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SHORTREPORTOpenAccessLimitedevidenceforevolutionarilyconservedtargetingoflongnon-codingRNAsbymicroRNAsBabakAlaei-MahabadiandErikLarsson*AbstractBackground:Longnon-codingRNAs(lncRNAs)areemergingasimportantregulatorsofcellphysiology,butitisyetunknowntowhatextentlncRNAshaveevolvedtobetargetedbymicroRNAs.
ComparativegenomicshaspreviouslyrevealedwidespreadevolutionarilyconservedmicroRNAtargetingofprotein-codingmRNAs,andhereweappliedasimilarapproachtolncRNAs.
Findings:WeusedamapofputativemicroRNAtargetsitesinlncRNAswheresiteconservationwasevaluatedbasedon46vertebratespecies.
Wecomparedobservedtargetsitefrequenciestothoseobtainedwitharandommodel,atvariablepredictionstringencies.
WhileconservedsiteswerenotpresentaboverandomexpectationinintergeniclncRNAsoverall,weobservedamarginalover-representationofhighlyconserved8-mersitesinasmallsubsetofcytoplasmiclncRNAs(12sitesin8lncRNAsat56%falsediscoveryrate,P=0.
10).
Conclusions:EvolutionaryconservationinlncRNAsisgenerallylowbutpatch-wisehigh,andthesepatchescould,inprinciple,harborconservedtargetsites.
However,whileouranalysisefficientlydetectedconservedtargetingofmRNAs,itprovidedonlylimitedandmarginallysignificantsupportforconservedmicroRNA-lncRNAinteractions.
WeconcludethatconservedmicroRNA-lncRNAinteractionscouldnotbereliablydetectedwithourmethodology.
Keywords:Longnon-codingRNA,lncRNA,microRNA,ComparativegenomicsFindingsBackgroundWhilesmallnon-codingRNAs,suchasmicroRNAs,havewell-establishedfunctionsinthecell,longnon-codingRNAs(lncRNAs)haveonlyrecentlystartedtoemergeaswidespreadregulatorsofcellphysiology[1].
Althoughearlyexampleswerediscovereddecadesago,large-scaletranscriptomicstudieshavesincerevealedthatmammaliangenomesencodethousandsoflong(>200nt)transcriptsthatlackcodingcapacity,butareotherwisemRNA-like[2-4].
Theirbiologicalimportancehasbeencontroversial,butnovelfunctionallncRNAswithroles,forexample,invertebratedevelopment[5],pluripotency[6]andgenomestability[7]arenowbeingdescribedatincreasingfrequency.
Afewrecentstudiesdescribeinteractionsbetweensmallandlongnon-codingRNAs,wherelncRNAsacteitherasregulatorytargetsofmicroRNA-induceddestabilization[8,9]orasmoleculardecoysofmicroRNAs[10-13].
Re-centresultsalsoshowthatstablecircularlncRNAscanbindandinhibitmicroRNAs[14,15].
Importantly,RNAi-basedstudies,includingsilencingof147lncRNAswithlentiviralshRNAs[6],showthatlncRNAsare,inprinciple,susceptibletorepressionbyArgonaute-smallRNAcom-plexes,despiteoftenlocalizingtothenucleus.
Inaddition,therearedatafromcrosslinkingandimmunoprecipitation(CLIP)experimentsthatsupportbindingofArgonauteproteinstolncRNAs[16,17].
Comparativegenomicshasrevealedthatmostprotein-codinggenesareunderconservedmicroRNAcontrol:conservedmicroRNAtargetsitesarepresentin3'un-translatedregions(UTRs)ofprotein-codingmRNAsatfrequenciesconsiderablyhigherthanrandomlyexpected,clearlydemonstratingtheimpactofmicroRNAsonmRNAevolution[18,19].
WhilelncRNAsingeneralareweaklyconserved,theymayhavelocalpatchesofstrongsequenceconservation[20].
