dFmr1 Plays Roles in Small RNA Pathways of Drosophila melanogaster

20 Pages·2017·1.94 MB·English

International Journal o f
Molecular Sciences
Review
dFmr1 Plays Roles in Small RNA Pathways of
Drosophila melanogaster
ValeriaSpecchia,SimonaD’Attis,AntoniettaPuricellaandMariaPiaBozzetti*
DipartimentodiScienzeeTecnologieBiologicheedAmbientali(DiSTeBA)—UniversityofSalento,73100Lecce,
Italy;[email protected](V.S.);[email protected](S.D.);[email protected](A.P.)
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:MartinPichler
Received:1March2017;Accepted:10May2017;Published:16May2017
Abstract: Fragile-Xsyndromeisthemostcommonformofinheritedmentalretardationaccompanied
by other phenotypes, including macroorchidism. The disorder originates with mutations in the
Fmr1 gene coding for the FMRP protein, which, with its paralogs FXR1 and FXR2, constitute
a well-conserved family of RNA-binding proteins. Drosophila melanogaster is a good model for
the syndrome because it has a unique fragile X-related gene: dFmr1. Recently, in addition to its
confirmedroleinthemiRNApathway,afunctionfordFmr1inthepiRNApathway,operatingin
Drosophilagonads,hasbeenestablished. InthisreviewwereportasummaryofthepiRNApathways
occurringingonadswithaspecialemphasisontherelationshipbetweenthepiRNAgenesandthe
crystal-Stellatesystem;wealsoanalyzetherolesofdFmr1intheDrosophilagonads,exploringtheir
geneticandbiochemicalinteractionstorevealsomeunexpectedconnections.
Keywords:FMRP/dFmr1;fragile-Xsyndrome;piRNApathway;crystal-Stellatesystem;dFmr1interactors
1. Introduction
1.1. TheFragile-XMentalRetardationGene
dFmr1istheDrosophilahomologofthegeneresponsibleforfragile-Xmentalretardationsyndrome,
one of the most frequent inherited causes of human mental retardation. Humans have three
homologousgenescodingfortheFragile-XMentalRetardationProteins: Fmr1,FXR1,FXR2,withhigh
sequencesimilarity;allofthemcodeforRNA-bindingproteins[1–3]. MutationsinFmr1,locatedon
theXchromosomeandcodingforFMRPproteins,areresponsibleforthesyndrome. FMRPandits
autosomalparalogs,theFragileX-RelatedproteinsFXR1PandFXR2P,constituteaconserved,small
familyofRNA-bindingproteins. FMRPhasbeenstudiedinhumans,mice,andDrosophila,principally
inrelationtoitsroleinthenervoussystem,atthesynapses,asatranslationalregulatorworkingwith
differenthypothesizedmechanisms: (i)byrepressingtranslationalinitiation[4–6];(ii)bythemiRNA
pathwaybindingtothe3(cid:48) endoftargetmRNAs[7–12];and(iii)bydirectinteractionwithtranslating
ribosomes[8,12–15]. ThehumanFMRPisexpressedindifferenttissuesduringdevelopment,with
apreferenceforthegonadsandthebrainand,asexpected,themaineffectsofitslossareinthebrain
andthetestes[16].
Drosophilamelanogasterisconsideredagoodmodelforfragile-Xsyndrome[17]becauseithas
asingle,well-conservedfragileX-relatedgenenameddFmr1[18,19],exhibitinghighsequencesimilarity
with all three human genes, and is predicted to be ancestral to the three mammalian members.
dFmr1mutantfliesexhibitdefectsinneuronalstructureandfunction,andingermlinedevelopment,
resemblingthoseobservedinFmr1mutationsinmiceandhumans[19,20]. ThehumanFMRPand
Drosophila dFmr1 exhibit similar structures and domains [21]; they contain two KH domains, two
Tudor domains, and an RGG box, in addition to a nuclear localization signal (NLS) and a nuclear
Int.J.Mol.Sci.2017,18,1066;doi:10.3390/ijms18051066 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2017, 18, 1066 2 of 20
resembling those observed in Fmr1 mutations in mice and humans [19,20]. The human FMRP and
IDntr.oJ.sMopohl.ilSac i.d2F01m7,r118 ,e1x0h66ibit similar structures and domains [21]; they contain two KH domains2, otfw2o0
Tudor domains, and an RGG box, in addition to a nuclear localization signal (NLS) and a nuclear
export signal (NES) [2,3,21–24] (Figure 1). The high conservation between the two proteins suggests
exportsignal(NES)[2,3,21–24](Figure1). Thehighconservationbetweenthetwoproteinssuggests
a common molecular role that is also supported by the phenotypes in the mutants.
acommonmolecularrolethatisalsosupportedbythephenotypesinthemutants.
FMRP has a wide spectrum of functions; however, its main role is exerted in different small
FMRP has a wide spectrum of functions; however, its main role is exerted in different small
RNA-mediated pathways in humans and in the mouse and Drosophila models. The majority of the
RNA-mediatedpathwaysinhumansandinthemouseandDrosophilamodels. Themajorityofthe
studies regarding the small RNA pathways in Drosophila have been conducted in the ovaries, leading
studiesregardingthesmallRNApathwaysinDrosophilahavebeenconductedintheovaries,leading
to the identification of specific molecular functions of dFmr1.
totheidentificationofspecificmolecularfunctionsofdFmr1.

