Guan, D., Altan-Bonnet, N., Parrott, A. M., Arrigo, C. J., Li, Q., Khaleduzzaman, M., Li, H., Lee

13 Pages·2008·1.53 MB·English

MOLECULARANDCELLULARBIOLOGY,July2008,p.4629–4641 Vol.28,No.14
0270-7306/08/$08.00(cid:1)0 doi:10.1128/MCB.00120-08
Copyright©2008,AmericanSocietyforMicrobiology.AllRightsReserved.
Nuclear Factor 45 (NF45) Is a Regulatory Subunit of Complexes with
(cid:1)
NF90/110 Involved in Mitotic Control †
Deyu Guan,1 Nihal Altan-Bonnet,3 Andrew M. Parrott,1 Cindy J. Arrigo,1‡ Quan Li,1
Mohammed Khaleduzzaman,1§ Hong Li,1 Chee-Gun Lee,1 Tsafi Pe’ery,1,2
and Michael B. Mathews1*
DepartmentsofBiochemistryandMolecularBiology1andMedicine,2NewJerseyMedicalSchool,UniversityofMedicineand
DentistryofNewJersey,185SouthOrangeAvenue,P.O.Box1709,Newark,NewJersey07103-1709,andDepartmentof
BiologicalSciences,RutgersUniversity,Newark,NewJersey071023
Received22January2008/Returnedformodification29February2008/Accepted28April2008
Nuclear factor 90 (NF90) and its C-terminally extended isoform, NF110, have been isolated as DNA- and
D
RNA-bindingproteinstogetherwiththeless-studiedproteinNF45.Thesecomplexeshavebeenimplicatedin o
w
generegulation,butlittleisknownabouttheircellularrolesandwhethertheyareredundantorfunctionally n
lo
distinct.Weshowthatheterodimericcorecomplexes,NF90-NF45andNF110-NF45,existwithinlargercom- a
d
plexesthataremorelabileandcontainmultipleNF90/110isoformsandadditionalproteins.Depletionofthe e
d
NF45subunitbyRNAinterferenceisaccompaniedbyadramaticdecreaseinthelevelsofNF90andNF110. fro
Reciprocally,depletionofNF90butnotofNF110greatlyreducesthelevelofNF45.CoregulationofNF90and m
NF45isaposttranscriptionalphenomenon,resultingfromproteindestabilizationintheabsenceofpartners. m
c
DepletionofNF90-NF45complexesretardscellgrowthbyinhibitionofDNAsynthesis.Giantmultinucleated b
.a
cells containing nuclei attached by constrictions accumulate when either NF45 or NF90, but not NF110, is s
m
depleted.ThisstudyidentifiedNF45asanunstableregulatorysubunitofNF90-NF45complexesanduncovered .o
their critical role in normal cell division. Furthermore, the study revealed that NF90 is functionally distinct rg
fromNF110andismoreimportantforcellgrowth. at U
M
D
N
Human nuclear factor 90 (NF90) and nuclear factor 45 GQSY region that can interact with nucleic acids. Although J
(NF45) were originally purified as a sequence-specific DNA characterizedasDNA-bindingproteins(17,36,40,41),NF90 -U
N
binding complex regulating the interleukin-2 (IL-2) promoter andNF45donotcontainarecognizedsequence-specificDNA- IV
E
(10, 17). NF90 is the founder member of a family of proteins binding domain and the complex containing NF90 and NF45 R
S
generated from differentially spliced transcripts of the ILF3 doesnotappeartointeractwithDNAdirectly.NF90andNF45 IT
Y
gene(12).NF90andNF110,whichdifferattheirCtermini,are have been purified in complexes containing the Ku proteins L
thetwomostprominentILF3isoformsincells(12,33,42,55). andDNA-proteinkinase(PK),aswellaseukaryoticinitiation IB
R
Bothhavebeenrepeatedlyisolatedindiversestudiesandhave factor2(eIF2),anditislikelythattheirinteractionswithDNA A
R
beengivenavarietyofnames.Forexample,MPP4(M-phase aremediatedbyKuorDNA-PK(5,50). IE
S
phosphoprotein 4) is similar, if not identical, to NF90 and is Ontheotherhand,thedirectbindingofNF90anditsrela- o
phosphorylatedduringMphase(23),andcloselyrelatedpro- tives to double-stranded and structured single-stranded RNA n J
teins4F.1and4F.2werecharacterizedinXenopusasdouble- hasbeenwellstudied(22,33,37)andmuchofthefunctional uly
stranded RNA (dsRNA)-binding proteins (3). NF90 is also research on NF90 and NF110 is based on their RNA binding 28
known as DRBP76, NFAR1, and TCP80 (34, 43, 55), and properties. NF90 and NF110 (as well as NF45) are mainly , 20
NF110isalsoknownasILF3,NFAR2,TCP110,andCBTF122 0
located in the nucleus through binding to RNA as well as to 8

(4, 43, 53, 55). Underlining the importance of these proteins,
protein (33). The two dsRBMs in NF90 and NF110 are re-
knockoutofthemouseILF3geneledtomuscledegeneration,
quiredfortheiractivityinregulatinggeneexpression(38),and
respiratoryfailure,anddeathsoonafterbirth(44).
dsRBMmutationsthatinterferewithRNAbindingreducethe
NF90 and NF110 contain two dsRNA binding motifs
stimulation of gene expression by NF110 (37). NF90 forms
(dsRBMs) which are responsible for their ability to interact
complexeswithanumberofsmallnoncodingRNAs(31,32)as
withstructuredRNA.TheyalsohaveanRGGdomainthatis
well as mRNAs. In activated Jurkat cells, NF90 is exported
capable of nucleic acid binding, and NF110 has an additional
fromthenucleustothecytoplasm,bindstoAREelementsin
the 3(cid:2) untranscribed region of IL-2 mRNA and stabilizes the
mRNA (45). Similarly, NF90 binds to the 3(cid:2) untranscribed
*Corresponding author. Mailing address: Phone: [phone]. region of MyoD and p21WAF1/CIP1 mRNAs and is implicated
Fax:[phone].E-mail:[email protected].
‡Presentaddress:DepartmentofBiology,NewJerseyCityUniver- instabilizingthesemRNAs(44).GeneticablationofNF90in
sity,JerseyCity,NJ07002. mice led to rapid degradation of MyoD and p21WAF1/CIP1
§Department of Pathobiological Sciences, School of Veterinary mRNAs. NF90 also binds several viral RNAs (16, 22, 26, 27,
Medicine,LouisianaStateUniversity,BatonRouge,LA70803.
