet1(Liuetal.,2016)(kindlyprovidedbyShawnLiu),replacingthedCas9codingsequence.Thisplasmidwasco-transfectedwithpiggyBacTransposaseexpressionvector(SystemBiosciences,PB210PA-1)intothePABPC1-AIDcellline(sC152-C16).48hraftertransfection,cellswereselectedin600µg/mlG418for10d.SinglecloneswereexpandedandcheckedforOsTIR1expression.TheclonewiththehighestOsTIR1expression(sC278-C2)waspickedforIAA-inducedPABPC1-AIDdegradation.Tominimizebackgrounddegradation,cellswereincubatedwith1µg/mldoxycyclineand0.2mMauxinole(Aobious,AOB8812),aninhibitorofOsTIR1(Yesbolatovaetal.,2019)for6hr,afterwhichfreshmediawith1µg/mldoxycyclineand0.5mMIAAwereadded.Cellswerecollectedafter0.5,1,and3hrofinduction.Forthenon-inducedcondition,freshmediawith1µg/mldoxycyclinebutnoIAAwereadded,andcellswerecollectedafter1hr.PlasmidsusedformakingthePABPC1-AIDcelllinesandtheirsequenceinformationwillbeavailableatAddgene. Topreparelysatefrommammaliancells,cycloheximide(CHX)wasaddedtoeachplateatfinalconcentration100µg/ml.Plateswereimmediatelymovedtoacoldroomandculturemediawereremoved.Cellswerewashedtwicewithice-coldPBSsupplementedwith100µg/mlCHX.Afterthelastwash,1mlbufferRLL(20mMHEPESpH7.5,100mMKCl,5mMMgCl2,1%(v/v)TritonX-100,100µg/mlcycloheximide,500unit/mlRNasinPlus[Promega,N2615],2mMDTT,cOmpleteproteaseinhibitorcocktail[Sigma,11836170001,1tabletper10mlbuffer])wasaddedtoeach15cmplate,andcellswerescrapedoffandincubatedonicefor10min.Theresultinglysateswerepassedthrougha26-gaugeneedlesixtimesandclearedbycentrifugationat1300gat4°Cfor10min.Topreparelysatefromfrogoocytes,injectedoocyteswerewashedoncewithcompleteOR-2bufferandthreetimeswithbufferRLL.Afterremovingallwashbuffer,oocyteswerelysedinbufferRLL(10µl/oocyte)byvigorousshakingandpipetting.Lysateswereclearedbycentrifugationat5000gat4°Cfor10min.Onetenthofeachclearedlysatewasaddedto10volumesofTriReagentfortotal-RNApreparation,followingthemanufacturer’sprotocol.Anothersmallportion(~10µl)wastakenforwesternanalysis.Theremainderwasaliquoted,flashfrozeninliquidnitrogen,andstoredat–80°C. Forribosomeprofiling,RNaseI(ThermoFisher,AM2294)wasaddedtolysate,using30unitsperOD260unitforlysatefrommammaliancellsand10unitsperOD260unitforlysatefromfrogoocytes.Afterincubationat23°Cfor30min,lysateswereloadedontoa10–50%sucrosegradient(20mMHEPESpH7.5,100mMKCl,5mMMgCl2,10–50%(w/v)sucrose,100µg/mlcycloheximide,20units/mlSUPERase•In,2mMDTT),centrifugedinaBeckmanultracentrifugewithSW41Tirotorat36,000rpmfor2hr,andthenfractionedonaBioCompgradientfractionator.Fractionscorrespondingto80Sribosomeswerecollected,exceptinexperimentsthatgenerateddataforFigure6AandB,inwhichfractionscorrespondingto40S,60S,and80Sribosomeswerecollectedsoastoavoidlossofthesmaller-sizedmitochondrialribosomes(Rooijersetal.,2013).CollectedfractionswereconcentratedwithanAmiconfilterunit(Millipore,UFC810024),andribosome-protectedRNAfragmentswerereleasedinabuffercontaining20mMHEPESpH7.5,100mMKCl,5mMMgCl2,2mMEDTA,20units/mlSUPERase•In,2mMDTT.ReleasedRNAfragmentswereincubatedwith0.2mg/mlProteinaseK(ThermoFisher,AM2548)inthepresenceof1%SDSat42°Cfor20min,andthenpurifiedbyphenol/chloroformextractionandisopropanolprecipitation. Sequencinglibrarieswerepreparedasdescribedpreviously(Subtelnyetal.,2014),exceptinexperimentsthatgenerateddataforFigure6AandB,inwhichdifferentsizemarkerswereusedtoisolateRNAfragmentsfromalargersizerange(20–40ntinthefirstgel)soastoavoidlossofmitochondrialribosome-protectedfragments(Rooijersetal.,2013). FormatchedRNA-seq,rRNAswereeitherdepletedwiththeRiboZeroGoldKit(Illumina,Figure2A,Figure2—figuresupplement1A-B,andFigure4—figuresupplement1B),depletedwiththeNEBNextrRNADepletionKit(Human/Mouse/Rat,NEB,E6310L,Figure6andFigure6—figuresupplement1–2),ornotdepleted(Figure2B,EandF,andFigure2—figuresupplement1C).BecausetheRiboZeroGoldKitdepletesmitochondrialmRNAs(Figure2—figuresupplement1C),mitochondrialmRNAswerenotusedfornormalizationofdatageneratedwiththiskit.TotalRNA(withrRNAdepletedornotdepleted)wasfragmentedbyincubatingat95°Cfor20mininRNAFragmentationbuffer(2mMEDTA,12mMNa2CO3,88mMNaHCO3)andthenethanolprecipitated.RNAfragmentsweresize-selectedandsequencedinparallelwithribosome-protectedfragments.Adetailedprotocolforribosomeprofilingisavailableathttp://bartellab.wi.mit.edu/protocols.html. SequencingwasperformedonanIlluminaHiSeq2500,withstandardrunsofeither40or50cycles.ReadsweretrimmedatbothendstoremovedadaptersequencesusingCutadapt(v1.18)(Martin,2011)andthenmappedtotheirrespectivegenomereferenceusingSTAR(v2.4.2a)withtheparameters‘--runModealignReads--outFilterMultimapNmax1--outReadsUnmapped