Localized mRNA translation mediates maturation of cytoplasmic … · 2020-04-21 · 2 spermatogenesis 3 4 Short Running title: mRNA localization in sperm cilia maturation 5 6 Jaclyn

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LocalizedmRNAtranslationmediatesmaturationofcytoplasmiccilia inDrosophila1

spermatogenesis2

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ShortRunningtitle:mRNAlocalizationinspermciliamaturation4

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JaclynMFingerhut1,2,*andYukikoMYamashita1,2,3,4,*6

7 1CellularandMolecularBiologyProgram,2LifeSciences Institute,3DepartmentofCelland8

Developmental Biology, 4Howard Hughes Medical Institute, University of Michigan, Ann9

Arbor,MI48109,USA10

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*Correspondence:[email protected],[email protected]

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.CC-BY-NC-ND 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted April 22, 2020. ; https://doi.org/10.1101/2020.04.21.054247doi: bioRxiv preprint

https://doi.org/10.1101/2020.04.21.054247

http://creativecommons.org/licenses/by-nc-nd/4.0/

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Abstract32

Cytoplasmiccilia,aspecializedtypeofciliainwhichtheaxonemeresideswithinthe33

cytoplasmratherthanwithintheciliarycompartment,areproposedtoallowtheefficient34

assemblyofverylongcilia.Despitebeingfounddiverselyinmalegametes(e.g.Plasmodium35

microgametocytesandhumanandDrosophilasperm),verylittleisknownaboutcytoplasmic36

ciliaassembly.HereweshowthatanovelRNPgranulecontainingthemRNAsforaxonemal37

dynein motor proteins becomes highly polarized to the distal end of the cilia during38

cytoplasmic ciliogenesis inDrosophila sperm. This allows for the localized translationof39

these axonemal dyneins and their incorporation into the axoneme directly from the40

cytoplasm.WefoundthatthisRNPgranulecontainstheproteinsReptinandPontin,lossof41

which perturbs granule formation and prevents incorporation of the axonemal dyneins,42

leading tosterility.Wepropose that cytoplasmic ciliarequire the local translationofkey43

proteinconstituentssuchthattheseproteinsareincorporatedefficientlyintotheaxoneme.44

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AuthorSummary46

Cytoplasmiccilia,whicharefoundinhumanandDrosophilasperm,areuniqueinthat47

the axoneme is exposed to the cytoplasm. The authors show that a novel RNP granule48

containing axonemal dynein mRNAs facilitates localized translation of these axonemal49

proteins,facilitatingcytoplasmicciliaformation.50

51

Abbreviations52

SCspermatocyte53

IFTintraflagellartransport54

ODAOuterdyneinarm55

IDAInnerdyneinarm56

RNPRibonucleoprotein57

smFISHsinglemoleculeRNAfluorescentinsituhybridization58

IFImmunofluorescent59

ICIndividualizationcomplex60

TEMTransmissionlightmicroscopy61

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Introduction63

Ciliaaremicrotubule-basedstructurespresentonthesurfaceofmanycells.These64

specializedcellularcompartmentscanbenon-motileprimaryciliathatlargelyfunctionin65

signaling,ormotileciliathatcaneithermoveextracellularmaterials(e.g.lungmulticiliated66

cells) or allow for cell motility (e.g. Chlamydomonas flagellum, sperm in many species)67

(IshikawaandMarshall,2011).Itiswellestablishedthatmostciliaareseparatedfromthe68

bulkcytoplasm(Fig.1A),whichservestoconcentratesignalingmoleculesforrapidresponse69

toextracellularsignalsreceivedbythecilia,andthattheciliarygateatthebaseofthecilia70

formsadiffusionbarrier,throughwhichmoleculesmustbeselectivelytransported(Reiter71

etal.,2012;Whewayetal.,2018).However,recentstudiesidentifiedanadditionaltypeof72

cilia,calledcytoplasmiccilia,inwhichtheaxoneme(themicrotubule-basedcoreofthecilia)73

isexposedtothecytoplasm(Fig.1A)(Avidor-Reissetal.,2017;Avidor-ReissandLeroux,74

2015; Dawson and House, 2010; Fawcett et al., 1970; Sinden et al., 1976; Tates, 1971;75

Tokuyasu,1975).CytoplasmicciliaarefoundinhumanandDrosophilaspermaswellasin76

PlasmodiumandGiardia.Therearetwoproposedadvantagestocytoplasmiccilia:1)faster77

assemblyasthecelldoesnotneedtorelyonciliarytransportmechanisms,allowingforthe78

assembly of longer cilia, and 2) proximity tomitochondria for energy (Avidor-Reiss and79

Leroux, 2015; Sinden et al., 2010).Despite being found acrossdiverse taxa, very little is80

known about how cytoplasmic cilia are assembled and whether their assembly bears81

similaritytothatoftraditionalcompartmentalizedcilia(Desaietal.,2018).82

Cytoplasmicciliogenesishasbeenproposedtooccurintwostages(Fig.1A)(Avidor-83

Reiss and Leroux, 2015). In the first stage, microtubules are polymerized in a small84

compartmentalized region, which is similar to canonical compartmentalized cilia, at the85

mostdistalendofthecilia(Gottardoetal.,2013;Tokuyasu,1975).Thisregionisgatedbya86

transitionzone(CaudronandBarral,2009;Kwitnyetal.,2010;Vieillardetal.,2016).This87

entirecompartmentalizedregion,calledtheciliarycaporthegrowingend,migratesaway88

fromthebasalbody,whichisdockedatthenuclearmembrane(Basirietal.,2014;Fawcett89

etal.,1970).Theciliarycapdoesnotchange insizeas theciliaelongates.Thecontinued90

polymerization of microtubules inside the ciliary cap displaces recently synthesized91

microtubulesoutofthecompartmentalizedregion,exposingthemtothecytoplasm(Fig.192

A).The secondstage is axonemematuration, inwhichadditional axonemalproteins (e.g.93



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axonemal dyneins, the motor proteins that confer motility by allowing axonemal94

microtubules to slide against each other, Fig. 1 B) are added to the bare microtubule95

structure after it emerges from the ciliary cap (Tates, 1971; Tokuyasu, 1975). Axoneme96

maturationwas inferred to occur in the cytoplasmbased on the dispensability of ciliary97

transportmechanismsandthe inefficiencyofrelayingondiffusionthroughthetransition98

zone(Avidor-ReissandLeroux,2015;Breslowetal.,2013;Briggsetal.,2004;Hanetal.,99

2003;Hoengetal.,2008;Keeetal.,2012;Linetal.,2013;Sarpaletal.,2003).However,how100

thismaturationprocessoccursinthecytoplasmiccompartmenttoallowforcytoplasmiccilia101

formationremainsunknown.102

Drosophilaspermatogenesisprovidesanexcellentmodelforthestudyofcytoplasmic103

ciliogenesis (Fig. 1 B), owing to rich cytological knowledge of spermatogenesis and the104

conservation of almost all known ciliary proteins (Zur Lage et al., 2019). Developing105

spermatidselongatefrom15μmto1,900μm(1.9mm)(Tates,1971;Tokuyasu,1975).Within106

mature sperm, the cytoplasmic cilia are 1,800μm and the ciliary caps (the107

compartmentalizedregion)areonly~2μm.Ciliogenesisstartsinpremeioticspermatocytes108

(SCs), which assemble short primary (compartmentalized) cilia (Fabian and Brill, 2012;109

Gottardoetal.,2013;Riparbellietal.,2012;Tates,1971).Priortoaxonemeelongation,these110

primarycilia,whichweredockedattheplasmamembraneinSCs,invaginate,formingthe111

ciliarycap.Duringaxonemeelongation,themajorityofthelengthoftheciliawillbeexposed112

tothecytoplasm,asdescribedabove.Accordingly,axonemeassemblyinDrosophiladoesnot113

requireintraflagellartransport(IFT)(Hanetal.,2003;Sarpaletal.,2003),theprocessused114

bytraditionalcompartmentalizedciliatoferryaxonemalproteinsfromthecytoplasminto115

the ciliary compartment for incorporation (Rosenbaum and Witman, 2002). Other116

cytoplasmicciliahavebeenfoundnottorequireIFTfortheirassembly(Avidor-Reissand117

Leroux,2015;Briggsetal.,2004;Hoengetal.,2008),leadingtotheappreciationofadistinct118

typeofcilia:basedonthedispensabilityofIFT,itwaspostulatedthataxonemematuration119

mustoccurinthecytoplasm,hencetheterm‘cytoplasmiccilia’.120

IthaslongbeenknownthatSCstranscribealmostallgeneswhoseproteinproducts121

areneededpost-meioticallyandthat thesemRNAsmaynotbetranslateduntildayslater122

when proteins are needed (Barckmann et al., 2013; Olivieri and Olivieri, 1965). We123

previouslyshowedthattheY-linkedtestis-specificaxonemaldyneinheavychaingeneskl-3124



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and kl-5, as well as the testis-specific axonemal dynein intermediate chain Dic61B, are125

transcribed inSCs (Fingerhutet al., 2019).However, axonemeelongationdoesnotbegin126

until after meiosis, suggesting that these mRNAs may not be translated until later in127

development. Intriguingly, we previously showed that kl-3 and kl-5 mRNAs localize to128

cytoplasmic granules in SCs. Ribonucleoprotein (RNP) granules (e.g. stress granules, P129

granulesandgermgranules)areknowntoplaycriticalrolesinmRNAregulation,suchas130

mediating thesubcellular localizationofmRNAsandcontrolling the timingof translation131

(AndersonandKedersha,2009;Buchan,2014;Medionietal.,2012).Therefore,wedecided132

toinvestigatetheroleofthisnovelRNAgranuleinthetranslationalregulatorymechanisms133

that ensure proper axoneme assembly and found that it plays an essential role in the134

incorporation of axonemal proteins, providing the first insights into the molecular135

mechanismof cytoplasmic ciliamaturation.We show four axonemal dynein heavy chain136

mRNAs,includingkl-3andkl-5,colocalizeinthesenovelgranulesinlateSCsalongwiththe137

AAA+ (ATPases Associated with diverse cellular Activities) proteins Reptin (Rept) and138

Pontin(Pont).TheseRNPgranulesaresegregatedduringthemeioticdivisionsandlocalize139

to the distal end of the cytoplasmic compartment as the axoneme elongates during140

spermiogenesis. We further show that Rept and Pont are required for RNP granule141

formation, and that RNP granule formation is necessary for robust translation and142

incorporation of the axonemal dynein proteins into the axoneme. We propose that143

cytoplasmicciliamaturationreliesonthe local translationofaxonemalcomponentssuch144

that theycanbe incorporated intothebaremicrotubulestructureas itemerges fromthe145

ciliarycap.146

147

Results148

AxonemaldyneinheavychainmRNAscolocalizeinRNPgranulesinspermatocytes149

Inourpreviousstudy,weanalyzedtheexpressionoftheY-linkedaxonemaldynein150

geneskl-3andkl-5andshowedthatthesetwomRNAslocalizedtocytoplasmicgranulesin151

lateSCs(Fingerhutetal.,2019).UsingsinglemoleculeRNAfluorescentinsituhybridization152