46), and a C-terminal variant of NF90 (NF90ctv or NF90c)
†Supplementalmaterialforthisarticlemaybefoundathttp://mcb
attenuateshumanimmunodeficiencyvirustype1replicationin
.asm.org/.
(cid:1)Publishedaheadofprinton5May2008. a stably transduced cell line. These observations suggest that
4629
4630 GUAN ET AL. MOL.CELL.BIOL.
NF90 is exploited during virus multiplication in mammalian 15mMMgCl,200mMNaCl,100(cid:4)g/mlcycloheximide,2(cid:4)g/mlleupeptin,2
2
cells. It is interesting that NF90 interacts and cofractionates (cid:4)g/mlaprotinin,1mMdithiothreitol)andcentrifugedat150,000(cid:3)gfor20hat
4°CinanSW41rotor(Beckman).Fractions(25(cid:3)200(cid:4)l)wereremovedby
with the eIF2 kinase protein kinase R (PKR), as well as with
aspiration.Proteinswereresolvedin7.5%sodiumdodecylsulfate(SDS)–poly-
eIF2, and is a substrate for phosphorylation by PKR (19, 29,
acrylamidegelsandanalyzedbyimmunoblotting.Agradientcontainingprotein
30,34,50). standards(50(cid:4)gbovineserumalbumin,100(cid:4)galdolase,and100(cid:4)gcatalase)
The properties of NF45 are less well understood, and its was centrifuged simultaneously, and fractions were analyzed by A and gel
280
specific function is unknown. NF45 is encoded by the ILF2 electrophoresisfollowedbystaining.
Gelfiltrationchromatography.A10-mlSephacrylS-400HRcolumn(Amer-
gene.Inmice,NF45isexpressedathighlevelsinbrain,kidney,
shamBiosciences)wasequilibratedwithrunningbuffer(50mMTris-HCl[pH
and testis (56), all of which also contain high levels of NF90
8.0],150mMNaCl,0.1mMEDTA).Cytoplasmicandnuclearextractsof293
and/orNF110(44),althoughtheirdistributionsinsomeother cells were loaded, and 30 100-(cid:4)l fractions were collected. The column was
tissuesdonotappeartomatch.NF45iscomplexedwithNF90 calibratedwithBio-Radstandardsasfollows(molecularmass,Stokesradius):
inmanycelllinesandinhumanplacenta(16,17,22,27,28,41, thyroglobulin(670kDa,8.5nm),bovinegammaglobulin(158kDa,5.34nm),
chickenovalbumin(44kDa,3.05nm),equinemyoglobin(17kDa,2.05nm),and
46, 50). In the HTB-14 glioma cell line, however, NF90 and
vitaminB (1.35kDa,0.16nm).ProteinsweredetectedbyA andgelelec-
NF45 were largely distributed in different fractions with little 12 280
trophoresisfollowedbystaining.VitaminB wasdetectedbyA .
12 361
overlapinsizeexclusionchromatography(27).TheN-terminal Immunoblottingandimmunoprecipitation.Cellswereharvestedbyscraping.
regionsofbothNF90/110andNF45containDZF(dsRBM-and Subsequentprocedureswerecarriedoutat4°Cexceptasnoted.LysatesinIP D
zincfingerassociated)motifswhicharesharedwiththeconserved (immunoprecipitation)buffer(50mMTris-HCl[pH7.4],150mMNaCl,1mM ow
EDTA,1%TritonX-100,2(cid:4)g/mlleupeptin,2(cid:4)g/mlaprotinin,1mMdithio- n
proteinZFRthatisimportantfordevelopmentoffliesandmice threitol)wereclarifiedat12,000rpmfor10min,and10to20(cid:4)gproteinwas loa
(25).OverexpressionofNF45enhancedthestimulationofcyto- d
loaded onto 7.5% SDS–polyacrylamide gels. Immunoblots were probed with e
mceeregvailsoiaveir)u,sNpFr4o5miontheirbibteydNPFK9R0,(3w8h)e.rIenasyeNaFst90(Saacctcihvaatreodmytcheiss a(mntoibuosed,y1a:g1a,i0n0s0t;NBFD90T(rraabnbsditu,c1t:i1o0n0)L(a3b3o),raNtoFr4i5es()r,aobbrita,c1t:i2n00()m(o3u3s)e,,D1R:2B,0P0706; d fro
m
kinase(30). Sigma) or with Omni-tag monoclonal antibody (mouse, 1:5,000; Santa Cruz). m
ItisnotclearwhetherthecellularrolesofNF90andNF110 Notethatanti-NF90andanti-DRBP76bothreactwithNF110.Forimmunopre- cb
areidenticalordistinctandwhetherNF45,NF90,andNF110 cwiapsitamtiioxend,awnittih-N1F090(cid:4)l(1p:r2o5t)e,iannAti--NSeFp4h5a(r1o:s5e0)b,eoardasn(tAi-Ommernshi-atamg(B1i:o5s0c0ie)nacnetsib)ofdoyr .asm
functionincellsasmonomersorascomplexes.Wecharacter- 2h.ThebeadswerewashedthreetimeswithIPbuffer,mixedovernightwith100 .o
ized complexes containing these proteins and used RNA in- (cid:4)lsupernatantofcellextract,andthenwashedthreetimesinIPbuffer.Proteins rg
a
terference to elucidate their function. NF90 and NF110 form wereresolvedin7.5%SDS–polyacrylamidegelsandanalyzedbyautoradiogra- t U
heterodimeric core complexes with NF45 and large in vivo phyorimmunoblotting. M
GST pull-down assays. Glutathione S-transferase (GST) and GST fusion D
complexes in which multiple NF90/110 isoforms interact with N
proteinswereproducedandusedinpull-downassaysasdescribedpreviously J
eachotherandwithadditionalproteins.DepletionofNF45led (29).Radiolabeledproteinsweresynthesizedinwheatgerm(PromegaTnT)with -U