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1.2. DrosophilaSmallRNAPathways: AnOverview
1.2. Drosophila Small RNA Pathways: An Overview
DifferentclassesofsmallRNAshavebeendescribedandcharacterizedinthelasttwodecades;
theyhDaivffeerdeinffte crelanstsbeiso ogfe snmesaisll aRnNdAfusn hcativoen bseaennd dbeeslcornibgetdo adnifdfe crhenatrascmtearlilzRedN Ain pthaeth lwasaty tswdoe dpeencaddinegs;
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apnrdotetrinans,s crfioprtmioinnagl tadrigffeetrerengt ulRatIiSoCn [2c5o]m. pTlhexeetsh retehamt aiinnistimatael l RpNatAhwpaaytsh wlaeyasd,inagls otcoa lleddiffRerNenAt
ipnotesrt-fetrraennsccer(iRptNioAnia)lp aanthdw traaynss,carriep:ti(oi)ntahle tasirRgNetA repgautlhawtioayn ([a2n5d]. tThheer tehlarteeed menaidno s-smiRaNll ARNpaAth pwatahyw)tahyast,
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bdieodgiecnaetesdis ;AirngoDnraousotpeh pilraottheien m[2iR6]N; A(iis) athree gmeinReNraAte dp,atvhiwaaayt wthoa-ts tuespesp rsomcaelsls ,RfNroAms eans dloonggen aosu tshlye
tsriaRnNscArisb, eadnpdr imexahriybimt iaR NveArsy. mpeiRcuNliAars gbuioidgeenAegsois1; ,itnh eDArrogsoopnhaiulat etphreo tmeiinRpNrAedso amrein agnetnlyeriantveodl,v vediai na
tthwisop-sattehpw payr,otcoesosb, tafrinoma treanndsolagteionnoaulsrleyp rtersasnioscnriobfetdh epmriRmNaAry tamrgiRetNsA[2s7., 2m8]i;R(iNii)Atsh egpuiiRdNe AApgaot1h,w tahye
tAhragtownaasutfier pstrodtiesicno vperereddominintahnetlgyo innavdosl,veadn dinw thasis ppraetdhowmaiyn,a tnot loybtcaoinns aid terraendsltahtieongaoln raedparle-ssspieocni fiocf
pthaeth mwRayN[A29 t–a3r2g]eetsv e[2n7t,h2o8]u; g(hii,i)r ethceen ptliyR,NthAe ppiaRtNhwAapya tthhawt awyahsa fsiraslts odibseceonveforeudn dini nthteh egonneravdosu, sansyds twemas
nporetdoonmlyininanDtrlyos coopnhisliadbeuretda ltshoe ignomnaicdea[l3-s3p,3e4c]if.iPc ipwait-hinwtearya c[2ti9n–g32R]N evAesn( pthiRouNgAhs, )reacreenlotlnyg, ethret hpainRNthAe
optahtehrwcalays hseass oaflssom baelelnR NfoAunsd(2 i3n– t3h2en ntelrovnogu)sa snydstPemiw in,oAt uobnelyrg iinn eD(rAosuobph),ilaan bduAt agloso3 ,idn emfiniceed [3a3s,3th4e].
PPiiwwii-siunbtefraamctiilnygp RroNteAinss (,pairReNthAesA) argreo nloanugteerp trhoatnei tnhsei novthoelvr ecdlaisnsetsh oisf ssimleanlcli RngNpAast h(2w3a–y3.2T nht elopniRg)N aAnds
pPriowtei,c tAaunbiemrgailncee l(lAs ufrbo)m, atnhde dAeg-ore3g, udlaetfiionnedo fast rathnes pPoiswoni ssuabnfdamotihlye rprreopteeitnitsi,v eargee ntheeti cAerlgeomneanuttse,
pprreosteerivnisn igngveonlvoemde inst tahbiisl istyile[n35c,i3n6g] .pAatlhthwoauyg.h Tthhee pthiRreNeAmsa pinroRteNcAt ainpiamthawl caeylslse fxrhoimbi tthsep edcei-fircegfeualtautiroens
aonf dtrcaonmsppoosnonens tasn,dth oertheeirs rseopmeetiteivveid geennceetitch aetletmheeyntssh, aprreesseormviencgo gmepnoonmeen ststaobrilfiutyn c[3ti5o,3n6s]w. Aitlhthootuhgehr
pthaeth twhraeyes mrealiante RdNtoAtih peaRthNwAayms eetxahbiobliits smpe[3ci7f–ic4 2fe].atures and components, there is some evidence that
they share some components or functions with other pathways related to the RNA metabolism [37–
1.3. piRNAGenesAreConservedduringEvolution
42].
Piwi proteins and the piRN A genes have been identified in model organisms and also in
humans. They are deeply conserved in evolution even though the molecular mechanisms in
which they are involved are not completely understood. The mouse is the animal in which many
studieshavebeenconducted,demonstratingthatthepiRNA-mediatedsilencingoftransposonsis
active in the male germline to ensure fertility [43]. The role of piRNAs and the conservation of

peculiar elements belonging to the piRNA pathway, such as the Piwi proteins, Vasa, Maelstrom,
Int.J.Mol.Sci.2017,18,1066 3of20
MOV10L1/Armitage, Tudor domain proteins, and others, suggest that they may act to protect
animalcellsfromtransposableelements,keepingthemsilencedandensuringgenomestabilityinthe
gonads[36,44–48]. AcomprehensiveanalysisandannotationofhumanpiRNAsinadulthumantestes
hasrevealedarelationshipbetweenthesmallRNAsandtransposons[49]. However,thedirectrole
ofthepiRNApathwayinthesilencingofTEshasnotbeenclarifiedsofar. AlinkbetweenthePiwi
humangenes(PIWIL1,PIWIL2,andPIWIL4)andthetransposonshasbeenindirectlydemonstrated
inthetumorigenesisofhumantestes. Inparticular,theepigeneticinactivationofthePiwiproteins
and the Tudor protein TDRD1 causes a reduction in piRNA expression in addition to the DNA
hypomethylationofLINE1,anactivehumantransposon[50].
2. ThepiRNAPathwayinDrosophilaGonads
ThepiRNA-mediatedpathwayisinvolvedinthetranscriptionalandpost-transcriptionalsilencing
ofthetransposableandrepetitiveelementsofthegenome. Itoccurspredominantlyinthegermcells
andintheirsomaticprecursorsinthegonadsofbothsexesandisrequiredforfertilitynotonlyin
flies but also in mammals [35,51]. Most of what has been found on the piRNAs biogenesis, their
roles,andthepiRNA-relatedgenesinDrosophilacomesmainlyfromacombinationofgeneticsand
deepsequencingapproaches. Genome-widescreenstogetherwithtranscriptomicanalyseshavebeen
performed with the aim to identify as many piRNA-related genes as possible [52–54]. Both germ
cells and their somatic precursors use the piRNA pathway to silence transposable elements (TE).
