total777mi.com
777mi.com 时间:2021-05-08 阅读:()
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.
Received:5June2013Accepted:29July2013Published:20August2013References1.
WangKC,ChangHY:MolecularmechanismsoflongnoncodingRNAs.
MolCell2011,43:904–914.
2.
CarninciP,KasukawaT,KatayamaS,GoughJ,FrithMC,MaedaN,OyamaR,RavasiT,LenhardB,WellsC,KodziusR,ShimokawaK,BajicVB,BrennerSE,BatalovS,ForrestAR,ZavolanM,DavisMJ,WilmingLG,AidinisV,AllenJE,Ambesi-ImpiombatoA,ApweilerR,AturaliyaRN,BaileyTL,BansalM,BaxterL,BeiselKW,BersanoT,BonoH,ChalkAM,etal:Thetranscriptionallandscapeofthemammaliangenome.
Science2005,309:1559–1563.
3.
CabiliMN,TrapnellC,GoffL,KoziolM,Tazon-VegaB,RegevA,RinnJL:IntegrativeannotationofhumanlargeintergenicnoncodingRNAsrevealsglobalpropertiesandspecificsubclasses.
GenesDev2011,25:1915–1927.
4.
DerrienT,JohnsonR,BussottiG,TanzerA,DjebaliS,TilgnerH,GuernecG,MartinD,MerkelA,KnowlesDG,LagardeJ,VeeravalliL,RuanX,RuanY,LassmannT,CarninciP,BrownJB,LipovichL,GonzalezJM,ThomasM,DavisCA,ShiekhattarR,GingerasTR,HubbardTJ,NotredameC,HarrowJ,GuigóR:TheGENCODEv7catalogofhumanlongnoncodingRNAs:Analysisoftheirgenestructure,evolution,andexpression.
GenomeRes2012,22:1775–1789.
5.
UlitskyI,ShkumatavaA,JanCH,SiveH,BartelDP:ConservedfunctionoflincRNAsinvertebrateembryonicdevelopmentdespiterapidsequenceevolution.
Cell2011,147:1537–1550.
6.
GuttmanM,DonagheyJ,CareyBW,GarberM,GrenierJK,MunsonG,YoungG,LucasAB,AchR,BruhnL,YangX,AmitI,MeissnerA,RegevA,RinnJL,RootDE,LanderES:lincRNAsactinthecircuitrycontrollingpluripotencyanddifferentiation.
Nature2011,477:295–300.
7.
HuarteM,GuttmanM,FeldserD,GarberM,KoziolMJ,Kenzelmann-BrozD,KhalilAM,ZukO,AmitI,RabaniM,AttardiLD,RegevA,LanderES,JacksT,RinnJL:AlargeintergenicnoncodingRNAinducedbyp53mediatesglobalgenerepressioninthep53response.
Cell2010,142:409–419.
8.
HansenTB,WiklundED,BramsenJB,VilladsenSB,StathamAL,ClarkSJ,KjemsJ:miRNA-dependentgenesilencinginvolvingAgo2-mediatedcleavageofacircularantisenseRNA.
EMBOJ2011,30:4414–4422.
9.
CalinGA,LiuCG,FerracinM,HyslopT,SpizzoR,SevignaniC,FabbriM,CimminoA,LeeEJ,WojcikSE,ShimizuM,TiliE,RossiS,TaccioliC,PichiorriF,LiuX,ZupoS,HerleaV,GramantieriL,LanzaG,AlderH,RassentiL,VoliniaS,SchmittgenTD,KippsTJ,NegriniM,CroceCM:UltraconservedregionsencodingncRNAsarealteredinhumanleukemiasandcarcinomas.
CancerCell2007,12:215–229.
10.
Franco-ZorrillaJM,ValliA,TodescoM,MateosI,PugaMI,Rubio-SomozaI,LeyvaA,WeigelD,GarciaJA,Paz-AresJ:TargetmimicryprovidesanewmechanismforregulationofmicroRNAactivity.
