Journal of Cell Science ura n giea 03 buz ta. 04 auae al., et Masuda 2004; al., 1998; et Abruzzi Aguilera, 2003; and Aguilera, and (Piruat export Huertas yeast) mRNA yeast) the in in of Stra Sub2 (Mex67 recruitment and NXF1 the (Yra1 for UAP56 responsible and are ALY complex which THO the of two association THO/ the the with by metazoans, formed In is 2005). complex al., it et TREX Masuda metazoans 2002; al., (Stra in et machinery Jimeno while splicing co- the mRNP is with the THO/TREX intronless associates yeast, to in are that recruited fact transcriptionally the This yeast in introns. reflected several Most is contain difference usually 2005). genes metazoans metazoans al., in whereas and et couple yeast Masuda to in the 2002; shown both in export been (Stra transport mRNA has evolutionarily and The further complex transcription ribosome. for THO/TREX the by messenger conserved (NPC) translation competent the and complex to cytoplasm transport nascent addressed pore be The a will 2009). nuclear that into Proudfoot, (mRNP) particle processed and ribonucleoprotein Moore is see co-transcriptionally mRNA review, recruited are a and (for interact other often each conserved, with well physically evolutionary general in are mRNA of polyadenylation 3 and the cleavage splicing, cap, methylguanosine o:10.1242/jcs.118000 doi: 2656–2667 126, ß Science Cell of Journal 2013 March 19 Accepted Universite Universite U964, INSERM oriae oiiain hticueadto fa5 a of and addition successive include undergoes that transcript transcription modifications II) pre-mRNA (Pol coordinated II nascent polymerase RNA the during eukaryotes, In Introduction NPC- an is TREX-2 functions. that basket-related show NPC results putative in our that involved Thus, reveal is NXF1. accumulation it words: nuclear from that Key suggest mRNA was basket differently and the of but (TREX-2) requires cells human profiles NPC , mammalian of 2 Differential the dynamics in with basket transcription. complex the TREX-2 complex of to of characterize export associated association independent we similarly the and Here, is pore Moreover, functions NPC. but basket. transcription nuclear the TREX-2 NPC TPR, and the The the and sites with NPC. to associates NUP153 transcription the stably between nucleoporins transcript the for it shuttle NXF1 that nascent to to receptor show and export and the (mRNP) NXF1 mRNA TREX-2 carry the with particles recruits interact that and to ribonucleoprotein export factors suggested mRNA messenger conserved to of transcription evolutionarily couples transport complex many THO/TREX involves The (NPC). export complex mRNA eukaryotes, In Summary ( correspondence for *Authors 3 2 1 Umlauf David with basket associated pore stably nuclear is the complex TREX-2 human The 2656 arn Brino Laurent mgreCluar tTsuar,UR14 ntttCre NS 6RedUm 54 ai ee 5 France 05, Cedex Paris 75248 d’Ulm, Rue Ge 26 de CNRS, Institut Curie, Facility, Institut Screening 144, Throughput UMR High Tissulaire, et Ge Cellulaire de Imagerie Institut Program, Dynamics Nuclear and Signaling Cellular 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. se n ut 00 tt ta. 00 ieoe l,2002; al., et Jimeno 2000; al., et Stutz 2000; Hurt, and ¨sser se n ut 00 ieoe l,20;Znlsne al., et Zenklusen 2002; al., et Jimeno 2000; Hurt, and ¨sser 9 n.Tecmlxsivle ntemtrto ftepre- the of maturation the in involved complexes The end. eSrsor,B 04 70 LKRHCdx Ud tabug France Strasbourg, de CU Cedex, ILLKIRCH 67404 – 10142 BP Strasbourg, de ´ H/RX X1 RX2 MX rncito,mN xot ula oecomplex pore Nuclear export, mRNA Transcription, AMEX, TREX-2, NXF1, THO/TREX, 1 3 aqe Bonnet Jacques , iirDevys Didier , [email protected] eSrsor,B 04 70 LKRHCdx Ud tabug France Strasbourg, de CU Cedex, ILLKIRCH 67404 – 10142 BP Strasbourg, de ´ ; 1, [email protected] n La and * 1 Franc , se n ut 2000; Hurt, and ¨sser ´ne ´szlo ) osWaharte ¸ois tqee eBooi Mole Biologie de et ´tique Tora ´ 1, ´ne 9 * tqee eBooi Mole Biologie de et ´tique 7- 2 ajreFournier Marjorie , atce oteNC(erfe l,19;Gru 1997; al., mRNP et transport (Segref to NPC suggested the the in were to actors particles and late be pathway to export shown been mRNA have Mex67 and NXF1 2005). ta. 02.I ua,dpeino otTE- subunits TREX-2 most other of all Jani depletion 2012; which human, al., onto et In Ellisdon 2012). 2009; scaffold al., al., et et the (Jani be assemble Sac3 Sem1 subunits to where that Structural yeast and shown to 2012). revealed similarly (Thp1 was molecules al., complex ENY2 DSS1 et two GANP-ENY2 binds (Jani and GANP recombinant hand PCID2 of other with analysis the and on homologues) hand homologues) Cdc31 one and (Sus1 on CETN2/CETN3 interact and homologue) ENY2 (Sac3 GANP with human and of regions NPC independent yeast. the in of described side functions the nuclear 2010). recapitulate the al., to at appears et localized AMEX Lu determined, predominantly 2007; be to is al., remains composition mRNA et exact and its (Kurshakova Anchoring Although for plants AMEX and called Export) is in complex isolated the been (where 2004; have al., complexes Orthologous et in 2009). Fischer NUP153 results 2002; subunits human al., TREX-2 et Rodrı the (Fischer any defects Nup1, of export Thp1, deletion the mRNA of Sac3, yeast, face basket In inner called the ortholog. the subunits with interacts via five it and NPC of Sem1 and TREX- composed yeast Sus1 The Cdc31, is gating. complex and shown export was mRNA 2 and in yeast