Itwasrecentlyshownthatde-velopmentaldefectscausedbyknockdownoflncRNAsinzebrafishcouldberescuedbyintroductionofputativehu-manorthologsidentifiedbasedonsuchshortpatches[5],*Correspondence:erik.
larsson@gu.
seDepartmentofMedicalBiochemistryandCellBiology,InstituteofBiomedicine,TheSahlgrenskaAcademy,UniversityofGothenburg,SE-40530,Gothenburg,Sweden2013Alaei-MahabadiandLarsson;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Alaei-MahabadiandLarssonSilence2013,4:4http://www.
silencejournal.
com/content/4/1/4supportingthatlncRNAfunctionsmaybeconservedoverlargeevolutionarydistancesdespitelimitedse-quencesimilarity.
ItisthusplausiblethatlncRNAsalsohaveevolvedtobetargetedbymicroRNAsdespitetheiroveralllowconservation,andthatthiswouldmanifestitselfthroughthepresenceoftargetsitesinlocalcon-servedsegments.
ResultsWeusedourpreviouslydescribedpipelinetomapandas-sesstheevolutionarilyconservationofputativemicroRNAtargetsitesinlncRNAs[21].
Briefly,wemappedcomple-mentarymatchestoestablishedmicroRNAseedfamiliesintheGENCODEv7lncRNAannotation,whichwasre-centlycharacterizedindetailbytheENCODEconsortium[4].
Conservationlevelsweredeterminedbasedona46-vertebratemultiplesequencealignment[22],andsiteswerescoredbasedontheirpresenceinprimates,mam-malsandnon-mammalvertebrates.
Thisallowedustovarythestringencytoconsiderprogressivelysmallersetsoftranscriptswithhigherconservationlevels.
Wecom-paredobservedsitefrequenciestoexpectedfrequenciesbasedonarandomdinucleotidemodel,inprotein-codinggenesandinsubsetsoflncRNAs(Figure1).
Ouranalysisrevealedwidespreadpresenceofcon-servedtargetsitesinmRNAs,whichrecapitulatesprevi-ousobservationsandestablishesourmethodology[18,19].
Dependingonpredictionstringency(conserva-tionlevelandseedtype),seedcomplementarymatchestoconservedmicroRNAfamilieswerepresentatupto6.
1*theexpectedfrequencyin3'UTRs,and1.
4*incodingregions(Figure2A).
Sitesfornon-conservedmicroRNAfamilies,whichwereincludedasanegativecontrol,wereobservedonlyatexpectedfrequencies(Figure2A).
Next,weinvestigatedsitefrequenciesinlncRNAs,spe-cificallyoftheintergenictypetoavoidconfoundinggen-omicoverlaps.
Inasetof2,121intergeniclncRNAgenes,weobservednosignificantenrichmentofsites(Figure2B).
Restrictingoursearchto3'or5'endsoftranscripts,orsubsetsofintergeniclncRNAspreviouslyfoundtohaveconservedpromoterregions[4],resultedinasimilarlackofenrichment(datanotshown).
ManydescribedlncRNAsparticipateintheassemblyofriboproteincomplexesinthenucleus[1],whilemicroRNAsareconsideredtobeactiveprimarilyinthecytoplasm.
WeusedsubcellularRNA-seqdatatonarrowdownouranalysistoasmallersetofcytoplasmiclncRNAs(n=169),whichwerealsoexpressedatcom-parativelyhighlevels(Figure2B).
Pan-mammaliancon-servedhigh-quality(8-mer)siteswerehereobservedat1.
8xtheexpectedfrequency(P=0.
10),whichcorre-spondstoafalsediscoveryrateof56%,butthenumberoftargetsandsiteswassmall(12sitesin8lncRNAgenes,Table1).
OneoftheeighttargetlncRNAs(AC010091.
1)showeddistanthomologytohumanpro-tocadherinFat4protein(maximum36%identityover94a.
a.
),andcouldthusrepresentanancientpseudogeneormisclassifiedcodinggene.
Allotherslackedhomologytoanyof565,000+knownsequencesinUniProtKB/Swiss-Prot,andsevenoutofeightwerealsoclassifiedaslongnon-codinginarecentRNA-seq-basedmappingofhumanlncRNAs[3].
ConservedtargetingoflncRNAsbymicroRNAsisplausible,giventhatLncRNAsaresusceptibletoAGO-mediatedrepression,andthattheyshowpatch-wisestrongsequenceconservation.
However,ouranalysisin-dicatesthatthisisnotawidespreadphenomenon,eventhoughasmallsubsetofcytoplasmictranscriptsshowedaweakenrichmentofconservedsitesatmarginalstatis-ticalsignificance.