todecreasedlevelsofNF90andNF110.Thiscoregulationisat [35S]methioninefromlinearizedplasmidsencodingNF90,NF110,andNF45or NIV
the protein level: the stability of NF90 family proteins is de- theirmutants.Plasmidsusedtoproducetruncatedproteinsweremadebydi- E
R
pendentonbindingtotheirNF45partner.Mostinterestingly, gestionofparentalvectorsatappropriatesiteswithrestrictionenzymesorby S
insertingPCRfragmentsintopcDNA-3.1. IT
cellgrowthisretardedandgiantmultinucleatedcellsaccumu- Y
latewhentheexpressionofNF45orNF90,butnotNF110,is seeMdeetdabinol1ic2-wlaebllelpilnagt.esFaotr4t(cid:3)hy1m0i4d/wineell.inActo4rp8ohraatfitoenrterxapnesfreimcteionnts(,HceeLllascweellrse) LIB
reducedinHeLacells. or seeding (cell lines expressing shRNAs), cells were labeled with 0.5 (cid:4)Ci R
A
[3H]thymidine/wellfor1handthenwashedthreetimeswithphosphate-buffered R
saline(PBS).Samplesoftrichloroaceticacidprecipitatesweredissolvedin0.1N IE
S
MATERIALSANDMETHODS NaOH and mixed with Ecolite for liquid scintillation counting. For protein o
Celllinesandplasmids.HeLaS3andHEK293cellswereculturedinDul- labeling,HeLacells(2(cid:3)105/wellinsix-wellplate)weretransfectedwithsiRNA. n J
At48hposttransfection,cellswerewashedtwicewithPBSandmediumwas u
becco’s modified Eagle’s medium (DMEM) (Mediatech, Inc.). 293 cell lines ly
expressingNF90borNF110boremptyvectorwerereportedpreviously(33). changedtoGIBCOmethionine-negativeDMEMcontaining10%fetalbovine 2
uCseilnlsgcLuilptuorfeedctianmsiinxe-w2e0ll0p0la(2te(cid:4)s(l;8I(cid:3)nv1it0r4o/gweenll)).wTehreerteraangesfnetctweads2i4nchuabfatteerdseweidthin1g pserroudmucatinodn5o0f(cid:4)cCelil[e3x5tSr]amctest.hFioonrin“ech.aCseel”lsiwnceurebactuioltnusr,etdhfeomr1edhiuamndwhaasrvreesmteodvefodr, 8, 20
(cid:4)gplasmidDNAor80pmolsmallinterferingRNA(siRNA)in100(cid:4)lDMEM thecellswerewashedthreetimeswithPBS,andmethionine-positivemedium 08
for 15 min at room temperature. Expression vectors pcDNA-NF45, pcDNA- containing10%fetalbovineserumwasadded.Incubationwasresumed,andcells
NF90a,pcDNA-NF90b,pcDNA-NF110a,pcDNA-NF110b,andpcDNA-GFP- wereharvestedathourlyintervals.
NF90chavebeenreportedelsewhere(37–39). Immunofluorescenceandlive-cellimaging.CellsexpressingshRNAscultured
RNA interference. The siRNA oligonucleotides C, D2, D3, D4, and D5 oncoverslipswerewashedoncewithPBSandfixedinfreshlymadePBScon-
(Dharmacon)wereusedatafinalconcentrationof25nM,exceptwherenoted taining 4% paraformaldehyde for 10 min at room temperature. After three
otherwise.Plasmidsd2,d3,d4,andd5,expressingshorthairpinRNA(shRNA) washeswithPBS,thecellswerepermeabilizedinPBScontaining0.2%Triton
correspondingtoD2,D3,D4,andD5andacontrolhairpinRNAcontaininga X-100for5min.AfterwashingthreetimesinPBS,thecellswereblockedwith
mismatchsequence(c;5(cid:2)halffromD2and3(cid:2)halffromD4)wereconstructed 5%donkeyseruminPBScontaining0.2%TritonX-100atroomtemperaturefor
according to the two-step method (48). Synthetic DNA sequences encoding 1h.Antitubulinantibody(Sigma)wasaddedinblockingbuffer(PBScontaining
shRNA was inserted between the ApaI and EcoRI sites of pSilencer 1.0-U6 0.2%TritonX-100and5%donkeyserum)at1:1,000(vol/vol)andallowedto
(Ambion).TheKpnI-EcoRIfragmentsfromtheresultantplasmidswereinserted reactat4°Covernight.Secondaryantibody(donkeyanti-mouseantibody;Jack-
intopcDNA3.1.Togeneratestablecelllines,HeLacellsweretransfectedwith son ImmunoResearch Laboratories, Inc.) was added at 1:800 (vol/vol), and
theseplasmidsandG418(GIBCO)wasaddedtothemediumtoafinalconcen- incubationcontinuedatroomtemperaturefor1h.Hoechst33342wasincluded
trationof1(cid:4)g/mlat24hposttransfection.Resistantcellswereclonallyselected withthesecondaryantibodyat100(cid:4)g/ml.Cellswereobservedbyfluorescent
10to14dayslater,screenedbyimmunoblotting,andcryopreserved.Cultures microscopyusinganoilimmersionlens(60(cid:3)10).Toobservemultinucleated
weremaintainedinthepresenceofG418forupto10passages. cells,thed5celllineculturedinatwo-wellchamberedcoverglass(Nunc)was
Glycerolgradientanalysis.Cytoplasmicandnuclearextracts(90(cid:4)leach)from transfectedwith1(cid:4)gpEGFP-N1(ClontechLaboratory,Inc.)orhistone2B-cyan
293cells(33)wereincubatedfor30minat37°Cintheabsenceorpresenceof fluorescent protein (histone-CFP) (1). At 48 h after transfection, cells were
RNasesAandT (0.01and0.5U/(cid:4)l,respectively)andthenloadedonto10to observedbyconfocalmicroscopy(ZeissLSM510META).Photobleachingwas
1
40%glycerolgradientsinpolysomeextractionbuffer(15mMTris-HCl[pH7.4], doneasdescribedpreviously(1).