role a in play characterized to initially was that complex cliee ellie(GM) NSUR70,ISR U964, INSERM 7104, UMR CNRS (IGBMC), Cellulaire et ´culaire oerecently, More another is (TREX-2) 2 complex export and transcription The ´ uzNvroe l,20;Wle ta. 08 aae al., et Faza 2008; al., et Wilmes 2004; al., et guez-Navarro cliee ellie(GM) NSUR7104, UMR CNRS (IGBMC), Cellulaire et ´culaire nvitro in 1 atiuStierle Matthieu , uldw sasdmntae httwo that demonstrated assays pull-down 1 eotFischer Benoit , eerhArticle Research tre l,1998). al., et ¨ter Drosophila 3 , Journal of Cell Science ags uui,NF n U13 thsbe rpsdthat proposed been has II it on polymerase NUP153, and Based RNA NXF1 the localization. Conversely, subunit, GANP, GANP 2012). largest between affect al., detected not et interactions did Jani the depletion with 2010; ENY2 associate localization al., ENY2 to et for required shown (Wickramasinghe is as GANP were where such envelope CETN2 subunits nuclear and Some ENY2 accumulation. nuclear GANP, mRNA in results EN Ges ta. 04 n AAseii antibodies specific SAGA and 2004) al., as et well as (Giessl developed PCID2 The and newly CETN3 GANP our against further S1B). antibodies using was polyclonal analysis TREX-2 rabbit Fig. and western-blot SAGA by both material confirmed with ENY2 (supplementary after of peptides association few digestion produce may protein GANP, was trypsin small DSS1 of this that as Note composed Spectrometry detected CETN3. or not be CETN2 Mass either would and ENY2 TREX-2 Our PCID2, that S1A). subunits suggest FLAG-HA-SPT3 Fig. TREX-2 analyses the material putative not all (supplementary In but recovered purifications. ENY2-FLAG-HA immunoprecipitation both in the identified contrast, were subunits Multi all SAGA to corresponding non-gel-based known peptides mass expected, or (MudPIT) As methods. Technology spectrometry LC-MS/MS Identification Protein by nanoflow either Dimensional analyzed extracts gel-based were proteins and nuclear associated by the (Nakatani and from immunoprecipitations 2003) HA Ogryzko, purified and subunit stably FLAG were SPT3 successive lines FLAG- specific and proteins ENY2-FLAG-HA cell SAGA peptide. HA-SPT3 FLAG-HA HeLa the a or with two tagged ENY2 generated either we expressing homologous interact their complexes, SAGA like linked and functionally yeast are TREX-2 and human other each the with whether determine To complex independently SAGA periphery the nuclear of the at localized is TREX-2 be Results to NPC- appears an export transcription. localization ongoing is mRNA of whose TREX-2 independent the complex Therefore, after knockdown. basket-associated observed NXF1 that receptor than but similar one milder highly were the basket strikingly depletion pore TPR) to or and ENY2) (NUP153 and comparable component (GANP of subunits export is TREX-2 mRNA dynamics after Interestingly, periphery defects the nucleoporins. scaffold nuclear that for show the described experiments Unexpectedly, at cells. imaging TREX-2 human cell in live stable extremely our is NPC the role with regulatory and/or structural complexes. important both ENY2/ an in that play a suggesting could 2010) around Sus1 al., wraps et fold (Ellisdon Ko ENY2/Sus1 ATXN7L3/Sgf11 2008; the and al., Strikingly, long 2010). et (Ko SAGA al., Zhao activity of et 2006; deubiquitination protein al., histone et its ATXN7L3/Sgf11 regulate the together for with TREX-2 recruit interacts to Ko SAGA yeast 2006; of (Rodrı specific ability of targeting the gating proper gene on that depends suggested genes was it mRNP and (SAGA) competent of acetyltransferase Spt-Ada-Gcn5 complex coactivator transcriptional shuttling NPC. the the to sites for transcription from required particles is TREX-2 eso eeta h soito fteTE- complex TREX-2 the of association the that here show We the with shared is Sus1 subunit TREX-2 the Interestingly, a hlxfudi ohGN/a3adteSG subunit SAGA the and GANP/Sac3 both in found -helix he ta. 08.I ua n es ENY2/Sus1 yeast and human In 2008). al., et ¨hler ´ uzNvroe l,20;Cble al., et Cabal 2004; al., et guez-Navarro he ta. 00 Samara 2010; al., et ¨hler ¨hler h noeosTE- ope splmnaymaterial (supplementary complex TREX-2 S3D). Fig. in integrated endogenous is EGFP-ENY2 the that showed evidence Biochemical xeiet.Tetge-N2dslydasrn and strong a (IF) displayed immunofluorescence tagged-ENY2 confocal complex The TREX-2 indirect the experiments. of performing localization cellular by the determine to material supplementary 1A,B; human S1). (Fig. SAGA. interaction Fig. both complexes an than of support two not part the abundant do between is and more complexes ENY2 TREX-2 that is and SAGA confirm TREX-2 data the nuclei these that Together, spectral cell indicated SAGA analyses during HeLa and MudPIT normalized TREX-2 the in for from and The determined 2006) as al., subunits S1). extracts et 1B; (Paoletti (Fig. Fig. factor complex nuclear abundance TREX-2 is material the RNA of the formation supplementary that the in in indicated addition involved not experiment without A This or immunoprecipitation. with RNase concentrations experiment same of salt the performed physiological TREX-2 we at the mediated between To RNA interactions and conditions. are the SAGA experimental subunits that possibility our that the under out suggesting no interact rule complex but not do subunits, TREX-2 TREX-2 SAGA the only FLAG-HA- of our identified In subunits 1A). we (Fig. 2004; 2009) purifications