Poly A tail length analysis of in vitro transcribed ... - Springer

mRNA that is produced synthetically through in vitro transcription (IVT) gains a poly A tail either by encoding the polyadenosine sequence into ... Skiptomaincontent Advertisement SearchSpringerLink Search AbstractThe3′-polyadenosine(polyA)tailofinvitrotranscribed(IVT)mRNAwasstudiedusingliquidchromatographycoupledtomassspectrometry(LC-MS).PolyAtailswerecleavedfromthemRNAusingribonucleaseT1followedbyisolationwithdTmagneticbeads.ExtractedtailswerethenanalyzedbyLC-MSwhichprovidedtaillengthinformationatsingle-nucleotideresolution.A2100-ntmRNAwithplasmid-encodedpolyAtaillengthsofeither27,64,100,or117nucleotideswasusedforthesestudiesasenzymaticallyaddedpolyAtailsshowedsignificantlengthheterogeneity.ThenumberofAsobservedinthetailscloselymatchedSangersequencingresultsoftheDNAtemplate,andevenminorplasmidpopulationswithsequencevariationsweredetected.WhentheplasmidsequencecontainedadiscreetnumberofpolyAsinthetail,analysisrevealedadistributionthatincludedtailslongerthantheencodedtaillengths.TheseobservationswereconsistentwithtranscriptionalslippageofT7RNAPtakingplacewithinapolyAsequence.ThetypeofRNAPdidnotaltertheobservedtaildistribution,andcomparisonofT3,T7,andSP6showedallthreeRNAPsproducedequivalenttaillengthdistributions.Theadditionofasequenceatthe3′endofthepolyAtaildid,however,producenarrowertaillengthdistributionswhichsupportsapreviouslydescribedmodelofslippagewherethe3′endcanbelockedinplacebyhavingaGorCafterthepolynucleotideregion. DeterminationofmRNApolyAtaillengthusingmagneticbeadsandLC-MS. Thisisapreviewofsubscriptioncontent,accessviayourinstitution. Accessoptions Buysinglearticle InstantaccesstothefullarticlePDF. 39,95€ PriceincludesVAT(Singapore) RentthisarticleviaDeepDyve. LearnmoreaboutInstitutionalsubscriptions Fig.1Fig.2Fig.3Fig.4Fig.5Fig.6Fig.7Fig.8 ReferencesHarrisonPF,PowellDR,ClancyJL,PreissT,BoagPR,TravenA,etal.PAT-seq:amethodtostudytheintegrationof3′-UTRdynamicswithgeneexpressionintheeukaryotictranscriptome.RNA.2015;21(8):1502–10.CAS  Article  GoogleScholar  AitkenCE,LorschJR.Amechanisticoverviewoftranslationinitiationineukaryotes.NatStructMolBiol.2012;19(6):568–76.CAS  Article  GoogleScholar  MoqtaderiZ,GeisbergJV,StruhlK.Secondarystructuresinvolvingthepoly(A)tailandother3′sequencesaremajordeterminantsofmRNAisoformstabilityinyeast.MicrobCell.2014;1(4):137–9.CAS  Article  GoogleScholar  SubtelnyAO,EichhornSW,ChenGR,SiveH,BartelDP.Poly(A)-tailprofilingrevealsanembryonicswitchintranslationalcontrol.Nature.2014;508(7494):66–71.CAS  Article  GoogleScholar  RatcliffeCD,SahgalP,ParachoniakCA,IvaskaJ,ParkM.Regulationofcellmigrationandbeta1integrintraffickingbytheendosomaladaptorGGA3.Traffic.2016;17(6):670–88.CAS  Article  GoogleScholar  BeilharzTH,PreissT.Widespreaduseofpoly(A)taillengthcontroltoaccentuateexpressionoftheyeasttranscriptome.RNA.2007;13(7):982–97.CAS  