VOL.28,2008 NF90/110 AND NF45 COREGULATION AND FUNCTION IN MITOSIS 4631
D
o
w
n
lo
a
d
e
d
fro
m
m
c
b
.a
s
m
.o
rg
a
t U
M
D
N
J
-U
N
IV
E
R
S
IT
Y
FIG. 1. AnalysisofNF45,NF90,andNF110complexesincells.(A)Glycerolgradientfractionationofnuclearandcytoplasmicextractsof293cells. L
Aftersedimentation,allfractionswereanalyzedbyimmunoblottingwithanti-NF90andanti-NF45antibodies.Fractionnumbersandthepositionsof IB
R
proteinstandardsareindicated.Nonreactiveregionsofthegradientarenotshown.IndistinguishableresultswereobtainedwithRNase-treatedextracts A
(notshown).BSA,bovineserumalbumin.(BandC)Gelfiltrationchromatographyofnuclearandcytoplasmicextractsof293cellsafter(B)orwithout R
(C)RNasetreatment.ProteinswereanalyzedasinpanelA.IgG,immunoglobulinG.(D)Coimmunoprecipitationresults.Immunoprecipitates(IP)were IE
S
preparedwithanti-NF45antibodyfromwhole-cellextractof293celllinesexpressingOmni-NF90borOmni-NF110borcontainingemptyvector(V). o
ImmunoblotsoftheIP(right)andinput(left)proteinswereprobedwithanti-Omnitag,anti-NF90,andanti-NF45antibodies.(E)IPwerepreparedwith n J
anti-OmnitagantibodyfromnuclearextractofcelllinesexpressingNF90b(left)andfromgelfiltrationfraction(Fr.)13plus14(middle)orglycerol u
ly
gradientfraction8(right)preparedfromthisextract.Immunoblots(IB)oftheIPandinputproteinswereprobedwithanti-DRBP76andanti-NF45 2
alinnteisboadnides.r(eFsoalvneddGb)yPrSoDteSingsealsseolecciatrteodphwoirthesNis.F9P0u/r1i1fi0e.dCeplrloutleairncsomweprleexestsawineerdeiwmitmhuSnYoaPffiRnOityRisuoblayteadnfdrovmisucaolniztreodl(wVit)hanadUOVmntir-aNnFsi9ll0ubm(i9n0abto)rc:elal 8, 20
representativegelisshown.ProteinsinOmni-NF90borOmni-NF110bcomplexeswereidentifiedbymatrix-assistedlaserdesorption–timeofflightmass 08
spectrometry.Theproteinsandnumbersofpeptidesidentifiedforeachproteinarelisted.*,novelNF90/110-associatedproteins;#,foundonlyin
complexesfromextractsthatwerenottreatedwithRNase.
RESULTS shape (47). This method requires the independent measure-
ment of sedimentation coefficient (e.g., by gradient sedimen-
Heterodimeric core complexes between NF45 and NF90 or
tation)andStokesradius(e.g.,bygelfiltration).InHeLaand
NF110.EvidencethatNF45associateswithNF90inavariety
293 cells, NF90 is distributed between the nuclear and cyto-
ofhumancellsandtissuesincludestheircopurificationthrough
plasmic fractions while NF110 is almost exclusively nuclear
conventional (17, 46, 50) and RNA affinity chromatography
(16, 22, 26) and coimmunoprecipitation (27–29, 38, 41). Al- (33). Because the great majority of NF90 and NF110 is com-
thoughlessextensivelydocumented,similarobservationshave plexed with RNA (33), we treated nuclear and cytoplasmic
been made for NF45-NF110 interactions (16, 28, 29, 41, 50). extractswithRNasepriortofractionation:completedigestion
Themolecularweightsandstoichiometriesofthesecomplexes ofRNAwasmonitoredbygelanalysis(datanotshown).NF90
have not been rigorously determined, however. To character- andNF110sedimentedthroughglycerolgradientsatapproxi-
ize the complexes, we used a method that allows molecular mately5.9Sand7.7S,respectively(Fig.1A),andgelfiltration
weighttobedeterminedwithoutassumptionsaboutmolecular results(Fig.1B)gaveestimatesof4.7nmand4.2nmfortheir
4632 GUAN ET AL. MOL.CELL.BIOL.
Stokes radii. Similar gel filtration behavior for NF90 was ob- lane 6). Thus, the large complexes contain additional mole-
servedinapreviousstudy(27).Inbothtypesoffractionation, culesofNF90/110,possiblyinteractingwitheachotheraswell
alloftheNF45cosedimentedwithNF90andNF110,indicat- aswithNF45andotherproteins.
ingthattheseproteinsarepredominantlycomplexedtogether To identify further components of the large complexes, we
incells.CombiningthedatawiththeSiegelandMontyequa- isolated NF90 complexes from NF90b cells by affinity purifi-
tionyieldsmolecularmassesof114kDafortheNF90-contain- cation using anti-Omni antibody cross-linked to beads. Sepa-
ing complex and 135 kDa for the NF110-containing complex, ration in SDS-polyacrylamide gels revealed the presence of
in close agreement with those calculated for 1:1 complexes many additional proteins in submolar yield relative to NF90
(119 kDa for NF90-NF45 and 138.5 kDa for NF110-NF45). and NF45 (Fig. 1F). The banding patterns obtained with
Weconcludethatbothcomplexesareheterodimericasprevi- Omni-NF110bweresimilarbutnotidentical.Individualbands
ouslyassumed,andthattheirinteractionisRNAindependent. wereanalyzedbymassspectrometry,andseveralproteinswere
Estimates of the frictional coefficient, f/f , gave values of 1.39 identified(Fig.1GandseeTableS1inthesupplementalma-
0
forNF90and1.33forNF110,implyingthatthecomplexesare terial).Asexpected,endogenousNF110waspresentinOmni-
nothighlyasymmetrical(14,47). NF90bimmunoprecipitatesandendogenousNF90waspresent
Higher-ordercomplexesincells.WhenRNasepretreatment in Omni-NF110b immunoprecipitates. NF45 was common to
was omitted, all three proteins eluted from gel filtration in NF90andNF110complexes,butotherpartnersweredistinct. D
earlier fractions and a small proportion of the NF110 was Some of the interactions have been reported previously, with ow
excludedfromthecolumn(Fig.1C),consistentwiththeirpres- RNA helicase A, exportin-5, and ADAR-1, while novel asso- nlo
a
enceinmuchlargercomplexes.OmittingRNasepretreatment ciationsweredetectedwithhnRNPproteins,additionalATP- d
e
didnotaffecttheirdistributioninglycerolgradients,however dependentRNAhelicases(DDX3XandDDX5/p68),andpro- d
(notshown).Whenafraction(fraction8)fromglycerolgradi- teins associated with cell structure and gene regulation fro
m
ent analysis performed without RNase treatment was sub- (matrin-3 and nucleolin). Interestingly, hnRNP U and NF90 m
jected to gel filtration, the proteins were all detected in frac- are both associated with 7SK complexes (52), consistent with cb
tions 20 to 24, indicating that the large RNA-dependent ourfindingof7SKincross-linkedcomplexeswithNF90(32). .as
m
complexesarenotstableinglycerolgradientconditions(data Mostoftheproteins’bindingpartnerswereobservedincom- .o
notshown).Withouttheirsedimentationcoefficients,wewere plexesderivedfromRNase-treatedextracts,implyingprotein- rg
a
unabletocalculatethemolecularweightsoftheselargecom- protein interactions. Two exceptions were ADAR-1 and t U
plexes by the Siegel and Monty method. From size exclusion DDX3X(seeTableS1inthesupplementalmaterial). M
D
data alone, we estimate that NF90 and NF110 are present in Interactions among NF90, NF110, and NF45 in vitro. Re- N
RNase-sensitivecomplexesof(cid:5)600kDa.Similarresultswere constitution experiments showed that NF90c and NF45, syn- J-U
N
obtainedinHeLacells(datanotshown). thesizedinbacteriaoraheterologouscell-freesystem,areable IV
We used stable cell lines expressing Omni-tagged forms of toassociateinvitro(29,30).However,NF90cwasnotfoundin E
R
NF90orNF110(33)toexaminethecompositionofthelarge cells (18, 39) and it differs at its C terminus from authentic S
IT
complexes. To verify that the exogenous tagged proteins par- NF90 and NF110, both of which occur in a and b isoforms Y
ticipateincomplexformationwithNF45,wefirstprobedanti- distinguished by the absence or presence, respectively, of a LIB
NF45 immunoprecipitates with antibody directed against the four-amino-acid(aa)insertbetweentheirdsRBMs.Todeter- R
A
Omni tag (Fig. 1D, right panel). Omni-NF90b and Omni- minewhethertheNF90/110isoformsallinteractwithNF45in R
IE
NF110bwereimmunoprecipitatedwithNF45fromtheNF90b vitro, we produced them in wheat germ and conducted GST S
and NF110b cell lines, respectively, but not from the control pull-down assays. Both NF110 isoforms and all three NF90 on
celllinecarryingemptyvector(toppanel).Probingwithanti- isoformsboundtightlytoGST-NF45invitro(Fig.2A).Quan- Ju
ly
NF90/110 antibody (middle panel) revealed the presence of titationindicatedthattheaandbisoformsofNF90andNF110 2
8
endogenousNF90andNF110inalloftheNF45immunopre- boundatleastthreefoldmoreefficientlythanNF90c(datanot , 2
cipitates, as well as the Omni-tagged forms in those from the shown),suggestingthatthecomplexisstabilizedbythenatural 00
8
NF90b and NF110b cell lines (although Omni-NF110b was C termini of the a and b isoforms of NF90 and NF110 or
difficulttodetectbecauseofitsweakexpressioninthecellline, destabilizedbytheacidicCterminusofNF90c.
