eeomn 3,13-61(2007)doi:10.1242/dev.002006 Development 134,1635-1641 1 as apotentiatorofWingless (Wg)signaling.Consistentwitharole New York, NY, USA). Drosophila example ofamicroRNA-modulatedsignalingpathway in temperature cyclesdisruptsregularommatidialpatterning.Another reported thatexposingdeveloping buffering underadversephysiological conditions.Indeed,Carthew robustness ondevelopmentaldecisionsandtoprovidegenetic suchas sensory organ precursorsin specify photoreceptors(LiandCarthew, 2005)andchordotonal epidermal growthfactorreceptor(EGFR)signalingpathways to described how for microRNAsarethemost-clearlyevident. microRNAs, siRNAsandpiRNAs(Fig.1)–thedevelopmentalroles classes ofsmallregulatoryRNAsdiscussedatthemeeting– Plasterk, 2006;Jones-Rhoadesetal.,2006).Ofthethreemain genetic studiesinmodelorganisms andinhumans(Kloosterman their diverseexpressionpatternsand,insomecases,confirmedby whose regulatoryrolesindevelopmenthavebeenimplicatedby Animals andplantshavehundredsofdistinctmicroRNA , Developmental functionsofmicroRNAs translation. structure, orpost-transcriptionally, byaffecting mRNA stabilityor regulation atthetranscriptionallevel,byaffecting chromatin specific nucleicacidsequences.ThesesmallRNAscanexert as regulatoryRNAsthatdirectthebindingofproteincomplexesto association withArgonaute-family proteinsandbytheirfunctions RNAs (piRNAs)aresmallregulatorythatunifiedbytheir MicroRNAs, smallinterferingRNAs(siRNAs)andPiwi-interacting Introduction elaboration ofmulticellularcomplexity. targets, theirmechanismsofaction,androlesinthe functions, theirbiogenesis,theidentificationofregulatory the diversityofsmallregulatoryRNAsandtheirdevelopmental RNAs (piRNAsandrasiRNAs).Amongthethemesdiscussedwere microRNAs, smallinterferingRNAs(siRNAs)andPiwi-interacting biological aspectsofsmallregulatoryRNAs,including siRNAs’ meetingbroughttogetherscientistsworkingondiverse Stephen FriendandJohnMaraganore).The‘MicroRNAs for Target Validation andasaTherapeutic’,organizedby Bartel andShivGrewal(andwasheldinconjunctionwith‘RNAi Biological FunctionsandMechanisms’wasorganizedbyDavid A recentKeystoneSymposiumon‘MicroRNAsandsiRNAs: AmbrosVictor genomes bysmallRNAs The regulation ofgenes and *Author forcorrespondence (e-mail:[email protected]) 92521, USA. Dartmouth MedicalSchool,Hanover, NH03755,USA. Richard Carthew(NorthwesternUniversity, Evanston,IL,USA) was discussedbyEricLai(Sloan-KetteringInstitute, miR-7 1, andXuemeiChen * functions inanintricatenetworkofNotchand miR-7 miR-315 function insuchnetworkstoconfer Drosophila a dniidi acell-basedscreen was identifiedin mir-7 . Carthewproposedthat 2 2 UC Riverside,CA uatflyeyesto mutant reported byRonald Plasterk(HubrechtLaboratory, Utrecht, role for cells (Poyetal.,2004).These new findingssuggestanadditional indicated thatitactsintheregulation ofinsulinsecretionbyislet cells. Previousoverexpression experimentswith architecture andadisruptionin the balancebetweenalphaandbeta mouse phenotypes,inwhichpancreatic isletsdisplayabnormal (also knownas Molecular SystemsBiology, Zurich,Switzerland)described modulate cMyblevelsatcrucialtimes. maturation issensitivetothedosageof development ofmaturelymphocytes,suggestingthatlymphocyte expression of target of possibly viatheregulationoftranscriptionfactorcMyb,adirect Genome Informatics)playsaroleinlymphocytematuration, showing that,inmouse, Bender’s laboratory(Harvard MedicalSchool,Boston,MA,USA) presented resultsofacollaborativeprojectfromhisandTimothy developmental pathways. particularly suitedtothecontrolofdelicatelyregulated lymphocyte developmentexemplifieshowmicroRNAsmaybe development. Chenpointedoutthatthisroleof a ‘rheostat’ to tuneT cellsensitivityatvariousstagesofT cell hence theirresponsetoantigens,indicatingthat in T cellsmodulatestheirsensitivitytoT cell-receptorsignaling, and of for Alto, CA,USA)reportedhowthemanipulationof antigen presentation.Chang-ZhengChen(StanfordUniversity, Palo a roleinthepositiveselectionofDP lymphocytesinresponseto (Neilson etal.,2007).Theseobservationsindicatethat and wasfoundbySharptobeenrichedindouble-positive(DP)cells role inmouselymphocytecellfatespecification(Chenetal.,2004), whichhadpreviouslybeenshowntoplaya Genome Informatics), particular notewas expression inverselycorrelatedwithitspredicted-target