UniversityofNaturalResourcesandAppliedLifeSciences
DepartmentofAppliedGeneticsandCellBiology
TheputativeRNAsilencingprotein ERL1isinvolvedinchloroplast
ribosomalRNAprocessinginplants
Doctoratethesis submittedby DIJuttaMariaHelm March,20111
2
Declaration
IherebydeclarethatIhavewrittenthisthesisindependently.Allresultspresentedin
theresultsectionhavebeenobtainedbymyownwork,probesandplasmidshave
beensharedbyseverallabmembers.Relevantresultsobtainedbycoworkersare
onlypresentedinthesupplementaryresultsectionwithpropercitations.Allintellec
tualpropertyusedforthepreparationofthisworkhasbeencitedproperly.
TheresultsobtainedfromtransgenicN.benthamianaplantsmisexpressingERL1in
cludingcrossing,lightmicroscopyaswellasphotosyntheticanalysis,andrRNA
cloningexperimentshavebeendescribedanddiscussedbyHeikoSchumacherin
2009.However,allexperimentalproceduresandplantmaintenancehavebeenexe
cutedbyme.Thechloroplasticlocalization,analysisofchloroplastrelatedtranscripts
andelectronmicroscopyexperimentshavebeenexecutedindependentlybyHeiko
Schumacherandmetopreparereplicatesofthefindings. March2011,Vienna
DIJuttaMariaHelm
3
4
AcknowledgementsThisworkwouldnothavebeenpossiblewithoutthesupportofvariouspeople.Es
peciallyIwouldliketothank
DrKritonKalantidisforbeingavaluableteacherandstillleavingmeagreatfree
domformyworkanddevelopment.
DrMarieTheresHauserforgivingmetheopportunitytolearninherlabandfor
allthesupportandgoodadvicefromfar.
allmembersofthePlantMolecularBiologyLaboratoryinCreteformakingita
placetoremember,Iwillkeepinmindthenicetimeswehad.
themembersofthePlantDevelopmentalGeneticsLaboratoryinViennaforwel
comingmesofriendlybeforeandaftermystayinGreece.
SergiaTzortzakaki,EvaPapadogiorgaki,KosmasHaralampidisandmystudents
Andreas,Evguenia,Giorgos,KalliaandRitsawhoallprovidedvaluablehelp.
HeikoSchumacherforteachingmealessonforlife.
allthepeoplewhohadbeenthereandwentthewayinCretealongwithmefora
while,forsomeIamespeciallyproudtocallthemfriends.
myfriendsbackathomeandaroundtheworldwhodidnotforgetmeevenfroma
distance,forkeepingcontactbySkype,email,etc.andforspendingtheirholidays
withme.
myparentsforlettingmegoandforgivingmethefeelingthatIcanalwayscount
onthemincaseofneed,despiteanylocaldistancebetweenus.
mybrotherAndreasforconstantlysolvingallmycomputerproblemsinnotime.
mysisterMartinaforalwaysbeingthere!
HaraldZwillingforbeingHaraldZwilling!5
6
AbstractDuringevolutioneukaryoteshaveacquiredasystemusingsmallRNAmolecules
(siRNAs)asnegativeregulatorsofendogenousandexogenousRNAsequences
calledRNAinterferenceorRNAsilencing.Thismechanismisalsousedinthede
fenseagainstpathogensthathavethereforedevelopedseveralstrategiestocounter
actitbyexpressionofviralsuppressorsofsilencing(VSRs).Aputativeendogenous
suppressorofsilencingmightbethe35exonucleaseERI1(enhancedRNAi)andits
homologuesinvariousspecies,whichspecificallybindanddegradesiRNAs.Re
centlyanadditionalconservedroleofERI1homologueshasbeenidentified,where
theycatalyzethefinalstepin5.8SrRNAprocessing.
InthisworktheplanthomologuetermedERL1(ERI1LIKE1)isanalyzed.Thepro
teinlocalizestothechloroplastandfailstoexhibitanyRNAsilencingsuppressor
activity.ThisfindingisnotsurprisinginthiscontextsinceRNAsilencingisrestricted
tothecytoplasm.AlsotheDrosophilamelanogasterhomologuedoesnotpossessthis
functionsuggestingtwofunctionallydistinctgroupsofERI1homologues.Constitu
tiveoverexpressionofERL1intransgenicNicotianabenthamianaplantsmanifestedin
variegatedphenotypescharacterizedbyableachingoftheplants.Theresultcouldbe
confirmedinArabidopsisthalianaplantsoverexpressingERL1.Theobservedpheno
typesreachedfrompalegreen,yellow,mosaicgreenandwhitetocompletelossof
chlorophyll.Theseverityofthebleachingcorrespondedtotheexpressionlevelsof
ERL1.Thetransgenicplantlinesshowedmorphologicalandtranscriptionalaltera
tionsreminiscentofreporteddefectsinchloroplasticribosomalRNAbiogenesis.In
deeditcouldbeshownthat5SrRNAisdownregulatedaftertransientandconstitu
tiveoverexpressionofERL1andelongatedbytwonucleotidesatits3endinafrac
tionoftheanalyzedsamples.Aputativeribonucleasehasbeenproposedearlierto
assistinchloroplasticrRNAprocessinginamutantbackgroundof
RIBONUCLEOTIDEREDUCTASE1(RNR1)inArabidopsisthalianawhichmaybe
constitutedbyERL1.Inadditionafractionof16SrRNAhasbeenelongatedbyone
nucleotideinArabidopsisinsertionmutantssuggestingalsoafunctioninmaturation
ofthischloroplasticrRNA.7
8
ZusammenfassungImLaufederEvolutionhabenEukaryonteneinSystemerworben,daskleineRNA
Molekle(siRNAs)alsnegativeRegulatorenvonendogenenundexogenenRNA
Sequenzenverwendet.DiesesogenannteRNAInterferenzoderRNASilencingwirkt
auchalsImmunsystemgegenKrankheitserreger,deshalbhabendieseverschiedene
Strategienentwickelt,umdiesemMechanismusdurchdieAusbildungvonviralen
SilencingSuppressoren(VSR)entgegenzuwirken.Einmutmalichereukaryotischer
endogenerSilencingSuppressorknntedie35ExonukleaseERI1sein,diespezi
fischsiRNAsbindenundabbauenkann.AuerdemkatalysiertERI1denletzten
SchrittbeiderReifungvon5.8SribosomalerRNA.
IndieserArbeitwurdedasPflanzenHomologERL1(ERI1like1)analysiert.Das
ProteinwirdinChloroplastengeschleustundbesitztkeineRNASilencing
SuppressorAktivitt.DiesesErgebnisistinsofernnichtberraschend,daRNA
SilencingaufdasZytoplasmabeschrnktist.DashomologeDrosophilaProteinzeigt
ebenfallsdiesesVerhalten;wahrscheinlichexistierenzweiunterschiedlicheGruppen
vonERI1HomologeninEukaryoten.berexpressionvonERL1intransgenenTa
bakundArabidopsisPflanzenresultierteinvielfltigenPhnotypen,diedurchein
BleichenderPflanzengekennzeichnetwaren:esreichtevonblassgrn,bergelb,
grnundweigesprenkeltbiszukomplettemChlorophyllVerlust.DerSchwere
graddesPhnotypsentsprachderberexpressionvonERL1.DietransgenenLinien
zeigtenmorphologischeundtranskritptionelleVernderungen,dieanbereitsbe
schriebeneMngelinderReifungribosomalerRNAsinChloroplastenerinnern.Es
konntetatschlichgezeigtwerden,dassdie5SrRNALevelsnachderberexpression
vonERL1verringertsindundam3EndeeinehufigeVerlngerungumzwei
Nukleotidebesaen.ERL1knntediemutmalicheRibonukleasesein,diederRibo
nukleotidreduktase1(RNR1)beiderVerarbeitungvonChloroplastenrRNAassis
tiert.Auerdemzeigteauchdie16SrRNAinArabidopsisMutanten,beidenenERL1
unterdrcktwar,teilweiseVerlngerungenumeinNukleotid;eventuellbesitztERL1
aucheineFunktioninderReifungdieserrRNA. 9
10
Abbreviations% C ME m M Ci g J L 3 3hExo 5 7mG A A.thaliana/At
A.tumefaciens aa ACS ADAR Ago/AGO APconjugate APS ARC Arg Asp ATP
RLI2 attsites Aub BLAST bp BSA C C.elegans/Ce CBC cDNA Chp1 Percent
DegreesCelsius Mercaptoethanol Micrometer(s) Micromolar
Microcurie(s) Microgram(s) Microjoule(s) Microlitre(s) 3prime
3primehistoneexonuclease 5prime 7methylguanosine Adenosine
ngstrm(s) Arabidopsisthaliana Agrobacteriumtumefaciens Aminoacid(s)
Acetosyringone AdenosinedeaminasethatactsonRNA Argonauteprotein
Alkalinephosphataseconjugate Ammoniumpersulfate
ACCUMULATIONANDREPLICATIONOF CHLOROPLASTS Arginine Aspartate
Adenosinetriphosphate RNASELINHIBITOR2 Attachmentsites Aubergine
BasicLocalAlignmentSearchTool Basepair(s) Bovineserumalbumin
Cytosine Caenorhabditiselegans Capbindingcomplex ComplementaryDNA
ChromodomainproteininS.pombe11
AbbreviationsCHS Ci CLP CLSY1 cm cm CTAB Cterminus D D.rerio
dATP DCL14 DCP2 DCR DCR1/2 dCTP ddC DDL DGCR8 dGTP DMSO DNA DnaQ
DNase dNTP dpi DRB4 DRD1 DRM2 dsDNA dsRBD dsRNA DTT dTTP Duf E
E.coli e.g. CHALCONESYNTHASE Curie(s) CASEINOLYTICPROTEASE CLASSY1
Centimeter(s) Squarecentimeter(s) Cetyltrimethylammoniumbromide
Carboxyterminus Aspartate Daniorerio Deoxyadenosinetriphosphate
DICERLIKE14 Decappingprotein2 Dicer DicerRelated1/2
Deoxycytosinetriphosphate Dideoxycytosine DAWDLE
DiGeorgesyndromechromosomalregion Deoxyguanosinetriphosphate
Dimethylsulfoxide Deoxyribonucleicacid
DNApolymeraseIIIepsilonsubunit Deoxyribonuclease
Deoxynucleosidetriphosphate Dayspostinfection/dayspostinfiltration
DOUBLESTRANDEDRNABINDINGPROTEIN4
DEFECTIVEINRNADIRECTEDDNAMETHYLATION1
DOMAINSREARRANGEDMETHYLTRANSFERASE2 DoublestrandedDNA
Doublestrandedribonucleicacidbindingdomain DoublestrandedRNA
Dithiothreitol Deoxythymidinetriphosphate Domainofunknownfunction
Glutamate Escherichiacoli exempligratia12
D.melanogaster/Dm Drosophilamelanogaster
AbbreviationsEDTA eIF4E ELSS ERI1/Eri1 ERL1 EST etal. EtBr
EXOIII fmol For/F FoRTH FRY1 G g GFP GW182 h H H.sapiens H2O HCPro
HEN1 HEPES HRP HYL1 i.e. IDN2 IGN IMBB IPTG K kb KD kDa KO KTN kV
Ethylenediaminetetraaceticacid Eukaryoticinitiationfactor4E
Extensivelocalsilencingspread EnhancedRNAi1 ERI1LIKE1
Expressedsequencetag andothers Ethidiumbromide
Exonuclease(III)domain Femtomole Forward
FoundationforResearch&TechnologyHellas
3(2),5BISPHOSPHATENUCLEOTIDASE/INOSITOL POLYPHOSPHATE1PHOSPHATASE
Guanosine;Glycine Gramm(s);relativecentrifugalforce
GreenFluorescentProtein Glycinetryptophanrepeatprotein182 Hour(s)
Histidine Homosapiens Water Helpercomponentproteinase HUAENHANCER1
4(2hydroxyethyl)1piperazineethanesulfonicacid Horseradishperoxidase
HYPONASTICLEAVES1 Idest INVOLVEDINDENOVOMETHYLATION2
Intergenicnoncodingtranscript
InstituteofMolecularBiology&Biotechnology
IsopropylS1thiogalactopyranoside Lysine Kilobase(s) Knockdown
Kilodalton(s) Knockout KATANIN Kilovolt(s)13
AbbreviationsL LB let7 lin4/14 LOQS M M.musculus mA MES Met mg
min miRNA miRNA* mL mM mm MMA MOPS mRNA MS N N.crassa N.tabacum/Nt
NADP NAT natsiRNA NCBI NEP ng NiNTA nm NPC NRPD1a/b nt Nterminus
O.sativa/Os OD600 Liter(s)
Lysogenybroth/Luriabroth/LuriaBertanibroth LEThal7
AbnormalcellLINeage4/14 Loquacious Molar Musmusculus Milliampere(s)
2(NMorpholino)ethanesulfonicacid Methionine Milligram(s) Minute(s)
MicroRNA MicroRNApassengerstrand Milliliter(s) Millimolar
Millimeter(s) MS/MES/acetosyringone
3(NMorpholino)propanesulfonicacid MessengerRNA Murashige&Skoog
Normal Neurosporacrassa Nicotianatabacum
Nicotinamideadeninedinucleotidephosphate Naturalantisensetranscript
NaturalantisensetranscriptderivedsiRNA
NationalCenterforBiotechnologyInformation Nucleusencodedpolymerase
Nanogram(s) NickelNitriloaceticacid Nanometer(s) Nuclearporecomplex
NUCLEARRNAPOLYMERASED1A/B Nucleotide(s) Aminoterminus Oryzasativa
Opticaldensityat600nm14
N.benthamiana/Nb Nicotianabenthamiana
AbbreviationsOH P.trichocarpa/Pt PAA PAGE PAP PAZ Pbody PCMP PCR
PEP pH PIPES piRNA Piwi PLMVd pmol PNK Pol PolII/IV/V PPR PPV
premiRNA PRG1 primiRNA PSRP1 PSTVd PTGS QDE2 QIP qPCR Qrich R2D2
RACE Ran rasiRNA RBCL RdDM RDR16/RdRP Rev/R Hydroxyl
Populustrichocarpa Polyacrylamide Polyacrylamidegelelectrophoresis
polyApolymerase Piwi/Argonaute/Zwille Processingbody
Plantcombinatorialandmodularprotein Polymerasechainreaction
Plastidencodedpolymerase pondusHydrogenii/potentiaHydrogenii
PiperazineN,Nbis(2ethanesulfonicacid) PiwiinteractingRNA
Pelementinducedwimpytestes Peachlatentmosaicviroid Picomole(s)
Polynucleotidekinase Polymerase RNApolymeraseII/IV/V
Pentatricopeptiderepeat Plumpoxvirus PrecursormiRNA
Piwirelatedgene1 PrimarymiRNA PHLOEMSMALLRNABINDINGPROTEIN1
Potatospindletuberviroid Posttranscriptionalgenesilencing
Quellingdeficient2 QDE2interactingprotein QuantitativePCR
Glutaminerich TwodsRNAbindingdomains,associatedwithDCR2
RapidamplificationofcDNAends Rasrelatednuclearprotein
RepeatassociatedshortinterferingRNA
Ribulosebisphosphatecarboxylase,largechain
RNAdirectedDNAmethylation RNADEPENDENTRNAPOLYMERASE16 Reverse15
AbbreviationsRf rgsCaM RISC RITS RNA RNAi RNase RNasin RNR1 RPB1
rpm RPOB RRF rRNA RT RuBisCo s/sec S S.bicolor S.pombe S.purpuratus
SAF SAP SDE3 SDN SDS SE SGS3 SINE siRNA siRNase SLBP SLSS snoRNA
Snp snRNA snRNP SOB sp. Nuclearrestorer
REGULATOROFGENESILENCINGCALMODULINLIKE RNAinducedsilencingcomplex
RNAinducedtranscriptionalsilencing Ribonucleicacid RNAinterference
Ribonuclease RNaseinhibitor RIBONUCLEOTIDEREDUCTASE1
RNApolymeraseIIlargesubunit Rotationsperminute
RNAPOLYMERASESUBUNITBETA RNAdirectedRNApolymerasefamily
RibosomalRNA Reversetranscription;Roomtemperature
Ribulose1,5bisphosphatecarboxylase/oxygenase Second(s)
Svedberg(sedimentationcoefficient) Sorghumbicolor
Schizosaccharomycespombe Strongylocentrotuspurpuratus
Scaffoldattachmentfactor SAFA/B,AcinusandPIAS SILENCINGDEFICIENT3
SMALLRNADEGRADINGNUCLEASE Sodiumdodecylsulfate SERRATE
SUPPRESSOROFGENESILENCING3 Shortinterspacedelement
SmallinterferingRNA Smallinterferingribonuclease
Stemloopbindingprotein Shortrangelocalsilencingspread
SmallnucleolarRNA Snipper SmallnuclearRNA
Smallnuclearribonucleoprotein Superoptimalbroth Species16
AbbreviationsSPT5 SSC ssDNA ssRNA T TAE Taq TAS3 tasiRNA TBE TE
TEM TEMED TIC/TOC Tm TNRC6A,B,C TRBP Tris tRNA TRV U UBA UTR UV V
V.vinifera v/v VCS VSR W W w/v X.laevis XGal xray XRN14 Y Z.mays/Zm
SuppressorofTyinsertion5 Sodiumchloride/sodiumcitratebuffer
SinglestrandedDNA SinglestrandedRNA Thymine Tris/Acetate/EDTA
Thermusaquaticus TRANSACTINGSIRNA3 TransactingsiRNA
Tris/Borate/EDTA TrisEDTA Transmissionelectronmicroscopy
Tetramethylethylenediamine
Transloconattheinner/outerenvelopemembraneofchloro plasts
Meltingtemperature Trinucleotiderepeatcontaining6A,B,C
TransactivatingresponseRNAbindingprotein
Tris(hydroxymethyl)aminomethan TransferRNA Tobaccorattlevirus
Unit(s) Ubiquitinassociated Untranslatedregion Ultraviolet Volt(s)
Vitisvinifera Volumepervolume VARICOSE Viralsuppressorofsilencing
Watt(s) Tryptophan Weightpervolume Xenopuslaevis
5Bromo4chloro3indolylDgalactopyranoside Roentgenrays
EXORIBONUCLEASE14 Tyrosine Zeamays17
18
TableofContent1.
Introduction.....................................................................................................................
25 1.1 RNAmoleculesandtheirlifebetweenDNAandprotein
.............................. 25 1.2
RNAsilencingnewrolesfortheintermediate...............................................
27 1.2.1
siRNAmediatedgenesilencing......................................................................
29 1.2.2
miRNAsmodulatetheexpressionofendogenoussequences....................
