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SmallAn(senseRNAsand RNAInterference BCMP200 November20,2015 BCMP200 - The Central Dogma 6. Protein translation (Melissa Leger-Abraham & Dipanjan Chowdhury) Cap AAAAAAA AAAAAAA AAAAAAA Cap AAAAAAA Cap AAAAAAA 5. Transcription/Processing (Stirling Churchman) Cap AAAAAAA Cap 4. Gene Regulation (Timur Yusufzai) 2. DNA Replication (Johannes Walter) 3. DNA Repair (Johannes Walter) 1. Guiding principles of protein and DNA interactions (Joseph Loparo) Cap OverviewofLecture Introduc)ontosmallan)senseRNAs Classifica)onofsmallan)senseRNAs miRNAs,siRNAsandpiRNAs Historicaliden)fica)onofthesmallan)sense RNAs • Processingandfunc)onofmiRNAs • ProcessingofsiRNAs • UsingsiRNAsasareversegene)ctool • • • • TheCentralDogmaandRNA MoleculesSoFar • Wehavebeenfollowingthenarra)veoftheflow informa)onfromDNAtoproteins • TimurandS)rlingmen)onedvarioustypesof RNAsofar: – mRNA*,messengerRNA(Module5) – rRNA,ribosomalRNA(Module6,transla)on) – tRNA,transferRNA(Module6,transla)on) – snRNA,smallnuclearRNA – snoRNA,smallnucleolarRNA SmallAn(senseRNAs InthislectureandinMonday’sresearchseminarwewillbe introducinganotherclassofRNA -mRNA -rRNA -tRNA -snRNA -snoRNAs -miRNAs,siRNAs,andpiRNAs ThemiRNAs,siRNAsandpiRNAsareallexamplesofsmall RNAsthataltergeneexpressionthroughinterac)onswith mRNAs.Theseareallan(senseRNAs. The“smallan(senseRNAs”are~21nt to~23ntinlength U1 snRNA smallan(senseRNAsare ~21to23nt(onaverage), butsomeaslarge31nt snRNAsareonaverage 150ntinlength snoRNAsareonaverage 60bpinlength ThesmallRNAsintoday’slecturearetypically~21ntto~23ntinlength. ThesesmallRNAsareinvolvedinpost-transcrip)onalgenesilencing. TheybindtomRNAthathasbeenproducedinthecellanddisrup)ontheexpressionof thatmRNA. Classifica(on • ThesmallRNAsareclassifiedintotwomajor groups: -miRNAs(microRNAs) -siRNAs(shortinterferingRNAs) • ThesiRNAscanalsobesub-dividedintoendosiRNAsandexo-siRNAs • TherearealsopiRNAs The21nt–23ntRNAscanbe characterizedbasedon4criteria ORIGINS PROCESSING • Whathostsequences(DNAorRNA) • Whereandhowaretheyprocessedto giverisetothesmallan(senseRNAs? becomematurean(senseRNAs? • Dotheycomefrominsidethecellor • Whatproteinsareinvolvedin dotheycomefromoutsidethecell? allowingthesmallRNAstomature? WHEREDOTHEYFUNCTION? • Wheredothesesmallan(senseRNAs func(oninsidethecell? • Canthesmallan(senseRNAsmove fromonecelltoanother? WHATEFFECTONTHETARGET? • Dothean(senseRNAsdegradethe targetmRNA?Doesitsequesterit? • Whatpartnersdoesthesmall an(senseRNAsworkwith? ORIGINS PROCESSING • Whathostsequences(DNAorRNA) • Whereandhowaretheyprocessedto giverisetothesmallan(senseRNAs? becomematurean(senseRNAs? • Dotheycomefrominsidethecellor • Whatproteinsareinvolvedin dotheycomefromoutsidethecell? allowingthesmallRNAstomature? WHEREDOTHEYFUNCTION? • Wheredothesesmallan(senseRNAs func(oninsidethecell? • Canthesmallan(senseRNAsmove fromonecelltoanother? WHATEFFECTONTHETARGET? • DoesthemiRNAdegradethetarget