神经科学论文 (4)

CellularandMolecularLifeSciences

REVIEW

Nonsense-mediatedmRNAdecayinhumancells:mechanisticinsights,functionsbeyondqualitycontrolandthedouble-lifeofNMDfactors

PamelaNicholson•HasmikYepiskoposyan•StefanieMetze•RodolfoZamudioOrozco•

¨hlemannNicoleKleinschmidt•OliverMu

Received:22July2009/Revised:16September2009/Accepted:6October2009/Publishedonline:27October2009

¨userVerlag,Basel/Switzerland2009ÓBirkha

AbstractNonsense-mediateddecayiswellknownbytheluciddefinitionofbeingaRNAsurveillancemechanismthatensuresthespeedydegradationofmRNAscontainingprematuretranslationterminationcodons.However,aswereviewhere,NMDisfarfrombeingasimplequalitycontrolmechanism;italsoregulatesthestabilityofmanywild-typetranscripts.WesummarisetheabundanceofresearchthathascharacterisedeachoftheNMDfactorsandpresentaunifiedmodelfortherecognitionofNMDsubstrates.ThecontentiousissueofhowandwhereNMDoccursisalsodiscussed,particularlywithregardtoP-bodiesandSMG6-drivenendonucleolyticdegradation.Inrecentyears,thediscoveryofadditionalfunctionsplayedbyseveraloftheNMDfactorshasfurthercomplicatedthepicture.Therefore,wealsoreviewthereportedrolesofUPF1,SMG1andSMG6inothercellularprocesses.KeywordsNMDÁNonsensemRNAsurveillanceÁPost-transcriptionalgeneregulationÁPTCÁmRNAturnover

Introduction

Thecascadeofeventsduringgeneexpression,fromtranscriptionoftheDNAencodedgeneticinformationto

P.NicholsonÁH.YepiskoposyanÁS.MetzeÁ

¨hlemann(&)R.ZamudioOrozcoÁN.KleinschmidtÁO.Mu

InstituteofCellBiology,UniversityofBern,Baltzerstrasse4,3012,Bern,Switzerland

e-mail:oliver.muehlemann@izb.unibe.ch

theeventualproteinsynthesis,arguablyrepresentssomeofthemostinfluentialbiochemicalpathwaysforalivingorganism.Naturally,diverseregulationmechanismshaveevolvedtoensuretheaccuracyofgeneexpressionatmultiplelevels,amongstthemisaprocesscommonlyreferredtoasnonsense-mediatedmRNAdecay(NMD)ormRNAsurveillance.Thirtyyearsago,itwasobs-ervedinSaccharomycescerevisiaethatnonsensecodonstruncatingtheopenreadingframe(ORF)oftheura3mRNAdramaticallyreducedtheRNA’shalf-life[1]andthatinb0-thalassemicpatientshomozygousforanon-sensemutationintheb-globingene,theb-globinmRNAwassubjectedtorapiddegradation[2].FastdegradationofmRNAsharbouringtruncatedORFsduetoprematuretranslationterminationcodons(PTCs)wassubsequentlydocumentedinmanyotherorganisms,anditisbelievedtooccurinmostifnotalleukaryotes(reviewedin[3]).ThenamescoinedtodescribethisPTC-associatedmRNAturnoverpathway,NMDandmRNAsurveillance,emphasiseitsqualitycontrolfunc-tioninpreventingtheproductionofpotentiallydeleteriousC-terminallytruncatedproteinstranslatedfromPTC-containingmRNAs.However,ithasbecomeclearduringrecentyearsthatmanyphysiologicalmRNAsarealsoNMDsubstrates,indicatingaroleforNMDbeyondmRNAqualitycontrolasatranslation-dependentpost-transcriptionalregulatorofgeneexpres-sion(reviewedin[4]).

Herein,wedescribedifferenttypesofNMDsubstratesandreviewrecentliteraturethathasledtoaunifiedmolecularmodelfortheidentificationofNMDelicitingmRNAs.Additionally,wesummariseourknowledgeaboutthetrans-actingNMDfactors,theirbiochemicalfeaturesandtheintriguingdouble-lifeofseveralofthesefactors.

678NMDtargetsbothaberrantandphysiologicaltranscripts

OneimportantgroupofNMDsubstratescomprisetran-scriptsharbouringaPTCthattruncatestheirORFs.Ifnotdetectedanddegraded,thePTC-containing(PTC?)transcriptscanresultintheaccumulationofpotentiallyharmfulC-terminallytruncatedproteins.PTC?transcriptsariseeitherbymutations(orrearrangementsinthecaseofimmunoglobulingenes)attheDNAlevel,orattheRNAlevelduetoerrorsintranscription,pre-mRNAprocessingandinparticularbyaberrantalternativesplicing(Table1).

OntheDNAlevel,directnonsensemutationsandmorefrequentlyframe-shiftingdeletionsandinsertionsgeneratePTCs.Moreover,mutationsatsplicesitesorsplicingregulatorysequencescanresultinaberrantlysplicedPTC?transcripts.TheprogrammedDNArearrangementsoccurringintheimmunoglobulinandT-cellreceptorgenesduringlymphocytematurationgeneratePTCsatahighfrequency,duetorandomdeletionsandtheadditionofnon-templatednucleotidesattherecombinationsites(reviewedin[5]).Hence,NMDisveryimportantforthedifferentiationandmaintenanceofhematopoieticcells[6].

ErrorsduringtranscriptioncanalsoproducePTC?transcripts,albeitlessfrequently.Incontrast,unproductive

Table1TranscriptsthatcanbetargetedforNMDinmammaliancellsPTC?

PTCsarisingatNonsensemutationsthatDNAlevel

directlygeneratePTCsNucleotideinsertionsanddeletionsthatshiftthereadingframe

Mutationsleadingtosplicesignalalterations

DNArearrangementsof

immunoglobulinandT-cellreceptorgenes

PTCsarisingatTranscriptionerrorsRNAlevel

FaultyoralternativesplicingPre-mRNAsthatescapednuclearretentionProgrammedframeshifts

PTC-PhysiologicalmRNAswithuORFmRNAs

mRNAswithintronsinthe30UTR

mRNAswithlong30UTRsSelenoproteinmRNAsmRNAsoftransposons,retrovirusesandpseudogenes

P.Nicholsonetal.

alternativesplicingisbelievedtoconstituteamajorsourceofPTC?mRNAsinmammals.Asmuchas95%ofmulti-exonhumangenesarealternativelyspliced[7],andtheaveragenumberofalternativelysplicedmRNAisoformspergeneisapproximately3.5inhumans[8].Usingbio-informaticsapproaches,itwasproposedthataboutone-thirdofthealternativelysplicedhumanmRNAscontainaPTC,implyingawidespreadcouplingofalternativesplicingandNMD[9].Forexample,severalstudieshaverevealedthatsomealternativesplicing(AS)eventsexploitNMDforpost-transcriptionalregulation(termedregulatedunproductivesplicingandtranslation:RUSTorAS-NMD[9]).Thistypeofregulationiscommonformanysplicingregulators,includingSRproteinsandhnRNPs,whichindicatesanimportantfeedbackregulationofsplicing(reviewedin[10,11]).Interestingly,suchPTC-introducingexonsoftencoincidewithultra-conservedgenomicele-ments,suggestingakeyroleforRUSTinvertebratebiology.However,amicroarrayprofilingstudydetermin-ingtherelativelevelsofPTC?comparedtothePTC-freesplicevariantsinavarietyofmammaliantissuetypesproposedthatmostPTC-generatingalternativesplicingeventslocatedwithintheORFofmammaliangenespro-ducePTC?mRNAsthatarenotunderstrongpositiveselectionpressureandhenceareunlikelytohaveimportantfunctionalroles[12].Altogether,thisadvocatesthatcellsproducealargenumberoffaultyPTC?mRNAsthatarerecognisedandeliminatedbyNMD.Nonetheless,severalstudieshaveinferredanevenbroaderroleoftheNMDpathwayinmutingthe‘‘transcriptionalnoise’’ofsuppos-edlynon-functionalRNAssuchastranscribedpseudogenes,ancienttransposonsormRNA-likenon-pro-teincodingRNAsfromintergenicregions[13–15].

AnothergroupofNMDsubstratesincludesphysiologi-calRNAtranscriptsthatencodefunctionalfull-lengthproteins.AcrucialroleofNMD,notonlyasavacuumcleanerforaberranttranscriptsbutalsoasaregulatorofphysiologicalmRNAabundance,becameapparentduetoseveralmicroarraystudiesindifferentorganisms(reviewedin[16]).Transcriptome-profilingwithNMD-deficientSaccharomycescerevisiae,DrosophilamelanogasterandHomosapienscellsrevealedthatNMDdirectlyandindi-rectlycontrolstheabundanceof3–10%ofallmRNAsintherespectivecells[14,17–21].Severalfeaturesofphys-iologicalmRNAscanrenderthemNMD-sensitive,whereassomemRNAshaveevolvedstabilisingelementsprotectingthemfromNMD.Notably,intronsinthe30untranslatedregions(30UTR),ORFslocatedupstreamofthemaincodingregion(uORFs),programmedframeshiftsandlong30UTRsallcanelicitNMD.MessengerRNAscontainingUGAtriplet(s)thatdirectselenocysteineincorporationrepresentaninterestingcaseofNMDsub-strates;whenseleniumisabundant,UGAcodesfor

NMDinhumancellsselenocysteine,butitisinterpretedasaPTCwhentheseleniumconcentrationislow[22](see‘‘Inselenium-deprivedcells,NMDreducestheselenoproteinencodingmRNA’’foradiscussionofselenoproteinsandNMD).ThelargeanddiverserepertoireoftranscriptscontrolledbyNMDreflectsthesignificantinfluenceofNMDonthemetabolismofthecellandconsequentlyinmanyhumandiseases.

NMDisimplicatedinthemodulationofmanygeneticdiseases

Therearenumerousexamplesofhumandiseasesassoci-atedwithmutationsthatresultinPTCs[23–25].Iftranslated,thePTC?mRNAswouldgiverisetotruncatedproteinsthathaveeithercompletelylosttheirfunction,arestillfunctional,haveacquireddominant-negativefunctionorhavegainednewfunctions.Asaconsequenceofthesedifferentpossibilities,NMDhasadouble-edgedeffectonthemanifestationofadisease:NMDisdetrimentalifitpreventstheproductionofproteinswithsomeresidualfunction,butitisbeneficialifitpreventsthesynthesisoftoxictruncatedproteins.Hence,NMDrepresentsacrucialmodulatoroftheclinicaloutcomeofmanygeneticdiseases.

