A perspective on mammalian upstream open reading frame function

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The

International

Journal

of

Biochemistry

&

Cell

Biology

j o u r n al ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / b i o c e l

Review

A

perspective

on

mammalian

upstream

open

reading

frame

function

Joanna

Somers

,

Tuija

Pöyry

,

Anne

E.

Willis

MedicalResearchCouncilToxicologyUnit,LancasterRd,LeicesterLE19HN,UnitedKingdom

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received27February2013

Receivedinrevisedform16April2013 Accepted17April2013

Available online 25 April 2013 Keywords:

Proteinsynthesis uORF

Upstreamopenreadingframe Translationalcontrol

a

b

s

t

r

a

c

t

Post-transcriptionalcontrolmakesamajorcontributiontotheoverallregulationofgeneexpression pathway.WithinthecytoplasmthisismediatedbyacombinationofregulatoryRNAmotifswithinthe 5and3untranslatedregionsofmRNAsandtheirinteractingprotein/RNApartners.Oneofthemost commonregulatoryRNAelementsinmammaliantranscripts(presentinapproximately40%ofallmRNAs) areupstreamopenreadingframes(uORFs).However,despitetheprevalenceoftheseRNAelementshow theyfunctionisnotwellunderstood.Ingeneral,theyacttorepresstranslationofthephysiologicalORF undercontrolconditions,andundercertainpathophysiologicalstressesthisrepressioncanbealleviated. Itisknownthatre-initiationfollowingthetranslationofanuORFisutilisedinsomesituationshowever therearenumerousalternativemechanismsthatcontrolthesynthesisofaproteinwhosemRNAcontains uORFs.Moreover,thetrans-actingfactorsthatarealsoinvolvedinthisprocessarenotwelldefined.In thisreviewwesummariseourcurrentunderstandingofthisareaandhighlightsomecommonfeatures oftheseRNAmotifsthathavebeendiscoveredtodate.

© 2013 The Authors. Published by Elsevier Ltd.

Contents

1. Introduction... 1690

2. Reinitiation... 1692

2.1. eIF2concentrationandreinitiation... 1693

3. Mechanismsotherthanreinitiation... 1694

3.1. Upstreamopenreadingframenucleotidedependence... 1694

3.2. Upstreamopenreadingframepeptideexpressionandfunction... 1695

3.3. Upstreamopenreadingframebypass... 1696

3.4. Internalribosomeentrysegments... 1697

3.5. Nonsensemediateddecay ... 1697

4. Non-AUGupstreamopenreadingframes... 1697

5. Upstreamopenreadingframesanddisease... 1698

6. Conclusion ... 1698

Acknowledgements... 1698

References... 1698

Abbreviations: 4EBPs,4E-bindingproteins;AdoMetDCS,adenosyl-methionine decarboxylase;Eif,eukaryoticinitiationfactor;EMCV,encephalomyocarditisvirus; IRES,internalribosomeentrysegment;NMD,nonsensemediateddecay;RRL,rabbit reticulocytelysate;SNP,singlenucleotidepolymorphism;THPO,thrombopoietin; uORF,upstreamopenreadingframe;UTR,untranslatedregion.

∗ Correspondingauthors.Tel.:+441162525611.

E-mailaddresses:[email protected](J.Somers),[email protected](T.Pöyry), [email protected](A.E.Willis).

1. Introduction

Geneexpressioncanberegulatedatmultiplelevelsincluding: transcription,mRNAprocessingandlocalisation,protein transla-tionandproteinstability.Itisnowwellacceptedthatcontrolof translationmakesamajorcontributiontotheoverallregulation ofgeneexpressionalthoughincomparisontotranscriptionithas beenlessextensivelystudied.Regulationattheleveloftranslation allowsarapidandusuallyreversibleresponsetovariousinternal orexternalsignalsinthelife-cycleofacell.

Translation can be considered as a three-step process that iscomprisedofinitiation,elongationandtermination.Although 1357-2725 © 2013 The Authors. Published by Elsevier Ltd.

http://dx.doi.org/10.1016/j.biocel.2013.04.020

Open access under CC BY-NC-ND license.

J.Somersetal./TheInternationalJournalofBiochemistry&CellBiology45 (2013) 1690–1700 1691

Fig.1. Translationinitiationanditsregulation.(A)Schematicoftranslationinitiationpathway.Twocomplexesarerequiredforinitiationoftranslation;theeIF4Fcomplex andtheeIF2-TC.eIF2-TCinteractswiththe40Sribosomalsubunittoformthe43Spreinitiationcomplexwhichtheninteracts(viaeIF3bindingtoeIF4G)witheIF4Fcomplex toformtheinitiationcompetent43Spreinitiationcomplex.TranslationinitiationcanbecontrolledbyregulatingthelevelsofeIF2-TCoreIF4Fcomplex.(B)eIF4Fcomplex isregulatedbycontrollingtheavailabilityofthecap-bindingproteineIF4E.DephosphorylationoftheeIF4Ebindingpartners,4EBPs,allowsthemtobindtoeIF4Eandso reducetheamountofthisproteinthatisavailableforeIF4Fcomplexformation.(C)eIF2-TCiscontrolledbyphosphorylationofthealphasubunitofeIF2byitskinaseswhich resultsinsequestrationofeIF2anditsGEFeIF2Binaninactivecomplex.ThislimitsGTP:GDPrecyclingoneIF2andthereforereducestheamountofeIF2-TCavailable. all three stages can be regulated, translation is primarily

reg-ulated at the initiation stage and this is assumed to be the rate-limiting step of the process (Sonenberg and Hinnebusch, 2009).Translationinitiationisanintricateprocessthatculminates inanelongation-competent80Sribosomeformingatthe initia-tioncodon.Essentiallytwocomplexesarerequiredforthisprocess whichfunctiontobindtothemRNA,viathem7_GTPcapatthe5

endofthemessage,recruittheribosomeandbringtheinitiator Met-tRNAi tothestartcodon.TheeIF4Fcomplexisrequiredfor

bothcap-bindingandribosomerecruitmenttothemessage.This complexiscomprisedofeIF4E(thecapbindingprotein),eIF4G(a scaffoldprotein)andeIF4A(aDEAD-boxhelicase)(Fig.1A).eIF4G alsocontains binding sitesfor eIF3and poly A bindingprotein (PABP).Theothercomplex, theeIF2ternarycomplex (eIF2-TC), containseIF2,GTPandtheMet-tRNAi.Onceformedthiscomplex