al., al., et et SPT3 Helmlinger Nagy 2001; 2008; al., al., et et Zhao Brand 1995; al., et (Mengus ifrn ellrlclztosadd o upr functional a support complexes. not two these do of and coupling have localizations SAGA cellular and TREX-2 different that FLAG- 1D; suggest S2A,B). and further (Fig. Fig. observations ENY2-FLAG-HA material These (supplementary staining in cells obtained antibodies expressing perinuclear were same HA-SPT3 results the al., a Similar using et S2). by displayed Resendes Fig. 2008; material also al., supplementary et both and Trojan earlier Finally, reported 1997; 2008) as staining. al., centriole et the nuclear at (Middendorp and localized periphery CETN3 cytoplasmic and nuclear the TREX-2 CETN2 the a at at different with found predominantly is along localized for PCID2 while is specific envelope GANP nuclear that antibodies showed using parental subunits in cells experiments IF different our HeLa having suggest addition, complexes In further different localizations. two data, cellular in biochemical present is above results, ENY2 FLAG the These that no cells. with HeLa whereas agreement parental staining weak the in nuclear in a detectable diffuse as was FLAG-HA-SPT3 and staining contrast, well faint In as a 1C). periphery showed (Fig. staining nuclear nuclear the diffuse at signal punctuated rtisatrtetetwt ml nefrn N (siRNA) RNA interfering endogenous small 3 the with corresponding targeting treatment the perinuclear their after of kept proteins depletion proteins upon fusion both localization material Moreover, supplementary S3). 2A; the Fig. (Fig. for IF PCID2 the by corresponding and obtained of expressing signal ENY2-FLAG-HA the distribution the a recapitulated than that showed protein and clones fluorescent levels selected were lower ones EGFP-PCID2 endogenous at Independent proteins protein. EGFP expressing and recombinant the the stably to at cells fused EGFP-ENY2 PCID2 localized HeLa or is generated ENY2 complex either we TREX-2 envelope, the nuclear where determine NPC the To at located is TREX-2 etw sdteEY-LGH n LGH-P3cells FLAG-HA-SPT3 and ENY2-FLAG-HA the used we Next RX2i ihl soitdwt Ps2657 NPCs with associated tightly is TREX-2 9 T ftecrepnigmN sebelow). (see mRNA corresponding the of UTR Journal of Cell Science 2658 yimnfursec.A oto,clswr permeabilized were cells labeled be control, the can a envelope at nuclear As the nuclear immunofluorescence. localized of face by the inner proteins 0.004% the not not conditions, but with but outer these cytoplasmic treated the Under the were membranes. permeabilize of cells to subunits localizes, digitonin complex such all EGFP. TREX-2 no that and alone, NPC. suggest the GANP at EGFP results localize 2A; between complex (Fig. expressing these detectable stably Altogether periphery was not nucleoplasm. cells did the colocalization nuclear in control NXF1 subunits TREX-2 However, In the tested the S3B). with Fig. colocalize at material EGFP-ENY2 with supplementary EGFP-PCID2 colocalizes al., NXF1 et that and (Wickramasinghe show TREX-2 NXF1 could receptor we that 2010), export mRNA showing the 2A). with (Fig. complete periphery nuclear almost the at was in NPCs detected with of ENY2 NPC colocalizes between be signal the extent also colocalization can and the the complex Moreover, significant cells. TREX-2 was intact cases periphery the nuclear the that the all and suggesting at EGFP-PCID2 subunits we In TREX-2 or NPC other EGFP-ENY2 the microscopy. between or colocalization complex confocal antibodies the specific anti-NPC of using or anti-TREX-2 subunits using IF other performed the to relative osuytedsrbto fEF-N2adEGFP-PCID2 and EGFP-ENY2 of distribution the study To ofrhrdtrieo hc ieo h ula neoethe envelope nuclear the of side which on determine further To interacts GANP that showing study former a with agreement In ora fCl cec 2 (12) 126 Science Cell of Journal iapaac fteprncerCT3adEF-N2signal EGFP-ENY2 and the CETN3 in perinuclear results the depletion protein of GANP scaffold disappearance Although the complex. GANP TREX-2 for of the cells localization depleted of the we more on aim influence this an To and have TPR. turn in NPC would basket NPC the with the are basket. interaction of TPR nuclear and the TREX-2 reduction NUP153 specifically cell that for strong indicate corresponding results the required These a in 3B). (Fig. EGFP-ENY2 was line of there staining periphery complete perinuclear nuclear Similarly, a the show from 3A). CETN3 TPR (Fig. by and or localization GANP Blobel, NUP153 of TREX-2 for and disappearance assessed knocked-down (Sukegawa and Cells 2002) basket IF. al., pore used nucleoporins et nuclear two we Frosst the TPR, 1993; NPC and to NUP153 the belong for at that cells function deplete to TREX-2 siRNA characterize better basket To the TPR on and depends NUP153 NPC nucleoporins the at localization TREX-2 r xlsvl oaie tteinrfc ftenuclear the of face inner S4). 