levels.Of and identifiedatleastonemicroRNA foreachstagewhose their computationallypredictedtargets duringT celldevelopment, MA, USA)comparedtheexpressionprofilesofmicroRNAsand let-7 respective wormandfly control eventsinthedevelopmentofadultnervoussystem.In (Reinhart etal.,2000),themainfunctionoffly controls developmentaltransitionsmainlyinhypodermalcells Hanover, NH,USA)revealedthat,unliketheworm of Victor Ambros’s laboratory(DartmouthMedicalSchool, organisms. Thegeneticanalysisof different geneticpathwaysanddevelopmentalprocessesindifferent (Pasquinelli etal.,2000),aconservedmicroRNA canregulate the deepphylogenicconservationofsomemicroRNA genes progression ofcertaincelllineagesandfates.Interestingly, despite developmental transformationsassociatedwithexcessWgactivity. presumptive thoraxofdevelopinglarvaecauseddramatic Notum. Moreover, theoverexpressionof Jan Krützfeldt(fromMarkusStoffel’s group,Instituteof Nikolaus Rajewsky(Max-Delbruck-Centrum,Berlin,Germany) Phillip Sharp(MassachusettsInstituteofTechnology, Cambridge, MicroRNAs playimportantrolesinthedevelopmental miR-315 miR-315 also differ. miR-375 miR-150 are thenegativeregulatorsofWgpathway, Axinand in promotingWgsignalingvivo,amongthetargets miR-150 Mirn375 in pancreaticdevelopment.Similar findingswere . Intransgenicmousemodels,moderateectopic miR-181 or agradedreductionofcMybblocksthe miR-150 – MouseGenomeInformatics)-knockout let-7 (also knownas pathways, thekeydirecttargets of (also knownas rspialet-7 Drosophila MEETING REVIEW miR-150 Mirn181a-2 miR-181 let-7 Mirn150 miR-315 miR-181 by NickSokol seems tobe miR-375 , whichmay miR-181 let-7 miR-181 may actas – Mouse – Mouse mir-375 , which in the levels 1635 had has in

DEVELOPMENT to leadtheproduction ofasecondrasiRNA. silencing. AnexistingrasiRNAguidesthecleavageofatargetRNAthrough thesliceractivityofassociatedPiwisubfamily Piwi,Aubergine(Aub)andAGO3–targethomologoustransposonsscattered around thegenome proteins – subfamily ofArgonaute associated siRNAs(rasiRNAs)in and thesiRNAscomplexedwithAGOactinaself-reinforcing looptomaintainheterochromatin attheloci. ( from repeated DNAortransposonsviatheactivitiesofRNA-dependentRNApolymerases(RdRP)andDicer. HistoneH3lysine9(H3- miRNA familymayhavedistinctdevelopmentalfunctions.( shared predicted targets,butgeneticstudiesreveal that developmental process. RelatedmiRNAs(lightanddarkblue)oftenhave that eachmiRNAisabletoregulate scores oftargettranscripts,geneticstudiesshowthatonemajorunderliestherole are boundbyaneffector protein Argonaute(AGO) andcausethecleavageortranslationalinhibitionoftargetmRNAs.Inanimals (miRNA)precursors, whichare processed byDROSHAandDICER(DICER1)intomiRNAs. (top) are transcribedbyPolIIintomicroRNA (Houbaviy etal.,2003).Homozygous mir-295 characterization ofaknockoutmousethatlacks embryonic stem(ES)cells?Sharpreportedtheinitial (Plasterk, 2006).WhatroledomicroRNAsplayinpluripotent considered tobepotentialdeterminantsoforgan orcelltypeidentity the specificityofthisphenotype. abnormalities inpancreaticisletorganization, stronglysupporting microRNA orthestemof with morpholinooligonucleotidesdirectedagainsteitherthemature Netherlands) forthezebrafish Fig. 1.ThebiogenesisandfunctionofendogenoussmallRNAsinanimals,plantsfungi. 1636 AGO-bound CA, USA)usedlentivirusvectors tooverexpress Gladstone InstitutesandUniversity ofCalifornia,SanFrancisco, Frasch, 2006).KathrynIvey(from DeepakSrivastava’s group, expression isevolutionarilyconservedamonganimals(Nguyen and and atparticularlyhighlevelsinmuscle.Thismuscle-specific of pluripotentcellsinembryosandthefemalegermline. indicating thatthesemicroRNAsmayfunctioninthemaintenance female survivorsareinfertileduetoanabsenceofgermcells, die asembryos,althoughsomegrowtoadulthood.Significantly, between specified bycooperative,followed byopposing,interactions ES cells,andshowedthatdistinct stepsinmuscledevelopmentare 133 precursors ABC miRNA miRNA Cell type-specificororgan-specific microRNAsaregenerally miR-133 during theinvitrodevelopment of embryoidbodiesfrommouse MEETING REVIEW Developmental miR-1 ( process 1 mir-290–295 and Key AGO miR-1 and Pol II Weak contribution Strong contribution MIR genes miR-133

