Post-transcriptionalgenesilencing(PTGS),whichwasinitiallyconsideredabizarrephenomenonlimitedtopetuniasandafewotherplantspecies,isnowoneofthehottesttopicsinmolecularBIOLOGy(1).Inthelastfewyears,ithasbecomeclearthatPTGSoccursinbothplantsandanimalsandhasrolesinviraldefenseandtransposonsilencingmechanisms.Perhapsmostexciting,however,istheemerginguseofPTGSand,inparticular,RNAinterference(RNAi)—PTGSinitiatedbytheintroductionofdouble-strandedRNA(dsRNA)—asatooltoknockoutexpressionofspecificgenesinavarietyoforganisms(reviewedin1-3).

HowwasRNAidiscovered?Howdoesitwork?Perhapsmoreimportantly,howcanitbeharnessedforfunctionalgenomicsexperiments?ThisarticlewillbrieflyanswerthesequestionsandprovideyouwithresourcestofindindepthinformationonPTGSandRNAiresearch.

Morethanadecadeago,asurprisingobservationwasmadeinpetunias.Whiletryingtodeepenthepurplecoloroftheseflowers,RichJorgensenandcolleaguesintroducedapigment-producinggeneunderthecontrolofapowerfulpromoter.Insteadoftheexpecteddeeppurplecolor,manyoftheflowersappearedvariegatedorevenwhite.Jorgensennamedtheobservedphenomenon"cosuppression",sincetheexpressionofboththeintroducedgeneandthehomologousendogenousgenewassuppressed(1-5).

Firstthoughttobeaquirkofpetunias,cosuppressionhassincebeenfoundtooccurinmanyspeciesofplants.Ithasalsobeenobservedinfungi,andhasbeenparticularlywellcharacterizedinNeurosporacrassa,whereitisknownas"quelling"(1-3).

Butwhatcausesthisgenesilencingeffect?Althoughtransgene-inducedsilencinginsomeplantsappearstoinvolvegene-specificmethylation(transcriptionalgenesilencing,orTGS),inotherssilencingoccursatthepost-transcriptionallevel(post-transcriptionalgenesilencing,orPTGS).Nuclearrun-onexperimentsinthelattercaseshowthatthehomologoustranscriptismade,butthatitisrapidlydegradedinthecytoplasmanddoesnotaccumulate(1,3,6).

IntroductionoftransgenescantriggerPTGS,howeversilencingcanalsobeinducedbytheintroductionofcertainviruses(2,3).Oncetriggered,PTGSismediatedbyadiffusIBLe,trans-actingmolecule.ThiswasfirstdemonstratedinNeurospora,whenCogoniandcolleaguesshowedthatgenesilencingcouldbetransferredbetweennucleiinheterokaryoticstrains(1,7).ItwaslaterconfirmedinplantswhenPalauquiandcolleaguesinducedPTGSinahostplantbygraftingasilenced,transgene-containingsourceplanttoanunsilencedhost(8).Fromworkdoneinnematodesandflies,wenowknowthatthetrans-actingfactorresponsibleforPTGSinplantsisdsRNA(1-3).

RNAiIsDiscoveredinNematodesThefirstevidencethatdsRNAcouldleadtogenesilencingcamefromworkinthenematodeCaenorhaBDitiselegans.Sevenyearsago,researchersGuoandKemphueswereattemptingtouseantisenseRNAtoshutdownexpressionofthepar-1geneinordertoassessitsfunction.Asexpected,injectionoftheantisenseRNAdisruptedexpressionofpar-1,butquizzically,injectionofthesense-strandcontroldidtoo(9).

Thisresultwasapuzzleuntilthreeyearslater.ItwasthenthatFireandMellofirstinjecteddsRNA—amixtureofbothsenseandantisensestrands—intoC.elegans(10).Thisinjectionresultedinmuchmoreefficientsilencingthaninjectionofeitherthesenseortheantisensestrandsalone.Indeed,injectionofjustafewmoleculesofdsRNApercellwassufficienttocompletelysilencethehomologousgene"sexpression.FurThermore,injectionofdsRNAintothegutofthewormcausedgenesilencingnotonlythroughouttheworm,butalsoinitsfirstgenerationoffspring(10).