isrecruitedtothe40SribosomalsubunitalongwitheIF1,eIF1A,

eIF3,andpossibly eIF5,formingthe43Spre-initiationcomplex. The43ScomplextheninteractswiththeeIF4FcomplexviaeIF3 bindingtoeIF4Gtoformthescanning competent43Scomplex (Fig.1A).Thiscomplexisadditionallystabilisedbytheinteractionof eIF4GwithPABPandofeIF4BwitheIF4AandPABP(Sonenbergand Hinnebusch,2009;Bushelletal.,2001).Thescanningmodelthen predictsthatthe43Scomplexprogressivelyscansalongthe mes-sageuntilasuitableinitiationcodonisrecognised.Althoughthe codonAUGisusedprimarilyasthe“startcodon”therearemany examplesofnon-AUGstartcodonsthatareusedincludingGUGand CUG(Tourioletal.,2003).Oncethestartcodonisrecognisedand a48Spreinitiationcomplexhasbeenformed,joiningofthe60S ribosomalsubunitoccurstoformthe80Sribosome(reviewedby Jacksonetal.,2010).

Overall translation ratescan beregulatedbycontrolling the levelsandactivitiesofthecomponentsoftheeIF4FandeIF2-TC

complexes. Thus the availability of eIF4E is dependent on the phosphorylation status of its interacting partners, 4E-binding proteins(4EBPs). Whenphosphorylated they release eIF4E and thisincreasesthenetamountofproteinthat isabletointeract witheIF4GtoformtheeIF4Fcomplex(Fig.1B).Signallingthrough the phosphoinositide 3-kinase pathway (PI3K) regulates 4EBP phosphorylation and when activated by growth factors, PI3K phosphorylatesAkt,whichinturnphosphorylatesthemammalian target of rapamycin (mTOR) leading to 4EBP phosphorylation and upregulation of translation. Since there is an increase in both the levelsand phosphorylation status of translation initi-ation factorsin cancers,drugs that targetthesepathwayssuch as rapamycin (which targets mTOR) are being investigated as potentialtherapeuticagents(BlagdenandWillis,2011).

eIF2-TCformationisregulatedbyalteringthephosphorylation statusofeIF2.Thisproteiniscomprisedof3subunitsand phos-phorylationoccursonthealphasubunitatpositionSer51(Dever and Sicheri, 2007). The phosphorylationof eIF2 inhibits trans-lationsince phosphorylated eIF2 binds withhighaffinity toits guaninenucleotideexchangefactoreIF2Bthatispresentinlimiting amounts.This prevents eIF2recycling, lowering theamount of eIF2-TCformation,andinhibitstranslation(Fig.1C).Therearefour kinases that phosphorylate eIF2 that are activatedby different externalstimuli(Spriggsetal.,2010).Forexample,GCN2(General controlnon-derepressible2)isactivatedfollowingnutrient star-vationandUVBexposure(Powleyetal.,2009;Spriggsetal.,2010), PERK(PKR-like ERkinase) is activated whenunfolded proteins accumulateinthecelle.g.inpriondisease(Morenoetal.,2012),PKR (RNA-dependentproteinkinase)isactivatedduringviralinfection, butalsoduringthedisruptionofsystemicmetabolichomeostasis (Hotamisligil,2006)andHRI(Haem-regulatedinhibitor)promotes survivaloferythroidprecursorswhenironlevelsarelow(Chen, 2007).

The main protein coding sequence of the mRNA is often flankedbyupstreamanddownstreamregions,whicharetermed untranslatedregions(UTRs).Theseregionsmaycontainregulatory elementssuchashairpins,proteinbindingsites,internalribosome entrysequences(IRES)elementsorupstreamopenreadingframes (uORF)inthe5UTRormiRNAbindingsites,localisationelements andpoly-Atailsignalsinthe3UTRs(Pichonetal.,2012).Herein, we willfocus ourattention ondiscussing the occurrences and behavioursofmammalianuORFs.

Severalbioinformaticandgeneticstudiesrevealedthat40–50% ofhumanandrodentmRNAscontainatleastoneuORF(Calvoetal., 2009;Iaconoetal.,2005;Matsuietal.,2007).Transcriptswithone uORFgenerallyhavea5UTRofatleast75nucleotides.The prob-abilitythatamRNAwillcontainmore thanoneuORFincreases withtheincreasinglengthofthe5UTR,althoughtheoccurrenceof upstreamAUGs(uAUGs)doesnotshowanycorrelationwith5UTR length(Fig.2)(Iaconoetal.,2005).BothuORFsanduAUGsoccurless frequentlythanexpectedbychanceimplyingthattheyareunder negativeselectionpressure(Calvoetal.,2009;Iaconoetal.,2005; Reschetal.,2009).However,thepresenceofmultipleuORFsseems tobeenrichedincertainsubgroupsofmRNAs,includinggenes cod-ingforgrowthfactors,transcriptionfactorsandproto-oncogenes (Davulurietal.,2000;Kozak,1987a).

BothuAUGoruORFs(whenuAUGisfollowedbyanin-frame stopcodon)aremajorregulatoryelementsinthe5UTRregions. Accordingtothescanningmodel the43Spreinitiationcomplex entersthemRNAatthe5capofthemRNAandscanssequentially alongthe5UTRuntilit encountersthefirstAUGcodon(Kozak, 1989b).Therearesomecircumstances,whichallowtheribosome toskipthefirstAUG,e.g.ifthefirstAUGisinasequencecontext which is not recognised efficiently by the scanning ribosome causingleakyscanning(discussedbelow)(Kozak,1986),orifthe firstAUGislocatedtooclosetothe5cap(Kozak,1991).However,

uAUGs/uORFslocatedinthe5UTRthatarerecognisedbythe40S ribosomal subunitwillby default downregulate thetranslation from the main open reading frame (mORF). The recent large scalegeneticandproteomicsstudyhasshownthatuORFsreduce proteinexpressionfromthedownstreammORFby30–80%(Calvo etal.,2009).