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Fig. neir cl as 5 bars: nuclear Scale the interior. represents cell. bar stained black corresponding The the in inset as shown an staining DAPI the in region highlighted yellow the in distribution fluorescence the indicate Charts a with stained were ( cells interior. nuclear bar the black represents The cell. corresponding of staining the DAPI the in highlighted yellow region the in distribution fluorescence C A PI2(90 and (2990) -PCID2 aetl LGH-P3and FLAG-HA-SPT3 Parental, ) FLAG-HA- mock, of blot Western ) a FA nioyadaaye by analyzed and antibody -FLAG a CT3antibodies. -CETN3 m B m. a GN (2988), -GANP etr blot Western ) D HeLa ) Journal of Cell Science motn o P oaiain(i.3.I sntwrh that the noteworthy influence cellular is the influence not It not do GANP 3). or does (Fig. TPR reciprocally NUP153, localization not of it depletion is TPR TREX-2 line, for that important suggesting cell TPR of corresponding localization the in ipnal o h erimn fNF-soitdmNsat mRNPs NXF1-associated of NPC. are recruitment This the nucleoporins the 3). basket (Fig. for nuclear and NXF1 dispensable TREX-2 receptor that export suggest mRNA would the of localization RX2i ihl soitdwt Ps2659 NPCs with associated tightly is TREX-2 as 5 bars: with stained and A/C, X-100 Lamin Triton 0.1% or digitonin units.( The the right chart). 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CETN3 and and showed PCID2 EGFP-ENY2 cells CETN3, staining GANP-depleted PCID2, perinuclear expected GANP, the transfected for showed using Cells siRNA subunits. approach scramble different knockdown with nuclear the the systematic against with siRNAs a interaction specific undertook the for we the and for basket complex important the subunits of TREX-2 stability the identify to Next, the with basket interaction pore TREX-2 for nuclear crucial are ENY2 and GANP with stained then were Cells hours. 72 for siRNAs a GANP or or siRNA TPR scramble NUP153, with targeting transfected basket were the (B) on cells stably-expressing depends TPR. NPC and the NUP153 at nucleoporins localization TREX-2 3. Fig. 2660 GN (2988), -GANP tiigy iN elto fEY e oasgiiatloss significant a to led ENY2 of depletion siRNA Strikingly, ora fCl cec 2 (12) 126 Science Cell of Journal a CT3and -CETN3 m m. a TRatbde n nlzdb confocal by analyzed and antibodies -TPR a ( A TRatbd eec-tie iha with co-stained were antibody -TPR , B eacls()o EGFP-ENY2 or (A) cells HeLa ) nvivo in a - r ipnal n ontifuneisstability. its influence that not CETN3 do and PCID2 and to dispensable opposed anchoring as are for basket crucial NPC are the to ENY2 TREX-2 and data GANP These that GANP S5). indicate the Fig. together of material staining (supplementary the subunits in ENY2 changes and observe not did we mRNAs, ugsigta N2i rca o h tblt fistwo its of stability the ENY2- The for 4D). ENY2, (Fig. crucial ATXN7L3 targeting and is GANP siRNA partners with interaction ENY2 treated that cells suggesting the almost was in ATXN7L3 of undetectable Indeed, stability ATXN7L3. the partner affect would interaction depletion its its whether tested we 2011) (Ko SAGA S5). unaffected reproducibly Fig. were material was levels supplementary level CETN3 4C,D; and (Fig. protein PCID2 cellular addition, GANP whereas In total diminished depletion unchanged. cells However, were ENY2 levels EGFP-ENY2. depleted upon protein by CETN3 in accompanied and GANP was reduction PCID2 TREX-2 that GANP similar of of investigated a reduction strong analysis further a revealed we Western-blot fluorescence knockdown, EGFP-ENY2 stability. the GANP of decrease upon a observed stability other’s we each Since influence ENY2 and GANP opeefursec eoeyi 0mnts(i.5) The 5A). (Fig. observed minutes 10 we in photobleaching recovery perinuclear fluorescence after NPC construct complete and typical a signal a with displayed like transfected cells of These dynamics transiently EGFP-NUP153. the cells expressing analyzed using we 3), protein (Fig. this NPC 2004). the the al., with et for association (Rabut described structure previously core its one NPC as the the basket with of pore range nucleoporins EGFP- nuclear the in and with is TREX-2 associated dynamics EGFP-ENY2 Therefore, stably slow. be of very to is eighty seems turnover periphery nuclear and the the at EGFP-ENY2 PCID2 NXF1, in that for indicate to fluorescence results These hours contrast a 5A). 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(Fig. staining similarly protein interior endogenous nuclear the the in and to periphery the nuclear localized the EYFP- EYFP-NXF1 at assessed cells, both expressing transfected first In photobleaching transiently experiments. after (FRAP) We recovery cells fluorescence performed using factors. and to NXF1 NXF1 export relation of in mRNA dynamics EGFP-PCID2 determine known and to sought EGFP-ENY2 other we of basket dynamics NPC the the at localizes TREX-2 highly As is basket pore nuclear stable the with hence association knockdown. 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NPC transcription D of the the distribution of with Actinomycin effect the TREX-2 inhibitors tested of we association dependent interact determine transcription stable To to 2012). the al., suggested et if been (Jani machinery recently transcription the has with complex TREX-2 The is basket pore independent nuclear transcription the with association TREX-2 2662 ora fCl cec 2 (12) 126 Science Cell of Journal a aaii ntenuclear the on -amanitin dnia eut eeosre hnlv el were in nuclear independent the transcription cells. cells unambiguously is with HeLa localization data associated this these that stably live and together is envelope Taken TREX-2 when cell. that to cells. the proteins demonstrate soluble of observed treated allowing out thus flow D the fixation were to Actinomycin prior on perinuclear permeabilized results and same D the cells untreated Identical Actinomycin displayed HeLa in CETN3 