Zalpha-domains: At the intersection between RNA editing and innate immunity

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SeminarsinCell&DevelopmentalBiologyxx(2011)xxx–xxx

ContentslistsavailableatScienceDirect

Seminars

in

Cell

&

Developmental

Biology

j ou rn a l h o m e pa g e :w w w . e l s e v i e r . c o m / loc a t e / s e m c d b

Highlights

SeminarsinCell&DevelopmentalBiologyxx (2011)xxx–xxx

Zalpha-domains:AttheintersectionbetweenRNAediting andinnateimmunity

AlekosAthanasiadis∗

ITheAtoIRNAeditingmodiﬁcationhasaregulatoryroleininnateimmunity.ITheRNAeditingenzymeADAR1increasesviralproliferation. IDistinguishingfeatureofADAR1isaZ-DNA/Z-RNAbindingdomain,Zalpha.IZalphadomainscharacterizeproteinsoftheinterferon responseinvolvedintherecognitionofforeignnucleicacids.

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Pleasecitethisarticleinpressas:AthanasiadisA.Zalpha-domains:AttheintersectionbetweenRNAeditingandinnateimmunity.SeminCell DevBiol(2011),doi:10.1016/j.semcdb.2011.11.001

ARTICLE IN PRESS

GModel YSCDB12701–6

SeminarsinCell&DevelopmentalBiologyxxx (2011) xxx–xxx

ContentslistsavailableatSciVerseScienceDirect

Seminars

in

Cell

&

Developmental

Biology

j our na l h o me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / s e m c d b

Review

1

Zalpha-domains:

At

the

intersection

between

RNA

editing

and

innate

immunity

2

Alekos

Athanasiadis

∗

Q1

3

InstitutoGulbenkiandeCiência,RuadaQuintaGrande6,2780-156Oeiras,Portugal

4 5

a

r

t

i

c

l

e

i

n

f

o

6 7 Articlehistory: 8 Available online xxx 9 Keywords: 10 DNAstructure 11 AtoIRNAediting 12 Interferonresponse 13 ADAR1 14 Zalpha 15 Z-DNA 16

a

b

s

t

r

a

c

t

TheinvolvementofAtoIRNAeditinginantiviralresponseswasﬁrstindicatedbytheobservationof

genomic hyper-mutationforseveralRNAvirusesinthecourseofpersistentinfections.However,inonly afewcasesanantiviralrolewaseverdemonstratedandsurprisingly,itturnsoutthatADARs–theRNA editingenzymes–mayhaveaprominentpro-viralrolethroughthemodulation/down-regulationofthe interferonresponse.AkeyroleinthisregulatoryfunctionofRNAeditingisplayedbyADAR1,aninterferon inducibleRNAeditingenzyme.AdistinguishingfeatureofADAR1,whencomparedwithotherADARs, isthepresenceofaZ-DNAbindingdomain,Zalpha.Sincetheinitialdiscoveryofthespecificandhigh affinitybindingofZalphatoCpGrepeatsinaleft-handedhelicalconformation,otherproteins, allrelated totheinterferonresponsepathway,wereshowntohavesimilardomainsthroughoutthevertebrate lineage.Whatisthebiologicalfunctionofthisdomainfamilyremainsunclearbutasignificantbodyof workprovidespiecesofapuzzlethatpointstoanimportantroleofZalphadomainsintherecognitionof foreignnucleicacidsinthecytoplasmbytheinnateimmunesystem.Herewewillprovideanoverview ofourknowledgeonADAR1functionininterferonresponsewithemphasisonZalphadomains.

Contents

17

1. Introduction... 00

18

2. LinksbetweenAtoIRNAeditingandimmunity... 00

19

2.1. Modiﬁcationofviralnucleicacids... 00

20

2.2. ADAR1asaregulatorofantiviralresponses... 00

21

3. TheADAR1Zalphadomain... 00

22

3.1. TheADAR1Zalphadomainstructureanditsnucleicacidscomplexes... 00

23

3.2. InterferonresponserelatedproteinswithZalphadomains... 00

24

3.3. Thenon-conformingZbeta... 00

25

4. InsearchfortheZalphadomaintargetanditsbiologicalfunction... 00

26

4.1. NucleicacidsboundbyZalphainvivo... 00

27 5. Concludingremarks... 00 28 Acknowledgements... 00 29 References... 00 30 31 1. Introduction 32