are expressedinmousemesodermallineages, ), anEScell-specificmicroRNA cluster AGO Developmental process 2 Drosophila . Thesefindings provideinsightinto miR-375 miR-375 AGO mir-290–295

are produced from amasterlocuscomposedof multipletransposons.ThesesmallRNAsbindthePiwi Knockdownof . precursor causedsimilar AGO-bound mir-290 precursor transcript Nascent miRNA siRNA miR-1 –/– animals often B ) EndogenoussiRNAsin through to and miR-375 Key Pol II AGO miR- Pol IV(?)inplants Dicer RdRP AGO AGO Repeated DNA H3-K9 methylation knockout ofonethetwo maintains differentiated muscleduringlarvalgrowth.Inmice, (Sokol andAmbros,2005;Kwonetal.,2005)suggestedthat published characterizationof roles maybedynamic,changingandcomplex.A previously other tissue-ororgan-specific microRNAs,wheretheirfunctional 2005). Crocereported thatboth for distinguishingthetissuesof originofhumantumors(Luetal., patterns appeartobemorereliable thanmRNA expressionpatterns prognostic markersforcancer, andhowmicroRNA expression OH, USA)discussedtheimportance ofreliablediagnosticand Carlo Croce(OhioStateUniversity CollegeofMedicine,Columbus, microRNAs andcancer response toserumstarvation. miR-206 the defects, includingapparenthyperplasiaofmyocytes.So,although Mirn1-2 muscle andshow C2C12 cellsareremovedfromserum,theyproducedifferentiated regenerative myogenesis–inculture(Buckingham,2006).When in themyogenicdifferentiation ofmouseC2C12cells–amodelfor Virginia, Charlottesville,VA, USA)reported a functionfor miR-1 identical betweeninsectsandmammals,itisnotyetclearwhether Heterochromatin mir-1 maintenance function isentirelyconserved.AnindyaDutta(Universityof S. pombe ), asreportedbySrivastava’s laboratory, leadstoheart sequence anditsmuscle-specificexpressionisessentially appears toberequiredformuscledifferentiation in and inplantsare generatedfrom longtranscripts i-,miR-133 miR-1, Aub C Pol II? transposons mir-1 Drosophila mir-1- ( i nukonmcaim repeat- anunknownmechanism, ) Via A Multiple Aub ) Inanimals,microRNA (MIR)genes Piwi MIR-15 loci ( and AGO3 Aub mir-1-2 miR-206 and Piwi ofArgonauteprotein Development 134(9) ofamiRNAin MIR-16 AGO3 Aub knockout mutants membersofa , despitethefact , alsoknownas K9) methylation Piwi The miRNAs upregulation. Homologous AGO3 transposon are tumor for miR-206 miR-1

DEVELOPMENT revealed arolefor Shah (MassachusettsGeneralHospital,Boston,MA,USA), Medical School,Boston,MA,USA),incollaborationwithKhalid better prognosisfarsoonerthanalternateassays. Stage-1 humanlungcancersofpoorprognosisfromthosewitha Similarly, measurementsof potentially permittingearlierinterventionwithappropriatetherapy. patterns distinguishanaggressivefromindolentformofCLL, ( target B-cellCLL/lymphoma2 suppressors inchroniclymphocyticleukemia(CLL),andthatthey Development 134(9) complementary sequencesin3 of animalmicroRNA target identificationistheoccurrenceof in animals),followedbyexperimentalvalidation.A centralprinciple near-perfect, asinplants,orpartialbase-pairing,ismorecommon include thecomputationalpredictionofbase-pairmatches(either Current strategiestoidentifytheregulatorytargets ofmicroRNAs animals Finding andvalidatingmicroRNA targetsin suppressive activity(Johnsonetal.,2005). conserved sitesfor 3 isinducedbythedeletionof A2 (HMGA2)inhumans oncogenic behaviorofcertainmutatedformsHighmobilitygroup (Whitehead InstituteandMIT, Cambridge,MA,USA).The may functiontorepressoncogenes,wasillustratedbyDavidBartel repression ofoneormoretumorsuppressorgenes. contribute tothemalignancyofthesetumorcells,probablyvia brain tumors,sotheseresultssuggestthat mice. oligonucleotides inhibitstheirtumorigenicitywhentransplantedinto treatment ofhumanbraintumorcellswithanti- Nomenclature Database)inmammalianbraintumors.Thepre- point inreviewingtheroleof than isoftencomputationallypredicted.Ambroselaborated this suggests agreaterfunctionalspecificityformicroRNA-targeting microRNA target sites. many animalgenesareunderselectivepressuretomaintain predicted interactionsareevolutionarilyconserved,indicatingthat targets beingpredictedforagivenmicroRNA.However, many microRNA. Theserelativelylaxcriteriacanresultinnumerous matches tothe‘seed’ region(nucleotides1-8,fromthe5 timing. Although approach toseed-match target prediction was presented.Both Cambridge, MA,USA)andBartel. Fromseparatestudies,arefined presented byChrisBurge (MassachusettsInstituteofTechnology, candidate targets, andimprovementsinthesemethodologies were the relativephenotypicimpactof specificpredictedinteractions. that geneticanalysismaybethe mostdefinitivewaytodetermine immediately apparentfromcomputationalpredictionsalone, and suggest thattheeffects ofmicroRNAs ongenenetworksmaynotbe predicted target, the mutant areefficiently suppressed bythemutationofasingle Drosophila al., 2000).Ambrosreportedthatthisalsoappliesto mutation ofjustoneparticulartarget (Ambros,1989;Slacket result fromthelossofeithermicroRNA canbesuppressedbythe conserved targets (Lalletal., 2006),thepleiotropicphenotypesthat Ј Anna Krichevsky(BrighamandWomen’s HospitalandHarvard The conversesituation,wheretumor-suppressive microRNAs Interestingly, asCarthewdiscussed,experimentalevidence Nevertheless, computationalmethods canefficiently identify UTR (Mayretal.,2007),whichcontainsseveralevolutionarily MIR-21 , wheretheseverebehavioraldefectsof overexpression isahallmarkofcertainclasses lin-4 MIR-21 LET-7 abrupt and (also knownas amicroRNA withpotentialtumor- , let-7 MIR-155 let-7 transcription factor. Theseresults Ј untranslated regions(UTRs),and are eachpredictedtohavemany and BCL2 and lin-4 ). microRNA expression LET-7 MIRN21 in wormdevelopmental MIR-21 levels distinguish let-7 – HumanGene could indeed let-7 Ј function in end) ofthe HMGA2 deletion MIR-21 from the5 which allowsthemRNA tobeslicedbetween10and11 nucleotides animal microRNAshavesufficient complementaritytothemRNA, transcriptional, andtheyinvolvedifferent mechanisms.A few 2006; Pillaietal.,2006).Bothoftheseeffects arepost- translational repressionandareductioninmRNA level(Carthew, microRNAs exerttwogeneraleffects ontarget mRNAs: expression microRNA-mediated inhibition oftargetgene interactions. the directvalidationoffunctionalendogenousmicroRNA-target identification ofspecificmicroRNA-target complexesshouldpermit conjunction withproteomics-basedassays,thebiochemical protein alongwithassociatedmRNAsfromculturedhumancells.In York, NY, USA)reportedtheco-immunoprecipitationofArgonaute from complextissues.ThomasTuschl (Rockefeller University, New homogeneous samples,suchasculturedcells,orsortedcelltypes downregulated. Thismethodmaybeaparticularlypowerfultoolfor changes, independentlyofwhetherthecorrespondingmRNA is this methodshouldpermitthedirectmeasurementofproteinlevel proteins areexpectedtobeupregulatedinthemutant.Inprinciple, spectrometry. Inthisdifferential approach,microRNA target separated by2Dgelelectrophoresisandanalyzedmass isotopes, andproteinfractionsfromthesepopulationsweremixed, type ormicroRNA-mutantwormswerelabeledwithheavylight proteomics-based validationmethodwasreportedbyPlasterk.Wild- microRNA-target complexformationinvivo.A promising requires protein-levelassaysandtheverificationoffunctional of whatconstitutesaneffective microRNA-mRNA interaction to characterizeallfunctionaltargets. Thus,acomprehensivepicture (without alsoassayingthecorrespondingproteins)isnotsufficient of mRNA downregulation. of complementaritytomRNAsconsistentlymediatedifferent levels also foundthatdifferent typesofseedmatcheswithspecificpatterns conserved favorablefeatures,suchasthosereportedabove.They conserved seedmatches,perhapsinpartduetotheevolutionarily seed matchestendtoprovidemoreefficient knockdownthannon- exogenous microRNAs.BothBurge andBartelfoundthatconserved microRNA-targeting followssimilaroridenticalrulestothatof position. Burge alsoreportedthatendogenousvertebrate an effective interaction,regardlessofthebase-pairingatthat an A orUatposition9ofthetarget sitewasanefficient predictorof of thesitefrommiddleUTR.Curiously, Burge foundthat sequences immediatelysurroundingtheseedmatch,anddistance and positionwithinthemicroRNA oftheseedmatch,A/U-rich Bartel reportedthatfavorabletarget-site featuresincludethelength site (ofaparticularmicroRNA)caninfluencetheseinteractions. level oftarget mRNA knockdown,revealshowfeaturesofthetarget The strengthofamicroRNA-target interaction,asrevealedbythe mRNA levelsweremonitoredbymicroarrayhybridizationanalysis. were perturbedinvariousways.Theresultantchangestarget groups analyzeddatafromexperimentsinwhichmicroRNA levels Rhoades etal.,2006). However, translationalrepressioncan occur targets andleadtomRNA cleavage (Tang etal.,2003;Jones- 1999). MostplantmicroRNAs havenear-precise matchestotheir and/or someformoftranslational repression(OlsenandAmbros adenylation anddecapping(Wu etal.,2006;Giraldez2006), destabilization byother(non-slicer) mechanisms,suchasde- microRNAs impreciselymatch their targets andcausetarget mRNA mediated RNA silencing(Yekta etal.,2004).However, mostanimal Ultimately, assayingonlythechangesintarget mRNAslevels Ј end ofthemicroRNA,asisseenintraditionalsiRNA- MEETING REVIEW 1637

