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Original citation:
Kumari, Pooja and Sampath, Karuna. (2015) cncRNAs :
Bi-functional RNAs with protein
coding and non-coding functions. Seminars in Cell & Developmental Biology, 47-48 . pp.
40-51.
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ContentslistsavailableatScienceDirect
Seminars
in
Cell
&
Developmental
Biology
jo u r n al h om ep age : w w w . e l s e v i e r . c o m / l o c a t e / s e m c d b
cncRNAs:
Bi-functional
RNAs
with
protein
coding
and
non-coding
functions
Pooja
Kumari
1,
Karuna
Sampath
∗DivisionofBiomedicalCellBiology,WarwickMedicalSchool,TheUniversityofWarwick,GibbetHillRoad,CoventryCV47AJ,UnitedKingdom
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Availableonline20October2015
Keywords:
cncRNAs Bi-functionalRNA Non-codingRNA Protein-coding RegulatoryRNA RNAstructure RNAprocessing DualfunctionRNA
a
b
s
t
r
a
c
t
Formanydecades,themajorfunctionofmRNAwasthoughttobetoprovideprotein-codinginformation embeddedinthegenome.Theadventofhigh-throughputsequencinghasledtothediscoveryofpervasive transcriptionofeukaryoticgenomesandopenedtheworldofRNA-mediatedgeneregulation.Many regulatoryRNAshavebeenfoundtobeincapableofproteincodingandarehencetermedasnon-coding RNAs(ncRNAs).However,studiesinrecentyearshaveshownthatseveralpreviouslyannotated non-codingRNAshavethepotentialtoencodeproteins,andconversely,somecodingRNAshaveregulatory functionsindependentoftheproteintheyencode.Suchbi-functionalRNAs,withbothproteincodingand non-codingfunctions,whichwetermas‘cncRNAs’,haveemergedasnewplayersincellularsystems.Here, wedescribethefunctionsofsomecncRNAsidentifiedfrombacteriatohumans.Becausethefunctionsof manyRNAsacrossgenomesremainsunclear,weproposethatRNAsbeclassifiedascoding,non-coding orbothonlyaftercarefulanalysisoftheirfunctions.
©2015TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).
1. Introduction
The ‘one gene one enzyme’ hypothesis proposed by Beadle
andTatumin1941[1]andtheelucidationofthedoublehelical
structure of DNA in 1953[2] ledCrick to propose of the
cen-traldogmaofmolecularbiologyplacingRNAatthecenterofthe
directionalinformation flow from genes totheir protein
prod-ucts[3].SubsequentidentificationofmessengerRNAs(mRNAs),
adaptorRNAmolecules(tRNA)andribonucleoprotein-dependent
catalysisofpolypeptidesynthesis(rRNA/ribosomes)validatedRNA
versatilityandeventuallyinspiredthefirstmodelofRNA-based
regulatorynetworksincellsofhigherorganisms[4–8].However,
thediscoveryofcis-regulatoryelementsinDNAcontrollinggene
expressionbyvirtueoftheirinteractionwithcognate
transcrip-tionfactorscaptured theimaginationand interest of scientists,
and for many years, the regulatory roles of RNA were largely
ignored.
Thisprotein-centricviewof gene regulation waschallenged
by the discovery of small regulatory RNAs (e.g., miRNAs) and
genesilencingbyRNAinterference(RNAi)[9–11].Subsequently,
the advent of high-throughput sequencing and transcriptome
∗Correspondingauthor.
E-mailaddress:[email protected](K.Sampath).
1 Present address: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse66,BaselCH4058,Switzerland.
analysisshowedthatthousandsofgenomiclociundergo
transcrip-tiontoproducelargetranscriptsthatmaynotcodeforproteins
[12,13](Fig.1A).These findings are supportedby the ENCODE
(EncyclopediaofDNAElements)projectwhichshowedthat∼80%
ofthemammaliangenomeistranscribed[14].Furthermore,the
ratioofnon-codingtoproteincodingtranscriptshasbeenproposed
toincreasewiththecomplexityoforganismsandapproximately
95%ofhumantranscriptsarethoughttobenon-codingRNAs[15].
Theregulatoryfunctionsoflongnon-codingRNAs(lncRNAs)are
underactiveinvestigationbyseveralgroupsandhavebeenrecently
reviewedin[16–18].Thephenomenalscaleofthenon-protein
cod-inggenomeshowsthatourcurrentunderstandingofRNA-based
generegulationisrathercursory.Studiesinavarietyoforganisms
overthelasttwodecadessuggestthatRNAmoleculescontainmany
morecis-andtrans-regulatoryfunctionsthanpreviouslythought.
Although initiallylncRNAs were thoughttofunction strictly as
RNAsandnotcodeforproteins,recentstudieshaveshowedthat
many previously annotated non-coding RNAs can recruit
ribo-somesandencodeshortpeptides[19–21].Inaddition,emerging
evidencesuggeststhatevenproteincodingmRNAscanhave
struc-turaland/orregulatoryfunctionsindependentoftheproteinthey
encode.[22].TheseadditionalfunctionsofRNAmayseem
surpris-ing,butarenotcompletelyunexpectedinlightoftheviewthatall
currentformsoflifemighthaveevolvedfromanRNAworld[23,24].
RNAisaversatilemoleculeinthatRNAcanstoregenetic
informa-tionsimilartoDNA,andcanalsoactasacatalystsimilartoproteins
[25,26].Inthisreview,wefocusonbi-functionalRNAswithboth
http://dx.doi.org/10.1016/j.semcdb.2015.10.024
Fig.1.Schematicshowingvariousclassesofnon-codingRNAs.(A)Transcription:thenandnow.Theconventionalconceptoftranscriptionsuggestedthatonlyspecificlociin thegenomearetranscribedtocodeforproteinswhilecurrentunderstandingpointstowardpervasivetranscriptionofthegenomeandwidespreadoccurrenceofnon-coding RNAs.Theschematicshowsagenomicregionwithtwogenes,A(exonsinblue)andB(exonsinyellow).Accordingtotheoldconceptthereisnotranscriptionintheintergenic regionbetweengenesAandB.ThecurrentconceptsupportsthepresenceofintergenicncRNAs(purple,betweengeneAandB),intronicncRNAs(green,betweenexonII andIIIofgeneA)andantisensencRNA(orange,inoppositeorientationinexonIIIofgeneA).AlternativesplicingmayalsoleadtodifferentisoformsofRNAsasshownin thesecondisoformofgeneAwhichlacksexonII.(B)Crosstalkbetweencodingandnon-codingRNAs.Thetranscriptomeismorecomplexthananticipated.Proteincoding pre-mRNAscangiverisetonon-codingRNAs.Longnon-codingRNAscanencodeforshortpeptidesandprotein-codingmRNAscanhaveadditionalregulatoryfunctions.(For interpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
proteincodingandnon-codingroles(cncRNAs).cncRNAs,carrying
bothproteincodingandRNA-intrinsicfunctions,callforreviewing
theconceptwheremRNAswereconsideredapassivestepinthe
transitionofgeneticinformationfromDNAtoprotein.Thesedual
functionRNAsalsopresentapotentialevolutionarylinkbetween
mRNAs and ncRNAs(miRNA, endo-siRNA,piRNA, lncRNA, etc.),
whichwerepreviouslythoughttobeinherentlydifferent(Fig.1B).