mRNA?Doesitsequesterit? • Whatpartnersdothesmallan(sense RNAsworkwith? microRNAs(miRNAs): Origins,Characteris(cs,andFunc(ons • WheredomiRNAscomefrom? • HowaremiRNAsprocessed?Whatcharacterizes a“mature”miRNA? • HowdoesanmiRNAinteractwithitstarget messengermRNA? • WhatistheeffectofthismiRNAonthemRNA? miRNA-mediatedgenerepression • miRNAsareafamilyof~21-23ntendogenousRNAsthatnega)vely regulategeneexpression • HundredsofmiRNAshavebeeniden)fiedinplantsandanimals • miRNAsexhibitcell-typespecificexpressionando\enregulate aspectsofdevelopment(over60%ofthecodinggenomecanbe regulatedbymiRNAs) • ThedegreeofcomplementaritybetweenamiRNAanditstarget, determinestheregulatorymechanism • Whenthereisamismatch betweenthemiRNAand themRNA,itleadsto repression • Whenthean)sense miRNAisfull complementarytothe mRNAtarget,itleadsto degrada)onofthemRNA lin-4,inC.elegans,wasthefirst miRNAdiscovered 7yearslaterthenextmiRNAwasdiscovered, andthisonewasconserved ThereareanumberofmiRNAsinvery diversespecies NorthernBlotsareanessen)altechniqueforstudying smallan)senseRNAs. Smallcomplementaryprobes(~20nt)areusedto iden)fyatargetmRNAonablotanditdependsonthe interac)onbetweentheprobeandtarget. RNAInterference(RNAi) shortinterferingRNAs(siRNAs) • siRNAsare~21-23ntinlength • siRNAsoriginatefromlongdsRNAintroducedtothecell eitherfromanexogenoussource(ourfocus)butalso some)mesfromexogenoussources • siRNAsusuallytargetthecomplementarymRNAfor degrada)on • siRNAsandmiRNAsareprocessedbysomeofthesame proteinsbuttherearesomeuniqueproteinsintheir pathwaysanddifferentfunc)onsatplayforthesame proteinsinthepathways piRNAs • Pi=Piwi(asubclassofthe Argonauteproteinfamily) • Piwi=p-elementinducedwimpy testes • piRNAsareaspecialclassofsmall an)senseRNAs,thatwerefirst iden)fiedfromthetestesof organismslikeDrosophila • Func)oninthegermlinetoprotect againstthemovementof transposableelements • Theprocessingmechanismfor piRNAshavenotbeenwell elucidated • ThepiRNAsareaslightlylargerclass ofsmallan)senseRNAs,usually rangingfrom26nt–31ntinlength PathwaysfortheprocessingofdoublestrandedRNAtoproducesmallan(senseRNAs miRNAsoriginatefromlongRNAs,transcribed byRNApolII,thatfoldintolargehairpins TheprocessingofmiRNAsbeginsin thenucleus. miRNAsareo\enfoundin intergenicspaceacrossthe genome. RNApolymeraseIIdrivesthe expressionofRNAmoleculesthat willfoldontothemselves. TheProteinDroshaTrimsTheFolded Moleculeintoashorterhairpinloop • Droshafunc)onsinthenucleustoconvertthe pri-miRNAtoapre-miRNA. DGCR8 TheProteinDicerRemovestheLoop toRetainaShortDouble-stranded StretchofRNA • Expor)n5andRan+GTP arenecessaryforthe transportofthe miRNAsfromthe nucleustothe cytoplasm • Inthecytoplasm,Dicer willcutthepre-miRNA toremovetheloop GTPasanenergycurrencymolecule • SofarwehaveseenATPasanenergy currencymoleculeinthecell. • ATPhydrolysisreleasesenergyandproduces ADPandaphosphategroup. • Thereleasedenergycanbeusedtodrive