(smFISH),wefoundthatmRNAsforfourtestis-specificaxonemaldyneinheavychaingenes153

(the Y-chromosome genes kl-2, kl-3, and kl-5, as well as the autosomal gene Dhc98D154

(Carvalho et al., 2000; Goldstein et al., 1982; Hardy et al., 1981; Zur Lage et al., 2019))155



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colocalizetogetherwithinRNPgranulesinthecytoplasmoflateSCs,witheachSCcontaining156

several of these cytoplasmic granules (Fig. 1 C and D). We termed these granules “kl-157

granules” after the three Y-linked constituent mRNAs. It should be noted that robust158

transcriptionofthesegenesisstillongoinginSCnuclei(visibleasbrightnuclearsignal,Fig.159

1CandD)butthesearenascenttranscriptsthatstillcontainintronicRNA,whereasthekl-160

granulesinthecytoplasmdonotcontainintronicRNA,asweshowedpreviously(Fingerhut161

et al., 2019). The present study focuses the fate of these cytoplasmic RNPs that contain162

maturemRNA.mRNAswithinakl-granulearespatiallysub-organized:kl-3andkl-5mRNAs,163

whichencodeouterdyneinarm(ODA)dyneinheavychainproteins,clustertogetherinthe164

coreofthekl-granulewhilekl-2andDhc98DmRNAs,whichencodeinnerdyneinarm(IDA)165

dyneinheavychainproteins,localizeperipherally(Fig.1E–G).Thisissimilartothesub-166

compartmentalizationobservedinotherRNPgranules,includingthegermgranulesinthe167

Drosophilaovary,stressgranules,Pgranulesandnucleoli(Boisvertetal.,2007;Jainetal.,168

2016;Trceketal.,2015;Wangetal.,2014).Wenotedthatkl-granuleformationisunlikely169

tobedependentuponanyonemRNAconstituentasRNAimediatedknockdownofkl-3,kl-5,170

kl-2, or Dhc98D (bam-gal4>UAS-kl-3TRiP.HMC03546 or bam-gal4>UAS-kl-5TRiP.HMC03747 or bam-171

gal4>UAS-kl-2GC8807orbam-gal4>UAS-Dhc98DTRiP.HMC06494)didnotperturbgranuleformation172

despiteefficientknockdown(Fig.S1).173

We conclude that mRNAs for the testis-specific axonemal dynein heavy chains174

localizetonovelRNPgranules,whichwetermedkl-granules,inlateSCs.175

176

Thekl-granulessegregateduringthemeioticdivisionsandlocalizetothedistalend177

ofelongatingspermatids178

Asthekl-granulescontainmRNAsforaxonemalproteinsthatareonlynecessaryfor179

spermiogenesis, we next followed the fate of the kl-granules through meiosis and into180

spermiogenesis.Thekl-granulessegregatethroughthetwosequentialmeioticdivisions(Fig.181

2A) such that each resultinghaploid spermatid receivesa relativelyequal amountofkl-182

granule(Fig.2B).Uponcompletionofmeiosis,theresultantspermatidsareinterconnected183

duetoincompletecytokinesisduringthefourmitoticdivisionsthatoccurearlyingermcell184

developmentandthetwomeioticdivisions,formingacystof64spermatids(Fuller,1993;185

Hime et al., 1996). As the axoneme starts to elongatewithin each spermatid, the nuclei186



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clustertotheproximalendofthecystwhiletheaxonemeelongatesunidirectionallyaway187

fromthenucleiwiththeciliarycapsclusteredatthedistalendofthecyst(Fig.1B)(Fabian188

andBrill,2012).Strikingly,wefoundthatthekl-granulesbecomelocalizedtothedistalend189

ofelongatingspermatidcysts(Fig.2C).Thispolarizedlocalizationremainsastheaxoneme190

continuestoelongate(Fig.2DandE).Atlaterstagesofelongation,thekl-granulesbeginto191

dissociateandthemRNAsbecomemorediffuselylocalizedatthedistalend(Fig.2DandE).192

Interestingly, some mRNAs dissociate from the kl-granules before others: kl-3 and kl-5193

mRNAs(encodingODAproteins)dissociateearlierthankl-2andDhc98DmRNAs(encoding194

IDA proteins) (Fig. 2 D and F). It is of note that the differential timing of dissociation195

correlateswiththesub-compartmentalizationofconstituentmRNAsdescribedabove:kl-3196

andkl-5localizetothecoreofthekl-granulesanddissociatefirst(Fig.1E),whereaskl-2and197

Dhc98D localize to the peripheryof the kl-granules (Fig. 1 F) and dissociate later. These198

results show that kl-granules exhibit stereotypical localization to the growing end of199

spermatidsafterbeingsegregatedduringmeiosis,implyingthatprogrammedpositioningof200

the kl-granules may play an important role during spermatid elongation and axoneme201

maturation.202

203

TheAAA+proteinsReptinandPontincolocalizewiththekl-granules204

Tofurtherunderstandhowkl-granulesformandtheirpotentialfunction,wesought205

toidentifyaprotein(s)thatlocalizetothekl-granules.Inourpreviousstudy,wescreened206

forproteinsinvolvedintheexpressionoftheY-linkedaxonemaldyneingenes(Fingerhutet207

al.,2019).Reptin(Rept)andPontin(Pont),twoAAA+proteins(Puchadesetal.,2020),were208

includedinthisscreenbecauseoftheirhighexpressioninthetestisandtheirinvolvement209

inRNPcomplexformationinothersystems(MaoandHoury,2017;Robinsonetal.,2013).210

Also,studiesinDrosophila,mouse,zebrafish,ChlamydomonasandXenopushavespecifically211

implicatedReptandPontinaxoneme/motileciliaassemblyand/orspermmotility,although212

theunderlyingmechanismremainsunknown(Dafingeretal.,2018;Huizaretal.,2018;Liet213

al.,2017;Stolcetal.,2005;TammanaandTammana,2017;Zhaoetal.,2013;ZurLageetal.,214

2018).215

We found that Rept and Pont colocalize in cytoplasmic granules in SCs through216

elongatingspermatids(Fig.3AandB).ImmunofluorescentstainingcombinedwithsmFISH217



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(IF-smFISH)showedthatReptandPontcolocalizewiththekl-granules.Pontfirstcolocalizes218

withDhc98DmRNAinearlySCs(Fig.3C)andwithallotherkl-granuleconstituentmRNAs219

inlaterSCs(Fig.3D)andthroughoutspermatidelongation(Fig.3E).Closeexaminationof220

thekl-granulesinlateSCsrevealedthatPontisnotevenlydistributedwithinakl-granule221

andratherconcentratesnearthecorewithkl-3andkl-5mRNAs(Fig.1EandFig.3F).In222

contrast,kl-2andDhc98DmRNAsoccupytheperipheryofthekl-granule(Fig.1F),where223

Pontislessconcentrated.224

WeconcludethatReptandPontlocalizetothekl-granulestogetherwithaxonemal225

dyneinheavychainmRNAs.Itisinterestingtonotethatpreviousstudieshaveproposedthat226

Rept and Pont function as chaperones in the assembly of axonemal dynein motors227

(complexes containing a combination of dynein heavy, intermediate, and light chains)228

(Huizar et al., 2018; Li et al., 2017; Zur Lage et al., 2018). It remains unknownwhether229

previouslyreportedRept-andPont-containingchaperoncomplexesalsocontainmRNA(see230

Discussion).231

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ReptinandPontinarerequiredforkl-granuleassembly233

ToexplorethefunctionofReptandPontinkl-granuleformation,weperformedRNAi234

mediatedknockdownofeitherreptorpont (bam-gal4>UAS-reptKK105732orbam-gal4>UAS-235

pontKK101103).Inadditiontoeliminatingthetargetedprotein,depletionofreptresultedinloss236

ofPontandviceversa,reminiscentoffindingsfrompreviousstudies,likelybecausethese237

proteinsstabilizeeachotherascomponentsofthesamecomplex(Fig.S2)(Goryniaetal.,238

2011;Lietal.,2017;Rivera-Calzadaetal.,2017;Venteicheretal.,2008).239

We next determinedwhether Rept and Pont are needed for kl-granule assembly.240

Indeed,knockdownofreptorpontresultedindisruptionofthekl-granules.smFISHclearly241

detectedthepresenceofdispersedkl-3andkl-5mRNAsinlateSCs,suggestingthatreptand242

pontarerequiredforkl-granuleformationbutnotforthestabilityoftheconstituentmRNAs243

(Fig.4A–C,notethatnuclearsignalwasoversaturatedinordertofocusonthedispersed244

cytoplasmicsignal).Thiseffectwasmorepronouncedinelongatingspermatidswherekl-3245

andkl-5mRNAswerediffusethroughouttheentirecystintheRNAiconditions(Fig.4D–F).246

RT-qPCRconfirmedthatmRNAlevelswerenotreducedcomparedtocross-siblingcontrols247

(Fig.4GandH),demonstratingthatkl-granuleformationisnotrequiredformRNAstability.248



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This is in accordancewith observations in other systems that suggest that RNA granule249

formation is not required for mRNA stability and may be more important for mRNA250

localizationortranslation(Bleyetal.,2015;Leeetal.,2020).251

Interestingly,knockdownofreptorponthadasomewhatdifferenteffectonkl-2and252

Dhc98DmRNAs.smFISHforkl-2andDhc98DfollowingRNAiofeitherreptorpontshowed253

lossofkl-granulelocalizationinlateSCssimilartothatseenforkl-3andkl-5(Fig.4I–K).254

However,inelongatingspermatids,kl-2andDhc98DmRNAsappearedtolocalizeproperly255

atthedistalendofthecyst(Fig.4L–N).ConsideringthatPontprimarilycolocalizedwith256

kl-3andkl-5mRNAs(Fig.3F),thismaysuggestthatotherproteinsparticipateinlocalizing257

kl-2andDhc98DmRNAstothekl-granule.258

Inconclusion,ReptandPontarecriticalforassemblingthekl-granules.259

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kl-granuleassemblyisrequiredforefficientKl-3translationandspermmotility261

Previous studies in Drosophila and mouse demonstrated that Rept and Pont are262

requiredformalefertility(Lietal.,2017;ZurLageetal.,2018).Weconfirmedthatseminal263

vesicles,wherematuremotilespermarestoredafterexitingthetestis,wereemptyinrept264

orpontRNAitestes(Fig.5A–C),aswasobservedforkl-3,kl-5,kl-2,orDhc98DRNAitestes265

(Fig.S3)(Fingerhutetal.,2019;ZurLageetal.,2018).266

We further characterized thesterilityphenotypeofrept andpontRNAi testesand267

found that spermiogenesis fails during individualization. As sperm develop as cysts, the268

processofindividualizationremovesexcesscytoplasmfromthespermatidsandseparates269

thecystintoindividualspermviaactin-richindividualizationcomplexes(ICs)(Fabianand270