ThebiogenesisofpiRNAsstartswiththetranscriptionofspecificgenomicclusterslocatedatdifferent
positions in the genome; they are composed of repetitive, non-functional relicts of transposable
elements [29]. Two main types of piRNA clusters have been described, both transcribed by RNA
PolymeraseII:uni-strandclusters,likeAT-X1andcluster2(germlinespecific)andflamenco(somatic
specific), transcribed as single-strand precursors; and dual-strand clusters, like 42AB and 80EF
(germline-specific)[55–57],transcribedfrombothstrands[29,31,51]. ClustersproduceRNAprecursors
thatareprocessedintopiRNAsintheperinuclearregionofthegonadalcellscalled“nuage”,which
surroundsthenuclearenvelope[58–62]. AlthoughtherolesofthepiRNA-relatedgenesarealmostthe
sameinthegonadsofbothsexes,somedifferencesexist[63,64].
2.1. ThepiRNAPathwayintheOvary
MostoftheknowledgeaboutthepiRNApathwaycomesfromstudiesintheDrosophilaovary.
Aftertheirtranscription,thepiRNAprecursorsenterdifferentpathwaysdependingonwhichpiRNA
will be produced and in which type of cell it will function, somatic or germline. In the somatic
primarypiRNApathwayoccurringinthefolliclecells,thepiRNAprecursorsundergoacleavageby
theendonucleaseZucchini,whichseemstohavearoleingeneratingboththe5(cid:48) andthe3(cid:48) endofthe
primarypiRNAs[29,40,65–67]. Theypossessastrong1Ubiasand,aftertheirproduction,theyare
boundtoPiwiandtootherproteins(HelicasesandTudordomainproteins)involvedinthepathway
andlocatedinthestructurecalledtheYb-body[68]. Afterthat,maturepiRNAsenterthenucleusto
exertTEtranscriptionalsilencing[65,66,69–72]. TheinteractionbetweentheTudorproteinYb,oneof
thekeycomponentsoftheYbbody,andtheRNAhelicaseArmitagehaveafundamentalroleinthe
entranceofPiwiintothenucleus[72],whereitisinvolvedintheH3K9me3-mediatedtranscriptional
silencingoftransposons. Aftertheirtranscription,theprimarypiRNAintermediatesplacethemselves
inawell-definedstructurecalledaflam-bodyorDotComadjacenttotheYb-body[73,74].
Theprimarypathwayalsooccursinthegermcells,nursecells,andoocyte,producingprimary
piRNAs. TheimmaturepiRNAstranscriptsaretransportedbytheDEADboxhelicaseUAP56[75,76],
to the perinuclear region of the cytoplasm, the nuage, where a multi-protein perinuclear complex
operates. ThePiwicladeproteinsAubergine(Aub)andArgonaute-3(Ago3)arethenboundtothe
primarypiRNAsandenterthegermlinespecialized“ping-pong”amplificationcyclewiththesetwo
proteinsasprotagonistsinthenuagetoproducethesecondarypiRNAsthatsilenceTEs[29,35,51,77,78].
ThepiRNAsproducedbytheping-pongamplificationloopshowconservedspecificsignatures,likeA
Int.J.Mol.Sci.2017,18,1066 4of20
in10thpositionoftheRNA(10Abias)[51]. Piwiseemsnottobeinvolvedinthegermline-specific
primarypathway[51,79–82].
Many proteins involved in piRNAs biogenesis and transposon silencing, such as Aubergine,
Ago3,Vasa,Krimper,Tudor,Spindle-E,Tejas,andKumo/Qinarelocalizedinthenuage[51,61,83–88].
AlthoughforsomeoftheseproteinsthemolecularmechanismofthepiRNApathwayisnotcompletely
clarified, the complex framework of interactions and actions continues to be enriched with new
discoveries. TheTudorproteinKrimper,forinstance,hasakeyroleintheformationofthenuage,
leadingtoaninteractionbetweenAubandAgo3[87,89],eventhoughitsroleseemstobedifferentin
thepiRNApathwaysofovariesandtestes,whereitisrequiredinassociationwithAubergineforthe
properlocalizationofitselfandAubergineatthenuage[63,64].
2.2. ThepiRNAPathwayintheTestesandItsRoleinthecrystal-StellateRegulation
The“crystal-Stellatesystem”wasfirstdescribedasanexampleofheterochromatin–euchromatin
interaction[90,91]. StellateandcrystalorSu(Ste)arehomologousrepetitivesequenceslocatedmainlyat
threedifferentpositionsonthesexualchromosomes: StellaterepeatsarelocatedontheXchromosome,
attheeuchromatic12Eregionandattheheterochromatich26region;crystalisontheYath11region.
Allofthemarerepetitivehomologoussequencesdifferinginsomefeatures[91–95]. Maleslacking
thecrystalregionexhibitcrystallineaggregatesintheirspermatocytes[96,97]. Inadditiontocrystals,
thesemalesalsoshowdefectsinchromosomecondensationandsegregation. TheStellateandcrystal
loci are normallysilent; however, deficiencies in the crystal region lead tothe de-repression ofthe
Stellate sequences in the germline. At the molecular level, the loss of the crystal region results in
the production of a testes-specific Stellate mRNA of 750 bases in length [93,98,99]. In 1995 we
discovered that the Stellate protein, produced by the Stellate mRNA, is the main component of
crystallineaggregates[100]. Anearlygeneticscreeningpermittedustoidentifysomemodifiersof
thecrystal–Stellateinteractionamongwhichareaubstingandhsp83scratch[98,99,101]. In2001itbecame
clear that the crystal-Stellate regulation occurs by the small-RNA-mediated pathway [30,102,103],
which, later, was identified as the specialized piRNA pathway [29,35,51,64,83,95]. In fly testes the
mostabundantpiRNAsassociatedwithAubergineandAgo3correspondto“crystal”piRNAs[83].