ThemajorityofPTC?disease-associatedallelesexerttheirnegativeeffectsduetoinsufficientproductionofafunctionalprotein.AnexamplewhereNMDaggravatestheclinicaloutcomeisprovidedbyseveraldiseasephenotypescausedbymutationsinthedystrophingene.Whilemostofthetruncatingmutationsinthedystrophingeneareasso-ciatedwithasimilarphenotype,theraretruncatingmutationsthatoccurnearthe30endofthedystrophingenecanresultinextremelyvariablephenotypes.Ithasbeensuggestedthatalltruncatedproteinsencodedbygeneswithmutationsnearthe30endwouldintheorybecapableofrescuingtheDMDphenotype,butwhenNMDpreventstheirsynthesis,theclinicalmanifestationsofthediseaseareaggravated[26].Conversely,NMDhasawell-docu-mentedbeneficialroleinthedegradationofPTC?b-globinmRNA,therebypreventingthesynthesisofC-terminallytruncatedb-globinthatwouldotherwisecausetoxicprecipitationtogetherwithsurplusa-globinchains.Inaheterozygotecontext,thesecondwild-typeallelesupportsalmostnormallevelsofb-globinsynthesis,contributingtothecorrecthaemoglobinassembly,whichisreflectedintherecessiveinheritanceofthisb-thalassemiatype.However,rareNMD-insensitivePTCsareresponsibleforthedominantformofb-thalassemia[27,28].

Giventhegeneralinspectionandclean-uproleofNMD,itisnotsurprisingthatdiseasesassociatedwithpre-maturetranslationterminationareremarkablydiverse.

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Furthermore,asalludedtoabove,thePTCpositiondeter-minesifNMDensuesandthiscontributestotheseverityoftheclinicalmanifestations.Tolistafewexamplesofthisdiversity:(1)thediseaseseverityintheconnectivetissuedisorderMarfansyndromecorrelateswiththeabundanceofthePTC?fibrillin1(FBN1)mRNA[29],(2)PTCsatdifferentpositionsinCFTR(cysticfibrosistransmem-brane-conductanceregulator)cancausemildtoseverecysticfibrosis[30],and(3)truncatedformsofIFNGR1(interferongammareceptor1)canresultinrecessivelyordominantlyinheritedsusceptibilitytomycobacterialinfections[31,32].

NMDalsoappearstoplayaprominentroleincarcino-genesis.PTCsinseveraltumoursuppressorgenes(BRCA1,p53,WT1)havebeenreportedtoresultinreducedabundanceoftheirmRNAsduetoNMD[33–36].Indeed,astrategyreferredtoas‘‘geneidentificationbyNMDinhibition’’(GINI)hasbeensuccessfullyusedtoidentifytumoursuppressorgenes[37,38],pointingtoacrucialroleofNMDineliminatingfaultytumoursup-pressortranscriptsandthusprotectingcellsfrommalignantgrowth.

Inadditiontodiseasesthatresultfromnonsensemuta-tions,defectsinNMDfactorscanalsocausedisease.ArecentstudyidentifiedmutationsintheNMDcorefactorUPF3BtoberesponsibleforX-linkedmentalretardation[39].Furthermore,thereisagrowingbodyofevidencethatmanyphysiologicaltranscriptsaresubjecttoNMDregu-lation.ItislikelythattheimpairmentofNMD-dependentregulationoftheabundanceofthesemRNAsisayettobeidentifiedcauseofvarioushumandiseases.

FormanydisorderscausedbyPTC-generatingmuta-tionstherearenoeffectivetreatmentsyetavailable.However,ininstanceswhereNMDeliminatestranscriptsthat,despitethePTC,wouldstillencodeafunctionalprotein,promisingresultshavebeenobtainedwithPTCread-throughapproaches.Remarkably,aslittleas1–5%ofnormalproteinlevelscangreatlyreduceoreliminatetheprincipalmanifestationsofPTC-associateddiseasessuchascysticfibrosisandHurlersyndrome[40–42].Amino-glycosideantibiotics(e.g.gentamicin)havebeenshowninvitrotosuppressnonsensemutationsbypromotingread-throughofterminationcodonsandhaveimprovedCFTRfunctioninclinicaltrialswithcysticfibrosispatients[43,44].However,veryhighaminoglycosideconcentrationsarerequiredandtheassociatedtoxicsideeffectshavelimitedtheirclinicaluse.Instead,anewsuppressorofPTCswasrecentlyreportedthatselectivelyinducesribo-somalread-throughonlyatPTCsandnotatnaturalterminationcodons[45].Thissmallmolecularcompound,calledAtaluren(formerlyPTC124),rescuedstriatedmus-clefunctioninmdxmice(amodelformusculardystrophy)andhasbeentestedinphaseIIclinicaltrialsoncystic

680fibrosispatients.TheoraladministrationofthedrugreducedtheepithelialelectrophysiologicalabnormalitiescausedbyCFTRdysfunction.Atalurenwasgenerallywelltoleratedwithinfrequentandmildtomoderateadverseeffects[46].Theseresultsareencouragingandtheappli-cationofAtalurenmightwellbeextendedtootherdiseasessuchasDMD.

Thepartslist:humanNMDfactors

Thefirsttrans-actingfactorsrequiredforNMDwereidentifiedthroughgeneticscreensinS.cerevisiae(calledUpfs,forup-frameshift;[47–50])andinC.elegans(calledSMGs,forsuppressorofmorphologicaldefectsongeni-talia;[51–53]).Thehumanorthologueswerelateridentifiedbasedonsequencesimilarities[54–61].TheNMDpathwayinhumancellscomprisesthefactorsUPF1,UPF2,UPF3A,UPF3B,SMG1,SMG5,SMG6andSMG7,withUPF1,UPF2andUPF3(AandB)beingthehomo-loguesofC.elegansSMG2,SMG3andSMG4,respectively(reviewedin[3];Table2).Morerecently,fouradditionalNMDfactorshavebeendescribed:NAG,DHX34,SMG8andSMG9.RNAi-mediateddepletionofNAGandDHX34,thehumanhomologuesofC.elegansSMGL-1andSMGL-2,increasedtheabundanceofaNMDreportermRNA[62].ThetwofactorsSMG8andSMG9wereshowntoregulateSMG1kinaseactivityinhumancellsandtheirknockdownsmoderatelystabilisedaPTC?b-globinreportertranscript[63].AninterspeciescomparisonofNMDfactorsrevealedanumberofinter-estingdifferenceswithprobablemechanisticconsequences(seebelow).WhileSMG7ispresentinmammalsandC.elegans,D.melanogasterappearstolackaSMG7homo-logue[],andincontrasttometazoans,S.cerevisiaeonlyrequiresUPF1,UPF2andUPF3forNMD.SMG5andSMG6homologuesarenotpresentinS.cerevisiaeanddeletionoftheSMG7homologueEbs1phasbeenshowntoonlyslightlyincreasetheabundanceofendogenousNMDtargets[65].

TheUPFproteinsconstitutethecoreNMDmachineryOfalltheUPFgenes,UPF1isfunctionallythemostimportantfactorforNMDandhenceisthemostconserved[3,66].UPF1isagroup1RNAhelicaseandnucleicacid-dependentATPase.TheATPaseactivityresidesintwoofthesevenhelicasemotifsinthemiddlesectionoftheproteinandislinkedtothe50-to-30helicaseactivity[67,68].ATPhydrolysisprovidestheenergytofacilitatemodulationsinthestructureofRNAorRNA–proteincomplexes[69].UPF1interactswithUPF2[56,60,70]throughitsN-terminalcysteine-andhistidine-rich(CH)

P.Nicholsonetal.

domain,whichdisplaysauniquecombinationofthreezinc-bindingmotifsarrangedintwotandemmodules[71].UPF1alsointeractswiththeeukaryotictranslationreleasefactorseRF1andeRF3[72,73],andKashimaetal.reportedbasedonimmunoprecipitationexperimentsthatUPF1formsacomplexwithSMG1,eRF1andeRF3(calledSURFcomplex[74]).UPF1isaphosphoproteinandsequentialphosphorylation/dephosphorylationcyclesareessentialforNMDinmammalsandC.elegansbecausetheycontributetoremodellingofthemRNAsurveillancecomplex[58].TheC-terminalserine-glutamine(SQ)motifsofUPF1aretargetsforphosphorylationbySMG1[55,61,75].InadditiontotheSMG1kinase,UPF1phosphorylationwasshowntorequireUPF2andUPF3[66,74,75],butmorerecentstudieshaveprovidedevidenceforbothUPF2-independentandUPF3-independentNMDpathways[73,76–79].ThephosphorylationofUPF1probablyinducesthedissociationofeRF3fromUPF1,sinceitwasfoundthattheover-expressionofaSMG1mutantdeficientforitskinaseactivitystronglyincreasedUPF1co-immunoprecipitationwitheRF3[74].Phosphor-ylatedUPF1interactswithSMG5,SMG6andSMG7,whichinturnpromotesdephosphorylationofUPF1bytheproteinphosphatasePP2A[58,80,81].

SimilartoUPF1,UPF2isalsoaphosphoprotein,bothinmammals[81]andinS.cerevisiae[82].PhosphorylationoftheyeastUpf2poccursatserineresiduesinitsN-terminaldomain.TogetherwithotherspecificaminoacidsintheN-terminalregion,theseserineresiduesareresponsibleforelicitingNMDandfortheinteractionofUpf2pwithHrp1p,anRNA-bindingproteinimplicatedinyeastNMD[82].TheN-terminalregionalsocontainsseveralnuclearlocalisationsignals(NLS),yettheproteinresidespre-dominantlyinthecytoplasm[56,57,60].BesidesbindingtoUPF1,UPF2alsointeractsthroughaseparatesurfacewithUPF3,therebyactingasabridgebetweenthesetwoproteins[56,60,83,84].ThetworegionsofhumanUPF2thatcontactUPF1havebeenmappedtoaminoacids94–133and1,085–1,124/1,167–1,194,withtheC-terminalregionofUPF2contributingmoretotheinteraction[60].ThepreciseinteractionoftheC-terminalregionofUPF2withtheCHdomainofUPF1hasrecentlybeendeterminedinastructuralstudy[70].Usinghighlyconserved,mainlynegatively-charged,residuesinthelastofitsthreeMIF4G(middleportionofeIF4G)domains,UPF2interactswithamainlypositively-chargedb-sheetsurfaceoftheRNPdomain(ribonucleoprotein-typeRNA-bindingdomain)ofUPF3B[85].Notably,UPF2aloneandtheUPF2-UPF3Bcomplex,butnotUPF3Balone,bindtoRNAinvitro[85].UPF3istheleastconservedoftheUPFproteins[3].Incontrasttoyeast,nematodesandflies,thehumangenomeencodestwoUPF3genes:UPF3Aonchromosome13andUPF3B(alsocalledUPF3X)ontheXchromosome[56,

Table2HumanNMDfactorsCharacteristicsUPF2SMG1SMG5aSMG6SMG7eRF1eRF3PP2AbBTZeUPF1UPF3AUPF3BeRF3cSMG1UPF2eRF3Y14dMAGOHdBTZeeIF4A3eSer/Thr-kinaseofPIKKfamily,phosphorylatesUPF1UPF2SMG8fSMG9fUPF1aSMG7PP2AUPF1PP2AUPF1SMG5PP2AffcProteinAlternativename/nameinotherspeciesDirectinteractionpartnersCellularlocalisationReferencesUPF1RENT1(human,mouse)NMDinhumancells