DEVELOPMENT human HuR(also knownasELAVL1 –HumanGeneNomenclature (Friedrich MeisnerInstitute, Basel, Switzerland),involvesthe factors. Onesuchexample,as discussedbyWitold Filipowicz translational repressiveactivity ofAGO2inatetheringassay. phenylalanine residueskeyto cap-bindingactivityimpairedthe displayed 7mGcap-binding activity, andthemutation of protein andthe7mGcap-bindingdomainofEIF4E.Indeed,AGO2 to aminoacidsimilaritiesbetweenaregionoftheHumanAGO2 University ofPennsylvania,Philadelphia,PA, USA)drewattention elongation. MarianthiKiriakidou(fromtheMourelatosgroup, 80S complexes,andrepressingtranslationalinitiationand/or the 60Sribosomalsubunit,preventingformationorstability of tethered toanmRNA viatheAgo2-microRNA complex,couldbind eIF6 Informatics) fromanimalcells.Moreover, RNAiknockdownof Argonaute 2(Ago2;alsoknown asEif2c2–MouseGenome the 60Sribosomalsubunit,canbeco-immunoprecipitated with Itgb4bp –MouseGenomeInformatics),alongwithcomponentsof eukaryotic translationinitiationfactor6(eIF6;alsoknownas de RegulacióGenòmica,Barcelona,Spain)reportedthatthe translational repressionbymicroRNAs.RaminShiekhattar(Centre interactions. primary modeofrepressionbysome,butnotall,microRNA-target (and subsequentdegradation)ofde-adenylatedmRNAsisthe adenylation ordecappingenzymes,whichindicatesthat certain targets wererepressedbyamicroRNA intheabsenceofde- capping enzymes(Behm-Ansmantetal.,2006).Significantly, only isolated componentsofP-bodies,andde-adenylationde- (RNAi) screensforproteinsrequiredmicroRNA function,and luciferase reportertoperformgenome-wideRNA interference Institute, Tuebingen, Germany)usednatural 3 dependent effects onknockdown?ElisaIzaurralde(MaxPlanck associated proteins. the microRNA-target duplexthatarerecognizedbymicroRNA- microRNA-target interactionmaydependonstructuralfeaturesof translational repression.Theseresultssuggestthattheoutcomeofa mRNA level,whereasothersled tomRNA reductionwithout found tocausepotenttranslationalrepressionwithoutaffecting otherwise matchedatallotherpositions.Certainstructureswere and mismatchesatpositions9,1011 ofamicroRNA that mutant reporterconstructs,Sharptestedalternativebase-pairings Using microRNAstransfectedintoanimalcellscontainingasetof microRNA causesRNA degradationortranslationalinhibition. an animalmicroRNA-target hybridaffect whetherornotthe translational inhibitionoftarget mRNAs. plant microRNAscanleadtobothtranscriptcleavageand microRNAs. Thepresenceofthisclassmutantssuggeststhat proteins, butnotofmRNAspredictedtobetargeted byother but notof One classofmutantsisolatedshowedelevatedlevelsGFP protein fluorescent protein(GFP)transgeneunderthecontrolof performed mutagenesisofan common despitetheprevalenceofnearlyperfectmatches.Hisgroup France) suggestedthattranslationalregulationinplantsmaybe Voinnet (InstitutdeBiologieMoléculairedesPlantes,Strasbourg, Sakai 2003;Chen2004;Arteaga-Vazquez etal.,2007).Olivier for certainmicroRNA-target interactionsinplants(Aukermanand 1638 measurable de-repressionofmicroRNA target expression. microRNA activitycanbemodulatedbyadditionalproteinco- Progress wasreportedinthesearchforpotentialmechanismsof Can specificmicroRNAsorassociated3 Sharp discussedhowthestructuralandbase-sequencefeaturesof in animalcells,aswell MEETING REVIEW GFP mRNA, aswellelevatedlevelsofendogenous Arabidopsis C. elegans Ј line bearingagreen UTRs havecontext- Ј UTRs fusedtoa , resultsinthe mir171 eIF6 , . of CAT-1 proteinproductionbyinterferingwiththerepressiveactivity andde-represses mRNA Human GeneNomenclatureDatabase) to anelementinthe3 in responsetostress.ThisElav-familyRNA-bindingproteinbinds Database) protein,whichisexportedfromthenucleusoflivercells in thecontextofsome3 of themore-effective sitesattheendsof3 A/U-rich sequencessurroundingthesiteandpreferentialplacement