Here,wedescribecncRNAsfroma varietyoforganismsranging
frombacteriatohumans,withemphasisonthestructuralor
regu-latoryfunctionsofprotein-codingRNAswithrolesindevelopment
anddisease.
2. SmallregulatoryRNAsinbacteria
Small non-coding RNAs have been shown to regulate
post-transcriptionalgeneexpressioninallkingdomsoflife,including
bacteria.Bacterialgenomesencodealargenumberofsmall
trans-cripts(sRNAs)intherangeof50–350nucleotides.BacterialsRNAs
canbegroupedintwoclasses:(1)antisenseRNAsthatfunction
viabase-pairingwiththeirtargets,and(2)protein-bindingsRNAs
[27].MostbacterialantisenseRNAsarenon-codingandarealso
called‘ribo-switches’or‘ribo-regulators’.However,inrecentyears
peptides[28].Here,wedescribethreebi-functionalbacterialsRNAs
thathavebeenfunctionallycharacterized.
2.1. RNAIII
Staphylococcus aureus RNAIII was the first bacterial sRNA
reportedtohavedualfunctions.S.aureusisapotentpathogenand
itsvirulenceisattributedtobothcellsurface-associatedproteins
andsecreted toxins.The5 regionofRNAIIIencodesa secreted
26aapeptide,␦-hemolysin (hld),which targetshostcell
mem-branes,causinglysis[29].␦-hemolysindoesnothaveanyknown
regulatoryfunctionsbutRNAIII,a514nucleotidelongsRNA,
reg-ulatesstabilityandtranslationofvirulencefactorsbydirectbase
pairingwiththecorrespondingtranscripts.Theexpressionofcell
surface-associatedfactorsisrepressedattheendofexponential
growthphasewhilethatofsecretedfactorsisstimulated[30,31].
Thisreciprocalregulationiscarriedoutbytheagrlocus.RNAIII
sRNAisthemajoreffectoroftheagrresponse[32].The3regionof
RNAIIIinhibitsribosomalbindingandtranslationinitiationof
coag-ulase(anenzyme),staphylococcalproteina(acellsurface-associated
factor),androt(atranscriptionfactor).Consistently,thisregionof
theRNAismoreconservedamongdifferentisolatesofS.aureus
[33–35].The5 regionof RNAIIIalsofunctionsbybase-pairing
andfacilitatesthetranslationof˛-hemolysin(hla),asecretedfactor
bypreventingtheformationofatranslationalinhibitorycomplex.
Thisregionoverlapswiththecoding sequenceofhld,hencethe
basepairingactivityofRNAIIIwithmRNAsmayprevent
transla-tionofhld[36].Suchamodeofregulationisconsistentwithadelay
inaccumulationof␦-hemolysinafterRNAIIIsRNAsynthesis[37].
Hence,itcanbeenvisagedthathldisregulatedatseverallevelsand
thereisapossibleinterplaybetweenproductionof␦-hemolysin
andtheantisensefunctionsofRNAIII.
2.2. SgrS
SgrS (Sugar transport related), a 227-nucleotide sRNA, is
induced during glucose-phosphate stress conditions resulting
fromdisruptionofglycolyticfluxandaccumulationof
glucose-6-phosphate.SgrSactivelyalleviatesstressbynegativelyregulating
thestability and translation of themajor glucose transporters,
ptsG and manXYZ, via base pairing [38,39]. In addition to this
base-pairingantisense activity,SgrS codes for a 43-aminoacid
peptide, SgrT [40]. Interestingly, SgrT also functions in the
glucose–phosphatestressresponse,butbyunrelatedmechanisms.
EctopicexpressionofSgrTfromconstructslackingthebase-pairing
sequenceseasesthestressresponsewhilethestabilityof
trans-portersisnotaffected.IthasbeensuggestedthatSgrTfunctions
byinhibitingtheactive componentsof glucosetransported but
theprecisemechanismsarenotclearlyunderstood[40].
Vander-poolandcolleaguesidentifiedahighly conserved15-nucleotide
sequenceatthe3endofSgrSfromseveralentericspecies,even
thoughtheoverallsequencewasratherdivergent[41].These
con-servednucleotidesarecomplementarytotheribosomalbinding
site in ptsGmRNA, and hencebase pairingleadsto translation
repressionandmRNAdegradation(Fig.2A)[42].IncaseofmanXYZ
polycistronicmRNAs,SgrSbasepairswiththecodingsequenceof
manXleadingtoRNAdegradation[38,39].IncontrasttoRNAIII,the
regulatorysequencesandcodingsequencesarespatiallyseparated
inSgrS.ThecoupleddegradationofSgrSduringribo-regulation
sug-geststhatthetwofunctionsofSgrSaremutuallyexclusiveandthe
sameRNAmoleculecannotserveasbothribo-regulatoranda
tem-platefortranslationofSgrT.Furtherinvestigationisrequiredto
studyifthereisanyrelationshipbetweentheregulatoryfunction
andtranslationofSgrS.
2.3. SR1
SR1isadualfunctionRNAidentifiedinthegram-positive
bac-terium,Bacillussubtilis.SR1repressesthetranslationofa
transcrip-tionalactivatorahrCthatregulatestheargininecatabolicoperons,
rhoABCandrhoDEF[43].Therearesevenregionsof
complemen-taritybetweenSR1andahrC.SR1bindinginhibitstranslationof
ahrCmRNAby inducing structural changesdownstream ofthe
ribosomalbindingsite [44].Ina questtodiscovermore targets
ofSR1,Brantlandcolleaguesdiscoveredthat SR1alsoregulates
theglycolyticgapAoperon[45].However,themechanismof
SR1-mediatedregulation ofgapA is notby basepairingoftheRNA.