coupledwiththeinefficientextractionofNF110fromnuclei). Becausethelargecomplexesincellextractscontaintwoor
Next, we conducted reciprocal experiments to examine anti- more molecules of NF90/110, we considered the possibility
OmniimmunoprecipitatesfromNF90bcellextracts.Strikingly, that members of this protein family can interact directly with
immunoblottingwithanti-NF90/110showedthatOmni-NF90b one another. In GST pull-down experiments, GST-NF90b
coimmunoprecipitatedwiththeendogenousNF90andNF110 boundtotheaandbisoformsofbothNF90andNF110(Fig.
isoforms (Fig. 1E, lane 2, top panel) as well as with NF45 2B),consistentwiththeresultsofyeasttwo-hybridexperiments
(bottompanel).SimilarresultswereobtainedwiththeOmni- (49).TheN-terminal335residueswerenotrequiredforbind-
NF110b(datanotshown),suggestingthatthelargecomplexes ing, but C-terminal truncations infringing on or removing the
obtainedbygelfiltrationcontainmultipleNF90/110proteins. dsRBMs were deleterious. Further experiments showed that
Consistent with this interpretation, endogenous NF90 and NF90 fragments 1 to 591 and 336 to 591 retained binding
NF110coprecipitatedwithOmni-NF90bfromhigh-molecular- activity (data not shown), mapping the region responsible to
weightgelfiltrationfractionsfromtheNF90bcellline(Fig.1E, the central part of the molecule containing its two dsRBMs
lane 4). Such coprecipitation was not observed from low-mo- (Fig. 2D). Binding was resistant to treatment with RNase or
lecular-weightgelfiltrationfractions(datanotshown)orfrom high salt concentrations (up to 750 mM NaCl). Thus, as with
glycerol gradient fractions from the NF90b cell line (Fig. 1E, interactionsbetweenNF90andPKR(29)thebindingofNF90
VOL.28,2008 NF90/110 AND NF45 COREGULATION AND FUNCTION IN MITOSIS 4633
D
o
w
n
lo
a
d
e
d
fro
m
m
c
b
.a
s
m
.o
rg
a
t U
M
D
N
J
-U
N
IV
E
R
S
IT
Y
L
IB
R
A
R
IE
S
o
n
J
u
ly
2
8
, 2
0
0
8

FIG. 2. BindingofNF45,NF90,andNF110invitro.(A)InteractionofNF90andNF110isoformswithGST-NF45.Equalamountsofthe
indicated35S-labeledproteinsexpressedinwheatgerm(bottompanel)wereboundtobeadscarryingGST-NF45(toppanel)orGSTalone(middle
panel).BoundproteinswereresolvedinSDS-polyacrylamidegelsandanalyzedbyautoradiography.(B)Interactionoffull-lengthandtruncated
formsofNF90andNF110withGST-NF90b.ExperimentswereconductedasinpanelA.(C)Interactionoffull-lengthandtruncatedformsofNF90
withGST-NF45.ExperimentswereconductedasinpanelA.(D)Summaryofpull-downassaysmappingthebindingsitesforNF45andNF90on
NF90/110.
with NF90 and NF110 is mediated by the proteins’ dsRBMs 334ofNF90/110.Littleornobindingwasobservedwithaa336
butisnotdependentonRNA. to 591. These findings are consistent with direct binding of
ImmunoprecipitationexperimentsshowedthattheN-termi- NF45 to the N-terminal region of NF90/110 containing the
nal region (aa 1 to 418) of NF90/110 binds strongly to NF45, DZFdomainandweakerindirectbindinginvivototheRNA-
whereas the C-terminal region (aa 403 to 670) displayed a binding domain mediated by interactions between NF90 iso-
weak interaction that could be indirect (38). To examine the forms.