anothernon-spontaneousreac)on. • ThesameistrueforGTP. ATPandGTPhydrolysis(spontaneous)canbe coupledwithnon-spontaneousreac(onsto drivethemforward ΔG=ΔH–TΔSGibbsFreeEnergy Onestrandoftheshort~21–23bp RNAassembleswiththeArgonaute proteininformingtheRISCcomplex • TheRISCcomplex includinganmiRNA, designatedanmiRISC complex,cantargetan mRNAsequencewith fullyorpar)ally complementary sequence TheRISCcomplexcantargetmRNAsequencesthatcontain sequencescomplementarytothe“guide”RNAintheRISCcomplex ThetargetmRNAsequenceis foundbasedoncomplementarity oftheguidemiRNAthatispresent intheRISCcomplex. TheArgonauteproteinisthe “Slicer”proteinthatisresponsible cugngtheRNAwhenitwillbe degraded. AlthoughthemiRNAis~21–23ntinlength,each miRNAincludesan8ntsequencecalledthe“seed” sequencewhichisessen(alfortheinterac(onwith thetargetmRNA OncethemiRISCcomplexbindstothe mRNAitcaneitherdirectrepressionor degrada(onofthetargetmRNA Ahighlyregulatednetworkoffactorsare involvedinthedegrada(onofmRNAstargeted bymiRNAswithfullcomplementarity • mRNAsaregivenstabilitybythepresenceofa 5’Capanda3’poly(A)tail • Degrada)onoffromthe5’endcanbedone decappingenzymes(Dcp1-2)anda5’–>3’ exonucleaseac)vity(XRN1) • Degrada)onfromthe3’endcanbedoneby deadenylaseswhichremovethepoly(A)tail andthe3’–>5’ac)vityoftheexosome WheredorepressedmRNAsgo? The3’UTRofthetargetmRNAsequence containsthetargetsiteformiRNAsthatrepress transla(on TheRISCComplex,whichincludesa“guide”miRNA, canalsocausethetargettobedegraded • Degrada)onofthemRNAtargetscaninvolve de-cappinganddeadenyla)on • Thenucleicacidsequenceisdegradedusing exonucleasespresentinthecellorthe exosome miRNA-mediateddecayinvolvesthedecapping Dcp1:Dcp2complexandCCR:Notdeadenylase complex miRNAsdownregulatelevelsoftarget mRNAs BasedonearlierworkitwasthoughtthatmiRNAsinhibitthe produc)onofproteinwithoutaffec)ngmRNAlevels. Introduc)onofsynthe)cmiR-124(brain-specific)ormiR-1(musclespecific)intoHeLacellsfollowedbygeneexpressionprofilingby microarrayanalysis. DownregulatedmRNAsareenriched in“seed”sequencesintheir3’UTR Re-capoftheProcessingofmiRNAs • RNAPolIItranscribesarela)velylongRNA sequencethatcanfoldbackonitselftoforma hairpin • Thelargesequenceisprocesseddowntoa smallerhairpinbyDrosha • Thehairpinexitsthenucleusandentersthe cytoplasmwhereDicerremovestheloop • Onestrandofthe~21–23ntdsRNAmolecule thatisproducedbyDicerisloadedtoaRISC complexwithArgonaute Re-capoftheAc(onofmiRNAs • miRNAs,whichare~21–23ntIengthandincludean8 ntseedsequence,guidetheRISCcomplextothetarget mRNA • ThismiRISCcomplexusuallyinteracts • ThedegreeofcomplementaritybetweenthemiRNA andthetargetmRNAdeterminestheeffectonthe mRNAmolecule:repressionvsdegrada)on • RepressedmRNAsaredirectedtothePbodiesand stressgranules • DegradedmiRNAsaredisassembledbycombina)ons oftheac)vityof5’decappingand3’deadenyla)on, andtheac)vityofendonucleasesandexonucleases Technique:Valida(ngmiRNAtarget genes