SR1encodesa 39-aapeptide(SR1P)thatstabilizesgapAoperon
RNA. SR1P was reported to directlybind to GapA protein, but
themechanismsunderlyingthismodeofregulationarenotfully
understood[45].
3. Bi-functionalRNAsinplants
Plantsexhibitaremarkabledevelopmentalplasticityand
exten-sively regulate their gene expression profiles in response to
environmentalcuesandstress.RNA-mediatedregulationappears
toplayasignificantroleinadaptationtovaryingenvironmental
conditions[46].Here, wediscuss twoplantRNAsthat codefor
peptidesandalsohaveintrinsicfunctionasRNAs.
3.1. ENOD40
Early nodulin 40 (ENOD40) is the best-studied example of a
cncRNAinplants.Itwasfirstidentifiedasageneexpressedduring
earlystages of root noduleformation, resulting from the
sym-biotic association of leguminous plants with rhizobial bacteria
[47].ENOD40is expressedindifferentiatingcellsofnodule
pri-mordiaandtheexpressionlevelsofENOD40positivelycorrelate
withtherateofnodulationintransgenicplants[48].Duetothe
absenceofanylongopenreadingframe(ORF)andthehighly
sta-blesecondarystructureoftheRNA,ENOD40wasproposedtobe
anon-codingRNA.However,themolecularmechanisms
under-lyingitsactivityremainedunclearformanyyears[49,50].Later,
studiesin Medicago truncatula(a model legumeplant) showed
that there are two conserved short ORFs in the ENOD40
tran-script and that the 5ORF is highly conserved [51]. Transient
expressionofENOD40in rootsresultedin corticalcelldivisions
athighfrequency.Bytargetingwildtypeandtruncated/mutated
ENOD40tothecorticalcellsinroots,Crespiandcolleaguesshowed
that translation of both short ORFs is required for theactivity
ofENOD40. Interestingly,deletionofaninter-ORFregionofthe
RNAwithapredictedsecondarystructurealsoaffectedthe
activ-ity of ENOD40, without altering translation of the ORFs. These
results emphasized the importance of both the RNA structure
andshortORFs,andimplyadualroleforENOD40 RNAinplant
roots.
Yeast-three-hybrid studies showed that a novel protein,
MtRBP1,interactswithENOD40RNA.MtRBP1wasfoundtobe
cyto-plasmicinnoduleprimordiacellsexpressinghighlevelsofENOD40,
whereasMtRBP1localizedtonuclearspecklesinotherrootcells.
Consistentwiththis,uponexpressionofENOD40inheterologous
cells,MtRBP1relocatedfromthenucleustothecytoplasm.While
theshortORFsencodedbyENOD40didnotplayarolein
local-izationofMtRBP1,theRNAwasfoundtobedirectlyrequiredfor
cytoplasmiclocalizationofMtRBP1.ThefunctionofthisRNP
(ribo-nucleoprotein)is stillunknown,althoughit hasbeenproposed
tofunctionasatranslationalregulatorinthecytoplasm[52].
Fur-thermore,insoybean,thetwoshortpeptidesencodedbyENOD40
Fig.2. RegulatoryfunctionsofcodingmRNAs.(A)BasepairingleadstoRNAdegradation/translationalregulation.Glucosephosphatestress(G-6-P)leadstoactivationof transcriptionofacncRNA,SgrS.The5regionofSgrSencodesforashortpeptide(SgrT)whilethe3regionregulatestheexpressionofptsGmRNAbybasepairing.Theminimal basepairingregionofSgrSRNAisunderlinedandtheShine–Dalagarno(SD)sequenceofptsGmRNAishighlighted.Thisbasepairingleadstotranslationalrepressionand RNAdegradation.(B)StructuralroleofRNAincytoskeletalorganization.HereaXenopuseggisdepictedalongtheanimal(A)–vegetal(V)axisandthevegetalcortex(boxed) isillustratedindetail.InXenopusoocytes,cytokeratin(greenfilaments)formacomplexinterconnectednetworkspanningbetweenthecorticalgranules(brown)andthe yolkgranules(yellow)atthevegetalcortex.Germplasmislands(pink)areanchoredatthevegetalpolebythecytokeratinnetwork.Germinalgranules(red)arelocated withintheseislands.ProperorganizationofcytokeratinnetworkrequiresVegTRNA(blue).InVegTdepletedoocytes,longcytokeratinfilamentsaredisintegrated,andthe fragmentedcytokeratinnetworkaffectsgermplasmdistributionsuchthattheislandsandindividualgerminalgranulesfuseintolargeraggregates.(C)RNAasascaffoldto assembleregulatorycomplexes.Severalco-regulatorsparticipateinnuclearreceptorsignaling.Inabsenceofligand(L),repressorssuchasSHARPandSLIRPbindthenuclear receptors(NR)andrepresstranscriptionbymobilizinghistonedeacytylases(HDAC).Uponligandbinding,therepressorsarereplacedbyco-activators(e.g.,SRC-1andp300), thatinturnrecruitRNApolymeraseIIandinitiatetargetgeneexpression.SRA,theRNAco-regulatoristhoughttofunctionasascaffoldandbringsthewholecomplex togetheratthenuclearresponseelement(NRE)andfacilitatesgeneregulation.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothe webversionofthisarticle.)
PhosphorylatedSUC1undergoesproteasomaldegradation.Thus,
ENOD40 peptides regulatethe turnoverof SUC1. These diverse
functionssubstantiatethebi-functionalnatureofENOD40.Recent
studiesinArabidopsisandricehaveidentifiedanumberofRNAs
similartoENOD40thatcancodeforshortORFs[53,54].Itis
con-ceivablethatthesearealsopotentialcncRNAs.
3.2. MtHAP2-1
IthasbeenobservedthatshortORFsinthe5UTR(upstream
ORF, uORF) of an RNA can contribute to gene regulation [55].
Forinstance,aHAP2familytranscriptionfactorinM.truncatula,
byitsuORF.MtHAP2-1isakeyregulatorinthenodulemeristem
andfunctionsinnoduledevelopment.Alternativesplicingofthe
firstintroninthe5UTRofMtHAP2ispredominantduring
nodula-tion,andresultsinproductionofuORF1p.UnlikeotheruORFsthat
regulatetranslationbyribosomalstalling,uORF1prepresses
trans-lationbybindingtothe5UTRofMtHAP2-1[56].Thisregulation
isimportantforspatialregulationofMtHAP2-1andnodulation.
Hence,MtHAP2isanexampleofacncRNAwhosealternative
splic-ingresultsindualfunctionsoftheRNA.