interactions in vitro, we used GST-NF45 to pull down frag- Previous GST pull-down assays gave no evidence of homo-
mentsofNF90.Theresults(Fig.2CandD)demonstratedthe meric NF45-NF45 interactions (29). Therefore, taken to-
existence of a strong site for NF45 binding between aa 1 and gether,ourresultssuggestthatNF90/110interactsdirectlyvia
4634 GUAN ET AL. MOL.CELL.BIOL.
D
o
w
n
lo
a
d
e
d
fro
m
m
c
b
.a
s
m
.o
rg
a
t U
M
D
N
J
-U
N
FIG. 3. EffectofsiRNAonexpressionofectopicNF90andNF110.(A)SchematicofNF110,NF90,andNF45showingregionstargetedby IV
siRNAsC,D2,D3,D4,andD5.NotethattheNF90aandNF110aisoformsareidenticaltoNF90bandNF110b,respectively,exceptfortheNVKQ E
sequencelackingintheaisoforms.(B)SequenceandtargetsofsiRNAs.(C)TimecourseofinhibitionbyD2siRNA.293cellsweretransfected R
S
with 25 nM siRNA together with 2 (cid:4)g pcDNA-NF90b or pcDNA-NF110b. Cells were harvested at the times indicated and analyzed by IT
ipmcDmNunAo-bElGotFtiPng-NwFi9th0catnotgi-eOthmerniwtiathgsaiRndNAanCtiaocrtinD2anotribwoidthieosu.t(sDiR)NSpAec((cid:7)ifi)c.itAyto4f8Dh2afstieRrNtrAa.ns2f9e3cticoenll,scwelelsrewetrraenosfbescetrevdedwbityhpphcaDseN-cAo-nEtrGasFtPanodr Y LIB
fluorescencemicroscopy.(E)CellstransfectedwithpcDNA-EGFP-NF90cwereharvested,andextractswereexaminedbyimmunoblottingusing R
A
anti-NF90andantiactinantibodies. R
IE
S
o
n
its N-terminal region with NF45 to form heterodimeric com- brokethroughtheinhibition.Toverifythespecificityofknock- Ju
ly
plexes.Furthermore,homomericNF90/110interactionsviaits down, cells were cotransfected with siRNA and plasmids ex- 2
8
dsRBMsmaycontributetotheformationofhigher-ordercom- pressing enhanced green fluorescent protein (EGFP)-NF90c , 2
plexes. orEGFPalone.D2silencedEGFP-NF90cexpression,whileC 00
8
Coregulation of NF90 and NF45. We designed a series of was ineffective (Fig. 3D), in keeping with immunoblotting re-
siRNAs to evaluate the biological effects of knocking down sults (Fig. 3E). Neither siRNA had any effect on EGFP fluo-
NF45andmembersoftheNF90family,individuallyorcollec- rescence. From our observations, it would appear that the
tively (Fig. 3A and B). D2 is directed against both NF90 and residual expression of exogenous NF90b that is resistant to
NF110, whereas D3, D4, and D5 selectively target NF90, knockdown by D2 comes from a low level of expression of
NF110, and NF45, respectively. Although designed to knock NF90binalargenumberofcellsratherthanfromafewcells
downNF90andNF110,RNACwasineffectiveandservedas thatarewhollyresistant.
a control. To optimize NF90 depletion, we cotransfected 293 Similarly,endogenousNF90andNF110werebothsilenced
cellswithD2andavectorexpressingOmni-NF90b.Increasing by D2 siRNA (Fig. 4A), albeit somewhat less efficiently than
concentrationsofD2siRNAprogressivelyreducedNF90bac- the exogenous proteins. As expected, siRNAs D3 and D4,
cumulation,anditwas(cid:6)90%effectiveat25nM(seeFig.S2in directedatspecificsequencesintheproteins’Ctermini,selec-
thesupplementalmaterial).Higherconcentrationsdidnotre- tively depleted endogenous NF90 and NF110, respectively.
sultinmorecompleteknockdown,andRNACwasineffective Likewise, D5 siRNA silenced the expression of endogenous
even at high concentrations (data not shown). D2 effectively NF45. Remarkably, we observed additional inhibitory effects.
depletedtaggedNF110baswellasNF90b(Fig.3C).Itssilenc- In particular, D2 and D3 (specific for NF90 and NF90/110)
ingactionwasobservedasearlyas16haftertransfectionand alsobroughtdownthelevelofNF45.Conversely,D5(specific
persisted for as long as 120 h, although synthesis gradually for NF45) also brought down the levels of both NF90 and
VOL.28,2008 NF90/110 AND NF45 COREGULATION AND FUNCTION IN MITOSIS 4635
siRNA target region at the NF90 C terminus (Fig. 3A) but
containstherestofthemolecule,includingtheNF45binding
site(Fig.2).Accordingly,NF90cproteinaccumulatedintrans-
fected d3 cells (Fig. 4C, top panel). Although the levels of
endogenousNF90andNF110wereunaffected(secondpanel),
NF45levelswererestoredbyNF90cexpression(thirdpanel).
Takentogether,theseresultsindicatethatthecellularcon-
centrationsofNF45andNF90/110arecoordinatelyregulated,
consistentwiththeirpresenceinheterodimericcomplexes.Ex-
ceptionally, the level of NF45 did not decline discernibly in
concert with reduced levels of NF110, even though NF45
knockdown caused a reduction in NF110 level. Interestingly,
expression of exogenous NF90 or NF110 did not result in
substantiallyelevatedlevelsoftotalNF90/110orofNF45(Fig.
1D and data not shown), suggesting that the cell has mecha-
nismslimitingtheoverallconcentrationsoftheseproteins. D
o
Mutual regulation of NF90 and NF45 is posttranslational. w
n
AlthoughtherewerenoobvioussimilaritiesbetweentheNF90 lo
a
andNF45targetsequences,weconsideredthepossibilitythat de
d
coregulation occurs by a mechanism operating at the mRNA fro
level. RNase protection assays were conducted with probes m
specific for NF45 and NF90/110 mRNAs and for glyceralde- m
c
hyde-3-phosphatedehydrogenase(GAPDH)mRNAasacon- b.a
trol.Nocross-inhibitionactivitywasobservedattheRNAlevel sm
(seeFig.S1inthesupplementalmaterial).Theresultsshowed .o
rg
that all of the siRNAs were specific for their target mRNAs, a
and no effects were observed on GAPDH RNA. Thus the t U
M
coregulationofNF90andNF45involvesaprocessdownstream D
N
ofthelevelofmRNAconcentrationinthecell,probablyeither J
-U
protein synthesis or protein stability. To distinguish between N
FIG. 4. Knockdown of endogenous NF45, NF90, and NF110. these alternatives, we first monitored the synthesis of NF90, IV
E
(A)TransientexpressionofsiRNA.HeLacellsweremocktransfected NF110, and NF45. Cells were pulse-labeled with [35S]methi- R
(m)ortransfectedwithcontrol(C),D2,D3,D4,orD5siRNA.Atthe onineat48haftertransfectionwithD3,D5,orcontrolsiRNA. SIT
timesindicated,cellswereharvestedandanalyzedbyimmunoblotting Y
withantibodiesagainstNF90,NF45,andactin.(B)Celllinesexpress- Labeled proteins were immunoprecipitated with antibodies L
ingshRNAs.Cellsfromthestablecelllinesc,d2,d3,d4,andd5were against NF90 and NF110 or against NF45, resolved by gel IBR
examined as in panel A. (C) Rescue of NF45 expression by ectopic electrophoresis, and observed by autoradiography (Fig. 5A). AR
NF90c.Cellsofthed3celllineweretransfectedwithincreasingcon- D3, which targets NF90 mRNA, inhibited the synthesis of IE
centrationsofpcDNA-NF90c,usingpcDNA3.1emptyvectortomain- S
tainaconstantamountoftransfectedDNA.Cellswereharvestedat NF90butnotofNF110orNF45.Correspondingly,D5,which on
48haftertransfectionforimmunoblottinganalysisasinpanelA. targetsNF45mRNA,inhibitedthesynthesisofNF45butnot J
u
of NF90 or NF110. These observations match the effects on ly
2
mRNAlevelsseeninRNaseprotectionassays(seeFig.S1in 8
NF110 (Fig. 4A). However, D4 (specific for NF110) elicited thesupplementalmaterial)andarguethatthecross-inhibition , 200
littleornodecreaseinNF45.ItisnotablethatwhenNF45was phenomenon is not exerted at the translational level. NF45 8
targeted by D5 siRNA, the level of NF45 protein fell rapidly synthesis was also unaffected by D2 and D4 siRNAs, which,
(by24h)andthelevelsofNF90andNF110declinedrelatively respectively, target both NF90 and NF110 and NF110 alone
slowly(visibleafter48h).Thiscross-inhibitiondidnotextend (datanotshown).
