Introduction to RNAi.docx
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Introduction to RNAi.docx
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IntroductiontoRNAi
IntroductiontoRNAi
ThisoverviewincludesadescriptionofthediscoveryofRNAinterferenceand“RNAsilencing”(amoregeneraltermforthevarietyofrecentlydiscoveredsequence-specificcellularresponsestoRNA),asummaryofcurrentthinkingonthemechanismofRNAi,andvariousapproachestoRNAiexperiments.Thematerialpresentedinthisoverview,whennotexplicitlyreferenced,iscoveredinoneofseveralrecentreviews[1–7].
RNAInterferenceandRNASilencing
RNAinterference(RNAi)istheprocessofmRNAdegradationthatisinducedbydouble-strandedRNAinasequence-specificmanner.RNAihasbeenobservedinalleukaryotes,fromyeasttomammals.ThepowerandutilityofRNAiforspecificallysilencingtheexpressionofanygeneforwhichsequenceisavailablehasdrivenitsincrediblyrapidadoptionasatoolforreversegeneticsineukaryoticsystems.
TheRNAipathwayisthoughttobeanancientmechanismforprotectingthehostanditsgenomeagainstvirusesandroguegeneticelementsthatusedouble-strandedRNA(dsRNA)intheirlifecycles.RNAiisnowrecognizedtobebutoneofalargersetofsequence-specificcellularresponsestoRNA,collectivelycalledRNAsilencing.TheseresponseshavebeenshowntoplayarolenotonlyinmRNAanddsRNAstability/degradation,butalsoinregulationoftranslation,transcription,chromatinstructure,andgenomeintegrity.Inplantsandanimals,RNAsilencinghasbeenadaptedtoplayacriticalroleinregulationofcellgrowthanddifferentiationusingaclassofsmallRNAscalledmicroRNAs(miRNAs).
InallRNAsilencingpathways,dsRNAisprocessedtoa21–30nucleotide-longRNA,whichthenfunctionsasacomponentofa“silencingcomplex”tospecificallyrepressexpressionorfunctionofatargetgeneorgenomicregion(Figure1).Specifically,intheRNAipathway,dsRNAisprocessedtoshortinterferingRNA(siRNA):
21–25bpdsRNAwithdinucleotide3'overhangs.OnestrandofthesiRNA(theguidestrand)isthenassembledintoanRNA-inducedsilencingcomplex(RISC)thatcleavesthetargetmRNA.ThesiRNAisthoughttoprovidetargetspecificitytoRISCthroughbasepairingoftheguidestrandwiththetargetmRNA.
Figure1.GeneralStepsandMethodsofRNASilencing.InallRNAsilencingpathways,double-strandedRNA(dsRNA)isprocessedtoasmallRNAwhichisassembledwithRISCintoasilencingcomplexthatspecificallyrepressesexpressionorfunctionofatargetgeneorgenomicregionbycleavingthecorrespondingmRNA.
WhenRNAiisartificiallyinducedforreversegenetics,scientistsuseoneofseveralformsofdsRNAtotriggerthepathway.Thistopicisdiscussedindetailinsubsequentsectionsofthisoverview.
ThesmallRNAsthatprovidetargetspecificitytothesilencingmachinery—shortinterferingRNAs(siRNAs),repeat-associatedsiRNAs(rasiRNAs),andmicroRNAs(miRNAs)—canbedistinguishedbytheirorigin.siRNAsareprocessedfromdsRNAprecursorsmadeupoftwodistinctstrandsofperfectlybase-pairedRNA,whilemiRNAsoriginatefromasingle,longtranscriptthatformsimperfectlybase-pairedhairpinstructures.siRNAswereoriginallyidentifiedasintermediatesintheRNAipathwayafterinductionbyexogenousdsRNA;however,endogenoussourcesofsiRNAshavenowbeenrecognized.ManyoftheseendogenoussiRNAsarederivedfromrepetitivesequenceswithinthegenome,hencethetermrepeat-associatedsiRNAs,orrasiRNAs.miRNAswerediscoveredthroughtheircriticalrolesindevelopmentandcellularregulation,andrepresentalargeclassofevolutionarilyconservedRNAs.miRNAshavealwaysbeenrecognizedasbeingofendogenousorigin.However,syntheticprecursorsandinhibitorsofmiRNAsareusedtounderstandandexploitthevariousfunctionsofthisimportantclassofsmallRNA.
AlthoughsmallRNAsdifferintheirorigins,someoftheproteinsinvolvedintheirproductionandfunctioninsilencingcomplexesarecloselyrelated,andinsomecasesareidentical.Theseincludethemostwellcharacterizedcomponentsofthesilencingmachinery,DicersandArgonautes.DicerproducessmallRNAsfromlongerprecursorsandplaysakeyroleinassemblingtheguidestrandofthesmallRNAwithapathway-specificcomplexofsilencingproteins.ArgonauteisacorecomponentofallsilencingcomplexescharacterizedsofarandassociateswiththesmallRNAinthesilencingcomplex.InthecaseofRNAi,ArgonautehasbeenshowntobethecatalyticengineofmRNAcleavage.
DiscoveryofRNAi
RNAinterferencewasfirstobservedinpetunias,whenNapolietal.(1990)[8]discoveredthatintroductionofapigment-producinggeneundercontrolofapowerfulpromotersuppressedexpressionofboththeintroducedgeneandthehomologousendogenousgene,aphenomenontheycalled“cosuppression.”Cosuppressionwassubsequentlyfoundtooccurinmanyspeciesofplantsandfungi(whereitwascalled“quelling”)andtooccuratthepost-transcriptionallevel[8–10].