ForanuORFtobeabletoregulatedownstreamprotein expres-sion,it hastoberecognised bythe 40Sribosomesubunit.The mostfavourablenucleotidecontextsurroundingastartcodonhas beenshowntobeGCCA/GCCAUGG(termedKozakconsensus)of whichAat−3andGat+4(theAattheAUGcodonbeing+1)are themostimportantnucleotides(Kozak,1986,1989b).TheKozak context hasbeen shownto beespecially importantaroundthe non-AUGstartsites(Kozak,1989a;Peabody,1989).TheAatthe −3positionwasdemonstratedtointeractwiththeeIF2␣subunit andtheGat+4withhelix44inthe18SrRNAinexperimentsusing UV-crosslinkingandpurifiedfactors.Boththeseinteractionsare proposedtopromoteAUGrecognitionatthestartsite(Pisarevetal., 2006).However,inadditiontoAUGcontext,thesecondary struc-tureofthetranscript,especiallyfollowingthestartsite,hasbeen showntoplayanimportantroleinstartsiteselection(Kozak,1986, 1991).Interestingly,ithasbeenshownthatabout90%ofthemORF AUGstartsitesareeitherinastrong(both−3A/+4G)oradequate (either−3or+4)nucleotidecontext;therewasnolikewise‘bias’ forthestartsitesofuORFs(Iaconoetal.,2005).

TheuORFsidentifiedinmRNAstodatevaryinawidenumberof ways.Thus,theydisplaydifferencesin(i)length,(ii)number,(iii) distancefromthecap,(iv)whethertheuORFiscompletelywithin the5UTRoroverlappingwiththemORFand(v)distancebetween theuORFstopcodonandthestartsiteinthemORF(Fig.2).How allthesedifferentparameterscontributetothedownregulation oftranslationfromthemORFisacomplexprocessthatisnotwell understood.Inthisreview,wesummarisethecurrentliteraturein ordertogeneratesomecommonrulesfromthestudiescarriedout thusfar.

2. Reinitiation

Theabilityofeukaryoticribosomestoreinitiateafter termina-tionwasfoundunexpectedlyduringstudiesthatgaverisetothe scanningribosomemodel.AnexperimentwhichinsertedanuAUG intothe5UTRofthe(pre)proinsulinmRNAreducedtheamountof proteinexpressedfromthismRNA.However,insertinganin-frame stopcodonaftertheuAUGincreasedtheyieldof(pre)proinsulin compared to the mRNA only having the uAUG (Kozak, 1984). Followingstudiesshowed thatthe reinitiationefficiency atthe downstreamAUGincreasedwithanincreasingintercistronic dis-tance(thedistancebetweentheuORF-stopandthe‘reinitiation’ startcodondownstream)(Kozak,1987b).Thissuggestedthatthe ribosomeneededtoreacquiresomeinitiationfactorsduringthe scanningbeforebecomingcompetenttoallowreinitiationatan AUGcodondownstream.Oneoftheobviousfactorslackingfrom theribosomewaseIF2-TC,whichislostfromtheribosome dur-ingthefirstinitiationevent.Subsequentstudiesbothinyeastand mammaliansystemshaveshownthattheavailableeIF2-TC con-centrationcorrelateswiththedistancethe40Sribosomalsubunit needstoscan beforeit is ableto reinitiate(Hinnebusch, 2005; VattemandWek,2004;discussedinmoredetailbelow).

Reinitiationefficiencyisverydifficulttodetermineaccurately asit ishard toassesshow much isreally truereinitiationor a combinationofleakyscanningandreinitiation.However,efficient reinitiationhasbeendetectedafterveryshortuORFs(upto50%for GCN4)(Hinnebusch, 2005).The reinitiationefficiency decreases withtheincreasinglengthoftheuORF(Luukkonenetal.,1995),or ifthetranslationoftheuORFissloweddownbystrongsecondary structurewhichcausesthetranslatingribosometopause(Kozak,

J.Somersetal./TheInternationalJournalofBiochemistry&CellBiology45 (2013) 1690–1700 1693

Fig.2.ManypropertiesmaycontributetoanuORF’sroleinthetranslationalcontrolofamORF.Theseincludethelengthofthe5_{UTR,thesecondarystructureandGC}

content.ConsiderationofwheretheuORFissituated,includingthedistancefromthecapandtheintercistronicdistancebetweentheterminationoftheuORFandthemORF (A).ThesequenceoftheuORFmightbeimportant,whetheritisanAUGornon-AUGinitiatorcodon,thestrengthofthesurroundingKozakcontextandtheuORFlengthand conservation.ConservationofuORFsmayindicatearoleforthepeptidecodingsequenceoftheuORF(B).Longer5_{UTRstendtohavemultipleuORFs,soconsiderationof}

thedistancebetweentheseuORFsisimportant(C).Lastly,someuORFsdonotterminatewithinthe5UTR,rathertheyoverlapwiththemORF.TheseuORFsifrecognisedby ribosomeswillcauserepressionofthemORF(D).

1987b,2001).Togetherthesefindingssuggestthatthetimetaken totranslateanuORFismorecriticalthanthelengthoftheuORF per se.This impliesthat somecritical initiation factorsneeded forreinitiationarelostduringtranslationoftheuORFratherthan duringtheribosome‘subunitjoining’stageofinitiation.Aseriesof experimentswerecarriedouttoaddresswhetherreinitiationwas dependentontheeIFsthatinitiatedatthefirstuORFstartcodon. Usinganinvitrotranslationassayinrabbitreticulocytelysate(RRL) efficientreinitiationoccurredat20–35%whenanuORF-containing constructwasdrivenbyeither scanningdependentmechanism orby theencephalomyocarditisvirusIRES (EMCV), which both requiredtheeIF4Fcomplexforinitiation(exceptthatEMCVdoes notneedeIF4E)(Pöyryetal.,2004).However,noreintiationwas observediftheclassicalswinefevervirusIRES(whichdoesnot requireanyoftheeIF4familymembers)orthecricketparalysis virusIRES (which is not dependent onany eIFs)were used in thetranslationassay.WhenanuORFconstructthathad 19xCAA-repeatsinthe5UTR, whichcaninitiatetranslation withoutthe eIF4Fcomplex(PestovaandKolupaeva,2002)wereusedefficient reinitiationonlyoccurredinRRLthatcontainedalleIFsorinan eIF4G-depleted lysate supplemented with recombinant eIF4G p50 fragment (eIF4G central domain which has both eIF3 and eIF4Abindingsites).Thesedataledtoamodelthatsuggeststhat reinitiation willonlyoccuriftheeIF4F complexparticipated in the primary initiation event and that an interaction between

40S/eIF3/eIF4Fis maintainedduringtheinitialtranslation.Only the40Sribosomesubunitsthathavekeptthisinteractionatthe uORFterminationcodonareabletoresumescanningandreinitiate atadownstreaminitiationcodon(Pöyryetal.,2004).