Brill,2012).TheICs formaroundthenucleiat theproximalendof thecystandprogress271

evenly towards the distal end (Fig. 5 D). It is well established that defects in axoneme272

assembly,includinglossofaxonemaldyneinmotorproteins,perturbICprogression(Fatima,273

2011;Fingerhutetal.,2019;Wangetal.,2019).WefoundthatRNAi-mediatedknockdown274

ofreptorpont,doesnotaffectICassemblybutdoesresultindisorganizedICprogression275

(Fig. 5 E – J), as is observed following knockdownofkl-3,kl-5,kl-2, orDhc98D (Fig. S3)276

(Fingerhutetal.,2019).277

Asprevious studieshave implicatedReptandPont inmale fertilityandaxonemal278

dynein motor assembly, and the observed individualization defects are characteristic of279



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axonemal defects, we analyzed Kl-3 protein levels following rept or pont RNAi.Western280

blottingusingtotaltestisextractsrevealedthatKl-3proteinlevelsaredrasticallyreduced281

followingknockdownofreptorpont(Fig.5K).Takentogether,ourresultsdemonstratethat282

Rept and Pont are required for mRNAs to congress in the kl-granule, which in turn is283

required forefficient translation.Thisdefect inaxonemaldynein expression is the likely284

causeofsterilityinreptandpontRNAitestes.285

286

kl-granuleformationandlocalizationarerequiredforcytoplasmicciliamaturation287

PrecisemRNAlocalizationandlocalizedtranslationarewidelyutilizedmechanisms288

toensurethatproteinsareconcentratedwheretheyareneeded(Glocketal.,2017;Medioni289

et al., 2012). As described above, the kl-granules localize to the distal end of elongating290

spermatids(Fig.2)wherebareaxonemalmicrotubulesarefirstexposedtothecytoplasm291

after being displaced from the ciliary cap as new microtubules are polymerized. We292

thereforepostulatedthat thekl-granulemayfunction incytoplasmicciliamaturation.We293

first determined whether the kl-granules localize within the ciliary cap or within the294

cytoplasmiccompartment.ByusingUnc-GFPtomarktheringcentriole,astructureatthe295

baseof the ciliary capat theboundarybetween the cytoplasmicandcompartmentalized296

regions(Bakeretal.,2004;Phillips,1970),wefoundthatthekl-granulesarelocatedwithin297

thecytoplasmiccompartment,immediatelyproximaltotheciliarycap(Fig.6A),suggesting298

thatthismaybethesiteoflocalizedKl-3translation.Indeed,wenotonlyfoundthatFLAG-299

taggedKl-3protein(expressedfromtheendogenouslocus,seeMethods)occupiesthesame300

regionproximaltotheciliarycapasthekl-granulesbutthatKl-3proteinisrestrictedtothe301

cytoplasmic compartment while the microtubules extend into the compartmentalized302

compartment(i.e.theciliarycap)(Fig.6B).Theseresultsindicatethatwhiletheaxonemal303

microtubulesarepolymerizedwithintheciliarycap,axonemematuration(theincorporation304

of axonemal dyneins and other axonemal proteins) may occur within the cytoplasmic305

compartment,ashasbeenproposed(Avidor-ReissandLeroux,2015).306

DetailedexaminationofKl-3proteinwithintheelongatingspermatidcystsprovided307

insights into where Kl-3 protein may be translated and incorporated into the growing308

axoneme (Fig. 6 C and D). At the distal end of the cyst, where the kl-granules are309

concentrated, Kl-3 protein was predominantly observed in the cytoplasm, while being310



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excluded from the axonemalmicrotubules (Fig. 6D, see the right panel for intensityplot311

showingmutuallyexclusivelocalizationofmicrotubulesandKl-3).ThissuggeststhatKl-3312

protein at the distal end may represent the pool of newly translated Kl-3 before it is313

incorporatedintotheaxoneme,whichisalsoconsistentwiththepresenceofkl-granulesat314

this location. In contrast to the distal end, Kl-3 proteinwas observed to colocalizewith315

axonemalmicrotubulesattheproximalend(Fig.6D,seetherightpanelforintensityplot316

showing colocalization ofmicrotubules and Kl-3), suggesting that Kl-3 protein has been317

successfully incorporated into the axoneme. These results suggest that Kl-3 protein is318

translatedatthedistalend,wherethekl-granuleslocalize,andthatthediffusecytoplasmic319

Kl-3 protein is the newly synthesized pool, which is subsequently incorporated into the320

axoneme.321

Following RNAi mediated knockdown of rept or pont, which prevents kl-granule322

formation(Fig.4)anddrasticallyreducesKl-3proteinlevels(Fig.5K),westillobservedKl-323

3proteininthecytoplasmatthedistalend(Fig.6FandH),althoughatamuchreducedlevel.324

However,Kl-3proteinwasneverobservedtocolocalizewiththeaxonemalmicrotubulesat325

theproximalenduponreptorpontRNAi(Fig.6EandG),suggestingthatReptandPontare326

requiredforincorporationofKl-3intotheaxoneme.327

Consistentwiththisnotion,transmissionelectronmicroscopy(TEM)revealedthat328

theODAsandIDAsarelargelyabsentfromtheaxonemesfollowingreptorpontRNAi.(Fig.329

6I–K).Additionalgrossaxonemaldefects(e.g.brokenaxonemes)werepresentintheRNAi330

conditions(Fig.6L–N),suggestingadditional impairmentstoaxonemeassembly.These331

resultssuggestthatlocalizedmRNAtranslationviaformationofthekl-granulesisrequired332

foraxonemaldyneinmotorproteinstoincorporateintotheaxoneme.333

334

Discussion335

Cytoplasmic cilia have been found in organisms as diverse as Plasmodium and336

humans(Avidor-Reissetal.,2017;Avidor-ReissandLeroux,2015;DawsonandHouse,2010;337

Fawcettetal.,1970;Sindenetal.,1976;Tates,1971;Tokuyasu,1975).While ithasbeen338

proposedthataxonemematurationproceedsthroughthedirectincorporationofaxonemal339

proteins from the cytoplasm, this model remained untested (Avidor-Reiss and Leroux,340

2015). Our study provides the first insights into the mechanism of cytoplasmic cilia341



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formation.OurresultsshowthatlocalizedtranslationofaxonemaldyneinmRNAsfacilitates342

thematurationofcytoplasmicciliabyallowingfortheefficientincorporationofaxonemal343

dyneinproteinsintobareaxonemalmicrotubulesdirectlyfromthecytoplasm.344

345

Mechanismforcytoplasmicciliamaturation346

Ithasbeenproposedthatcytoplasmicciliaassembleintwosteps(Avidor-Reissand347

Leroux,2015):first,microtubulesarepolymerizedwithinasmallcompartmentalizedregion348

ofthecilia,then,asthebaremicrotubulesaredisplacedfromthisregion,axonemalproteins349

areincorporateddirectlyfromthecytoplasmduringthematurationstep.Previousstudies350

thathaveshownthatIFT,theprocessusedbytraditionalcompartmentalizedciliatoferry351

axonemal proteins into the ciliary compartment, is dispensable for Drosophila352

spermiogenesis,andthatthegenomesofsomeotherorganismsknowntoformcytoplasmic353

cilia(e.g.Plasmodium)donotencodeIFTand/ortransitionzoneproteins(Avidor-Reissand354

Leroux,2015;Breslowetal.,2013;Briggsetal.,2004;Hanetal.,2003;Hoengetal.,2008;355

Keeet al.,2012;Linetal.,2013;Sarpalet al., 2003).Thesestudies ledto thenotionthat356

maturationofcytoplasmicciliaoughttohappeninthecytoplasm,althoughdirectevidence357

hasbeenlacking.358

Our study, which identified a novel RNP granule, the kl-granule, composed of359

axonemal dynein heavy chainmRNAs and the proteinsRept andPont, provides the first360

molecularinsightsintocytoplasmicciliamaturation.Ourresultsshowthataxonemaldynein361

heavychainmRNAs(kl-3,kl-5,kl-2,andDhc98D)congressintokl-granulesinSCs.Wefurther362

showthatReptandPontarerequiredforkl-granuleassemblyandthepropertranslationof363

axonemal dynein mRNAs. We demonstrate that the polarized localization of kl-granule364

mRNAswithinthecytoplasmiccompartmentpromoteslocalizedtranslationandallowsfor365

the incorporationof their encodedproteins into theaxoneme, facilitating thematuration366

stepincytoplasmicciliaassembly(Fig.7).Ourresultsrefinetheproposedtwostepmodel367

for cytoplasmic cilia assembly by demonstrating that concentrating axonemal proteins368

withindistalregionsofthecytoplasmiscriticalformaturation.Thus,axonemematuration369

proceedsinastepwisefashion,allowingfortheefficientassemblyofthisverylongcilia.This370

model implies that theproximal regionof theaxonemeshouldbemoremature than the371

distal region, a notion that is supported by previous studies that looked at axoneme372



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ultrastructureandtubulindynamicswithintheaxoneme(Noguchietal.,2011;Sindenetal.,373

2010;Tokuyasu,1975).374

375

FunctionofReptinandPontinindyneinassembly376

AwiderangeoffunctionshavebeenassignedtoRept-andPont-containingcomplexes377

includingrolesinchromatinremodeling,transcriptionregulation,DNArepairandribosome378

assembly(MaoandHoury,2017).Theycanactalone,togetheroraspartoflargercomplexes379

(Huenetal.,2010;KakiharaandSaeki,2014).Amongthese,previousstudieshaveproposed380

thatReptandPontaredyneinarmpreassemblyfactors–chaperonesthattakeindividual381

dyneinmotorsubunits(i.e.theheavy,intermediate,andlightchainproteins)andstabilize382

andassemblethemintoamotorunitinthecytoplasmthatisthenferriedintotheciliafor383

incorporation(Desaietal.,2018;FabczakandOsinka,2019;FowkesandMitchell,1998).384

These assembly factors include R2TP and R2TP-like complexes (which include Rept385

(RUVBL2) and Pont (RUVBL1)) in association with dynein axonemal assembly factors386

(DNAAFs)(FabczakandOsinka,2019).Whilepreviousstudieshaveclearlydemonstrated387

thatRept,Pont,R2TP,andDNAAFsareneededforaxonemaldyneinproteinstabilityand388

incorporation(Huizaretal.,2018;Lietal.,2017;Liuetal.,2019;Yamaguchietal.,2018;Zhao389

et al., 2013; Zur Lage et al., 2018), our study is the first to demonstrate involvement of390

axonemaldyneinmRNAswiththesecomplexes,showingthatReptandPontarerequiredfor391

axonemaldyneinmRNAs to localize to thekl-granules. It remainsunknownwhether the392

Rept-andPont-associateddyneinarmpreassemblycomplexesreportedinpreviousstudies393

also contain dynein mRNAs. However, important differences exist between these394

preassemblycomplexesandthekl-granules.Firstly,whilekl-3mRNAispresentinthekl-395

granules, no puncta are observed for Kl-3 protein, indicating that Kl-3 protein does not396

concentrate within the kl-granules (or another granule) as dyneins do in the dynein397

preassemblycomplexesreportedinothersystems(Dafingeretal.,2018;Huizaretal.,2018).398