Inbiologywhatyouseeisnotalwayswhatyouget.The dis-33

coveryofRNAeditinginXenopuseggsin1988[1]andextensive 34

research since have challenged a deeply rootedbelief that the 35

Abbreviations:ADAR,adenosine deaminase actingonRNA;AtoI,adenosineto inosine;PKR,proteinkinaseRNAdepended;DAI,DNAdependedActivatorofIRFs; IFN,interferon;CNS,centralnervoussystem;eIF2a,eukaryoticinitiationfactor2a; HIV,humanimmunodeﬁciencyvirus.

∗ Tel.:+351214464648;fax:+351214407970. E-mailaddress:alekos@igc.gulbenkian.pt

sequence of transcribed nuclear RNA is a faithful copy of the 36 genomicsequence.ThisRNAmodificationwasfoundtobepresent 37 inallmetazoansand wastermedAtoIRNAeditingbecauseit 38 consistsin thehydrolyticdeaminationofadenosines (A)toino- 39 sine(I)withinRNAs.Inosinebase-pairswithcytosineinsteadof 40 thymineandsoforanybiologicalprocessthatinvolvesrecognition 41 oftheRNAsequencethroughbasepairing,inosineisanequivalent 42 ofguanosine.Asaconsequence,AtoIeditingisabletorecode 43 aminoacidsequences[2],toalter splicesites[3], createmiRNA 44 bindingsitesandchangethespecificityofmiRNAsthemselves[4]. 45 ResponsibleenzymesforthisRNAmodificationareadenosine 46 deaminases called ADARs that show limited target sequence 47

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2 A.Athanasiadis/SeminarsinCell&DevelopmentalBiologyxxx (2011) xxx–xxx speciﬁcity[5]. Instead, theirspeciﬁcity is determinedprimarily

48

bythesecondarystructureoftheRNAsubstrate[6].WhileADARs 49

modifyaperfect RNAduplex ina promiscuousmannerleading 50

upto50%ofmodiﬁedadenosines,themodiﬁcationcanbehighly 51

speciﬁctowardscomplexRNAstructurescontainingbulges and 52

internalloops[6]. 53

ThediscoverythatthisRNAmodiﬁcationleadstofunctionally 54

criticalaminoacidchangesinvertebrateneurotransmitter

recep-55

torsliketheGluR-2,GluR-5and-6subunitsofAMPAreceptors,

56

theSerotonin-2C(reviewedin[2,7])andthe␥-aminobutyricacid 57

(GABA)receptors[8]aswellasininvertebratechloridechannel 58

subunits[9],nicotinicacetylcholinereceptor(nAChR)subunits[10]