32 1.2.3
ThepiRNApathwayprotectsthegermlinefromtransposonactivity...... 36
1.2.4
PlantspossessahighdiversityofsiRNAmolecules....................................
38 1.2.4.1 CisactingsiRNAsmediatechromatinsilencinginplants
...................... 41 1.2.4.2
TransactingsiRNAs......................................................................................
42 1.2.4.3 NaturalantisensesiRNAs
............................................................................
44 1.2.5 PeculiaritiesofplantmiRNAs
.........................................................................
45 1.2.6
SpreadingofRNAsilencinginplantsresemblesanimmunesystem....... 46
1.2.7
ViralstrategiestosuppressRNAsilencinginplants...................................
49 1.2.8
RepressingtherepressorsendogenoussuppressorsofRNAsilencing.. 50
1.2.9 ERI1isanexampleforanendogenoussuppressorofRNAsilencing.....
51 1.2.10 ERI1LIKE1,theplanthomologueofERI1
................................................ 56 1.3
Chloroplasts
...........................................................................................................
61 1.3.1 Photosynthesis
...................................................................................................
62 1.3.2 SpecificitiesofchloroplasticRNAs
.................................................................
64 1.4 Thesisobjectives
....................................................................................................
66 2. MaterialsandMethods
..................................................................................................
69 2.1 Materials
.................................................................................................................
69 2.1.1 Instruments
........................................................................................................
69 2.1.2 Chemicals
...........................................................................................................
71 2.1.3 Consumables&kits
..........................................................................................
74 2.1.4 Solutions
.............................................................................................................
76 2.1.5 Others
..................................................................................................................
84 2.1.5.1
Enzymes..........................................................................................................
84 2.1.5.2 Sizemarkers
...................................................................................................
85 2.1.5.3
Bacterialstrains..............................................................................................
85 2.2 Methods
..................................................................................................................
85 2.2.1 Standardmolecularbiologymethods
............................................................ 85
2.2.1.1
Cultivation......................................................................................................
85 2.2.1.2 Chemicallycompetentcells
.........................................................................
86 2.2.1.3 Transformation
..............................................................................................
86 2.2.1.4
Plasmidpreparation......................................................................................
87 2.2.1.5
Agarosegel.....................................................................................................
88 2.2.1.6 Gelextraction
.................................................................................................
88 2.2.1.7 Digest
..............................................................................................................
88 2.2.1.8 Ligation
...........................................................................................................
88 2.2.2 Planttransformationtechniques
.....................................................................
88 2.2.2.1
Plantcultivation.............................................................................................
88 2.2.2.2
LeafDisctransformation..............................................................................
8919
TableofContent2.2.2.3
FloralDip........................................................................................................
89 2.2.2.4
Agroinfiltration..............................................................................................
89 2.2.3 Southernanalysis
..............................................................................................
90 2.2.3.1
DNAextraction..............................................................................................
90 2.2.3.2 Southernanalysis
..........................................................................................
90 2.2.3.3 Capillaryblot
.................................................................................................
91 2.2.4 Northernanalysis
..............................................................................................
91 2.2.4.1 RNAextraction
..............................................................................................
91 2.2.4.2
Denaturingagarose/formaldehydegels.....................................................
91 2.2.4.3 PAGEnorthern
..............................................................................................
92 2.2.4.4
Semidryblot..................................................................................................
92 2.2.5
Hybridization.....................................................................................................
92 2.2.5.1 Randomprimedlabelling
............................................................................
92 2.2.5.2 Endlabelling
..................................................................................................
93 2.2.5.3 Hybridization,washesanddeveloping
..................................................... 93 2.2.6
Westernanalysis................................................................................................
93 2.2.6.1 Proteinextraction
..........................................................................................
93 2.2.6.2 SDSPAGE
......................................................................................................
93 2.2.6.3 Electroblot
.....................................................................................................
94 2.2.6.4
Westerndetection..........................................................................................
94 2.2.7 rRNAcloning
.....................................................................................................
94 2.2.7.1 SelfLigation
...................................................................................................
94 2.2.7.2 LinkerLigation
..............................................................................................
94 2.2.7.3 ReverseTranscription(RT)
..........................................................................
95 2.2.7.4
PolymeraseChainReaction(PCR)..............................................................
95 2.2.8
RapidAmplificationofcDNAends(RACE).................................................
96 2.2.9 QuantitativerealtimePCR(qPCR)
................................................................ 96
2.2.10 Protoplasts
..........................................................................................................
96 2.2.11 Preparationformicroscopicanalysis
............................................................. 97
2.2.12
Fluorescencemeasurement..............................................................................
97 3. Results
..............................................................................................................................
99 3.1 Localization
............................................................................................................
99 3.2
RACE.....................................................................................................................
101 3.3 TransgenicNicotianabenthamianaplants
...................................................... 103 3.3.1
KnockdownofERL1......................................................................................
103 3.3.2 OverexpressionofERL1
.................................................................................
104 3.3.2.1 Microscopy
...................................................................................................
107 3.3.2.2 EffectofERL1overexpressiononchloroplastmRNAs
......................... 110 3.3.2.3
EffectofERL1overexpressiononthephotosyntheticapparatus......... 112
3.4
TransgenicArabidopsisthalianaplants...........................................................
115 3.4.1
KnockdownofERL1......................................................................................
115 3.4.2 OverexpressionofERL1
.................................................................................
117 3.5 EffectofERL1onsilencing
................................................................................
11920
TableofContentCrosses
..............................................................................................................
119 LNA159
.............................................................................................................
120 3.6 EffectofERL1onribosomalRNA
....................................................................
121 3.6.1
rRNAblots........................................................................................................
121 3.6.2 Linkerligations
................................................................................................
123 3.6.2.1 Effecton5.8SrRNA
....................................................................................
123 3.6.2.2 EffectonchloroplasticrRNAs
...................................................................
124 4. Discussion
......................................................................................................................
129 4.1 ImplicationsofplantERL1inRNAsilencingprocesses
............................... 129 4.2
InvolvementsofERL1inchloroplastmetabolism..........................................
131 4.3
SeverephenotypicalterationsafteroverexpressionofERL1suggestanin
volvementinchloroplastdevelopment
...........................................................................
133 4.4
ERL1isinvolvedinchloroplasticribosomalRNAprocessing.....................
135 4.5 Conclusions
..........................................................................................................
140 5. References
......................................................................................................................
143 6. Supplements
..................................................................................................................
171 6.1 Supplementarymethods
....................................................................................
171 6.1.1 Virus/viroidinfectionsinNicotianasp.plants
............................................. 171 6.1.2
Invitrotranscription........................................................................................
171 6.1.3
PurificationofrecombinantERL1protein...................................................
171 6.1.4
InvitroassaysforrecombinantERL1protein.............................................
172 6.2
Supplementaryresults........................................................................................
172 6.2.1 PSTVdderivedsiRNAsaresuppresseduponERL1overexpression
..... 173 6.2.2
ERL1failstoaffectRNAsilencinginAgrobacteriumcoinfiltrationassays
174 6.2.3 ExogenouslyinducedsilencingspreadmaybesuppressedafterERL1
overexpression
..............................................................................................................
176 6.2.4
ERL1overexpressingplantsarehypersensitivetowardsviralinfection 177
6.2.5
InvitroexperimentswithERL1.....................................................................
178 6.2.6
PreparationofaNbERL1suppressionconstructandanalysisofitseffects
aftertransientandtransgenicexpression
.................................................................
179 6.3
Oligonucelotides..................................................................................................
181 6.4
Vectormaps..........................................................................................................
183 6.4.1
AtERL1GFP....................................................................................................
183 6.4.2 AtleaderGFP
..................................................................................................
183 6.4.3 AtERL1over
...................................................................................................
184 6.4.4 NtERL1hp
......................................................................................................
184 6.5
Sequences..............................................................................................................
185 6.5.1
Newlyidentifiedsequences...........................................................................
185 6.5.2 Publishedsequencesusedforinsilicoanalysisandprimerdesign
......... 186 6.6 Curriculumvitae(March,2011)
........................................................................
19621
3.5.1 3.5.2
TableofContentListofFigures:
Figure1.1:Ghildiyal&Zamore,2009.................................................................................