Additionally, dynein preassembly complexeswere found to contain proteins (e.g.Wdr78399

(Huizaretal.,2018)),wheretheDrosophilahomolog(Dic61B)mRNAsarenotconstituents400

ofthekl-granules(Fig.S4,seebelow).Therefore,thekl-granulemaybeanoveladaptation401

of a Rept and Pont containing dynein arm assembly complex specifically found in402

cytoplasmicciliaandisdistinctfromitsroleasadyneinpreassemblyfactorinothersystems.403



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It is likely that additional protein components of the kl-granule remain to be404

discovered. Structural analyses inpreviousstudieshave identifiedmechanismsbywhich405

otherproteinsinteractwithReptandPont(Rivera-Calzadaetal.,2017),however,Reptand406

PontdonothaveanyRNAbindingdomains(MaoandHoury,2017).Therefore,itislikely407

thatadditionalproteins,notReptandPontthemselves,physicallyinteractwithconstituent408

mRNAsforkl-granuleformation.Ourdataalsosupportstheexistenceofadditionalproteins409

governing kl-granule dynamics. For example, as spermatids elongate, the ODA and IDA410

mRNAsseparateslightlyfromeachotherwhileremainingpolarizedatthedistalend(Fig.2).411

Moreover,intheabsenceofReptandPont,theIDAmRNAsarestillabletocongressatthe412

distal end of the elongating spermatid cyst, after failing to form kl-granules in SCs. In413

contrast,localizationoftheODAmRNAsentirelydependsonReptandPont,asODAmRNAs414

remaindiffusethroughoutspermatogenesisfollowingreptorpontRNAi.Finally,Pontmore415

stronglycolocalizeswiththeODAmRNAswithinthekl-granule(Fig.3F),whichaltogether416

suggests that there are additionalproteins that can sort and specify the fate of these kl-417

granulemRNAsbothalongsideor in theabsenceofReptandPont.The identityof these418

additionalproteinsisthesubjectoffurtherstudy.Inaddition,determiningtheinvolvement419

oftheotherdyneinarmpreassemblyfactorsisofparticularinterest,especiallyconsidering420

the existence of multiple dynein arm assembly complexes that have been shown to421

differentiallyregulateIDAandODAassembly(FabczakandOsinka,2019;Yamaguchietal.,422

2018).Itisalsoappealingtoposittheexistenceoftestis-specificfactorswhichmayhelpto423

distinguish the role of Rept and Pont in cytoplasmic cilia formation from its role in the424

assemblyofothercellularbodies.425

426

PurposeofmRNAlocalizationtokl-granules427

Interestingly,we found thatnotallmRNAs foraxonemal/spermiogenesisproteins428

localize to the kl-granules (Fig. S4). mRNAs for other axonemal proteins (the dynein429

intermediatechainDic61B,thedyneinheavychainCG3339,andtheODAdockingcomplex430

componentCG17083(ZurLageetal.,2019))aswellasmRNAsforotherY-linkedtranscripts431

(CCYandPPR-Y(Carvalhoetal.,2001))andanon-axonemalspermatidprotein(fzo(Hales432

andFuller,1997))didnotlocalizetothekl-granules.Insteadtheyremainevenlydistributed433

throughouttheSCcytoplasm,despitealsobeingimportantforspermmaturation(Fig.S4).434



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Additionally,wepreviously reportedthatmRNAs for theY-likedgeneORY alsogather in435

cytoplasmicRNAgranulesinlateSCs(Fingerhutetal.,2019),however,theseRNAgranules436

aredistinctfromthekl-granule(Fig.S4G).437

Inparticular,itisintriguingthatmRNAforDic61B,anIDAintermediatechainthat438

needs tobind to the IDAheavychainsKl-2andDhc98D, is locateddifferently (diffusely)439

within the spermatid cyst. Dynein preassembly is believed to be important for dynein440

proteinstabilityandaprerequisiteforaxonemalincorporation(FabczakandOsinka,2019;441

FowkesandMitchell,1998).Anintriguingpossibilityisthattemporal/spatialregulationof442

dyneinmRNAsplaysaroleinhelpingtheorderedassemblyofdyneincomplexes.Itwillbe443

offutureinteresttodeterminewhenandwhereduringspermiogenesisdyneincomplexes444

areformedinthecytoplasmicciliaaswellaswhatfactorsarenecessaryfortheirformation.445

Acomprehensiveunderstandingofkl-granulemRNAsandproteinswouldallowforfurther446

studyintothistemporal/spatialregulatorymechanismandamorethoroughunderstanding447

ofhowthekl-granulesfunctioninthematurationofcytoplasmiccilia.448

449

Insummary,ourstudyprovidesthefirstinsightsintothemechanismofcytoplasmic450

ciliamaturation:mRNAsforaxonemaldyneinmotorproteinsarelocalizedatthedistalend451

oftheaxonemewithinthecytoplasmiccompartment,whichallowsforefficientmaturation452

ofcytoplasmicciliathroughlocalizedtranslation.453

454

MaterialsandMethods455

Flyhusbandry456

AllflystockswereraisedonstandardBloomingtonmediumat25°C,andyoungflies457

(1-to5-day-oldadults)wereusedforallexperiments.Fliesusedforwildtypeexperiments458

werethestandardlabwildtypestrainyw(y1w1).Thefollowingflystockswereused:bam-459

GAL4:VP16 (BDSC:80579), UAS-kl-3TRiP.HMC03546 (BDSC:53317), UAS-kl-5TRiP.HMC03747460

(BDSC:55609), UAS-Dhc98DTRiP.HMC06494 (BDSC:77181), and C(1)RM/C(1;Y)6, y1w1f1/0461

(BDSC:9460) were obtained from the Bloomington Stock Center (BDSC). UAS-kl-2GC8807462

(VDRC:v19181), UAS-reptKK105732 (VDRC:v103483), and UAS-pontKK101103 (VDRC:v105408)463

wereobtainedfromtheViennaDrosophilaResourceCenter(VDRC).unc-GFP(GFP-tagged464

unc expressed by the endogenous promoter) and Ub-a-tubulin84B-GFP were a gift of465



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CayentanoGonzalez (Baker et al., 2004; Rebollo et al., 2004) andbam-gal4was a gift of466

DennisMcKearin(ChenandMcKearin,2003).Thekl-3-FLAGstrainwasconstructedusing467

CRISPRmediatedknock-inofa3X-FLAGtagattheC-terminusofkl-3aspreviouslydescribed468

(Fingerhutetal.,2019).469

470

SinglemoleculeRNAfluorescentinsituhybridization471

AllsolutionsusedforRNAFISHwereRNasefree.Testesfrom2-3dayoldflieswere472

dissected in1XPBSandfixed in4%formaldehyde in1XPBSfor30minutes.Thentestes473

werewashedbrieflyin1XPBSandpermeabilizedin70%ethanolovernightat4°C.Testes474

werebrieflyrinsedwithwashbuffer(2Xsaline-sodiumcitrate(SSC),10%formamide)and475

then hybridized overnight at 37°C in hybridization buffer (2X SSC, 10% dextran sulfate476

(Sigma, D8906), 1mg/mL E. coli tRNA (Sigma, R8759), 2mM Vanadyl Ribonucleoside477

complex (NEB S142), 0.5% bovine serum albumin (BSA, Ambion, AM2618), 10%478

formamide).Followinghybridization,sampleswerewashedthreetimesinwashbufferfor479

20minuteseachat37°Candmounted inVECTASHIELDwithDAPI(VectorLabs). Images480

wereacquiredusinganuprightLeicaTCSSP8confocalmicroscopewitha63Xoilimmersion481

objectivelens(NA=1.4)andprocessedusingAdobePhotoshopandImageJsoftware.482

Fluorescentlylabeledprobeswereaddedtothehybridizationbuffertoafinalconcentration483

of100nM.Probesagainstkl-3,kl-5,kl-2,Dhc98D,CG3339,Dic61B,CG17083,CCY,PPR-Y,ORY484

and fzomRNAsweredesignedusing theStellaris®RNAFISHProbeDesigner (Biosearch485

Technologies,Inc.)availableonlineatwww.biosearchtech.com/stellarisdesigner.Eachsetof486

customStellaris®RNAFISHprobeswaslabeledwithQuasar670,Quasar570orFluorescein487

(TableS1).488

For strains expressing GFP (e.g. unc-GFP or Ub-a-tubulin84B-GFP), the overnight489

permeabilizationin70%ethanolwasomitted.490

491

Immunofluorescencestaining492

Testesweredissectedin1XPBS,transferredto4%formaldehydein1XPBSandfixed493

for30minutes.Testeswerethenwashedin1XPBST(PBScontaining0.1%Triton-X)forat494

least60minutesfollowedbyincubationwithprimaryantibodiesdilutedin1XPBSTwith495



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3%BSAat4°Covernight.Sampleswerewashedforatleast1hourin1XPBST,incubated496

withsecondaryantibodyin1XPBSTwith3%BSAat4°Covernight,washedasabove,and497

mountedinVECTASHIELDwithDAPI(VectorLabs).Imageswereacquiredusinganupright498

LeicaTCSSP8confocalmicroscopewitha63Xoilimmersionobjectivelens(NA=1.4)and499

processedusingAdobePhotoshopandImageJsoftware.500

Thefollowingprimaryantibodieswereused:anti–α-tubulin(1:100;mouse,Sigma-501

AldrichT6199),anti-FLAG(1:500;rabbit,InvitrogenPA1-984B),anti-Reptin(1:200;rabbit,502

gift of Andrew Saurin (Diop et al., 2008)), anti-Pontin (1:200; guinea pig, this study),503

Phalloidin-Alexa546or488(1:200;ThermoFisherA22283orA12379).ThePontinantibody504

wasgeneratedbyinjectingapeptide(CKVNGRNQISKDDIEDVH,targeting18aafromthec505

terminal end of Pontin) in guinea pigs (Covance). Alexa Fluor-conjugated secondary506

antibodies(LifeTechnologies)wereusedatadilutionof1:200.507

AmodifiedversionofStefanini’sfixative(4%formaldehyde,0.18%w/vPicricAcid508

(RiccaChemical5860),0.3MPIPESpH7.5(AlfaAesarJ63617),0.05%Tween-20)wasused509

in order to detect Kl-3 (Muller, 2008). No signal was detectable using traditional510

formaldehydefixation.511

512

Immunofluorescence staining with single molecule RNA fluorescent in situ513

hybridization514

TocombineimmunofluorescentstainingwithsmFISH,testesfrom2-3dayoldflies515

weredissectedin1XPBSandfixedin4%formaldehydein1XPBSfor30minutes.Thentestes516

werewashedbrieflyinPBSandpermeabilizedin70%ethanolovernightat4°C(unlessfrom517

astrainexpressingGFP,inwhichcasethisstepwasomitted).Testeswerethenwashedwith518

1XPBSandblockedfor30minutesat37°Cinblockingbuffer(1XPBS,0.05%BSA,50µg/mL519

E.colitRNA,10mMVanadylRibonucleosidecomplex,0.2%Tween-20).Primaryantibodies520

weredilutedinblockingbufferandincubatedat4°Covernight.Thetesteswerewashedwith521

1XPBScontaining0.2%Tween-20,re-blockedfor5minutesat37°Cinblockingbufferand522

incubated 4°C overnight in blocking buffer containing secondary antibodies. Then testes523

werewashedwith1XPBScontaining0.2%Tween-20andre-fixed for10minutesbefore524

continuingthesmFISHstartingfromthebriefrinsewithwashbuffer.525



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526

RT-qPCR527

Total RNA from testes (50 pairs/sample)was extracted usingTRIzol (Invitrogen)528

accordingtothemanufacturer’sinstructions.1μgoftotalRNAwasreversetranscribedusing529