SMG2(C.elegans)PhosphorylatedatserineresiduesinC-terminalSQmotifsRNAhelicase,nucleicacid-dependentATPaseandRNA-bindingproteinShuttlingprotein;atsteady-statepredominantlyinthecytoplasm[48–50,54,56,58–61,66–71,73,74,80,83,92,96,102,103,188,225–227]UPF2RENT2(human,mouse)Nmd2p(S.cerevisiae)PromotesphosphorylationofUPF1AssociateswiththeEJCPromotesphosphorylationofUPF1Phosphoprotein,phosphorylatedatserineresiduesinN-terminalpartCytoplasmic;mainlyperinuclear[20,49,50,56,57,60,70,71,74,79,82,83,85,,92,226,228,229]SMG3(C.elegans)UPF3AandUPF3BUPF3X(=UPF3B)SMG4(C.elegans)Shuttlingprotein;atsteady-stateprimarilyinthenucleus(UPF3B)[39,49,56,60,79,83,85–,92,93,226,228]SMG1ATXUPF1Cytoplasmandnucleus[55,61,63,74,75,94,106,222,224]SMG5EST1BRequiredforUPF1dephosphorylation,directsPP2AtophosphorylatedUPF1Ribonucleasewithendonucleolyticactivity.RequiredforUPF1dephosphorylationRequiredforUPF1dephosphorylationCytoplasm;co-localiseswithSMG7toP-bodies[58,,80,96,97,99,202,203]SMG6EST1ACytoplasm[,81,96,99–101,202,203]SMG7EST1CEbs1p(S.cerevisiae)Cytoplasm;co-localiseswithSMG5toP-bodies[53,58,,65,96,97,202,203]681

682)s]e2c9n[ereAf]]]]3e33228R6666[[[[E–A28E4)1]8Y5d[nnsaodiitacAsa86iloEaddddnceeee–iosssslyyyymAlllla6r6aaaaa3lnnnnuaaaa6DllttttleooooaHCNNNNnOimGrAet-MN(siss’1ylFaPnUalfanononoiitotciaaddtteureelessemtnyylldis(drffaatnsnayce11nailenGGasnerttyiroovliDaMMtoaitpSSNNntcaaetiplpsaiencr1ysoet1r,GinitvGp1arMimaoFtotMsunPtScucaShnnmumUahh.iarftetsinydmoidwitawiDgenienDvMleaBtixiakxMstny3ceencN.CobFislp1lpNaarinPdctmGmgeodawUniresfnittsoMoolalaipohhicfecttrSciaisihiiel.ltcstasaaennaewwctiCasistherBndp3xmxrbmmenAssoeeaiunFllhrhrsooahsNEuPppCFnIFERm]6Ummm2ooco]ci02t8[nnyniieb[eomsaitdd))nisceteassntnnnwaiavrcc/aaogeeeteehertetmlesggseenciddaeeelli,.;;g.g;nceenSgnnge..CeiinsnissivpCCns((siisssiiistmniiare12nnhLLwwmmmretoGGnoonnohoonthliionMMsAtsitticcti––SSayyccllaarnraenrertooettenntinniindnietoottcituittccecncceerreiiaarirtdrriineedddorttrconnrorifioffotf2tfcfocffen4i3eeoooroolebtGGGXrriioroPDrPPraoDPTrMMAHPSSNDabcdefP.Nicholsonetal.

60].BothgenesgeneratetwoalternativelysplicedmRNAs,resultinginfourUPF3isoforms.InUPF3B,skippingofexon8leadstoaproteinlackingaminoacids270–282oftheotherwise483aminoacidscontainingpolypeptide,andexon4skippingofUPF3Aresultsina420aminoacidscontainingUPF3ASprotein,lackingaminoacids117–149ofthe452aminoacidslongUPF3ALisoform[60].TwoproteinscorrespondinginmasstoUPF3ASandUPF3ALhavebeendetectedusinganUPF3A-specificanti-serum[86],buttheshortformisprobablynotubiquitouslypro-duced[87].UPF3AandUPF3Bshareanoverallsimilarityof60%,withtheN-terminalaminoacids38–236(com-prisingtheRNPdomainandanuclearexportsignal)beingthemostconserved(86%similarity).TheC-terminalhalves(aminoacids202–453)areconsiderablymoredivergentandharbouroneorseveralNLSs[56,60].Despitetheextensivesimilarity,tetheringassayshaveshownthatUPF3BismoreeffectivethanUPF3Aattrig-geringNMDandstimulatingtranslation[88].HighactivityinNMDcorrelateswithashortC-terminalsequencemotifthatiswellconservedinUPF3Bofdifferentspecies,butnotinUPF3Aproteins[88].Recently,itwasshownthatUPF3AandUPF3BcompeteforbindingtoUPF2andconsequently,whenUPF3Blevelsarelow,moreUPF3AcanbindtoUPF2,whichstabilisesUPF3AbecauseUPF3Aaloneislikelytobeinherentlyunstable.Ontheotherhand,whenUPF3Blevelsarehigh,lessUPF3AcanbindtoUPF2andthereforetheUPF3Alevelsdecrease.MostlikelyitiscrucialthatUPF3AisquicklydegradedwhenUPF3BlevelsarehighbecauseUPF3AisalessefficientNMDactivator[87].TheUPF3proteinsarecomponentsoftheexon-junctioncomplex(EJC)[].TheC-terminalregionofUPF3interactswithacompositebindingsiteoftheEJCcore[90,91]comprisingpartsofY14,MAGOHandeIF4A3[88,92,93].

TheSMGproteinsdeterminethephosphorylationstatusofUPF1

Aspreviouslymentioned,SMG1isaproteinkinasethatcanphosphorylateUPF1[55,61].SMG1belongstothephosphatidylinositol3-kinase-relatedproteinkinase(PIKK)superfamilyandfunctionsspecificallyasaserine-threoninekinase(reviewedin[94]).RegulationofUPF1’sphosphorylationstateduringNMDisprobablylimitedtometazoansbecauseS.cerevisiaeappeartolackorthologuesofSMG1,SMG5andSMG6,andNMDisonlymoderatelyaffectedbyadeletionoftheSMG7homologueEbs1p[65].BiochemicalstudieshaverevealedSMG1indifferentmulti-proteincomplexes:inadditiontotheSURFcomplex,whereSMG1isassociatedwithUPF1,eRF1andeRF3,SMG1alsoco-immunoprecipitateswithEJCcomponents(eIF4A3,Y14,MAGOH)andNMDfactors(UPF1,UPF2,

NMDinhumancellsUPF3A,UPF3BandSMG7)inHeLacellextracts[74].ThereisevidencethatUPF1phosphorylationrequirestheassociationbetweentheSURFcomplexandtheEJC,andthatthisassociationismediatedbydirectinteractionsofSMG1withUPF2and/orY14[74].

C.elegansmutatedforSMG5,SMG6orSMG7aredeficientinNMDandaccumulatehyper-phosphorylatedSMG2(theUPF1orthologue),suggestingthatthesefactorsarerequiredfordephosphorylationofSMG2[66].More-over,SMG5interactsspecificallywithphosphorylatedSMG2,withSMG7,andwiththestructuralandcatalyticsubunitsofproteinphosphatase2A(PP2A),inferringthatSMG5maygivePP2AspecificityforUPF1[80].Similarly,humanSMG6wasalsoshowntoco-purifywiththecata-lyticsubunitofPP2A,SMG1,UPF1,UPF2andUPF3B,andalsotospecificallytargetdephosphorylationofUPF1,butnotofUPF2[81].(PleasenotethatSMG6istermedSmg5/7ainthisstudy.)

SMG5,SMG6andSMG7shareasimilardomainorganisation:allcontaintwotetratricopeptide(TPR)repeats,eitherattheN-terminus(SMG5andSMG7)orinthemiddlesection(SMG6).TPR-containingdomainsconsistof34aminoacidslongTPRrepeatsthatusuallyfunctionasmediatorsofprotein–proteininteractions[95].ForSMG7,theTPRswereshowntoadoptasimilarfoldto14-3-3,whichisasignal-transductionproteinthatbindsphosphoserine-containingpolypeptides.Sequencesimilar-itiessuggestconservationofthis14-3-3-likedomainstructureinSMG5andSMG6[96],andinSMG7thisdomainisresponsibleforbindingtoUPF1[96]aswellascontributingtoaninteractionwiththecorresponding14-3-3-likedomainofSMG5[97].

SMG5andSMG6containaPIN-likedomain(forPilTNterminus)intheirC-termini.Generally,PIN-likedomainsfunctionasphosphodiesterasesandoftenexhibitnucleaseactivity[98].DespitethefactthatthePIN-likedomainsofSMG5andSMG6adoptasimilaroverallfoldthatisrelatedtoribonucleasesoftheRNaseHfamily,SMG6harboursthecanonicaltriadofacidicresiduescrucialforRNaseHactivity,whereasSMG5lackstwoofthesethreekeycatalyticresidues[99].Thesestructuraldifferencesarereflectedatthemolecularlevel,asonlythePINdomainofSMG6hasnucleaseactivityonsingle-strandedRNAinvitro[99],anditwasrecentlydemonstratedinhumanandDrosophilacellsthatSMG6istheendonucleasethatcaninitiatecleavageofnonsensemRNAnearthePTC[100,101](seebelow).

IntracellularlocalizationofNMDfactors

Reliabledeterminationoftheintracellulardistributionofanyproteinbyimmunofluorescencemicroscopydependsontheavailabilityofhighlyspecificantibodies.Moreover,

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plasmid-basedexpressionoftaggedproteinsorGFPfusionproteinscanproducenon-physiologicallocalisationpat-terns.Withthiscaveatinmind,immunolocalisationstudiesofNMDfactorsinhumancellsrevealedthatUPF1local-isespredominantlytothecytoplasm[56,60].However,biochemicalevidenceimpliesthatUPF1shuttlesbetweenthecytoplasmandthenucleus[102],whichisconsistentwithitsroleinbothnuclearandcytoplasmicprocesses([103,104]andseebelow).UPF2exhibitsperinuclearcytoplasmiclocalisationandUPF3isanucleo-cytoplasmicshuttlingproteinwithsteady-statenuclearlocalisation[56,60].TheSMG5,SMG6andSMG7proteinsarealllocal-isedtothecytoplasm.Over-expressedSMG5andSMG7,togetherwithUPF1,co-localisewithP-bodies,andtheC-terminusofSMG7hasbeenshowntoberequiredforthislocalisation[96,97,105].Incontrast,itwasshownthatover-expressedSMG6accumulatedinseparatecyto-plasmicfoci[97].Biochemicalfractionationandimmuno-fluorescencemicroscopyrevealedthatSMG1localisestoboththecytoplasmandthenucleus[106].