ThepotencyofRNAiinspiredFireandTimmonstotryfeedingnematodesbacteriathathadbeenengineeredtoexpressdsRNAhomologoustotheC.elegansunc-22gene.Surprisingly,thesewormsdevelopedanunc-22null-likephenotype(11-13).FurtherworkshowedthatsoakingwormsindsRNAwasalsoabletoinducesilencing(14).Thesestrategies,wherebylargenumbersofnematodesareexposedtodsRNA,haveenabledlarge-scalescreenstoselectforRNAi-defectiveC.elegansmutantsandhaveledtolargenumbersofgeneknockoutstudieswithinthisorganism(15-18).

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RNAiinDrosophilaRNAihasalsobeenobservedinDrosophila.AlthoughastrategyinwhichyeastwereengineeredtoproducedsRNAandthenfedtofruitfliesfailedtowork,microinjectingDrosophilaembryoswithdsRNAdoeseffectsilencing(2).Silencingcanalsobeinducedby"shooting"dsRNAintoDrosophilaembryoswitha"genegun"orbyengineeringfliestocarryDNAcontaininganinvertedrepeatofthegenetobesilenced.Overthelastfewyears,theseRNAistrategieshavebeenusedasreversegeneticstoolsinDrosophilaorganisms,embryolysates,andcellstocharacterizevariousloss-of-functionphenotypes(2,19-23).

SohowdoesinjectionofdsRNAleadtogenesilencing?Manyresearchgroupshavediligentlyworkedoverthelastfewyearstoanswerthisimportantquestion.AkeyfindingbyBaulcombeandHamiltonprovidedthefirstclue.TheyidentifiedRNAsof~25nucleotidesinplantsundergoingcosuppressionthatwereabsentinnon-silencedplants.TheseRNAswerecomplementarytoboththesenseandantisensestrandsofthegenebeingsilenced(24).

FurtherworkinDrosophila—usingembryolysatesandaninvitrosystemderivedfromS2cells—shedmorelightonthesubject(3,25,26).Inonenotableseriesofexperiments,ZamoreandcolleaguesfoundthatdsRNAaddedtoDrosophilaembryolysateswasprocessedto21-23nucleotidespecies.TheyalsofoundthatthehomologousendogenousmRNAwascleavedonlyintheregioncorrespondingtotheintroduceddsRNAandthatcleavageoccurredat21-23nucleotideintervals(26).Rapidly,themechanismofRNAiwasbecomingclear.

CurrentModelsoftheRNAiMechanismBothbiochemicalandgeneticapproaches(see"TheGenesandEnzymesInvolvedinPTGSandRNAi"belowforadiscussionofgeneticapproachesusedtoundersandRNAi)haveledtothecurrentmodelsoftheRNAimechanism.Inthesemodels,RNAiincludesbothinitiationandeffectorsteps(27,seealsoaFlashanimationof"HowDoesRNAiWork?",fromreference3).

Intheinitiationstep,inputdsRNAisdigestedinto21-23nucleotidesmallinterferingRNAs(siRNAs),whichhavealsobeencalled"guideRNAs"(reviewedin3,18,27).EvidenceindicatesthatsiRNAsareproducedwhentheenzymeDicer,amemberoftheRNaseIIIfamilyofdsRNA-specificribonucleases,processivelycleavesdsRNA(introduceddirectlyorviaatransgeneorvirus)inanATP-dependent,processivemanner.SuccessivecleavageeventsdegradetheRNAto19-21bpduplexes(siRNAs),eachwith2-nucleotide3"overhangs(27,28).