28 Figure1.2:MacReaetal.,2006
.............................................................................................
28
Figure1.3:Argonauteproteins............................................................................................
29
Figure1.4:Okamuraetal.,2007...........................................................................................
31
Figure1.5:Carthew&Sontheimer,2009............................................................................
35 Figure1.6:Zamore,2010
......................................................................................................
38
Figure1.7:Ghildiyal&Zamore,2009.................................................................................
40 Figure1.8:Matzkeetal.,2009
..............................................................................................
39 Figure1.9:Allen&Howell,2010
........................................................................................
43 Figure1.10:Kalantidisetal.,2008
.......................................................................................
47
Figure1.11:Cheng&Patel,2004.........................................................................................
51
Figure1.12:AlignmentofpublishedERI1homologs.....................................................
56
Figure1.13:AlignmentofERL1homologuesinvariousplantspecies.........................
57 Figure1.14:Winteretal.,2007
.............................................................................................
57 Figure1.15:Winteretal.,2007
.............................................................................................
58 Figure1.16:Campbelletal.,1996
........................................................................................
63 Figure1.17:Sternetal.,2010
................................................................................................
64 Figure3.1:LocalizationofplantERL1
.............................................................................
100
Figure3.2:AlignmentsofNicotianasp.ERL1sequence.................................................
101
Figure3.3:AnalysisofpresumableERL1suppressorplants........................................
101
Figure3.4:AnalysisofNicotianabenthamianaplantsoverexpressingERL1
............... 104
Figure3.5:TEManalysisofphenotypesofNicotianabenthamianaplantsoverexpress
ingERL1................................................................................................................................
108
Figure3.6:PhenotypicanalysisbylightmicroscopyofNicotianabenthamianaplants
overexpressingERL1
..........................................................................................................
107 Figure3.7:Northernanalysisofselectedchloroplastrelatedgenes
........................... 109
Figure3.8:DeterminationofphotosyntheticparametersinNicotianabenthamiana
plantsoverexpressingERL1
..............................................................................................
111
Figure3.9:CharacterizationofselectedpubliclyavailableArabidopsisthalianaERL1
knockoutplants
..................................................................................................................
116
Figure3.10:AnalysisofArabidopsisthalianaplantsoverexpressingERL1..................
118
Figure3.11:EffectofplantERL1ondifferentmoleculesoftheRNAsilencingappara
tus
..........................................................................................................................................
117
Figure3.12:NorthernanalysisofchloroplasticribosomalRNAsandcytosolic5.8S
rRNAfollowingoverexpressionofERL1
........................................................................
122
Figure3.13:Alignmentofcytosolic5.8SrRNAfromNicotianabenthamianaplants
overexpressingERL1
..........................................................................................................
123
Figure3.14:AlignmentofchloroplasticrRNAsofArabidopsisthalianaandNicotiana
benthamianaplantsmisexpressingplantERL1
................................................................
126
Figure4.1:SecondarystructuresofchloroplasticrRNA(predictedbyRNAfold;Gru
beretal.,2008).
.....................................................................................................................
135
22
TableofContentFigure6.1:ComparativeagroinfiltrationtimecourseinsystemicallyPSTVdinfected
tobacco.
.................................................................................................................................
174
Figure6.2:AgrobacteriumcoinfiltrationassaysinN.benthamianaline16CtotestERL1
forRNAsilencingsuppressoractivity
.............................................................................
175
Figure6.3:SilencingoftheERL1phenotypeinducedbyagroinfiltration..................
177
Figure6.4:ERL1overexpressorplantsarehypersensitivetowardsinfectionbyPPV
................................................................................................................................................
178 Figure6.5:AnalysisofthesuppressionofERL1inNicotianabenthamiana.
................ 180 ListofTables:
Table1.1:predictedlocalizationofplantERL1homologs
.............................................. 58
Table3.1:SegregationofNicotianabenthamianaT2plantlinestransformedwitha
hairpinconstructdesignedfordownregulationofERL1..............................................
104
Table3.2:SummaryofsegregationandphenotypicpatternofNicotianabenthamiana
T1plantlinesoverexpressingERL1
.................................................................................
107
Table3.3:SummaryofcharacteristicsofArabidopsisthalianaERL1knockdownplant
lines........................................................................................................................................
117
Table3.4:SummaryofsequencealterationsinchloroplasticrRNAafterERL1misex
pressioninNicotianabenthamiana(Nb)andArabidopsisthaliana(At)plants...............
125
23
24
1. Introduction
SinceitsdiscoveryDNAhasbeenconsideredasaverystablemoleculewiththeabil
itytoencodeforaninfinitenumberofproteins,whicharefactorswithanunlimited
versatilityforcatalyticprocesses.TheintermediaryforminthisprocessisRNA,lack
ingboththestabilityandtheflexibilityoftheabovementionedkeyplayers.
Thisprocess,however,isatypicalchickenandeggparadox,sincenucleicacidsare
requiredforproteinsynthesiswhileproteinsarenecessaryfornucleicacidproduc
tion.OnehypothesistryingtoovercomethisparadoxconsidersRNAasthemolecule
bothstoringgeneticinformationandcatalyzingchemicalreactionsinprimitivecells
ofthissocalledRNAworld.DNAhasthendevelopedasamoresuitablemolecule
forstoragesinceitsstabilityincreasedthemaximumsizeofthehereditarymolecules.
Thismighthavehappenedinparallelwiththenecessityforstorageofincreased
amountsofgeneticinformationafteraccumulationofadditionalproteincatalysts
(Albertsetal.,2008).
1.1
RNAmoleculesandtheirlifebetweenDNAandproteinmRNA(messengerRNA)isthefirstmoleculeinvolvedintheinformationflowfrom
DNAtoprotein.AnRNApolymeraseguidesthetranscriptionofadefinedgenomic
stretchbycatalyzingtheformationofphosphodiesterbondsbetweenribonucleo
tides.Thepolymeraserecognizesacertainpromotersequencewhereitbindstothe
DNAstrand.ItsynthesizesasinglestrandedmRNAmoleculein5to3direction
andstopstheelongationwhenreachingtheterminatorsequence.
Thisgeneralprocessismorecomplexineukaryotes.TheypossessthreeRNApoly
meraseswithPolIIbeingresponsibleformostproteincodinggenesandPolIand
PolIIItranscribinggenesforotherRNAmolecules.Thetranscriptioninitiationre
quirestheactionofgeneraltranscriptionfactorsfacilitatingthebindingtothepro
motersequenceandformingatranscriptioninitiationcomplex.Thesynthesized
25
1.IntroductionmRNAstrandismodifiedatits5endbyadditionofacapof7methylguanosine
(7mG)whichbindsaproteincomplexcalledcapbindingcomplex(CBC).
Eukaryoticproteincodinggenestretchescontainexpressedsequences(exons)and
noncodinginterveningsequences(introns).Theremovalofthelatterfromthepre
cursormRNAstrandisperformedbythespliceosome.Theycontainanothertypeof
RNA,thefivesmallnuclearRNAs(snRNAsU1,U2,U4,U5andU6)togetherwithat
leastsevenproteinsformingthesmallnuclearribonucleoproteincomplex(snRNP).
Theintronicsequenceiscircularizedintoastructurecalledlariat,excisedandthe
endsoftheexonsarethenjoinedtogethertoacontinuouscodingsequence.
ProteinfactorsaccompanyingthetranscribedmRNAmoleculerecognizethe3end
ofthestrandandleadtoitscleavage.ThepolyApolymerase(PAP)thenaddsap
proximately200adenosinenucleotides,thefinallengthofthispolyAtailisdeter
minedbypolyAbindingproteins.
AberrantRNAsaredegradedbythenuclearexosomeconsistingof3to5RNAex
onucleases.OnlysplicedmaturemRNAwitha5capanda3polyAtailareex
portedtothecytoplasmthroughthenuclearporecomplexes(NPCs).
ThenucleotidesequenceofthemRNAistranslatedintoaminoacidsbycodonscon
sistingofthreeconsecutivenucleotides.ThenecessaryRNAmoleculesforthisproc
essaretRNAs(transferRNAs)whichareextensivelystructuredLshapedadaptor
moleculeswithunusualbasesorganizedintothreesinglestrandedloops.Oneof
themcontainstheanticodonwhichbindstothemRNAsequence.Therelevantamino
acidisconnectedtothe3endofthetRNAmoleculebyaminoacyltRNAsyntheta
ses.Thepolypeptidechainissynthesizedbythestepwiseadditionofaminoacidsto
itsCterminalendthatisactivatedbythebindingtoatRNAmolecule(whichisthen
calledpeptidyltRNA).
Theabovedescribedprocesstakesplaceintheribosomes,acatalyticmachinerycon
sistingofribosomalproteinsandribosomalRNAs(rRNA).Theyconstituteupto80
%ofthetotalRNAofcellsandareencodedinmultiplerRNAgeneswhichareoften
arrangedintandemsandtranscribedbyPolI.26
1.IntroductionLongrRNAprecursormoleculesareextensivelymodifiedatpositionsthatarespeci
fiedbyguideRNAs,alsoknownassmallnucleolarRNAs(snoRNAs).Theyareoften
encodedinandfurtherexcisedfromintronicsequences,especiallyfromribosomal
proteins.ThenucleolusisadistinctstructureinthenucleuswhererRNAprocessing
andribosomeassemblytakeplace.Thetworibosomalsubunitsarethenexportedto
thecytoplasmwheretheyjointoformthematureribosome.Theprokaryotic70Sri
bosomeconsistsofa50Ssubunit(with5Sand23SrRNA)anda30S(with16SrRNA).
Theeukaryotic80Sribosomeconsistsofa60Ssubunit(with5S,5.8Sand23SrRNA)
anda40Ssubunit(with18SrRNA).Thematureribosomecontainsfourbindingsites
forRNA,oneformRNAandthreefortRNA:thelatterareboundattheAsite,the
aminoacidsarethenconnectedtogetheratthePsiteandtheemptytRNAfinally
getsreleasedattheEsite.ThemajorcatalyticreactionsarecarriedoutbytherRNA
moleculeswhichcanbeconsideredasribozymes(Albertsetal.,2008).
1.2
RNAsilencingnewrolesfortheintermediateFormanyyearsthecentraldogmaofmolecularbiologywaspostulatedbyFrancis
Crick,statingthatthegeneticinformationonlyflowsfromnucleicacidstoproteins.
TheimportanceofRNAhadbeenboostedsignificantlybythefinding,thatdouble
strandedRNAisatriggerofgenesilencingandthereforeprovidinganegativefeed
backmechanismwhichisindependentofproteinsynthesis(Fireetal.,1998).
ThefirstobservationofanRNAsilencingmechanismwasreportedin1928when
newlyemergingleavesofTRVinfectedNicotianatabacumplantswerefoundtobe
freeofinfectionsymptoms(Wingard,1928).Manyotherimportantobservations
whichfinallyleadtothediscoveryoftheRNAsilencingmechanismhadalsobeen
madeinplants:afterexpressionofantisenseRNAthetobacconopalinesynthasewas
inhibited(Rothsteinetal.,1987).Thisphenomenoncalledcosuppression,wherean
endogenousgeneisdownregulatedafterstrongoverexpressionofthesamese
quence,wasdiscoveredbytwogroupstryingtoincreasethepurplepigmentationof
petunia.TheoverexpressionofCHALCONESYNTHASE(CHS),anenzymeofthe
27
1.Introductionanthocyaninpathway,resultedinalargefractionofplantswithwhitepetals(Napoli
etal.,1990;vanderKroletal.,1990).
SeveralclassesofsmallRNAshavebeenidentifiedtoresultinvarioussilencingpro
cedures.Commonfeaturesaretheinvolvementof1835nucleotidelongsmallRNAs
whicharecomplementarytotargetRNAs.Bindingresultsintargetrepressioneither
bytranslationalarrestorbycleavageofthetarget.Inaddition,chromatinremodeling
canoccurinsomespecies.ThemajorplayersintheRNAsilencingmechanismare
smallinterferingRNAs(siRNAs),microRNAs(miRNAs)andPiwiinteractingRNAs
(piRNAs),thelatterbeingrestrictedtotheanimalkingdom.Moreover,membersof
theArgonaute/Piwi(AGO)proteinfamiliesandtheRNaseIIItypeproteinDicer
functioninallsilencingpathways.