2.1. eIF2concentrationandreinitiation

Asdiscussedabove,phosphorylationoftheeIF2␣subunitisan important mechanismtoregulate proteinsynthesis in different stressconditionswithinthecell.HoweveralthougheIF2␣ phos-phorylation in generalreduces protein synthesis in the cell, it simultaneouslyinducesproteinsynthesisofasubsetofmRNAse.g. ATF4andATF5.

Thebestunderstoodmechanismofmammaliantranslational controlby uORFsisreinitationonthehumanATF4mRNA. This transcriptencodesabasiczipper(bZIP)transcriptionfactorandhas 2uORFsinits5UTRofwhichtheuORF1isonly3aminoacidslong andtheuORF2is59aminoacidslongandoverlapswiththeATF4 mORF(Fig.3)(VattemandWek,2004).Undernormalconditions (eIF2-TCabundant)uORF1isefficientlytranslated.Any40S riboso-malsubunitsthatresumescanningaftertheuORF1stopcodonwill reacquireeIF2-TCbeforereachingtheuORF2AUGcodonandthus reinitiateatuORF2(Fig.3A).BecausetheuORF2ismuch longer andmoreimportantly,overlapswiththemORF,themajorityofthe ribosomeswillberecycledattheuORF2terminationcodon,leading

Fig.3.ReinitationmechanismofATF4mRNAtranslation.(A)Undernormalconditions(abundanteIF2-TC),ribosomesthattranslateuORF1(3codons)mayreinitate,thatis the40Sremainsattachedtothetranscriptandresufmesscanningdownstream.eIF2BactsasaGEFtoeIF2-GDP,exchangingtheGDPforGTP.DuetotheabundanteIF2-TC availabilitythe40SribosomereacquiresaternarycomplexbeforeuORF2allowingreinitationatuORF2AUGcodon.Hence,preventingtranslationoftheATF4mORF.(B) Understressconditions,whichelevateeIF2␣phosphorylationuORF1intranslatedasdescribedabove.However,phosphorylatedeIF2␣onSer51bindsmorestronglyto eIF2BinhibitingitsGEFfunctionandreducingtheavailablepoolofeIF2-TC.Thisleadstothe40Sribosomesthatresumescanningtoprogressfurtheralongthetranscript beforereacquiringeIF2-TC,thusbypassingthestartcodonofuORF2toreinitiateattheATF4mORF.

torepressionoftheATF4mORF.Duringstressconditions(i.e.ER stress)eIF2␣subunitphosphorylationisincreasedwhichreduces theeIF2-TCconcentration.Now,the40Sribosomalsubunitsthat resumescanningwillbypass theuORF2-AUGcodondue tothe longertimeittakestoreacquireeIF2-TC,thusleadingtoreinitation attheATF4mORF(Fig.3B)(VattemandWek,2004).Interestingly, intheATF4andATF5transcriptsboththeuORFsandthenucleotide distancebetweenthemareconservedamongdifferentvertebrate speciesthatsuggestsacommonregulatorymechanisminthese mRNAs(VattemandWek,2004;Zhouetal.,2008).

Ithasbeenshownthatthereiscoordinatedtranslational upre-gulation of a subset of mRNAs that contain uORFs following exposureofcells toUVBlight.Inthis case,aspartofthestress response,theeIF2␣subunitisphosphorylated and thereduced eIF2-TClevelsleadtotheselectivesynthesisofproteinsthatare requiredaspartoftheDNAdamageresponseincludingthecritical DNArepairenzymesERCC1,ERCC5andDDB1(Powleyetal.,2009). However,thisappearstobespecifictotheDNArepairenzymes sinceATF4wasnotfoundtobeupregulated(Powleyetal.,2009). Furtherworkisneededtounderstandtheprecisemechanismof thisselectivetranslationalcontrol.

Ingeneral,mostmammalianuORFsarepermissivefor reiniti-ationexceptforsomerarecaseswhere,forexample,thenascent peptidechaincausesribosomestallingattheterminationcodon (discussedbelow).Incontrast,mostoftheuORFsinyeastappear tobenonpermissiveforreinitiation.TheonlynativeuORFsinyeast thathave been shownto bepermissive for reinitiation arethe firstuORFintheGCN4mRNAandtheonlyuORFinYAP1mRNA (Hinnebusch, 2005;Munzarovaet al.,2011;Vilela etal., 1998). Inaddition,nucleotidesequencesbothupstreamandimmediately downstreamoftheuORF1intheGCN4mRNAhavebeenshownto interactwiththeyeasteIF3andthatthisinteractionisimportant forreinitiationafteruORF1translation(Munzarovaetal.,2011; Szamecz etal., 2008).Interestingly,mammalianeIF3 and eIF4F

interactdirectlywitheach other,and this interactionhasbeen suggestedtobeimportantforreinitiationinmammaliansystems (Pöyryetal.,2004),butinyeastonlyanindirectinteractionbetween eIF3andeIF4F(byeIF1andeIF5)hasbeenshown(Hinnebusch, 2006).

3. Mechanismsotherthanreinitiation

Thepremiseofreinitiationdoesnotaccountfortheobserved behaviourofalluORFs.Therearealsoanumberof,asyet,notfully understoodmechanismsbywhichanuORFmayfunction(Fig.4). Herein,wediscussthepossibilitiesthatcanoccurconcurrentlyor independentlytoreinitiationincluding;uORFnucleotide depend-ence, uORF peptideexpression and functionality, uORF bypass, non-sensemediatedmRNAdecay(NMD),andinterplaywithan IRES.

