REVIEWOpenAccessReverse-geneticsstudiesoflncRNAs—whatwehavelearntandpathsforwardFanGao,YeCai,PhilippKapranov*andDongyangXu*AbstractLongnon-codingRNAs(lncRNAs)representamajorfractionofthetranscriptomeinmulticellularorganisms.Althoughahandfulofwell-studiedlncRNAsarebroadlyrecognizedasbiologicallymeaningful,thefractionofsuchtranscriptsoutoftheentirecollectionoflncRNAsremainsasubjectofvigorousdebate.HerewereviewtheevidenceforandagainstbiologicalfunctionalitiesoflncRNAsandattempttoarriveatpotentialmodesoflncRNAfunctionalitythatwouldreconcilethecontradictoryconclusions.Finally,wediscussdifferentstrategiesofphenotypicanalysesthatcouldbeusedtoinvestigatesuchmodesoflncRNAfunctionality.IntroductionAtthebeginningofthiscentury,thecentraldogmaofbiologythatpositsgeneticinformationflowfromDNAtoRNAtoproteinwaschallengedbythediscoveryofpervasivetranscriptioninthehumangenome[1,2].Longnon-coding(lnc)RNAsaccountformostofthecomplexityofhumantranscriptome[3]andrepresenttranscriptsover200nucleo-tidesinlength[4]withnoobviousprotein-codingpotentialandanumberofadditionalfeatures(i.e.,abundance,sequenceconservation,splicingefficiency,subcellularlocalization,andothers)thatdistinguishthemfromthecanonicalrealmofprotein-codingmRNAs[3,5–7].Inthepastdecade,biologicalfunctionsandmolecularmechanismsoflncRNAshaveattractedsignificantinterestfromthescien-tificcommunity[8–12].AlthoughanumberoflncRNAshavebeenassociatedwithdiversebiologicalprocessesandfunctions[13–15],formostpart,thesetranscriptsremainen-igmatic.ThemostcriticalandyetthemostcontroversialissuecentersonbiologicalsignificanceofthelncRNAclassoftranscriptsandthefractionoftrulyfunctionalmembersitcontains.Indeed,thereisagrowingbodyofcontradictoryevidencebasedonreverse-geneticsassaysthateithersupportsorquestionsthebroadbiologicalfunctionalityofthisclassofRNAsasdescribedbelow.Thisleadstoagreatdealofconfusionwhilealsofuelingthedebateaboutfunc-tionalityofthesetranscripts.OnesideinthisdebatearguesthatmostofthecurrentlyannotatedlncRNAsarenotfunc-...