59

andseveralotherproteinsinvolvedinfastsynaptictransmission

60

[11],ledtothenotionthattheprimaryroleofAtoIRNA edit-61

ingistoprovidethemuchneededdiversityintheproteomeof 62

theanimalcentral nervoussystem(CNS).Thisnotionhoweverleft 63

unexplainedtheobservedabundanceofRNAeditingactivity out-64

sideoftheCNS. 65

Indeed,inrecentyearstranscriptomeanalysishasshownthat 66

ADARs,theAtoIRNAeditingenzymes,areresponsible for exten-67

sivemodiﬁcationof3UTRandintronicsequencesofthousandsof

68

humangenes[12–15]targetingsecondarystructuresoftencreated 69

bypairsofinvertedrepetitiveelements. Theseﬁndingsindicate

70

thatanyextensiveand stablepre-mRNAstem-loopstructure is 71

accessibletoADARsand apotential targetfor RNAediting and

72

haveledtoawiderethinkingregardingtheprimaryroleofthis 73

post-transcriptionalmodiﬁcation. 74

Double stranded RNA in the cytoplasm is known to repre-75

sentadangersignalindicatingviralinfectionandseveralantiviral 76

pathwaysaretriggeredbydsRNAincludingtheRNAinterference 77

pathwayininvertebrates[16]andtheinterferonresponsein ver-78

tebratecells[17].ThroughtheintroductionofmismatchesinRNA 79

duplexes, A toI RNA editing canreduce theirdouble stranded 80

natureandmarksuchduplexeswithInosine.CouldnuclearAto 81

IRNAediting preventtheexposureofcellularmRNAs

contain-82

ingdsRNAstructurestosuchantiviralpathways?IsRNAediting 83

inthecytoplasmresponsibleforlimitingthedurationand/orthe 84

intensityofactiveantiviralresponses?WhileevidencelinkingA 85

toIRNAeditingtoantiviralresponsesdatesbacktoitsdiscovery, 86

intherecentyearsseverallinesofresearchconvergeonan unex-87

pectedrolefortheeditingmachineryinthemodulationofcellular 88

responsestoforeignnucleicacids. 89

2. LinksbetweenAtoIRNAeditingandimmunity 90

Theﬁrstevidencefor a roleof AtoIRNA editingin immu-91

nitycamewiththediscoveryofmodiﬁcationsinseveralviralRNA 92

genomesand viraltranscripts.Thesemodiﬁcations wereshown 93

totaketheformeitherof genomichyper-editing inthecourse 94

ofpersistentviralinfectionsormorespeciﬁcRNAeditingevents 95

instructuredviralmRNAs.Asecondandindependentlineof evi-96

dencefora roleofRNAeditingininnateimmunitycomesfrom 97

workimplicatingtheinterferoninducibleADAR1editingenzyme 98

intheregulationoftheantiviralinterferonresponse. 99

2.1. Modiﬁcationofviralnucleicacids 100

Viral nucleic acids have been among the ﬁrst known sub-101

stratesofADARs:thediscoveryofhyper-mutatedRNAgenomes 102

ofmeaslesvirus[18]andothermembersoftheparamyxoviridae 103

familywasfollowedbysimilarobservations forVesicular Stomati-104

tisVirusandmRNAsofthemousepolyomaDNA-virusreviewedin 105

[19–21].Morerecentlysimilarhypermutationwasobservedinthe 106

invertebratesigmaRNA-virus[22]involvingtheADARenzymeof 107

drosophilids.Theseﬁndingsweretakentosuggestanantiviralrole 108

ofADARmediatedRNAediting,mechanisticallybasedonscram- 109 blingtheinformationcontentofviralnucleicacids,notunlikethe 110 roleoftheAPOBEC3 familyofcytidine deaminasesinretroviral 111 restriction.However,inonlyfewcasesanantiviralroleofADARs 112 hasbeendemonstratedand even inthesecases itstill remains 113 unclearifthisisadirectresultofhyper-editingofviralnucleicacids 114 ortheresultoftheinﬂuenceofADARsonelementsoftheinnate 115 immunesystem.Indeed,knockdown ofADAR1in anADAR2−/− 116 backgroundincellcultureisshowntoalterthecytopathiceffects 117 ofpolyomavirusinfection[23]inamannerindependentofthe 118

modiﬁcationofviralRNA. 119

Specificeditingwithinawell-definedsequenceandstructure 120 contextofviralnucleicacidswasfirstdemonstratedforHepati- 121 tisDvirus(HDV)[24].Surprisingly,HDVwasshowntomakeuse 122 ofthecellularRNAeditingmachineryforitsownneeds:a stop 123 codoninHepatitisdeltaantigenisbeingchangedtoatryptophan 124 codonbyRNAeditingallowingtheswitchfromthereplicationstage 125 tothe packagingstage, thusenabling, viral proliferation.Inter- 126 estingly, HIV replication is alsoshown to beenhanced in cells 127 over-expressingADAR1,andHIVtranscriptsofRevandTatcod- 128 ingsequencesaswellastheTARRNAareshowntobeeditedby 129 ADAR1.However,itremainsunclearwhethertheincreasedrepli- 130 cationiseditingdepended[25–27].SpecificRNAeditinghasalso 131 beenfoundintranscriptsoftheKaposisarcomaassociatedvirus 132

(KSHV)[28,29],whereagainRNAeditingappearstobeadaptedby 133 thevirusasthelevelsofeditingcorrelatewiththereplicativestate 134

ofthevirus[29]. 135

Insummary,forboththehyper-editingandthemorespeciﬁc 136 editingeventsobservedinviralnucleicacidsthereisnoclearevi- 137 dencethatthesebasemodiﬁcationsoverallrepresentanantiviral 138 actionofADARs.Onthecontrary,forviruseslikeHepatitisD,RNA 139 editingrepresentsavaluablecellularcontribution. 140