in of and CETN3 localization GANP and effect the 6B). GANP TREX-2 (Fig. the assess endogenous endogenous To of tested on 6A). localization (Fig. we inhibition EGFP- NPC transcription subunits the and of at EGFP-ENY2 influence stable both remained as PCID2 distribution TREX-2 affect soitdwt h P.Teeoe eivsiae whether stably investigated is we TREX-2 Therefore, that NPC. show the results our with In model 2012). associated al., this et Jani to 2010; contrast al., interacts et it is (Wickramasinghe where NXF1 NUP153 NPC to the with bound and nucleoplasm TREX-2 the that between proposed shuttling was It NXF1. receptor h itiuino poly(A) of of analyzed We transport basket. distribution pore nuclear nucleocytoplasmic the the at the directly particles facilitate mRNP could TREX-2 RX2hsbe eotdt eivle nteepr of export the in involved be to reported TPR poly(A) been and NUP153 has with observed TREX-2 those mRNA as similar defects in export results knockdown subunit TREX-2 eetasetytasetd cl as 5 constructions bars: other Scale whereas transfected. lines transiently cell were stable EGFP-PCID2 and EGFP-ENY2 the every on acquisition z acquired an with were hours Images 24 minutes. for 5 imaged and were EGFP-ENY2 Cells mitosis. stable during a cells to ( opposed signal. EYFP-NXF1 as perinuclear of nucleus EGFP-PCID2 fluorescence the minutes, from 10–15 disappears After totally seconds. carried 30 of was every panels) Photobleaching out pre-bleach EYFP-NXF1. the and in EGFP-PCID2 indicated EGFP-ENY2, the as of (rectangles photobleaching repetitive area Representative during nucleoplasmic fluorescence FLIP. nuclear by the analyzed show were images cells ( expressing is panel. EYFP-NXF1 photobleaching each seven after of Time top arrows). on (white indicated photobleached shown a are at region recovery perinuclear cells. fluorescence expressing of EGFP-NUP153 images fifteen Representative stable and highly EGFP-PCID2 is six basket EGFP-ENY2, pore nuclear the on homogenous vivo with effect in association an TREX-2 an have 5. to Fig. NUP153, of seems cells of HeLa Instead in depletion ENY2 cells. nucleus, or GANP the TPR, HeLa in throughout higher in consistently accumulation was than ENY2 and HCT116 GANP tested downregulation TPR, other after NUP153, the retention of and nuclear we NXF1 mRNA lines, cell although between in both proteins difference seen In clear that 7). a to (Fig. cells similar observed knockdown very TPR was or depletion NUP153 ENY2 or observed pattern GANP FISH after the Interestingly retention. nuclear mRNA ietdaantNF n bevdamsieacmlto of siRNA accumulation used massive we a defect, observed poly(A) and export NXF1 mRNA against for cells. directed control HCT116 basket and positive the HeLa a as both As in well TPR as and subunits NUP153 nucleoporins TREX-2 different of knockdown fluorescence fGN rEY ed oamle u erdcbepoly(A) reproducible but milder a to leads depletion contrast, ENY2 In or factor. export GANP mRNA of main the of one being sak o GPEY r hw.Teeeprmnswr efre on performed were experiments These shown. are EGFP-ENY2 for -stacks . ( A + + RPeprmnswr efre nnn YPNF,five EYFP-NXF1, nine on performed were experiments FRAP ) RAtruhisitrcinwt h RAexport mRNA the with interaction its through mRNA RAi ohcl ye ossetwt hsprotein this with consistent types cell both in mRNA nsitu in yrdzto FS)floigsiRNA following (FISH) hybridization B C i GPEY,sxEF-CD and EGFP-PCID2 six EGFP-ENY2, Six ) ielpeiaigo EGFP-ENY2 of imaging Time-lapse ) + RAi h ulu by nucleus the in mRNA z ai ihase f1 of step a with -axis m m. m .Selected m. + Journal of Cell Science n nlzdb ofclmcocp.Saebr 5 bar: Scale co- microscopy. then confocal were by Cells analyzed formaldehyde. and 2% using for fixed an X-100 with then Triton stained and 0.1% ice using on first minutes Triton permeabilized 5 0.1% were using were cells permeabilized Cells or then applied. for and X-100 formaldehyde then 5-FU 2% were with with protocols incubated fixed different and either hours Two 4–6 minutes. for 20 D another actinomycin with treated were Uatbd.Clswr nlzduigcnoa irsoy ( microscopy. confocal using analyzed were Cells antibody. FU 0mnts noprto nonsettasrpswsrvae sn an using revealed was for transcripts 5-FU nascent with into cells Incorporation the minutes. incubating 20 by or monitored D was actinomycin inhibition with Transcription treated were cells stable uui ncdwsmmctemN ula retention TREX-2 nuclear as and mRNA 3) (Fig. the versa mimic vice not to knockdowns localization but TREX-2 subunit basket for necessary NPC are the TPR and components. after NUP153 export the observed mRNA As emphasizing those not between further was differences in component which functional death TREX-2 shown) cell any not above of of rate (data knockdown the high types a with cell to both Consistent led depletion S7). NXF1 observations, Fig. material (supplementary h ubradtesz ftencerpoly(A) nuclear the of size the and number the of independent is basket II. pore polymerase nuclear RNA the by at transcription localization TREX-2 6. Fig. a 5F nioyand antibody -5-FU a GN 28)or (2988) -GANP ( A GPEY rEGFP-PCID2 or EGFP-ENY2 ) a mntnfr46hours. 