3.1. Upstreamopenreadingframenucleotidedependence

ForcertainuORFsthenucleotidesequenceisthemost impor-tantdeterminantofuORFbehaviour.Methioninesynthasecontains 2inhibitoryuORFs.Interestingly,theseconduORFcontains6rare codonsforeitherarginineorprolinewithinits30codonsequence. Thiswouldbepredictedtopotentiallyslowdownorevenstallthe ribosomeduringuORFtranslation.Indeed,synonymous replace-mentoftheadjacentrareargininecodonsintheN-terminusofthe peptidewithamoreabundantcodon(eitherarginineoralanine) alleviated repression (Fig. 4A) (Col et al., 2007). A nucleotide sequencethatallowedforRNAsecondarystructuretoformand/or bindingmotifswithinthe5UTRsequencecouldalsoaffectuORF behaviour and mRNA translation. For example, the pausing of theribosomecaused by thepresence ofhighly structuredRNA couldallowincreasedtimetorecogniseanAUGinamechanism thatcouldbesimilartothewayinwhichpseudoknotsareused

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Fig.4.TherolesofuORFsintranslationalcontrol.Inadditiontoreinitiation(Fig.2)uORFshavebeencharacterisedtoperformanumberofdifferentroles.(A)Thenucleotide sequencecanhaveapredominantroleonuORFtranslatability,forinstancebyencodingforrarecodonsthatcausetheribosometostall(methioninesynthase)orthepotential involvementofsuchsequencesinsecondarystructureorRNAmotifs(UCP2).ThepeptideproductofuORFscanhavecisregulatoryfunctions,forinstancecausingthestalling ofribosomes(AdoMetDCandCHOP)(B),orbythetransrepressionofthemORF(AS,␤2adrenergicreceptorandvasopressinV1Breceptor)(C).(D)BypassofaninhibitoryuORF hasbeenobservedunderstressconditionsandisdependentoneIF2␣phosphorylation.Aninterestingpossibilityforhowthismayoccuristheloadingofthe40Swithout aneIF2-TCwhichitacquiresasitscans(C/EBP␣and␤,CHOP,GADD34,proteinkinaseCisoform␩).(E)InteractionsthatinvolvebothuORFsandIRESelementswithin5_UTRs

cancauseexpressionofthemORF(Cat-1)orrepressionofparticularsplicevariants(VEGF-A).(F)Approximately35–50%ofuORFcontainingtranscriptsundergoselective degradationbyNMD(e.g.ATF4,CHOPandIFDR1).

toachieveframe-shiftingincoronaviruses(Robertsetal.,2009). Mutationalanalysisof the36codonuORF inhibitorypeptideof UCP2demonstratedthattheuORFC-terminalnucleotidesequence wasimportantforitsinhibitorybehaviour,althoughthe mecha-nismisnotyetunderstood(Hurtaudetal.,2006;Pecqueuretal., 2001).

3.2. Upstreamopenreadingframepeptideexpressionand function

ThedetectionofendogenousuORFpeptideexpressionhasbeen limited.Globalscreensofsmallpeptideexpressionwithinhuman cellsbymassspectrometryonlyidentified7uORFpeptides(Oyama

etal.,2004,2007).Amorerecentpeptidomicsapproachdetected 15uORFpeptides(Slavoffetal.,2013).Theselownumberssuggest thateitherthemajorityofuORFproductsareexpressedatlevels currentlyundetectableand/orthattheyareselectivelydegraded withincells(Oyamaetal.,2007;Slavoffetal.,2013).WhenuORF peptideexpressiondoesoccuritmaybespatiallyand/or devel-opmentallyregulated.ForinstanceMYCNOTexpression,theuORF productoftheMYCN1btranscript,wasdetectablewithinfoetalbut notadultbrains(Besanconetal.,2009).Furtherconfusionarisesas tothefunctionofsuchuORFpeptides,withmanycontainingunique domainsandmotifs(Besanconetal.,2009;Oyamaetal.,2007). Differencesinsubcellularlocalisation;the2uORFpeptidesofthe MKKSgenebothlocalisetothemitochondria,whichisincontrast tothecytoplasmiclocationoftheMKKSprotein(Akimotoetal., 2012).Ortheirassociationwithothercellularfactors,theuORF2 peptideoftheGRtranscriptvariant1Ainteractswitha number ofproteinsinthecytoplasmbutnotmembrane-boundGR(Diba etal.,2001).Collectively,theseobservationshintattheprospect thatuORFpeptidesrefractorytorapidproteolysismayalsofulfil rolesadditionaltotranslationalcontrol.

FunctionalcharacterisationhasshownthatuORFpeptide prod-ucts can produce cis- or trans-regulation on mORF translation (Fig.4BandC).S-adenosyl-methioninedecarboxylase(AdoMetDC) uORF-mediatedrepressionisregulatedbypolyamine concentra-tion,whichdeterminesthedurationofribosomalpausingatthe C-terminusof the uORF sequence, thus obstructing passage of ribosomestothemORF(Lawetal.,2001;Nishimuraetal.,1999; Raneyetal.,2000).AdoMetDCencodesaninhibitory6codonuORF (Hill and Morris, 1992).Detailed molecularstudiesboth in cell andcell-freesystemssupportthehypothesisthattheAdoMetDC uORFproteinsequenceisresponsibleforthisinhibition,withthe codon identity at the fourth and fifth positions as wellas the lengthof thispeptide beingessential forits repressive proper-ties(HillandMorris,1993;Mizeetal.,1998;Raneyetal.,2000). DespitetheabilityoftheAdoMetDCuORFtoinhibitareporter con-structinyeast,thepeptideshowsnosequencesimilaritytoyeast uORFsthatlikewisepromoteribosomalpausing/stalling suggest-ingthesepeptidesassociatewithdifferentmoleculartargetswithin theribosomeorassociatedfactors(Lawetal.,2001;Mizeetal., 1998).

SeveralexamplesofanuORFpeptideactinginatrans-manner havebeendemonstrated.Argininosuccinatesynthase(AS) trans-criptsdiffer intheir5UTRlengthduetothe usageofdifferent transcriptionalstartsites.Themajortranscripts(presentat>90% ofallAStranscriptswithin acell)contain ashort UTRwithno uAUG,whereasthelongerUTRsoftheminorproductscontainan inhibitoryuORF(Pendletonetal.,2002).TransfectionoftheASuORF constructintobovineaorticendothelialcellsrepressedendogenous ASprotein expression, with mutational analysis demonstrating that both the uORF peptide sequence and lengthbeing essen-tial determinants of repression (Pendleton et al., 2005). Other examplesof trans-repression have thus far been demonstrated bytheadditionofhighconcentrationsofuORF peptideswithin cell-free systems. These include themammalian ␤2 adrenergic receptoruORFpeptidesequence,whichencodesanumberof con-servedarginineresidues.However,thiseffectwasrecreatedwith anunrelated peptide of equivalenthighbasic charge, although arginine alone proved insufficient (Parola and Kobilka, 1994). Anotherexample involvesthelast uORFpeptideofVasopressin V1breceptor (whoseendogenous expressionhasbeendetected within cells) that was able to re-establish translational inhibi-tionwhenaddedexogenouslytoacellfreesystemexpressinga transcriptinwhichthisuORFAUGhadbeenmutated( Rabadan-Diehlet al.,2007).Theseinteresting examplesofuORF peptide functionalityhighlighttheimportantofexperimentaldesignand appropriate controlswhen dissecting out an uORF’sbehaviour,

includingtheneedforconsiderationofanycoexpressedtranscript variants.