2.2. ADAR1asaregulatorofantiviralresponses 141

In vertebrate specieswe ﬁndtwo genesencoding for active 142 dsRNA dependent adenosine deaminases: ADAR1 and ADAR2. 143 ADAR1 was the ﬁrst to be discovered and expression of an 144 N-terminallyextendedformoftheproteinwasshowntobeup- 145 regulated during viral infection. Indeed, ADAR1 comes in two 146

isoforms:ashort,constitutivelyexpressedandnuclearform(P110) 147 andalonger,mainlycytoplasmicform(P150)whichistranscribed 148 fromadifferentpromoterresponsivetotypeIandtypeIIinter- 149

ferons [30] (Fig.1A). The homozygous knockoutof the ADAR1 150 geneinmiceisembryoniclethalanditsphenotypiccharacteriza- 151 tionshowsthatitslethalityisassociatedwithextensiveapoptosis 152 in thehematopoietic tissue [31,32]. Furthercharacterization of 153 hematopoieticstemcellsderivedfromaninducibleADAR1gene 154 knockoutinmiceshowsaglobalupregulationofinterferonrespon- 155 sivegenesduringembryonicdevelopment[33]pointingtoaroleof 156 ADAR1asasuppressorofinterferonsignaling.Selectiveknockout 157 oftheinterferoncontrolledisoformP150[34]suggeststhatthisiso- 158 formisresponsiblefortheobservedembryoniclethality,although 159

thereisanongoingdebateonthismatter. 160

The mechanism through which ADAR1 exerts control over 161 innateimmuneresponseshasnotbeenyetclariﬁed.Clueshowever, 162

havebeenobtainedinstudiesshowingthatADAR1canantago- 163 nizeprotein kinaseR(PKR)[35,36],a keyprotein ofinterferon 164 responsethatmediatesshutdownofcellulartranslationthrough 165 thephosphorylationof eukaryoticinitiationfactoreIF2a during 166 infection.PKRisactivatedbydsRNAandADAR1caninhibititsacti- 167 vationeitherthroughthesequestrationofdsRNAorbyrendering 168 suchdsRNAunrecognizablebyPKRthroughmodiﬁcation.Indeed, 169

ectopicADAR1expressionresultsinageneralincreaseinprotein 170 translationthroughtheinhibitionofPKR[37,38]andpossiblyof 171 otherdsRNAactivatedeffectorproteinsoftheinterferonresponse 172

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Fig.1.DomainorganizationofproteinswithZalphamotifs(A).ADAR1isoformsP150andP110areindicated.RBDstandsfordsRNAbindingdomain.Thedomainorganization ofPKRisshownforcomparisonwithPKZ.ConservedinteractionsbetweenZalphadomainsandZ-DNA(B).Ty177,Asn173andTrp195positionedtorecognizethecharacteristic Z-DNAphosphatebackboneandaguanosineinSynconformation.