LncRNAsarecurrentlydefinedsolelybasedonlengthandcodingcapacity,andareassuchlikelytorepresentahighlyfunctionallydiversegroup.
Itisthuspossiblethatother,notyetdefined,subfamilieshaveevolvedtobemicroRNAtargets,butthatthissignalistoodilutedtobedetectableinourcurrentanalysis.
ItshouldbenotedthattheGENCODEannotationusedhereisoneofseveralpublishedlncRNAsets,andFigure1WorkflowtodetectconservedmicroRNAtargetingoflongnon-codingRNAs(lncRNAs).
ConservedmicroRNAtargetsites(complementaryseedmatches)wereidentifiedintheGENCODEhumangeneannotationbasedona46-speciesmultiplesequencealignmentasdescribedpreviously[21].
Atotalof1,267microRNAfamilieswereconsidered.
DifferentsubsetsoflncRNAswereanalyzedforover-representationofsitescomparedtoarandombackgroundmodel.
Alaei-MahabadiandLarssonSilence2013,4:4Page2of5http://www.
silencejournal.
com/content/4/1/4whilecomprehensive,itdoesnotcoverallknowntran-scribedloci[3].
Likewise,thereareseveralapproachestotargetsitepredictionanddetailedresultsmayvary.
Not-ably,ouranalysiswasdesignedtocaptureanoverallsig-natureofconservedtargeting,andwhenappliedtomRNAsitefficientlyrecapitulatedastrongenrichmentsignal.
Differentimplementationsandannotationscouldgivevariableresultsatthelevelofindividualtranscriptsandsites,butthemainconclusionisunlikelytodependontheseparameters.
WhilesomeestablishedmicroRNA-lncRNAinter-actionsitesareconservedtovariousextents,inprincipleenablingdetectionbycomparativegenomicsapproaches[8-10],otherslackconservationdespitehavingexperi-mentallyconfirmedfunctions[12,13].
Thisisconsistentwithdatashowingthatmanynon-conservedhumanmicroRNAsitescanmediatetargeting[23].
Notably,evenwell-characterizedlncRNAs,suchasHOTAIRandXIST,haveoftenevolvedrapidly,andmayshowconsid-erablefunctionalandstructuraldifferenceswithinthemammalianlineage[24,25].
Ourcomparativegenomicsmethodologythereforedoesnotexcludethatnon-conservedandrecentlyevolvedtargetingcouldbecom-monplace,andthismotivatesfurthercomputationalandexperimentalstudies.
MethodsWereliedontheGENCODEcoding/non-codingclassifi-cation,andconsideredaslncRNAsgenesthatonlypro-ducedtranscriptsofthe'antisense','lincRNA','non_coding'ABFigure2RatiosbetweenobservedandexpectedmicroRNAtargetsitefrequenciesincodinggenesandlongnon-codingRNAs(lncRNAs).
(A)Ourmethodologywasfirstestablishedoncodinggenes.
The3'untranslatedregions(UTRs)andcodingsequences(CDS)wereanalyzedseparately.
Wecomparedobservednumbersofseedmatches(inparentheses)torandomlyexpectednumbersbasedonsetsofsyntheticseedsthatpreservedthedinucleotidefrequenciesoftheactualseeds.
Differentpredictionstringencies(siteconservationlevelandseedquality)wereapplied,furtherexplainedwithingrayboxes.
TheanalysisfocusedonhighlyconservedmicroRNAfamilies(n=87),butnon-conservedfamilieswereincludedasacontrol.
Barsshowmeanobserved-to-expectedratiosfrom20repeatedtrials.
(B)SimilaranalysisbasedonintergeniclncRNAsandcytoplasmicintergeniclncRNAs.
Placentalmammalconserved8-mersiteswerepresentaboveexpectationinasmallsubsetofcytoplasmicintergeniclncRNAs(12sitesfor11microRNAfamilies,in8lncRNAgenes).
SubcellularlocalizationwasdeterminedbasedonRNA-seqlibrariesfromsevenfractionatedcelllines.
*,empiricalP1wererequiredforsignificanceattheempiricalP≤0.
05level,and18/20forP=0.
10.
MicroRNAfamilydefinitionsandconservationclassifi-cationswerederivedfromTargetScan[18].