Thisfindinghasreinforcedtherelationshipbetweencrystal-StellateregulationandthepiRNApathway.
Indeed,thede-regulationofthecrystal–Stellateinteractionrepresentsasimplereadouttoidentifygenes
involved in the piRNA pathway in testes [95,98,99,101,104,105]. Many genes have been assigned
to the piRNA pathway, and the majority of them came from studies in ovaries [51,77,106,107].
Eventhoughnosystem-widescreeninghasbeenperformedtodateinsearchingforpiRNA-mediated
genes in testes, our group and others analyzed a wide variety of mutations of piRNA genes in
relation to their role in the crystal–Stellate interaction. The main features of several piRNA genes
identifiedduringtheyearsaresummarizedinTable1,wheretheirdomains,functions,localization,
cellular sub-localization, and especially their relationship with the crystal-Stellate regulation are
highlighted[32,35,38,53,57,61,64–66,69–71,73,75,76,78,81–86,88,95,99,101,104,105,108–128]. From the
analysisofthetableandfromFigure2,whereaVenn-likediagramisreported,showingthedistribution
of the genes listed in Table 1, it is clear that all the crystal-Stellate modifiers are piRNA-related
genes, although the opposite is not true. A crucial point regards Piwi that is not involved in the
piRNA-mediatedsilencingofStellateinthetestes: nonetheless,itisfundamentaltopiRNAbiogenesis
intheovaries. SincePiwiseemsnottoberequiredinthegermlineprimarypathway,atleastinthe
ovary[64,79–82,129],itisconceivablethatthesilencingoftheStellatesequencesmainlyrequiresthe
germlineprimarypathway;thisisalsosupportedbytheobservationthattheStellate-relatedpiRNAs
sharesomesignaturesoftheping-pongpathway[64,83,95,128]. However,manygenesrequiredforthe
ping-pongamplificationintheovaryarealsorequiredforthesilencingoftheStellatesequencesandof
thetransposonsintestes(Table1). Furthermore,theobservationsthatPiwiandRhinoarerequiredin
thefirststepofthepiRNAbiogenesisintheovaries[82]andthatbothproteinsarenotrequiredfor
Stellate-relatedpiRNAbiogenesisintestesindicatethatdistinctpathwaysoccurinthegonadsofboth
Int. J. Mol. Sci. 2017, 18, 1066 5 of 20
that the Stellate-related piRNAs share some signatures of the ping-pong pathway [64,83,95,128].
However, many genes required for the ping-pong amplification in the ovary are also required for the
silencing of the Stellate sequences and of the transposons in testes (Table 1). Furthermore, the
oInbt.sJe.rMvoalt.iSocni.s2 0t1h7a,t1 8P,i1w06i6 and Rhino are required in the first step of the piRNA biogenesis in the ov5aorife2s0
[82] and that both proteins are not required for Stellate-related piRNA biogenesis in testes indicate
that distinct pathways occur in the gonads of both sexes (the mechanism of primary piRNA
sexes(themechanismofprimarypiRNAproductioninthegermlinecouldbedistinctfromthepiRNA
production in the germline could be distinct from the piRNA production in the ovary) [63,87,89].
productionintheovary)[63,87,89].

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akrrmimitpaegre,,z ukrcicmhipneir,,q zinu/ckcuhminoi,, dqFinm/rk1u,mshou, tddFomwnr1,,h ssph8u3td.o(vwrnet,e nhsop,8p3ap. i(,vergegtelensos,, rphaipnio, ,emggaelelsstsr,o rmh)inhoa, vmeaneolsttbroemen)
haanvaely nzoedt bineerne laantiaolnyzteodt hine rcerylasttiaol-nS tteol ltahtee rcergysutlaalt-iSotnel.late regulation.
Int.J.Mol.Sci.2017,18,1066 6of20
Table1. piRNA-relatedgenesandthecrystal-Stellateregulation. Thebest-knownpiRNAgenes,withtheirdomainsandlocalizationsreported. Abbreviations:
N=nuclear;C=cytoplasmic;ND=notdetermined;theasterisks(*)indicatethattheproteinislocatedatthepiNGbodies;“+”indicatesthepresenceoftheprotein
and/orofthecrystals;“++”indicatesahighpresenceoftheproteinand/orofthecrystals;“++++”indicatesaveryhighpresenceoftheproteinand/orofthecrystals,
“++”,whereindicatedtheinformationareontheRNAcodingforcrystals;“?”indicatesthattheinformationarenotavailable.
Stellate-Made
Mutants ProteinDomains ProteinLocalization Testes Ovaries ProteinFunction Transposons References
Crystals
ArgonauteProteins
ping-pongpathway,
aubergine PAZ,MID,PIWI germline(C-nuage);germgranules +* + germinal + [35,38,78,83,88,108]
primarypathway
ago3 PAZ,MID,PIWI germline(C-nuage);germgranules +* + ping-pongpathway germinal + [35,70,71]
piwi PAZ,MID,PIWI soma+germline(nuclear) + + primarypathway germinal+somatic - [83,84,109]
ago1 PAZ,PIWI soma,germline(C-nuage) +* + piRNApathway,miRNApathway somatic + [105,111]
Helicases
armitage RNAhelicase(SDE3) soma(C-Ybbodies)+germline(C) + + primarypathway germinal+somatic + [70,71,112–114]
germline(Amplifier
vasa DEADRNAhelicase +* + primary+ping-pongpathway germinal+somatic ++(RNA) [84,86]
Complex-C-nuage);germgranules
UAP56 DEADRNAhelicase germline(N)+N/Cboundary ND + primary+ping-pongpathway germinal+somatic ND [73,75]
TudorProteins
DEADRNAhelicase+ primarypathway(singlestrand
Yb soma(C-Ybbodies) ND + somatic - [32,70,71,113,114]
Tudordomain clusters(flamenco))
DEADRNAhelicase+
spindle-E germline(C-nuage) +* + primarypathway germinal + [61,84,104]
Tudordomain
germline(C-nuage)+soma
krimper Tudordomain + + germinal ++++ [61,115]
(krimperbody)
germline(C-nuage)soma;
tudor Tudordomain +* + germinal - [116–118]
germgranules
tapas Tudordomain+Lotus germline(C-nuage) + + ping-pong?,primary? germinal ++++(RNA) [113,119]
Tudordomain+Lotus
tejas germline(C-nuage) + + ping-pong?,primary? germinal ++++ [84]
domain
Tudordomain+ soma(C)+germline(C-nuage)
zucchini + + primarypathway germinal+somatic + [65,66,69,71,81]
nuclease mitocondrial
Tudordomain+ germline(C-nuage);
squash +* + germinal+somatic + [69,81]
nuclease NDinthesoma
Tudordomain+ germinal(seenonlyin
vreteno soma(C)+germline(C-nuage) + + primarypathway ND [113,114]
RRMdomai ovaries)+somatic
Int.J.Mol.Sci.2017,18,1066 7of20
Table1.Cont.