Intheeffectorstep,thesiRNAduplexesbindtoanucleasecomplextoformwhatisknownastheRNA-inducedsilencingcomplex,orRISC.AnATP-dependingunwindingofthesiRNAduplexisrequiredforactivationoftheRISC.TheactiveRISCthentargetsthehomologoustranscriptbybasepairinginteractionsandcleavesthemRNA~12nucleotidesfromthe3"terminusofthesiRNA(3,18,27,29).Althoughthemechanismofcleavageisatthisdateunclear,researchindicatesthateachRISCcontainsasinglesiRNAandanRNasethatappearstobedistinctfromDicer(27).

BecauseoftheremarkablepotencyofRNAiinsomeorganisms,anamplificationstepwithintheRNAipathwayhasalsobeenproposed.AmplificationcouldoccurbycopyingoftheinputdsRNAs,whichwouldgeneratemoresiRNAs,orbyreplicationofthesiRNAsthemselves(see"PossibleRoleforRNA-dependentRNAPolymerase"below).Alternativelyorinaddition,amplificationcouldbeeffectedbymultipleturnovereventsoftheRISC(3,18,27).

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PossibleRoleforRNA-dependentRNAPolymeraseGeneticscreensinNeurospora,C.elegans,andArABIdopsishaveidentifiedseveralgenesthatappeartobecrucialforPTGSandRNAi.Severalofthese,includingNeurosporaqde-1,ArabidopsisSDE-1/SGS-2andC.elegansego-1,appeartoencodeRNA-dependentRNApolymerases(RdRPs).Atfirstglance,itmightbeassumedthatthisisproofthatanRdRPactivityisrequiredforRNAi.CertainlytheexistenceofanRdRPmightexplaintheremarkableefficiencyofdsRNA-inducedsilencingifitamplifedeitherthedsRNApriortocleavageorthesiRNAsdirectly.Butmutantsofthesegeneshavevaryingphenotypes,whichmakestheroleofRdRPinRNAidifficulttodiscern(1,3,17,18).

InC.elegansego-1mutants("ego"standsfor"enhancerofglp-1"),RNAifunctionsnormallyinsomaticcells,butisdefectiveingermlinecellswhereego-1isprimarilyexpressed.InArabidopsisSDE-1/SGS-2mutants("SGS"standsforsuppressorofgenesilencing),siRNAsareproducedwhendsRNAisintroducedviaanendogenouslyreplicatingRNAvirus,butnotwhenintroducedbyatransgene.IthasbeenproposedthatperhapstheviralRdRPissubstitutingfortheArabidopsisenzymeinthesemutants(1,3,17,18).AlthoughnohomologofanRdRPhasbeenfoundinfliesorhumans,anRdRPactivityhasrecentlybeenreportedinDrosophilaembryolysates(30).Onemodelofamplification,termedthe"randomdegradativePCR"model,suggeststhatanRdRPusestheguidestrandofansiRNAasaprimerforthetargetmRNA,generatingadsRNAsubstrateforDicerandthusmoresiRNAs(27,30).Evidencesupportingthismodelhasbeenfoundinworms,whereasexperimentalresultsrefutingthemodelhavebeenobtainedfromDrosophilaembryolysates(26,27).

RNAiInitiatorsTwoC.elegansgenes,rde-1andrde-4("rde"standsfor"RNAideficient"),arebelievedtobeinvolvedintheinitiationstepofRNAi.MutantsofthesegenesproduceanimalsthatareresistanttosilencingbyinjectionofdsRNA,butsilencingcanbeeffectedintheseanimalsbythetransmissionofsiRNAfromheterozygousparentsthatarenotsilencingdeficient.TheC.elegansrde-1geneisamemberofalargefamilyofgenesandishomologoustotheNeurosporaqde-2("qde"standsfor"quellingdeficient")andtheArabidopsisAGO1genes("AGO"standsfor"argonaute";AGO1waspreviouslyidentifiedtobeinvolvedinArabidopsisdevelopment).AlthoughthefunctionofthesegenesinPTGSisunclear,amammalianmemberoftheRDE-1familyhasbeenidentifiedasatranslationinitiationfactor.Interestingly,ArabidopsismutantsofAGO1,whicharedefectiveforcosuppression,alsoexhibitdefectsinleafdevelopment.ThussomeprocessesorenzymesinvolvedinPTGSmayalsobeinvolvedindevelopment(1,3,17,18).