Weuseddatafromapreviousstudy[4]todefinesubsetsoflncRNAswithconservedregulatoryregions.
The500or250mostconservedintergeniclncRNAsbasedoneitherpan-mammalorpan-vertebratepromoterconservationscores(intotal,foursets)wereanalyzedasdescribedabove.
RNA-seqdata(fastqfiles)producedwithintheENCODEproject[26]bytheGingeraslaboratory(ColdSpringHarborLaboratories,ColdSpringHarbor,NY,USA)wereobtainedthroughtheUCSCFTPserver.
Atotalof1.
71billion76ntreadpairsfrompolyA+nu-clearandcytoplasmicfractionsfromsevenhumancelllines(Gm12878,HelaS3,HepG2,Huvec,H1hesc,NhekandK562)werealignedtothehumanhg19referencegenomewithTophat[27].
ThealignerwassuppliedwithGENCODEgenemodelsusingthe-Goption.
GeneswerequantifiedusingtheHTSeq-countutility(http://www-huber.
embl.
de/users/anders/HTSeq).
Cyto-plasmictranscriptsweredefinedashavinganormalizedcytoplasm/nucleusratio>1.
Atotalofatleast20mappedreadsacrossallconditionswasrequired,toavoidunreliablecytoplasm/nuclearratiosinthelow-abundancerange.
Ethicalapprovalorpatientconsentwasnotrequiredforthisstudy.
AbbreviationsCDS:Codingsequence;CLIP:Crosslinkingandimmunoprecipitation;LncRNA:Longnon-codingRNA;UTR:Untranslatedregion.
Table1Pan-mammalianconserved8-merputativemicroRNAtargetsitesincytoplasmicintergeniclongnon-codingRNAs(lncRNAs)TargetGENCODEIDTargetsymbolMicroRNAfamilySitechromosomeSitegenomepositionCabilietal.
lincRNAaUniProtKB/Swiss-ProtBLASTbENSG00000226856.
1AC093901.
1miR-182chr2118940821YesNohitsENSG00000231532.
1AC022311.
1miR-133abcchr24676715YesNohitsENSG00000231532.
1AC022311.
1miR-22/22-3pchr24676706↑↑ENSG00000231532.
1AC022311.
1miR-383chr24676629↑↑ENSG00000233491.
2AC010091.
1miR-133abcchr781218260YesE=4e-5(HumanFAT4)ENSG00000233491.
2AC010091.
1miR-9/9abchr781218258↑↑ENSG00000236719.
2RP11-522D2.
1miR-30abcdef/30abe-5p/384-5pchr1180535222YesNohitsENSG00000245017.
1AC013418.
2miR-138/138abchr1298879829YesNohitsENSG00000248927.
1CTD-2334D19.
1miR-135ab/135a-5pchr5120126269YesNohitsENSG00000248927.
1CTD-2334D19.
1miR-19abchr5120126442↑↑ENSG00000250366.
1AL133167.
1miR-218/218achr1496389499YesNohitsENSG00000253507.
1CTD-2501M5.
1miR-146ac/146b-5pchr8132329800NoNohitsaAnnotatedasalongnon-codingRNAinCabiliMN,TrapnellCetal.
,GenesandDevelopment(2011).
bHitswithBLASTE-value<0.
5.
Repeatmaskingwasperformedtoavoidmatchesto,forexample,translatedSINEsinSwissProt.
GenomiccoordinatesrefertotheHg19assembly.
Alaei-MahabadiandLarssonSilence2013,4:4Page4of5http://www.
silencejournal.
com/content/4/1/4CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Author'scontributionsELdesignedthestudy,analyzeddata,andwrotethemanuscript.
BAanalyzeddata.
Bothauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsWewouldliketoacknowledgeDrs.
AndersJacobsenandDeboraS.
Marksforhelpfulcommentsanddiscussions.
ThisworkwassupportedbygrantsfromtheSwedishMedicalResearchCouncil;theSwedishCancerSociety;theAssarGabrielssonFoundation;theMagnusBergvallFoundation;thekeWibergfoundation;andtheLarsHiertaMemorialFoundation.
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doi:10.
1186/1758-907X-4-4Citethisarticleas:Alaei-MahabadiandLarsson:Limitedevidenceforevolutionarilyconservedtargetingoflongnon-codingRNAsbymicroRNAs.