Fastx--outFilterTypeBySJout--outSAMattributesAll--outSAMtypeBAMSortedByCoordinate’.Mappedreadswerecountedforeachgenewithhtseq-count(0.11.0).ForbothribosomeprofilingandRNA-seq,onlyreadsthatuniquelymappedtocodingregionsofannotatedgenes(excludingthefirst15codonsandthelastfivecodons)wereincludedindownstreamanalyses.ForTEanalyses,anexpressioncutoffof30RNA-seqreadswasappliedforeachgene,withnocutoffforribosome-footprintreads.NormalizationsofRNA-seqandribosome-footprintreadswereperformedwithDESeq2(Loveetal.,2014),consideringreadsforallgenespassingthecutoff,exceptwhenabsoluteTEcomparisonsweremadebetweentwosamples—inwhichcase,RNA-seqandribosome-footprintsdatawerenormalizedbyonlyconsideringreadsfrommitochondrialgenesusingsoftwareDESeq2(Loveetal.,2014).WhenonlyrelativeTEswereconsidered,TEsweremanuallycenteredat0. OurimplementationofTAIL-seq(Eisenetal.,2020)resembledthatofmTAIL-seq(Limetal.,2016)inthatitusedsplintligationtoappendthe3′adapter,asinPAL-seqv1(Subtelnyetal.,2014).TotalRNA(1–30µg)wasmixedwithtwosetsoftail-lengthstandards(0.1ngper1µgtotalRNAforeachset)(Subtelnyetal.,2014),andtrace5′-radiolabeledmarkerRNAs(Eisenetal.,2020),whichwereusedtoevaluatetail-lengthmeasurementsand3′ligationefficiency,respectively.TheseRNAswereligatedtoa3′adapterina64µlreactioncontaining1.5µM3′adapter,1.25µM3′splintoligo,0.2mMATP,oneunit/µlRNasinPlus(Promega,N2615),10mMMgCl2,1xT4RNALigasetworeactionbuffer(NEB,M0239S),and0.5units/µlT4RNALigase2(NEB,M0239S),withtheenzymeaddedaftertheothercomponentshadbeenmixedandincubatedat23°Cfor5min.Theligationreactionwasincubatedat18°Cfor18hr.Smallportions(2µl)wereremovedatthestartandendofthereactionforexaminingtheligationefficiency.Afterligation,RNAwasextractedwithphenol/chloroform,precipitatedwithethanol,resuspendedin10µlwater,andmixedwith90µl1xRNASequencingBufferfromtheRNaseT1kit(ThermoFisher,AM2283).Afterincubationat50°Cfor5minandonicefor5min,1µlRNaseT1(oneunit/µl,ThermoFisher,AM2283)wasadded,andthereactionwasincubatedat23°Cfor15min,followedbyphenol/chloroformextractionandprecipitationwiththePrecipitation/InactivationBufferintheRNaseT1kit.RNAwasresuspendedin12µlRNAGelLoadingDyeandRNAfragmentsrangingbetween100and760ntwerepurifiedonan8%acrylamidedenaturinggel,capturedonstreptavidinbeads,5′phosphorylated,andligatedtoanequalmixoffourphased5′adaptersasdescribed(Eisenetal.,2020).cDNAwasgeneratedusingSuperScriptIII(ThermoFisher,18080044)withDNAprimerscontainingbarcodesusedformultiplexing,elutedfrombeadsbybasehydrolysisofRNAandresolvedon6%urea-acrylamidedenaturinggelsasdescribed(Subtelnyetal.,2014),exceptthatcDNAfragmentswithsizesbetween150ntand760ntwereselected.cDNAsweresequenceddirectlyusingaHiSeq2500machineinapaired-end50-by-250runinnormalmodewithav3kitasdescribed(Eisenetal.,2020).Poly(A)-taillengthsshowninFigure2,Figure2—figuresupplement1,Figure4,Figure4—figuresupplement1andFigure4—figuresupplement2C–Gweremeasuredwiththismethod.SequencesofadaptersandotheroligosusedforTAIL-seqareprovidedinSupplementaryfile1. BecauseTAIL-seqappearstorequirean8-laneflowcell(Eisenetal.,2020)andthusrequiresmanysamplesforefficientimplementation,westoppedusingthismethodandswitchedtoPAL-seq.PAL-seqv3differsfromPAL-seqv2(Eisenetal.,2020)intwomajorways:(1)Barcodeswereembeddedinthe3′adapterssothatdifferentsamplescouldbepooledfordownstreamstagesoflibraryconstruction.(2)SequencingwasperformedwithcDNAratherthanPCR-amplifiedcDNA.Eachreactionwassetupinatotalvolumeof20µlbymixingtotalRNA(0.3–3µg)withtwosetsoftail-lengthstandards(0.1ngofeachsetper1µgtotalRNA)(Subtelnyetal.,2014),twosplintDNAoligos(55pmolA-splintand2.75pmolU-splint,whichallowedpolyadenylatedRNAswithaterminaluridinetobemoreefficientlycaptured)andonebarcoded3′adapter(50pmol).Poly(A)-selectedmRNAfromzebrafishZF4cellline(0.1ngperµgtotalRNA)wasalsoaddedtomammalianRNAsamplestoenableadditionalassessmentoftail-lengthmeasurementreproducibility.TheRNA–DNAmixwasincubatedat65°Cfor5min,andthetemperaturewasgraduallyloweredto16°C(0.1°Cpersec)beforeothercomponentwereaddedtoavolumeof30µlcontaining0.2mMATP,oneunit/µlRNasinPlus,10mMMgCl2,1xT4RNALigase2reactionbufferand0.5units/µlT4RNALigase2.Afterincubationat16°Cfor16hr,EDTA(1µl,0.5M,pH8.0)wasaddedtostopligationandsamplestobesequencedtogether(withdifferentbarcodes)werecombined,phenol/chloroformextracted,andprecipitatedwithethanol.LigatedRNAwasresuspendedin20µlwater,mixedwith180µlRNaseT1buffer(20mMsodiumcitratepH5.0,1mMEDTA,7Murea),heatedto50°Cfor5min,andchilledonicefor5min,beforeadding1.6µlRNaseT1(oneunit/µl,ThermoFisher,AM2283).Afterincubatingthereactionat23°Cfor30min,RNAwasextractedwithphenol/chloroformandprecipitatedusingthePrecipitation/InactivationBufferintheRNaseT1kit.Fragmentsbetween150and760ntweregel-purified,selectedonbeads,5′-endphosphorylated,ligatedtoa5′adapter,andusedforcDNAsynthesisinthesamewayasinourimplementationofTAIL-seq,exceptoneofthefourphased5′adapterswasusedandcDNAswithlengthsbetween200and760ntwereselected.SequencingwasperformedsimilarlyasinPAL-seqv2(Eisenetal.,2020)butwithsomedifferences:(1)1fmolcDNAratherthanPCR-amplifiedcDNAwasusedperlane.