Download Small AnDsense RNAs and RNA Interference

SmallAn(senseRNAsand
RNAInterference
BCMP200
November20,2015
BCMP200 - The Central Dogma
6. Protein translation
(Melissa Leger-Abraham & Dipanjan Chowdhury)
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5. Transcription/Processing
(Stirling Churchman)
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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)
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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.