4–6 for amanitin m m. a CT3antibodies -CETN3 + RAfoci mRNA B eacells HeLa ) a -5- epcierlsi RAepr a euculd Our uncoupled. be their can that export suggesting NXF1 mRNA to in opposed as roles NPC respective the but to GANP only bound not SAGA. within stabilize partner to interaction data, crucial our its is of ATXN7L3, support ENY2 also a In that 2012). with al., show et GANP we contrasts (Jani for NPC important This the not at is localization TREX-2. ENY2 that of suggesting report and previous localization stability have other’s the we each influence Furthermore therefore ENY2 and dispensable. whereas GANP the NPC that appear the shown for CETN3 at crucial complex and the are of PCID2 nuclear ENY2 localization the the and at and GANP stability proteins basket TREX-2, these seems Within of the TREX-2 basket. localization turn the requires in for whereas dispensable TPR association and of NUP153 This TREX-2 independent nucleoporins and that transcription. stable extremely show is ongoing We NPC the using approaches. with complex association cellular TREX-2 human and the biochemical characterized is have we export Here mRNA the in Discussion basket. that NPC TPR the conceivable and at is anchoring NUP153 TREX-2 it on of dependent TPR, function and correct NUP153 with observed iial oohrnceprn uha P n P2.(iv) GP120. and TPR as any such (iii) nucleoporins G1 compartments. poly(A) excluding early other not in two to NPC the does fraction these similarly with re-associates ENY2 between ENY2 nucleoplasmic mitosis, TREX-2 Following of its of fraction shuttling with NPC-associated the contrast the In to exchange (ii) opposed structure. NXF1, as this NPC with to NXF1 the highly of with association a TREX-2 dynamic uncovered of basket: unambiguously NPC association all the experiments stable at that FRAP function evidence TREX-2 Our for of (i) model lines unique a several to by point supported are conclusions tteNCbse o ute export. further TREX-2 for to basket binds NPC particles the possibility the mRNP the of at NXF1-bound out depletion of rule is fraction after cannot a NXF1 even we that NPC of However, the basket. fraction pore at significant nuclear localized a be 2007; Therefore, al., to 2008). et export expected core (Yao al., the basket mRNA with et NPC interact the Yao the of can independent of orthologue pre-60S NUPs yeast independent scaffold the its of that is export basket and the that pathway in or particle involved GANP studies is subunits ribosomal NXF1 Previous that that localization. TREX-2 shown NXF1 have in affect showed of However, not study did cells. depletion we nucleoporins depleted former NXF1 contrast, a 2010) in with striking altered not agreement al., is In localization et 2012). our al., by (Wickramasinghe supported et not are (Jani NPC the results and mammalian likely transcription active in of more sites TREX-2 and of S8). Fig. specificity properties material (supplementary general type cells the cell lines above cell reflecting the human excluding different of two be in therefore Most to obtained (vii) were found results transcription. was TREX-2 described NPC ongoing or the of at nucleoporins independent localization altered basket TREX-2 not (vi) either was subunits. NXF1 of are of depleted depletion localization NXF1 nucleoporins in following perinuclear seen The basket that (v) than or milder cells. much subunits and similar TREX-2 highly of knockdown u nlsscnitnl eosrt htTE- stightly is TREX-2 that demonstrate consistently analyses Our rvosmdl ugsigta RX2i htln between shuttling is TREX-2 that suggesting models Previous RX2i ihl soitdwt Ps2663 NPCs with associated tightly is TREX-2 + RAnceracmlto bevdafter observed accumulation nuclear mRNA Journal of Cell Science 2664 n motneo lentv prahssc slv cell live as poly(A) such factors. 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NXF1 after seen that than milder is that knockdown u IHdt hwcmaal ula cuuainof accumulation nuclear comparable show data FISH Our interactions tight suggests experiments imaging cell live Our + ora fCl cec 2 (12) 126 Science Cell of Journal RAuo AP N2 U13adTPR and NUP153 ENY2, GANP, upon mRNA ssae ewe h w opee.Hwvr h available the However, complexes. two that the Sus1 between through mediated shared be Ko is to 2006; suggested al., was et interaction be physical (Cabal Such to gating proposed a gene been in Furthermore, has involved TREX-2 periphery. re- and gene SAGA nuclear on between effect interaction the an had at SAGA or localization TREX-2 is either mRNA of subunits of all subset specific a depletion. if its or of by function export affected global mRNA a by on explained whether TREX-2 is determined phenotype be export export- to mRNA of remains the It transport particles. basket-associated nucleo-cytoplasmic mRNP NPC the competent an is helps TREX-2 that which complex in model Thus, a types. favor cell we those subunits between TREX-2 differences or functional nucleoporins suggesting basket either of depletion after 02.Oreprmnsi eaadHT1 el reveal cells HCT116 and been HeLa has poly(A) in in what differences experiments quantitative al., et from Jani Our 2010; al., different 2012). et export Wickramasinghe are 2007; al., mRNA et but (Kurshakova for in 2003) essential published al., previously not et are showing studies (Green nucleoporins previous with basket agreement good that in are data These omrsuisi es eotdta eeino oebtnot but some of deletion that reported yeast in studies Former Drosophila 10 bars: Scale microscopy. by confocal analyzed were Cells probe. poly(T) using Alexa-Fluor-488-labelled FISH an by analyzed was mRNA the poly(A) and of hours distribution 72 cellular for NXF1 or ENY2 GANP, TPR, NUP153, siRNAs targeting or siRNA scramble