(2)Asingleprimerwasusedtoobtaintworeads.(3)Afterclustergenerationandsequencing-primerhybridization,andbeforeextensionoftheprimerthroughthepoly(A)-tailregionusingtheKlenowfragment,16darkcycleswereperformedinordertoextendthesequencingprimerpastthebarcodeandconstantregionsofthe3′adaptersaswellaspasttwonucleotidescorrespondingtotheRNA3′termini.(4)40cyclesofstandardsequencing-by-synthesiswereperformedtoyieldthefirstsequencingread(read1).(5)Afterobtainingread1,theflowcellwasstripped,thesamesequencingprimerwasannealed,and260cyclesofstandardsequencing-by-synthesiswereperformedtoreadthebarcode(sixnt,whichrequiredsevencycles),aconstantsegmentofthe3′adapter(eightnt,whichrequiredonlysevencyclesbecauseofanextracycleinthebarcoderegion),andthesequenceoftheRNA,beginningatits3′terminus,whichrevealedwhethertheRNAhadaterminaluridineandprovidedinformationusedtomeasurethelengthofthepoly(A)tail.Poly(A)-taillengthsshowninFigure3,Figure3—figuresupplement1,Figure4—figuresupplement2A–BandFigure5weremeasuredwiththismethod.SequencesofadaptersandotheroligosusedforPAL-Seqv3areprovidedinSupplementaryfile1. WefoundthatinPAL-seqv3,somebarcoded3′adaptersgaverisetomuchlowernumbersofmappedreads,possiblyduetolowligationefficiency.Therefore,wedevelopedthePAL-seqv4protocol,inwhichwemadeseveralchanges.The3′-ligationreactionwasessentiallythesame,exceptthatone3′adapterwasusedforallsamples.Aftertheligation,eachsamplewasprocessedseparatelyratherthanmixed.LigatedRNAwasresuspendedin10µlwaterandfragmentedina100µlratherthan200µlvolume,andfragmentswithsizesbetween130and760ntwerethengelpurified.Forreversetranscription,differentprimerscontainingthemultiplexingbarcodesequencesaswellasaregionwithfiverandomnucleotideswasused.AftercDNAelutionfromthebeads,halfofeachsamplewassavedat–20°Cforfutureuse.cDNAsamplestobesequencedonthesamelaneinaflowcellwerecombined,ethanol-precipitatedandgel-purifiedasinPAL-seqv3,exceptcDNAfragmentswithsizesbetween190ntand800ntwereselected.SequencingclustergenerationwasperformedsimilarlytoPAL-seqv3,using0.3fmolcDNAmixtureforeachlane.Read1startedwith12cyclesofstandardsequencing-by-synthesisthatfirstsequencedthe5-ntrandomregion(usedtocallclusters)andthensequencedthe6-ntbarcoderegion(whichrequiredsevencycles)withacustomprimer.Theflowcellwasstripped,asecondsequencingprimerannealed,andtwodarkcycleswereperformedinordertoextendthisprimerpastthetwonucleotidescorrespondingtotheRNA3′termini.Thecustomextensionoftheprimerthroughthepoly(A)tailregionwithKlenowwasthenperformed,asinPAL-seqv2(Eisenetal.,2020)andv3,followedby40cyclesofstandardsequencing-by-synthesistocompletetheread1,whichwasgeneratedusingtwosequencingprimersandhadatotallengthof52nt(12cyclesbeforeand40cyclesaftertheKlenowreaction).Theflowcellwasthenstripped,andthesecondsequencingprimerwasusedfor255cyclesofstandardsequencing-by-synthesistogenerateread2.Poly(A)taillengthsshowninFigure6EandFigure6—figuresupplement2weremeasuredwiththismethod.SequencesofadaptersandotheroligosusedforPAL-Seqv4areprovidedinSupplementaryfile1. Bothhuman(release25,GRCh38.p7,primaryassembly)andmouse(release10,GRCh38.p4,primaryassembly)genomicsequencesweredownloadedfromtheGENCODEwebsite.Sequencesofmitochondrialpseudogenes(Supplementaryfile4)inhumanandmousegenomesweremaskedtoavoidlosingmitochondrialmRNAreadsduetomulti-mapping.X.laevisgenomicsequencesweredownloadedfromtheXenbasewebsite(v9.1assembly,repeatmasked),andscaffoldswereremovedsothatonlychromosomalsequenceswouldbeconsidered.TheX.laevismitochondrialgenomicsequencewasobtainedfromNCBIwebsite(NC_001573.1)andappendedtotheX.laevisgenome. Bothhuman(release25,GRCh38.p7,mainannotation)andmouse(release10,GRCh38.p4,primaryassembly)geneannotationsweredownloadedfromtheGENCODEwebsite.X.laevisgeneannotations(v9.1assembly,v1.8.3.2primarytranscript)weredownloadedfromtheXenbasewebsite,andonlychromosomalannotationswereused.X.laevismitochondrialgeneannotationswerecuratedbasedontheinformationobtainedfromtheNCBIwebsite(NC_001573.1)andappended.Forallspecies,annotationsforprotein-codinggeneswereextracted,andforeachgene,theisoformwiththelongestopenreadingframe(ORF)wasselectedtorepresentthatgene.IncasesinwhichmultipleisoformshadORFsofthesamelength,theisoformwiththelongesttranscriptwasselectedasthereferenceannotation.ForTAIL-seqandPAL-seqanalyses,thedatabasesweresupplementedwithsequencesandannotationsoftail-lengthstandards,eGFP,Rluc,Fluc,andNluc.ForribosomeprofilingandmatchedRNA-seqanalyses,thedatabasesweresupplementedwithcodingsequencesandannotationsofeGFP,Rluc,Fluc,andNluc.ForRNA-seqanalysesusedtodeterminehalf-lives,thedatabasesweresupplementedwithcodingsequencesandannotationsofAcGFP(Eisenetal.,2020),Rluc,andFluc.AllsupplementedsequencesareprovidedinSupplementaryfile3. EachPAL-seqtagcorrespondingtoanmRNAprovidesthesiteofcleavageandpolyadenylation,whichenabledannotationofmRNA3′-endisoforms.UniquelymappedtagsfromallPAL-seqdatasetsinthesamecelltype(eitherHeLaorfrogoocyte)weremerged.RNA-seqreadsgeneratedforcomparisontoribosomeprofilingweresimilarlymerged.SoftwareHOMER(Heinzetal.,2010)wasusedtocallpeaksbyusingthemergedRNA-seqdataasthebackgroundandthemergedPAL-seqdataasthesignal,withthefollowingparameters‘-stylefactor-oauto-strandseparate-fdr0.001-ntagThreshold50-fragLength40-size40-inputFragLength30-center’.ThepeaksthatintersectedwithannotatedproteinexonswereretainedasuniquemRNA3′ends.Theannotated3′endsareprovidedinSupplementaryfile5. Phasedconstantsequencesatthe5′endandpoly(A)sequencesatthe3′endofreadonewereremovedwithacustomscript(Eisenetal.,2020).ThetrimmedsequencesweremappedtoreferencegenomeswithSTAR(v2.4.2a)withtheparameters‘--runModealignReads--outFilterMultimapNmax1--outReadsUnmappedFastx--outFilterTypeBySJout--outSAMattributesAll--outSAMtypeBAMSortedByCoordinate’.Mappedsequenceswereintersectedwithsequencesofprotein-codinggenesbybedtools(v2.26.0)withtheparameters‘intersect-wa-wb-bed-s’,retainingonlythosesequencesthatwereassignedtoasinglegene.Remainingclusterswerethenfiltered,requiringatleastfivecombinedNandTbasesinthefirst6nucleotidesofread2.Foreachlibrary,1%ofthefilteredreadclusters(butnomorethan50,000andnolessthan5000)wererandomlypickedasthetrainingsetfordeterminingtheHiddenMarkovModel. ForeachIlluminasequencingcluster,averageintensitiesofeachchannelfromposition15–50ofreadonewereusedtonormalizeintensitiesofeachchannelinread2.ThenaT-signalvalueforeachcycleofreadtwoineachclusterwascalculatedbydividingnormalizedintensityfromTchannelbythesumofnormalizedintensitiesfromtheotherthreechannels.IftheT-signalwas0foraspecificcycle,theT-signalvaluesfromneighboringcycles(upto10,minimum5)wereaveragedtoinferthevalueforthatcycle.Ifaclusterhadmorethanfivecycleswitharead2T-signalvalueof0,theclusterwasdiscarded.Afive-statemixedGaussianHiddenMarkovModel(frompythonghmmpackage)wasthenusedtodecodethesequenceofstatesthatoccurredinread2.Itconsistedofaninitiationstate(state0),astrongpoly(A)-tailstate(state1),aweakpoly(A)-tailstate(state2),aweaknon-poly(A)-tailstate(state3)andastrongnon-poly(A)-tailstate(state4).Allreadsstartedinstate0,andallstateswereonlyallowedtogoforward(from0to4).Themodelwasinitializedwiththefollowingtransitionprobabilitymatrix(fromstateinrowtostateincolumn): 0.040.930.020.010.000.000.940.030.020.010.000.000.500.400.100.000.000.000.600.400.000.000.000.001.00 Theemissionmatrixforthemixedpopulationonewasinitializedwith(statesinrow,mean,varianceandpopulationfractionincolumn): 100.001.001.001.501.500.951.501.500.751.501.500.501.501.500.25 Theemissionmatrixforthemixedpopulationtwowasinitializedwith(statesinrow,mean,varianceandpopulationfractionincolumn): 0.001.001.00-1.001.500.05-1.001.500.25-1.001.500.50-1.001.500.75 Aftermodelinitialization,allclustersfromthetrainingsetwereusedtoperformunsupervisedtraining,andthenthetrainedmodelwasusedtodecodethesequenceofstatesforallretainedclusters.Foreachcluster,thepoly(A)taillengthwasdeterminedbysummingthenumberofstatesinstate1and2.Eachclusterassignedtoaspecificgenebyreadonewasconsideredasapoly(A)tag.Whenevaluatingtail-lengthdistributionsofmRNAsfromindividualgenes,onlyresultsfromgeneswithatleast100tagswereconsidered.NotethattheHiSeq2500machineinhigh-throughputmoderaisesthelaserintensitiesafter50cyclesinread2,causingirregularT-signalatthispositionandlower-than-expectedstatetransitionpredictedbytheHiddenMarkovModel.Thisledtoamilddepletionofpoly(A)tagswithtaillengthscalledat50nt,butitdidnotaffectresultsandconclusionsmadefromoveralltail-lengthdistributions. Fornormalizationofpoly(A)tagsamongsamples,DESeq2(Loveetal.,2014)wasusedwithallspike-intail-lengthstandardstoobtainthescalingfactorforeachdataset.Whenanalyzingmediantaillengths,onlygeneswithpoly(A)tagcountsexceedinganindicatedcutoffwereincludedintheanalyses. ForPAL-seqv3,readonesequencesweremappedtoreferencegenomeswithSTAR(v2.4.2a)withtheparameters‘--outFilterMultimapNmax1--outFilterTypeBySJout--outSAMattributesAll--outSAMtypeBAMSortedByCoordinate’.Mappedsequenceswereintersectedwithannotationsforprotein-codinggenesbybedtools(v2.26.0)withtheparameters‘intersect-wa-wb-bed-S’,retainingonlythosetagsforwhichthereadonesequencewasassignedtoasinglegeneortoanmRNAisoform,whenmRNA3′-endannotationswereused.Remainingclusterswerethenfilteredbyfirstremovingthefirst14basesofread2(whichcorrespondedtoaconstantregionandthebarcoderegion)andthenrequiringatleastfivecombinedNandTbaseswithinthenext6nucleotidesofread2.