al., 2005).Bartelcommentedthattwofeaturesoffunctionalsites– to itscomplementarysequences(RobinsandPadgett,2005;Zhaoet target site,andhowthiscouldaffect theaccessibilityofmicroRNA secondary structureofthemRNA inthevicinityofmicroRNA 2006). presence ofagerm-cell-specificfactorthatblocks Haven, CT, USA)reportedhowthe mRNA. Similarly, AntonioGiraldez (Yale SchoolofMedicine,New translated, perhapsviathedisplacementofmicroRNA fromthe whereas, uponHuRbinding, activity leadstothesequestrationof activity of Japan) reportedprogressinunderstandingthebiogenesis and USA) andMikikoSiomi(UniversityofTokushima, Tokushima, Hannon (ColdSpringHarborLaboratory, ColdSpringHarbor, NY, Although piRNA functioningametogenesisisstillunclear, Gregory 2006; Aravinetal.,GirardLau2006). family proteins(Saitoetal.,2006;Vagin etal.,2006;Grivna small RNAs–piRNAshavebeenfoundtobeassociatedwith Piwi (Cox etal.,1998;Kim,2006);inthepastyearorso,anewclass of essential forspermatogenesisinmiceandgametogenesisflies mechanisms (Fig.1).ThePiwiclassofArgonaute proteinsis small RNA-guidedgeneregulationbyArgonaute-based Recent advanceshaveilluminatedthesurprisinglydiversemodesof siRNAs Gene regulation bypiRNAsandendogenous serves asamastercontrollocus tosilence transposon-rich heterochromatic locusontheflyXchromosomethat function ofaclusterrasiRNAsfromthe are similartomammalianpiRNAsinmanyrespects. natural 3 influence ofatarget secondarystructurecanalsobeobservedfor non-cleaving activityofmicroRNA-guidedRISC,andwhetherthe be excitingtoseeifthesebiochemicalanalysescanadaptedthe accessibility onRISCbindingwasconvincinglydemonstrated.Itwill predicted target secondarystructures,astrongeffect of target the RISC-cleavagereaction,andbiochemicalverificationof (IMBA, Vienna, Austria).Bycarefulquantitationofthevelocity cleavage experimentsperformedincollaborationwithJavierMartinez from RNA-inducedsilencingcomplex(RISC)-bindingandtarget- Schroeder (MaxPerutzLaboratories,Vienna, Austria)reportedresults the accessibilityofmicroRNA-bindingsitein3 of the3 somatic cells,butnotinthegermline.Theeffect isduetoaproperty repression ofa pong’ mechanismthatoccurs withouttheinvolvementofDicer of theserasiRNAsrevealaspects oftheirbiogenesisviaa‘ping- homologous transposonsequences throughoutthegenome.Features spawns rasiRNAsthatpresumably interactwith,andsilence, transposons scatteredaround the genome.The One areathatisbeginningtogarnermoreattentiontheintrinsic Hannon presentedresultsfromstudiesonthebiogenesisand miR-122 Ј Ј UTR outsideofthe UTR substrates. Drosophila ( MIRN122A mir-430 Ј repeat-associated siRNA (rasiRNAs),which UTR of ) (Bhattacharyyaetal.,2006). Ј family microRNA sensorinzebrafish UTRs, butnotothers(Mishimaetal., miR-430 CAT-1 CAT-1 mRNA exitstheP bodiesandis sites andisconsistentwiththe Nanos CAT-1 (also knownas Ј gypsy UTR –couldinfluence 3 Development 134(9) Ј mRNA inP bodies, UTR mediatesthe flamenco , miR-430 flamenco ZAM Ј UTR. Renée SLC7A1 and other miR-122 locus, a activity locus –

DEVELOPMENT are foundinthemainbody(not atthepromoteror3 restricted tosmallregions(within 1kb)inplants.H3K27me3marks PRC2 complex,andthismightexplainwhyH3K27me3tendsto be Pho-repressive complex(PhoRC).Plants,however, onlypossessthe complexes; Polycombrepressivecomplex1(PRC1),PRC2, and gene promoters,andareregulatedbythreePolycombrepressor developmentally importantloci,suchastheHoxclustersandcertain importance ofaclasssmallRNAs (University ofRochester, Rochester, NY, USA)illustratedthe ATPase Mit1(Sugiyamaetal.,2007).MartinGorovsky activities ofthehistonedeacetylaseClr3andaSNF2family machinery andeffects transcriptionalgenesilencingviathe binds theheterochromatinplatformestablishedbyRNAi connects theRNAipathwaytochromatinremodeling.SHREC complex in Jia, 2007).Grewalintroducedamulti-enzymesilencingeffector order tofendoff futureinvasionbysimilarsequences(Grewaland bound RNAifactorsmayselectivelyproducerepeatsiRNAsin domains thatcontaincertainrepeats.Theseheterochromatin- components stablytetherRNAimachineryacrosschromosomal of aself-reinforcingloopmechanism,inwhichheterochromatin (NIH), Bethesda,MD,USA]providedevidencefortheexistence role inthisprocess.ShivGrewal[NationalInstitutesofHealth stabilization ofgeneexpression,andsmallRNAsplayacentral perhaps piRNAs,inheterochromatinformation. 