Silence20134:4.
SubmityournextmanuscripttoBioMedCentralandtakefulladvantageof:ConvenientonlinesubmissionThoroughpeerreviewNospaceconstraintsorcolorgurechargesImmediatepublicationonacceptanceInclusioninPubMed,CAS,ScopusandGoogleScholarResearchwhichisfreelyavailableforredistributionSubmityourmanuscriptatwww.
biomedcentral.
com/submitAlaei-MahabadiandLarssonSilence2013,4:4Page5of5http://www.
silencejournal.
com/content/4/1/4
ComparativegenomicshaspreviouslyrevealedwidespreadevolutionarilyconservedmicroRNAtargetingofprotein-codingmRNAs,andhereweappliedasimilarapproachtolncRNAs.
Findings:WeusedamapofputativemicroRNAtargetsitesinlncRNAswheresiteconservationwasevaluatedbasedon46vertebratespecies.
Wecomparedobservedtargetsitefrequenciestothoseobtainedwitharandommodel,atvariablepredictionstringencies.
WhileconservedsiteswerenotpresentaboverandomexpectationinintergeniclncRNAsoverall,weobservedamarginalover-representationofhighlyconserved8-mersitesinasmallsubsetofcytoplasmiclncRNAs(12sitesin8lncRNAsat56%falsediscoveryrate,P=0.
10).
Conclusions:EvolutionaryconservationinlncRNAsisgenerallylowbutpatch-wisehigh,andthesepatchescould,inprinciple,harborconservedtargetsites.
However,whileouranalysisefficientlydetectedconservedtargetingofmRNAs,itprovidedonlylimitedandmarginallysignificantsupportforconservedmicroRNA-lncRNAinteractions.
WeconcludethatconservedmicroRNA-lncRNAinteractionscouldnotbereliablydetectedwithourmethodology.
Keywords:Longnon-codingRNA,lncRNA,microRNA,ComparativegenomicsFindingsBackgroundWhilesmallnon-codingRNAs,suchasmicroRNAs,havewell-establishedfunctionsinthecell,longnon-codingRNAs(lncRNAs)haveonlyrecentlystartedtoemergeaswidespreadregulatorsofcellphysiology[1].
Althoughearlyexampleswerediscovereddecadesago,large-scaletranscriptomicstudieshavesincerevealedthatmammaliangenomesencodethousandsoflong(>200nt)transcriptsthatlackcodingcapacity,butareotherwisemRNA-like[2-4].
Theirbiologicalimportancehasbeencontroversial,butnovelfunctionallncRNAswithroles,forexample,invertebratedevelopment[5],pluripotency[6]andgenomestability[7]arenowbeingdescribedatincreasingfrequency.
Afewrecentstudiesdescribeinteractionsbetweensmallandlongnon-codingRNAs,wherelncRNAsacteitherasregulatorytargetsofmicroRNA-induceddestabilization[8,9]orasmoleculardecoysofmicroRNAs[10-13].
Re-centresultsalsoshowthatstablecircularlncRNAscanbindandinhibitmicroRNAs[14,15].
Importantly,RNAi-basedstudies,includingsilencingof147lncRNAswithlentiviralshRNAs[6],showthatlncRNAsare,inprinciple,susceptibletorepressionbyArgonaute-smallRNAcom-plexes,despiteoftenlocalizingtothenucleus.
Inaddition,therearedatafromcrosslinkingandimmunoprecipitation(CLIP)experimentsthatsupportbindingofArgonauteproteinstolncRNAs[16,17].
Comparativegenomicshasrevealedthatmostprotein-codinggenesareunderconservedmicroRNAcontrol:conservedmicroRNAtargetsitesarepresentin3'un-translatedregions(UTRs)ofprotein-codingmRNAsatfrequenciesconsiderablyhigherthanrandomlyexpected,clearlydemonstratingtheimpactofmicroRNAsonmRNAevolution[18,19].
WhilelncRNAsingeneralareweaklyconserved,theymayhavelocalpatchesofstrongsequenceconservation[20].