NatGenet2007,39:1033–1037.
11.
CazallaD,YarioT,SteitzJA:Down-regulationofahostmicroRNAbyaHerpesvirussaimirinoncodingRNA.
Science2010,328:1563–1566.
12.
WangJ,LiuX,WuH,NiP,GuZ,QiaoY,ChenN,SunF,FanQ:CREBup-regulateslongnon-codingRNA,HULCexpressionthroughinteractionwithmicroRNA-372inlivercancer.
NucleicAcidsRes2010,38:5366–5383.
13.
WangY,XuZ,JiangJ,XuC,KangJ,XiaoL,WuM,XiongJ,GuoX,LiuH:EndogenousmiRNAspongelincRNA-RoRregulatesOct4,Nanog,andSox2inhumanembryonicstemcellself-renewal.
Developmentalcell2013,25:69–80.
14.
MemczakS,JensM,ElefsiniotiA,TortiF,KruegerJ,RybakA,MaierL,MackowiakSD,GregersenLH,MunschauerM,LoewerA,ZieboldU,LandthalerM,KocksC,Le-NobleF,RajewskyN:CircularRNAsarealargeclassofanimalRNAswithregulatorypotency.
Nature2013,495:333–338.
15.
HansenTB,JensenTI,ClausenBH,BramsenJB,FinsenB,DamgaardCK,KjemsJ:NaturalRNAcirclesfunctionasefficientmicroRNAsponges.
Nature2013,495:384–388.
16.
JalaliS,BhartiyaD,LalwaniMK,SivasubbuS,ScariaV:SystematictranscriptomewideanalysisoflncRNA-miRNAinteractions.
PLoSOne2013,8:e53823.
17.
ParaskevopoulouMD,GeorgakilasG,KostoulasN,ReczkoM,MaragkakisM,DalamagasTM,HatzigeorgiouAG:DIANA-LncBase:experimentallyverifiedandcomputationallypredictedmicroRNAtargetsonlongnon-codingRNAs.
NucleicAcidsRes2013,41:D239–D245.
18.
FriedmanRC,FarhKK,BurgeCB,BartelDP:MostmammalianmRNAsareconservedtargetsofmicroRNAs.
GenomeRes2009,19:92–105.
19.
LewisBP,BurgeCB,BartelDP:Conservedseedpairing,oftenflankedbyadenosines,indicatesthatthousandsofhumangenesaremicroRNAtargets.
Cell2005,120:15–20.
20.
GuttmanM,GarberM,LevinJZ,DonagheyJ,RobinsonJ,AdiconisX,FanL,KoziolMJ,GnirkeA,NusbaumC,RinnJL,LanderES,RegevA:Abinitioreconstructionofcelltype-specifictranscriptomesinmouserevealstheconservedmulti-exonicstructureoflincRNAs.
NatBiotechnol2010,28:503–510.
21.
JeggariA,MarksDS,LarssonE:miRcode:amapofputativemicroRNAtargetsitesinthelongnon-codingtranscriptome.
Bioinformatics2012,28:2062–2063.
22.
BlanchetteM,KentWJ,RiemerC,ElnitskiL,SmitAF,RoskinKM,BaertschR,RosenbloomK,ClawsonH,GreenED,HausslerD,MillerW:Aligningmultiplegenomicsequenceswiththethreadedblocksetaligner.
GenomeRes2004,14:708–715.
23.
FarhKK,GrimsonA,JanC,LewisBP,JohnstonWK,LimLP,BurgeCB,BartelDP:ThewidespreadimpactofmammalianMicroRNAsonmRNArepressionandevolution.
Science2005,310:1817–1821.
24.
SchorderetP,DubouleD:Structuralandfunctionaldifferencesinthelongnon-codingRNAhotairinmouseandhuman.
PLoSGenet2011,7:e1002071.
25.