Intriguingly,inArabidopsisthaliana,mostofUPF3andasubstantialfractionofUPF2wererecentlyreportedtolocalisetothenucleolus,andnucleolarfractionswerehighlyenrichedinaberrantmRNAsthatareNMDsub-strates[107].

Distinguishingbetweenproperandaberranttranslationtermination

NMDstrictlydependsonreadingframerecognitionandthereforeontranslation.ThedecisionofwhetherNMDistobeinitiatedornotismadewhenaribosomestallsatanyofthethreeterminationcodons(UAG,UGAandUAA).ThelocalmRNPenvironmentoftheterminationcodoninfluencestheprocessoftranslationtermination(reviewedin[108–110]).Duringnormaltranslationtermination,eRF1recognisestheterminationcodonintheA-siteofthestallingribosomeandformsacomplexwiththeGTPaseeRF3tocatalysepeptiderelease[111–113].ThroughitsC-terminalregioneRF3interactswiththeC-terminusofeRF1,whiletheN-terminalregionofeRF3interactswiththeC-terminaldomainofpoly(A)-bindingprotein(PABP)[114–116],whichisbelievedtostimulateproperandeffi-cienttranslationtermination[117](Fig.1).IthasbeenshowninyeastthatwhentheinteractionbetweentheeRF3orthologueSup35andPab1pisimpaired,theterminatingribosomecannotefficientlydissociatefromthemRNA[118].Consistentwiththecorrespondingroleofmamma-liancytoplasmicpoly(A)bindingprotein1(PABPC1)instimulatingtranslationtermination,itwasrecentlydem-onstratedthatmammaliancellslackingPABPC1exhibitedincreasedread-throughofterminationcodons[73].Inthe

684Fig.1SchematicmodelofefficienttranslationterminationinthepropermRNPenvironment.ThemodelpostulatesthatnormaltranslationterminationinvolvesaninteractionbetweenPABPandeRF3,whichbyacurrentlyunknownmechanismpromotesfastpolypeptiderelease,disassemblyoftheribosomalsubunitsandre-initiationoftheribosomeatthestartcodon.Thepropertermination-stimulatingmRNPenvironmentischaracterisedbyaproteincomplexinvolvingPABP,eIF4G,thecapbindingfactor(eIF4EorCBP80/CBP20)andadditionalfactorsthatbringthe50andthe30endsofthemRNAincloseproximityandconstrainthemRNPinacircularstructure

caseoftranscriptsthataresubjectedtoNMD,theinter-actionbetweeneRF3andPABPC1islessefficientandisantagonisedbyUPF1recruitment[119](Fig.2).InS.cere-visiae,D.melanogasterandhumancells,NMDcanbesuppressedbytetheringPABPnearthePTC[73,118–122],whichfurthersupportstheNMDantagonisingfunctionofPABP.

ThekeyNMDfactorUPF1hasbeenshowntointeractwitheRF3andeRF1[72],butitiscurrentlyunknownhowUPF1isrecruitedtotheterminatingribosome,andifitispresentinallterminationevents.ThereisevidencethatthedecisionofwhetherNMDistobetriggeredornotreliesuponcompetitionbetweenUPF1andPABPforbindingtoeRF3ontheterminatingribosome[119].TheoutcomeofthiscompetitionislargelyinfluencedbythestructureofthemRNP[121].Extensiveresearchusingavarietyofmodelsystemshasprovidedamultitudeofdataregardingthe

P.Nicholsonetal.

NMD-triggeringcharacteristicsofmRNPs.OneclearlydefinedNMD-elicitingfeatureisthelengthofthe30UTR,whichproposesthatthephysicaldistancebetweentheterminationcodonandthepoly(A)tailofthetranscriptdeterminesthefateofthemRNA.Experimentscarriedoutinyeast,worms,plants,humanandflycelllinesrevealedthatterminationcodonsarerecognisedbytheNMDmachinerywhentheyaresituatedtoofarupstreamfromthepoly(A)tail[52,62,118–121,123–126].The‘‘faux30UTRmodel’’wasfirstcoinedtodescribeNMDinyeast,proposingthatprematureterminationisintrinsicallyabnormalsincethelong30UTRisnotproperlyconfiguredtobringPab1pintotheproximityofterminationcodontoensureefficienttranslationtermination[118].Basedontheaforementionedstudiesconductedinavarietyoforgan-isms,weadvocateextensionofthe‘‘faux30UTRmodel’’toaunifiedmodelthatexplainsthebasicmechanismforrecognitionofNMDsubstratesinallorganisms[4](Fig.2).InthesituationoftheNMD-inhibitingsignalfromPABPbeingabsent,thenUPF2and/orUPF3canbindtoUPF1.Inmammals,UPF1ispresentintheSURFcomplexandUPF2and/orUPF3BarethoughttopromoteSMG1-mediatedphosphorylationofUPF1[74].Subsequently,SMG5,SMG6and/orSMG7proteinscanbindtophos-phorylatedUPF1,eventuallyleadingtothedegradationofthetranscript.TheresultsfromKashimaetal.arguethatphosphorylationofUPF1requiresbothUPF2andUPF3B[74].Yet,recentdatafromotherlaboratoriesunveiledtheexistenceofbothUPF2-independentandUPF3-indepen-dentbranchesoftheNMDpathway[73,76–79].

Numerousinvestigationshaveshownthatnotonlythephysicaldistanceofthe30UTRbutalsothespecificRNAsequencesthatitcontainscandeterminethefateofthemRNA.Forexample,itwasproposedthatthesurveillancecomplexscansandidentifiesadownstreamsequenceele-ment(DSE)thatstimulatesNMDinyeast,butthiselementhasremainedpoorlydefined[127,128].Despitethefactthatsimilarsequenceelementshavenotbeenfoundinmammals,itispossiblethatalonger30UTRprovidesabetterplatformforbindingoftrans-actingfactorsthatimproveNMDefficiency.Theaverage30UTRlengthinhumansis700–800nucleotides,yetexperimentswithreportermRNAsharbouring30UTRsthatareonly200–300nucleotidesinlengthwerealreadyabletodisplaysomedestabilisationduetoNMDinhumancellculture[121].AsproposedbySinghetal.,mRNAscontaininglong30UTRshaveperhapsevolvedmechanismstoevadeNMD[119].Thestabilitycanpossiblybeachievedbyhighlystructured30UTRs,whereRNAbasepairingandinternalloopscanbringthepoly(A)tailclosertotheterminationcodon[121].Furthermore,specificRNAsequencesorsecondarystruc-turesmightrecruitNMDantagonisingfactorstothevicinityofthestopcodon.

NMDinhumancells685

Fig.2Modelforaberranttranslationtermination,whichleadstotheassemblyofamRNAsurveillancecomplexthatmarksthemRNAforsubsequentdegradation.WhenthestopcodonislocatedinanmRNPenvironmentwhereitfailstoreceivethePABP-mediatedtermination-stimulatingsignal,theribosomestallsforaprolongedperiodoftimeatthestopcodon,whichallowsbindingofUPF1toeRF3.TheassemblyofthisSURFcomplexmarksthemRNAforNMD.ThemodelfurtherpostulatesthatthismarkingstepisstillreversibleandthatthemRNAisonlyirreversiblycommittedtoNMDafterUPF1phosphorylation(licensingstep).UPF2and/orUPF3arenecessaryforSMG1-mediatedUPF1phosphorylation.ThepresenceofanEJCinthe30UTRservesasastrongenhancerofNMD(EJC-enhancedlicensing),becauseUPF2and/orUPF3interactionwiththeSURFisgreatlyfacilitatedbyvirtueoftheircloseproximity,whereasUPF2and/orUPF3recruitmenttakelongerintheabsenceofanEJC(EJC-independentlicensing),resultinginoveralllessefficientNMD.FollowingphosphorylationandpossiblyinducedbyATPhydrolysis,UPF1undergoesaconformationalchangethatincreasesitsaffinityforRNAandisthenreadyforinteractionwithSMG5–7,whichinitiatemRNAdegradation(seeFig.3)

Mammaliantissueculturestudieshaveestablishedthesplicing-dependentEJC[129,130]asanotherimportanttrans-actingcomponentforNMDinitiation.Themamma-liantranscriptomeiscomplexwithalargenumberofmulti-exongenesandextensivealternativesplicing.One-thirdofallalternativelysplicedtranscriptsareexpectedtobeNMDsubstrates[9].Thecouplingofasplicing-dependentsignaltoNMDfacilitatesefficientrecognitionofthisgroupofNMDsubstratesandincreasestheaccuracyofgeneexpression[131–133].Indeed,ifaterminationcodonoccursprematurelyinthemRNA,itisprobablethatanexon–exonjunctionwillbepresentdownstreamofthe

686PTC,andthisprobabilityincreasesthefurtherupstreamthePTCislocated.Duringpre-mRNAsplicing,EJCsaredeposited20–24nucleotidesupstreamofexon–exonjunctions[129].TheEJCisadynamicstructurewithaheterogeneousproteincomposition,whereintheproteinsY14,MAGOH,eIF4A3andBarentsz(Btz)remainasso-ciatedwithmRNAafterexportintothecytoplasmandconstitutethestablecoreoftheEJC[90,91].TheNMDfactorsUPF2andUPF3havebeenfoundassociatedwithEJC,whichhintedforaroleoftheEJCinNMD[].ItisthoughtthatEJCslocatedwithinanORFareremovedbyelongatingribosomesinaprocessthatinvolvestheribo-some-associatedproteinPYM[134].Incontrast,EJCslocateddownstreamoftheterminationcodonremainassociatedwiththemRNP.ThepresenceofanEJCdownstreamoftheSURFcomplexgreatlyfacilitatesUPF2and/orUPF3interactingwiththeSURFbyvirtueoftheircloseproximity.Accordingtothismechanisticmodel,thepresenceofanEJCdownstreamoftheterminationcodonservesasapotentenhancerofNMD(Fig.2;reviewedin[110]).

AnotherinterestingobservationwithregardstoNMDefficiencyasafunctionofPTCpositionisthatinseveralmRNAsPTCsclosetothetranslationinitiationcodonfailtoelicitefficientNMD[36,121,135–138].ItisknownthatPABPC1canbindtoeukaryoticinitiationfactor4G(eIF4G)whichactstobridgethe30and50endsofthemRNAintoacircularstructurethatenhancestranslation[139,140].Inthisclosed-loopstructure,theNMDsup-pressorPABPC1wouldalsobeinclosedistancetothe50regionofanmRNAwhereitcanantagoniseNMDinitia-tionatearlyPTCs,regardlessofthepresenceofdownstreamEJCs[122,141].Inaddition,theribosomecouldinitiateatanin-frameAUGdownstreamoftheearlyPTCandsoremovetheremainingEJCsfromthemRNA,whichwouldeliminateapotentpromoterofNMD.