3.3. miRNA-encodedpeptides(miPEPs)
ArecentreportfromCombierandcolleaguesshowsthatsome
pre-miRNA transcriptsinplants have functionalORFs [57].The
highlyconservedpre-miRNAsequenceofM.truncatulamiR171b
withonly 0.85% SNPs, suggested thepossibility of ORFs in the
sequence.Indeed,twoORFswerefoundinthe5 regionof
pre-miRNA 171b, encoding short peptides of 5 and 20 amino acid
residues, respectively. Further analysis with a -glucuronidase
(GUS)reportershowedthatonlytheORFencoding20-aminoacid
peptidenamedasmiPEP171bisexpressedandtranslatedatthe
lateralroot initiation site. Interestingly, miPEP171b specifically
enhancestheexpressionofmiR171b,andnotothermiRNAswhen
overexpressedaspre-miRNAinM.truncatularootsandintobacco
leaves.AdditionofsyntheticmiPEP171btotheseedlingsofM.
trun-catulaincreasedthelevelsofmiR171bandaffectedlateralroot
development.Analysisof50pre-miRNAsequencesfrom
Arabidop-sisthalianashowedpresenceofatleastoneORFineachsequence
[58].Interestingly,overexpressionofvariousmiPEPsencodedby
pre-miRNAof differentclassedin M.truncatulaand A. thaliana,
positivelycorrelatedwithaccumulationofcorrespondingmiRNAs.
InhibitionofRNAsynthesisduringoverexpressionofmiPEPsand
analysisinRNApolymerasesubunitmutantssuggestthatmiPEPs
functionastranscriptionalregulatorsofthecorrespondingmiRNAs
[57].Furtherstudiesarerequiredtounderstandhowcytoplasmic
translationofpre-miRNAandnuclearmaturationofmiRNAsis
reg-ulated.ThediscoveryofmiPEPsfurtherstrengthenstheconceptof
bi-functionalcncRNAs,and itwillbeinterestingtodetermineif
miPEPsexistinotherorganisms.
4. Bi-functionalRNAsinanimaldevelopment
Earlyembryogenesisofmanyanimalsreliesonalargenumberof
transcriptsmaternallydepositedintheoocytesandmediatingfirst
stepsofdevelopmentpriortocommencementofthezygoticgene
expressionprogram.SomeofthesematernalRNAsarerequiredfor
oocytematurationwhileothersarestoredintheformofmRNPs
and are translatedand/or degradedin an orchestrated manner
duringearlyphasesofembryonicdevelopment.Hence,maternally
depositedRNAsareundertightpost-transcriptionalregulationthat
includesregulatedprocessing,localizationandtranslation[59–63].
Itiswidelybelievedthatthemajorbiologicalfunctionof
localiza-tionandtranslationalcontrolofRNAsinoocytesandembryosis
spatialandtemporalregulationofthecorrespondingprotein
prod-uct.However,studiesinXenopus,Drosophilaandmorerecentlyin
zebrafishsuggestthatbesidescodingforproteins,localizedRNAs
canhaveadditionalnon-codingfunctions.
4.1. XenopusVegT
VegTwasidentifiedasamaternalRNAlocalizedtothevegetal
cortexXenopuslaevisoocytes.VegTcodesforaT-boxtranscription
factorthatpatternsthemesendodermalongthedorso-ventralaxis
[64].Heasmanetal.firstreportedthatdepletionofVegTmRNA
leadstodisruptionofvegetallocalizationofmaternalmRNAssuch
asVg1[65].Followingthis,Klocandcolleaguesdiscoveredthat
VegTmRNAand anon-codingRNAXlsirts, playstructuralroles
in theorganizationof thecytoskeletonat thevegetalcortex of
Xenopusoocytes,andthatthevegetalcytoskeletonisimportantfor
anchorageofgerm-linespecificRNAsandformationofthegerminal
granules[66].DepletionofeitherVegTorXlsirtsRNAbyinjection
ofantisenseoligonucleotidesspecificallydisruptedthecytokeratin
networkatthevegetalcortex.However,translation-blocking
anti-sensemorpholinosagainstVegTmRNAdidnotaffectcytokeratin
structure.Additionally,uponinjectionofsyntheticVegTmRNAinto
theVegTdepletedoocytes,thecytokeratinstructurewasrestored.
TheselinesofevidencessuggestedthatVegThasanmRNA-intrinsic
function.[66,67].FurtherstudiesbyKlocandcolleaguesto
ana-lyzethethreedimensionalultra-structureofcytoskeletonshowed
thatVegT mRNAmoleculesareintegrated intothemultilayered
cytoskeletonwhichcollapsesanddisintegratesintheabsenceof
RNA.Theintegrityofthecytoskeletonisimportantforcorrect
dis-tributionofthegermplasmandgerminalgranulesatthevegetal
cortex(Fig.2B).Basedonthesefindings,VegTmRNAhasbeen
sug-gested tohave a structuralfunction in germ-linedevelopment,
independentofthefunctionofVegTproteiningermlayer
pattern-ing[67].