RNAiEffectorsImportantgenesfortheeffectorstepofPTGSincludetheC.elegansrde-2andmut-7genes.ThesegeneswereinitiallyidentifiedfromheterozygousmutantwormsthatwereunabletotransmitRNAitotheirhomozygousoffspring(16).Wormswithmutatedrde-2ormut-7genesexhibitdefectiveRNAi,butinterestingly,theyalsodemonstrateincreasedlevelsoftransposonactivity.Thus,silencingoftransposonsappearstooccurbyamechanismrelatedtoRNAiandPTGS.Althoughtherde-2geneproducthasnotyetbeenidentified,themut-7geneencodesaproteinwithhomologytothenucleasedomainsofRNaseDandaproteinimplicatedinWernersyndrome(arapidagingdisease)inhumans(1,3,17,18,31).PerhapsthisproteinisacandidateforthenucleaseactivityrequiredfortargetRNAdegradation.

PTGSHasAncientRootsDiscoveriesfrombothgeneticandbiochemicalapproachespointtothefactthatPTGShasdeepevolutionaryroots.ProposalshavebeenputforththatPTGSevolvedasadefensemechanismagainsttransposonsorRNAviruses,perhapsbeforeplantsandanimalsdiverged(1,3,17,18).

Interestingly,itwasnotedbymanyresearchersthatdisruptionofgenesrequiredforRNAioftencausesseveredevelopmentaldefects.ThisobservationsuggestedalinkbetweenRNAiandatleastonedevelopmentalpathway.

AgroupofsmallRNAmolecules,knownassmalltemporalRNAs(stRNAs),regulatesC.elegansdevelopmentaltimingthroughtranslationalrepressionoftargettranscripts.ResearchindicatesthattheC.eleganslin-4andlet-7stRNAsaregeneratedfrom70-nttranscriptsfollowingthefoldingoftheselongertranscriptsintoastem-loopstructure.ThefoldedRNAmoleculesarecleavedtoproduce22-ntstRNAsbytheenzymeDicer(calledDCR-1inC.elegans).ThusDicergeneratesbothsiRNAsandstRNAs,andrepresentsanintersectionpointfortheRNAiandstRNApathways(32-34).

Recently,nearly100additional~22ntRNAmolecules,termedmicroRNAs(miRNAs),wereidentifiedinDrosophila,C.elegans,andHeLacells(35-38).Muchlikelin-4andlet-7,thesemiRNAsareformedfromprecursorRNAmoleculesthatfoldintoastem-loopsecondarystructure.Thenewlydiscovered~22ntmiRNAsarebelievedtoplayaroleinregulationofgeneexpression,andatleasttwoofthemareknowntorequireDicerfortheirproduction(37).ItappearsthattheuseofsmallRNAsforbothgeneregulationandRNAiisacommonthemethroughoutevolution.

Non-specificGeneSilencingbyLongdsRNAsWhilethenaturalpresenceofRNAihadbeenobservedinavarietyoforganisms(plants,protozoa,insects,andnematodes),evidencefortheexistenceofRNAiinmammaliancellstooklongertoestablish.TransfectionoflongdsRNAmolecules(>30nt)intomostmammaliancellscausesnonspecificsuppressionofgeneexpression,asopposedtothegene-specificsuppressionseeninotherorganisms.Thissuppressionhasbeenattributedtoanantiviralresponse,whichtakesplacethroughoneoftwopathways.

Inonepathway,longdsRNAsactivateaproteinkinase,PKR.ActivatedPKR,inturnphoshorylatesandinactivatesthetranslationinitiationfactor,eIF2a,leADIngtorepressionoftranslation.(39)Intheotherpathway,longdsRNAsactivateRNaseL,whichleadstononspecificRNAdegradation(40).