SuperScript III®ReverseTranscriptase(Invitrogen) followedbyqPCRusingPowerSYBR530

Green reagent (AppliedBiosystems)on aQuantStudio 6Real-TimePCR system (Applied531

Biosystems).PrimersforqPCRweredesignedtoamplifyonlymRNA.Thegenesanalyzedby532

qPCRareallpredictedtocontainmegabasesizedintrons,andprimersweredesignedtospan533

theselargeintronssuchthataproductwouldbedetectonlyiftheintronhadbeenspliced534

out(Fingerhutetal.,2019).RelativeexpressionlevelswerenormalizedtoGAPDHandcross-535

siblingcontrols.Allreactionsweredoneintechnicaltriplicateswithatleasttwobiological536

replicates.Graphicalrepresentationwasinclusiveofallreplicates.Primersusedarelistedin537

TableS1.538

539

Westernblot540

Testes(40pairs/sample)weredissectedinSchneider’smediaatroomtemperature541

within30minutes,themediawasremovedandthesampleswerefrozenat-80°Cuntiluse.542

Afterthawing,testeswerethenlysedin200uLof2XLaemmliSampleBuffer+βME(BioRad543

161-0737).ForKl-3,sampleswereseparatedonaNuPAGETris-Acetategel(3-8%,1.5mm,544

Invitrogen) and for Rept and Pont, sampleswere separated on a Novex Tris-Glycine gel545

(10%,1mm,Invitrogen)withtheappropriaterunningbufferinaXcellSureLockmini-cell546

electrophoresissystem(Invitrogen).ForKl-3,proteinsweretransferredusingtheXCellII547

blot module (Invitrogen) onto polyvinylidene fluoride (PVDF) membrane (Immobilon-P,548

Millipore)usingNuPAGEtransferbuffer(Invitrogen)withoutaddedmethanol.ForReptand549

Pont,transferbuffercontained20%methanol.Membraneswereblockedin1XTBST(0.1%550

Tween-20) containing 5% nonfat milk, followed by incubation with primary antibodies551

diluted in 1XTBST containing 5%nonfatmilk.Membraneswerewashedwith 1XTBST,552

followedbyincubationwithsecondaryantibodiesdilutedin1XTBSTcontaining5%nonfat553

milk.Afterwashingwith1XTBST,detectionwasperformedusingthePierce®ECLWestern554

Blotting Substrate enhanced chemiluminescence system (Thermo Scientific). Primary555

antibodies used were anti–α-tubulin (1:2,000; mouse, Sigma-Aldrich T6199), anti-FLAG556



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(1:2,500;mouse,Sigma-AldrichF1804),anti-Reptin(1:2000;rabbit,giftofAndrewSaurin),557

anti-Pontin (1:2000; guinea pig, this study), anti-Vasa (1:3000; rabbit, Santa Cruz558

Biotechnology D-260). The secondary antibodies were horseradish peroxidase (HRP)559

conjugatedgoatanti-mouseIgG,anti-rabbitIgG,oranti-guineapigIgG(1:10,000;Abcam).560

561

Phasecontrastmicroscopy562

Seminal vesicles were dissected in 1X PBS and transferred to slides for live563

observationbyphasecontrastonaLeicaDM5000Bmicroscopewitha40Xobjective(NA=564

0.75)and imagedwithaQImagingRetiga2000RFast1394MonoCooledcamera. Images565

wereadjustedinAdobePhotoshop.566

567

Transmissionelectronmicroscopy568

Testeswerefixedforonehourorovernight(at4°C)with2.5%glutaraldehydein0.1M569

Sorensen’s buffer, pH7.4. Samples were rinsed twice for 5 minutes each with 0.1 M570

Sorensen’sbufferandpostfixedforonehourin1%osmiumtetroxidein0.1MSorensen’s571

buffer.Next,testeswererinsedtwiceindoubledistilledwaterfor5minuteseachandenbloc572

stainedwith2%uranylacetateindoubledistilledwaterforonehour.Thesampleswere573

them dehydrated in increasing concentrations of ethanol, rinsed with acetone, and574

embedded in Epon epoxy resin.Thin sectionsweremounted on Formvar/carbon-coated575

slottedgridsandpost-stainedwithuranylacetateandleadcitrate.Sampleswereexamined576

onaJEOL1400transmissionelectronmicroscopeandimagescapturedusingasCMOSXR401577

customengineeredopticcamerabyAMT(AdvancedMicroscopyTechniquesCorp.).578

579

Onlinesupplementalmaterial580

Fig.S1showsefficiencyofRNAiknockdownofkl-3,kl-5,kl-2,andDhc98DbysmFISH581

and lack of dependence upon a single one of those transcripts for kl-granule formation582

(relatedtoFig.1).Fig.S2showsthatRNAiofreptorpontresultsinefficientknockdownof583

both(relatedtoFig.4).Fig.S3showsthesterilityphenotypeofkl-3,kl-5,kl-2,andDhc98D584

RNAi flies (related to Fig. 5). Fig. S4 shows smFISH for other axonemal, Y-linked, and585

spermatid-essentialtranscripts(relatedtoDiscussion).586

587



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Acknowledgements588

We thank Drs. Dennis McKearin, Cayentano Gonzalez, and Andrew Saurin, the589

Bloomington Stock Center, and the ViennaDrosophila Resource Center for reagents.We590

thankSashaMeshinchiandtheUniversityofMichiganMicroscopyCore forhelpwithEM591

experiments. We thank the Yamashita laboratory and Drs. Sue Hammoud and Joshua592

Bembenekfordiscussionandcommentsonthemanuscript,Drs.TomerAvidor-Reissand593

TonyMahowaldforhelpfulsuggestions,andDr.JiandieLinforsharingequipment.Thiswork594

wassupportedbytheHowardHughesMedicalInstitute(toYMY)andtheNIHCellularand595

Molecular Biology Training Grant T32-GM007315 (to JMF). The authors declare no596

competingfinancialinterests.597

598

AuthorContributions: J.M.Fingerhut conceived theproject andconductedexperiments.599

J.M.FingerhutandY.M.Yamashitadesignedexperiments,analyzedthedata,andwrotethe600

manuscript.601

602

Figures603

604



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605



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Figure 1: Axonemal dynein heavy chain mRNAs colocalize in an RNP granule in606

spermatocytes.607

(A) Diagram comparing and contrasting traditional compartmentalized cilia and608

cytoplasmiccilia.Nucleus(darkgray),cytoplasm(lightgray),basalbody(green),transition609

zone(orange)andaxoneme(blue).(B)DiagramofDrosophilaspermiogenesiswithstagesof610

spermatid elongation. Nucleus (dark gray), cytoplasm (light gray), basal body & ring611

centriole (green), ciliary cap (orange)andaxoneme (blue).Axonemecross section image612

showinglocationofaxonemaldyneinarms.Microtubulesandotherstructuralcomponents613

(gray),innerdyneinarm(green)andouterdyneinarm(magenta).(CandD)smFISHagainst614

axonemaldyneinheavychaintranscriptsinSCsshowingkl-3,kl-5,andkl-2mRNAs(C)orkl-615

3,kl-2,andDhc98DmRNAs(D)inkl-granules.kl-3(blue),kl-2(green),kl-5(red,C),Dhc98D616

(red, D), DAPI (white), kl-granules (yellow arrows), SC nuclei (yellow dashed line),617

neighboringSCnuclei(whitedashedline).Bar:10µm.(EandF)smFISHagainstkl-3,kl-5,618

andkl-2(E)orkl-3,kl-2,andDhc98D(F)showingasinglekl-granule.kl-3(blue),kl-2(green),619

kl-5(red,E),Dhc98D(red,F).Bar:1µm.(G)Tablelistingthegenesfocusedoninthisstudy,620

theirfunctionandtheirlocalizationwithintheaxoneme.621

622



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623 Figure2:kl-granulessegregateduringthemeioticdivisionsandlocalizetothedistal624

endofelongatingspermatids.625



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(A) smFISH against kl-3 and kl-5 during meiosis. kl-3 (blue), kl-5 (red), a-tubulin-GFP626

(green),DAPI(white)andkl-granules(yellowarrows).Bar:10µm.(B–E)smFISHagainst627

kl-3, kl-5, and kl-2 during spermiogenesis. The round spermatid (B), early elongating628

spermatid(C),midelongatingspermatid(D)andlateelongatingspermatid(E)stagesare629

shown.kl-3(blue),kl-5(red),kl-2(green),DAPI(white),spermatidcyst(cyandashedline).630

Bar:10µm(B),25µm(CandE)or50µm(D).(F)smFISHagainstkl-3,Dhc98D,andkl-2in631

midelongatingspermatids.kl-3(blue),Dhc98D(red),kl-2(green),DAPI(white),spermatid632

cyst(cyandashedline).Bar:25µm.633

634

635 Figure3:ReptinandPontincolocalizewiththekl-granules.636



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(AandB)ReptandPontcolocalizationinSCs(A)andearlyelongatingspermatids(B).Rept637

(red),Pont(green),DAPI(white),SCnuclei(yellowdashedline,A),neighboringSCnuclei638

(whitedashedline,A),kl-granules(yellowarrows,C)spermatidcyst(cyandashedline,B).639

Bar:10µm(A)or25µm(B).(C–E)IF-smFISHforPontproteinandkl-3andDhc98DmRNAs640

inearlySCs(C),lateSCs(D)andearlyelongatingspermatids(E).Pont(green),kl-3(blue),641

Dhc98D(red),DAPI(white),SCnuclei(yellowdashedline,CandD),neighboringSCnuclei642

(white dashed line, C andD), kl-granules (yellow arrows, C andD) spermatid cyst (cyan643

dashedline,E).Bar:10µm(CandD)or25µm(E).(F)IF-smFISHforPontproteinandkl-3644

andDhc98DmRNAsinasinglekl-granule.Pont(green),kl-3 (blue),Dhc98D (red)andkl-645

granuleboundary(yellowdashedline).Bar:1µm.646

647



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648 Figure4:ReptinandPontinarerequiredforkl-granuleassembly.649



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(A – F) smFISH against kl-3 and kl-5 in control (A and D), rept RNAi (bam-gal4>UAS-650

reptKK105732,BandE)orpontRNAi(bam-gal4>UAS-pontKK101103,CandF)SCs(A–C,singlez651

plane)andearlyelongating spermatids (D–F, z-projection).kl-3 (blue),kl-5 (red),DAPI652