(ThisfilteringstepwasskippedforanalysesofterminaluridylationinFigure5andFigure5—figuresupplement1).Foreachlibrary,1%ofthefilteredreadclusters(butnomorethan50,000andnolessthan5000)wererandomlypickedasthetrainingsetfordeterminingtheHiddenMarkovModel.Thesequenceofpoly(A)stateswasdeterminedsimilarlyasforTAIL-seq,exceptasingleGaussianHiddenMarkovModelwasused,andtheemissionmatrixwasinitializedwith(statesinrow,meanandvarianceincolumn): 100.01.02.00.51.00.5-1.00.5-2.00.5 Fornormalizationofpoly(A)tagsamongsamples,DESeq2(Loveetal.,2014)wasusedwithallspike-intail-lengthstandardstoobtainthescalingfactorforeachdataset.Whenanalyzingmediantaillengths,onlymRNAisoformswithpoly(A)tagcountsexceedinganindicatedcutoffwereincludedintheanalyses.DataanalysesforPAL-seqv4werethesameasforPAL-seqv3,exceptbeforemapping,thefirst12ntofreadoneweretrimmedtoremovetherandomregionandthebarcoderegion,andnonucleotidesofreadtwoweretrimmed.NotethattheHiSeq2500machineinrapid-runmoderaisesthelaserintensitiesafter101cyclesinread2ofaPAL-seqv4run,causingirregularT-signalatthispositionandlower-than-expectedstatetransitionpredictedbytheHiddenMarkovModel.Thisledtoamilddepletionofpoly(A)tagswithtaillengthscalledat101nt,butitdidnotaffectresultsandconclusionsmadefromoveralltail-lengthdistributions. AvariantHeLacellline(sC262-4)wasusedfordatashowninFigure5D–FandFigure5—figuresupplement1.ThisFlp-InT-RexHeLacelllinehadanOsTIR1geneknocked-in.AnRPL3-3xHAgenewasinsertedusingtheFlp-In(ThermoFisher)systemandasingleclonewaspickedafterselectionwith400µg/mlHygromycinB(ThermoFisher10687010)for10days.Wehavenoreasontosuspectthattheuniquefeaturesofthislineaffectedanyresultsshown.DatawereprocessedasinPAL-seqv3,exceptthatmappedreadswereintersectedwithHeLamRNA3′-endannotations(seeAnnotationofmRNA3′-endisoforms).Allpoly(A)tagswithpoly(A)-taillengths ≥ 2ntwereusedwhenexaminingthepresenceofUnucleotidesneartheendsofpoly(A)tails. Forty-eighthraftersiRNAtransfection,HeLacellswereincubatedwithpre-warmedfreshmediawith400µM5-ethynyluridine(5EU,JenaBiosciences)for1,2,4,and8hr.Cellswereremovedfromplatesbytreatmentwithtrypsin,washedoncewithicecoldPBSandlysedwith200µlicecoldbufferRL.Lysateswereclearedbycentrifugingat1300gfor5min.Supernatantsweretransferredto2mlTriReagent,andtotalRNAwaspreparedaccordingtothemanufacturer’sprotocol. Biotinylationof5EU-labeledRNAandpurificationofbiotinylatedRNAwereperformedatdescribed(Eisenetal.,2020).RNA-seqlibrarieswerepreparedfrompurified5EU-labeledRNAandfromtotalRNAwiththeNEXTflexRapidDirectionalmRNA-seqKit(BiooScientific,5138–10).SequencingwasperformedonanIlluminaHiSeq2500withastandard40-cyclerun.SequencingreadsweremappedwithSTAR(v2.4.2a)withparameters‘--runModealignReads--outFilterMultimapNmax1--outReadsUnmappedFastx--outFilterTypeBySJout--outSAMattributesAll--outSAMtypeBAMSortedByCoordinate’.Exon-mappedreadswerecountedforeachgenewithhtseq-count(0.11.0). Relative5EU-labeledmRNAlevelsforeachgeneateachtimepointwereobtainedbynormalizingreadcountsbasedonthecountsfora5EU-containingGFPRNAstandardthathadbeenspikedintoeachsamplepriorto5EUbiotinylation(Eisenetal.,2020).Thesteady-statemRNAlevelsofeachgeneweremeasuredastheaverageofnormalizedreadcountsobtainedfromsequencingtheinputRNAforthe1and8hrtimepoints.Atotalof987geneswithvaluesthatdifferedby >2-foldatthesetwotimepointswereexcludedfromanalysis.ThenlspackageinRwasusedtofitthefollowingequationforeachgene: y=C+log21-e-kx-t0, wherexisthelabellingtimeinhours(using9999hasthelabelingtimeforthesteady-statedatapoint),yislog2valueofthenormalizedreadcount(leveloflabeledRNA),andt0isthetimeoffset.k isthedecayconstant,whichwasusedtodeterminethehalf-lifet1/2using, t1/2=ln⁡2k,andC isacoefficientdeterminedbybothkandthesynthesisrate S,suchthat, C=log2Sk. Tofitt0foreachcondition,thevalueoft0wasvariedfrom0.05to0.7,withanintervalof0.05,andthevaluethatgavethesmallestmeansquare-lossofywhenfittingtodatafromallgeneswasused.Whenfittingtoresultsforeachgene,C wasboundby(0,Inf),andk wasboundby(0,Inf).C wasinitializedwithmax(y),andk