Itwasrecentlyshownthatde-velopmentaldefectscausedbyknockdownoflncRNAsinzebrafishcouldberescuedbyintroductionofputativehu-manorthologsidentifiedbasedonsuchshortpatches[5],*Correspondence:erik.
larsson@gu.
seDepartmentofMedicalBiochemistryandCellBiology,InstituteofBiomedicine,TheSahlgrenskaAcademy,UniversityofGothenburg,SE-40530,Gothenburg,Sweden2013Alaei-MahabadiandLarsson;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Alaei-MahabadiandLarssonSilence2013,4:4http://www.
silencejournal.
com/content/4/1/4supportingthatlncRNAfunctionsmaybeconservedoverlargeevolutionarydistancesdespitelimitedse-quencesimilarity.
ItisthusplausiblethatlncRNAsalsohaveevolvedtobetargetedbymicroRNAsdespitetheiroveralllowconservation,andthatthiswouldmanifestitselfthroughthepresenceoftargetsitesinlocalcon-servedsegments.
ResultsWeusedourpreviouslydescribedpipelinetomapandas-sesstheevolutionarilyconservationofputativemicroRNAtargetsitesinlncRNAs[21].
Briefly,wemappedcomple-mentarymatchestoestablishedmicroRNAseedfamiliesintheGENCODEv7lncRNAannotation,whichwasre-centlycharacterizedindetailbytheENCODEconsortium[4].
Conservationlevelsweredeterminedbasedona46-vertebratemultiplesequencealignment[22],andsiteswerescoredbasedontheirpresenceinprimates,mam-malsandnon-mammalvertebrates.
Thisallowedustovarythestringencytoconsiderprogressivelysmallersetsoftranscriptswithhigherconservationlevels.
Wecom-paredobservedsitefrequenciestoexpectedfrequenciesbasedonarandomdinucleotidemodel,inprotein-codinggenesandinsubsetsoflncRNAs(Figure1).
Ouranalysisrevealedwidespreadpresenceofcon-servedtargetsitesinmRNAs,whichrecapitulatesprevi-ousobservationsandestablishesourmethodology[18,19].
Dependingonpredictionstringency(conserva-tionlevelandseedtype),seedcomplementarymatchestoconservedmicroRNAfamilieswerepresentatupto6.
1*theexpectedfrequencyin3'UTRs,and1.
4*incodingregions(Figure2A).
Sitesfornon-conservedmicroRNAfamilies,whichwereincludedasanegativecontrol,wereobservedonlyatexpectedfrequencies(Figure2A).
Next,weinvestigatedsitefrequenciesinlncRNAs,spe-cificallyoftheintergenictypetoavoidconfoundinggen-omicoverlaps.
Inasetof2,121intergeniclncRNAgenes,weobservednosignificantenrichmentofsites(Figure2B).
Restrictingoursearchto3'or5'endsoftranscripts,orsubsetsofintergeniclncRNAspreviouslyfoundtohaveconservedpromoterregions[4],resultedinasimilarlackofenrichment(datanotshown).
ManydescribedlncRNAsparticipateintheassemblyofriboproteincomplexesinthenucleus[1],whilemicroRNAsareconsideredtobeactiveprimarilyinthecytoplasm.
WeusedsubcellularRNA-seqdatatonarrowdownouranalysistoasmallersetofcytoplasmiclncRNAs(n=169),whichwerealsoexpressedatcom-parativelyhighlevels(Figure2B).
Pan-mammaliancon-servedhigh-quality(8-mer)siteswerehereobservedat1.
8xtheexpectedfrequency(P=0.
10),whichcorre-spondstoafalsediscoveryrateof56%,butthenumberoftargetsandsiteswassmall(12sitesin8lncRNAgenes,Table1).
OneoftheeighttargetlncRNAs(AC010091.
1)showeddistanthomologytohumanpro-tocadherinFat4protein(maximum36%identityover94a.
a.
),andcouldthusrepresentanancientpseudogeneormisclassifiedcodinggene.
Allotherslackedhomologytoanyof565,000+knownsequencesinUniProtKB/Swiss-Prot,andsevenoutofeightwerealsoclassifiedaslongnon-codinginarecentRNA-seq-basedmappingofhumanlncRNAs[3].
ConservedtargetingoflncRNAsbymicroRNAsisplausible,giventhatLncRNAsaresusceptibletoAGO-mediatedrepression,andthattheyshowpatch-wisestrongsequenceconservation.