suchas the 2-5-oligoadenylate synthetase(OAS). A key ques-173

tionthatarisesiswhetherandtowhatextendcellulartranscripts 174

containingdsRNAcanerroneouslyactivatedsRNAdependent path-175

waysandwhethertheobservedextensiveeditinginsuchcellular 176

RNAduplexescanhaveapreventiverole.Experimentsaimingto 177

answerthisquestionshowthat asingletranscript containinga 178

longdsRNAhairpin activatesinterferonresponseonlyat

abnor-179

mallyhighexpressionlevels[39].However,giventhatthereare 180

possiblythousandsofgenesproducingtranscriptswithembedded 181

dsRNAstructures,theactivationofinterferonresponsebycellular 182

RNAsseemspossible. 183

AnotherdsRNAdependentpathway thatactsas anantiviral 184

responseininvertebratesbutalsopresentinvertebrates,isRNA 185

interference(RNAi).AninteractionbetweenADARsandtheRNAi 186

pathwaywasﬁrstsuggestedinexperimentsinCaenorhabditis ele-187

gansshowingthattheADARknockoutphenotypecanberescued 188

bytheablationofcomponentsoftheRNAimachinery[40].More 189

recentworkalsoinC.elegansprovidessupportforaspeciﬁcrole 190

ofADARstoprotectanextensivepopulationofdsRNAincellular 191

transcriptsfromRNAi[41].RNAiofdsRNAinmammaliancellular 192

transcriptshasbeenrecentlydemonstratedinoocytes[39]aswell 193

astheabilityof ADARs,andinparticularofcytoplasmicADAR1 194

P150tobindsiRNAandlimittheefﬁcacyoftheRNAipathway[42]. 195

Thus,invertebratesADAR1isshowntoantagonizebothRNA 196

interferenceandtheinterferonresponse.Indoingso,ADAR1may 197

limitdsRNAdependentresponsesnotonlyagainstcellularRNAbut 198

alsoagainstviralRNA.Indeed,inasurveyfortheeffectsof380 199

IFN-regulatedgenesonviralproliferation,isshowntosigniﬁcantly 200

enhancethereplicationratesofalltestedviruses[43]supportinga 201

pan-viralmechanismofenhancement. 202

Anattractivemodelbasedontheavailabledataascribesadual 203

function of ADAR1: in the nucleus, it keeps under control the 204

amountofcellulardsRNAthatcanreachdsRNAsensorsinthe cyto-205

plasmwhileuponprolonged activationoftheinterferonpathway 206

the P150 isoform migrates to the cytoplasm where it down-207

regulatesantiviralresponsesbylimitingtheavailabledsRNA.This 208

modelestablishesADAR1asacentralnodewithinafeedbackmech- 209 anismthatlimitsrunawayantiviralresponsesandtheircytopathic 210

effects. 211

WhatarethemoleculardetailsofADAR1actioninthecytoplasm 212 andwhatareitsinteractions withthecytoplasmicnucleicacids 213 sensors?Theanswertothesequestionsmustlargelybefoundatthe 214 296aminoacids(inhumans)longextensionattheN-terminusof 215 theinterferoninducibleP150isoform.Theuniquerecognizableand 216 conservedfeatureofthissegmentofADAR1isaDNA/RNAbinding 217 domainofsofarunknownbiologicalfunction,theZalpha. 218

3. The ADAR1Zalphadomain 219

TheN-terminaldomainofADAR1wasthesurprisingyieldofan 220 invitroscreenforZ-DNAbindingdomainsinbloodnuclearextracts 221

[44]andfromthisdiscoveryderivesitsname:Zalpha.Itwasshown 222 thatthis78aadomainofADAR1bindswithhighafﬁnity[45]toCpG 223 repeatsandthatthebounddsDNAformsaleft-handedZ-DNAhelix, 224

analternativeDNAconformationoriginallydescribedmorethan30 225 yearsago[46].WhileinvitroformationofZ-DNArequireshighsalt 226 concentrations,theZalphadomainisshowntostabilizethiscon- 227 formationonCpGrepeatsunderphysiologicalsaltconcentrations. 228 ThismakesZalphatheﬁrstknownspeciﬁcZ-DNAbindingprotein 229

domain. 230

3.1. TheADAR1Zalphadomainstructureanditsnucleicacids 231

complexes 232

The crystal structure of the ADAR1 Zalpha domain with a 233 T(CG)3dsDNAcomplexshowedthatitbelongstothelargerfam- 234 ilyofwinged-Helix-Turn-Helixdomains(wHTH),acommonDNA 235 bindingmotifusedinthiscaseinauniqueconﬁguration [47].A 236 keyroleintherecognitionoftheZ-DNAbackboneisplayedbya 237 tripletofresidues:Tyr177,Asn173andTrp195(allaminoacidref- 238 erencesarebasedonhumanADAR1-Zalphanumbering),whichare 239