Stellate-Made
Mutants ProteinDomains ProteinLocalization Testes Ovaries ProteinFunction Transposons References
Crystals
Tudordomain+RING soma(N)+germline(Amplifier primarypathway(dualstrand
qin/kumo + + germline+somatic + [75,85,120]
domain Complex-C-nuage) clusters)
Tudordomain,KH,
dFmr1 soma+germline(N+C) + + germinal+somatic + [105]
RGG,NLS,NES
Tudordomain+KH
papi soma(N)+germline(C-nuage) ND + primary+ping-pongpathway germinal+somatic ND [121]
domain
eggless Tudordomain+SET H3K9methyltransferase
germline(N)+soma(N) ND + germinal+somatic ND [122]
(dSETDB1) domain (TEregulation)
NonTudorProteins
chromo/shadow primary+ping-pongpathway
rhino germline+soma(N) ND + germinal - [57,76,82]
domain (dualstrandclusters)
primary+ping-pongpathway
cutoff DXO/Dom3Zfamily germline(C-nuage)+N ND + germinal - [57,76]
(dualstrandclusters)
Maelstromdomain
germline+soma:shuttle
maelstrom (DNA/RNAbinding); + + primary+ping-pongpathway germinal+somatic ND [123]
nucleus-cytoplasm(nuage)
HMGprotein
capsuleen PRMT germline(C-nuage) + + argininemethyltransferase germline + [124,125]
FKBP6family soma(YB-bodies)+
shutdown + + primarypathway+ping-pong germinal+somatic + [115,127]
(co-chaperone) germline(N+C)
hsp83 heatshockprotein soma+germline(N+C) + + primarypathway+ping-pong germinal+somatic + [70,78,99,101]
Int. J. Mol. Sci. 2017, 18, 1066 8 of 20
Int.J.Mol.Sci.2017,18,1066 8of20
3. dFmr1 Participates in the piRNA Pathway Occurring in Gonads
3. dFmr1ParticipatesinthepiRNAPathwayOccurringinGonads
Recently our group has demonstrated that dFmr1 plays a role in the piRNA pathway in testes
and ovaries of Drosophila melanogaster [105]. This study originates from the observation that dFmr1, a
RecentlyourgrouphasdemonstratedthatdFmr1playsaroleinthepiRNApathwayintestes
panrodteoivna irnivesolovfeDd rions oRpNhiAla mmeeltaanbooglaissmter, [w10a5s ]f.oTuhnids stotu bdey aosrsiogciinaateteds ifnr oam cotmhepolebxs ewrvitaht icoonmthpaotndenFtms ro1f,
the RNA interference pathway [7,8]. In addition, the complexity of the symptoms exhibited by
aproteininvolvedinRNAmetabolism,wasfoundtobeassociatedinacomplexwithcomponentsof
fragile-X patients and by Drosophila mutants in the gonads and in the nervous system, along with the
theRNAinterferencepathway[7,8].Inaddition,thecomplexityofthesymptomsexhibitedbyfragile-X
dpiastcieonvtesryan odf bpyiRDNrAosso pinhi tlahem nuetravnotsuisn styhsetegmo noafd Dsraonsodpihniltah aennde hrvuomuasnssy s[t3e3m,3,4a,1lo3n0–g1w33it]h, atlhloewdiesdc ouvse troy
hoyfppoiRthNeAsiszein tthhaet nmeruvcohu smsoyrset ermemoafiDnrso stoop bhiel adainscdohvuermedan asb[o33u,t3 4th,1e3 0m–u13lt3i]p,lael lfouwnecdtiounsst oofh ythpeo tFhMesRizPe
protein.
thatmuchmoreremainstobediscoveredaboutthemultiplefunctionsoftheFMRPprotein.
33..11.. ddFFmmrr11’’ss RRoollee iinn tthhee TTeesstteess
TThhee rroollee ooff ddFFmmrr11 iinn tthhee ppiiRRNNAA ppaatthhwwaayy wwaass ddeemmoonnssttrraatteedd aatt ffiirrsstt,, bbyy tthhee pprreesseennccee ooff tthhee
SStteellllaattee--mmaaddee ccrryyssttaalllliinnee aaggggrreeggaatteess iinn ssppeerrmmaattooccyytteess ooff ddFFmmrr11∆∆5500,, ddFFmmrr11∆∆11113 3mmuutatannt tmmaalleess aanndd iinn
RRNNAAii--ddFFmmrr11 mmaalleess iinn wwhhiicchh tthhee ffuunnccttiioonn ooff tthhee pprrootteeiinn wwaass sseelleeccttiivveellyy rreedduucceedd iinn ddiiffffeerreenntt cceellllss ooff
tthhee ggoonnaaddss ((ggeerrmmiinnaal loorr ssoommaatitci)c )(F(Figiguurer e3,3 r,erde dbobxoexse)s. )T.hTeh perepsreensecne coef ocrfycsrtyaslsta wlsaws aacscaocmcopmanpieadn ibeyd
tbhye tdhreasdtrica srteicdurecdtiuocnt ioofn tohfe tShteelSlatteel-lraetela-rteelda tpeidRNpiARsN. ATsh.e Tchonefciromnfiatrimona toiof nthoef rtohlee roofl edoFfmdrF1 minr 1thine
pthiReNpAiR NpaAthpwaatyh wina tyesinteste, sctaems,ec farmome ftrhoem obthseervoabtsieornv tahtiaotn int hdaFtmirn1 dmFmutra1nmts uatnadn tdsFamnrd1-RdFNmAr1i -mRaNleAs,i
smevaleersa,ls etrvaenrsaplotrsaanbslpe oeslaebmleeneltesm aerne tsacatrievaatcetdiv awteitdhw eiftfhecetfsf eoctns othnet hfeerfteilrittiyli toyf otfhteh eininddivivididuuaalsl s[[110055]]. .