(white),SCnuclei(yellowdashedlines),neighboringSCnuclei(narrowyellowdashedlines),653

SCkl-granules(yellowarrows)andspermatidcyst(cyandashedline).Bar:Bar:10µm(A–654

C)or25µm(D–F).(GandH)RT-qPCRinreptRNAi(bam-gal4>UAS-reptKK105732,G)orpont655

RNAi(bam-gal4>UAS-pontKK101103,H)forkl-3,kl-5,andkl-2usingtheindicatedprimersets656

(seeTableS1).DatawasnormalizedtoGAPDHandsiblingcontrols.(I–N)smFISHagainst657

kl-2andDhc98Dincontrol(IandL),reptRNAi(bam-gal4>UAS-reptKK105732,JandM)orpont658

RNAi(bam-gal4>UAS-pontKK101103,KandN)SCs(I–K,singlezplane)andearlyelongating659

spermatids(L–N,z-projection).kl-2(green),Dhc98D(red),DAPI(white),SCnuclei(yellow660

dashedlines),neighboringSCnuclei(narrowyellowdashedlines),SCkl-granules(yellow661

arrows)andspermatidcyst(cyandashedline).Bar:Bar:10µm(I–K)or25µm(L–N).662

663



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664 Figure 5:kl-granule assembly is required for efficient Kl-3 translation and sperm665

motility.666

(A–C)Phasecontrastimagesofseminalvesiclesincontrol(A),reptRNAi(bam-gal4>UAS-667

reptKK105732,B)andpontRNAi(bam-gal4>UAS-pontKK101103,C).Maturesperm(cyanarrows).668

Bar:100µm.(D)SchematicofICprogressionduringindividualization.Nucleus(darkgray),669

axoneme(blue),ICs(red)andcytoplasm(lightgray).(E–J)Phalloidinstainingofearlyand670



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lateICsintheindicatedgenotypes.Phalloidin(Actin,red)andDAPI(white).Bar10µm.(K)671

WesternblotforKl-3-3XFLAGintheindicatedgenotypes.672

673



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674



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Figure 6: kl-granule formation and localization are required for cytoplasmic cilia675

maturation.676

(A)smFISHagainstkl-3infliesexpressingUnc-GFP.kl-3(magenta),Unc-GFP(ringcentriole,677

green),transitionzone(yellowarrows),andspermatidcyst(cyan,dashedline:cytoplasmic678

region,solidline:compartmentalizedregion).Bar:20µm.(B)Kl-3-3XFLAGproteininflies679

expressingUnc-GFP.Kl-3(red),Unc-GFP(ringcentriole,green),a-tubulin(white),transition680

zone(yellowarrows),andspermatidcyst(cyan,dashedline:cytoplasmicregion,solidline:681

compartmentalizedregion).Bar:20µm.(C–H)Kl-3-3XFLAGproteinexpressionincontrol682

(CandD),reptRNAi(bam-gal4>UAS-reptKK105732,EandF)andpontRNAi(bam-gal4>UAS-683

pontKK101103,GandH)proximal(C,EandG)anddistal(D,FandH)regionsoflateelongating684

spermatids. Kl-3 (red), a-tubulin-GFP (white), transition zone (yellow arrows), and685

spermatidcyst(cyan,dashedline:cytoplasmicregion,solidline:compartmentalizedregion).686

Intensityplotsareshownfortheregionswithintheyellowrectangles.Bar:5µm(C,EandG)687

or25µm(D,FandH).(I–N)TEMimagesofcontrol(IandL),reptRNAi(bam-gal4>UAS-688

reptKK105732, JandM)andpontRNAi(bam-gal4>UAS-pontKK101103,KandN)axonemes.Pink689

arrows:ODA,greenarrows:IDA,yellowarrows:brokenaxonemesbrokenaxonemes.The690

controlsingledoubletenlargedimageisduplicatedtotheleftofthefigureandcoloredto691

matchthediagram.Bar:50nm(I–K)or200nm(L–N).692

693



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694 Figure7:Modelforcytoplasmicciliamaturation.695

Thekl-granule(lightpurple)localizesimmediatelyproximaltotheciliarycap(orange)and696

ringcentriole(green)withinthecytoplasmiccompartment.ConstituentmRNAs(purple)are697

locallytranslated(ribosomes,limegreen)andtheirproteins(axonemaldyneins,yellow)are698

incorporatedintotheaxoneme(blue)asthemicrotubulesaredisplacedfromtheciliarycap.699

Inthisway,cytoplasmicciliamaturationisprogressivewithaxonemalproteinsbeingadded700

tothebaremicrotubulesaselongationproceeds.701

702



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703 FigureS1:kl-granuleformationisnotdependentuponanyonemRNAconstituent.704

(A–D) smFISHagainsteachknownkl-granulemRNAconstituent followingRNAiof that705

constituentshowssuccessfulknockdown(noremainingcytoplasmicsignal).Notethatwe706

usemultiplesmFISHprobesetsforsomemRNAstargetedagainstdifferentregionsofthe707

transcript(seeTableS1).(A)kl-3exon1(blue),kl-3exon14(red)andDAPI(white).(B)kl-708

5exons1-6(red),kl-5exons16-17(green)andDAPI(white).(C)kl-2exons1-2(green),kl-709

2exons10-12(blue)andDAPI(white).(D)Dhc98D(red)andDAPI(white).Forall,SCnuclei710



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(yellowdashed line) and neighboring SC nuclei (white dashed line). Bar: 10µm. (E –H)711

smFISHagainsttheotherthreeconstituentmRNAsafterRNAiofthefourthmRNA.Notethat712

thecolorusedtorepresenteachsmFISHprobecorrespondstotheprobesetsinA–D.(E)713

kl-5(green),kl-2(blue),Dhc98D(red)andDAPI(white).(F)kl-3(blue),kl-5(green),Dhc98D714

(red)andDAPI(white).(G)kl-3(blue),kl-5(green),Dhc98D(red)andDAPI(white).(H)kl-715

3 (blue),kl-5(red),kl-2 (green)andDAPI(white).Forall,SCnuclei(yellowdashedline),716

neighboringSCnuclei(whitedashedline),andkl-granules(yellowarrows).Bar:10µm.717

718

719 FigureS2:RNAiofreptorpontresultsinlossofbothproteins.720

(A–C)ReptandPontproteinexpressioninSCsincontrol(A),reptRNAi(bam-gal4>UAS-721

reptKK105732,B)orpontRNAi(bam-gal4>UAS-pontKK101103,C).Rept(red),Pont(green),DAPI722

(white), SC nuclei (yellow dashed line), neighboring SC nuclei (white dashed line), kl-723

granules(yellowarrow).Bar:10µm.(D)Westernblot forPontandRept in the indicated724

genotypes.725

726



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727 FigureS3:RNAiofkl-3,kl-5,kl-2orDhc98Dresultsinthesamesterilityphenotypeseen728

inreptorpontRNAitestes.729

(A – D) Phase contrast images of seminal vesicles in kl-3 RNAi (bam-gal4>UAS-kl-730

3TRiP.HMC03546,A),kl-5RNAi(bam-gal4>UAS-kl-5TRiP.HMC03747,B),kl-2RNAi(bam-gal4>UAS-kl-731

2GC8807, C) andDhc98D RNAi (bam-gal4>UAS-Dhc98DTRiP.HMC06494, D). Bar: 100µm. (E – L)732

PhalloidinstainingofearlyandlateICsintheindicatedgenotypes.Phalloidin(Actin,red)733

andDAPI(white).Bar10µm.734

735



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736 Figure S4: Transcripts for other axonemal, Y-linked, and spermatid proteins don’t737

localizetokl-granules.738

(A–G)smFISHagainstotheraxonemal(A–C),Y-linked(D–F)orspermatid-essential(G)739

transcripts. (A) Dic61B (dynein intermediate chain, red) and DAPI (white). (B) CG3339740

(axonemaldyneinheavychain,red)andDAPI(white).(C)CG17083(ODAdockingcomplex,741

red)andDAPI(white).(D)CCY(red)andDAPI(white).(E)PPR-Y(red)andDAPI(white).742

(F)kl-5 (red),ORY (blue)DAPI (white)andkl-granules(yellowarrow).(G) fzo (red)and743

DAPI(white).Forall,SCnuclei(yellowdashedline)andneighboringSCnuclei(whitedashed744

line).Bar:10µm.745

746

747

748

749

750



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TableS1:smFISHprobesandRT-qPCRprimersusedinthisstudy751

Probe Target Fluorophore 5’-Sequence-3’ CG17083, All exons

Quasar® 670 aacttcttgatttcctccat, tttccaggaaggcaagcttg, aattccttgtgcttcagcag, ctggacaggatcttcttgta, caaagctggcctcgatgatg, gcgattgttctgttttttgt, tcgacatggcagcgaatcag, tcagagagttaatgtcctgc, ccttctccatgttggaaatc, tatttgcttggacatgcgac, gctgatccggaatggttaaa, cgcattacataggcctgatg, ctccaaaatggtcatccttt, gtaaaaccgcactcctgacg, ataagctgctctcgcagatc, gaaggtgcgatgattcagga, gcaccattttggtgtatgaa, tactttttctcgctgttcag, gagcgtattcgatcagatct, aatgcagccatcgaaggtat, ccttttggccaacaaatcaa, atctccacacgtctcatatc, tgccacctttcgcataatac, caggaaggcggtgttatcac, ctcgaggcttagacttgcaa, tgatctttttgttgtgcacc, tggagaatcttctgcagcac, ccatattgctagattccgta, gctgaaacttatcgatcacc, cgagtaatagagactctcct, catttcgttggcatagttga, attcagcatagttatgtggt, caaacaggcgattcaccgaa, gtgttggagttatccttttt, ggaaatctgatccagttggt, tgttggattcctgtttgtta, agattctccaaacgtgcatc, tctatggaacacatctcgca, tcaccaaggagtttggtcag, taaccagattgacgtgggtg, cgagcagcttcaagaatcga, cgctccatcacataaacact, tgaccacatagtcgaagcgg, tcgcagatcttctcgatctg, gggtgagcacaatgtcattg, atccatgttgaaggcttcac, tgtggacaaggacaccatcg, taaccttctcgtacagcttc

CG3339, Exons 1-19

Quasar® 570 agtcgtgaagctggaagagg, tattgcggcgcacaaagtag, cgctccaagacaatgtcatg, cagtgttatgtactttggct, ccgaattaaatgcggcagtc, tttgggtgcttcgatagtat, gaaaagttccagaaccgcga, cttgtagtccaacattcgtt, agcactttaaactcgccgaa, cattcggctgaagaacaggg, cgcaatcggtggaaagttgt, tgctcaacgagttggcttaa, agagcaaggaattccactgt, ccatgagatccagattttca, tcaagtagattgcgaccgta, agaagttggcaatcagcagg, tggagtacagctgaaactcc, tcctccggaaattcacgaat, cgctggccaaatatttgttg, ttgctgaaggactaacctcg, tccattttttgactgtacgc, acttcgttgtagttctcgat, tgagcggcttgaacacattg, tgcttaacggtactcgagtg, catacatcgtagacttgcgg, tgcaaccgaaggtagacttc, ctggatgtcaagaagtcggg, ttgtcatctagggtttcgat, caatcagagtatcgatgcct, aactgcagcaattgggcaag, agaagcgtggaaaggccaat, gttaccgttggacaagatgt, tttgatgaggtgtcggtcaa, acgaagggcacatactcatc, acgataaacgccggttgatc, ctcctcatttgctggataat, gccgaaataattcgtgcagg, ttcgcaaagcaatcctcatc, actcgtaggcataccgaaac, ggtgatataacagcgatccg, tcctttgtaaatgttgccac, cacgcagatgcgattgaact, caaatcaaggcgaagtccgg, aaatatgggtacatcctccg, aagaggtcaccaatgagacc, ttctcgaactcaaagacccg, gaaaaccgagtgtcgcacat, atccacatgggatcaatgtc