wasinitializedwith0.23.Geneswithoutaconvergedfit(27of9644,17of9646,and12of9728inanalysisofthesiControl,siPABPC1,andsiPABPC1&4samples,respectively)wereomittedfromdown-streamanalyses.mRNAhalf-lifevaluesarereportedinFigure4—figuresupplement3—sourcedata1. GraphsweregeneratedandstatisticalanalyseswereperformedusingR(RDevelopmentCoreTeam,2013).Statisticalparametersincludingthevalueofn,statisticaltest,andstatisticalsignificance(pvalue)arereportedinthefiguresortheirlegends.Nostatisticalmethodswereusedtopredeterminesamplesize.Statisticaltestsforcorrelations(betweennonoverlappingdependentgroups)wereperformedbasedonapublishedmethod(SilverandHittner,2004)usingRpackagecocor(DiedenhofenandMusch,2015).Forluciferaseassaysofinjectedoocytes,eachreplicatereferstoagroupof7–10oocytes.For35Slabelingofinjectedoocytes,eachreplicatereferstoagroupof5–8oocytes.Forthepuromycin-basedtranslationassay,eachreplicatereferstoaseparatetransfectionexperiment. RawandprocesseddatafromsequencingweredepositedintheGEOdatabase(GSE166544).TAIL-seqandPAL-seqanalyseswereperformedusingacustomscriptwritteninPython2.7andavailableathttps://github.com/coffeebond/PAL-seq,(copyarchivedatswh:1:rev:b0aa1ba99cc89ce2080069b50cd441a7718b7b03, Xiang,2021a, Xiang,2021b). Addacomment +Openannotations.Thecurrentannotationcountonthispageisbeingcalculated. RawandprocesseddatafromsequencingweredepositedintheGEOdatabase(GSE166544).TAIL-seqandPAL-seqanalyseswereperformedusingacustomscriptwritteninPython2.7andavailableathttps://github.com/coffeebond/PAL-seq(copyarchivedathttps://archive.softwareheritage.org/swh:1:rev:b0aa1ba99cc89ce2080069b50cd441a7718b7b03). 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Oureditorialprocessproducestwooutputs:(i)publicreviewsdesignedtobepostedalongsidethepreprintforthebenefitofreaders;(ii)feedbackonthemanuscriptfortheauthors,includingrequestsforrevisions,shownbelow.Wealsoincludeanacceptancesummarythatexplainswhattheeditorsfoundinterestingorimportantaboutthework. Acceptancesummary: Thismanuscriptaddressesalong-standingquestion,namelyhowdoesthepoly(A)tailinfluencetranslationalefficiency?Itwillthereforebeofbroadinteresttoreadersfrommanyareasofmolecularbiologyincludingthoseinterestedintranslation,mRNAstability,developmentandgeneexpressioningeneral.Theauthorsconvincinglysetoutthreecriteriathatmustbemetforcouplingofpoly(A)taillengthwithtranslation. Decisionletterafterpeerreview: Thankyouforsubmittingyourarticle"Themolecularbasisofcouplingbetweenpoly(A)-taillengthandtranslationalefficiency"forconsiderationbyeLife.Yourarticlehasbeenreviewedby3peerreviewers,includingTimothyWNilsenastheReviewingEditorandReviewer#1,andtheevaluationhasbeenoverseenbyJamesManleyastheSeniorEditor. Thereviewershavediscussedtheirreviewswithoneanother,andtheReviewingEditorhasdraftedthistohelpyoupreparearevisedsubmission. Essentialrevisions: Allthreereviewerswerequitepositiveaboutthework.Neverthelessreviewer3raisedafewconcernsandreviewer2hadsomerelativelyminorsuggestions.Pleaseaddresstheseissuesasthoroughlyaspossible.Itseemslikerevisioninthetextwillbesufficient. Reviewer#1(Recommendationsfortheauthors): ThisisanexcellentmanuscriptinwhichXiangandBarteluseanabundanceofapproachestoprovidecompellingevidencerelevanttothecouplingbetweenpoly(A)-taillengthandtranslationalefficiency.Withoutreiteratingtheresults,thedataareconvincingandthepaperisclearlywritten.Anyconcernsaretootrivialtoarticulate.Publicationoftheworkisrecommendedasis. Reviewer#2(Recommendationsfortheauthors): Figure1–thereissubstantialvariabilityacrossdifferentextracts.Forexample,thedifferenceintranslationofshortvs.longtailreporteris15.2-fold(1B),33.8-fold(1C),and7.4-fold(1E).Whatisthesourceofthisvariability?Also,somepanelshaveerrorbarsandothersdonot.Canerrorbarsbeincludedinallpanels? Figure1Cand1E–HowdoestheoverexpressionlevelofPABPCcomparetoendogenouslevels? Pleasedefine"poly(A)tags"thefirsttimeitismentioned(Figurelegend2?) Figure2c–arethemoreprominentbandseGFPandPABPC1?Ifso,pleaselabelthem. Thereareseveraldifferentsequencingmethodsused(TAIL-seq,PAL-seqv3,PAL-seqv4)–whywasagivenmethodchosen?Itwouldbehelpfultomentionwhichmethodwasusedinthefigurelegends. Reviewer#3(Recommendationsfortheauthors): 1.NoerrorbarsarepresentedinFigure1A-B,whyisthisthecase? 2.TheauthorsconcludethelossofPABPCincelllinesresultsintheselectiveeliminationoftranscriptswithshortpoly(A)tails.Cantheauthorsexcludearoleforpoly(A)tailelongationintheabsenceofPABPC? 3.Theauthorsconcludebroadrulesfromasinglecoupledsystemandcelllineexperiments,wherethedepletionofPABPChasdrasticconsequencestothetranscriptome(~70%reductioninmRNAabundance)andlikelytothecell.Theauthorsshouldacknowledgetheselimitations. 