ddFFmmrr11 iiss eexxpprreesssseedd iinn tthhee ggeerrmm cceelllsl soof faadduultl ttetsetsetse sasa scocnocnlculduedde dfrofrmom itsi tcsocloolcoacliazlaiztiaotnio wniwthi tVhaVsaa,s aa,
gaegrmerlminlein-sep-sepciefcicifi mcamrkaerrk [e8r6,[18364,1],3 a4s], parsevpiroeuvsiloyu dsleyscdriebsecdri b[1e0d5][.1 I0t5 i]s. alIstoi spraelssoenpt riens tehnet piniNtGhe bpoidNieGs
(bFoidguierse( F4iAgu–Cre),4 Are–pCr)e,sreenptriensge nstpinecgifsipce gciifianctg ibaondtibeosd oief stohfet hneunaguea,g ew,hwehree resosmome eppiRiRNNAA ccoommppoonneennttss
ppeerrffoorrmm tthheeirirf ufuncntcitoinon[1 3[153].5d]. FdmFrm1lro1c aloliczaaltiizoantiiosnp riesd opmreidnoamntilnyacnyttloyp claystmopicla,semveinc, theovueng htahonuugchle aar
nlouccalleiazra tlioocnalaiznadtifounn catniodn fuhnacvteiobne ehnavreep boeretned re[1p3o6r,t1e3d7 []1.36,137].

FFiigguurree 33.. GGeermrmlilninee anandd sosmomataicti crerqeuqiureirmemenetn otfo dfFdMFMR1R i1n itnhet hteestteesst. eAs. scAhesmchee mof ethoef athpeicaalp picaarlt poaf rat
toefstais teisst isreipsorretpedo.r teRde.d Raerdrowarsr owansda nbdoxbeos xeinsdiincdaitcea tteheth eefefeffcet ctofo fththee kknnoocckkddoowwnn aanndd//oorr tthhee
oovveerreexxpprreessssiioonn ooff ddFFmmrr11 iinn tthhee hhuubb aanndd iinn tthhee ggeerrmmlliinnee oobbttaaiinneedd uussiinngg uuppddGGaall44 ((hhuubb)) aanndd nnaannoossGGaall44
((ggeerrmmlliinnee)) ddrriivveerrss.. TThhee ggrreeeenn aarrrrooww aanndd bbooxx iinnddiiccaatteess tthhee eeffffeeccttss ooff tthhee oovveerreexxpprreessssiioonn ooff aauubbeerrggiinnee. .
GGSSCC ssttaannddss ffoorr ggeerrmm sstteemm cceellllss;; GGBB ssttaannddss ffoorr ggoonniiaallbbllaassttss..
Int.J.Mol.Sci.2017,18,1066 9of20
Int. J. Mol. Sci. 2017, 18, 1066 9 of 20

Figure4. dFmr1andVasaimmunolocalizationinwtadulttestes. Maxintensityoftripleconfocal
Figure 4. dFmr1 and Vasa immunolocalization in wt adult testes. Max intensity of triple confocal
sections(40×)ofawildtypetestislabeledwith(A)anti-dFMR1,(B)anti-Vasa,(C)Mergewiththe
sections (40×) of a wild type testis labeled with (A) anti-dFMR1, (B) anti-Vasa, (C) Merge with the
DAPIchannel.
DAPI channel.
ddFFmmrr11 wwaass aallssoo ffoouunndd ttoo iinntteerraacctt ggeenneettiiccaallllyy aanndd bbiioocchheemmiiccaallllyy wwiitthh ttwwoo AArrggoonnaauuttee pprrootteeiinnss::
AAuubbeerrggiinnee ((aaP Piwiwipi rportoetinei)n()F i(gFuigreur3e, g3r,e genrebeonx b)oanx)d aAngdo 1A(gaon1A (gaon pArogtoe ipnr),obteoitnh),o fbowthhi cohf awrehliochca taerde
alotctahteedn uaat gtheeo nfuthaegeg eorfm thcee gllesr.mT hceelglse. nTehteic giennteertaicc tiinotnerwacittihonA wubitehr gAinuebearngdinAe gaon1d aAlsgooo1c aclusros oicncuthres
nine rtvhoe unsesrvysotuesm syastttehme naet uthroe mneuuscroomlarujsucnoclatiro jnusn[c1ti0o5n].s [105].