toproteinssuchasactin(Fig.3and4),EGFP(Fig.4),tubulin, NF45isrelativelyunstableandprotectedbyNF90.Wenext
ornucleolin(datanotshown).Themutualregulationobserved examinedtheeffectofNF90depletiononthestabilityofNF45
between NF90/110 and NF45 is consistent with a secondary protein,andviceversa.Todeterminethehalf-lifeofNF45,we
effectduetocomplexformation. conducted pulse-chase experiments in cells transfected with
Wefurtherevaluatedthespecificityofthecross-inhibitionin varioussiRNAs(Fig.5B).NF45wasimmunoprecipitatedfrom
cell lines that stably express corresponding shRNAs. Cross- cell extracts immediately after labeling or after 1 to 3 h of
regulationbetweenNF90andNF45wasseeninthed2,d3,and subsequent“chase”withunlabeledaminoacids.Asexpected,
d5 cell lines (corresponding to D2, D3, and D5 siRNAs, re- nolabelingofNF45wasdetectedincellstransfectedwithD5
spectively), but not between NF110 and NF45 in the d4 line siRNA. The half-life of NF45 was only 1.42 h, and it was
(corresponding to D4 in Fig. 4B). This mirrors the results of furthershortenedbyD2andD3siRNAto0.53hand0.37h,
the silencing experiments (Fig. 4A). Furthermore, we con- respectively (Fig. 5B). D4 siRNA elicited a smaller effect on
ducted a rescue experiment. The d3 cell line was transfected thehalf-lifeofNF45,whichwasreducedto0.95h.Evidently,
withavectorexpressingtheNF90cisoform,whichlackstheD3 NF90depletionacceleratesNF45turnoverandNF110deple-
4636 GUAN ET AL. MOL.CELL.BIOL.
D
o
w
n
lo
a
d
e
d
fro
m
m
c
b
.a
s
m
.o
FIG. 5. NF90depletiondestabilizesNF45.(A)D3andD5siRNAsspecificallyreducethesynthesisofNF90andNF45,respectively.HeLacells rg
transfectedwithC,D3,orD5siRNAwerelabeledwith[35S]methioninefor1hat48hposttransfection.Immunoprecipitates(IP)preparedwith a
antibodiesdirectedagainstNF90andNF110(upperpanel)orNF45(lowerpanel)wereanalyzedbygelelectrophoresisandautoradiography. t U
(B)Pulse-chaseanalysisofNF45incellstransfectedwithC,D2,D3,D4,orD5siRNA.Cellswerelabeledandchasedasrepresentedschematically M
D
inthediagram.NF45wasimmunoprecipitatedanddetectedasinpanelA(leftpanel)andquantifiedbyPhosphorImager(rightpanel).NF45 N
half-life(inset)wascalculatedaccordingtoequationsdrawnfromrespectivecurvesusingtheExcel2003TRENDfunction. J-U
N
IV
E
R
tionalsohasaneffectonNF45stability,eventhoughthisdid blueexclusion:(cid:8)2%ofthecellswerestainedineachcellline, S
IT
notregisterasadetectabledecreaseinNF45level(Fig.4A). indicatingthatlittlenecroticorapoptoticcelldeathwastaking Y
ComparableanalysesofeffectsonthestabilityofNF90and place. Fluorescence-activated cell sorting (FACS) analysis of LIB
NF110wereprecludedbythelonghalf-livesoftheseproteins, the cell lines confirmed that each cell line had a similar, low R
A
estimatedat2.4and1.8days,respectively,inHeLacells(data percentageofapoptoticcells,andnosignificantaccumulation R
notshown).Slowturnoverrates(half-livesof(cid:5)5and(cid:5)3days) ofcellswasobservedinanycompartmentofthecellcycle(Fig. IES
havealsobeenreportedfortheseproteinsinHepG2cells(55). 6D to F), arguing against a block in progression in the cell on
Furtherexperiments,usingtheinhibitorMG-132,indicatethat lines. On the other hand, the incorporation of [3H]thymidine Ju
ly
NF45isprobablydegradedbytheproteasome(seeFig.S3in into DNA was reduced in cells transfected with or stably ex- 2
8
thesupplementalmaterial).WeconcludethatNF45isashort- pressing D2, D3, or D5 (Fig. 6A and B, insets). Thus, slow , 2
lived protein that is stabilized by binding to its partners and growth correlated with inhibition of DNA synthesis. Anoma- 00
speculatethatoneofitsrolesistostabilizeNF90andNF110. lously, [3H]thymidine uptake was increased by D4, although 8
Cell division is slowed by knockdown of NF90 or NF45. cellgrowthwasmarginallyreduced.Weconcludethattheslow
TransfectionofD2,D3,orD5significantlyreducedthegrowth growth brought about by depletion of NF90 and NF45 is ac-
ratesofcellculturesincomparisonwiththoseofcellsthatwere companied by reduced DNA synthesis but not by a cell cycle
mocktransfectedortransfectedwithcontrolsiRNA(Fig.6A). block.