Dhc98D, Exons 10-16 (isoform B, these exons are

Quasar® 570 tcatactggacgaacacgcg, tcgtcaagcatgcgatagac, aacatgtcatagtcgacgct, ttgttgaacaactccagcga, ttgatgctttggtcgatcac, tcatagcattgaccaccaag, cagtaggttctcatgtttgg, gcgtttatctcggtcacaaa, gtcttgagctttttacctag, aagagctgcgctttaggaag,



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common to all three isoforms)

ctcgaagagcaccttttcaa, tacgtggcgttctgtatgaa, tgtagattccttgcatatgc, gtcatttgcaattcaggtcg, ctgtaggcactaaacgcttc, ttcttttgtgatgtgtcacc, ttactgaccatccacatgat, ataaacgctgtgcattcctg, acatctttgggcttcttatc, cagcagatattgggactctg, aagacatcgcgcttcagatt, actgctgagcttcaaacagg, gactgcgggaagtgtaaagc, gcaggtcctttttcataatc, ttgacctatacctgtcaacg, gtaggaagatcacctctgac, cacgcccatttggataaaga, tatcgatacgagagcgcaga, gtagatacgattcgccagag, gacatggatatatgccggat, ggagtcgtaaagcttcttca, ttccagaaaatgtgcccaac, tacacttgaatgtctctcct, ggcggttgatatagcatttt, gacatcaggagaacagcgtt, tgaaccgaaggctcaaggac, ggtcatccaacgggtgaaaa, ggattacgtcctcatagtac, tgaccagatccacaatggat, ccactctagaagccaaatcg, tgaaactcttctcgaaggcc, ctcatcgatttcgatgagcg, gtttccacaatggacgagta, cacacttgatgtcctggaac, cagcaagggctccaaattta, cctcatgttatcgttgacat, gtggtcttgttcagattcag, atttcgtggattttctgctc

Dic61B, Exon 4 (isoform A, common with other isoform)

Quasar® 670 gttcccagttaatagtttca, cttcgagagcctcaacagtg, aaggacttggtatacggctt, gacttgcgtaaggtgatctt, gtctggctgtgaatttcgaa, cagatactcgtagtttcggt, aattttgcctttccctattg, tctgagcgtcattgtctgag, gagtggtgaagagcagagta, ggttatgaggatggtgttga, aacgtaagagctgacggtgg, gcgtgtcgtacatctcaaag, actccttggtgatattgttc, cgaactcatagtggctgttg, tcgtcgtcctcaaaggtatc, cttgtctttttcggcaacgg, cggaacaggcgctctatctg, atgatggcgttgcgaaactc, tcgtttgtgttgctcgagag, ggtcaaagttacggaagcgt, catcgagtagacgtactcgg, taccagccgaaagagcaagt, cggcagaaatcaatgtcgct, tgagtacagtccgtaggcta, cggtacgtgctgtgaggaaa, acaggaaaacgctgccggag, ctcgcctggatttttaatgc, acatcgtagtagtatgcgcg, aagtttattgccgttactgg, aaggatggcaagaatggcga, tcgtacagaccaatggcgag, tgcaaactgctcacatctcg, aaacttgtttgatccagcgg, tgtctgtttcatgatcgtcg, gagaggctcaggaatggatc, tgataatccggaagcgggtg, aaccaagcaggaagggactt, actcgctccagaatcatttg, gaacgaaaatgccctcagga, ttagactcgcaggggatacg, gtagtgatgcttaagccttg, atgtccttgtgcagaggatg, tcatctgtcagcacgtagta, gattgagcacttgtggatgc, ccaagtactgatgctgataa, cgtcgtgacaacgcaggact, gaactccattacggtgacac, aggtgaggaagagtttggga

fzo, All exons Quasar® 570 aaacgaggacaccgacgacg, gcgaagacgacgatgaggat, gcgtccacaaactcacttaa, tatatatcctgcagttctgt, tttaacaggacagtctcctc, aaagaacctttgcaatggcc, gtccaaaaaatgccaccttc, cattgatcacggcacttttt, gcaggattttttcatgcaga, aaaacagctggtggtatggc, cagagttgagggttttaggg, atccaatatcgagcaacggt, tagctatctaggcaatcgtc, actcggcgttgagaactaga, ttgaagaactgcctttccac, agagatttggacgcgagagt, cccatcgattgttgagtata, caacgttccatatgctgatc, acacctaattcatccacgag, aacatgatagatcctttccc, cgtaatctgaccattcctta, gaaactcctgatatcgctgt, accgcgaggcaattcgaaaa, acttagcaagtgtggaccaa, ctctatttcggcattcaagt, tctatcctcttgcattagta, gtcatttcagtcagttcttc, tctggtcgttcaaaacgcac,



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gatagcacagacgagggtat, ctgggaattccgggtgaaat, ataacgagcgttggtactgg, ggggtatggagagacattga, aagcccaatttctttctcta, tagattagttgccaatcgca, tctggctgaaagtcactcat, ttcgaaatggacttgcgtgc, tccgttaactaaaggtggca, ttcaccaaagattccagcat, tcagtaaaacggtgcccaag, ccagttgaacgaacggatgg, cccaccaaggatcaatacaa, cttgaaacttcgctcttggg, ctaacagtttgctgcacatc, gtcgcaactggagctcaaaa, attcagtgtctcattggact, tgtatttgtttggtcagctc, gaaacttcttcaggctgagc, taggtctcttgaaagtctcc

kl-2, Exons 1 & 2 Fluorescein Cgatcagtctcagtactttc, ttcacatattcaacaagccc, tctgttgaattgatcaacca, gtgagaacatcgcaatcgta, acaggaaatccaaggcaacc, cataaatcaataaccggggc, ttatttctttagaggaccga, cgaagttaaccatgtcgtga, caacatcatcttgcacagta, aaattttcaataggcagcca, tgggagcatatataagctct, cccattccataaaatttcga, aacgttgcttcacattgtct, atttatccagggacgtacag, ttgctgttaagattcctaga, aactgtcatgccagacattt, ctcttttgctatttctttga, attctctgtcgtacgatgaa, cgcaatacactccaagttct, aattcgaataagcgtagccc, tggtaagggtcttgtcattt, ctcatccccaacactaatat, accgatatagccagaactcg, tgcgcatttagtccttgaag, ttgaacatccacttgatcca, gggtacttcgtagaactctt, attagttcatcgatctgttt, ccatcagttcatgtgtagat, atttgatgttttccatagct, gttgtgaacatggttgcatt, gtgagacaaaagttgggctt, tcaagtgaattgttcgggga, gtattgttcagagtttaacc, ccagttattaaatcacgtct, ccttttcagtacaaaaccgt, tccaaaccttgaacttcctg, attcctttataagtcaggca, ttttgcatgggtttttgaca, ccaacctattcgtatgttta, aatcattgcattgtcaaggc, ctccataaaggcatcgacat, gtcttccgaaaaccatcatt, gtatactctctgattcgtca, taccaccgaattgaggctta, gcttcaaattctgtaccact, attttcaacgttgtcggcag, ataatgccgtaagagtcacc, atgatctctttggaatccgt

kl-2, Exons 10-12 Quasar® 670 tcgttcgagtaggactgtat, cattccagccaagttctaaa, ggagtgtcttcatattcatt, agatactttaaagctcccca, ccgtaatttactcctgctat, caatcgtcggtaatgtgtcc, gttattaatagtcggcgatc, tgcaatgcttggtcacagaa, aatatgattgcacatcgccg, gggaaacatttgtatttggt, gtccaaaagcatcaggctta, tgacgctatatcggcatttg, aagcattcttgtttctccta, ttgcatagaaagcagagcct, acttgtgctattagtctgga, ttgtctcaccgttttcatta, tttatctcatccggtgtatt, aatttttgccgtctgttcat, gggagttcgattgattccaa, agcgctcaatttcttgaagt, gagtggacatgtcaacgaga, ccacgtcttaagtcacgtaa, attacaacaagtccctgtat, atatcctctaagtccgaact, ccttcagagacagctagata, ttaaccattgtaatggcacc, tgccgctaatggtttcaatg, atgtattaagtctctagccc, gcccaactattaaaatgtcc, taatattggagggcggagtg, cgtgtaagctgcaagccaaa, gctgtaacaaatccagttgg, ctcgagctgaagtttgtagt, tcccaagagagttcatcaat, ggcagtatcttcttcaacaa, ttcccttattatacgagctg, tcgaatgtaaacgcctcctc, gccaccctccaaaaacaaac, attggtttttcctcaaccat, atcggtagtggatcctgaag, gtgtattactggtaatggac, taggttttctactggcttaa, aacaccacgacatcgttttt, ataatatgcgggacactggt,