Inmammaliancells,NMDhasbeenproposedtooccurduringtheso-calledpioneerroundoftranslation,beforeeIF4Ereplacesthecap-bindingcomplex(CBC)[142].CBP80,whichisaconstituentoftheCBC,hasbeenshowntointeractwithUPF1andtheknockdownofCBP80reversedNMD,butnottherepressionofsteady-statetranslationthroughthetranslationinhibitor4E-BP1([143,144],reviewedin[10]).Incontrast,NMDoccursmainlyoneIF4E-boundmRNAsinS.cerevisiae:CBC-boundtranscriptsarelargelyinsensitivetoNMDandtheyeasthomologueofCBP80isdispensableforNMD[145–147].Insummary,webelievethatmostofthecurrentlyavailableexperimentaldatasupportaNMDmodelinwhichthecompetitiveinteractionbetweenUPF1andPABPwiththeribosome-boundreleasefactorsisthekeydeterminantforNMDinitiation.Alternativemodelsthatremaintobeexperimentallytestedwererecentlyproposed

P.Nicholsonetal.

byBrognaandWen[148].Itisnotyetpossibletounam-biguouslypredictwhichmRNPsareabletoelicitNMDandtoexplaineachofthediscussedNMD-triggeringfea-turesofmRNPs.Avarietyofstudieshaveshownthatcertaincis-actingelements,suchaslong30UTRsorthepresenceofEJCsdownstreamoftheterminationcodon,aresufficientontheirowntotriggerNMD,probablybecause,inbothcases,UPF1bindingtoeRF3andsubsequentlytoUPF2and/orUPF3isfacilitated.Nonetheless,neitheralong30UTR,adownstreamEJCnoranyotherreportedNMDtriggeringfeatureonmRNAisabsolutelyrequiredforNMD.Therefore,theonlycertainrequirementforNMDsofaristhetranslation-dependentrecruitmentofUPF1tothemRNA.

DegradationofNMDtargets

Evidenceforendonucleolyticandexonucleolyticdecaypathways

MessengerRNAturnoverinvolvesanimportantandtightlyregulatedsetofribonuclease-mediateddegradationpathwaysusedbyeukaryoticcellstoregulatenormalgeneexpression,togetridofaberrantmRNAs(e.g.thosecontainingPTCs)andtoeliminateparasiticmRNAspe-cies(viralmRNAs,transposons).Duetotheircriticalfunctions,manyoftheribonucleasesresponsibleforRNAdegradationarehighlyconservedamongsteukaryotes[149–152].DegradationofnormalmRNAsisgenerallyinitiatedbytheremovalofthepoly(A)tailfromthe30endofmRNAs.Inyeast,thedeadenylationreactioniscatalysedbytheCcr4p/Caf1pcomplex,whileinmam-malsthepoly(A)tailisshortenedbytheconsecutiveactionoftwodifferentcomplexes:thePAN2/PAN3complexfirstshortensfull-lengthpoly(A)tailsofapproximately200basestoapproximately110bases.Theseintermediatepoly(A)tailsaresubsequentlytargetedbytheCCR4/CAF1complexthatremovestheremainingadenines[153]andfollowedbyexonucleolyticdegrada-tionofthetranscriptin30-to-50directionbytheexosome[154].ConcomitantwithCCR4/CAF1-mediateddead-enylation,thedecappingenzymeDCP1/DCP2removesthe7-methylguanosine(m7G)capstructure,leavinganunprotected50endthatisaccessibleforrapiddegradationbytheabundant50-to-30exonucleaseXRN1.

AnimportantquestiontoaddressiswhatpathwayofdegradationNMDfollows.Forinstance,arethegeneralmRNAturnoverpathwaysutilisedafterimpropertransla-tiontermination,orisNMDinitiatedbyspecialnucleases?InS.cerevisiae,degradationofPTC?mRNAshasbeenshowntorelyonthegeneralmRNAturnoverpathwayandamodificationthereofthatistypifiedbydeadenylation-

NMDinhumancells

Fig.3ModelfordegradationofNMDsubstrates.ThemodelpositsthatUPF1-boundmRNAscanbedegradedbytwo

differentpathways,dependingonwhethertheSMG5/SMG7heterodimerortheendonucleaseSMG6bindstophosphorylatedUPF1.InteractionofSMG5/SMG7withphospho-UPF1promotesdeadenylationfollowedbydecappingandexonucleolyticRNAdecayfrombothends(leftbranch).InteractionofSMG6withphospho-UPF1leadstoaSMG6-mediated

endonucleolyticcleavageneartheaberrantterminationsite,followedbytheexonucleolyticdegradationofthetwoRNAfragmentsfromtheinitialcleavagesite

687

independentremovalofthem7G-capstructure,followedbysubsequentXrn1p-mediated50-to-30exonucleolyticdecay[155,156].However,NMDsubstrateswerealsofoundtobechannelledintoadegradationpathwaythatinvolvesdeadenylationfollowedby30-to-50exonucleolysisbytheexosomeandSkicomplexes[155,157].Incontrast,NMDinD.melanogasteriselicitedbyendonucleolyticcleavageofthePTC?mRNAinthevicinityofthePTC.Theresulting50and30decayintermediatesarethenrapidlydegradedinthe30-to-50directionbytheexosomeandinthe50-to-30directionbyXRN1,respectively,andcanonlybedetectedincellsdepletedfortheaforementionedexonuc-leases[158].Inhumancells,thesituationislessclearandprobablymorecomplex.DegradationofNMDreportertranscriptsviatheconventionalmRNAturnoverpathway,

startingwithdeadenylation,followedbydecappingandXRN1-mediatedexonucleolyticdecay,hasbeenreported[159–161](Fig.3;SMG5/SMG7mediatedexonucleoly-sis).ConsistentwitharoleofdecappinginhumanNMD,UPF1andDCP1ahaverecentlybeendocumentedtointeractviaaproteincalledPNRC2[162].Ontheotherhand,endonucleolyticcleavagenearthePTChasrecentlybeendemonstratedinhumancellsdepletedforXRN1[100].Moreover,thePINdomainofSMG6wasshowntopossesstheendonucleaseactivityresponsibleforinitiatingNMDinbothDrosophilaandhumancells[100,101].Therefore,itseemsthatmammalianNMDtargetscanbedegradedbybothaSMG6-dependentendonucleolyticpathwayandadeadenylation-anddecapping-dependentexonucleolyticpathway(Fig.3;SMG6mediated

688endocleavage),whereasDrosophilaNMDisconfinedtotheformerandyeastNMDtothelatterdecaypathway.Consistentwiththisconclusion,mammalspossessbothSMG6andSMG7,whereasDrosophilalacksaSMG7homologueandyeasthasnoSMG6equivalent.FurtherworkisrequiredtodeterminetherelativecontributionsofthetwodecaypathwaysinvolvedinmammalianNMDandtounderstandwhatdetermineswhichdecayrouteistakenbythedifferenttypesofmRNAsdirectedtotheNMDpathway.

DoeshumanNMDtakeplaceinP-bodies?

Prominentcytoplasmicfocicalledprocessingbodies(P-bodies,alsocalledDCP1-orGW-bodies)havereceivedampleattentionduringthelastfewyearsbecausetheyseemtorepresentimportantsitesfortranslationalrepressionandmRNAdecay[163–165].Thesehighlydynamicgranulesareenrichedforcomponentsofthedecappingand50-to-30degradationmachinery,includingDCP1/DCP2,thedecappingactivatorsRCK/p54,Hedls/Ge-1,EDC3andPat1,theLsm1-7complexandthe50-to-30exonucleaseXRN1.MammalianP-bodiesalsocontainthedeadenylasesCCR4/CAF1andPAN2/PAN3[166],aswellascomponentsofthemiRNApathway[1].Additionally,ithasbeenfoundthat,undercertaincon-ditions,theNMDfactorsUPF1,UPF2,UPF3,SMG5andSMG7arelocalisedinP-bodies[97,105,162,167].Theco-localisationofthemRNAdegradationmachineryandofvariousNMDfactorsinP-bodieshasstimulatedresearchonthefunctionalsignificanceofP-bodiesinNMD.Inyeastcells,Upf1p,Upf2pandUpf3paccumu-lateinP-bodiesinDdcp1,Ddcp2orDxrn1strains[167].Furthermore,aNMDreportertranscriptwasshowntolocalisetoP-bodiesinanUpf1p-dependentmannerandmoreNMDreportertranscriptaccumulatedinP-bodiesofDupf2orDupf3strains[167].TheseobservationsarguethatNMDinyeastoccurswithincomplexesthatcanaccumulateintoP-bodies.Incontrast,NMDwasnotaffectedinDrosophilaandhumancellsbyRNAi-medi-ateddepletionofGW182andGe-1,atreatmentthatpreventedtheformationofanymicroscopicallyvisibleP-bodies[168,169].TheseresultsindicatethatNMDdoesnotrequirethepresenceofP-bodiesinmetazoans.Moreover,SMG6doesnotco-localisewithP-bodies[97],whichisconsistentwiththeviewthatmostNMDinhumancellsmightoccuroutsideofP-bodieswhereitisinitiatedbyadecapping-independentSMG6-mediatedendonucleolyticcleavageofthesubstrateRNA[100].Conversely,thisobservationdoesnotruleoutthepossi-bilitythat,underphysiologicalconditions,NMDmayoccurtosomeextentinP-bodies,inkeepingwiththereportedlocalisationofseveralNMDfactorsandaNMD

P.Nicholsonetal.

reportertranscripttoP-bodiesinover-expressioncondi-tions[97,105,162].

AnextensivearrayofphenotypesareobserveduponinactivationofNMDeffectors

WhilethemechanisticdetailsofNMDarebeinggraduallyunravelled,thephysiologicalroleofNMDstillremainslargelyunknown.Thephenotypesobserveduponinacti-vationofNMDeffectorsvaryconsiderablyamongstdifferentorganisms.NMDmutantsinS.cerevisiaeonlyshowapartialimpairmentinrespiratorygrowthwhichisenhancedatlowtemperatures[48],whereasC.elegansmutantshavedefectsinthemalebursaandthehermaph-roditevulva[51,52].Incontrast,UPF1andUPF2areessentialfortheviabilityofD.melanogasterlarvae[170],anddepletionofNMDfactorsimpairsproliferationofDrosophilaembryo-derivedcells(S2cells)[19].Likewiseinzebrafish,UPF1,UPF2,SMG5andSMG6wereshowntobeessentialforembryonicdevelopmentandsurvival[171].Inmice,knockoutofUPF1isembryoniclethalandisolatedblastocystsundergoapoptosisafteronlyafewdaysinculture[172].Likewise,noUPF2knockoutmouseembryoscouldbedetectedatday9.5postcoitus,andconditionalknockoutsofUPF2inhematopoieticprecursorsledtothecompleteextinctionofhematopoieticstemandprogenitorcells[6].Similarly,inArabidopsisthaliana,UPF1isnecessaryforseedlinggrowth[173].