3.3. Upstreamopenreadingframebypass

uORF bypass hasbeen usedto describe thederepression of a mORF downstream of one or more inhibitory uORFs. It has beensuggestedthatthisarisesfromanincreaseinleakyscanning ofuORFstartcodons.However,asdiscussedbelowthe require-mentfortheuORFsduringderepressionshowsthisexplanation islikelytobetoosimplistic(Leeetal.,2009;Raveh-Amitetal., 2009).Currentknownexamplesofthisphenomenonoccur dur-ingelevatedlevelsofeIF2␣phosphorylation(Fig.4D)(Calkhoven etal.,2000;Leeetal.,2009;Palametal.,2011;Raveh-Amitetal., 2009).WhetherthismechanismisduetothereductionofeIF2-TC availabilityorachangeinanothertranslationfactoriscurrently unknown.

TheconservedCCAAT/enhancerbindingprotein(C/EBP)␣and ␤isoformscontainanumberofpotentialinitiatorcodons, includ-inga shortuORF positionedupstreamof themORF. Unlikethe otherexamplesthisuORFpromotesreinitiation,butits surround-ingsequenceactstoreducethelikelihoodofinitiationfromthefull lengthmORFinfavourofinitiationataninternalAUGthatencodes atruncatedC/EBPproduct(Calkhovenetal.,1994,2000;Lincoln etal.,1998).Indeed,intheabsenceoftheuORFnotruncated prod-uctisproduced(Calkhovenetal.,1994,2000).Translationofthefull lengthortruncatedproductsisregulatedbychangesinthe avail-abilityofinitiationfactors.InadditiontoeIF2␣phosphorylation, areductionoffreeeIF4EbyrapamycincausesbypassoftheuORF AUGinfavouroftranslationfromthefulllengthmORF(Calkhoven etal.,2000).

CHOPmRNAcontainsaconservedrepressiveuORF,with2 in-frameAUGs creatinga 31 or34 codon product inrodents and humans (Jousse et al., 2001).Mutagenesis studies have shown thattheC-terminalpeptideregionisessentialfortheinhibitory propertiesofthisuORF. Theabsenceof atrans-effectledtothe supposition that thepeptide sequence actsin a cis-manner to impederibosomalmovementeitherduringelongationor termina-tion,althoughfutureexperimentsareneededtoconfirmsuchan occurrence(Jousseetal.,2001;Palametal.,2011).However, follow-ingERstresswheneIF2␣isphosphorylated,CHOPistranslationally upregulated,bothinthepresenceorabsenceoftheuORF.Therefore up-regulationofCHOPproteinrequireseIF2␣phosphorylation,but isnotnecessarilydependentontheuORFs.Itwassuggestedthat undertheseconditionstheuORFstartcodons(2in-frame)become lesswellrecognisedbytheribosomecomplex(Palametal.,2011). FurthersupportforthistheorywasshownbymutationoftheuORF toastrongKozakconsensus(whichwouldbeexpectedtodecrease leakyscanning)reducedCHOPtranslationalupregulation(Palam etal.,2011).

Otherexamplesofpotentialbypassrequirethepresenceofan uAUGforthederepression.GADD34mRNAundergoesboth trans-criptionalandtranslationalupregulationafterERstress.The5UTR ofGADD34mRNAcontains2conserveduORFsthatareinhibitory. Derepressionafterthapsigargintreatment(whichinducesERstress PERKactivationand eIF2␣phosphorylation) wasreliantonthe presenceoftheseconduORFstartcodon,whereasthefirstuORF wasdispensable(Leeetal.,2009).Similarly,proteinkinaseC iso-form ␩ mRNA contains 2 conserved uORFs that are inhibitory tomORF translation, especially uORF2that hasa strong Kozak consensus sequence (A−3/G+4). Derepression after amino acid starvationrequiredbothGCN2kinaseandthepresenceofeither uORF.Interestingly,mutationoftheuORF2stopcodon,sothatit nowterminateddownstreamofthemORFstartcodonhadnoeffect (Raveh-Amitetal.,2009).Thisimpliesthatitisthestartcodonsof

J.Somersetal./TheInternationalJournalofBiochemistry&CellBiology45 (2013) 1690–1700 1697 theuORFsthatarerequiredandrecognisedduringuORFbypass

events,ratherthanuORFtranslation. 3.4. Internalribosomeentrysegments

IRES elements provide a structured RNA context that can recruitribosomestoinitiatetranslationinternallyandthus inde-pendentlyfromcap-dependenttranslationalevents(seereview, Pichonetal.,2012).Acoupleofexampleshavebeendemonstrated in mammalian transcripts where translational control involves coordinationbetweenanuORFandanIRESelement(Fig.4E).Cat-1 mRNAundergoes IRES-mediated translationalupregulation fol-lowingaminoacidorglucosedeprivation.Interestingly,translation ofa48codonuORFisrequiredformaximalIRESactivity(Fernandez etal.,2001,2002a,2002b;Yamanetal.,2003).Detailed mutagen-esisstudiesledtoamodelwhichpredictsthattheCat-1IRESis inaninactiveconformationwhichisrestructuredintotheactive IRESduringtranslation oftheuORF.Although,it isnotyetfully understoodhowtheuORFisinitiallytranslated(asthesequence ofthe5endoftheUTRisinhibitorytoribosomalmovement)or howtheIRESismaintainedinitsactivecontextduringstress stim-ulation(Yamanetal.,2003).TheVEGF-Ageneundergoesanumber oftranscriptionalandtranslationalregulatorystepsthatgiverise to9proteinisoforms.Thepresenceof2IRESelementswithinthe 5UTRallowstranslationinitiationfromeitherthemORFAUGor fromanuCUGcodonthatproducesaN-terminallyextendedprotein thatissubsequentlycleaved.VEGF-Aisoformsarise from differ-entialsplicing,withthe121aminoacidisoform,unliketheother commonlyexpressed165and189aminoacidisoforms,onlybeing translatedfromtheuCUGcodon.Interestingly,cap-independent translationofanuORFupstream(situatedbetweentheuCUGand mainAUGcodons)actstorepressIRES-mediatedexpressionofthe 121isoformfromtheAUGcodon.Thisdemonstratesthe occur-renceofwidespreadcisregulatoryactionsbetweentheuORF,the IRESelementanddownstreamRNAsequences(Bastideetal.,2008). 3.5. Nonsensemediateddecay