NesterovaTB,SlobodyanyukSY,ElisaphenkoEA,ShevchenkoAI,JohnstonC,PavlovaME,RogozinIB,KolesnikovNN,BrockdorffN,ZakianSM:CharacterizationofthegenomicXistlocusinrodentsrevealsconservationofoverallgenestructureandtandemrepeatsbutrapidevolutionofuniquesequence.
GenomeRes2001,11:833–849.
26.
MyersRM,StamatoyannopoulosJ,SnyderM,DunhamI,HardisonRC,BernsteinBE,GingerasTR,KentWJ,BirneyE,WoldB,CrawfordGE:Auser'sguidetotheencyclopediaofDNAelements(ENCODE).
PLoSBiol2011,9:e1001046.
27.
TrapnellC,PachterL,SalzbergSL:TopHat:discoveringsplicejunctionswithRNA-Seq.
Bioinformatics2009,25:1105–1111.
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.
Received:5June2013Accepted:29July2013Published:20August2013References1.
WangKC,ChangHY:MolecularmechanismsoflongnoncodingRNAs.
MolCell2011,43:904–914.
2.
CarninciP,KasukawaT,KatayamaS,GoughJ,FrithMC,MaedaN,OyamaR,RavasiT,LenhardB,WellsC,KodziusR,ShimokawaK,BajicVB,BrennerSE,BatalovS,ForrestAR,ZavolanM,DavisMJ,WilmingLG,AidinisV,AllenJE,Ambesi-ImpiombatoA,ApweilerR,AturaliyaRN,BaileyTL,BansalM,BaxterL,BeiselKW,BersanoT,BonoH,ChalkAM,etal:Thetranscriptionallandscapeofthemammaliangenome.
Science2005,309:1559–1563.
3.
CabiliMN,TrapnellC,GoffL,KoziolM,Tazon-VegaB,RegevA,RinnJL:IntegrativeannotationofhumanlargeintergenicnoncodingRNAsrevealsglobalpropertiesandspecificsubclasses.
GenesDev2011,25:1915–1927.
4.
DerrienT,JohnsonR,BussottiG,TanzerA,DjebaliS,TilgnerH,GuernecG,MartinD,MerkelA,KnowlesDG,LagardeJ,VeeravalliL,RuanX,RuanY,LassmannT,CarninciP,BrownJB,LipovichL,GonzalezJM,ThomasM,DavisCA,ShiekhattarR,GingerasTR,HubbardTJ,NotredameC,HarrowJ,GuigóR:TheGENCODEv7catalogofhumanlongnoncodingRNAs:Analysisoftheirgenestructure,evolution,andexpression.
GenomeRes2012,22:1775–1789.
5.
UlitskyI,ShkumatavaA,JanCH,SiveH,BartelDP:ConservedfunctionoflincRNAsinvertebrateembryonicdevelopmentdespiterapidsequenceevolution.
Cell2011,147:1537–1550.
6.
GuttmanM,DonagheyJ,CareyBW,GarberM,GrenierJK,MunsonG,YoungG,LucasAB,AchR,BruhnL,YangX,AmitI,MeissnerA,RegevA,RinnJL,RootDE,LanderES:lincRNAsactinthecircuitrycontrollingpluripotencyanddifferentiation.
Nature2011,477:295–300.
7.
HuarteM,GuttmanM,FeldserD,GarberM,KoziolMJ,Kenzelmann-BrozD,KhalilAM,ZukO,AmitI,RabaniM,AttardiLD,RegevA,LanderES,JacksT,RinnJL:AlargeintergenicnoncodingRNAinducedbyp53mediatesglobalgenerepressioninthep53response.
Cell2010,142:409–419.
8.
HansenTB,WiklundED,BramsenJB,VilladsenSB,StathamAL,ClarkSJ,KjemsJ:miRNA-dependentgenesilencinginvolvingAgo2-mediatedcleavageofacircularantisenseRNA.
EMBOJ2011,30:4414–4422.
9.