Collectively,theseverityofthephenotyperesultingfrominactivationofthevariousNMDfactorscorrelateswiththeoverallcomplexityoftheorganismanditsextentofalternativesplicing.ThisallowsforthespeculationthattheaccumulationofaberrantlysplicedmRNAsandtheresultingproductionofdetrimentalC-terminallytruncatedproteinsmaybethecauseoftheobservedphenotypes.Ontheotherhand,itmaybethatNMDregulatestheexpres-sionofanessentialproteininmiceandfliesbutnotinyeastandworms.Moreover,severaloftheproteinscharacterisedasNMDfactorshavealsobeenreportedtofunctionincellularprocessesseeminglyunrelatedtoNMD,anditisthereforeequallyplausiblethattheobservedphenotypesaretheconsequenceofdisruptingtheseNMD-independentmechanisms.

Inselenium-deprivedcells,NMDreducestheselenoproteinencodingmRNA

Selenium(Se)isanessentialmicronutrientthatislinkedtomanyaspectsofhumanhealth.Auniqueclassofproteinscalledselenoproteins,mostofwhichareinvolvedinpro-tectingthecellfromoxidativestress,incorporate

NMDinhumancellsselenocysteine(SecorU)atin-frameUGAcodonsinarchaea,prokaryotesandeukaryotes.Unliketheotheraminoacids,SecismadeonitstransferRNA(tRNA)andselenocysteyl-tRNA[ser]secisdeliveredtotheA-siteoftheribosomebyaspecificelongationfactorcalledEFsec[174].ThefactthattheUGAcodonspecifiesSecincorporationratherthantranslationterminationischieflyduetothepresenceofspecificsecondarystructuresinselenoprotein-encodingmRNAstermedselenocysteineinsertion(SECIS)elements,andthetrans-actingfactorsthatassociatewiththeseSECISelements.Ineukaryotes,theSECISelementislocatedinthe30UTR(reviewedin[175]).AproteincalledSECISbindingprotein2(SBP2)bindstoSECISelementsandtothelargeribosomalsubunitandrecruitstheeEFsec-selenocysteyl-tRNA[ser]seccomplextotheribosome,ensuringefficientSecincorporationinvivoandinvitro[175,176].Moreover,SBP2canactuallysuppresstheterminationpromotingfunctionofeRF1[177].

TheefficiencyofSecincorporationvariesdependingonthenatureoftheSECISelement,thepositionoftheUGAintheORF[178]andthenucleotidessurroundingit[177,179],aswellastheintracellularseleniumconcentration[22].ItwasobservedthatSedeficiencydecreasestheabundanceofmRNAsencodingtheselenoprotein,gluta-thioneperoxidase1,andthatthisispossiblymediatedbytheNMDpathway[22].ThemRNAcodingforphospho-lipidhydroperoxideglutathioneperoxidase(PHGPx)wasalsoreportedtobeanNMDtargetinNIH3T3fibroblastsorH35hepatocytesunderSe-deprivedconditions[180],butnocorrespondingchangeinPHGPxmRNAabundanceintheliverortestisofSe-deficientratswasobserved[181].ItwassuggestedthatamechanismabsentinmouseNIH3T3fibroblastsandratH35hepatocytesmaskstheNMDofPHGPxmRNAintheratliverandtestis[180].Giventheimportanceofselenoproteinstotheviabilityoftheanimalandtheirspecialisedfunctions,thefactthattheseproteinsarehighlyconservedandancient,thecomplexcis-actingelementsandtrans-actingfactorsthatensurethattheUGAcodonisnotreadasastopcodonandtheirintricatehier-archyofexpressionwhichmatchestheSeintake,itmaybethat,underphysiologicalconditions,NMDincombinationwithadditional,presentlyunknownprocessesactstoreg-ulatethestabilityofselenoproteinencodingmRNAs.

ThemultiplepersonalitiesoftheNMDfactorsUPF1,SMG1andSMG6

Staufen-1-mediatedmRNAdecay(SMD)andNMDarecompetitivepathways

Thedouble-strandedRNAbindingproteinStaufenwasoriginallyidentifiedasamaternalfactorrequiredforthe

6

correctformationoftheanterioposterioraxisintheD.melanogasterembryo[182,183].StaufenisprincipallyknowntobeinvolvedinthetransportofmRNAstoulti-matelyachievetheirlocalisedtranslation[184,185],inmice[186]andinhumans[186,187].MammalianStaufenproteinhasfourdouble-strandRNAbindingdomains(dsRBDs)andcontainsaputativemicrotubule-bindingdomainofmicrotubule-associatedprotein1BinitsC-ter-minusthatisnotpresentintheStaufenproteinofD.melanogasterorC.elegans[187].

InadditiontoStaufen’spreviouslycharacterisedfunc-tions,mammalianStaufenwasfoundtoplayaroleinmRNAdecay[188].Yeasttwo-hybridanalysisrevealedthatStaufen-1(STAU1)interactswithUPF1,andthisinteractionwasconfirmedbyGSTpull-downsandfarwesternblotting(Table3).TetheringofSTAU1toareportermRNAcaninduceUPF1-dependentandtransla-tion-dependentmRNAdegradationcalledSMD(forSTAU1-mediatedmRNAdecay)[188].NaturaltargetsofSMDareexpectedtobindSTAU1downstreamofthetranslationterminationcodonand,upondepletionofeitherSTAU1orUPF1,themRNAshouldbestabilised.InHeLacellsdepletedforSTAU1,approximately1%ofthetran-scriptomewasmorethantwofoldup-regulatedwhilstapproximately1%wasmorethantwofolddown-regulated,inferringthatSTAU1potentiallyactstoregulateamulti-tudeoffunctionallyunrelatedphysiologicaltranscriptsandcaninfluencemanymetabolicpathways[1].

InadditiontoSTAU1,thereisasecondStaufenproteincalledSTAU2thatisencodedbyadifferentgeneandshares51%aminoacididentitywithSTAU1[190].Con-sideringthehighsequencesimilaritybetweenSTAU1andSTAU2andalsotheevidencethatSTAU2co-immuno-precipitateswithmRNAfromhumancells[190,191],agenome-wideapproachwasundertakentoexamineifSTAU1andSTAU2canbindtoandregulateasimilarcollectionofmRNAs.DistinctbutoverlappingsubsetsofcellularmRNAswerefoundtoassociatewithSTAU1andSTAU2containingRNPcomplexes[190].Tofurtherelu-cidatetheinvolvementoftheStaufenproteinsinmammalianRNAmetabolism,thestructureoftheSTAU1andSTAU2bindingsitesandtheirpositionrelativetothetranslationstartandstopcodonswereexamined.ThepositionoftheSTAU1bindingsiteiscriticalasitdeter-mineswhetherthemRNAistargetedforenhancedtranslation[192]orifitistobedegradedbySMD[188].ItappearsthatSMDisamechanismthatdependsontrans-lationandiselicitedwhenSTAU1bindstoitshairpinbindingsiteapproximately25–30nucleotidesdownstreamfromthetranslationterminationcodon.FurtheranalysishasindicatedthattheSTAU1bindingsiterequiresmoreelaboratesecondarystructurethanasinglehairpinelement[1].STAU1isthoughttorecruitUPF1tothe30UTRvia

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Table3SummaryofadditionalNMDindependentfunctionsforUPF1,SMG1andSMG6indifferentcellularpathwaysFactorUPF1

Reportedroles

Staufen-mediateddecaypathway:STAU1bindsthe3’UTRandrecruitsUPF1toelicitdecayofthemRNADNAreplication,repairandstabilitypathways:UPF1contributestothecompletionofDNAreplicationandpropercellcycleprogression

Notesofinterest

NMDandSMDarecompetitivepathways,UPF1bindseitherUPF2orSTAU1butnottobothsimultaneously

UPF1depletionresultsinanearlyS-phasearrest.UPF1associateswithchromatininacellcycleregulatedmannerandwhenATRisdepleted,chromatinloadingofUPF1isimpaired.c-irradiationincreasesUPF1chromatinassociation.UPF1

co-immunoprecipitateswithp66subunitandp125subunitsofDNApolymerasedUPF1depletionstabiliseshistonemRNAafterHUtreatmentandattheendof

S-phase.ImmunoprecipitationshowedthatUPF1interactswithSLBP/HBP

UPF1isacomponentoftheHIV-1RNPanditsfunctionrequiresitsATPaseactivity.ItsrecruitmentmaybemediatedbySTAU1.over-expressionofUPF1resultsinup-regulationofHIV-1expressionattheRNAandproteinlevel

Detectedintelomericchromatinfractions.DepletionofUPF1increasesTERRAattelomeresandleadstotelomeredamageSMG1isactivatedbyDNAdamageandstimulatedbyUVorc-radiation.DepletionofSMG1resultsinDNAdamageandincreasedsensitivitytoradiation.Phosphorylatesp53invitroanduponc-radiationinvivo.AlongwithATM,SMG1canphosphorylateUPF1inresponsetodsDNAbreakscausedbyc-radiation.SMG1isimportantforoxidativestressprotectioninC.elegansandprotects

againstTNF-ainducedapoptosisinhumancells.SMG1alongwithATMcanmodulatep21levelstoinhibitcelldivision,induceDNArepairorblockapoptosisDetectedintelomericchromatinfractions.DepletionofSMG1increasesTERRAsignalsandleadstotelomeredamageDetectedintelomericchromatinfractions.Physicallyinteractswithtelomerase.DepletionofSMG6increasesTERRAsignalsandleadstotelomeredamage.Over-expressionresultsinend-to-endchromosomefusionsandalteredtelomerelengths

P.Nicholsonetal.

References[188,1,193]

[103,104,207,208]

ReplicationdependenthistonemRNAmetabolism:UPF1isimportantforhistonemRNAdegradation

HIV-1metabolism:possiblyUPF1

stabilisesHIV-1RNAininfectedcells

[214]

[194]

Telomeremetabolism:possiblyUPF1facilitatesTERRAreleasefromtelomeres

SMG1

Protectionofgenomicstability:SMG1initiatescellularstressresponseswhengenomeintegrity,mRNAtranslationornutrientavailabilityiscompromised

[103,104,202,203]

[106,222,224]

Telomeremetabolism:possiblySMG1facilitatesTERRAreleasefromtelomeres

SMG6

Telomeremetabolism:possiblySMG6facilitatesTERRAreleasefromtelomeres

[103,104,202,203]

[103,104,202–204]

directinteraction[188]and,therefore,SMDmaybetrig-geredbyUPF1interactingwiththeeRFsatthetranslationterminationcodonanalogoustoNMD.Withthisinmind,itwillbeinsightfultodetermineifSMDalsoinvolvestheUPF1bindingendonucleaseSMG6,whenitelicitsdegra-dationofitstargettranscripts.Interestingly,theSTAU1andUPF2-bindingsiteswithinUPF1haverecentlybeenshowntooverlap,renderingSTAU1andUPF2bindingtoUPF1mutuallyexclusive[193].DuringthedifferentiationofC2C12myoblaststomyotubes,NMDandSMDpath-waysappeartobeincompetition:SMDactivityincreaseswhiletheactivityoftheUPF2-dependentNMDpathwaydecreases[193].Insupportofthephysiologicalimportanceofthiscompetition,SMDtargetsPAX3mRNAwhose

NMDinhumancellsdecaypromotesmyogenesis,whilethemRNAencodingformyogenin,aproteinrequiredformyogenesis,isanNMDtarget[193].