AnumberofgroupshaveshownthatthedsRNA-inducedantiviralresponseisabsentfrommouseembryonicstem(ES)cellsandatleastonecelllineofembryonicorigin.(41,42)ItisthereforepossibletouselongdsRNAstosilencespecificgenesinthesespecificmammaliancells.However,theantiviralresponseprecludestheuseoflongdsRNAstoinduceRNAiinmostothermammaliancelltypes.

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siRNAsBypasstheAntiviralResponseInterestingly,dsRNAslessthan30ntinlengthdonotactivatethePKRkinasepathway.Thisobservation,aswellasknowledgethatlongdsRNAsarecleavedtoformsiRNAsinwormsandfliesandthatsiRNAscaninduceRNAiinDrosophilaembryolysates,promptedresearcherstotestwhetherintroductionofsiRNAscouldinducegene-specificsilencinginmammaliancells(43).Indeed,siRNAsintroducedbytransienttransfectionwerefoundtoeffectivelyinduceRNAiinmammalianculturedcellsinasequence-specificmanner.TheeffectivenessofsiRNAsvaries—themostpotentsiRNAsresultin>90%reductionintargetRNAandproteinlevels(44-46).ThemosteffectivesiRNAsturnouttobe21ntdsRNAswith2nt3"overhangs.SequencespecificityofsiRNAisverystringent,assinglebasepairmismatchesbetweenthesiRNAanditstargetmRNAdramaticallyreducesilencing(44,47).Unfortunately,notallsiRNAswiththesecharacteristicsareeffective.Thereasonsforthisareunclearbutmaybearesultofpositionaleffects(46,48,49).ForcurrentrecommendationsondesigningsiRNAs,see"siRNADesign".

AlthoughthehistoryandmechanismofRNAiandPTGSarefascinating,manyresearchersaremostexcitedaboutRNAi"spotentialuseasafunctionalgenomicstool.AlreadyRNAihasbeenusedtoascertainthefunctionofmanygenesinDrosophila,C.elegans,andseveralspeciesofplants.WiththeknowledgethatRNAicanbeinducedinmammaliancellsbythetransfectionofsiRNAs,manymoreresearchersarebeginningtouseRNAiasatoolinhuman,mouseandothermammaliancellculturesystems.

Inearlyexperimentswithmammaliancells,thesiRNAsweresynthesizedchemically(AmbionisoneofseveralcompaniesthatoffercustomsiRNAsynthesis).Recently,Ambionintroducedakit(theSilencer™siRNAConstructionKit)toproducesiRNAsbyinvitrotranscription,whichisalessexpensivealternativetochemicalsynthesis,particularlywhenmultipledifferentsiRNAsneedtobesynthesized.Oncemade,thesiRNAsareintroducedintocellsviatransienttransfection.Duetodifferencesinefficacy,mostresearcherswillsynthesize3–4siRNAstoatargetgeneandperformpilotexperimentstodeterminethemosteffectiveone.Transientsilencingofmorethan90%hasbeenobservedwiththistypeofapproach(44-46,48,49).

Sofar,injectionandtransfectionofdsRNAintocellsandorganismshavebeenthemainmethodofdeliveryofsiRNA.Andwhilethesilencingeffectlastsforseveraldaysanddoesappeartobetransferredtodaughtercells,itdoeseventuallydiminish.Recently,however,anumberofgroupshavedevelopedexpressionvectorstocontinuallyexpresssiRNAsintransientlyandstablytransfectedmammaliancells(50-56).SomeofthesevectorshavebeenengineeredtoexpresssmallhairpinRNAs(shRNAs),whichgetprocessedinvivointosiRNAs-likemoleculescapableofcarryingoutgene-specificsilencing(50,53,54,56).ThevectorscontaintheshRNAsequencebetweenapolymeraseIII(polIII)promoteranda4-5thymidinetranscriptionterminationsite.Thetranscriptisterminatedatposition2oftheterminationsite(polIIItranscriptsnaturallylackpoly(A)tails)andthenfoldsintoastem-loopstructurewith3"UU-overhangs.TheendsoftheshRNAsareprocessedinvivo,convertingtheshRNAsinto~21ntsiRNA-likemolecules,whichinturninitiateRNAi(50).ThislatterfindingcorrelateswithrecentexperimentsinC.elegans,Drosophila,plantsandTrypanosomes,whereRNAihasbeeninducedbyanRNAmoleculethatfoldsintoastem-loopstructure(reviewedin3).