CalinGA,LiuCG,FerracinM,HyslopT,SpizzoR,SevignaniC,FabbriM,CimminoA,LeeEJ,WojcikSE,ShimizuM,TiliE,RossiS,TaccioliC,PichiorriF,LiuX,ZupoS,HerleaV,GramantieriL,LanzaG,AlderH,RassentiL,VoliniaS,SchmittgenTD,KippsTJ,NegriniM,CroceCM:UltraconservedregionsencodingncRNAsarealteredinhumanleukemiasandcarcinomas.
CancerCell2007,12:215–229.
10.
Franco-ZorrillaJM,ValliA,TodescoM,MateosI,PugaMI,Rubio-SomozaI,LeyvaA,WeigelD,GarciaJA,Paz-AresJ:TargetmimicryprovidesanewmechanismforregulationofmicroRNAactivity.
NatGenet2007,39:1033–1037.
11.
CazallaD,YarioT,SteitzJA:Down-regulationofahostmicroRNAbyaHerpesvirussaimirinoncodingRNA.
Science2010,328:1563–1566.
12.
WangJ,LiuX,WuH,NiP,GuZ,QiaoY,ChenN,SunF,FanQ:CREBup-regulateslongnon-codingRNA,HULCexpressionthroughinteractionwithmicroRNA-372inlivercancer.
NucleicAcidsRes2010,38:5366–5383.
13.
WangY,XuZ,JiangJ,XuC,KangJ,XiaoL,WuM,XiongJ,GuoX,LiuH:EndogenousmiRNAspongelincRNA-RoRregulatesOct4,Nanog,andSox2inhumanembryonicstemcellself-renewal.
Developmentalcell2013,25:69–80.
14.
MemczakS,JensM,ElefsiniotiA,TortiF,KruegerJ,RybakA,MaierL,MackowiakSD,GregersenLH,MunschauerM,LoewerA,ZieboldU,LandthalerM,KocksC,Le-NobleF,RajewskyN:CircularRNAsarealargeclassofanimalRNAswithregulatorypotency.
Nature2013,495:333–338.
15.
HansenTB,JensenTI,ClausenBH,BramsenJB,FinsenB,DamgaardCK,KjemsJ:NaturalRNAcirclesfunctionasefficientmicroRNAsponges.
Nature2013,495:384–388.
16.
JalaliS,BhartiyaD,LalwaniMK,SivasubbuS,ScariaV:SystematictranscriptomewideanalysisoflncRNA-miRNAinteractions.
PLoSOne2013,8:e53823.
17.
ParaskevopoulouMD,GeorgakilasG,KostoulasN,ReczkoM,MaragkakisM,DalamagasTM,HatzigeorgiouAG:DIANA-LncBase:experimentallyverifiedandcomputationallypredictedmicroRNAtargetsonlongnon-codingRNAs.
NucleicAcidsRes2013,41:D239–D245.
18.
FriedmanRC,FarhKK,BurgeCB,BartelDP:MostmammalianmRNAsareconservedtargetsofmicroRNAs.
GenomeRes2009,19:92–105.
19.
LewisBP,BurgeCB,BartelDP:Conservedseedpairing,oftenflankedbyadenosines,indicatesthatthousandsofhumangenesaremicroRNAtargets.
Cell2005,120:15–20.
20.
GuttmanM,GarberM,LevinJZ,DonagheyJ,RobinsonJ,AdiconisX,FanL,KoziolMJ,GnirkeA,NusbaumC,RinnJL,LanderES,RegevA:Abinitioreconstructionofcelltype-specifictranscriptomesinmouserevealstheconservedmulti-exonicstructureoflincRNAs.
NatBiotechnol2010,28:503–510.
21.
JeggariA,MarksDS,LarssonE:miRcode:amapofputativemicroRNAtargetsitesinthelongnon-codingtranscriptome.
Bioinformatics2012,28:2062–2063.
22.
BlanchetteM,KentWJ,RiemerC,ElnitskiL,SmitAF,RoskinKM,BaertschR,RosenbloomK,ClawsonH,GreenED,HausslerD,MillerW:Aligningmultiplegenomicsequenceswiththethreadedblocksetaligner.