STAU1andUPF1arebothinvolvedinHIV-1metabolism

UPF1andSTAU1werealsoreportedtofunctiontogetherinHIV-1RNAmetabolism[194–196](Table3).STAU1wasshowntoassociatewithHIV-1genomicRNAandtwotofiveSTAU1proteinsareincorporatedperHIV-1virion[197].STAU1over-expressioncanincreasetheabundanceofHIV-1genomicRNAandofSTAU1proteinpackagedintothevirion[196].RNAi-mediateddepletionofSTAU1resultedinasubstantialdecreaseofviralinfectivity[195].Moreover,STAU1wasshowninanRNA-dependentmannertodirectlyinteractwiththenucleocapsiddomainofthepr55Gag,akeymediatorofHIV-1genomicRNAen-capsidation[195].Therefore,theHIV-1RNPcontainsthemajorstructuralproteinpr55Gag,viralgenomicRNAandthehostproteinSTAU1,andithasbeenproposedthatSTAU1togetherwithpr55Gagplaysanimportantroleinviralassembly,genomicRNAencapsidationandthegen-erationofinfectiousviralparticles[195].

Recently,UPF1wasalsoidentifiedasacomponentoftheHIV-1RNP[194].ThepresenceofUPF1withtheHIV-1RNPismostlikelymediatedbySTAU1anditdoesnotrequireanyinteractionsbetweenUPF2andUPF3.KnockdownofUPF1ledtoalargereductioninsteady-stateHIV-1RNAandpr55Gagproteinlevels,andover-expressionofUPF1resultedinup-regulationofHIV-1expressionatthelevelofbothRNAandprotein.TheeffectsofUPF1onHIV-1RNAstabilityweredependentonitsATPaseactivityandrequiredongoingtranslation[194].ItwassuggestedthattargetingofUPF1functionmightrepresentasuitableapproachtoarrestHIV-1lateinthereplicationcycle[194].FurtherworkwillshedlightonhowUPF1isrecruitedtotheHIV-1RNP,itsexactroleandalsoifUPF1functionisutilisedbyotherretroviruses.NMDfactorsfunctionattelomeres

Telomeresaretheheterochromaticstructureslocatedattheterminioflinearchromosomes.Theycompensateforincompletesemi-conservativeDNAreplicationandalsoprotectthechromosomalendsagainstrecombinationwitheachotherandwithdouble-strandbreaks(DSBs)insidethechromosomes.Whilerepetitivesequencescoveranaverageof350basepairsinyeasttelomeres,humantelomeresexceedseveralkilobasescomposedofTTAGGGrepeats[198,199].

ThefirstconnectionbetweenNMDfactorsandtelomerefunctionwasreportedwhenitwasdiscoveredthat691

mutationsofUpf1p,Upf2pandUpf3pinyeastledtotelomereshorteningandde-repressionofsilencedtelo-mericloci[200,201].ConsistentwiththeideathatNMDaffectstheexpressionofgenesimportantfortelomerefunction,itwasfoundthatNMDmutantstrainshadincreasedlevelsofmRNAsencodingthetelomerasecata-lyticsubunit(Est2p),regulatorsoftelomerase(Est1p,Est3p,Stn1p,Ten1p)andproteinsimplicatedinregulationoftelomericchromatinstructure(Sas2p,Orc5p)[200].However,thepicturebecamemorecomplicatedwhenabioinformaticssearchforhumanhomologuesoftheS.cerevisiaeevershortertelomeres1(Est1)geneidentifiedthesamethreeproteinsthatwereindependentlyidentifiedashumanorthologuesoftheC.elegansNMDfactorsSMG5(=EST1B),SMG6(=EST1A),andSMG7(=EST1C)[202,203].Moreover,EST1A/SMG6andEST1B/SMG5werefoundtoassociatewithtelomeraseactivityinhumancellextractsandover-expressionofEST1A/SMG6ledtoend-to-endchromosomefusionsandalteredtelomerelengths[202,203](Table3).EST1A/SMG6interactswithtelomerasebybindingtothetelo-meraseRNAwithhighaffinitybutlowspecificity,andalsobymakingprotein–proteincontactswithtelomerasereversetranscriptase(TERT)[204].

Foralongtime,telomereswerebelievedtobetrans-criptionallyinactive.However,recently,asetofpolIItranscriptscalledtelomericrepeat-containingRNA(TERRA),alsoknownasTelRNA,havebeendiscoveredinhuman[104,205]andyeastcells[206](reviewedin[207]).Fascinatingly,enrichmentofNMDfactorswasdetectedintelomericchromatinfractions,andthedepletionofUPF1,SMG1andEST1A/SMG6(andtoalesserextentUPF2andEST1C/SMG7)increasedTERRAsignalsandtriggeredtelomeredamage,includingcompletetelomereloss[103,104].DespitethepresenceofoneUAGstopcodonineachtelomericrepeatsequence,itseemsthattheroleoftheNMDfactorsismostlikelynottostimulateTERRAdeg-radationbutrathertoreduceitsassociationwithtelomeres[104].IftheincreasedlevelofTERRAattelomeresisthecauseofthetelomerede-protectionobservedinNMD-deficientcells,thiswouldsuggestthatTERRAisunfavourablefortelomerestability.Ontheotherhand,ifNMD-depletiontriggerstelomeredamageindependentlyofaneffectonTERRA,theincreasedassociationofTERRAwithtelomerescouldbestimulatedbythecellinanattempttostrengthentelomereprotection.Similarly,theincreasedlevelsofTERRAobservedwiththermalshockmayhelptoprotecttelomeresagainststress-mediateddamage[205].TERRAmightalsobeinvolvedintheregulationoftelo-meraseactivity.EST1A/SMG6physicallyinteractswithtelomerase[204]and,invitro,TERRAprobablyinhibitstelomerasebyRNAduplexformationinthetemplateregionofthetelomeraseRNAcomponent(TERC)[205].

692TheeffectofTERRAontelomeraseremainstobetestedinvivo.

TherolesofUPF1inDNAreplicationandrepairEvidencefromseveraldifferentstudieshasconvergedinrecentyearstoshowthatUPF1playsasignificantroleinDNAandRNAmaintenancepathwaysthatarenecessaryforthecelltoaccomplishDNAreplication(Table3).UPF1depletionwasreportedtoresultinanearlyS-phasearrest,inwhichthecellscouldfirethestartofDNArep-licationbutwereunabletocompleteDNAreplication,andinducedanATR-dependentDNA-damageresponse[103].ThecellsaccumulatednuclearfocicomprisingtheS-phasemarkerproliferationcellnuclearantigen(PCNA),indi-catingthatUPF1isessentialforaccomplishingDNAreplicationduringS-phaseofthecellcycle.Notably,knockingdownUPF2causednoadverseeffecttoS-phaseprogression,suggestingthatUPF1’sfunctioninDNAreplicationisunrelatedtoNMD[103].Itwasfurtherobservedthathyper-phosphorylatedUPF1wasassociatedwithchromatinandthattheamountofchromatin-associ-atedUPF1vastlyincreasedduringS-phaseandalsouponc-irradiation[103].WhenATRwasdepleted,chromatinloadingofUPF1wasimpaired,whereasNMDwasnotaffectedundersuchconditions[103].Thisimpliesthat,eitherduringDNAreplicationand/oraDNArepairresponse,ATRphosphorylatesUPF1leadingtoitsasso-ciationwithchromatin,oralternativelythatUPF1ispresentonthechromatinduringreplicationandbecomesphosphorylatedbyATRinresponsetoDNAdamage.EvidencefortheinvolvementofUPF1inDNAreplicationandrepairiscompoundedfurtherbythefindingthatUPF1co-immunoprecipitateswiththep66subunitandp125catalyticsubunitofthereplicativeDNApolymerased.Incontrast,UPF2wasnotabletoco-immunoprecipitatewithp125DNApolymerased[103,208].ThefunctionofUPF1inDNAreplicationandcellcycleprogressionbecomesevenmoreintricatebyevidenceshowingthatUPF1isinvolvedinthedegradationofreplicationdependenthis-tonetranscriptsuponDNAreplicationinhibitionandattheendofS-phase[209].

UPF1actsinthedegradationofreplication-dependenthistonemRNAs

Thereplication-dependenthistonegenesprovidethelargeamountofhistoneproteins(108moleculesofeachcorehistoneprotein)requiredforgenomeduplication[210].HistoneproteinsareproducedduringS-phaseofthecellcycleandtheirexpressionisco-ordinatelyregulatedandfinelybalancedwithDNAreplication.Threemajorpro-cesses,transcription,mRNA30endprocessingandmRNAP.Nicholsonetal.

stabilitycontrol,contributetothecomplexregulationofhistonegeneexpressionduringS-phase[211].Thiscombinationoftranscriptionalandpost-transcriptionalmechanismsactstoregulatethe35-foldincreaseofhistonemRNAlevelsascellsprogressfromG1-intoS-phaseandensuresthatthemRNAabundancereturnstobaselinelevelsasthecellsexitS-phase.Thehalf-lifeofhistonemRNAsdecreasesfrom45–60minduringS-phasetoapproximately10minattheendofS-phaseduetoareg-ulatorymechanismthatactstorapidlyeliminatehistonemRNAsfromthecytoplasmwhenDNAsynthesisiscompletedorinhibited.SincehistonegenetranscriptionisonlyincreasedthreetofivefoldduringS-phase,thepost-transcriptionalregulationaccountsfortheremainingfivetosixfoldincreaseinhistonemRNAlevelsandfortheirrapideliminationwhenDNAreplicationiscompletedorinhib-ited(reviewedin[211–213].

HistoneproductionandDNAsynthesisareintimatelycoupledbyapoorlyunderstoodmechanisminvolvingcheckpointkinases,suchasATRandDNA-activatedpro-teinkinase(DNA-PK)[214,215].Additionally,UPF1hasalsobeenreportedtoplayaroleinhistonemRNAdesta-bilisation[214–216](Table3).RNAi-mediatedknockdownofUPF1(butnotofUPF2)orover-expressionofdominant-negativeUPF1mutants(K498AandR843C)resultedinanincreasedhistoneH2AmRNAlevelafterinhibitionofDNAsynthesisbyhydroxyurea(HU)treat-mentorattheendofS-phase[214].Furthermore,aweakinteractionbetweenUPF1andthehistonestem-loopbindingprotein(SLBP;alsocalledhairpinbindingprotein,HBP)wasdetectedinimmunoprecipitationexperiments.Recently,ithasbeenrevealedthatthedegradationofhis-tonemRNAsrequiresmanyofthefactorsinvolvedindegradationofpolyadenylatedmRNA,suchasLSM1,DCP2,XRN1andtheexosomecomponentsPM/Scl-100(RRP6)andEXOSC4(RRP41)[217].Furthermore,ithasbeenreportedthathistonemRNAdegradationbeginswiththeadditionof8–12uridinesbyuridylyltransferases(TUTases)tothe30endofthehistonemRNA[217].TheconcentrationofoligouridylatedhistonemRNAsincreasedstrongly15minafterHUtreatment,andknockdownofTUTases1and3reducedtherateofhistonemRNAdeg-radation[217].UPF1hasbeenproposedtobeinvolvedintherecruitmentoftheTUTase1and3tothehistonemRNA[217].Alternatively,ithasalsobeensuggestedthattheRNAhelicaseactivityofUPF1mayremodelthemRNPcomplextoallowtheTUTasestobindthe30endofthemRNA[218].Sofar,thereisnodirectevidenceforeitheroftheserolesbyUPF1,anditsfunctioninhistonemRNAmetabolismremainsunclear.