However,ouranalysisin-dicatesthatthisisnotawidespreadphenomenon,eventhoughasmallsubsetofcytoplasmictranscriptsshowedaweakenrichmentofconservedsitesatmarginalstatis-ticalsignificance.
LncRNAsarecurrentlydefinedsolelybasedonlengthandcodingcapacity,andareassuchlikelytorepresentahighlyfunctionallydiversegroup.
Itisthuspossiblethatother,notyetdefined,subfamilieshaveevolvedtobemicroRNAtargets,butthatthissignalistoodilutedtobedetectableinourcurrentanalysis.
ItshouldbenotedthattheGENCODEannotationusedhereisoneofseveralpublishedlncRNAsets,andFigure1WorkflowtodetectconservedmicroRNAtargetingoflongnon-codingRNAs(lncRNAs).
ConservedmicroRNAtargetsites(complementaryseedmatches)wereidentifiedintheGENCODEhumangeneannotationbasedona46-speciesmultiplesequencealignmentasdescribedpreviously[21].
Atotalof1,267microRNAfamilieswereconsidered.
DifferentsubsetsoflncRNAswereanalyzedforover-representationofsitescomparedtoarandombackgroundmodel.
Alaei-MahabadiandLarssonSilence2013,4:4Page2of5http://www.
silencejournal.
com/content/4/1/4whilecomprehensive,itdoesnotcoverallknowntran-scribedloci[3].
Likewise,thereareseveralapproachestotargetsitepredictionanddetailedresultsmayvary.
Not-ably,ouranalysiswasdesignedtocaptureanoverallsig-natureofconservedtargeting,andwhenappliedtomRNAsitefficientlyrecapitulatedastrongenrichmentsignal.
Differentimplementationsandannotationscouldgivevariableresultsatthelevelofindividualtranscriptsandsites,butthemainconclusionisunlikelytodependontheseparameters.
WhilesomeestablishedmicroRNA-lncRNAinter-actionsitesareconservedtovariousextents,inprincipleenablingdetectionbycomparativegenomicsapproaches[8-10],otherslackconservationdespitehavingexperi-mentallyconfirmedfunctions[12,13].
Thisisconsistentwithdatashowingthatmanynon-conservedhumanmicroRNAsitescanmediatetargeting[23].
Notably,evenwell-characterizedlncRNAs,suchasHOTAIRandXIST,haveoftenevolvedrapidly,andmayshowconsid-erablefunctionalandstructuraldifferenceswithinthemammalianlineage[24,25].
Ourcomparativegenomicsmethodologythereforedoesnotexcludethatnon-conservedandrecentlyevolvedtargetingcouldbecom-monplace,andthismotivatesfurthercomputationalandexperimentalstudies.
MethodsWereliedontheGENCODEcoding/non-codingclassifi-cation,andconsideredaslncRNAsgenesthatonlypro-ducedtranscriptsofthe'antisense','lincRNA','non_coding'ABFigure2RatiosbetweenobservedandexpectedmicroRNAtargetsitefrequenciesincodinggenesandlongnon-codingRNAs(lncRNAs).
(A)Ourmethodologywasfirstestablishedoncodinggenes.
The3'untranslatedregions(UTRs)andcodingsequences(CDS)wereanalyzedseparately.
Wecomparedobservednumbersofseedmatches(inparentheses)torandomlyexpectednumbersbasedonsetsofsyntheticseedsthatpreservedthedinucleotidefrequenciesoftheactualseeds.
Differentpredictionstringencies(siteconservationlevelandseedquality)wereapplied,furtherexplainedwithingrayboxes.
TheanalysisfocusedonhighlyconservedmicroRNAfamilies(n=87),butnon-conservedfamilieswereincludedasacontrol.
Barsshowmeanobserved-to-expectedratiosfrom20repeatedtrials.
(B)SimilaranalysisbasedonintergeniclncRNAsandcytoplasmicintergeniclncRNAs.
Placentalmammalconserved8-mersiteswerepresentaboveexpectationinasmallsubsetofcytoplasmicintergeniclncRNAs(12sitesfor11microRNAfamilies,in8lncRNAgenes).
SubcellularlocalizationwasdeterminedbasedonRNA-seqlibrariesfromsevenfractionatedcelllines.
*,empiricalP1wererequiredforsignificanceattheempiricalP≤0.