transfected with were cells (B) HCT116 nuclear accumulation. mRNA in results TREX-2 subunits of Knockdown 7. Fig. + m RAnceraccumulation nuclear mRNA m. 2o ua C16cells HCT116 human or S2 ( A he ta. 2008). al., et ¨hler , B ea()or (A) Hela ) + Journal of Cell Science 02.I diin eso eeta RX2adthe yeast in and reported been is each TREX-2 It has cells. gating with and human that gene in although interact coupled that here functionally al., not noteworthy not likely et show do are Jani two and SAGA other 2012; we the al., co-activator of addition, et interaction transcriptional Ellisdon In direct 2009; a al., 2012). et support (Jani not complexes do data structural h te nioisue nti td eedsrbdeswee TRRAP elsewhere: 2987: described were and study this 2988 in using used All purified serum instructions. were antibodies manufacturer’s the 2990 other to GANP according but the (Pierce) the Gel sera CETN3 ovalbumine: Coupling coupling All Sulfolink ITINQYLQQVYE(C); for 2985:(C)EINQEEFIAIMTGDI. used 2990: activated was serum serum that PCID2 following cysteine to (C)SSLLNKSSPVKKPSL; the added Wolfrum. against Uwe an raised Dr indicates peptide were from CETN3 (C) gift antibodies The where kind polyclonal Cruz. peptides a Santa rabbit was from specific Abcam. was immunofluorescence TREX-2 (sc27793) for was from and CETN2 epitope used HA (B2531) were to antibody the antibody Antibody against (ab24609) Roche. Antibody anti-5FU Sigma-Aldrich. complex from (F7425), from epitope pore were (T6557) NXF1 FLAG nuclear Tubulin the (ab5131), the against (ab8984), II and A/C Antibodies Lamin polymerase (ab290) (ab24276), RNA NUP358/RANBP2 EGFP (ab84516), (ab113295), TPR GANP (ab50609), against Antibodies Antibodies Erasmus For proteomics the 2010). the by al., of IGBMC. processed et the were center Krebs samples of 2006), in platform proteomics al., described et (as (Paoletti the analyses Rotterdam MudPIT at in LTQ- Center spectrometer an Medical on University mass LC-MS/MS nanoflow thermo by Orbitrap analysed analyses was spectrometry material mass Immunoprecipitated MudPIT and LC-MS/MS Nanoflow mM 10 in volume MgCl to mM volume 1.5 resuspended 8.0, pH were Tris-HCl cells immunoprecipitation For Immunoprecipitation to processes. 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MMgCl mM 1.5 Glycerol, 25% vrl,w rps htTE- saNCascae complex NPC-associated a is TREX-2 that propose we Overall, Drosophila 2 m 0 lcrl .%N4;03m T;10m C;Roche KCl; mM 100 DTT; mM 0.3 NP40; 0.1% glycerol; 10% ; /l ula xrcswr rprdi 0m rsHlp 8.0, pH Tris-HCl mM 20 in prepared were extracts nuclear g/ml) 2 5 MNC,1m CaCl mM 1 NaCl, mM 450 , uhamcaimwsnvrdsrbdin described never was mechanism a such , 2 0 lcrl .%N4;03m T;50m KCl; mM 500 DTT; mM 0.3 NP40; 0.1% glycerol; 10% ; 2 5 MNC,1m CaCl mM 1 NaCl, mM 150 , 2 0m C,poes niios(rmRoche) (from inhibitors protease KCl, mM 10 , ˚ ihat-Aspaoebeads sepharose anti-HA with C 2 rtaeihbtr fo Roche) (from inhibitors protease , ˚ ihat-LGM Sepharose M2 anti-FLAG with C 2 .%IEA CA- IGEPAL 0.1% , ojgae eodr nioisfo ako muoeerhLaboratories. peroxydase Immunoresearch using Alexa Jackson revealed were from were study antibodies blots this al., secondary Western 2009). in conjuguated et Invitrogen. al., used from et (Nagy 2001); antibodies Antibodies (Nagy 2678 2461 secondary Fluor al., SGF29 ADA3 all et 1995); 2008); immunofluorescence al., (Brand et For al., (Mengus 2GC-2C11 et 22TA-2A1 TAF12 GCN5 2ATX-2B1(Zhao 2009); and 2004); 2325 al., ATXN7L3 et (Helmlinger 2TRR-2D5 eonto ie n ucoe nothe into subcloned and sites recognition a incorporating primers with amplified the PCR First, steps. was two protein in EGFP- EGFP generated by HeLa 5 were the generated 2003). lines of Ogryzko, then cell and cDNA stable were (Nakatani EGFP-PCID2 lines in and N-terminal cell described ENY2 or Stable as (ENY2) infection sequence. C-terminal retroviral peptide a containing FLAG-HA vector (SPT3) retroviral pREV-HTF the pg/ 100 containing (50% buffer buffer hybridization hybridization in in overnight hour hybridized 1 were for 1 cells prehybridized in 2 and X-100 formamide, Triton citrate) 0.5% 1 sodium with in minutes ice 5 on minutes 5 eu n nuae o ora 37 at hour 1 for incubated and in minutes serum 5 times three fixed washed and minutes, 10 coverslips for glass PBS 1 on 1X in grown formaldehyde were 2% with cells immunofluorescence indirect For Immunofluorescence SPT3 and ENY2 of version 37 tagged FLAG-HA at the grown expressing were cells S3 HeLa treatments and culture Cell fEY rPI2wsapiidb C ihpiesincorporating primers with PCR cDNA length by full amplified the was step, PCID2 second a or As ENY2 (Invitrogen). of vector pcDNA3.1+ the of site a by followed sequence primers with PCR by amplified were SPT3 and ENY2 of incorporating clones cDNA line Human cell stable of Establishment Alexa-Fluor-488-oligo(dT) for transfected then and in hours fixed 24 were Cells for FISH. RNA 1 poly(A)+ coverslips in performing formaldehyde to glass 4% prior siRNA on with hours grown 72 were Cells FISH RNA Poly(A)+ Lipofectamine siRNA using All cells Dharmacon. anti-CETN3 HeLa from instructions. anti-TPR and in manufacturer’s purchased ON- L-013680-01) L-018808-01), transfected were as No.: (CatNO.: were siRNA (Cat well L-011832-00) anti-ENY2 anti-NXF1 as (CatNO.: L-010548-00), pool 2010). D-005283-01) No.: al., SMART No.: (Cat et (Cat plus (Wickramasinghe siRNA TARGET elsewhere anti-NUP153 described Ambion Additionally, were