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4 A.Athanasiadis/SeminarsinCell&DevelopmentalBiologyxxx (2011) xxx–xxx

a␲-bondwiththeguanosineintheSynconformation character-241

isticforZ-DNA,theonlydirectbasecontactmadebytheprotein 242

andwhichcontributestothediscriminationbetweentheB-DNA 243

andZ-DNAconformations.Mutationofanyresidueinthistriplet 244

eliminatesDNAbinding[48].Theremainingprotein/DNAcontacts

245

arewiththecharacteristicallyshapedZ-DNAphosphatebackbone, 246

allowingfurtherdiscriminationofthetwoDNA-conformers. Inter-247

estingly,twoZalphadomainsinteractwiththeDNAduplexwithout

248

formingaphysicaldimersuggestingacooperativemodeof DNA-249

binding,afeature thatis sofarseen inall structuresofZalpha 250

domains. 251

FormationofZ-DNArequires adinucleotiderepeatedunit of 252

alternatingpurine/pyrimidinebasessuggestingthatZalphacould 253

interactwithCA/TGrepeatsinadditiontoCG.Suchrepeats how-254

evershowlowerpropensityofZ-DNAformationandtheco-crystal 255

structure of Zalpha with these type of sequences had to wait 256

untilrecently[49].HowcanZalphabindCpGrepeatsinthe con-257

textofa largerDNAsegmentthatcannotbeentirelyintheleft 258

handedconformation? The answerto this question came with 259

thecrystalstructureofaZalphadomainboundtoaB–Zjunction 260

formedattheboundariesofthelefthandedhelix[50] demonstrat-261

ingthatsuchjunctions areformedbytheextrusionofa single 262

basepairatthejunctionsite.Similarly,Z–Zjunctionsareformed 263

whenZalphabindstoimperfectCpGrepeatswithdisruptionsof 264

thedinucleotiderepeat[51].Thesecrystal structuresdocument 265

themoleculardetailsonhowZalphadomainsareabletobind Z-266

DNAformingsegmentswithinnaturalDNAmoleculesofvariable 267

sequence. 268

CpGrepeatsindsRNAalsoadoptaleft-handedhelical confor-269

mation[52] knownas Z-RNA and consequently,this raisesthe 270

possibilitythattheinvivotargetofZalphadomainscouldbedsRNA. 271

Indeed,CD-spectroscopyconﬁrmedtheinteractionofZalphawith 272

RNA[53]andthecrystalstructureofADAR1ZalphawithanRNA 273

(CG)3oligonucleotiderevealedaZ-RNAstructure[54]with

signif-274

icantsimilaritiestoZ-DNA,makingZalphaoneoftherareifnot 275

uniquedomains capabletobinddsDNAand dsRNAofthesame 276

sequence. 277

Thisextensivestructuralanalysisof therecombinant ADAR1 278

Zalphadomaindemonstratedaconsistentbehaviorandconserved 279

interactionsofthedomainwithCpGsequencesinavariablenucleic 280

acidscontext. 281

3.2. InterferonresponserelatedproteinswithZalphadomains 282

SequencebasedidentiﬁcationofZalphadomainsinother pro-283

teinsledtothedelineationoftheZalphadomainfamily(SMARTID: 284

SM00550),asubfamilyofthewinged-Helix-Turn-Helixdomains. 285

Interestingly, all proteins sharing this motif were found to be 286

involvedintheinterferonresponsepathway,andparticularly com-287

ponentsofthisantiviralpathwaythatrelatetotheintracellular 288

recognitionofforeignnucleicacids.TwoZalphadomainsarefound 289

inDNA-dependentactivatorofIFN-regulatoryfactors(DAI),a cyto-290

plasmic receptor for foreign nucleic acids and activator of the 291

productionoftypeIinterferons,inZ-DNAdependentproteinkinase 292

(PKZ),aﬁshorthologofthemammalianPKR,andthepox-viral 293

inhibitorofinterferonresponseE3L(Fig.1A). 294

Intriguingly,isolatedZalphadomainsfromeachofthese pro-295

teinswerenotonlyshowntosharehighafﬁnityforCpGrepeats 296

butalsoahighly conservedmodeofinteractionwithZ-DNA as 297

demonstratedbythecrystalandNMRstructuresofbothDAIZalpha 298

domains[55–57],ofYatapoxvirusE3L[58]andbiochemicaldata 299

fromtheZalphadomainofPKZ [59].Suchresultspointtoa com-300

monfunctionofZalphadomainsandafunctionalinterconnection 301

oftheproteinsthatcontainthem. 302

DAI(also knownasDLM-1and ZBP-1) wasshown toactas 303

asensorfor dsDNAinthecytoplasmactivatedbyTpAand toa 304

lesserextentbyCpGrepeats[60].AthirdDNAbindingdomain(D3) 305 wasshowntobeprimarilyresponsiblefortherecognitionofTpA 306 repeatsanditstwoZalphadomainsexpectedlyplaynoroleinits 307 abilitytobindB-formDNA,neverthelesstheyappeartoberequired 308 fortheactivationofthesignalingpathwaythroughtheinterferon 309 regulatoryfactor 3(IRF3)[61].ThefunctionofDAIseemstobe 310

specialized,playingrolesinspeciﬁccelltypesandforviralinfec- 311 tionsbycytomegalovirus[62]andHerpessimplexvirus[63]but 312 notforexamplebyintracellularbacteriaasLegionellapneumophila 313