Article  GoogleScholar  PengJ,MurrayEL,SchoenbergDR.Invivoandinvitroanalysisofpoly(A)lengtheffectsonmRNAtranslation.MethodsMolBiol.2008;419:215–30.CAS  Article  GoogleScholar  KoskiGK,KarikoK,XuS,WeissmanD,CohenPA,CzernieckiBJ.Cuttingedge:innateimmunesystemdiscriminatesbetweenRNAcontainingbacterialversuseukaryoticstructuralfeaturesthatprimeforhigh-levelIL-12secretionbydendriticcells.JImmunol.2004;172(7):3989–93.CAS  Article  GoogleScholar  BernsteinP,PeltzSW,RossJ.Thepoly(A)-poly(A)-bindingproteincomplexisamajordeterminantofmRNAstabilityinvitro.MolCellBiol.1989;9(2):659–70.CAS  Article  GoogleScholar  BernsteinP,RossJ.Poly(A),poly(A)bindingproteinandtheregulationofmRNAstability.TrendsBiochemSci.1989;14(9):373–7.CAS  Article  GoogleScholar  ChangH,LimJ,HaM,KimVN.TAIL-seq:genome-widedeterminationofpoly(A)taillengthand3′endmodifications.MolCell.2014;53(6):1044–52.CAS  Article  GoogleScholar  JanickeA,VancuylenbergJ,BoagPR,TravenA,BeilharzTH.ePAT:asimplemethodtotagadenylatedRNAtomeasurepoly(A)-taillengthandother3'RACEapplications.RNA.2012;18(6):1289–95.CAS  Article  GoogleScholar  MurrayEL,SchoenbergDR.Assaysfordeterminingpoly(A)taillengthandthepolarityofmRNAdecayinmammaliancells.MethodsEnzymol.2008;448:483–504.CAS  Article  GoogleScholar  MeijerHA,BushellM,HillK,GantTW,WillisAE,JonesP,etal.Anovelmethodforpoly(A)fractionationrevealsalargepopulationofmRNAswithashortpoly(A)tailinmammaliancells.NucleicAcidsRes.2007;35(19):e132.Article  GoogleScholar  SallesFJ,StricklandS.RapidandsensitiveanalysisofmRNApolyadenylationstatesbyPCR.PCRMethodsAppl.1995;4(6):317–21.CAS  Article  GoogleScholar  MinasakiR,RudelD,EckmannCR.Increasedsensitivityandaccuracyofasingle-strandedDNAsplint-mediatedligationassay(sPAT)revealspoly(A)taillengthdynamicsofdevelopmentallyregulatedmRNAs.RNABiol.2014;11(2):111–23.CAS  Article  GoogleScholar  MuddimanDC,NullAP,HannisJC.Precisemassmeasurementofadouble-stranded500base-pair(309kDa)polymerasechainreactionproductbynegativeionelectrosprayionizationFouriertransformioncyclotronresonancemassspectrometry.RapidCommunMassSpectrom.1999;13(12):1201–4.CAS  Article  GoogleScholar  HannisJC,MuddimanDC.Detectionofdouble-strandedPCRampliconsattheattomolelevelelectrosprayedfromlownanomolarsolutionsusingFT-ICRmassspectrometry.FreseniusJAnalChem.2001;369(3–4):246–51.CAS  Article  GoogleScholar  EckerDJ,SampathR,MassireC,BlynLB,HallTA,EshooMW,etal.IbisT5000:auniversalbiosensorapproachformicrobiology.NatRevMicrobiol.2008;6(7):553–8.CAS  Article  GoogleScholar  WolkDM,KaletaEJ,WysockiVH.PCR-electrosprayionizationmassspectrometry:thepotentialtochangeinfectiousdiseasediagnosticsinclinicalandpublichealthlaboratories.JMolDiagn.2012;14(4):295–304.CAS  Article  GoogleScholar  BeverlyM,DellA,ParmarP,HoughtonL.Label-freeanalysisofmRNAcappingefficiencyusingRNaseHprobesandLC-MS.AnalBioanalChem.2016;408(18):5021–30.CAS  