4.Theauthorsfailtointegratepreviousfindingsinthemodelpresentedinfigure7.Morganetal.,2017haveshownthatlossofTUT4andTUT7inseveralcellsystemsandmousetissuesdoesnotimpactthetranscriptome.Theauthorsneedtodiscussthesefindingsandintegratethemintotheirmodel. https://doi.org/10.7554/eLife.66493.sa1 Reviewer#2(Recommendationsfortheauthors): Figure1–thereissubstantialvariabilityacrossdifferentextracts.Forexample,thedifferenceintranslationofshortvs.longtailreporteris15.2-fold(1B),33.8-fold(1C),and7.4-fold(1E).Whatisthesourceofthisvariability?Also,somepanelshaveerrorbarsandothersdonot.Canerrorbarsbeincludedinallpanels? Theexperimentthatproducedthe7.4-folddifferencewasperformedinoocytesratherthaninextracts,whichmighthelptoexplainwhyitsfold-changediffered.Forexperimentsdoneinextracts,onesourceofvariabilitymighthavebeenthedifferentbatchesofoocytelysates.Anothermighthavebeenthedifferentbufferconditionsthatresultedfromprotein-storagebufferbeingaddedtothereactionsthatdifferedby33.8-foldbutnottothosethatdifferedby15.2-fold. Toaddressthequestionofexperimentalvariabilityandlackoferrorbars,thesethreeexperimentswererepeatedwithreplicates.Whenrepeatingtheseexperiments,weusedNluc/RlucreportersandanFlucnormalizationcontrolthathadahistonemRNA3′-endstem-loop.Thenewresultsresembledtheoriginalonesandarenowshownasthe(Figure1B,C,EandFigure1—figuresupplement1B),whereasoneoftheoriginalpanelshasbeenmovedtothesupplement(Figure1—figuresupplement2A). Figure1Cand1E–HowdoestheoverexpressionlevelofPABPCcomparetoendogenouslevels? WetriedtoquantifytheoverexpressionofePABrelativeitsendogenouslevelbywestern,buttheePABantibodydidnotseemtorecognizethetaggedePABproteinweoverexpressed.WeobservedasignalfortheFLAGtagbutnotforoverexpressedePAB,whichmigratedsloweronthegel.Nonetheless,wewereabletodetectoverexpressedPABPC1(asshowninFigure1—figuresupplement1E)andestimatedthatitreachedalevelmorethansixtimesthatofendogenousPABPC1.Thisinformationisnowmentionedanddiscussedinourreviseddiscussion. Pleasedefine"poly(A)tags"thefirsttimeitismentioned(Figurelegend2?) We’veaddedthedefinitionintheFigure2legend. Figure2c–arethemoreprominentbandseGFPandPABPC1?Ifso,pleaselabelthem. Yes.We’venowlabeledtheseprominentbands. Thereareseveraldifferentsequencingmethodsused(TAIL-seq,PAL-seqv3,PAL-seqv4)–whywasagivenmethodchosen?Itwouldbehelpfultomentionwhichmethodwasusedinthefigurelegends. We’veaddedreasonsforchoosingthesequencingmethodstothemethodssectionandstatewhichmethodwasusedineachfigurelegend. Reviewer#3(Recommendationsfortheauthors): 1.NoerrorbarsarepresentedinFigure1A-B,whyisthisthecase? Whenperformingtheseexperimentswithdifferentextracts,weobservedconsistenteffectsofaddingPABPCbutvariablebaselineratios,presumablybecauseofvariablequalityoftheovaryweobtainedfromthevendor.Toassesstechnicalvariability,werepeatedtheexperimentsintriplicateusingthesamebatchofoocyteextract.We’vereplacedtheoriginalFigure1A–Bwiththeresultsofthesenewexperiments,witherrorbarsshowingstandarddeviationforthetechnicalreplicates,andwe’vemovedtheoriginalpanelAtoFigure1—figuresupplement2A. 2.TheauthorsconcludethelossofPABPCincelllinesresultsintheselectiveeliminationoftranscriptswithshortpoly(A)tails.Cantheauthorsexcludearoleforpoly(A)tailelongationintheabsenceofPABPC? Theideathatshortpoly(A)tailsmighthavegloballyelongatedwithnochangeinmRNAstabilityisnotsupportedbyourfindingsthat(1)absolutelevelsoflong-tailedmRNAs(determinedusingnormalizationtospike-instandards)remainedconstantanddidnotincrease,(2)mRNAhalf-livesdiddecrease,and(3)TUT4/7,whichhaveknownrolesinmRNAdegradationbutnottaillengthening,wererequiredforthelossofshorttails. 3.Theauthorsconcludebroadrulesfromasinglecoupledsystemandcelllineexperiments,wherethedepletionofPABPChasdrasticconsequencestothetranscriptome(~70%reductioninmRNAabundance)andlikelytothecell.Theauthorsshouldacknowledgetheselimitations. ThereasonwecreatedthePABPC-AIDfusionandexaminedtheeffectsofPAPBCdepletionbeforelargechangestothetranscriptomecouldoccurwastoavoidthesecondaryeffectsofconcerntothereferee.Nonetheless,wenowacknowledgethelimitationofexaminingonlyafewsystemsinthelastparagraphofourdiscussion. 4.Theauthorsfailtointegratepreviousfindingsinthemodelpresentedinfigure7.Morganetal.,2017haveshownthatlossofTUT4andTUT7inseveralcellsystemsandmousetissuesdoesnotimpactthetranscriptome.Theauthorsneedtodiscussthesefindingsandintegratethemintotheirmodel. WementiontheresultsofMorganetal.andintegratethemintoourmodelinourreviseddiscussion.ThefindingthatlossofTUT4andTUT7insomaticmousecellsdoesnotinfluencemRNAabundanceisconsistentwithourhypothesisthatPABPCisnotlimitinginthesecells.Accordingtoourmodel,inthesecellsinwhichPABPCisnormallynotlimiting,PABPCcoatsthemRNApoly(A)tailstherebyprotectingthemfromTUT4andTUT7. https://doi.org/10.7554/eLife.66493.sa2 Authordetails KehuiXiang HowardHughesMedicalInstitute,Cambridge,UnitedStates WhiteheadInstituteforBiomedicalResearch,Cambridge,UnitedStates DepartmentofBiology,MassachusettsInstituteofTechnology,Cambridge,UnitedStates Contribution Conceptualization,Resources,Datacuration,Software,Formalanalysis,Validation,Investigation,Visualization,Methodology,Writing-originaldraft,Projectadministration,Writing-reviewandediting Competinginterests Nocompetinginterestsdeclared "ThisORCIDiDidentifiestheauthorofthisarticle:" 0000-0002-3770-1367 