Figure1.1:Ghildiyal&Zamore,2009
(A)ThesiRNApathwayischaracterizedbyadsRNAwhichisprocessedbyDicerintosiRNAs.They
areincorporatedintotheRISCandleadtotargetcleavage.(B)InthemiRNAspathwayahairpin
shapedprecursormoleculeisexportedintothecytoplasmandprocessedbyDicer.ThemiRNAis
loadedintothemiRISCandleadstotranslationalrepressionofthetarget.(C)InthepiRNApathwaya
singlestrandedprecursorRNAiscleavedbyaPIWIproteinintopiRNAswhichgetmethylatedatthe
3end.TheymayinitiatetheproductionofsecondarypiRNAs. 28
1.Introduction1.2.1
siRNAmediatedgenesilencingsiRNAswereidentifiedtodirectendonucleolyticcleavageofthetargetRNAsin
plants(Hamilton&Baulcombe,1999)andanimals(Zamoreetal.,2000;Hammondet
al.,2000).TheyareproducedfromlongdsRNAmoleculesbytheenzymeDicer,
whichisadsRNAspecificribonucleaseoftheRNaseIIIfamily(Bernsteinetal.,
2001).ThefamilyisclassifiedintothreeclasseswithDicerbeingamemberofclassIII
(Nicholsonetal.,1999).Itusuallyconsistsofsixdistinctdomains,somememberslack
oneormoreofthem.ThePAZ[(Piwi/Argonaute/Zwille)(Ceruttietal.,2000)]domain
functionsinbindingofsingleanddoublestrandedRNAandDNAwithaprefer
enceforsinglestrandedRNAsmoleculesorsinglestranded3overhangs(Lingelet
al.,2003).ItisdirectlyconnectedtotheendonucleolyticRNaseIIIdomainresponsible
forRNAcleavage(Robertsonetal.,1968).AdditionaldoublestrandedRNAbinding
domains(dsRBDs)mayfunctioninthebindingofdoublestrandedRNA.TheN
terminusiscomprisedofaDExDhelicasedomain(Bernsteinetal.,2001)andado
mainofunknownfunction(Duf283)thatmaybeinvolvedinstrandselection(Dlaki,
2006).CharacterizationoftheDicerhomologueofGiardiaintestinalisrevealedthata
functionalenzymeonlyrequiresthecoreproteinconsistingofPAZandtwoRNase
IIIdomainswhichdimerizeanduseatwometalionmechanismforRNAcleavage
(MacReaetal.,2006).
ThenumberofDicerproteinsvariesbetweenspecies.Vertebratesandnematodes
possessasingleDicerprotein(Carmell&Hannon,2004);DrosophilahastwoDicer
proteinswithDCR1beingresponsibleformiRNAproduction(seechapter1.2.2)and
DCR2producingsiRNAs(Leeetal.,2004a).InArabidopsisfourdifferentDicerlike
proteinshavebeenidentified[(Baulcombe,2004)(seechapter1.2.4)].
Theycleavetheirtargetapproximatelyevery21nucleotidesintoadoublestrandof
19nucleotidesandtwonucleotideoverhangsatthe3ends(Elbashiretal.,2001a),the
strandspossessa5phosphateanda3hydroxyl(Elbashiretal.,2001b).Thelength
oftheproducedsmallRNAisdeterminedbythedistancebetweenthePAZandthe
RNaseIIIdomains(MacReaetal.,2006).29
1.Introduction(A) (B)
Figure1.2:MacReaetal.,2006 (A)DomainorganizationofhumanDicerwith
Helicase,DUF283,PAZ,twoRNaseIIIandthe
dsRBDdomainsandtheminimalDicerofGiardia
intestinaliswiththePAZandthetwoRNaseIII
domains.(B)Resolvedcrystalstructureofthe
GiardiaDicerwiththeNterminalplatformdomain
(blue),thePAZdomain(orange)connected(red)to
theRNaseIIIadomain(yellow),whichhasabridge
(grey)totheRNaseIIIbdomain(green).
Thedoublestrandisseparateddependingontherelativethermodynamicstabilityof
thetwoendsoftheduplex(Khvorovaetal.,2003;Schwarzetal.,2003).Thepassenger
strandisdestroyed(Matrangaetal.,2005;Leuschneretal.,2006)andtheguidestrand
isincorporatedintotheRNAinducedsilencingcomplex(RISC).Itischaracterized
bytheguidestrandthatisboundtoanArgonauteproteinandauxiliaryproteinsde
pendingonthespeciesandthetypeofsmallRNA(Hammondetal.,2000).
Argonauteproteinscontainfourdomainswithpartlyunidentifiedfunctions(see
Figure1.3a,b):anNterminaldomain,thePAZdomainwhichbindsssRNA(Lingel
etal.,2003),themiddledomainwithresemblancetothesugarbindingdomainofthe
lacrepressor(Friedmanetal.,1995)andthePiwi[(PElementinducedwimpytestis
domain)(Lin&Spradling,1997)]withanRNaseHfoldthatfunctionsasaribonucle
aseandconfersthecleavageofssRNA,whichisalsonamedslicing(Songetal.,2004).
ThesilencingcomponentofArgonauteproteinscanbesubdividedintotwogroups:
theAGOcladeconsistsofmembersthataresimilartoAGO1ofArabidopsisthaliana.
TheybindsmallRNAsderivedfromdsRNAintheRISCandareexpressedubiqui
tously.IncontrastthePiwicladeconsistsofthreeproteinswhichareprimarilyex30
1.Introductionpressedingonadaltissues(seeFigure1.3c):Piwi,theDrosophilaPelementinduced
wimpytestesprotein(Lin&Spradling,1997),Aubergine(Aub)firstidentifiedbyits
roleindorsoventralpatterning(Schpbach&Wieschaus,1991)andArgonaute3
(AGO3).
TheincorporatedsiRNAbindstoitstargetsequenceandtherespectiveArgonaute
proteincleavesthephosphodiesterbondofthetargetbetweenthetenthandeleventh
nucleotideoftheboundguidestrand.Thecleavedtargetisthenreleasedofthema
tureRISC(Elbashiretal.,2001b).
HumanspossesseightArgonauteproteinsbutonlyAgo2showsSliceractivity(Liuet
al.,2004;Meisteretal.,2004),DrosophilamelanogasterhasfiveArgonauteproteinsand
allofthempossesstheabilitytoslice(Miyoshietal.,2005).Caenorhabditiseleganshas
thelargestnumberofArgonauteproteinswith27differentmembers(Yigitetal.,
2006).Arabidopsisthalianapossesses10Argonauteproteins[(Vaucheret,2008)(see
chapter1.2.4)]. (A) (C)
(B)
Figure1.3:Argonauteproteins
(A)DomainorganizationofhumanAGO2withtheNterminalPAZdomain,theMiddomaininclud
ingthecapbindinglikeMCdomainandtheCterminalcleavagecompetentPIWIdomain(B)Crystal
structureoftheArgonauteofPyrococcusfuriosusincludingthesiRNA(purple)andmRNA(turquoise)
duplex.ActiveresiduesofthePIWIdomain(aDDHmotif)areshowninred.(Hutvagner&Simard,
2008)(C)MultiplesequencealignmentrevealedthreecladesofArgonauteproteins(Tolia&Joshua
Tor,2007). 31
1.Introduction1.2.2
miRNAsmodulatetheexpressionofendogenoussequencesThefirstreportofamicroRNAgenewaslin4whichhadtheabilitytorepresscell
proliferationinC.elegans(Chalfieetal.,1981).Althoughencodedbyagene,itwas
latershowntobeonlytranscribedintoanoncodingRNAwithsomecomplementar
itytothe3UTRofanothermRNAtranscript,lin14,whichconsequentlybecame
downregulated(Leeetal.,1993).Firstconsideredasauniquephenomenonsome
yearslaterlet7wasdiscoveredtousethesamemechanism(Reinhartetal.,2000).
SubsequentlyitwasshownthatmiRNAsareanabundantclassofsmallRNAmole
culesresponsibleformanyintracellularregulationprocesses(reviewedinCarthew&
Sontheimer,2009).
AnimalmiRNAsarehighlyconservedbetweenspecieswhichhasalsobeenusedfor
theiridentificationinthepast(Ambrosetal.,2003).ManynewmiRNAshavebeen
identifiedbydeepsequencingtechnologieswithnow15172entriesinmiRBasere
lease16,Sept2010(GriffithsJonesetal.,2008).
GenomicregionscodingformiRNAscanbelocatedinproteincodinggenesorin
intergenicregions.Itwasshownthattheycontainstandardpromoterelements.Con
sequentlytheyaretranscribedbyRNAPolymeraseII(PolII)(Leeetal.,2004b)into
primarymiRNA(primiRNA)transcriptswhichareusuallyhighlystructured(Leeet
al.,2002).Thefirstmaturationstepisalwayslocatedinthenucleusandexecutedby
theRNaseIIIendonucleaseDrosha(Leeetal.,2003)assistedintheMicroprocessor
complexbyadsRNAbindingprotein[Pashainflies(Denlietal.,2004)andDGCR8
inmammals(Gregoryetal.,2004;Hanetal.,2006)].ItresultsintheprecursormiRNA
(premiRNA)whichiscomprisedofanimperfecthairpinstructure(Leeetal.,2002).
InanimalsthemoleculeissubsequentlyexportedintothecytoplasmbyExportin5
andtheGTPaseRan(Yietal.,2003).Thesecondmaturationstepisexecutedbya
Dicerenzyme(Grishoketal.,2001;Hutvgneretal.,2001;Kettingetal.,2001)again
assistedbyadsRNAbindingprotein[infliesR2D2fordsRNA(Liuetal.,2003)and
LOQSforstructuredloci(Frstemannetal.,2005;Saitoetal.,2005)andTRBPin
mammals(Chendrimadaetal.,2005;Haaseetal.,2005)].ItgeneratesamiRNA/
32
1.IntroductionmiRNA*duplexofapproximately21ntand2ntoverhangsatthe3end.Although
resemblingsiRNAsinthisstep,theycanbedistinguishedbytheimperfectbinding
betweenthetwostrands(Leeetal.,2002).TheduplexisloadedintotheRISCandthe
miRNA*stranddegradedbytherespectiveAgoprotein(Filipowiczetal.,2008).
510%ofallmiRNAgeneswithlowexpressionarelocatedinintronswhichfoldinto
shorthairpins.Thesesocalledmirtronsarefirstprocessedbythesplicingmachinery
andthenlinearizedbythelariatdebranchase.Theyfurtherfoldintoahairpinsimilar
topremiRNAswhichareprocessedasdescribedabove(Okamuraetal.,2007;Ruby
etal.,2007).
Recentlyadistinctbiogenesismechanismhasbeendiscoveredinmice(Cheloufiet
al.,2010)andzebrafish(Cifuentesetal.,2010)formiR451whichpossessesanun
usualsecondarystructurewithashortstemof17nucleotides.ThepremiR451is
cleavedbyAgo2andtheresultingintermediatesarepolyuridylatedandfurther
processedbyyettobedefinednucleasesintothematuremiRNA(Cheloufietal.,
2010;Cifuentesetal.,2010). Figure1.4:Okamuraetal.,2007
ThecanonicalmiRNApathwayconsistsofaPolII
transcriptwhichfoldsintoahairpin.ThisprimiRNA
isprocessedbytheDroshacomplexintothepre
miRNAhairpin.Inthemirtronpathway,ashortin
tronissplicedandbranchedintoahairpin.Thepre
miRNAsareexportedtothecytoplasmbyEportin5 andcleavedbyDicer.
33
1.IntroductionAnimalmiRNAsbindtheirtargetsinadifferentmannerthansiRNAs:onlyaseed
regionofapproximatelysixnucleotidesaroundtheusualcleavagesiterequiresper
fectcomplementaritytothe3UTRsoftheirtargets,therestofthemiRNAspos
sessesmismatchesandfrequentnonconventionalbasepairing(G:Uwobbles)with
thetargetedmRNA.Theexactmechanismofsuppression,however,isstillunder
investigation.TheimperfectbindingpreventstargetcleavagebyArgonautes.One
modelproposescompetitionwiththecapbindingproteineIF4Eandsubsequentin
hibitionofthetranslationinitiation.Anotherhypothesisaccountsforthefactthat
manymiRNAregulatedmRNAsaredeadenylated.ItproposesRISCtostimulate
deadenylationandsubsequentmRNAdecay.Athirdmodelusesthefindingthat
RISChassomebindingaffinitytothe60Sribosomalsubunitandmaythereforepre
venttheassemblyoftheribosome(reviewedinCarthew&Sontheimer,2009).
ArgonauteproteinsinvolvedinthemiRNApathwayinteractwithGW182proteins
(BehmAnsmantetal.,2006).Theycontainfrequentglycine(G)andtryptophan(W)
repeats(Eystathioyetal.,2002)organizedinthreedistinctregions.TheNterminal
GWrepeatregionisfollowedbyaubiquitinassociated(UBA)likedomainanda
glutaminerich(Qrich)region.ThemiddleandaCterminalGWrepeatregionare
separatedbyaRNArecognitionmotif(reviewedinDing&Han,2007;Eulalioetal.,
2007).Whilethereexistthreeparaloguesinvertebrates(TNRC6A,BandC),fungi
havenoGW182proteinsandDrosophilapossessesonlyoneorthologuemakingita
goodmodelforstudyingtheirfunction(BehmAnsmantetal.,2006).TheC.elegans
orthologuesAIN1andAIN2containonlytheNterminalGWrepeatregionbut
functionalsoinmiRNAsilencing(Dingetal.,2005;Zhangetal.,2007).Bindingof
ArgonautebyGWcontainingproteinshasbeenalsoidentifiedinplants(NRPD1b
andSPT5liketranscriptionelongationfactor)(ElShamietal.,2007;BiesEtheveetal.,
2009)andS.pombe[(Tas3)(Partridgeetal.,2007;Tilletal.,2007)].
GW182proteinsexhibitsomeintrinsicsilencingactivity(Eulalioetal.,2009a;Zip
prichetal.,2009).InadditionlossofGW182suppressesmiRNAsilencing(Rehwinkel
etal.,2005),butitactsdownstreamofmiRNAprocessingandloadingintotheRISC34
1.Introduction(Eulalioetal.,2009a;Miyoshietal.,2009).TheNterminalregionisrequiredforthe
bindingtoAGO1(BehmAnsmantetal.,2006)andtogetherwiththeQrichregion
responsibleforlocalizationtothePbodies[(processingbodies)(Eulalioetal.,
2009b)].TheyarecytoplasmicgranuleswheretranslationallyrepressedmRNAscan
concentrate.Allfactorsinvolvedin53exonucleolyticdecayofmRNA,including
thedecappingenzymeDCP2andthemaincytoplasmic53exoribonucleaseXRN1
colocalizetothePbodiesinthecytoplasm.TheyarenotrequiredformiRNAsilenc
ingbutcanbeformedasaconsequenceofit(reviewedinEulalioetal.,2007).Acon
servedmotifofapproximately40residuesinsidethemiddleGWrepeatregionhas
recentlybeenidentifiedasaPolyAbindingproteininteractingmotif(Fabianetal.,
2009;Zekrietal.,2009),whichappearstobecriticalformiRNAmediatedsilencing
(Huntzingeretal.,2010).