4.2. Drosophilaoskar
Oskar(osk)wasidentifiedasamaternal-effectgenerequired
forantero–posteriorpatterningduringDrosophilaembryogenesis
[68].Duringearlyoogenesis,oskmRNAistransportedfromnurse
cellstothedevelopingoocyte.Subsequently,oskmRNAisactively
transportedtotheposteriorpole,whereOskproteinisexclusively
synthesizedfromlocalizedoskRNA.Priortolocalization,oskmRNA
istranslationallyrepressedbyCup,a4Ebindingprotein.Cup
regu-latesoskmRNAbyinteractingwithanRNA-bindingprotein,Bruno,
whichrecognizesspecificsequencemotifsinoskmRNA.Cup
com-peteswitheIF4GforbindingtoeIF4E,theproteinthatbindstothe
7-methyl-guanosinecapstructureinmRNAs.Interactionsbetween
eIF4Gand eIF4Earerequired for ribosomestoloadonmRNAs,
sosequestrationofeIF4EbyCupblockstranslation[69].Posterior
localizationandlocalizedtranslationofoskmRNAdeterminesthe
siteforformationofprimordialgermcellsandtheabdomen.Osk
proteinisknowntoregulateitsownRNAlocalizationand
func-tionsasascaffoldfortheassemblyofthegermplasm[70,71].The
classicaloskmutantsidentifiedinthematernaleffectscreenthat
producedembryoslackingabdomenandgermcellslacked
func-tionalOskproteinbutstillexpressedmRNA[72].Surprisingly,two
newoskalleleswithreducedornooskmRNAshowedmoresevere
andearlierdefectsduringoogenesiscomparedtooskallelesthat
expressmRNA.FemalesharboringRNAnullmutationsfailedtolay
eggsandweresterileasaresultofanearlyarrestduringoogenesis
[73].Theoogenesisarrestwascomplementedbynonsensemutant
alleleswhichstillexpressedoskmRNA,suggestingthattheearly
oogenesisfunctionofoskismediatedbyoskRNAandnotOsk
pro-tein.Toconfirmthispossibility,inaseriesofelegantexperiments,
Ephrussiandcolleaguesshowedthatoverexpressionofmerelythe
osk3UTRwassufficienttorescuetheegglessphenotypeofosk
RNA-nullmutants.Therefore,theysuggestedthattheosk3UTR
mightfunctionasascaffoldtoassembleRNPcomplexesthatare
requiredforoocytedevelopment[73].Inagreement,arecentstudy
showsthatlossofoskarRNAleadstoaccumulationofgermline
reg-ulatoryfactorsinthesomaticfolliclecellsandspecificelementsin
theoskar3UTRsequesterthetranslationregulator,Brunointhe
oocyte[74].Takentogether,thesestudiesshowthatoskfunctions
asaproteincoding-mRNAduringembryogenesisandanon-coding
RNAduringearlyoogenesis,andhencequalifiesasacncRNA.The
molecularmechanismsunderlyingthenon-codingfunctionofosk
4.3. Zebrafishsquint
Squint(Sqt)isaNodal-relatedsignalingmoleculebelongingto
thetransforming growthfactor beta(TGF) superfamily.Nodal
signalingplays important roles duringembryonic development
with essential functions in germ layer patterning [75,76]. The
roleofNodalsignalinginmesendoderminductionandpatterning,
specificationoftheventralneuraltube,andleft–rightaxis
spec-ificationhasbeenwellstudied [75,77–81].In additiontothese
knownroles,wediscoveredanovelnon-codingfunctionof
asym-metricallylocalizedmaternalsqt/nodaltranscriptsindorsalaxis
specification [82,83].In matureoocytes,sqt transcriptsare
dis-tributeduniformlythroughouttheyolk,andformdiscretepuncta
uponeggactivationandfertilization.Subsequently,thesesqtRNA
punctaformbiggeraggregatesandtranslocatetotheblastoderm
byamicrotubule-dependentmechanism[84].Bythe4-cellstage,
sqtRNAisasymmetricallylocalizedtooneortwocellsandthe
cellsacquiringsqt RNAarerequiredfortheformationofdorsal
structures [82].Removal of sqt-containing cells or depletionof
maternalsqtbyanti-senseoligonucleotidesresultedinembryos
with severe deficiencies in embryonic dorsal structures. These
experimentssuggestedthatasymmetricallylocalizedsqtRNAmay
functionindorsalaxisspecification.However,embryosobtained
fromhomozygousinsertionmutantsaffectingsqtexhibitmild
dor-saldefects,raisingquestionsregardingthecontributionofmaternal
sqtindorsalspecification[85,86].Interestingly,weobservedthat
whiletheinsertionmutantsforsqtdo notmake functional
pro-tein,mutantsqt RNAisexpressedandlocalizedinhomozygous
sqtinsertionmutantembryos.Furthermore,mutantsqttranscripts
expanddorsalprogenitorsinearlyzebrafishembryos.Usinga
vari-etyofmutationsthatdisruptSqtprotein,weshowedthatsqtRNA
functionsintheinitiationofembryonicdorsal,independentofSqt
protein.Over-expressionofthesqt3UTRsequencesrescuesthe
dorsaldefectsresultingfromdepletionofmaternalsqt.Subsequent
analysisof sqtRNAfunctioninmaternalmutantsaffectingWnt
andNodalsignalingshowedthatthedorsalizingfunctionofthesqt
3UTRrequiresWnt/cateninsignaling[83],butNodalsignaling
perseisnotrequiredforinitiationofdorsalspecification.These
findingsareconsistentwiththerequirementofNodalreceptorsand
theNodalco-receptor,One-eyedpinhead(Oep),fromlateblastula
stages[87,88].Basedontheseresultsweproposedarolefor
mater-nalsqtRNAinbindingandtransportingfactor(s)viaits3UTR,tothe
futuredorsalsideduringearlyblastulastagespriortothesignaling
functionsofSqtprotein.Suchabindingfactor(orcomplex)islikely
tofunctionviathecanonicalWnt/cateninpathway.Identification
ofthefactorsthatbindtosqt3UTRcanprovideinsightsintothe
mechanismsbywhichsqtRNAandparticularlythe3UTRcontrols
dorsalaxisformationviaWntsignaling.
We also uncovered another level of regulation that likely
controlsthecodingandnon-codingfunctionsofsqt inaspatial
andtemporal manner.Consistentwiththenon-codingfunction
ofsqtRNAinearlyembryos,maternalsqtRNAistranslationally
repressedduringearlycleavagestages[89].Y-boxbindingprotein
1(Ybx1),aconservednucleicacidbindingprotein,isrequiredfor
dorsal localizationof sqt and translationalcontrol of Sqt/Nodal
signalinginearlyzebrafishembryos.Ybx1bindstoalocalization
elementinthesqt3UTR, andtocap-bindingproteineIF4E,and
preventsSqt proteintranslation in earlyembryos.Maternal sqt
RNA is deposited in an unprocessed form in the egg, i.e., it is
un-spliced andnon-polyadenylated[83,90].TheRNA gets
com-pletely processedonly bythe 16-cellstage. In contrast,spliced
andpolyadenylatedsqt wasdetectedinembryosobtainedfrom
homozygousybx1femalesasearlyastheone-cellstage,indicating
prematureprocessingofthemRNAinmutantembryos.Consistent
withthis observation, Sqt proteinis precociously translated in
maternal ybx1 mutants compared to wild-type embryos. This
Fig.3.RNAprocessingfacilitatesdualfunctionofcncRNAs.(A)Regulatedsplicing, polyadenylation,andtranslationintemporalpartitioningofnon-codingversus cod-ingfunctionsofsqtRNAinzebrafish.A4-cellstagezebrafishembryoisdepictedwith dorsalprogenitorcells(D)attherightside.Inwild-typeembryos,bythe4-cellstage, sqttranscriptsareactivelylocalizedto1or2cells.Intheschematicrepresentation ofsqtRNA,blacklinesrepresentUTRs,blueboxesrepresentthe3codingexonsand thebluelinesrepresenttheintrons.MaternalRNAisnotcompletelyspliced,lacks apolyAtailandistranslationallyrepressedbyYbx1.Ybx1sequesterseIF4E(4E) eitherdirectlyorinacomplexwithaneIF4Ebindingprotein(4EBP)toprevent for-mationoftheeIF4translationpre-initiationcomplexandrecruitmentofribosomes (R).Inmaternalybx1mutant(Mybx1)embryos,sqtRNAfailstolocalizeandforms aggregatesintheyolk.MaternalsqtRNAisprecociouslyspliced,polyadenylated, andSqtproteinistranslatedprematurelyinMybx1mutantembryos.Thisleadsto prematureactivationoftheNodal/SquintpathwayinMybx1mutants.(B) Alterna-tivetranscriptionstartsites,intronretentionandalternativesplicingresultcoding andnon-codingisoformsofSRARNA.TheSRAgenomiclocusconsistsoffivecoding exons,andexonIhastwoin-framestartcodons(redasterisks).Thereisan alter-nativetranscriptionalstartsite(TSS)inexonIwhichleadstotheproductionofa non-codingisoformofSRA.AlternativesplicingleadstoretentionofintronIand sometimesintronIII,andcanalsoproducenon-codingSRAisoforms.(For interpre-tationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothe webversionofthisarticle.)