Asmentioned,thecouplingofDNAreplicationandhistonemRNAstabilityisthoughttobemediatedbyPIKKsactingupondownstreameffectors.Inadditionto

NMDinhumancellsSMG1(alsocalledATX),themammalianPIKKsincludeATM,ATR,mTOR/FRAPandDNA-PK.CheckpointregulationinmammalsisintricateandultimatelyunevenlydividedbetweenATMandATR[219].ATM,ATRandDNA-PKareactivatedbyvariousformsofDNAdamage.ATRisactivatedbyaberrantDNAstructuresinducedbyUVlightorreplicationalstresscausedbyDNAreplicationinhibitors,bothleadingtostallingofthereplicationfork.ATMismainlyactivatedbyDSBspredominatelyafterexposuretoionisingradiation.DNA-PKisrequiredforDSBrepairbynon-homologousend-joining(NHEJ)andtelomeremaintenance.Inarecentstudy,KaygunandMarzluffreportedthatATRbutnotATMisrequiredforhistonemRNAdegradationafterinhibitionofDNArepli-cation[209].Mu

¨lleretal.reportedthatATRisnotfunctionallylimitingfortherateorextentofhistonemRNAdecayinducedbyreplicationstressandcannotfullyaccountforthecouplingbetweenDNAreplicationandhistonemRNAstability.ExposureofcellstoaninhibitorofDNA-PKunveiledDNA-PKalsotobeinvolvedinlinkinghistonemRNAabundancewithDNAreplication[215].DNA-PKisactivatedduringreplicationstressandtheDNA-PKpathwayisenhancedwhenATRsignallingfails,highlightingthecomplexnatureofsuchsignallingpath-waysandshowingthat,invivo,therelativecontributionfromeachsignallingpathwaywouldbebasedonthenatureoftheDNAlesiongeneratingthereplicationstress.SinceSMG1interactswithandphosphorylatesUPF1,andbecauseofitsresemblancetotheseotherPIKKswithdocumentedrolesinregulationofcellcycleandDNAreplication,itistemptingtospeculatethatSMG1mayalsocontributetothecomplexsignallinginvolvedinthecou-plingofDNAreplicationandhistonemRNAstability.AroleforSMG1insafeguardinggenomeintegrityThehumanmembersofthePIKKfamilyallcompriseFAT,FATCandPI3Kdomains.SMG1differsfromtheotherPIKKsbecausethePI3KdomainisseparatedfromtheFATCdomainbyalargeinsertofover1,000aminoacids,whereasonlyapproximately100aminoacidssepa-ratethesetwodomainsintheotherPIKKs[61,75,220].InC.elegansSMG1,this1,000aminoacidssectiondoesnotexist.Possibly,thisportionappearedduringmetazoanevolutionandallowsSMG1tointeractwithabroaderrangeofupstreamregulatoryproteinsanddownstreamtargets[221].Inhumancells,depletionofSMG1resultsinspontaneousDNAdamageandahugelyincreasedsensi-tivitytoionisingradiation[106](Table3).ExposureofcellstoUVlightorionisingradiationstimulatesSMG1kinaseactivity.Moreover,likeATM,SMG1isabletophosphorylatep53atserine15andexpressionofSMG1isrequiredforoptimalp53activationaftergenotoxicstress.

693

Therefore,similartoATMandATR,SMG1iscentraltotheregulationofthegenotoxicstressresponsemachineryinmammaliancells.RecentreportshavealsoimplicatedSMG1tobeimportantintheresponsetooxidativestress[222].SMG1hasbeenobservedtoinitiatep53phosphor-ylationduringtheearlystagesofoxidativestress,andATMactstomaintainthep53phosphorylationovertime.Currently,itisnotclearwhytheactivationofSMG1precedesthatofATMinhyperoxiaconditions.Experi-mentsusingcellsdepletedofATMalsoindicatedthatSMG1andATMactsequentiallyandindependentlyofeachothertoregulatetheG1-checkpointduringprolongedoxidativestress[222].Potentially,SMG1andATMmayrecogniseandrespondtodifferentlesionsthatareproducedduetoprolongedhyperoxia.Phosphorylatedp53activatestranscriptionofp21and,interestingly,itwasshownthatthedestabilisationofp21duringhyperoxiacouldberestoredbytreatmentwithwortmannin,implyingtheinvolvementofPIKKs[222].HumanSMG1andATMalsoact,independentlyofp53,totargetp21fordegradationbytheproteasome.Hence,thisdualfunctionofSMG1andATMwhichcanregulateboththesynthesisanddegrada-tionofp21,therebyfinelycontrollingthep21levelsnecessarytoinhibitcellproliferation,induceDNArepairandblockapoptosis[222].

SimilarlySMG1wasalsofoundtobeimportantinrespondingtooxidativestressinC.elegans.InastudyinvestigatinglifespanregulationinC.elegans,SMG1wasidentifiedinascreenforgenesthatprolonglifespaninadaf-18-dependentmanner[223].FurtheranalysisrevealedthattheSMG1functioninlifespancontrolrequirescep-1,theC.elegansorthologueofp53.Moreover,theroleofSMG1inlifespancontrolisduetoitsfunctioninoxidativestressresponse.Interestingly,itwasalsoreportedthatinS.pombe,Upf1pandUpf2parerequiredforsurvivalofoxidativestress[21].ToinvestigatethepossibilitythatSMG1mayhaveafunctioninstresssignallinginducedbycytokines,awiderangeofagentsthatcaninducecelldeathwereexaminedinSMG1-,ATM-,ATR-,UPF1-orUPF2-depletedcells[224].OnlydepletionofSMG1,butnotoftheotherfactors,substantiallyincreasedtherateandtheextentofapoptoticcelldeathmediatedbytumournecrosisfactor-alpha(TNF-a)[224].SMG1,likeotherPIKKs,functionstoinitiatecellularstressresponseswhengenomeintegrity,mRNAtranslationornutrientavailabilityiscompromised,andtheroleofSMG1inNMDrepresentssimplyjustoneofitsmanyroles.

Conclusionandfuturedirections

Apartfromincreasingourunderstandingofthefunda-mentalmolecularmechanismsthatcontroltheextentand

694accuracyoftheexpressionofourgeneticinformation,aprincipalgoalofstudiesonNMDistoeventuallyutilisetheknowledgeforcuringdiseasesandimprovinghealth.Inspiteofsignificantadvancesduringrecentyears,wearestillfarfromunderstandingthecompletedetailsregardinghowmRNAsarerecognisedasNMDtargets,theirsub-sequentdegradationandthephysiologicalimportanceofNMDfactorsregulatingupto10%ofthetranscriptome.TheunifiedNMDmodelmakesmanytestablepredic-tionsandwillhopefullyprovideausefulframeworkforfuturemechanisticinvestigations.Themodelemphasisestherequirementofaproperlyconfigured30UTRfornormaltranslationtermination.ThepresenceofPABPnearbyaterminatingribosomeisonecrucialcharacteristicforaproperlyconfigured30UTR,butadditionalfeaturesareexpectedtoexist.Deviationsfromthis30UTRconfigura-tionorspecificfactorsthatprohibitthetermination-promotinginteractionofPABPwiththetranslationterminationcomplexsignalsananomalyinthemRNAmoleculeandtriggersNMD.ThemechanisticdetailsofUPF1recruitmentontotheSURFcomplexanditssub-sequentassemblywiththeribosomeremaintobeworkedout.Inaddition,severalopenquestionsregardingthedeg-radationpathway(s)ofNMDsubstratesneedtobeaddressed.Forexample,doesmammalianNMDreallycomprisetwoindependentwaystoinitiateRNAdecay:anendonucleolyticSMG6-dependentrouteandadecapping-dependentexonucleolyticroute?

Finally,ithascometolightinrecentyearsthatseveralfactorswhichwereinitiallyidentifiedandcharacterisedfortheirrolesintheNMDpathwayhavehighlyimportantfunctionsinthecellthatareindependentofNMD.ItisbecomingmoreevidentthatUPF1playsmanyroleswithregardstotheregulationofS-phaseprogression,consid-eringithasbeenreportedtobeinvolvedinreplication-dependenthistonemRNAmetabolism,thereplicationmachinery,andinconjunctionwithSMG6intelomeremaintenance.Additionally,thereisanincreasedassocia-tionofUPF1ontochromatinduringS-phaseanddepletionofUPF1leadstoanearlyS-phasearrest.Likewise,therecentlyreportedfunctionsofSMG1alsoindicatethisproteintobeultimatelyrequiredforgenomestability.ItismoredifficulttorelatethefunctionofUPF1inSMDandinHIV-1RNAmetabolismtoitsS-phaseandtelomerefunctions.ItisattractivetospeculateinanevolutionarycontextaboutwhenUPF1acquiredthesefunctionsinadditiontoitsroleinNMD.Replication-dependenthis-tonesinyeastarepolyadenylatedanddonotcontainSLBP/HBP;perhapsthisiswhydepletionofUPF1isdetrimentaltohumancellsbutnottoyeastcells.AnimportantgoalforthefutureistobegintodecipherwhatconstitutesadirecteffectandanindirecteffectwhentheNMDfactorsaredepletedandgenome-widemicroarrayprofilingis

P.Nicholsonetal.

conducted.ThefuturewillalsobeaboutdeterminingtheparametersforeachofthemanydifferentrolesthatUPF1carriesout,forinstanceitscellularlocation,itsroleincellcyclecontrol,whereandwhenitisphosphorylated,whe-therthereisaspecificphosphorylationpatterndirectingUPF1toitsfunction,itsstabilityandwhereandwhenitsmanyinteractionpartnersbind.MakingsenseofallthemolecularfunctionsofUPF1willnotonlybecriticaltounderstandingthemechanismofNMD,butalsotoeluci-dationofalloftheotherprocesseswhereUPF1hasbeenfoundtoplayarole.

AcknowledgmentsTheresearchoftheauthorsissupportedbygrantsfromtheEuropeanResearchCouncil,theSwissNationalScienceFoundation,theNovartisFoundationforBiomedicalResearch,theHelmutHortenFoundationandalsobytheKanton

Bern.O.M.isafellowoftheMaxCloe

¨ttaFoundationandR.Z.O.issupportedbyafellowshipfromCONACYTMe

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