Figure1.5:Carthew&Sontheimer,2009
miRNAdependenttranslationalrepressionmaybemediatedbycompetitionwithcaporribosome
binding,circularizationmaybeblocked,ortheribosomescoulddropoffaftertranslationinitiation.
AlternativelydeadenylationandsubsequentmRNAdegradationmightbeinduced.
35
1.IntroductionmiRNAsthemselvesareveryoftenundercontroloftheirtargetsinanegativefeed
backloop.AnothercontrolpointistheregulationofmiRNAprocessing,wheremany
factorshavebeenalreadyidentified,ortheregulationoftheeffectorproteinsofthe
miRISC(reviewedinKroletal.,2010).Afirstreportofexonucleasesdegrading
miRNAscomesfromplants(Ramachandran&Chen,2008)andahomologuehas
alsobeenidentifiedinC.elegans(Chatterjee&Grosshans,2009).
UndercertainconditionsthemiRNAloadedRISChasbeenshowntoactivatetrans
lationbuttheexactreasonandmechanismisnotclear(Vasudevanetal.,2007;Henke
etal.,2008;rometal.,2008).
1.2.3
ThepiRNApathwayprotectsthegermlinefromtransposonactivityInDrosophilamelanogasteralargefamilyofsmallRNAswithalengthof2326ntwas
identifiedtomaptorepetitiveheterochromaticregionsandtransposableelements,
theywerecalledrepeatassociatedsmallinterferingRNAs(rasiRNAs)(Aravinetal.,
2001)andcouldlateralsobeidentifiedinzebrafish(Chenetal.,2005).
PiwianditsorthologueswereshowntobindrasiRNAsand,inmammals,smallRNA
speciessimilartorasiRNAsbutnotderivedfromrepeatandtransposonsequences.
ThenewlyidentifiedfamilyofsmallRNAswasfurthercalledPiwiinteractingRNAs
[(piRNAs)(Malone&Hannon,2009)].Theyhave2Omethylated3ends,whichis
depositedbyanorthologueoftheplantmethyltransferaseHEN1(Kirino&Mourela
tos,2007;Oharaetal.,2007).Inaddition,theyhaveastrongbiasfora5uridineresi
dueandaDicerindependentbiogenesispathwayresultinginalargersizecompared
totheothersmallRNAs.Theyallarisefromchromosomalclustersandhaveasingle
strandedRNAprecursor(reviewedinKlattenhoff&Theurkauff,2008).Thereisevi
dencethatthe21URNAsofC.elegansarealsopiRNAssincetheycontaina5uridine
(Rubyetal.,2006),theysuppresstransposonmobility(Dasetal.,2008)andtheyin
teractwiththePiwirelatedgenePRG1(Wang&Reinke,2008).
InDrosophilapiRNApopulationscanbematchedtotransposons,usuallyenriched
forsequencesantisensetotransposons.TheseantisensepiRNAsareboundbyPiwi
andAubwhereasthesenseorientatedfractioninteractswithAGO3.Thetwoclasses36
1.IntroductionofpiRNAshaveoverlapping5endsseparatedbytennucleotides(Brenneckeetal.,
2007,Gunawardaneetal.2007),suggestingprocessingbyPiwiwhichhadbeen
showntocleaveitstargettennucleotidesfromthe5endoftheguidestrand(Saitoet
al.,2006).ThebiogenesisandamplificationofpiRNAsfollowsthesocalledping
pongcycle.ItisinitiatedbyprimaryantisensepiRNAswhichtargetthecleavageof
transposonmRNA.ThisresultsinsecondarysensepiRNAswhichareboundby
AGO3anddirectthecleavageofantisensetransposonsequences(Brenneckeetal.,
2007,Gunawardaneetal.2007).Partsofthiscyclehavealsobeendetectedinzebraf
ish(Houwingetal.,2007)andmice,althoughthecyclethereseemstobeinitiatedby
sensepiRNAsandisonlypresentinthemalegermline(Aravinetal.,2007).Thecycle
sharessimilaritieswiththesilencingofheterochromaticregionsbyRNAdirected
DNAmethylationinplantsandS.pombe(seechapter1.2.4.1).Inthelatterthetran
scriptionofcentromericrepeatsleadstosiRNAswhichdirecttheAGOorthologueto
cleavetargettranscriptsofthislocus.ThereactionactivatestheRNAdependent
RNApolymerasecomplexwhichgeneratesfurthersiRNAs.FinallythesesiRNAs
directthemodificationofhistones(Moazed,2009).
AdditionalfactorshavebeenidentifiedtoberequiredforthepiRNApathway,muta
tionsintheputativenucleasesZucchiniandSquashdisruptpiRNAproductionand
releasetransposonsilencing(Paneetal.,2007).Thesamecanbeobservedafteraloss
oftheputativehelicasesArmitage(Vaginetal.,2006)andSpindleE(Aravinetal.,
2004).Theyareallpartofnuage,whichisagermlinespecificperinuclearstructure
implicatedinRNAprocessing.ThissuggestsacompartmentalizationofpiRNAbio
genesisandaction(Lim&Kai,2007;reviewedinKlattenhoff&Theurkauff,2008).
C.elegans21URNAsarealsorequiredforfertility(Batistaetal.,2008)andthegerm
linefunction(Wang&Reinke,2008).
InDrosophilamainlyAubandAGO3associatedpiRNAstakepartinthepingpong
cycle,PiwiassociatedpiRNAsmayonlycomprisetheprimarypiRNAsinthegerm
linespecificcycle.However,anadditionalpathwayofpiRNAfunctionhasbeendis
coveredrecentlyinsomaticovarianfolliclecells,dependingexclusivelyonPiwiand37
1.IntroductiontheflamencopiRNAcluster(Maloneetal.,2009a,b).Althoughinitiallymorefactors
hadbeenidentified,recentfindingssuggestthefollowingproteinsbeingindispensa
bleforthesomaticpiRNApathway:Zucchini,Armitageandtheputativehelicase/
tudordomainproteinYbarerequiredforsilencingofthegypsytransposoninthe
somaticcells.Thelatterproteinsarebothlocalizedtocytoplasmicfoci.Piwiaccumu
latesinthecytoplasmintheabsenceofZucchini,whichmaybeconfersshuttlingbe
tweenthecytoplasmandthenucleus.InarmitagemutantsnopiRNAsaccumulate
suggestingafunctionearlyinthepathway(Olivierietal.,2010;discussedinZamore,
2010).
Figure1.6:Zamore,2010
InsomaticcellspiRNAsaresynthesizedbyaPIWIdependentlinearpathwaywithoutamplification,
whereasinthegermlinetheprimarypiRNAsaredependentonAubergineandgetamplifiedviathe
socalledpingpongcycleandAgo3.
1.2.4
PlantspossessahighdiversityofsiRNAmoleculesPlantsdisplayanastonishingvarietyofsiRNAtypesandproteinswhichareneeded
fortheirgeneration.ArabidopsisthalianahasfourDicerlikeandtenArgonautepro
teinswithdistinctmolecularfunctions.
EverymemberoftheDICERLIKEproteinfamilyinArabidopsishasdistinctfunctions
althoughsomeredundancieshavealsobeenidentified:DCL1isthemainDicerin38
1.IntroductionvolvedinmiRNAprocessingwhichcannotbecompensatedbytheothermembers
(Parketal.,2002;Reinhartetal.,2002;Pappetal.,2003).Inaddition,DCL1canprocess
somenatsiRNAsfromendogenousinvertedrepeatsequences(Borsanietal.,2005;
KatiyarAgarwaletal.,2006).DCL2alsoparticipatesinthelatterfunctionandgener
ates22ntsiRNAsfromvirussequences(Xieetal.,2004).DCL3produces24nt
siRNAswhicharemainlyinvolvedinchromatinsilencing(Xieetal.,2004).DCL4is
themainplantDicerfortheproductionofviral21ntsiRNAs(Dunoyeretal.,2005;
Bouchetal.,2006;Delerisetal.,2006).Moreover,itisinvolvedintasiRNAmetabo
lism(Gasciollietal.,2005;Xieetal.,2005b)andintheproductionofmiR822and
miR839(Rajagopalanetal.,2006).AfterprocessingbyDCLproteinsinplantsthere
sultingsmallRNAsaremethylatedattheir3endsbytheSadenosyldependentme
thyltransferaseHUAENHANCER1(HEN1)whichprotectsthemfromuridylation
andfurtherdegradation(Lietal.,2005;Yangetal.,2006)
PlantscontainahighnumberofArgonauteproteinswithtenmembersinArabidopsis
thalianaand19membersinOryzasativa.Theformerwillbediscussedinmoredetails
(reviewedinVaucheret,2008).
AGO1isthefoundingproteinforthewholeArgonauteproteinfamilyanditspleio
tropicdefectshavefirstbeendescribedin1998(Bohmertetal.,1998).Ithadlaterbeen
identifiedtoactinRNAsilencing(Fagardetal.,2000).AGO1predominantlyactsin
themiRNApathwaybutitcanalsobindseveralclassesofsiRNAs(Baumberger&
Baulcombe,2005).ItisitselfregulatedbyafeedbackmechanismthroughmiR168
(Vaucheretetal.,2006).AGO10(originallytermedPINHEAD/ZWILLE)istheclosest
paralogueofAGO1andpartlyshowsredundantfunctionindevelopment(Lynnet
al.,1999).RecentstudiesimplicateAGO10asanegativeregulatorofAGO1(Mallory
etal.,2010).AGO5isthethirdmemberofthisgroup.Itsexactfunctionisnotclear,
butithasbeenshowntopreferentiallyinteractwitha5cytosine(Takedaetal.,2008).
AnothercladeofArabidopsisAGOproteinscontainsAGO7whichisinvolvedinthe
TAS3biogenesispathway[(Montgomeryetal.,2008a)(seechapter1.2.4.2)].AGO2
39
1.IntroductionandAGO3arehighlysimilarproteinswithcurrentlyunknownfunction.Theformer
isprobablyregulatedbymiR403(Allenetal.,2005).
ThethirdcladeiscomprisedofAGO4,themajorproteininvolvedintranscriptional
genesilencing[(Zilbermanetal.,2003)(seechapter1.2.4.1)].AGO6hasapartialre
dundantactivitywithAGO4(Zhengetal.,2007).Theroleoftheothertwoproteinsof
thisclade,AGO8andAGO9,isstillunknown,buttheirsimilaritysuggestsaredun
dantfunction.SincetheAGO8expressionisverylowithasbeensuggestedtobea
pseudogene(Takedaetal.,2008).
Ithasbeenshownthatthe5nucleotideofthesmallRNAspeciesactsasasorting
signalintothedifferentAGOproteinsinplants:AGO2andAGO4preferentiallyin
teractwithadenosine.AGO1prefersuridinewhichisthepredominant5nucleotide
ofplantmiRNAs.FinallyAGO5bindssmallRNAswithaterminalcytosine(Mietal.,
2008).
Figure1.7:Ghildiyal&Zamore,2009
(A)InplantscisactingsiRNAprecursormoleculesaretranscribedbyPolIVandadsRNAgenerated
byRDR2.DCL3cleavesthe24ntcasiRNAswhichassociatewithAGO4.(B)InthetasiRNApathway
aprecursormoleculeissubjecttomiRNAmediatedcleavage.RDR6generatesadsRNAwhichis
processedbyDCL4intotasiRNAswhichassociatewithAGO1/7.(C)natsiRNAsderivefromoverlap
pingtranscriptswhichareprocessedintoadsRNA.AdicermoleculethengeneratesthenatsiRNAs.
40
1.Introduction1.2.4.1
CisactingsiRNAsmediatechromatinsilencinginplants
RNAdirectedDNAmethylation(RdDM)isanepigeneticsiRNAmediatedmodifica
tioninplants(reviewedinMatzkeetal.,2009;Law&Jacobsen,2010).Inplantsitis
responsiblefor30%ofdenovomethylationofcytosinesinheterochromaticandsome
euchromaticregionssuchastransposons(Cokusetal.,2008;Listeretal.,2008).This
methylationisdepositedbyDOMAINSREARRANGEDMETHYLTRANSFERASE2
(DRM2).InadditionRdDMrequirestwoplantspecificPOLIIrelatedRNApoly
merases,POLIVandPOLV(Pikaardetal.,2008,Wierzbickietal.,2008).
Theirlargest(NRPD1andNRPE1)andsecondlargestsubunits(thesharedsubunit
NRPD2/NRPE2)areuniquewiththelatterbeingalsorelatedtothelargestsubunitof
POLII(RPB1)(Wierzbickietal.,2008;Reametal.,2009).Theyactincomplexes
Figure1.8:Matzkeetal.,2009
dsRNAisprocessedbyDCL3/HEN1into24ntsiRNAswhichareloadedontoAGO4.Transcription
byPolVfacilitatesdenovomethylationatthesiRNAtargetedsite.PolIVtranscribesthemethylated
DNAwhichisfurthercopiedbyRDR2intodsRNA.