[64]. 314

PKZisanothernucleicacidssensorwithtwoN-terminalZal- 315 phadomains.Thisproteinwasfoundsofaronlyinfishspecieslike 316 zebrafish,Atlanticsalmonandgoldfishandisanorthologofthe 317 dsRNAdependentproteinkinasePKR.PKZshowsaconservedand 318 functionalkinasedomainbutthedsRNAbindingdomainsfound 319 inPKRarereplacedbyZalphadomains[59].Thiskinaseappears 320 tomaintaintheabilitytophosphorylateeIF2a likePKR[65]andto 321 blockcellulartranslation.UnlikePKRhowever,itsactivitydepends 322 onthepresenceofCpGdsDNAandnotpolyI:CdsRNA[65].While 323 bothDAIandPKZappearresponsivetoCpGDNAtheabilityofCpG 324 dsRNAtoactivatetheseproteinshasnotbeentested,leavingopen 325 thepossibilitythatthesesensorproteinsmayrespondtobothDNA 326

andRNA. 327

GiventheroleofZalphadomainsinantiviralresponses,itshould 328 notcomeasasurprisethataZalphadomainisfoundinE3L,aviral 329 inhibitorofinterferonresponse.E3Lisasmalltwo-domainprotein 330 foundinallpoxviruseswhoseC-terminaldsRNAbindingdomain 331 blocksPKRactivationandisrequiredforviralproliferation.ItsN- 332 terminalZalphadomainappearsdispensableforviralreplicationin 333 cellculturebutcrucialforpathogenicityinmice[66].Interestingly, 334

itisshownthatreplacementoftheE3LZalphadomainwiththat 335 ofADAR1orDAIfullysupportsviralpathogenicity,suggestingthat 336 Zalphadomainsarefunctionallyinterchangeable[66].TheE3LZal- 337 phadomaincouldfunctionthroughcompetitionfornucleicacids 338 withDAIsimilartothemechanismproposedfortheinhibitionof 339 PKRbyitsC-terminaldsRNAbindingdomain.Indeedmicro-array 340 analysisshowsthattheZalphadomainofE3Lblocksadistinctpart 341 oftheinterferonsignalingcascade[67]whichoverlapswiththe 342 IRF3-TANK-bindingkinase1(TBK1)signalingpathwayshownto 343

beactivatedbyDAI[60]. 344

3.3. Thenon-conformingZbeta 345

TheuniformbehaviorofZalphadomainshasasingleexception: 346 AdomainhomologoustoZalphais alsopresentintheconstitu- 347 tivelyexpressedisoformofADAR1.ItwasnamedZbetareflecting 348 itspositionwithinADAR1.Thisdomaininisolationdidnotdemon- 349 strateanyaffinityforCpGrepeatsandinmammalianADAR1Zbeta 350 domains,thecriticalaminoacidscorrespondingtoZalphaTyr177 351 andAsn173aremutated.ThecrystalstructureofZbeta[68]con- 352 firmed that this domainsharesthesame architecturewiththe 353 Zalphadomainbut italsoshows that Zbetacannotform inter- 354 actionswithDNAequivalenttoZalpha.Itisunfortunatethatin 355 proteinsthat containmorethan one Zalphadomain(DAI,PKZ) 356 thetermZbetahasbeenhistoricallyusedtodescribethesecond 357 domainalthoughthemammalianADAR1Zbetaappearstohave 358 uniquepropertiesnotsharedbyanyothermemberofthefamily. 359 Indeed,unliketheADAR1ZbetadomainthesecondZalphadomain 360 ofDAIdoesinteractwithCpGrepeatsasitisdescribedinthestruc- 361

turesofitscomplexeswithDNA[56,57]. 362

4. InsearchfortheZalphadomaintargetanditsbiological 363

function 364

Despitetheveryextensiveandsuccessfulworkcharacterizing 365 thenucleicacidsbindingpropertiesofZalphadomains,theinvivo 366