Suchpost-transcriptionalgenesilencing(PTGS)wasshowntobemediatedbyadiffusible,trans-actingmoleculeinbothNeurosporaandplants[9,11].Thecrucial1998discovery byFireetal.,thatinjectionofdsRNA—amixtureofbothsenseandantisensestrandsofthetargetmRNA,ratherthaneitherstrandalone—intogonadsofthenematodeCaenorhabditiselegansresultedinextremelypotentsilencingunequivocablyidentifieddsRNAastheinducerofRNAinterference[12].
Short(~25nt),antisenseRNAswerefirstimplicatedinPTGSinplants[13].FurtherworkinDrosophila-derivedinvitrosystemsshowedthatlongdsRNAwasprocessedtoshortinterferingRNAs(siRNAs),21–23ntlong,thatcouldmediatetargetmRNAcleavageintheregioncorrespondingtotheintroducedsiRNA[14–16].CurrentthinkingaboutthemechanismofRNAiandcomponentsoftheRNA-inducedsilencingcomplexisdescribedmorefullyinthetext.
InC.elegans,thedemonstrationthatsilencingcouldbeinducedbysimplyfeedingnematodeswithbacteriathathadbeenengineeredtoexpressdsRNAhomologoustothetargetgene,orbysoakingwormsinsuchdsRNA,droverapidadoptionofRNAiasatechnologyforreversegeneticsattheorganismallevel[17–19].Likewise,technologywasquicklydevelopedforinducingRNAiinDrosophilacellculturebybathingortransfectionwithdsRNA(seeArmknechtetal.2005[20]).
EffortstouseRNAiinmammaliancellswerehamperedatfirstbecauseofanonspecific,interferon-mediatedresponsetodsRNAlongerthan30 bpthatisseeninmostmammaliancelllines.Withthedemonstrationthat21–22ntsiRNAs,eitherchemicallysynthesizedorexpressedfromaplasmidvector,couldefficientlyinduceRNAiinmammaliancellswithoutinducingtheinteferonresponse,thedoorwasopenedfordevelopmentofRNAitoolsinmammaliansystems[21,22].
WhilesomeresearcherswerestudyingthephenomenonofRNAiandworkingtoexploititsuseasatoolforreversegenetics,othersweretryingtounderstandadistinctbutrelatedsetofsmallRNAs—microRNAs(miRNAs).ThefirstmiRNAstobediscovered,lin-4andlet-7,wereidentifiedthroughloss-of-functionmutationsaffectingcontrolofpostembryonicdevelopmentinC.elegans[23,24].OthermiRNAsweresoonidentifiedinC.elegans,Drosophila,andmousebycombinationsofforwardgenetics,cDNAcloning,bioinformatics,andreversegenetics.By2002,miRNAswerefirmlyestablishedasalargeclassofconservedregulatorymoleculesinanimalsandplants[25–29].miRNAshavebeenshowntoplayaroleindevelopmentaltiming,celldeath,cellproliferation,andoncogenesis[30–32].ThisclassofsmallRNAmayrepresent2–3%ofthetotalnumberofgenesinhumans[4,33],andestimatesofmiRNAtarget-bindingsitesindicatethatmiRNAsmayplayaroleinregulatingasmanyas30%ofmammaliangenes[34].
AformalconnectionbetweenthephenomenonofRNAinterferenceandmiRNAswasmadewhenthesameenzyme,Dicer,wasshowntoprocessbothlongdsRNAintosiRNAsandcytoplasmicmiRNAprecursors(pre-miRNAs)intomaturemiRNAs[35–38].OthercomponentsoftheRNAiandmiRNAsilencingpathwayshavebeenshowntobecloselyrelated,mostprominentlyArgonaute,whichisakeycomponentoftheRNA-inducedSilencingComplex(RISC).ItnowappearsthatmiRNAsfunctioninasilencingcomplexthatissimilar,ifnotidentical,toRISCtoregulateexpressionoftargetgeneseitherthroughcleavageofmRNAortranslationalrepression:
ifthemiRNAexhibitsperfectcomplementaritytoitstargetmRNA,themRNAiscleaved(typicallyinplants);ifthereisonlypartialcomplementary,translationalrepressionoccurs(typicallyinanimals).ThefurtherdiscoveryofendogenoussmallRNAs,distinctfrommiRNAs,thatfunctionintranscriptionalsilencingandgenomestabilityhasdriventheadoptionofthemoregeneralterms“RNAsilencing”and“smallRNAs”todescribethecollectionofrelatedsilencingpathwaysandtheirRNAguides.Theterm“RNAinterference”(RNAi)continuestodescribemRNAcleavagethatisinducedbyadsRNAtrigger.
SmallRNAs:
siRNAs,rasiRNAs,andmiRNAs
siRNAsandrasiRNAs
ShortinterferingRNAs(siRNAs)wereoriginallyidentifiedasintermediatesintheRNAinterferencepathway.siRNAsare21–25 bpdsRNAwithdinucleotide3'overhangsthatareformedinthecellfromlongerdsRNAmolecules.ThefullyassembledRNA-inducedsilencingcomplex(RISC)containsonlyonestrandofthesiRNA,theguidestrand.TheguidestrandisthoughttoprovidetargetspecificityforRISC-mediatedcleavagethroughperfectbasepairingwiththemRNAtarget.
LongdsRNA(severalhundredbasepairs)canbeusedtoartificiallyinducetheRNAipathwayinfungi,plants,insects,andworms.Inmammaliancells,dsRNA>30 bpinducesanonspecific,antiviralinterferonresponse.However,syntheticsiRNAs,~21 bpwit
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