UniversityofNaturalResourcesandAppliedLifeSciences DepartmentofAppliedGeneticsandCellBiology

TheputativeRNAsilencingprotein ERL1isinvolvedinchloroplast ribosomalRNAprocessinginplants

Doctoratethesis submittedby DIJuttaMariaHelm March,20111

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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

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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

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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

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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

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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

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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

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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

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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

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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/

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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.

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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.

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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

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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.

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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