AfterprimaryRdDMPolIVtranscribesthemethylatedtemplateanddownstreamsequenceswhich
resultinsecondaryRdDM. 41
1.IntroductionincludingSNF2likechromatinremodelingfactors:POLIVtogetherwithCLASSY1
(CLSY1)isinvolvedintheinitiationofsiRNAbiogenesisbytranscribingalongsin
glestrandedRNA.POLVtogetherwithDEFECTIVEINRNADIRECTEDDNA
METHYLATION(DRD1)(Pikaardetal.,2008)identifiesandmaybealsotranscribes
lowabundanceintergenicnoncodingtranscripts(IGN)(Wierzbickietal.,2008).
RNADPENDENTRNAPOLYMERASE2(RDR2)producesdsRNAfromthesingle
strandedPOLIVdependenttranscriptswhicharefurtherprocessedbyDCL3into
24ntheterochromaticsiRNAs(Mosheretal.,2008).TheseareboundbyAGO4[(or
sometimesAGO6(Zhengetal.,2007)]whichcaninteractwithPOLVthroughacon
servedGW/WGmotif(ElShamietal.,2007).TheIGNtranscriptsareprobablyrecog
nizedbytheAGO4boundsiRNAs(Wierzbickietal.,2008).RecentlyPOLII
dependentnoncodingtranscriptshavebeenidentifiedtoalsorecruitRdDMfactors
(Zhengetal.,2009).INVOLVEDINDENOVOMETHYLATION2(IDN2)mayinter
actwiththesiRNARNAduplexandrecruitRDM2(Ausinetal.,2009).
TheactionofRDR2mayleadtotheproductionofsecondarysiRNAsandfurtherme
thylationspreading(Daxingeretal.,2009).siRNAsalsoappeartoguideactivede
methylation(Zhengetal.,2008).TheRdDMmechanismisconservedinS.pombeas
wellwhereitleadstoheterochromatinization(reviewedinMoazed,2009).
1.2.4.2 TransactingsiRNAs
TransactingsiRNAs(tasiRNAs)areaplantspecifictypeofsmallRNAsgenerated
fromspecificTASloci.ThereexistfourTASfamilieswhichcanbefurthersubdivided
intotwoclasses:oneconsistsofTAS1,TAS2andTAS4whichrequireonemiRNA
bindingsitefortasiRNAbiogenesis.TheotheriscompromisedbytheTAS3family
whichrequirestwomiRNAbindingsitesfortasiRNAbiogenesis(Allen&Howell,
2010).
TheTAS1family,consistingofthreeloci,andTAS2werethefirstidentifiedTASloci
(Peragineetal.,2004;Vazquezetal.,2004).TheydependonthecleavagebymiR173
(Yoshikawaetal.,2005),whereasTAS4dependsonthecleavagebymiR828(Ra
jagopalanetal.,2006).TheinitialPOLIItranscriptsareboundbythemiRNAwith42
1.Introductionunusualmismatchesintheseedregion,substitutionofthesemismatchesabolishes
theproductionoftasiRNAs(Montgomeryetal.,2008b;Felippes&Weigel,2009).The
transcriptiscleavedbyAGO1(Vazquezetal.,2004)andboundbySUPPRESSOROF
GENESILENCING3(SGS3)(Peragineetal.,2004;Vazquezetal.,2004).Itcaninteract
withtheRNADEPENDENTRNAPOLYMERASE6(RDR6)(Kumakuraetal.,2009)
whichsynthesizesthecomplementarystrandfromthe3endtowardsthe5cleavage
site.TheresultingdsRNAisfurtherprocessedbyDCL4fromthemiRNAcleavage
siteinto21ntlongsiRNAs(Peragineetal.,2004;Vazquezetal.,2004).Thedominant
phasingpatterncanalsodriftforoneortwonucleotides,probablybytheredundant
Figure1.9:Allen&Howell,2010
InthefirstpathwaymiR173/828guidesthecleavageoftheTAStranscriptbyAGO1.Incontrastinthe
secondpathwaymiR390bindstwicetotheTAS3transcriptandguidesitscleavagebyAGO7atthe3
site.ThecleavedtranscriptissynthesizedbyRDR6intoadsRNAwhichisprocessedbyDCL4intothe
tasiRNAs. 43
1.IntroductionfunctionofotherDCLproteins(Howelletal.,2007).TheTAS3familyconsistsof
threeloci(Howelletal.,2007),allcharacterizedbytwobindingsitesformiR390
whichflankthetasiRNAproducingsequence(Allenetal.,2008).Fromtheseonlythe
3siteiscleavedanddeterminestheresultingtasiRNAs.The5sitepossessescritical
mismatchesanditisnotclearifitisalsocleaved.miR390wasshowntointeractwith
AGO7.Whilethecleavageatthe3sitecanbeaccomplishedbyanothermiRNA
AGOpair,thebindingofAGO7tothe5siteisindispensablefortasiRNAproduc
tion(Montgomeryetal.,2008a).
ThetasiRNAsaresortedintothecorrespondingArgonauteproteinsaccordingto
their5nucleotide(Mietal.,2008).tasiRNAscanbeconsideredasanamplificationof
aninitialsilencingsignal.Inadditiontheyarenoncellautonomousandcancreatea
silencinggradientacrossneighboringcells(Chitwoodetal.,2009;Schwabetal.,2009).
IdentifiedtargetsarePentatricopeptiderepeatproteins(Peragineetal.,2004;
Vazquezetal.,2004),MYBtranscriptionfactors(Rajagopalanetal.,2006)andAuxin
responsivefactors(Allenetal.,2005;Williamsetal.,2005;Adenotetal.,2006;Fahl
grenetal.,2006;Garciaetal.,2006). 1.2.4.3 NaturalantisensesiRNAs
NaturalantisensetranscriptderivedsiRNAs(natsiRNAs)havefirstbeendescribed
asaresultofsaltstressinArabidopsis(Borsanietal.,2005).Thereexisttwodifferent
typesofnaturalantisensetranscripts(NATs):cisNATshaveahighsequencecom
plementaritysincetheyaretranscribedfromopposingstrandsofthesamelocus.In
contrast,transNATsaretranscribedfromdifferentlociresultinginshortandimper
fectcomplementarityofthedoublestrand(Jinetal.,2008).
FactorsrequiredforthesynthesisoftheprimarynatsiRNAsareDCL2and/orDCL1,
RDR6,SGS3andPOLIV(Borsanietal.,2005;KatiyarAgarwaletal.,2006;Zhang&
Trudeau,2008).Theyguidethecleavageofthecomplementarytranscript.Although
ratherrareunderphysiologicalconditions,theymaysubstantiallycontributetothe
smallRNApopulationduringstressconditions,sinceNATpairscompromisemore
than7%ofalltranscriptionalunitsinArabidopsisthaliana(Henzetal.,2007).44
1.Introduction1.2.5
PeculiaritiesofplantmiRNAsThefirstplantmiRNAshavebeenidentifiedin2002(Parketal.,2002;Reinhartetal.,
2002).ExamplesofevolutionaryconservedmiRNAsbetweenanimalsandplantsare
rare,thereforeitisbelievedthattheyevolvedindependentlyinthetwogenera(Ax
tell&Bowman,2008).PlantmiRNAsconsequentlypossessseveralspecialfeatures
comparedtoanimalmiRNAs(reviewedinVoinnet,2009).
MostplantMIRgenesarelocatedinintergenicregions;thereexistmorethan100
familiesofrelatedmiRNAswhichareusuallyconservedbetweenangiosperms(Ax
tell&Bowman,2008)andpartlyalsobacktomoss(Garcia,2008).Anothergroupof
miRNAsareevolutionaryyounger,thereforelessconservedandwithahighlydi
versetargetrange(Zhangetal.,2006).MIRgenesaretranscribedbyPOLIIintopri
miRNAswhichgetcappedatthe3endandpolyadenylatedatthe5endlike
mRNAtranscripts.Thestrandfoldsintoanimperfecthairpinwhichisprobablysta
bilizedbytheRNAbindingproteinDAWDLE(DDL).ItalsobindsothermRNAs
andmightbeinvolvedinsiRNAbiogenesistoo(Yuetal.,2008).TheprimiRNAsare
furtherprocessedbyDCL1intomaturemiRNAsinatwostepprocess.Thefirsttran
sitiontopremiRNAstakesplaceinnuclearprocessingcenters(Dbodies)where
DCL1interactswiththedoublestrandedRNAbindingproteinHYPONASTIC
LEAVES1(HYL1)andtheZincfingerproteinSERRATE(SE),thelatteralsohavinga
roleinmRNAsplicing(Fang&Spector,2007;Kuriharaetal.,2006).Afterthesecond
processingstepbyDCL1,thematuremiRNAduplexesarestabilizedbymethylation
attheir3endswhichistransferredbyHEN1(Lietal.,2005;Yangetal.,2006).The
miRNAsareexportedintothecytoplasmbythenuclearporecomplexHASTY,al
thoughotherexportmechanismscannotbeexcluded(Parketal.,2005).
ThefirstidentifiedmodeofactionofplantmiRNAsischaracterizedbyextensive
complementaritytotheirtargetmRNAs.ThemiRNAsareloadedintotheArgonaute
proteinAGO1oftheRISCwhichslicesthemRNAtargetsbetweenthetenthand
eleventhnucleotide(Rhoadesetal.,2002).Lateramechanismoftranslationalrepres
sionsimilartothemiRNAmechanisminanimalshasbeenidentifiedinmiRNA
45
1.Introductionactiondeficient(mad)classIIImutants;itprobablyalsorequiresthefunctionof
AGO1aswellasamicrotubuleseveringenzymeKATANIN(KTN)andthePbody
componentVARICOSE(VCS)whichisrequiredformRNAdecapping(Brodersenet
al.,2008).TheextentofmiRNAexpressionisregulatedondifferentlevels:MIRgenes
havetheirownpromoters(Xieetal.,2005a)whichshowahighdegreeoftranscrip
tionfactorbindingsites(Megrawetal.,2006).Tissuespecificdifferencesinprotein
expressioncanaccountforalteredmiRNAaction.HighDCL3levelscompetewith
DCL1formiRNAprocessing,resultingin24ntproductswhicharepredominantly
sortedintoAGO4andthereforenotavailablefortheRISC(Vazquezetal.,2008).
Abundanttranscriptionofshortinterspacedelements(SINE)RNAcancompetefor
theessentialfactorHYL1(PouchPelissieretal.,2008).PlayersofthemiRNApath
wayarefrequentlyitselftargetsofmiRNAregulation,suggestingafeedbackregula
tion(Xieetal.,2003,Vaucheretetal.,2004).MoreoversinglestrandedmiRNAsare
alsosubjecttodegradationbymembersoftheSMALLRNADEGRADING
NUCLEASE(SDN)familyofexonucleases(Ramachandran&Chen,2008).
1.2.6
SpreadingofRNAsilencinginplantsresemblesanimmunesystemInplantsRNAsilencingactsassortofanimmunesystemprovidingresistanceto
viruses.Sincevirusesreplicaterapidlyandareabletospreadthroughoutthewhole
plant,thehostneedsamechanismtransmittingtheinitialimmunityofRNAsilenc
ingtootherleaves.ForstudyingthisphenomenonA.thalianaisusuallyreplacedby
Nicotianasp.asamodelorganism,sincetheyhostawiderangeofvirusesandhavea
lifecycleandsizemorefavorableforstudyingtheeffectsofviralinfections.Amajor
disadvantage,however,isthemissinggenomicinformationofNicotianasp.
TheinitiationofsilencingismediatedbytheoverexpressionofexogenousRNA
whichistransformedintodsRNAbytheactionofRNAdependentRNApoly
merases[(RDRs)(Wassenegger&Krczal,2006)].Theiractivityisstimulatedbythe
presenceofaberrantRNAwithmissing5capstructuresor3polyadenylation(Herr
etal.,2006;Luo&Chen,2007).RDR6andRDR1arethemajorplayersinantiviralsi
lencingmechanisms(Schwachetal.,2005;DiazPendonetal.,2007;Quetal.,2008)46
1.Introduction
Figure1.10:Kalantidisetal.,2008
ExamplesofsilencingspreadofaGFPtransgene:(A)notsilenced(B)spontaneousshortrangelocal
silencing(C)inducedshortrangelocalsilencing(D)fullysilenced(E)systemicsilencing(F)extensive
localspread(left)andsystemicsilencing(right).
togetherwiththecofactorsSILENCINGDEFICIENT3(SDE3),aputativeRNAheli
case(Dalmayetal.,2001)andSUPPRESSOROFGENESILENCING3(SGS3),a
coiledcoileddomainprotein(Mourrainetal.,2000;Kumakuraetal.,2009).Silencing
ofDNAvirusesandtheRNATobaccorattlevirus(TRV)additionallyrequiresRDR2
(Donaireetal.,2008).
Exogenousfactorssuchastemperature(Szittyaetal.,2003)andlight(Kotakisetal.,
2010)caninfluencetheefficiencyofsilencingonset.Endogenoussequencescannot
serveassubstratesforRDRs(Himberetal.,2003;Schwachetal.,2005;Bleysetal.,
2005).
ThespreadingofRNAsilencingcanbedifferentiatedintothreedifferentstages,
shortrangelocalspread(SLSS),extensivelocalspread(ELSS)andsystemicsilencing.