Nonsense mediated decay(NMD) allows for theelimination oftranscriptsthatencodepeptideswithaprematuretermination codon.Thepresentmodelpredictsthatduringtheinitialroundof translationmessageswithastopcodonmorethan50nucleotides upstreamofanexon–exonjunctionwillnothavetheexonjunction complexesdisplacedandthisdirectssuchmessagestobe recog-nisedandselectively removedby NMD(Fig.4F)(Rebbapragada andLykke-Andersen,2009).ManytranscriptswithuORFswould fitthis criterionandthusbecandidatesforthispathway. How-ever,examinationofHeLatranscriptomechangesfollowingsiRNA treatmentofUPF1,PNRC2orCTIF,allinvolvedinNMD,identified 35–50%ofupregulatedtranscriptscontainedanuORF(Kimetal., 2012;Mendelletal.,2004).However,40–50%ofhumanandmouse transcriptscontainatleastoneuORF(Calvoetal.,2009;Iaconoetal., 2005;Matsuietal.,2007),suggestingthatthereisnopredisposition forNMDbyuORFcontainingtranscripts.

ATF4andCHOPtranscripts(ATF4Reinitiation,CHOPuORFBypass) aretargetsofNMD,illustratingtheinvolvement ofmultifaceted pathwaysinuORFtranslationalcontrol(Gardner,2008;Mendell etal.,2004).Interestingly,duringhypoxiaeIF2␣phosphorylation causedarelocalisationofNMDcomponentstostressgranulesand inhibitionofNMD(Gardner,2008).Similarly,ithasbeen demon-stratedthattranscriptturnoverofanotherNMDtarget,theuORF containingIFDR1,wasreducedfollowingeIF2␣phosphorylation (Zhaoetal.,2010).Suchamechanismwouldbeexpectedto aug-menttranslationoftheseproteinsduringstressconditions.

TheverycomplexUTRofthrombopoietin(THPO)transcript con-tainsmultipleuORFs.TheseventhuORF,whichoverlapswiththe

mORF,fitsthecriterionforaNMDtarget.However,thistranscript avoidsNMDandonlybyexpandingtheseventhuORFfrom27to40 codonswasitpossibletoreducetranscriptlevels,whichwereonly inpartreversiblebyNMDinhibition(Stockklausneretal.,2006). Thisobservationfitswiththehypothesisthatproposesthatthe lengthofshortORFsmaybeakeydiscriminationfactorinNMD. Studiesusingmutant betaglobintranscriptsdemonstratedthat shorterORFsweremoreinsensitivetoNMD,andthisoccurred inde-pendentlyofanyreinitationevents(Inacioetal.,2004;Silvaetal., 2006).

4. Non-AUGupstreamopenreadingframes

Ribosomeshavebeenshowntostarttranslationatnon-AUG start codons, although this occurs with much lower efficiency than at AUGcodons both withinmammalian cells and invitro translationassaysusingRRL(Kozak,1989a;Peabody,1987,1989). Near-cognatenon-AUG startcodons(whichdifferfromAUGby one nucleotide) within a strong Kozak context were tested in RRL (Peabody, 1989).All variations caused a certain degree of leaky scanning, with the ACG and CUG being the best recog-nisedinitiationcodonsand AGGand AAG theleastrecognised. In another study, GUG was found to be the most efficient non-AUG start codon. Furthermore, it was observed that the nucleotide context surrounding the GUG had a stronger influ-encethanforanAUGcodonimplyingthatthenearbynucleotides compensatefortheweakercodon–anticodoninteraction(Kozak, 1989a).

Arecentnewtechnologytermedribosomeprofiling,involves deepsequencingofribosome-protectedmRNAfragments,allowing thedetectionofribosomeoccupancyinanygivenmRNAgenome wide.Thus,inprincipalonecandetectwhichpartofthemRNA istranslated(Ingoliaetal.,2009,2011).Bytheuseoftwodrugs; harringtonine, which actsby stalling 80Sribosomes at transla-tioninitiationstartsites,andcycloheximidethatarrestselongating ribosomes,thestartsitesandreadingframesofatranscriptcan beidentified.Ribosomeprofilinginmouseembryonicstemcells revealedhigh-levelribosomeoccupancyinuORFsthathad non-AUG start codons (Ingolia et al., 2011). In fact the majority of the all uORFs detected had near-cognate start sites of which; CUG was used the most (30–35%), GUG and AUG (20%), UUG (15%)andACG(10%).ManymRNAshadmultipleuORFswithboth AUGandnon-AUGstartsites.AlltheseuORFswereaccompanied byelongatingribosomefootprints,whichweredetectedwithout harringtoninetreatmentindicatingthattheuORFswereactively translated(Ingoliaetal.,2011).WhenATF4mRNAwasexamined, ribosomefootprintswerefoundatbothstartcodonsandwithin bothuORFs,howevernoribosomefootprintswereobservedatthe mORFstartcodonindicatingtranslationalsuppression.Incontrast, inmRNAscontainingnon-AUGuORFs,ribosomefootprintswere seenatboththestartcodonsandwithinthereadingframesof non-AUGuORFsaswellasatthemORFssuggestingthatthesenon-AUG uORFsmighthaveaweakerregulatoryfunctionthanuORFswith AUGstartcodons.

Arecentproteomicsstudyidentifiedshorttranslatedpeptides bymassspectrometryandfoundthatsomeuORFpeptideswere initiated froma non-AUGstartcodon.Duetothelimitationsof thetechnology,thesepeptideswereonaverage75aminoacids. Interestingly,manyofthesenon-AUGuORFsoverlappedthemORF. Tovalidatethesefindings,theuORFandtheadjoiningmORFwere taggedandexpressionofboththenon-AUGuORFandmORFwas confirmedusingimmunofluorescence.Mutationofthenon-AUG toanAUGcodon,completelyrepressedtranslationofthemORF indicatingthatthemORFwasbeingtranslatedbyleakyscanning (Slavoffetal.,2013).Howsuchnon-AUGcodonsarerecognised is not fully understood.However, it has beenshown that eIF1

playsanimportantrole instartcodonselection andthatinthe absenceofeIF1thescanningribosomeisunabletodiscriminate betweenanAUGandnon-AUGcodonandisalsoindiscriminate tothesurroundingKozakcontext(PestovaandKolupaeva,2002). Morerecently,overexpressionofeIF1incellsabolishedinitiation fromnon-AUG codons. Furthermore,it wasproposedthat eIF1 concentrationisinvolvedinanautoregulatoryfeedbackloop con-trollingitsowntranslation,asthemORFAUGcodonoftheeIF1 transcriptispositionedwithinapoorKozakcontext(Ivanovetal., 2010).