AnothersiRNAexpressionvectordevelopedbyadifferentresearchgroupencodesthesenseandantisensesiRNAstrandsundercontrolofseparatepolIIIpromoters(52).ThesiRNAstrandsfromthisvector,liketheshRNAsoftheothervectors,have5thymidineterminationsignals.SilencingefficacybybothtypesofexpressionvectorswascomparabletothatinducedbytransientlytransfectingsiRNA.

TherecentstudiesonRNAihavetakentheresearchworldbystorm.Theabilitytoquicklyandeasilycreateloss-of-functionphenotypeshasresearchersrushingtolearnasmuchastheycanaboutRNAiandthecharacteristicsofeffectivesiRNAs.Inthefuture,RNAimayevenholdpromisefordevelopmentofgene-specifictherapeutics.Muchhasbeenlearnedaboutthispowerfultechnique,butadditionalinformationbecomesavailableonanalmostdailybasis(seeTheRNAInterferenceResourcetolearnabouttheverylatestRNAiresearchandtools).ItisnotanunderstatementtosaythatthefieldoffunctionalgenomicsisbeingrevolutionizedbyRNAi.

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RNAinterferencewww.nature.com/nature/fow/000316.html

FlashAnimation:HowDoesRNAiWork?Hammond,S.M.,Caudy,A.A.,Hannon,G.J.(2001)Post-transcriptionalGeneSilencingbyDouble-strandedRNA.NatureRevGen2:110-119.www.nature.com/nrg/journal/v2/n2/animation/nrg0201_110a_swf_MEDIA1.html

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Cosuppression-Silencingofanendogenousgenecausedbytheintroductionofatransgeneorinfectionbyavirus.Thisterm,whichcanrefertosilencingatthepost-transcriptional(PTGS)ortranscriptional(TGS)level,hasbeenprimarilyadoptedbyresearchersworkingwithplants.

Post-transcriptionalGeneSilencing(PTGS)-SilencingofanendogenousgenecausedbytheintroductionofahomologousdsRNA,transgeneorvirus.InPTGS,thetranscriptofthesilencedgeneissynthesizedbutdoesnotaccumulatebecauseitisrapidlydegraded.ThisisamoregeneraltermthanRNAi,sinceitcanbetriggeredbyseveraldifferentmeans.

Quelling-PTGSinNeurosporacrassainducedbytheintroductionofatransgene.

RISC-RNA-inducedsilencingcomplex.Anucleasecomplex,composedofproteinsandsiRNA(seebelow),thattargetsanddestroysendogenousmRNAscomplementarytothesiRNAwithinthecomplex.

RNAinterference(RNAi)-Post-transcriptionalgenesilencing(PTGS)inducedbythedirectintroductionofdsRNA.Theterm"RNAinterference"wasfirstusedbyresearchersstudyingC.elegans.

siRNAs-SmallinterferingRNAs.CurrentmodelsofPTGSindicatethatthese21-23nucleotidedsRNAsmediatePTGS.IntroductionofsiRNAscaninducePTGSinmammaliancells.siRNAsareapparentlyproducedinvivobycleavageofdsRNAintroduceddirectlyorviaatransgeneorvirus.AmplificationbyanRNA-dependentRNApolymerase(RdRP)mayoccurinsomeorganisms.siRNAsareincorporatedintotheRNA-inducedsilencingcomplex(RISC),guidingthecomplextothehomologousendogenousmRNAwherethecomplexcleavesthetranscript.