TThhee pprreesseennccee ooff tthhee TTuuddoorr//AAggeenneett ddoommaaiinn iinn ddFFmmrr11 ssuuggggeessttss tthhaatt iitt mmaayy bbiinndd tthhee AArrggoonnaauuttee
pprrootteeiinnss bbyy tthheeiirr ssyymmmmeettrriiccaallllyy ddii--mmeetthhyyllaatteedd aarrggiinniinneess ((ssDDMMAAss)),, llooccaatteedd aatt tthhee NN--tteerrmmiinnaall ssiittee ttoo
aaccttiivvaattee tthheemm [[110099,,112211,,113388]].. HHoowweevveerr,, iinn ssppeecciifificc ccaasseess,, tthhee iinntteerraaccttiioonn bbeettwweeeenn AArrggoonnaauuttee pprrootteeiinnss
aanndd ssoommeeT uTduodropr rpotreoitnesiniss inisd einpdenepdeenndtfernotm frtohmes DthMe AsDs,MasAdse,m aos ndstermatoednsftorrattehde ifnotre rtahceti oinntbeeratwcteioenn
Abeutbweergenin eAaunbdeKrguinmeo a[1n2d0 ]Kourmfoor A[1g2o03] aonrd fKorri mApgeor3[ 8a9n].dI nKtrhiemcpaesre o[8f9d]F. mInr 1tahned cAasueb eorfg idnFem,itr1se eamnds
mAuobreerligkienley, tiht asetethmesi nmteorraec ltiikoenlyo ctchuart sthined ienpteernadcetinotnly ofcrcoumrst hinedseDpMenAdsenretlsyid furoesmo tfhAe usbDeMrgAinse rbeescidauusees
iotf hAausbbeeregnindee bmeocanusstrea itte dhatsh bateetnh edbeimocohnesmtraicteadl itnhtaetr athctei obnioocfhtehmeictwalo inptreoratecitniosni sonf othtel itmwiote pdrototetihnes
Nis -nteortm liimnaitleddo tmo athine oNf-Ateurmbeirngailn deo[1m0a5i]n. of Aubergine [105].
33..22.. ddFFmmrr11’’ss RRoollee iinn tthhee OOvvaarryy
RReecceennttllyy,, ddFFmmrr11 hhaass eemmeerrggeedd aass aa mmeemmbbeerr ooff ppiiRRNNAA--mmeeddiiaatteedd ppaatthhwwaayyss iinn oovvaarriieess [[110055,,113399]]..
TThhee fifrirsstti nidnidciactaiotinono fothf ethinev oinlvveomlveenmteonftd Fomf rd1Fimnrt1h einp iRthNeA ppiRaNthAw apyaothfwthaeyo voafr ythwe aosvthareya cwtiavsa titohne
oacfttirvaantsiopno soonf stcraaunssepdosboyntsh ecaloussseodf dbFym trh1e. Tlhosesl eovfe ldoFfmtrar1n.s cTrihpet iolenvoefl spofe ctirfiacntsrcarnipsptioosno nosfi nscpreecaisfeics
strigannsifipcoasnotnlsy iinncrtehaesedsF msirg1nimficuatnantlty oivna trhiees daFcmcor1m mpauntaiendt ,oavsaerixeps eacctecodm, bpyanthieed,r eadsu ecxtpioenctoedf,f ebryti ltihtye.
Mredoruecotvioenr, oitf ifseirntitleitryes. tMinogretoovneort,e itth isa tinthteereexstpinregs stioo nnoptaet ttherant sthoef edxFpmrer1ssainond pthaettperiRnNs oAf -dreFlmater1d apnrdot tehine
ApiuRbNerAg-irneelactoe-dlo pcraolitzeeinin Asupbeecrifigicntee rcroi-tloorcieaslizoef tihne spovecairfyic[ t1e0r5r]i.tories of the ovary [105].
TThhee DDrroossoopphhiillaao ovvaarryyi sisc coommppoosesdedo fo1f 212to to16 1o6v oavraiorlieosl.esIn. Iena cehacohn oe,nteh, ethmeo mstoaspt iacaplicsatrlu scttruurcetuisreth ies
gtheerm gaerrimumar,iuwmhe, rweshteerme csetlelmsa rceelllosc aatreed .loTchaetedde.v Telhoep idnegveeglogpcihnagm ebgegr pcahsasmesbtehrr opuagshsetsh ethgreorumgahr iuthme
agnerdmparroicuemed santhdr opurgohceietsdds etvheroloupgmhe intst, dfoervmelionpgmaelinnte, afrocrhmaiinng. daF lminre1aarn cdhAaiunb. edrFgminre1c oa-nlodc Aaliuzbeeirngtihnee
gcoe-rlmocaarliiuzme ,inw htheree gtheremyaarcicuumm,u wlahteeraet tthheeyp oascitciuomnuoflatthee astt etmhec eplloss.iTtihoen towfo tphero steteinms acleslolsc. oT-lhoec atlwizoe
ipnrothteeinnsu aaglseoo cfoth-leocnaulrizsee cinel ltshaen ndu,aingeth oef lathteer nstuargsees c,ethllesy aonvde,r ilnap thine tlhaetecry stotapgleass,m thoefyt hoevoeorlcaypte in[1 0th5e].
Tcyhteorpellaastmion osfh itpheb eotwoceyetne d[1F0m5r]1. Tanhde trheelaptiioRnNsAhipp abthewtwaeyeins adlFsomcro1n afinrmd etdheb ypitRheNiAnt epraatchtiwonayb eitsw aelesno
dcoFnmfirr1maendd bPyi wthie, winhtiecrhacatciotnto bgeettwheerenin dtFhmehr1e taenrodc hPriwomi, awtihcigchen aects itloengectinhgeri nins othme ahtiectecreollcsharonmdathtiec
tgreannes psoilseonncisnilge ninci nsogminatgiecr cmelclse lalsnd[1 4th0e]. tIrnanpsaprtoicsuolna rs,iiltenhcaisnbge einn gdeermmo ncesltlrsa t[e1d40t]h.a Itnd pFamrtri1cuislaers,s eitn htiaasl
fboeretnh deecmororencsttrloatceadli zthataito dnFomfHr1P i1s, eisnstehnetihael tfeorro tchher ocmorarteicnt olofcfaollilzicalteiocnel olsf [H13P91],. iInt itshke nhoewtenrotchhartoHmPa1tiins
aofh ifgohllliyclceo cneslelsrv [e1d39p]r.o Itte iins ikdnenotwifine dthaast aHcPru1c iiasl aco hmigpholnye nctonofsethrveehde tperrooctehirno midaetnintifwieidth aasr oal ecrinuctihael
PcoomsitpioonneEnftf eocft Vthaer iehgeatetiroonch(ProEmVa),tinno wwiathls oa rreocloeg niniz tehde aPsopsliatyioinng Erfofelecst iVnamriaengyatoiothne (rPpErVoc),e snsoews[ 1a4ls1o].