5. Upstreamopenreadingframesanddisease

Asthe preceding exampleshave shown, uORFs can play an integralpartinthetranslationalcontrolofgeneexpression.Itis thereforenotsurprisingthatanincreasingnumberofdiseasesare beinglinkedtochangesinuORFfunctionality.Herewewill dis-cussexamplesoftheconsequences ofalternative5UTRs,single nucleotidepolymorphisms(SNPs)anduORFmutationsindisease andtreatment.

Manygenesareencodedbyalternativetranscriptisoformsthat canarisefromtheusageofdifferentpromoters,transcriptionstart sitesand/ordifferentialsplicing.Strictlyspeaking,changesin iso-formabundanceincancerareduetoaberrationsintranscription. However,asisoformscandifferinboththeiruntranslatedregions and coding sequences, theymay produce a change to transla-tionalpotential.MDM2,anoncogenethatbindsandinactivatesthe tumoursuppressorp53,isencodedby2transcriptsashortandlong version,transcribedfromdifferentpromoters.Translationfromthe longtranscriptisdampenedbythepresenceof2inhibitoryuORFs, whichareabsentfromtheshortertranscript.Inhumansofttissue tumoursoverexpressionofMDM2proteincanoccurfrom enrich-mentofthemorehighlytranslatedshorttranscript(Brownetal., 1999).Alteredpromoterusagehasalsobeenidentifiedinbreast cancersthatdisplayreducedexpressionoftheDNArepair gene BRCA1.IncreasedtranscriptionofBRCA1transcriptswithalonger inhibitory5UTRareobservedinthesecells.Thelowertranslation ofthemORFinthesetranscriptsarisesfrombothanincreasein sec-ondarystructureandthepresenceof3uORFsinthe5UTR(Sobczak andKrzyzosiak,2002).Inheadandneckcancerachangein splic-ingcausedtheretentionofalongerinhibitory5UTRofthetumour suppressorhyaluronidasetobethepredominanttranscript pro-ducedinthesecancers.ThistranscriptcontainsnumerousuAUGs thatreducedexpressionofthemORF(Frostetal.,2000).

AsuORFsoftenactasinsulatorelementsonthedownstream translationofagene,thepossibilityofaSNPcreatingorremoving anuORFwouldbeexpectedtohaveseriousconsequencesonmORF translation.Over 509humangeneshave beenidentified where SNPsoccurtocreate/deleteanuORF.Furthertestingof5ofthese genesbyreporterassayconfirmedthatthelossoftheuORFsinall casesleadtoincreasedtranslationalefficiency(Calvoetal.,2009). TheexaminationofSNPswithinuORFsequencesalsoneedsfuture consideration.ASNPthatcreatesamissensechangewithinthe seconduORFoftheserotoninreceptorgeneHTR3Ahasbeen sig-nificantlylinkedwithbipolaraffectivedisorder.Reporterassays showedthisSNPtodoubleluciferaseactivity(Niesleretal.,2001). FamilialuORFmutationshavebeenreportedforanumberof diseases. In hereditary melanoma certain families show a G-T mutation,which createsanuAUGwithin theCdk4/Cdk8kinase inhibitorCDKN2AmRNA.ThisuORF,whichisefficientlyrecognised byribosomes,reducesCDKN2AmRNAexpressionandconsequently alleviatesitsrestrictiononG1progressioninthecellcycle(Liuetal., 1999).Thehumanhairlessgenecontains4uORFs,butitisthe sec-ond(andlongest)uORFinwhicharangeofmutationshasbeen identifiedinfamilieswithMarieUnna hereditaryhypotrichosis.

The mutations identified were either missense or nonsense or removedeithertheuORFinitiationorterminationcodon,asuORF2 isin-framewiththemORFthis wouldproduceanN-terminally extended protein.Alltestedmutationswereshown toenhance translationfromareportergene.Furthermore,theobservationthat missensemutationsaugmenttranslationsuggeststhatthepeptide sequenceoftheuORF2isimportantfor translationalrepression (Wen et al., 2009). Enhanced translation of the THPO gene is observedinhereditarythrombocythemia,the5UTRofwhich con-tains7uORFs.Onefamilialmutationinvolvesanucleotidechange thatcreatesanewsplicedonorsite,whicheliminatesalluORFs fromthesplicedmessage(Wiestneretal.,1998).Othermutations affecttheseventhuORF,whichoverlapswiththemORF.The intro-ductionofanonsensemutationupstreamofthemORF,allowed reinitiationbyribosomesafteruORF7translation(Ghilardietal., 1999).Alternatively,anucleotidedeletioninuORF7,whichplaces it in-framewiththe coding sequence,produced a N-terminally extendedprotein(GhilardiandSkoda,1999;Kondoetal.,1998).

Collectivelytheseexampleshighlightthegrowingemphasison identifyingmutationsorSNPswithinthe5UTRthatmayinfluence diseaseprogressionoraetiology.Thenumerouswaysinwhichan uORFmayregulatetranslationwillrequirecarefulstudyintohow aberrantuORFregulationaffectstranslation.

6. Conclusion

GiventheprevalenceofuORFsinmammaliantranscriptsitis perhapssurprisingthatstillrelativelylittleisknownabouthow theseelementsareregulated.Thecurrentdatastronglysuggest thattheirregulationislikelytobecomplexandthatseveral inde-pendentmechanismsexisttoallowtheirrepressiveeffectsunder normalconditionsandselectivetranslationofsubsetsofdefined mRNAs under situations of pathophysiological cell stress. Fur-therstudiesareneededtoaddresshowmutations/polymorphisms within uORFs/AUGs may contribute to an individual’s disease response.

Acknowledgements

WewouldliketothankThomasSbarratoforusefuldiscussions. ThisworkwassupportedbyfundingfromCRUK(JS),BBSRC (Pro-fessorialfellowshipAEW),MRC(TP).

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