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target(s)ofthese domainsremainelusive. Thisispartly dueto 367

thedynamicandtransientnatureofZ-DNAformationandpartly 368

becausetheinvolvementofthesedomainsininnateimmunity sug-369

geststhattheirnucleicacidstargetmayonlybepresentduring 370

speciﬁchost–pathogeninteractions.Theinformationavailableso 371

farcomesfrominvivocrosslinkingexperimentsandtheeffects 372

ofmutationsoftheZalphadomainsonthelocalizationofthe pro-373

tein. 374

4.1. NucleicacidsboundbyZalphainvivo 375

Pull-downexperimentsofgenomicDNAsegmentsprobedfor 376

Zalphabindingrevealanenrichmentincentromericrepeats[69]

377

andprobablyreﬂectregionsofZ-DNAformationratherthana func-378

tionalassociationbetweenthemostlycytoplasmicproteinsand 379

centromers. 380

Similarexperiments where Zalpha is cross-linked in vivoto

381

cellular RNA identiﬁed speciﬁc regions of ribosomal RNA of 382

bothbacterialandeukaryoticribosomes[70]associatedwiththe 383

domain.Zalphamediated bindingof ADAR1to theribosomeis 384

showntoinhibittranslationandtheauthorsproposethatthe trans-385

lationofADAR1-boundmRNAsmaybespeciﬁcallyinhibitedthis 386

way.Suchaninterferencewiththetranslationmachinerywould 387

beinagreementwiththeﬁndingthattheZalphadomainofDAI 388

isresponsibleforanassociationofDAIwithstressgranulesthat 389

containstalledtranslationpreinitiationcomplexes[71].Howsuch 390

actioncouldbereconciledwithaproviralroleofADAR1andthe 391

generalupregulationoftranslationthroughthemodulationofPKR 392

activityremainstobeseen. 393

TheaccumulationofnegativesupercoilingintroducedbyRNA 394

polymerases during transcription plays a key role in the sta-395

bilization of Z-DNA in vivo [72]. In recent years it has been 396

shownthatdetectionofcertaintypesofdsDNAinthecytoplasm 397

proceedsthroughitstranscriptionbyRNApolymeraseIIIand sub-398

sequentrecognitionoftheresulting5-triphosphate-RNAbythe 399

RIG-Ihelicase[73].ItisconceivablethatPolIIItranscriptioncan 400

bethesourceofnegative supercoilingthat gives risetoZ-DNA 401

formationandthesiteofattachmentforZalpha-containing pro-402

teins.AlthoughE3LisshowntoinhibitRIG-Imediatedactivation 403

of interferons against poly-AT DNA, this activityappears tobe 404

independentfromitsN-terminalZalphadomain[74].Thestudy 405

of othertypes of DNA activatorsequences maybenecessaryto 406

reveal an involvement of Zalpha domains on the Pol III path-407

way. 408

5. Concludingremarks

409

Theuniversalchemicalandstructuralnatureofnucleicacids 410

presentsagreatchallengeforcellsthathavetodistinguishbetween 411

selfandnon-self.CpGsequencesandtheiruniquestructural fea-412

turesappeartohaveacentralroleinthisdistinctionbothforToll 413

mediatedrecognitionandforcytoplasmicreceptorswhereZalpha 414

domainsmayhaveacentralrole.Deregulationofthisrecognition 415

andoftheRNAeditingprocessnotonlycanaffecttheprogress 416

ofpathogen-induceddiseasebutmayalsohaveacrucialrolein 417

autoimmunediseasesandcancer. 418

Acknowledgements

419

I thank Dr. Vasco Barreto, Krzysztof Kus and Dr. Matteo 420

deRosa for valuable comments onthemanuscript. The author 421

would like to acknowledge funding support from the Marie 422

CurieIRGprogram[PIRG03-GA-2008-23100]andthePortuguese 423

Foundation for Research and Technology (FCT) [PTDC/BIA-424

PRO/112962/2009].

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