05level,and18/20forP=0.
10.
MicroRNAfamilydefinitionsandconservationclassifi-cationswerederivedfromTargetScan[18].
Weuseddatafromapreviousstudy[4]todefinesubsetsoflncRNAswithconservedregulatoryregions.
The500or250mostconservedintergeniclncRNAsbasedoneitherpan-mammalorpan-vertebratepromoterconservationscores(intotal,foursets)wereanalyzedasdescribedabove.
RNA-seqdata(fastqfiles)producedwithintheENCODEproject[26]bytheGingeraslaboratory(ColdSpringHarborLaboratories,ColdSpringHarbor,NY,USA)wereobtainedthroughtheUCSCFTPserver.
Atotalof1.
71billion76ntreadpairsfrompolyA+nu-clearandcytoplasmicfractionsfromsevenhumancelllines(Gm12878,HelaS3,HepG2,Huvec,H1hesc,NhekandK562)werealignedtothehumanhg19referencegenomewithTophat[27].
ThealignerwassuppliedwithGENCODEgenemodelsusingthe-Goption.
GeneswerequantifiedusingtheHTSeq-countutility(http://www-huber.
embl.
de/users/anders/HTSeq).
Cyto-plasmictranscriptsweredefinedashavinganormalizedcytoplasm/nucleusratio>1.
Atotalofatleast20mappedreadsacrossallconditionswasrequired,toavoidunreliablecytoplasm/nuclearratiosinthelow-abundancerange.
Ethicalapprovalorpatientconsentwasnotrequiredforthisstudy.
AbbreviationsCDS:Codingsequence;CLIP:Crosslinkingandimmunoprecipitation;LncRNA:Longnon-codingRNA;UTR:Untranslatedregion.
Table1Pan-mammalianconserved8-merputativemicroRNAtargetsitesincytoplasmicintergeniclongnon-codingRNAs(lncRNAs)TargetGENCODEIDTargetsymbolMicroRNAfamilySitechromosomeSitegenomepositionCabilietal.
lincRNAaUniProtKB/Swiss-ProtBLASTbENSG00000226856.
1AC093901.
1miR-182chr2118940821YesNohitsENSG00000231532.
1AC022311.
1miR-133abcchr24676715YesNohitsENSG00000231532.
1AC022311.
1miR-22/22-3pchr24676706↑↑ENSG00000231532.
1AC022311.
1miR-383chr24676629↑↑ENSG00000233491.
2AC010091.
1miR-133abcchr781218260YesE=4e-5(HumanFAT4)ENSG00000233491.
2AC010091.
1miR-9/9abchr781218258↑↑ENSG00000236719.
2RP11-522D2.
1miR-30abcdef/30abe-5p/384-5pchr1180535222YesNohitsENSG00000245017.
1AC013418.
2miR-138/138abchr1298879829YesNohitsENSG00000248927.
1CTD-2334D19.
1miR-135ab/135a-5pchr5120126269YesNohitsENSG00000248927.
1CTD-2334D19.
1miR-19abchr5120126442↑↑ENSG00000250366.
1AL133167.
1miR-218/218achr1496389499YesNohitsENSG00000253507.
1CTD-2501M5.
1miR-146ac/146b-5pchr8132329800NoNohitsaAnnotatedasalongnon-codingRNAinCabiliMN,TrapnellCetal.
,GenesandDevelopment(2011).
bHitswithBLASTE-value<0.
5.
Repeatmaskingwasperformedtoavoidmatchesto,forexample,translatedSINEsinSwissProt.
GenomiccoordinatesrefertotheHg19assembly.
Alaei-MahabadiandLarssonSilence2013,4:4Page4of5http://www.
silencejournal.
com/content/4/1/4CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Author'scontributionsELdesignedthestudy,analyzeddata,andwrotethemanuscript.
BAanalyzeddata.
Bothauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsWewouldliketoacknowledgeDrs.
AndersJacobsenandDeboraS.
Marksforhelpfulcommentsanddiscussions.
ThisworkwassupportedbygrantsfromtheSwedishMedicalResearchCouncil;theSwedishCancerSociety;theAssarGabrielssonFoundation;theMagnusBergvallFoundation;thekeWibergfoundation;andtheLarsHiertaMemorialFoundation.
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