leadstoprematureandderegulatedSquint/Nodalsignaling,which
is catastrophic for embryonic development and maternal ybx1
mutantstypicallydonotsurvivebeyondearlygastrulastages[89].
Thus, sqt mRNA presentsan example of a cncRNA where RNA
processing and translation regulate thecoding and non-coding
functionsoftheRNA,suchthattheyaretemporallydistinctevents
duringembryonicdevelopment(Fig.3A).
5. EpigeneticregulationbyRNAs
RNAmoleculesactivelyparticipateinepigeneticregulationby
physicallyinteractingwithchromatinmodifyingenzymes.They
areinvolvedinmodificationofhistonesandDNAmethylation[91].
StudiessofarsuggestthatRNA-mediatedepigeneticregulationis
carriedoutforthemostpartbynuclearlncRNAsandlncRNAshave
beenshowntofunctionasbothactivators(HOTTIP,Mistral,etc.)and
repressors(HOTAIR,ANRIL,Xist,etc.)(reviewedin[92,93]).
How-ever,recentdatafromCoolenandEstellerlaboratoriessuggests
thatcoding RNAsmayalsobeinvolved inepigeneticregulation
[94]. Byspecifically lookingforRNAsthat arebound toSUZ12,
chemically cross-linked samplesof humanprostate cancercell
lines,Coolenandcolleaguesidentifiedanumberofproteincoding
RNAsthatbindtoSUZ12withaffinitiescomparabletothatof
lncR-NAs[94].Theyalsore-analyzedasimilardatasetfromtheEsteller
lab where RNAs bound to EZH2, another component of PRC2,
wereimmune-precipitatedandsequenced,andidentified
protein-codingRNAs[95].Thisstudywasperformedinhumancolorectal
cancercelllines.AnalysisofRNA-sequencingdataobtainedfrom
mouseembryonicstemcellsalsoidentifiedprotein-codingRNAs
thatbindtoEZH2[96].Althoughthefunctionalsignificanceofsuch
PRC2-mRNAinteractionareyettobediscovered,takentogether
thesestudiessuggestthatevenprotein-codingRNAscan
partici-pateinepigeneticregulation.
6. Bi-functionalRNAsindisease
MutationsleadingtodysfunctionalRNAscanleadtoavarietyof
humandiseasesrangingfromneuro-degenerationtocancer.Here,
wedescribethepathologicalfunctionofsomeRNAsindependent
oftheirproteinfunction.
6.1. SRA,adualfunctionco-regulatoroftranscriptionfactors
SteroidreceptorRNAActivator(SRA)wasthefirstmammalian
RNAtobediscoveredwithdual roles,proteincoding and
non-coding,inmyogenicdifferentiation[97].SRAwasinitiallyidentified
asapartnerofprogesteronereceptorwithco-regulatoryfunctions
[98].DespitethepresenceofalongORF,Lanzandcolleaguesdid
notdetectaproteinproductencoded bySRAmRNA. Theythen
testedtheabilityofSRAtoco-activateglucocorticoidreceptorin
thepresenceofadenovoproteinsynthesisinhibitor,
cyclohex-amideandconcludedthatSRAfunctionsasanRNAco-activatorof
nuclearreceptors[98].Soonthereafter,anumberofstudies
demon-stratedthatSRAco-activatesmanynuclearreceptorsincludingthe
estrogen,androgen, gluco-corticoid and retinoic acidreceptors.
Secondary structure prediction of SRA RNA followed by
muta-tionalanalysissuggestedthepresenceofmultiplestemloopsin
SRARNAthatarerequiredforitsactivity[99].SRA functionsas
ascaffoldthatbringstogethertranscriptionalco-activators,RNA
polymerase as well as gene insulators/repressors (reviewed in
[100–102])(Fig.2C).Theactivityoftranscriptionfactorssuchas
MyoDandGATA-3isalsoenhancedbySRARNA[103,104].
Sub-sequentsequenceanalysistoidentifythetranscriptionstartsite
showedthepresenceofanovelisoformofSRA,containingan
addi-tional5 exon.The5exoncontainstwoATGstartcodonsinthe
sameframethatcouldpotentiallyleadtothetranslationofeither
a224ora236-aaSRApeptide(SRAP).Theauthorsconfirmedthe
presenceofthecodingRNAisoformand doubletof
correspond-ingpeptides byreverse transcriptionand westernblotanalysis
respectively[97].Differentialtranscriptionalstartsiteand
alter-nativesplicing,resulting ineither retentionor exclusion ofthe
firstand sometimesthirdintron, determineswhetherSRA
func-tionsasacodingoranon-codingRNA(Fig.3B)[105,106].SRAPis
conservedamongchordatesandoneofthedomainsfoundinall
annotatedSRAPscontainsaRNArecognitionmotif(RRM),a
puta-tivenuclearlocalizationsignalandamotifthatmightinteractwith
nuclearreceptors[107].Usingsilentmutationsthatdisrupted
reg-ulatorymotifsinSRARNAandnonsensemutationsthatdisrupted
SRAP,itwasestablishedthatSRAPfunctionsinbothactivatorand
repressorcomplexesofnuclearreceptors,independentofSRARNA
[108–110].Incontrast,muscledifferentiationstudiesshowedthat
SRAPpreventedSRARNA-dependentactivationofMyoD[103].