recogInnitzheedo avsa pryla,ydiFnmg rr1oliessa ilnso minavnoyl voethdeirn ptrhoecmesiscerso R[1N41A].- mediatedpathway. Thetightlinkbetween
dFMIRn1 tahned othvaermy,i RdNFmArp1 aitsh walasyo hinasvoelmveedrg eind tfhroem mtihceroiRntNerAa-cmtioedniabteetdw epeanthtwheayF.r aTghilee -tXigphrto tlienink
abnetdwteheen bdaFnMtamR1m ainRdN tAhe tmoicRoNntAro plagtehrwmaliyn heasst eemmecregllesdi nfrothme tohvea irnyteorfaDctiroosno pbheitlwae[1en42 t]h.eI nFrhaugimlea-Xn
cperloltleiinne sa,nFdM tRheP binantetraamc tmswiRiNthAc otmo pcoonnternotls goefrtmhelinmei RstNemA pcealtlhs wina yt,hseu ochvaarsy Doifc eDrr1oasonpdhiAlag [o1142[1].4 3In].
human cell lines, FMRP interacts with components of the miRNA pathway, such as Dicer1 and Ago1
Int.J.Mol.Sci.2017,18,1066 10of20
InDrosophila,Ago1isalsorequiredforthebiologicalfunctionofdFmr1inneuraldevelopmentand
synaptogenesis[9].
4. GeneticandBiochemicalInteractorsofdFmr1
In the last part of this review we will examine the multiple roles and functions of the dFmr1
protein,followingthethreadsofitsgeneticandbiochemical/physicalinteractorsinthegonadsand
in the germ granules representing a bridge from oocyte to embryos in the fly and other animals
(miceandhumans).
dFmr1hasrecentlybeenassociatedwiththepiRNApathwayinthegonadsofbothsexes[105,139]
andtheinteractionwithsomeoftheArgonauteproteinssupportsthesefindings. dFmr1interacts
with Aubergine, allowing the correct silencing of the transposons and repetitive sequences [105],
andwithPiwi[139],cooperatinginthepiRNA-mediatedtranslationalsilencingofthetransposons.
ThesetwoproteinsbelongtothePiwi-subfamilyoftheArgonauteproteins,andwhereasAubergine
is cytoplasmic as well as the most common localization of dFmr1, Piwi is predominantly nuclear,
confirminganuclearfunctionandlocalizationofdFmr1. Ago1,belongingtotheAgo-subfamilyofthe
Argonauteproteins,isabiochemicalandgeneticinteractorofdFmr1inthepiRNApathway[105],as
wellasinthemiRNApathwayintheovary[9,142–147].
TheDEADboxRNAhelicaseVasaisalsoconsideredakeycomponentofthepiRNApathway;
it is located at the nuage of ovaries and testes [35,51,83,134,148–152] and has been considered
a“biochemicalplatform”toputtogetherthekeycomponentsofthepiRNAamplificationmachinery
(theAmplifiercomplex)innuage[86]. dFmr1hasbeendemonstratedtoco-localizewithVasainthe
testesandovaries[105].ThefunctionofthedFmr1geneisalsorequiredforpolecellsformation,located
attheposteriorpoleoftheearlyembryo,inwhichtheVasaproteinisoneofthemaincomponents
ofthepolargranules[134,153,154]. ThesefindingssupportthehypothesisthatdFmr1andVasamay
exerttheirroleinthesamepathwayfromthegermcellsintheovarytothepolecellsintheembryoin
thesilencingofthetransposableelementsmaintaininggenomestabilityinthesetissues.
Anothercomponentofthegermgranulesatthepoleplasm,wherethepolecellswillform,isthe
RNAbindingproteinCup[155–157]. ThegenecodingforCuphasbeenfoundinageneticscreening
forinteractorsofdFmr1[158]. ItcodesforanRNAbindingproteinpromotingtheaccumulationof
thegermplasmcomponentslikeOskar,VasaandStaufenattheposteriorpoleoftheoocyteanditis
requiredforthecorrectpolargranulesdeposition[157,159].Duringthedevelopmentoftheearlystages
ofoogenesis,Cupco-localizesandinteractswithHsp83[157]. Hsp83hasawell-demonstratedrolein
thesilencingoftransposonsandrepetitivesequencesrelatedtothegenomestability[95,101,160–162].
TheP-bodiescomponent,Me31BDEADboxhelicase,representsaveryintriguinglinkbetween
Cup, Hsp83 and dFmr1 [163]. Me31B co-localizes with dFmr1 and Cup in these cytoplasmic
structuresimplicatedinRNAmetabolismlikestorage,translationalrepression,andRNAdegradation.
Me31B also interacts with Aubergine and Vasa, and is required to recruit dFmr1 to the neuronal
granules[164–167],whichmaybeconsideredaneuronalcounterpartofthepolargranules.
AnemergingfunctionwhichhasbeendemonstratedforthemammalianformofFmr1protein
is related to its capacity to recognize the G-quadruplets, a specific structure in RNAs [168]. It is
interesting to note that the G-quadruplets have been shown to be present in the precursors of the
piRNAs[47]. TheconnectionbetweentheDrosophiladFmr1andtheG-quadrupletscanbesupported
bythefindingthatoneofthetargetsofdFmr1inthenervoussystemistheproteinFutsch,thehomolog
ofthemammalianMAP1B,exhibitingaputativeG-quadrupletsinits5(cid:48) end[19,144,153]. Thisspecific
aspecthasnotyetbeencompletelyelucidatedinDrosophila.
NuclearInteractorsintheSmallRNAPathway(intheGonads)
In additionto thecytoplasmic functionof dFmr1, anuclear functionhas been suggested due
not only to the NLS and NES domains in the protein (Figure 1) [1,24,169], but also to some of its
nuclear interactors. One of the known nuclear interactors in mammals is NUFIP (Nuclear Fmr1