DavidPBartel HowardHughesMedicalInstitute,Cambridge,UnitedStates WhiteheadInstituteforBiomedicalResearch,Cambridge,UnitedStates DepartmentofBiology,MassachusettsInstituteofTechnology,Cambridge,UnitedStates Contribution Conceptualization,Resources,Supervision,Fundingacquisition,Investigation,Projectadministration,Writing-reviewandediting Forcorrespondence [email protected] Competinginterests Nocompetinginterestsdeclared "ThisORCIDiDidentifiestheauthorofthisarticle:" 0000-0002-3872-2856 DavidPBartel DavidPBartel KehuiXiang Thefundershadnoroleinstudydesign,datacollectionandinterpretation,orthedecisiontosubmittheworkforpublication. WethankSEichhorn,TEisen,ASubtelny,SGupta,XWu,SMcGeary,WFang,KLin,JSmith,JKwasnieskiandHSiveforvaluablediscussions;SEichhorn,TEisen,KLin,andSGuptaforsharingimprovedmethodsforpoly(A)-tailprofiling;KMcKinley,KSu,MKanemaki,AHolland,A-BShyuforsharingcelllinesandplasmids;NGrayandMBrookforantibodies,andtheWhiteheadInstituteGenomeTechnologyCoreforsequencing.ThisworkissupportedbyNIHgrantGM118135.KXisaCancerResearchInstituteIrvingtonFellowsupportedbytheCancerResearchInstitute.DPBisaninvestigatoroftheHowardHughesMedicalInstitute. JamesLManley,ColumbiaUniversity,UnitedStates TimothyWNilsen,CaseWesternReserveUniversity,UnitedStates TimothyWNilsen,CaseWesternReserveUniversity,UnitedStates Received:January13,2021 Accepted:May21,2021 VersionofRecordpublished:July2,2021(version1) ©2021,XiangandBartelThisarticleisdistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduseandredistributionprovidedthattheoriginalauthorandsourcearecredited. 4,160 Pageviews 625 Downloads 15 Citations Articlecitationcountgeneratedbypollingthehighestcountacrossthefollowingsources:Crossref,PubMedCentral,Scopus. Atwo-partlistoflinkstodownloadthearticle,orpartsofthearticle,invariousformats. Downloads(linktodownloadthearticleasPDF) ArticlePDF FiguresPDF Opencitations(linkstoopenthecitationsfromthisarticleinvariousonlinereferencemanagerservices) Mendeley Citethisarticle(linkstodownloadthecitationsfromthisarticleinformatscompatiblewithvariousreferencemanagertools) KehuiXiang DavidPBartel (2021) Themolecularbasisofcouplingbetweenpoly(A)-taillengthandtranslationalefficiency eLife10:e66493. https://doi.org/10.7554/eLife.66493 DownloadBibTeX Download.RIS Categoriesandtags ResearchArticle ChromosomesandGeneExpression regulationoftranslation regulationofmRNAstability terminaluridylation poly(A)tail PABPC1 Xenopusoocytes Researchorganisms Human Mouse Xenopus Relatedto Furtherreading Furtherreading Longerpoly(A)tailsimprovetranslationinearlydevelopment,butnotinmaturecellsthathavehigherlevelsoftheproteinPABPC. CohesinfoldschromosomesviaDNAloopextrusion.Cohesin-mediatedchromosomeloopsregulatetranscriptionbyshapinglong-rangeenhancer–promoterinteractions,amongothermechanisms.Mutationsofcohesinsubunitsandregulatorscausehumandevelopmentaldiseasestermedcohesinopathy.VertebratecohesinconsistsofSMC1,SMC3,RAD21,andeitherSTAG1orSTAG2.Toprobethephysiologicalfunctionsofcohesin,wecreatedconditionalknockout(cKO)micewithStag2deletedinthenervoussystem.Stag2cKOmiceexhibitgrowthretardation,neurologicaldefects,andprematuredeath,inpartduetoinsufficientmyelinationofnervefibers.Stag2cKOoligodendrocytesexhibitdelayedmaturationanddownregulationofmyelination-relatedgenes.Stag2lossreducespromoter-anchoredloopsatdownregulatedgenesinoligodendrocytes.Thus,STAG2-cohesingeneratespromoter-anchoredloopsatmyelination-promotinggenestofacilitatetheirtranscription.Ourstudyimplicatesdefectivemyelinationasacontributingfactortocohesinopathyandestablishesoligodendrocytesasarelevantcelltypetoexplorethemechanismsbywhichcohesinregulatestranscription. Invertebrates,condensinIandcondensinIIcooperatetoassemblerod-shapedchromosomesduringmitosis.AlthoughthemechanismofactionandregulationofcondensinIhavebeenstudiedextensively,ourcorrespondingknowledgeofcondensinIIremainsverylimited.ByintroducingrecombinantcondensinIIcomplexesintoXenopuseggextracts,wedissecttherolesofitsindividualsubunitsinchromosomeassembly.WefindthatoneoftwoHEATsubunits,CAP-D3,playsacrucialroleincondensinII-mediatedassemblyofchromosomeaxes,whereastheotherHEATsubunit,CAP-G2,hasaverystrongnegativeimpactonthisprocess.ThestructuralmaintenanceofchromosomesATPaseandthebasicaminoacidclustersofthekleisinsubunitCAP-H2areessentialforthisprocess.DeletionoftheC-terminaltailofCAP-D3increasestheabilityofcondensinIItoassemblechromosomesandfurtherexposesahiddenfunctionofCAP-G2inthelateralcompactionofchromosomes.Takentogether,ourresultsuncoveramultilayeredregulatorymechanismuniquetocondensinII,andprovideprofoundimplicationsfortheevolutionofcondensinII. 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