D4, which knocks down NF110 alone, had little or no effect. Multinucleated cells arise when NF90 or NF45 is knocked
Similarly,thestablecelllinesd2,d3,andd5grewmoreslowly down. The d3 and d5 lines displayed a markedly increased
than the d4 or control cell lines (Fig. 6B). Hence, the expan- proportion of cells with polyploid DNA content (11.2% and
sion of cultures is slowed by the knockdown of endogenous 17% compared to 6.8% in control) (Fig. 6D to F). This pop-
NF90, NF90, and NF110 or NF45, indicating that coordinate ulationcouldincludecellaggregatesaswellasmultinucleated
reduction of NF45 and NF90 is associated with growth retar- cells. However, microscopic examination revealed that deple-
dation.Correspondingly,rescueofNF45byNF90cwasaccom- tion of NF90 and NF45 caused a striking accumulation of
paniedbyacceleratedgrowthofthed3cellline(Fig.6C). multinucleated cells. This phenomenon can be visualized in
We considered two possible explanations for this apparent cells stained simultaneously with Hoechst dye for DNA and
slowing of cell growth: (i) that cell death is more frequent in with antitubulin antibody (Fig. 7A). Multinucleated cells ac-
cells depleted of NF90 and NF45 or (ii) that such cells are countedfor(cid:5)5%ofthepopulationinthed2andd3celllines
blocked in the cell cycle. Cell viability was assessed by trypan andasmanyas(cid:5)12%inthed5line,comparedto(cid:8)1%inthe
VOL.28,2008 NF90/110 AND NF45 COREGULATION AND FUNCTION IN MITOSIS 4637
D
o
w
n
lo
a
d
e
d
fro
m
m
c
b
.a
s
m
.o
rg
a
t U
M
D
N
J
-U
N
IV
E
R
S
IT
Y
L
IB
R
A
R
IE
S
o
n
J
u
ly
2
8
, 2
0
0
8

FIG. 6. EffectsofNF45,NF90,andNF110knockdownoncellgrowth.Cellgrowthcurvesweremeasuredforcellsdescribedinthelegendto
Fig.4.(A)HeLacellsmocktransfected(m)ortransfectedwithcontrol(C),D2,D3,D4,orD5siRNA.(B)Stablecelllinesc,d2,d3,d4,andd5.
(C)d3cellstransfectedwithpcDNA-NF90c.For[3H]thymidineincorporation(insets),theaverage(controlandmockvaluessetat100U)and
standarddeviationwereobtainedfromthreeexperimentsintriplicate.(DtoF)Cellcycleanalysiswasperformedonc,d3,andd5cellsstained
withpropidiumiodideusingaBDFACSCaliburcellsorter.ThepercentagesofcellsintheG,S,G /M,andpolyploidcompartmentsareindicated
1 2
(lefttoright).*,P(cid:8)0.01relativetocontrolsat48handalllatertimepoints(exceptford4at48hinpanelB)byStudent’sttests.
4638 GUAN ET AL. MOL.CELL.BIOL.
D
o
w
n
lo
a
d
e
d
fro
m
m
c
b
.a
s
m
.o
rg
a
t U
M
FIG. 7. MorphologicalchangesincelllinesexpressingshRNA.(A)Formationofmultinucleatedcellsind2,d3,andd5celllines.(Toppanel) D
Cellsstainedwithpropidiumiodideobservedatlowpower(10(cid:3)40).Multinucleatedcellsareindicatedbyarrowheads.(Lowerpanels)Cells N
J
stainedwithHoechst33342andantitubulinantibodywereobservedathighmagnification(10(cid:3)60),andtheimagesweremerged.(B)Multinu- -U
cleatedcellswerecountedinthreefieldsof100cellseach.Thepercentageofsuchcells(withstandarddeviation)isplottedforeachcellline. N
IV
(C) The numbers of nuclei in multinucleated d2 cells (25 cells), d3 cells (22 cells), and d5 cells (72 cells) were counted. The percentage of E
multinucleatedcellswithaparticularnumberofnucleiisplotted. R
S
IT
Y
L
IB
control and d4 lines (Fig. 7B). The number of nuclei per wedidnotdetectmonomericNF90orNF45inthenucleusor R
A
multinucleated cell ranged from 2 to 11 (Fig. 7C). Of the cytoplasm. Our hydrodynamic data show that the core com- R
multinucleatedd2andd3cells,themajority((cid:6)90%)had2to plexes obtained from cells after RNase treatment are het- IES
6 nuclei. In d5 cells, however, over 15% had seven or more erodimeric: NF90-NF45 and NF110-NF45. Native complexes on
nuclei,indicatingthatNF45depletionresultsinamoresevere are much larger because of association with both RNA and Ju
phenotype. The rare multinucleated cells in control and d2 protein components. They are stable under conditions of gel ly 2
culturesusuallyhadonlytwonucleipercell(datanotshown). filtrationandimmunoprecipitationbutlabile,forreasonsthat 8, 2
Toverifythatthemultiplenucleiliewithinthesamecell,we areunclear,tosedimentationthroughglycerolgradients.The 0
0
conducted photobleaching experiments. Bleaching within a composition of these large complexes is not yet fully defined, 8
small localized area of the cytoplasm of a multinucleated d5 althoughmanyoftheircomponentsareknown.SeveralRNAs,
cellledtothelossinfluorescenceofallofthecytoplasmofthe bothsmallnoncodingRNAsandmRNAs,havebeenfoundin
multinucleated cell, whereas adjacent cells were unaffected complexeswithNF90.TheRNasesensitivityofthelargecom-
(Fig.8A).Observationunderthefluorescentmicroscopesug- plexes implies that RNA molecules contribute to the stability
gested that the multiple nuclei were often connected as visu- ofhigher-ordercomplexes,consistentwiththeRNA-mediated
alized in serial z-sections of live cells expressing fluorescent interactionofADAR1withNF90,NF110,andNF45(28)and
histone-CFP (Fig. 8B). Thus the cells are not true syncytia. withourfindingofADAR1andtheDDX3Xhelicaseincom-
TheseobservationssuggestthatNF90andNF45arerequired plexesfromuntreatedbutnotRNase-treatedextracts.Several
forthefinalstagesofnuclearseparationandcytokinesisduring proteins have been reported to associate with NF90/110, and
mitosis. additional partners are reported here. In keeping with the
involvement of NF90/110 in RNA metabolism, these include
twofurtherRNAhelicases,nucleolin,andseveralhnRNPpro-
DISCUSSION
teins.ThepresenceinNF90complexesofmatrin-3withstruc-
Structure of NF90/110 complexes with NF45. NF90 and turalrolesinthecellmaybecorrelatedwiththenewlydiscov-
NF110 occur in complexes with NF45 and several other pro- eredfunctionofNF90andNF45inmitosis.
teinsinmanycells.Inaccordwithpublishedobservations(27), Thehigher-ordercomplexesarelikelytobeheterogeneous