Article  GoogleScholar  GilarM.Analysisandpurificationofsyntheticoligonucleotidesbyreversed-phasehigh-performanceliquidchromatographywithphotodiodearrayandmassspectrometrydetection.AnalBiochem.2001;298(2):196–206.CAS  Article  GoogleScholar  ApffelA,ChakelJA,FischerS,LichtenwalterK,HancockWS.AnalysisofoligonucleotidesbyHPLC-electrosprayionizationmassspectrometry.AnalChem.1997;69(7):1320–5.CAS  Article  GoogleScholar  HuberCG,OberacherH.Analysisofnucleicacidsbyon-lineliquidchromatography-massspectrometry.MassSpectromRev.2001;20(5):310–43.CAS  Article  GoogleScholar  HailME,ElliottB,AndersonK.High-throughputanalysisofoligonucleotidesusingautomatedelectrosprayionizationmassspectrometry.AmBiotechnolLab.2004;22:12–3.CAS  GoogleScholar  HaukanesBI,KvamC.Applicationofmagneticbeadsinbioassays.Biotechnology(NY).1993;11(1):60–3.CAS  GoogleScholar  BerensmeierS.Magneticparticlesfortheseparationandpurificationofnucleicacids.ApplMicrobiolBiotechnol.2006;73(3):495–504.CAS  Article  GoogleScholar  AdamsNM,BordelonH,WangKK,AlbertLE,WrightDW,HaseltonFR.ComparisonofthreemagneticbeadsurfacefunctionalitiesforRNAextractionanddetection.ACSApplMaterInterfaces.2015;7(11):6062–9.CAS  Article  GoogleScholar  BlowerMD,JambhekarA,SchwarzDS,ToombsJA.CombiningdifferentmRNAcapturemethodstoanalyzethetranscriptome:analysisoftheXenopuslaevistranscriptome.PLoSOne.2013;8(10):e77700.CAS  Article  GoogleScholar  CabadaMO,DarnbroughC,FordPJ,TurnerPC.Differentialaccumulationoftwosizeclassesofpoly(A)associatedwithmessengerRNAduringoogenesisinXenopuslaevis.DevBiol.1977;57(2):427–39.CAS  Article  GoogleScholar  ParkJE,YiH,KimY,ChangH,KimVN.Regulationofpoly(A)tailandtranslationduringthesomaticcellcycle.MolCell.2016;62(3):462–71.CAS  Article  GoogleScholar  JacobsenN,NielsenPS,JeffaresDC,EriksenJ,OhlssonH,ArctanderP,etal.Directisolationofpoly(A)+RNAfrom4Mguanidinethiocyanate-lysedcellextractsusinglockednucleicacid-oligo(T)capture.NucleicAcidsRes.2004;32(7):e64.Article  GoogleScholar  PhelanD,HondorpK,ChoobM,EfimovV,FernandezJ.MessengerRNAisolationusingnovelPNAanalogues.NucleosidesNucleotidesNucleicAcids.2001;20(4–7):1107–11.CAS  Article  GoogleScholar  HoltkampS,KreiterS,SelmiA,SimonP,KoslowskiM,HuberC,etal.Modificationofantigen-encodingRNAincreasesstability,translationalefficacy,andT-cellstimulatorycapacityofdendriticcells.Blood.2006;108(13):4009–17.CAS  Article  GoogleScholar  Triana-AlonsoFJ,DabrowskiM,WadzackJ,NierhausKH.Self-coded3′-extensionofrun-offtranscriptsproducesaberrantproductsduringinvitrotranscriptionwithT7RNApolymerase.JBiolChem.1995;270(11):6298–307.CAS  Article  GoogleScholar  WagnerLA,WeissRB,DriscollR,DunnDS,GestelandRF.TranscriptionalslippageoccursduringelongationatrunsofadenineorthymineinEscherichiacoli.NucleicAcidsRes.1990;18(12):3529–35.CAS  Article  GoogleScholar  MolodtsovV,AnikinM,McAllisterWT.ThepresenceofanRNA:DNAhybridthatispronetoslippagepromotesterminationbyT7RNApolymerase.JMolBiol.2014;426(18):3095–107.CAS  