Interestingly,SRARNAisexpressedathigherlevelsinhuman
breast tumors as compared to adjacent tissues, and the levels
increasewithtumorprogression[111,112].SRAncRNAandSRAP
co-existinbreastcancercelllinesandtherelativeexpressionofthe
twomoleculesdiffersindifferentphenotypes,withhigherlevelsof
non-codingSRAdetectedininvasivecelllines.Thissuggeststhat
thebalancebetweenthetwoisoformsmightdefinetumor
phen-otypesandaltergeneexpressionduringtumorprogression[113].
Thesedataalsohighlighttheroleofalternativesplicingintumor
metastasis.SRAPisknowntofunctionasaco-activatorofandrogen
receptorsinprostatecancer.However,itspreciseroleintumor
pro-gressioninthiscontextisnotfullyunderstood,anditisunknown
ifnon-codingSRARNAhasaroleinprostratetumors[108,114].
TakentogetherthesestudiesshowthattheSRAlocuscodesfor
variousSRARNAisoformsthathaveeithercodingornon-coding
functions,andthatinsomecontexts,thecodingandnon-coding
functionscanbeintertwined.Importantly,thebalanceofSRA
iso-formsisrelevanttobothnormaldifferentiationanddisease.
6.2. DMPKinmyotonicdystrophy
Myotonicdystrophy(DM)isanautosomaldominantinherited
diseasecharacterizedbyslowprogressingmulti-systemic
symp-tomslikemusclewasting,myotonia,cardiacdefectsandreduced
cognitiveability.Bypositionalcloning,theDM1mutation
associ-atedwithtype1myotonicdystrophywasidentifiedasavariable
lengthpolymorphismwhichresultedfromincreasednumberof
trinucleotideCUGrepeatsinthe3UTRofDMproteinkinase(DMPK)
expressedin tissuesaffected bymyotonic dystrophy [115,116].
Theseverityofclinicalsymptomsofmyotonicdystrophycorrelates
withthenumberofCTGrepeatsfoundinpatients[117,118].
Unaf-fectedindividualshavelessthan38repeatswhereaspatientshave
between50and1500repeats.MutantDMPKmRNAwithexpanded
CUG repeats (CUGexp-RNAs) is transcribed but the transcripts
aresequesteredasdiscrete fociinnucleileadingtocytoplasmic
depletionofDMPKmRNA[119].Haplo-deficiencyofDMPKprotein
and/orSIX5encoded bythedownstreamgene leadstodelayed
onset ofmild symptoms, but werenot foundtobecompletely
responsibleforDM1phenotypes[120,121].However,recent
evi-dence suggests that the DM1 pathology involves a toxic gain
offunctionbymutantCUGexp-RNA.Structuraland biochemical
experimentsshowedthattheCUGrepeatsformastablehairpin
structure [122,123]. Moreover, CUGexp-RNA is not transported
tothecytoplasmandformsdiscreteaggregatesattheperiphery
ofnuclearspeckles,whicharestructuresenrichedwithsplicing
related factors[124].The hairpinstructure sequesters
develop-mentallyregulatedsplicingfactorslikeMBNL(Muscleblindlike)
[125].Anothersplicingfactor,CELF1(CUGBP1)alsobindtosingle
strandedCUGsequencesbutdonotco-localizewiththenuclear
aggregates of CUGexp-RNAs. However, expression of
CUGexp-RNA leadsto hyper-phosphorylationand stabilizationof CELF1
[126,127].Mis-regulationofMBNLandCELF1disruptssplicingof
asubsetofRNAsandleadtoembryonicsplicingpatternsinadult
tissue,andhencehasaprimaryroleindevelopmentofmyotonic
dystrophy [128]. Thus, repeat expansion of certain nucleotides
canconvertanmRNAintoafunctionalRNAimplicatedinprotein
sequestrationandhumandisease(Fig.4A).Expansionofsimilar
triplets(CGG, GAA),whicharecapableofbasepairing,inother
RNAshavebeenfoundassociatedwithanumberofotherhuman
diseasessuchasfragileXtremorataxiasyndromeandFriedreich
ataxia[129].
6.3. p53RNAinmammalianbreastcancercells
Disruptionofp53,acriticaltumorsuppressorgene,isthemost
frequent singlegene eventleading to humancancers. Thep53
proteinispost-translationallymodifiedandrenderedactiveasa
transcriptionfactorinresponsetostressessuchasDNA-damage,
Fig.4.cncRNAsindisease.(A)SequestrationandmodulationofregulatoryproteinsbyDMPKmutantRNAintypeImyotonicdystrophy.Variablelengthpolymorphismresulting fromincreasednumberofCUGrepeatsin3UTRofDMPKgeneleadstotranscriptionofatoxicformofRNA(DMPKCUGexp-RNA).TheCUGrepeatsformastablestem
loop(thehairpinintheDMPKCUGexp-RNA).ThesplicingfactorMNBL(yellow)directlybindstotheCUGrepeats.SequestrationofMBNLmakesitinactive.DMPKCUG-exp RNAstabilizesanothersplicingfactor,CELF1byindirecthyperphosphorylation.Thisleadstomis-regulationofalternativesplicingandmanifestationoftypeImyotonic dystrophy(DMI)symptoms.(B)ceRNAsasmiRNAsponge.AgroupofmRNAssharingaparticularmiRNAresponseelement(MRE)functionasceRNAsandinfluenceeach other’stranslation.Inanormalstate,alimitedpoolofmiRNAscanregulatethetranslationofanumberofmRNAsinaceRNAnetwork.WhentheexpressionofonemRNAis changed,theredistributionofavailablemiRNAmoleculeswillresultinachangeintranslationaloutputofothermRNAsinthenetwork,potentiallyleadingtodiseasestates. Inthisschematic,ORF2functionsasamiRNAspongetoregulatethetranslationaloutputofORF1andORF3.WhenORF2expressionisreduced(diseasestatei)miRNAthat wasboundtoORF2willbeavailabletotargetotherRNAsleadingtorepressionofORF1andORF3whilewhenORF2isoverexpressed(diseasestateii),translationalrepression ofORF1andORF3bymiRNAwillbealleviatedduetopresenceofmoreMREs.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothe webversionofthisarticle.)