TheshortrangelocalsilencingspreadisnotlimitedtoexogenousRNAandcanalso
affectendogenoussequences.Afterinitiationofsilencinginacellthesignalistrans47
1.Introductionferredto1015cellssurroundingtheinitialsourceofsilencingwithoutamplification
(Himberetal.,2003).Theexactnatureofthesignalcouldnotbediscoveredyet,butit
probablyreliesonpassivediffusionthroughtheplasmodesmata(Voinnetetal.,1998;
Himberetal.,2003;Kalantidisetal.,2006)withamobilitycomparabletosolublepro
teinsof2754kDa(Kobayashi&Zambryski,2007).Severalfactorshavebeenshown
tobeindispensablefortheshortrangespread:lossofDCL4abolishesthemechanism
suggestinganinvolvementof21ntsiRNAswhicharetheproductsofDCL4action
(Dunoyeretal.,2005).SeveralotherproteinsofthevariousRNAsilencingmecha
nismshavebeenshowntobeaprerequisitefortheshortrangespreadincluding
HEN1,DRB4,AGO1,thePOLIVsubunitNRPD1a,RDR2anditspresumableinter
actingproteinCLSY1(Hiragurietal.,2005;Adenotetal.,2006;Yangetal.,2006;
Dunoyeretal.,2007;Nakazawaetal.,2007;Smithetal.,2007).Thismultitudeofin
volvedproteinssuggestsanintensivecrosstalkofthedistinctRNAsilencing
mechanisms.
Extensivelocalspreadofsilencingischaracterizedbythefactthatthesignalexceeds
thelimitof1015cellsbutdoesnotspreadtothewholeplant.Itisaccompaniedby
amplificationoftheinitialsignalasaresultofanRDR6dependentmechanism
(Himberetal.,2003;Schwachetal.,2005).OtherrequiredfactorsappeartobeDCL4
andtheputativeRNAhelicaseSDE3(Dalmayetal.,2001).Itisunclearwhichfactors
definetheonsetofextensivelocalspread;itwasproposedthatacertainthreshold
hastobeexceededtotriggerastrongerreaction.Itisrestrictedtosinktissueswhich
receivethesignalfromthesourcetissuesoftheleaveswhichhadinitiallybeenchal
lengedwiththeexogenousRNA(Kalantidisetal.,2006).
Theleastunderstoodmechanismissystemicspreadwhichisprobablyaccompanied
bythetransportofthesilencingsignalthroughthephloem(Voinnet&Baulcombe,
1997;Fagard&Vaucheret,2000;Mlotshwaetal.,2002;Tournieretal.,2006).Itsaction
mightbeexecutedbyavirusspecificsiRNARISC(Lakatosetal.,2006).Thesignal
transmittingthesilencingspreadofviralsequencesisbelievedtobeanRNAmole
cule(Jorgensenetal.,1998),butithasbeenprovennottobeofthesizeofsiRNAs48
1.Introduction(Malloryetal.,2003).Anotherproteinnecessaryforbindingandfacilitatingthe
movementofRNAmoleculesbetweencellshasbeendiscoveredincucurbits(Yooet
al.,2004).However,sofarnohomologuesofthisPHLOEMSMALLRNABINDING
PROTEIN1(PSRP1)havebeenidentifiedinArabidopsisthalianaorNicotianasp.
1.2.7
ViralstrategiestosuppressRNAsilencinginplantsVirusesalsopossessstrategiestocounteracttheRNAsilencingmechanismsusedfor
theirclearancefromtheplantgenomes.Theyencodeforproteinsactingasviralsup
pressorsofsilencing(VSRs)inaverydiversemanner.Morethan35VSRfamilies
couldbefoundinallplantvirustypes,theycanbeclassifiedintothreedifferent
categories:classIVSRssuppressthelocalsilencingoftheviralRNAs,classIIVSRs
suppressthelocalsilencingspreadandclassIIIVSRsarethelargestclasssuppress
ingthesystemicspreadingofsilencing(reviewedinDazPendn&Ding,2008).
TheclassIsuppressorpotyviralhelpercomponentproteinase(HCPro)isamulti
functionalproteinwhoseperformancepartlydependsonitssilencingsuppression
activity(Kasschau&Carrington,2001).Itinterfereswithmethylation(Ebhardtetal.,
2005)andpreventsRISCassembly(Meraietal.,2006;Yuetal.,2006;Shibolethetal.,
2007).ThepoleoviralP0isalsoamemberofclassIVSRsandappearstomediatethe
degradationofAGO1whichabolishesintracellularRNAsilencingprocesses(Pfeffer
etal.,2002;Pazhouhandehetal.,2006;Baumbergeretal.,2007;Bortolamioletal.,
2007).Thetobamoviralproteinp126containsmethyltransferaseandhelicasedo
mainsandispresentinacomplexwithp183(Komodaetal.,2007).Itbindsduplex
siRNAsandinterfereswithmethylationbyHEN1(Blevinsetal.,2006;Csorbaetal.,
2007;Vogleretal.,2007)whichhasalsobeenshownforp21(Yuetal.,2006).Several
othervirusencodedproteinshavebeenshowntopossessviralRNAsilencingsup
pressoractivitywithyetunknownmechanisms,suchasthetranscriptionfactor
AL2/AC2/C2(Trinksetal.,2005;Yangetal.,2007),thetranslationalenhancerP6
(Loveetal.,2007)andthep23proteinwhichcontrolsviralRNAaccumulation(Sat
yanaryanaetal.,2002;Luetal.,2004).
49
1.IntroductionManyviralmovementproteinshavebeenshowntobemembersofclassIIVSRs.The
potexviralp25isanRNAhelicasethatinterfereswiththeplasmodesmata(Bayneet
al.,2005).Thetymoviralp69appearstotargetplantsilencingupstreamofRDR
dependentdsRNAsynthesis(Chenetal.,2004).p50inhibitssystemicspreadofsi
lencing(Yaegeshietal.,2007),whilep25targetsdownstreamofdsRNAsynthesis
(Voinnetetal.,2000;Bayneetal.,2005;Moissiardetal.,2007).
MostviralsuppressorsofsilencingbelongtoclassIII.Thetomoviralp19bindsshort
dsRNAmoleculeswithhighaffinity.ThesequesteringofsiRNAduplexesmaypre
ventRISCfunctionality(Silhavyetal.,2002;Vargasonetal.,2003;Yeetal.,2003;Laka
tosetal.,2004;Omarovetal.,2007).Therelatedp14alsobindsdsRNAmolecules
(Haveldaetal.,2003,Meraietal.,2005;Pantaleoetal.,2007).Thecucumoviral2bpro
teinmayhaveadualrolesinceitweaklysuppressesintracellularsilencinginaddi
tiontoitspotentinhibitionofRNAsilencingspreadwhichallowslongdistancevi
rusmovement(Guo&Ding,2002).ItblockstheproductionofRDR1dependentsec
ondaryviRNAs(Caoetal.,2005;Yaegeshietal.,2007).Someviralcoatproteinsalso
exhibitVSRfunction,thecarmoviralp38proteinisabletoreplacep19(Qu&Morris,
2002),itcanselectivelyinhibitDCL4andalsosuppress22ntsiRNAswhichare
DCL2products(Meraietal.,2006).
1.2.8
RepressingtherepressorsendogenoussuppressorsofRNAsilencingThepowerofthesmallRNAmoleculesandthepresenceofVSRswhichspecifically
sequestersmallRNAssuggestthattherealsoexistendogenoussuppressorsofRNA
silencing.AfirstreportwastheCa2+sensorproteinREGULATOROFGENE
SILENCINGCALMODULINLIKE(rgsCAM)whichbecameupregulateduponHC
Proexpression.EctopicoverexpressionofrgsCAMmimicsthesymptomsofHC
Procontainingviruses(Anandalakshmietal.,2000).RNaseLinhibitor2(RLI2)has
beenfoundtobeupregulatedwhentransgenicplantsaresubjecttoRNAinterference
(Brazetal.,2004).Laterithasbeenshownthatuponsimultaneousoverexpressionit
reducestheamountsofsiRNAs(Sarmientoetal.,2006).Thecytoplasmicexonuclease
XRN4hasbeenproposedasanendogenoussuppressorofsilencingsinceinthemu50
1.Introductiontantbackgroundxrn4RDRdependentsilencingwasincreased(Gazzanietal.,2004).
Inaddition,overaccumulationofmiRNAcleavageproductscouldbedetected
(Souretetal.,2004).Similarresultshavebeenobtainedforthenuclearexonucleases
XRN2andXRN3(Gyetal.,2007).Inthesamescreenthe3,(2),5bisphosphatenu
cleotidase/inositolpolyphosphate1phosphataseFIERY1(FRY1)hasbeenidentified
asasuppressorofvirusandtransgeneinducedposttranscriptionalgenesilencing
(PTGS)(Gyetal.,2007).
InC.elegansthelossoftheputativeRNAdirectedRNApolymeraseRRF3leadto
hypersensitivitytoRNAi(Simmeretal.,2002).Fornoneoftheabovedescribedpro
teinsaspecificeffectonsiRNAshasbeenprovenandsecondaryeffectscannotbe
excluded.
FormiRNAsithasrecentlybeenshownthattheyarespecificallydownregulatedbya
familyofexoribonucleasescalledSMALLRNADEGRADINGNUCELASE(SDN)in
plants(Ramachandran&Chen,2008)andC.elegans(Chatterjee&Grosshans,2009)
1.2.9
ERI1isanexampleforanendogenoussuppressorofRNAsilencingInCaenorhabditiseleganstheneuronalcellsarerefractorytoRNAinterference.Ina
geneticscreeneri1nullmutantswereidentifiedtopossessenhancedsensitivityto
dsRNAsthroughoutthewholeorganism.Themutantswereviableandshoweda
weakphenotypeexceptforsterilityduetoadefectinspermfunction.ERI1ismainly
localizedinthecytoplasmofdevelopingsomaticgonadsandinasubsetofneurons.
Itisanevolutionaryconservedproteinwithnucleicacidbindingproperties(con
ferredbyaSAP/SAFboxdomain)andaDEDDhlike35exonucleasedomain.It
partlydegradeddoublestrandedsiRNAswith2nt3overhangsinvitro.Inaddition
eri1mutantsaccumulatedmoresiRNAsafteringestionoflongdsRNAsorinjection
ofsiRNAs(Kennedyetal.,2004).Thefirstidentificationofanendogenousinhibitor
ofsilencinginC.eleganswastheproteinRRF3whichissimilartoRNAdependent
RNApolymerasesRdRPs).rrf3mutants(Sijenetal.,2001;Simmeretal.,2002)exhib
itedasimilarphenotypetoeri1mutants,includingtheenhancedRNAiphenotype
(Timmons,2004).BothproteinswerefoundinascreenforDCR1interactingpro51
1.Introductionteins.Inadditiontwonewlyidentifiedgeneseri3anderi5wereinteractingwith
DCR1.TheyalsoshowedanenhancedRNAiphenotypeandpromotedtheDCR
1/ERI1interaction.OnlythelongisoformofERL1(ERI1b)couldbedetectedin
DCR1immunoprecipitatessuggestingdistinctmolecularfunctionsofthetwoiso
forms(Duchaineetal.,2005).
AhomologueofERI1hadbeendescribedearlierbyDominskietal.,2003.Theyiden
tifiedaproteinbindingthehighlyconservedstemloopstructureofmetazoanhis
tonemRNAs.3hExocontainsaSAP(Kippetal.,2000)anda35exonucleasedo
main.Itbindsthe3terminalACCCAofthestemloopanddegradesit,unlessthe
histonemRNAisprotectedbythestemloopbindingproteinSLBP(Dominskietal.,
2003).TheconservedstemloopsequenceofhistonemRNAsisnecessaryforitsselec
tivedegradationandconfersthesamereactionwhenintroducedtoothermRNAsat
their3ends.The3hExoGFPproteinpredominatelyaccumulatedinthecytoplasm
andthenucleoli.DeletionoftheSAPdomainabolishedbindingof3hExotothe
stemloopRNAbuttheresidualexonucleaseexhibitedenzymaticactivity.There
placementofArg105intheSAPdomaineliminatedbindingtothestemloop.Asp234,
Asp298andMet235areindispensiblefortheenzymaticactivityof3hExo.Mutationof
thelatteraminoacidleadstoglobalstructuralchangesunabletobindthestemloop
(Yangetal.,2006).3hExorequiresaterminalhydroxylgroupandcannotprocess
RNAsterminatingwithaphosphategroup(Dominskietal.,2005).Itcanremovethe
2ntoverhangsandthefirstnucleotideofthedoublestrandedregionofthesiRNAsin
vitro(Yangetal.,2006).
Thecrystallographicstructureoftheexonucleasedomainof3hExoboundtorAMP,
areactionproductoftheenzyme,hasbeenresolvedataresolutionof1.6inthe
presenceofMg2+.Itiscomposedofasixstranded,twistedsheetwhichisbracketed
byninehelices(Cheng&Patel,2004).ThestructureissimilartoDnaQlike35
exonucleaseswhichusuallybindtoDNAandproducehydrolyticproductsreleasing
anucleotide5monophosphateandleavinga3hydroxylonthepenultimatenucleo
tide(Viswanathan&Lovett,1999).Theactivesiteiscomprisedoftheconserved52
1.IntroductionacidicDEDDmotifwhichbindstwomagnesiumions.Thesetwotogetherwiththe
conservedHistidineareindirectcontactwiththemonophosphateofrAMP.