DeterminingtheBindingSiteofan miRNA • PAR-CLIP=Photoac)vatable-RibonucleosideEnhancedCrosslinkingandImmunoprecipita)on • HITS-CLIP=High-throughputsequencingofRNA isolatedbycrosslinkingimmunoprecipita)on (HITS-CLIP)(alsocalledCLIP-Seq) • iCLIP=individualnucleo)deresolu)onCrosslinkingandimmunoprecipita)on) PAR-CLIP Determiningthe BindingSiteofan miRNA: PAR-CLIP,HITS-CLIP andiCLIP ProcessingofsiRNAs • siRNAsaresome)mesdescribedasendosiRNAsandexo-siRNAs • Thefocusofthecontetinthislecturewillbe onexo-siRNAs • ButallsiRNAsoriginatefromlongdsRNA molecules ProcessingofsiRNAs miRNAsaregeneratedfrom hairpinsthatoriginateinthe nucleus. siRNAsareprocessedfrom verylongdsRNAthatis presentinthecytoplasm. ThesiRNAsaregenerallyfully complementarytothetarget sequence The21nt–23ntRNAscanbe characterizedbasedon4criteria ORIGINS PROCESSING • Whathostsequences(DNAorRNA) • Whereandhowaretheyprocessedto giverisetothesmallan(senseRNAs? becomematurean(senseRNAs? • Dotheycomefrominsidethecellor • Whatproteinsareinvolvedin dotheycomefromoutsidethecell? allowingthesmallRNAstomature? WHEREDOTHEYFUNCTION? • Wheredothesesmallan(senseRNAs func(oninsidethecell? • Canthesmallan(senseRNAsmove fromonecelltoanother? WHATEFFECTONTHETARGET? • DoesthemiRNAdegradethetarget mRNA?Doesitsequesterit? • Whatpartnersdothesmallan(sense RNAsworkwith? ExogenousRNAusedareverse gene(ctechnique InanorganismlikeC.elegans,dsRNA(witha sequencewhichiscomplementarytoatarget mRNAofinterest)canbeusedtodownregulate thatmRNAexperimentally. InC.elegans,thedsRNAcanbeintroducedto theorganismthrough3methods. MethodsofIntroducingdsRNAfor reversegene(csinC.elegans SystemicRNAiisonlyknowntotakeplacein selectorganismslikeplantsandC.elegans InC.elegans,dsRNA introducedinonepartof thebodycanbespread toothercellsinthe body. Ithasbeenshownthat twoproteins,SID-1and SID-2,areinvolvedinthis transmission. Dipanjan’sResearchSeminar AdiscussionofhowcellusemiRNAstochoose betweentheHRandNHEJdouble-strandbreak repairpathwaysinresponsetoDSB CoreConcepts Smallan)senseRNAsareusedtoregulatetheexpressionoftarget mRNAs.Thesean)senseRNAsaretypcially~21–23ntinlength,but someareabitlarger. TherearethreeclassesofthesmallmiRNAs,siRNAsandpiRNAs. miRNAsareendogenoussmallRNAmoleculesthatarederivedfrom RNApolIImiRNAgenes(usuallypresentinintergenicspace)andthat caninteractwithtargetmRNAswithfullcomplementaritytocause degrada)onorwithsomemismatchtocauserepression. siRNAsmayoriginatefromendogenousorexogenoussources.The siRNAs.siRNAscausethedegrada)onoftheirtargetmRNAs. CoreConcepts DroshaisneededfortheprocessingofmiRNAsbutnot siRNAs. DicerandArgonauteareneededfortheprocessing/ac)vityof bothmiRNAsandsiRNAs. miRNAtargetscanbeiden)fiedusingthePAR-CLIP,HITS-CLIP oriCLIPtechniqueswhichdependonthecrosslinkingofthe RISCcomplextothetargetmRNAtodeterminethebinding site. Insome,butnotallorganisms,smalldsRNAscanmove betweencellsgivingrisetosystemicRNAinterference.