1 (HP1)inyeasttwo-hybridassays,whichimplicatesPiwi,and CN, USA),PiwiwasfoundtointeractwithHeterochromatinprotein collaborative studywithHaifanLin(Yale University, NewHaven, Aubergine arerequiredforrepeat-inducedgenesilencing.Ina University, StLouis,MO,USA) showedthat FlyBase) mRNA/proteininflytestes.SarahElgin(Washington methylation. correlate withthepresence of smallRNAs,norwithDNA developmental roles.Curiously, theH3K27me3marksdo not expressed inatissue-specific mannerandprobablyhave on euchromaticchromosomal arms. H3K27me3genestendtobe in et al.,2006)andhistoneH3lysine27(H3K27)trimethylation(me3) USA) presentedgenome-wideprofilesofDNA methylation(Zhang generated throughDiceractivity. generating double-strandedRNA (dsRNA),fromwhich,siRNAsare sustain RNA-dependentRNA polymerase(RdRP)activityin contain shortinternaltandemrepeats,whicharethoughttohelp and DNA transposons.Manyof the PolIVb-silencedlocialso include repeats,longterminalrepeats(LTRs) ofretrotransposons, polymerase (Pol)IVb-dependentmanner. ThePolIVb-silencedloci methylated andsilencedbyendogenoussiRNAsinanRNA (Fig. 1).Matzkereportedtheidentificationoflocithatare UK) andMarjoriMatzke(GregorMendelInstitute,Vienna, Austria) as discussedbyDavidBaulcombe(SainsburyLaboratory, Norwich, nucleus. direct histonemodificationsinthenewlyreplicated‘somatic’ conjugative reproductionintheprotist dependent mannertosilence locus, whichgeneratesrasiRNAsthatactinanAubergine- cleavage oftarget transcripts. Slicer-mediated suggest that,onewaytogeneraterasiRNAsisby activity andresultsintheamplificationofrasiRNAs.These Development 134(9) Arabidopsis Histone modificationiscrucialtotheregulationand Steven Jacobsen(UniversityofCalifornia,LosAngeles,LA, Plants alsousesimilarmechanismstomaintainheterochromatin, Siomi presentedsimilarfindingsforthe Schizosaccharomyces pombe . H3K27me3marksinanimalsspanlarge regionsof Stellate T. thermophila (also knownas ( scanRNAs) forproper (termed SHREC)that Suppressor ofstellate Drosophila Ј end) ofgenes . scanRNAs Piwi and SteXh – recently identifiednon-conservedmicroRNA lociin University, Corvallis,OR,USA)revealedhowhislaboratory to driveanimalandplantdiversity. JamesCarrington(OregonState rapidly evolving,suggestinganongoingpotentialformicroRNAs evolution ofcomplexanddiverseorgans andbodyplans. transcriptional andcell-cellsignalingpathwaysrequiredforthe regulators playedaroleintheadaptivediversificationofpre-existing an urbilaterianancestorsuggeststhatthesepost-transcriptional cniderians. TherapidexpansionofmicroRNA numberandtypein conserved familiesevolvedearlier, inanancestortobilateriansand of allbilaterians(the‘urbilaterian’),whereasperhapsjustthree at least27oftheconservedmicroRNA familiesaroseintheancestor animals (Sempereetal.,2006;Prochnik2007)indicatesthat forms. ThephylogeneticdistributionofconservedmicroRNAsin perhaps inconjunctionwiththeacquisitionofcomplexmulticellular emerged earlyduringtheevolutionofbothplantsandanimals, Bartel, 2005),indicatingthatmicroRNA-basedgeneregulation their respectivekingdoms(Pasquinellietal.,2000;Axtelland Several plantandanimalmicroRNAsarehighlyconservedwithin Evolution ofsmallregulatory RNAfunction variations ingeneexpression. natural selectioncouldmaintainfavorableepigeneticallydetermined to ongoing,epigeneticandheritablegenesilencing,and,therefore, (DRH-3). Mellosuggestedthatmuchofthewormgenomeissubject RdRp andthattheydependontheactivityofDicerrelatedhelicase reported thatendogenoussiRNAsarealsoproduceddenovoby Craig Mello(UniversityofMassachusetts,Worchester, MA,USA) siRNAs areproducedbydenovosynthesisofshortRNAsRdRp. Alto, CA,USA)resultsindicatethatthemajorityofsecondary 2007; PakandFire,2007).AndrewFire’s (StanfordUniversity, Palo siRNAs, whichinducelong-lasting,heritablesilencing(Sijenetal., generated bymechanismssimilartothosethatproducesecondary RNAi (Leeetal.,2006).EndogenouswormsiRNAsareprobably of introns,often withinprotein-codinggenes, andreportedon that mostmammalianmicroRNAs areencodedinthesensestrand a microRNA tothecorrectArgonaute effector complex.Kim noted features ofmicroRNA precursorsinfliesthatgovernthesortingof microRNA functionandevolution.Zamorereportedstructural microRNA expressionandactivity, andhencearecrucialfactorsin described