TheexonucleasedomainappearstolackabindingpocketaccommodatingRNAs
longerthandinucleotides.ProbablytheSAP(SAFbox,AcinusandPIAS)
DNA/RNAbindingdomainpositionsthe3overhangswithintheactivesite(Cheng
&Patel,2004).
Figure1.11:Cheng&Patel,2004
ThepositioningoftherAMPsubstrateinthe3hExoreactioncenterrequirestwoMagnesiumions
andisconferredbytwohydrogenbonds.
Thehomologueof3hExoinDrosophilamelanogasterisnamedSnipper(Snp),ahighly
activeandpromiscuous35exonuclease.Ithasabroadsubstratespecificityandde
gradesinvitrosinglestrandedanddoublestrandedDNAandRNAwiththere
quirementofaminimal25nt3flank.Itdoesnotrequirea2OHforsubstraterec
ognition,catalysisorproductrelease.3hExoandERI1share38%sequenceidentity
and60%sequencesimilarity,Snpshares31%sequenceidentitywithERI1andhas
acharacteristicDEDDhmotif(compareFigure1.12).Snpisamoreefficientnuclease
than3hExotowardhistonestemloopRNAssinceitcanalsocleavethedouble
strandedstemportion.OnereasonmaybetheabsenceoftheSAPdomainwhich53
1.Introductionmightbindthestemintheotherhomologuesandtherebyprotectsitfromdegrada
tion.SnpcancleaveDNAsubstratesandisalsoabletodegradethestemportionof
theDNAhairpin.Theminimumlengthofthe3flankforassociationinmobilityshift
assayswasfoundtobeatleasttwonucleotides.HomozygousSnpmutantsshowed
noincreasedRNAifunction,italsodoesnotplayamajorroleintheclearanceof
apoptoticDNAinDrosophila(Kupscoetal.,2006).
InNeurosporacrassaRNAiisessentialforthedsRNAortransgene(quelling)induced
genesilencing,itscomponentsareQDE2,anArgonauteproteinassociatedwith
siRNAsandtwoDicerproteins.ThehypotheticalproteinQIPcopurifiedwithQDE
2andcontainsa35exonucleasedomainbelongingtotheDEDDhsuperfamily.In
QIPKOssiRNAlevelsweresignificantlyhigherthaninwildtype.Genesilencing
wasimpairedintheabsenceofQIP,suggestingthatQIPisessentialforfunctional
RNAi.ItisrequiredforefficientprocessingofsiRNAduplexes,butactingdown
streamofQDE2.siRNAduplexesfromqipKOswerelessstableandsinglestranded
at57C,unlikesiRNAduplexesfromqdeKOs,suggestingthatinqipKOsthe
siRNAshadalreadybeenpreprocessed.QIPprobablyfunctionsintheRISCactiva
tionprocessbyremovingthenickedpassengerstrandfromthesiRNAduplexes
(Maitietal.,2007).
InfissionyeastheterochromatinassemblyrequirestheRNAimachineryandisiniti
atedbysiRNAs.Theyarederivedfromheterochromaticregionsandprocessedby
theRNAinducedtranscriptionalsilencing(RITS)complexwhichcontainsAgo1,
Chp1andTas3.DeletionofEri1,thesingleS.pombeorthologue,causesanincreasein
siRNAsassociatedwiththeRITScomplexandenhancesheterochromaticsilencing.It
containsconservedSAPandDEDDhexonucleasedomainsandshowsmorethan30
%identitytoC.elegansERI1.ItdegradesdsRNAwith2ntoverhangsandtheRNA
moietyofRNADNAhybrids.InanelectrophoreticmobilityshiftassaytheSAP
domainefficientlybounddsRNAandRNADNAhybridsbutnotssRNA,dsDNAor
ssDNA.LossofEri1whichpredominatelylocalizestothecytoplasmdidnotaffect
normalcellulargrowthbuttheoverexpressionofEri1causedaseveregrowthdefect.54
1.IntroductionTheamountofcentromericsiRNAswasconsiderablygreaterthaninwildtypecells
(Iidaetal.,2006).NewlygeneratedsiRNAscanalsorecruitheterochromatinproteins
andinitiatedenovosilencingintrans,butthisintranssilencingisstronglyinhibited
byEri1(Bhleretal.2006).
InvivosubstratesofERI1inCaenorhabditiselegansandSchizosaccharomycespombe
havelongbeenpoorlyunderstooduntilthediscoverythat5.8SrRNAineri1null
mutantwormsislongerthaninwildtype.Atleastoneadditionalnucleotideatthe3
endcouldbedetectedinallmutantworms;asubstantialfractioncontainedtwoto
four.ThesamewasfoundforS.pombeeri1KOswherethe5.8SrRNAhadtwoto
eightadditional3nucleotides,suggestingacommonancestorforthisfunctionin
animalsandfungi.BothERI1isoformsrescuedthe5.8SrRNAlengthinvivo,but
onlyERI1bwasfunctionalinRNAirescuewhileitalsofailedtorescuetherRNA
processinginvitro.MostrRNAprocessingoccursinthenucleolus.Inthematureri
bosomethe3endof5.8Sispairedwiththe5endofthe2528SrRNA,reminiscent
ofthehistonemRNAstemloopandsiRNAstructures.MutationsinH317andD321
completelydisruptedthefunctionofC.elegansERI1(Gabel&Ruvkun,2008).
SuppressionofthemouseorthologueofERI1increasedtheeffectofRNAi.After
introductionofhighamountsofexogenoussiRNAs,mouseERI1andADAR1
(adenosinedeaminasesactingonRNAwhichconvertadenosineintoinosine)tran
scriptlevelsareincreased,maybeleadingtotheobservablereboundaftertheinitial
RNAiinducedtargetsuppression.(Hongetal.,2005).
InmiceERI1isubiquitouslyexpressed,withmaximainspleen,thymusandtestis.It
ispresentinthecytoplasm,nucleusandslightlyenrichedinthenucleolus.Thebirth
weightofEri1KOmiceisreducedandthisremainedsignificantinthe10%surviv
ingadultmice.Growthdefectswerealsoobservedforcellsculturedinvitro.ERI1
wasfoundtobindindependentlytoeachribosomesubunit(40Sand60S).Under
stringentlysisconditionsonly5.8SrRNAwasabletocoimmunoprecipitatewith
endogenousERI1.InEri1KOmicethe3endsof5.8SrRNAwerevariablewith1or
2nt3extensions.PointmutationsshowedthatthecatalyticallyinactiveD130andE13255
1.Introductionmutantsstillbound5.8SrRNA,whereaslinkerregionmutantsK107andK108showed
impairedbinding.TheSAPandlinkerdomainshavesupportivefunctioninrRNA
bindingbutarenotcrucialfor5.8SrRNAinteraction.WildtypeERI1wasableto
converttheabnormal5.8SrRNAofpurifiedribosomesinvitro.Thenaturallyoccur
ring5.8S28Sduplexissufficient,butefficientprocessinginvolvesinteractionwith
otherfeaturesoftheribosome(Anseletal.,2008).C. elegans H. sapiens
M. musculus D. rerio X. laevis D. melanogaster S. pombe Consensus
C. elegans H. sapiens M. musculus D. rerio X. laevis D.
melanogaster S. pombe Consensus C. elegans H. sapiens M. musculus
D. rerio X. laevis D. melanogaster S. pombe Consensus C. elegans H.
sapiens M. musculus D. rerio X. laevis D. melanogaster S. pombe
Consensus C. elegans H. sapiens M. musculus D. rerio X. laevis D.
melanogaster S. pombe Consensus (1) (1) (1) (1) (1) (1) (1) (1)
(99) (83) (79) (69) (80) (20) (27) (101) (196) (176) (172) (164)
(175) (113) (92) (201) (277) (256) (252) (244) (255) (205) (192)
(301) (376) (331) (327) (319) (330) (274) (262) (401) 1 100
MSADEPSPEDEKYLESLRDLLKISQEFDASNAKQNDEPEKTAVEVESAETRTDESEKSIDIPREQQLLPSERVEPLKSMVEPEYVKKVIR--QMDTMTAE
-------MEDPQSKEPAGEAVALALLESPRPEGGEEPPR--PSPEETQQCKFDGQET-----KGSKFITS----SASDFSDPVYKEIAITNGCINRMSKE
-------MEDERGRE---RGGDAAQQKTPRPECEESRP---LSVEKKQRCRLDGKET-----DGSKFISS----NGSDFSDPVYKEIAMTNGCINRMSKE
-------METKEKSR------------KPPNKTPQSEG-----DQEDQPCPDTSCEK-----NEDQEPSSP---KQGEFSDPVYKEIALANGAINRMNRE
-------MEEQKENRP-LDTEDSVVEEDLCKKLSRNLD----LVGVKQRCRFDGQED-----NGTSTVSS----NTSDFSDPVYKEIAIANGCVNRMTKD
---------------------------------------------------------------------------------MALIKLARQLGLIDTIYVD
--------------------------------------------------------------------------MESPVQILVWPFPCDEMNQKTPSTVE
MED Q CR D E ISS SDFSDPVYKEIAI NG INRMTKE 101 * * 200
QLKQALMKIKVSTGGNKKTLRKRVAQYYRKENALLNRKMEPNADKTARFFDYLIAIDFECTCVEIIY---DYPHEIIELPAVLIDVREMKIISEFRTYVR
ELRAKLSEFKLETRGVKDVLKKRLKNYYKKQ--KLMLKESNFADS---YYDYICIIDFEATCEEGNPP--EFVHEIIEFPVVLLNTHTLEIEDTFQQYVR
ELRAKLSEFKLETRGVKDVLKKRLKNYYKKQ--KLMLKESSAGDS---YYDYICIIDFEATCEEGNPA--EFLHEIIEFPVVLLNTHTLEIEDTFQQYVR
ELRAKCTELKLDTRGVNDVLRKRLKSYYKKQKLMHSPAAEGNSDM---YFDYICVVDFEATCEENNPP--DYLHEIIEFPMVLIDTHTLEIVDSFQEYVK
ELKAKLVEHKLDTRGVKDVLRKRLKNYYKKQKLTHALHKDSNTDC---YYDYICVIDFEATCEAGNSL--DYPHEIIEFPIVLLNTHTLEIEDVFQCYVR
GARPDPNNDPEESFNEDEVTEANSVPAKSKK-------SRKSKRLAMQPYSYVIAVDFEATCWEKQAPPEWREAEIIEFPAVLVNLKTGKIEAEFHQYIL
EIRIALQELGLSTNG-----------------------NK---------R-YLLIVDVEATCEEGCGF--SFENEIIELPCLLFDLIEKSIIDEFHSYVR
ELRAKL E KLETRGVKDVLRKRLKNYYKKQ D YYDYICIIDFEATCEEGN DF
HEIIEFPVVLLNTHTLEIED FQ YVR 201 * * 300
PVRNPKLSEFCMQFTKIAQETVDAAPYFREALQRLYTWMRKFN-------------------LGQKNSRFAFVTDGPHDMWKFMQFQCLLSNIRMPHMFR
PEINTQLSDFCISLTGITQDQVDRADTFPQVLKKVIDWMKLKE-------------------LGTK-YKYSLLTDGSWDMSKFLNIQCQLSRLKYPPFAK
PEVNAQLSEFCIGLTGITQDQVDRADAFPQVLKKVIEWMKSKE-------------------LGTK-YKYCILTDGSWDMSKFLSIQCRLSRLKHPAFAK
PVLHPQLSEFCVKLTGITQEMVDEAKTFHQVLKRAISWLQEKE-------------------LGTK-YKYMFLTDGSWDMGKFLHTQCKLSRIRYPQFAR
PEINPQLSEFCVNLTGITQDTVDKSDTFPNVLRSVVEWMREKE-------------------LGSK-YKYAILTDGSWDMSKFLNMQCRISRLKYPRFAK
PFESPRLSAYCTELTGIQQKTVDSGMPLRTAIVMFNEWLRNEMRARNLTLPKMN--------KSNILGNCAFVTWTDWDFGICLAKECSRKGIRKPAYFN
PSMNPTLSDYCKSLTGIQQCTVDKAPIFSDVLEELFIFLRKHSNILVPSVDEIEIIEPLKSVPRTQPKNWAWACDGPWDMASFLAKQFKYDKMPIPDWIK
P INPQLSEFCI LTGITQDTVDKA F QVLKKVIEWMR KE LGTK YKYAFLTDGSWDMSKFL
QCKLSRIKYP FAK 301 * 400
-SFINIKKTFKEKFNGLIKGNGKSGIENMLERLDLSFVGNKHSGLDDATNIAAIAIQMMKLKIELRINQKCSYKENQRSAARKDEERELEDAANVDLTSV
-KWINIRKSYGNFYKVPRS---QTKLTIMLEKLGMDYDGRPHCGLDDSKNIARIAVRMLQDGCELRINEKMHAGQ---------------------LMSV
-KWINIRKSYGNFYKVPRS---QTKLTIMLEKLGMDYDGRPHSGLDDSKNIARIAVRMLQDGCELRINEKILGGQ---------------------LMSV
-KWINIRKSYGNFYKVPRT---QTKLICMLENLGMEYDGRPHCGLDDSRNIARIAIHMLKDGCQLRVNECLHSGE---------------------PRSV
-KWINIRKSYGNFYKVPRT---QTKLTTMLEKLGMTYNGRLHSGLDDSKNIARIAAHMLQDGCELRVNERMHAGQ---------------------LMTV
-QWIDVRAIYRSWYKYRPCN-----FTDALSHVGLAFE