lead to cancer. Activated p53 initiates a program of cell cycle
arrestand apoptosis[130,131].Thep53proteinis expressedas
at-leastfourdifferentisoformsresultingfromalternative
initia-tioncodons.Theseisoformswerefounddifferentiallyexpressed
inhumanbreastcancersamplesascomparedtonormalbreast
tis-sue[132,133].Mdm2,anE3ubiquitinligaseisamajorregulatorof
p53proteinandpreventsexcessiveandpersistentp53activation
viaafeedbackregulation[134,135].Recently,itwasshownthat
thereisanadditionalfeedforwardregulation,whereinp53mRNA
interactswithMdm2andleadstoenhancedp53translationand
stabilization[136].Mdm2associateswithp53polysomesviaits
RINGdomainandprobablyenhancestranslation.Consistentwith
thispossibility,aCUAtoCUGmutationinp53wasidentifiedin
Fig.5. Binaryphenotypes–proteinnullversusRNAnull.NormaltranscriptionandtranslationofacncRNAwillresultinwild-typephenotype.Mis-senseorinsertion mutationsinthegenomecanresultinmutantRNA(redasterisk)thatmightbestableifnottargetedbynon-sensemediateddecaypathway,andcancarryoutthenon-coding function.So,aproteinmutantphenotypewillbeobservedwithoutaffectingtheactivityoftheRNA.Butmutationsthateliminatethetranscript(transcriptionstartsiteorTSS mutationsandgenedeletions)orantisenseoligosthatdegradeRNAwillleadtoabinaryphenotyperesultingfromlossofbothRNAandproteinfunction.(Forinterpretation ofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
impairMdm2-mediatedenhancementofp53translation[137].p53
mRNArecruits Mdm2top53polysomes wherethelatter likely
functionsasachaperoneforp53proteinfolding.Duringthis
pro-cess,theE3ligaseactivityofMdm2isinhibited[136,138].Thus,
p53mRNAactsasaswitchthatcontrolsMdm2regulationofp53
protein.Interestingly,theregionofp53mRNAthatencodesforthe
Mdm2bindingsite inp53protein,alsointeractswiththeRING
domainofMdm-2[136].Thisisanexamplewherethesameregion
ofRNAmediatesbothcodingandnon-codingfunctions.Mutations
insuchRNAsshouldbedesignedcarefullybecausetheyhavethe
potentialtoaffectboththeactivityoftheRNAandtheencoded
proteinandleadtobinaryphenotypes.
6.4. CompetingendogenousRNAsincancer
Codingandnon-codingtranscriptscanfunctionasaspongeto
bindmiRNAsandalleviatetherepressiveactivityofmiRNAsonthe
targetmRNAs.SuchRNAsthatregulatetheactivityofotherRNAs
bydirectlycompetingformiRNAbindingarenamedascompeting
endogenousRNAs(ceRNAs)andanyperturbationintheirlevels
canleadtodiseasestates[139](Fig.4B).Oneofthebest-studied
examplesofceRNAregulatorynetworksisoneencompassingthe
tumorsuppressorgenePTEN.PTENencodesaphosphatase that
antagonizes the highly oncogenic PI3K/Akt signaling pathway.
Variousco-expressedRNAslikeVAPA,CNOT6LandPTENP1(a
non-codingpseudogeneofPTEN)werefoundtosharemiRNAresponse
elements(MREs)withPTEN.TheirRNAswereshowntorelieve
miRNA-mediatedrepression ofPTEN. Consistently,Pandolfi and
colleaguesshowedthatcopynumberlossoftheseceRNAsduring
cancerpromotestumorigenesisbyrepressingPTEN.These
interac-tionswereshowntobereciprocalasPTENmRNAcanalsoregulate
theexpressionofVAPAprotein[140].Thus,theceRNAsexhibita
regulatoryfunctioninadditiontotheirproteincodingfunction.
ManysuchRNAsthatfunctioninamiRNAdependentcross-talk
and theirregulatoryfunctionsin tumorsuppressionhave been
identified(reviewedin[141,142]).
7. Conclusionandperspectives
Here,wereviewedtheemergingclassofbi-functionalRNAsthat
combineprotein-codingandnoncodingfunctionsinasingleRNA
molecule. Thecurrent listof thesemoleculesmight belimited,
butphylogeneticanalysisandRNAstructurepredictionssuggest
thatthislistislikelytoexpandinthefuture[143,144].Indeed,a
largenumberofncRNAslackingcanonicalORFsaretranscribedby
polymeraseII,spliced,cappedandpolyadenylatedjustlikemRNAs
[145,146].Itremainsanopenquestionwhatfractionofthesecan
betranslatedintoshortfunctionalpolypeptides.Ontheotherhand,
theprotein-centricview that hasdominated molecularbiology
sinceitsinceptionmighthavebiasedcharacterizationofmRNAs
totheir‘informationmessenger’role leavinga wealthof
struc-turaland/orregulatoryfunctionslargelyunexplored.Animportant
futurechallengewillbetounderstandhowthesecncRNAsbalance
theircodingversusnon-codingcapacities.Dotheypartitionthetwo
functionstophysicallydistinctdomains(asexemplifiedby
bacte-rialSgrS,Drosophilaoskandzebrafishsqt)oralternatively,dothey
utilizetheprimarysequenceforencodingproteinswhilereserving
thesecondaryor tertiarystructure ofthesameregionfor
non-codingroles?DeterminingcncRNAconformationusingemerging
experimentalapproaches [147,148] should improve our
under-standingofhowthesevariedfunctionsareelicited.Inaddition,for
manyoftheknowncncRNAssuchasSRARNA,regulatedprocessing
eventssuchasalternativesplicing,cleavageandpolyadenylation
underlietheirabilitytoperformcodingversusnon-coding
func-tions.Arecentstudysuggestedthatabout300alternativelyspliced
bi-functionalRNAsmightexistinthehumangenome[149].
There-fore, we propose that the lossof RNAfunction phenotypes be
examinedforidentifyingnewcncRNAloci,asprotein-null
phen-otypesmightbedistinctfromtheRNA-nullmutants(Fig.5).An
importantfuturedirectionwillbeteasingapartprotein-codingand
non-codingfunctionsforcncRNAloci usingappropriategenome
editing methods [150,151]. This would require careful design
of genomic lesions to specificallytest phenotypicconsequence
characterizationofnovelcncRNAsandthemechanismsbywhich
theyelicittheirvariousfunctions,canprovidenewinsightsinto
generegulationinthecontextofnormalhomeostasisanddisease
states.
Acknowledgements
PKissupportedbytheFriedrichMiescherInstitutefor
Biomed-icalResearch and KSis supportedby WarwickMedical School.
WethankJonathanMillarforcoiningtheterm“cncRNA”,andour
colleaguesinSingapore,Warwick,and Baselfordiscussionsand
suggestions.
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