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aatgatcctgacctaacggg, ctttaagtccacggctatta, agtagtcagccttttcatta, taaaagtgcagtacctcgct

kl-3, Exon 1 Quasar® 670 taacattcctttctggatcc, cgcgaaacgccaaagagttt, gcagcacgctttaacatgtt, ttggtcacttacactaggtc, tatcgtcttctttgttggtc, cctcatttctcgaagtaact, caggcttgaaatccgttgtt, aaacataccgctggtttggg, atacccaacaaatctgcaca, gttactatttcctcaggatc, ttgctttcatccacaatacc, accatttacattctcaacat, gggacctttttcctcaaata, cgttacttatcattatggcc, cggaatcagttggatatcct, tttaagcttttcctggtagc, agagcgttgaatttcagtgt, actagatccgacctcaaaca, gtcgatatacaacagtccac, accgaacggttatcaatcga, aaatattgccacctcatcac, aagcaagagtttcgctcttc, cggctttaaaacgtgatcca, caaattcggtcacagcttct, tgagtttgctctttttctgc, acagttgcttcagatgattt, cctttaaatagttcatgcga, tttgccatctcacaagtaat, gtatttttgaactggcttca, caaccagtcgaactcgtgta, gccattgctcaaaataacgt, gtataccttgaatttgtcga, gtatttgttttccctctact, ctacatcgggtgtatctctt, ccaattgaccaacatttgca, tatatctggccaacatacgc, gtaacaaactccgttatggt, gtggttattaaatgctcggg, agataatgtcaagcaatcct

kl-3, Exon 14 Quasar® 570 gtactttgacatagccatgg, aagatttgcctttaagggca, tgatatttagcctcttgcac, ctgcttcttgtagatcactt, cgttttctttttgttgcagt, tttttggcctcgtctaatac, aaccaccaataagagcggtt, ttcagtccatcggatttttt, cggtcggtctcacttttaaa, ggagaataacatctccgacc, tcttgattaaatggtcccgt, atgttccactcaccaatttg, ttgacagttcatctgttggt, ttttaatccacacttttccc, taatcggaattcccatgcta, taactcctctgcaacatctt, aaggttgtccaagcaaggat, tccaatccacttctttatca, gattgggtagctttgttgta, cgcgcaaatatttcaggtgt, ccacgcattgtcacagtaaa, gaacccgttcatcttctaat, tttcatgtttccagtcacag, ttcctttcgtagtggacaac, cctcaatcactgtaacgtcg, tccttaacttcaatggcagt, cagcgtttatttttgcttct, acactacctcttgtagcaac, gcatcaaagcgctcaaggaa, tcctctagatttgtagcgat, ttaactcaagctggcgatca, cgcaccgccttttataaata, caccgaaacggaacaggagg, tataccacgtaaaccaagcc, ccatacaccacgaacgaaca, agtttagtactactggctcg, aatcattggcataagttccc, agctcattttttttggcgag, cttggcccatagaaatagga, cgtcttctaggcaggataat, tcctaagtgacagttttgca, actgtaagctctaccatgta, agcaggtggttcattagtat, tttttagtccagcacgtatt, tggagattgcgaatagtcca, tgcataccagtcggatgaat, tcctgctccttaaaactgta, cgtttgccatgtcatcaata

kl-5, Exons 1-6 Quasar® 570 cttcttttccttttcgtcag, aaaaactccggacggttgtc, ttgtcttggttaggtagttc, ccacttatccagcttaagac, cctaaactcgttggttgtta, atacgtttcttgttgggatt, ccaccggaattgattgtgaa, ctggaaagctgtaggatgga, agcaactttataccgtggtc, gaagtggtgttaagtaccga, atttgctaatgggttcggta, atcccacttgatttactgtg, tctgtcttcatatcgtttgc, tccatttcgcatttcttgag, aacgagacctttcatctggg, catgacttcgtccatgcaaa, gcttcataagagggttaacc, caagccactttacaaccata, tttacgaggtcttcaacgga, tgcctctggtagtggaaaat,



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caagttctcaaggttttcca, agtctttatacgcctatctc, tctgcgataagaatgctcga, acttagttatgtctctagcc, gtccatatcgtttgtttcaa, gtcgtatatctgatcggttc, cactaatccaattgtcagca, tgaaaataccgcgagtgacc, gttctgttgacacagtcgat, ggaatatggagtctggttct, ttataggcttcgtcgacatc, tgtaatactctaggtgctgc, cagtcctgcgagttaaatgt, atcgtccgaatatttcatcc, tcttttagctcttccaatcg, ccccataacaattttttcca, aagggcgaaagttatctgcc, tgctgttgtactcttcaaga, aatatttgtccactcacggt, acagagaattttctgcgtcc, tggccttgaagcttaaacga, tgaaacgataccatgcgctc, atgtgtgtcttcaagacctt, gcgaagaagtaaggagcctg, gggttcgacatgttttttga, atcatccacaatgacatgca, taacgcacatgatctcttcc, cgagcgccgtaaaaacgttt

kl-5, Exons 16-17 Fluorescein ttgtggtccgaatttcctac, taaacgggtagctacggttc, tgatattgtcaaatcgccca, ccaggtaattgtacagcaca, caaggtactctggtattggc, ccatacataatctctccgaa, ccagtcgtctgtaatgtgac, gataagttcggcataagcgg, agctctggctgcataaattc, aaaccctggcaatactctag, atttaagtattcctggagcc, atgtaattgtggtagccagt, gagatggactttcagacgga, gcatttgaatgaaggccgta, cgtagttaggaacccaatct, attcggaagagtcgttcaga, tctcggctgcaactcgaaaa, aaactgtctcaccaccacta, gttttttataatgtcttcct, aatggtgtgggagttttgtc, cgcgacccattagttctaaa, atgtatggacttcggtcttc, cgctcacactcttgaaatgc, gcttcaattcagtcataaga, aggtcaagctcattcagaga, agtcaattctcctttaaggc, ccattaaatcctccataact, gcacttggtccatgtaaaga, aaccaggattgtagccctag, cagccgcaacattaaatcgg, aggcatgcgaaagtcggcaa, atcctgccaaccaaattgat, aggagcgactgaggattaaa, cgtctgttgcatgatagctg, gacacattctatcgagaggc, ttccactttttggtgacatc, ttgatataggcaccctctcg, tgctccctccatgaaaagac, aatggttcccattttcatgt, gttcctttaaaaaggcgtct, agaacaggcatagcaggaaa, cttgggttactgcctttatg, gacatttttaatatcctgct, cggattttgtaaactgggca, caaacgaaggtgggtcctcg, ctctcggcttttcaagttaa, ctagagtccacttacttgct, tgcaagagaagacaaacccc

ks-1 (ORY), All exons

Quasar® 670 tttttggctttctttctgtc, aaaagttgaggctccgagtt, cgtttaattcgcgatgcttc, cttcatctacataccgacga, tccgatgttgaagtcagttc, cccccaacaaatctttaagt, tgatgcatctgattctttcc, tcttgcactaactgttctcg, cagttgtaccactatttcgg, ttagctcgagaagccatttt, attcaacgtttaggcgttcg, ccattgtcttcatcaaagct, tttccatgtgcttctttttg, ctctattcgcaatatccagt, agtttacgtgtcgtttctga, gcattgccttatttaatgcg, gccttaactgctttatcatt, aagcctgctcgttaattgag, ttgtacattcttgttgtcgc, catccttctccagagaatta, gacgaatatcatccgttcgg, cacgttgcaatgtctctttg, atgttttaagtccgccatag, tctttctctgctttggattt, cgtcttcagagagatttggt, tcgtgtaacttttccgtgag, acggactccaactgtttttt, cgctgaagcatcattttctc, ttttacttcgtttgcgctta, gccactaagttttctttgtt, tttctctcatatccttacgt, caatccgactaggttacgtt, aacaatctcgtcattccgtc, gggcattttgtgcaatttga, aaggcattcactttcagtgt, ctcatgtcagcagtactttt, tcttcagttagagctcgtat, caacgatgtactcctgtagg,



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aatttcttaggatcttcgcc, tattgactgctttagggagc, agcctcacacagtttatttt, gcatatgagatagcatcctt, taatgaacgctctgctgcta, gtcttgatttaagttccacc, aattgacagctctccgattt, tatttcgtttttcccatctg, atccacattccatatactct, atatccgttaacttcgcaca

ks-2 (CCY), Exon 1

Quasar® 570 tctttttgtgtggagaacgc, gttaagctcataactttcgt, attaacgcaaactccagtgc, cgatcgtcgattgaagatgg, ttcttcgttttctgaccatg, tcggcaacaaaacgcgattt, tggattgttacatgccacta, ttcgtttccatgctttacaa, cagcaggagattactaccat, ccgcatttattaagctgtct, gtaaccaatcgattgcgtca, gctggttgttaaggatgttt, agcttcatgtctgattcagg, cgcgttatccatttttatcg, gcgttgatttataatcctct, cctagcattatctcgattgt, ccatttagctttacttgcag, tcattctttgagcttatcca, ttcttttagttccttctctt, ccatgtcatcgatagttctt, cctcaagtaaatcctgggtt, gctttaggtaagcattttcc, cggcatcgaatggattgttt, tttgctcgggattaatttct, gaaggtactcaacgtctgtg, gccatttcgttaatttttcg, gccgaagtagcgataattct, cttataagcatatcttcggt, ttctacacatttgtgcgcac, cttgaaagcttcgttgcact, gcattattcatatagacacc, tttcttgtagaattctctgt, actttttacgcagagcatct, caagcgcttccagattttta, tgttctagggcattaagctt, aatgttttctgcctcatttg, attgtcttcaagatcctcag, gtttatgtattggttctgct, atccatttcctgctgaatat, agtttctctaatcctttcgc

Ppr-Y, All exons Quasar® 570 atttatttgatcgtcttccat, aataacttcatccacaagggg, tattccaggttcaatatctgg, agctttcaatcaaggaacggt, caccacgatgaacgtgtttta, cagttgatgtaaacgtcttcc, atttgttccaatacaacaggc, ttaaactccaaacgcattgtt, atccataagtgatcaatacgt, taaggcaaagcttggtcagat, aatggtctctattttgttgca, ggtgtcaagattttcgatctt, gacagaacttccaaatttact, ctcaatagcttcaattttgtt, agcatggttaacatatcgata, tatttccaaggctgataatta, tccttcaactgtgtcaattaa, ttcataaaccgaaatcgttca, gataggattaccttccaaatt, ctgatatgtactttaacaggc, cggagttcactcttaatgaaa, agtataaattacaggcttctg, tcttcaatttcccgaatctcc, cttgtatctccttttcttgtt, tctgattgttcacgttcaaga, aacttgaggctaatcgttttg, tgatgaccatccaaatgttca, cacgccaaagtgagtcaaaca, caacaagcatcagcacacgac, atatccttatcgtattcgtcg, gtaaatttcttgcgtaagttc, gtaaatctttctaagccaagt, tctcgaatttcttcatcgcgc, tatttgaagttcttcttggcc, acataagctccgcaatttttc, atgcttgagattgttctacaa, tcgagtcaatatttcggtcat, gtcttgaagtgcaaatttcgt, tcggagttcaataggaacgtc, ttaaaatggcgtcacggtcat, ctcgctcatctaatcgcaaag, gtctacaaattcctttgttcg, ctgtttagttggtctatcata, aatttttgaccgatgtcgttc, catcggtcatcatttctacaa, cttacatcgtgtggtaaggat, acataatcttcggctatcagc

PrimerName 5’-Sequence-3’ Gapdh-qF TAAATTCGACTCGACTCACGGT

Gapdh-qR CTCCACCACATACTCGGCTC

kl-3_exon1_qF CCCGAGCATTTAATAACCACAAG

kl-3_exon2_qR AACGGACATTATCCTTAGCTTCA



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kl-3_exon15_qF GCCACGAGCTCGATGAATA

kl-3_exon16_qR AGTACCTTCAACGGCAAGAA

kl-5_exon1_qF ATGCGTCTTAAGCTGGATAAGT

kl-5_exon2_qR TGTCCACCGGAATTGATTGT

kl-5_exon16_qF GCCTCTCGATAGAATGTGTCTT

kl-5_exon17_qR TTTCATGTCCCATCGTGCT

kl-2_exon1_qF AATGACAAGACCCTTACCAGTC

kl-2_exon2_qR TTTGTTGAACATCCACTTGATCC

kl-2_exon5_qF CGTCGGACTTTGCCCTTAAT

kl-2_exon6_qR GCTCCAAAGTGAAGTTCTTCGAG

752

753

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