Article  GoogleScholar  MacdonaldLE,ZhouY,McAllisterWT.TerminationandslippagebybacteriophageT7RNApolymerase.JMolBiol.1993;232(4):1030–47.CAS  Article  GoogleScholar  AnikinM,MolodtsovV,TemiakovD,McAllisterWT.Transcriptslippageandrecoding.In:AtkinsJF,GestelandRF,editors.Recoding:expansionofdecodingrulesenrishesgeneexpression,Nucleicacidandmolecularbiology:Springer;2010.p.409–32.ElangoN,ElangoS,ShivshankarP,KatzMS.OptimizedtransfectionofmRNAtranscribedfromad(a/T)100tail-containingvector.BiochemBiophysResCommun.2005;330(3):958–66.CAS  Article  GoogleScholar  GrierAE,BurleighS,SahniJ,CloughCA,CardotV,ChoeDC,etal.pEVL:alinearplasmidforgeneratingmRNAIVTtemplateswithextendedencodedpoly(A)sequences.MolTherNucleicAcids.2016;5:e306.CAS  Article  GoogleScholar  MockeyM,GoncalvesC,DupuyFP,LemoineFM,PichonC,MidouxP.mRNAtransfectionofdendriticcells:synergisticeffectofARCAmRNAcappingwithpoly(A)chainsincisandintransforahighproteinexpressionlevel.BiochemBiophysResCommun.2006;340(4):1062–8.CAS  Article  GoogleScholar  DownloadreferencesFundingSupportforthisworkwasprovidedbyDARPAgrantHR-0011-13-3-0003.AuthorinformationAuthorsandAffiliationsNovartisInstitutesofBiomedicalResearch,700MainStreet,Cambridge,MA,02139,USAMichaelBeverly, CaitlinHagen & OlgaSlackAuthorsMichaelBeverlyViewauthorpublicationsYoucanalsosearchforthisauthorin PubMed GoogleScholarCaitlinHagenViewauthorpublicationsYoucanalsosearchforthisauthorin PubMed GoogleScholarOlgaSlackViewauthorpublicationsYoucanalsosearchforthisauthorin PubMed GoogleScholarCorrespondingauthorCorrespondenceto MichaelBeverly.Ethicsdeclarations Conflictofinterest Theauthorsdeclaretheyhavenoconflictofinterest. Disclaimer Theviews,opinions,and/orfindingexpressedarethoseoftheauthorsandshouldnotbeinterpretedasrepresentingtheofficialviewsorpoliciesoftheDepartmentofDefenseortheUSGovernment. Electronicsupplementarymaterial ESM1(PDF1.04 mb)RightsandpermissionsReprintsandPermissionsAboutthisarticleCitethisarticleBeverly,M.,Hagen,C.&Slack,O.PolyAtaillengthanalysisofinvitrotranscribedmRNAbyLC-MS. AnalBioanalChem410,1667–1677(2018).https://doi.org/10.1007/s00216-017-0840-6DownloadcitationReceived:10August2017Revised:03November2017Accepted:17November2017Published:08January2018IssueDate:February2018DOI:https://doi.org/10.1007/s00216-017-0840-6SharethisarticleAnyoneyousharethefollowinglinkwithwillbeabletoreadthiscontent:GetshareablelinkSorry,ashareablelinkisnotcurrentlyavailableforthisarticle.Copytoclipboard ProvidedbytheSpringerNatureSharedItcontent-sharinginitiative KeywordsmRNAPolyATaillengthMassspectrometrySlippageInvitrotranscription Accessviayourinstitution Accessoptions Buysinglearticle InstantaccesstothefullarticlePDF. 39,95€ PriceincludesVAT(Singapore) RentthisarticleviaDeepDyve. LearnmoreaboutInstitutionalsubscriptions Advertisement