GenomeRes2004,14:708–715.
23.
FarhKK,GrimsonA,JanC,LewisBP,JohnstonWK,LimLP,BurgeCB,BartelDP:ThewidespreadimpactofmammalianMicroRNAsonmRNArepressionandevolution.
Science2005,310:1817–1821.
24.
SchorderetP,DubouleD:Structuralandfunctionaldifferencesinthelongnon-codingRNAhotairinmouseandhuman.
PLoSGenet2011,7:e1002071.
25.
NesterovaTB,SlobodyanyukSY,ElisaphenkoEA,ShevchenkoAI,JohnstonC,PavlovaME,RogozinIB,KolesnikovNN,BrockdorffN,ZakianSM:CharacterizationofthegenomicXistlocusinrodentsrevealsconservationofoverallgenestructureandtandemrepeatsbutrapidevolutionofuniquesequence.
GenomeRes2001,11:833–849.
26.
MyersRM,StamatoyannopoulosJ,SnyderM,DunhamI,HardisonRC,BernsteinBE,GingerasTR,KentWJ,BirneyE,WoldB,CrawfordGE:Auser'sguidetotheencyclopediaofDNAelements(ENCODE).
PLoSBiol2011,9:e1001046.
27.
TrapnellC,PachterL,SalzbergSL:TopHat:discoveringsplicejunctionswithRNA-Seq.
Bioinformatics2009,25:1105–1111.
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
RAKsmart：美国洛杉矶独服,E3处理器/16G/1TB,$76.77/月;美国/香港/日本/韩国站群服务器,自带5+253个IPv4
RAKsmart怎么样？RAKsmart机房即日起开始针对洛杉矶机房的独立服务器进行特别促销活动：低至$76.77/月，最低100Mbps带宽，最高10Gbps带宽，优化线路，不限制流量，具体包括有：常规服务器、站群服务器、10G大带宽服务器、整机机柜托管。活动截止6月30日结束。RAKsmart，美国华人老牌机房，专注于圣何塞服务器，有VPS、独立服务器等。支持PayPal、支付宝付款。点击直达...
修罗云50元/月起香港大宽带NAT VPS，香港沙田建站2核2G5M仅70元/月起
修罗云怎么样?修罗云是一家国内老牌商家，修罗云商家以销售NAT机器起家，国内的中转机相当不错，给的带宽都非常高，此前推荐的也都是国内NAT VPS机器。今天，云服务器网(www.yuntue.com)小编主要介绍一下修罗云的香港云服务器，适合建站，香港沙田cn2云服务器，2核2G，5M带宽仅70元/月起，同时香港香港大带宽NAT VPS低至50元/月起，性价比不错，可以尝试一下!点击进入：修罗云官...
恒创新客(317元)香港云服务器 2M带宽 三网CN2线路直连
恒创科技也有暑期的活动，其中香港服务器也有一定折扣，当然是针对新用户的，如果我们还没有注册过或者可以有办法注册到新用户的，可以买他们家的香港服务器活动价格，2M带宽香港云服务器317元。对于一般用途还是够用的。 活动链接：恒创暑期活动爆款活动均是针对新用户的。1、云服务器仅限首次购买恒创科技产品的新用户。1 核 1G 实例规格，单个账户限购 1台；其他活动机型，单个账户限购 3 台（必须在一个订单...
777mi.com为你推荐
-
华师京城虚拟仿真实验室软件phpadmin下载phpmyadmin怎么安装啊？可以直接下载安装吗？还需要下载其他数据库吗？360邮箱360免费申请邮箱在那里什么是支付宝支付宝是什么意思？支付宝账户是什么什么是企业支付宝账户抢米网会知道怎样抢小米的请进来说一下。12306.com注册12306邮箱地址怎么写12306.com12306身份信息待核验要多久？审核要多久curl扩展linux上怎么操作开启php curl扩展团购程序团购系统软件有哪些？一般需要考虑那几点？