aspectsofmicroRNA biogenesisthatprofoundlyaffect USA) andNarryKim(SeoulNationalUniversity, Seoul,Korea) poised fornaturalselection. potentially importantmicroRNA-target regulatoryinteractions exciting ideassuggestthatorganisms containavastreservoirof targets ofconservedmicroRNAs couldbesimilarlyneutral.These neutral. Rajewskypointedoutthatsomeofthenon-conserved and, hence,someofthesemicroRNAscouldbeevolutionarily species-specific microRNAsareexpressedatrelativelylowlevels evolutionary pathtoelimination.Carthewfoundthat have anypredictedtargets, suggesting thattheycouldbeonan conserved microRNAs(Fahlgrenetal.,2007);indeed,somedo not seem tobefarlesslikelytarget transcriptionfactorsthan Carrington notedthat,inplants,therecentlyevolvedmicroRNAs DCL1activityasthehairpinRNA accumulatesmismatches. via generation ofsiRNAs,andmicroRNAswouldsubsequentlyevolve Dicer-like 4(DCL4)-processingofthenewhairpinleadsto inverted duplicationoffounderloci,producingahairpinRNA. supporting theviewthatnewplantmiRNA locimayevolvefromthe Many microRNAsandtheirtarget interactionsappeartobe Worms alsocarryoutongoingsilencingofendogenousgenesby Phil Zamore(UniversityofMassachusetts,Worchester, MA, MEETING REVIEW Arabidopsis Drosophila 1639 ,

DEVELOPMENT Ambros, V. References supported bygrantsfrom NIH andNSF. owing tospaceconstraints.V.A. issupportedbygrantsfrom NIH.X.C.is on themanuscript.We apologizetothosewhoseworkcouldnotbediscussed unpublished results; andJ.Carrington,N.LauBrennecke forcomments speakers fortheirexcellentpresentations andforpermittingustodiscusstheir We thankD.BartelandS.Grewal fororganizingthe meetingprogram; allthe Keystone Symposiumnextyear. to theseandotherquestionsmayemerge whenwereturntothe al., 2000;Sempereet2006;Prochnik2007).Theanswers certain microRNAsforhundredsofmillionsyears(Pasquinelliet mechanisms thathavefixedtheentire21nucleotidesequenceof challenge forthefuturewillbetounderstandevolutionary repression? CanmicroRNAsactivatetheexpressionofatarget? One non-specific, modulatemicroRNA target recognitionand efficiency andstabilityoftarget mRNAs?Whatfactors, specific or By whatmechanismsdomicroRNAsinfluencethetranslational small RNAsmediatetheepigeneticregulationofgeneexpression? of piRNAs,rasiRNAsandendogenoussiRNAs.Preciselyhowdo developmental rolesofpiRNAs,andthemechanismsbiogenesis years. Someoftheimmediatechallengesaretoelucidate phenomena willcontinuetoblossominthecomingmonthsand regulatory mechanisms,anditislikelythatthecomplexityofthese continue tobesurprisedbythediversityofsmallRNAsandtheir The fieldofsmallregulatoryRNAsisenjoyingrapidprogress.We Conclusion worms. hallmarks ofendogenoussiRNA clustersthatareseeninplantsand genome, butexpressesaclassofendogenoussiRNAsthatbearsthe Chlamydomonas reinhardtii Baulcombe issuedacautionarytaleoftheunicellularalgae (reported bySharp)didnotrevealendogenoussiRNAs.However, Even thedeep-sequencingofsmallRNAsfrommouseEScells existence ofaprecursordsRNA oranRdRP-basedmechanism. rasiRNAs fromthe 2006). TheknownfeaturesofmammalianpiRNAsand are reportedtocontainsuchgenes(CeruttiandCasas-Mollano, products ofRdRPs;interestingly, amonganimals,onlynematodes plants andanimalsindicatethatmanyendogenoussiRNAsare comparative genomicsofRNAipathwaysandproteinsbetween processing oftheintronicmicroRNA. RNASEN –HumanGeneNomenclatureDatabase)-mediated mRNA splicingofthehostgeneandDROSHA (alsoknownas studies ofthemechanisticandregulatoryrelationshipbetweenpre- 1640 Axtell, M.J.andBartel,D.P. Arteaga-Vazquez, M.,Caballero-Perez, J.P. J.andVielle-Calzada, Aravin, A.,Gaidatzis,D.,Pfeffer, S.,Lagos-Quintana,M.,Landgraf,P., Bhattacharyya, S.N.,Habermacher, R.,Martine,U.,Closs,E.I.and Behm-Ansmant, I.,Rehwinkel,J.,Doerks, T., Stark,A.,Bork,P. and Aukerman, M.J.andSakai,H. developmental switchinC.elegans. 20 2730-2741. organ identitybyaMicroRNA anditsAPETALA2-like targetgenes. family ofMicroRNAs present inplantsandanimals. mouse testes. Nakano, T. etal. Iovino, N.,Morris,P., Brownstein, M.J.,Kuramochi-Miyagawa,S., human cellssubjected tostress. Filipowicz, W. CCR4:NOT deadenylaseandDCP1:DCP2 decappingcomplexes. Izaurralde, E. land plants. Are endogenoussiRNAsbroadlyconservedinanimals?The ,1885-1898. MEETING REVIEW (1989). 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