anti- from purchased and siRNA were siRNA ID:s31532) Anti-GANP ID:s226899) ID:s31531, ID:s32447, (ID:s32449, (ID:s31530, ENY2 anti-PCID2 (AM4635), Scramble transfection siRNA upeetdwt / lcs,1%FSadgnayi.HL el sdfor used cells HeLa gentamycin. and 37 FCS at 10% grown were glucose, immunofluorescence g/l 4 with supplemented Ivtoe)splmne ih1glguoe 0 ea afsrmand serum for calf incubated fetal were 10% cells 20 containing experiments glucose, medium with inhibition g/l hours 1 proteasome 15 For with gentamycin. supplemented (Invitrogen) iewdho 0pxl a used. was pixels a analysis, 20 distribution of fluorescence width For (NIH). line were software Images imageJ (Sigma-Aldrich). the Digitonin using (2990) of 1 analyzed 0.004% in with permeabilized formaldehyde PCID2 permeabilized 2% and further with and anti fixed formaldehyde first were the 4% cells in experiment, For fixed 1 Laboratories). alternatively with were (Vector cells Vectashield antibody on mounted rtnX10adicbtdwt h eodr nioycnuae oAlexa to conjugated antibody secondary the 1 with in incubated times three and washed X-100 then Triton were Cells buffer. blocking lo 8 rAeaFur54(nirgn nbokn ufrfr1hu t37 at hour 1 for buffer blocking in (Invitrogen) 594 Fluor Alexa or 488 Fluor ia aho iue n1 in minutes Laboratories). 5 (Vector of wash final a eete lce 0mntsi lcigbfe otiig1 containing buffer blocking in minutes 30 blocked then were S,tretmsi 0.5 in times three SSC, niiineprmnsclswr nuae – or ih5 with hours 4–6 incubated were cells experiments inhibition el eete ahdtretmsi 1 in times three washed then were Cells SgaAdih r50 or (Sigma-Aldrich) D sesdb tiigteclswt nat -Uatbd rmSigma-Aldrich. from antibody 5-FU anti an with was 5-fluorouridine cells nucleotide mM the modified 1 the staining of with by Incorporation assessed incubated minutes. further 15–20 for were (Sigma-Aldrich) cells inhibition transcription 6 9 Hin B n emaiie o 0mntsi .%Tio -0 n1 in X-100 Triton 0.1% in minutes 10 for permeabilized and PBS IIrsrcinst olwdb h Appiesqec n 3 a and sequence peptide HA the by followed site restriction dIII RX2i ihl soitdwt Ps2665 NPCs with associated tightly is TREX-2 6 B otiig05 rtnX10 nteDgtnnpermeabilization Digitonin the In X-100. Triton 0.5% containing PBS 6 Xho 6 S,1m/lBA gm RA 0 eta uft) The sulfate). dextran 10% tRNA, mg/ml 1 BSA, mg/ml 1 SSC, B olwdb iue n2 in minutes 5 by followed PBS I/ Not ˚ nDlec’ oiidEgesmdu (Invitrogen) medium Eagle’s modified Dulbecco’s in C 6 6 eonto ie n ucoe nothe into subcloned and sites recognition I Xho B o 0mnts ahdi 1 in washed minutes, 10 for PBS S alwse t37 at washes (all SSC 50 m ie C rdcswr lndit the into cloned were products PCR site. I ahdtretmsi 2 in times three washed , g/ml 6 B eoemutn h el sn Vectashield using cells the mounting before PBS a aaii SgaAdih.T vlaefor evaluate To (Sigma-Aldrich). -amanitin m ˚ ˚ nDlec’ oiidEgesmedium Eagle’s modified Dulbecco’s in C G3 clice) o transcription For (calbiochem). MG132 M 6 ihtepiayatbde iue in diluted antibodies primary the with C Xho B otiig01 rtnX10and X-100 Triton 0.1% containing PBS I/ Xba ˚ o iue ah olwdby followed each) minutes 5 for C ie ftepDA.+vector. pcDNA3.1+ the of sites I 6 6 B.Clswr incubated were Cells PBS. S 03MNC;00 M 0,03 NaCl; M (0.3 SSC 6 6 H 6 00acrigt the to according 2000 S,tretmsi 1 in times three SSC, B n permeabilized and PBS 6 B otiig0.1% containing PBS 6 B o 0minutes 10 for PBS m B n 0 goat 10% and PBS /lactinomycin g/ml Xho I/ 6 Hin Not B.Cells PBS. Xho dIII/ ie of sites I 9 I/ linker Xba Xho ˚ C. m 6 I I l Journal of Cell Science noi,W,Elneg .adDlz E. Dultz, and J. Ellenberg, W., Antonin, M. Rosbash, and S. Lacadie, C., K. Abruzzi, References (LEA- CNRS at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.118000/-/DC1 and online available material Supplementary UBican) and (2008 LSHG-CT-2007-037445 INCA SkinChroma). EUTRACC, la Pour (SAGATAC, EPIDIACAN), Fondation EU the from the ANR-09-BLAN-0266), fellowship ANR-10-INTB-1201; a (ANR-09-MNPS-023; of Me recipient Recherche a was experiments, J.B. designed L.T. FLIP Funding and manuscript. D.D. the and wrote 7. and Fig. FRAP data analysis in the throughput MudPIT analyzed high analyzed obtained the the data performed analyzed and of L.B. and and performed performed B.F. M.F. experiments. initial D.U. F.W. the with experiments performed lines. and FLAG-HA-SPT3 and cell established ENY2-FLAG-HA the HeLa M.S. wrote the of and characterization and experiments J.B. all analyzed manuscript. performed, designed, D.U. The contribution interest. Author experiments. of the conflict FLIP in no and have FRAP help they in for that help declare for for Kessler platform authors Institute P. microscopy Curie Duval PICT-IBISA the and the of Koch G. and Demmers IGBMC. M. experiments J. antibody, the microscopy synthesis; analysis, CETN3 of the proteomic for facility Wolfrum peptide for U. culture pEGFP-C1-NUP153 to cell grateful for are the We and and Eberling antibodies, pEYFP-C1-NXF1 generating C.L. P. plasmid; the pREV critical the plasmids; for for for Shav-Tal Hamiche Y. A. Tomasetto manuscript; and the Torres-Padilla of M.E. reading to grateful are We Acknowledgements cells experiments imaging cell live Lab-Tek For on camera. grown EMCCD were 897+ iXon ANDOR microscope 63 an Leica Apo 6000 Pl DMI HCX a a using using analyzed were cells immunofluorescence, For Microscopy EGFP-ENY2 further monoclonal and with lines. isolated made cell were were EGFP-PCID2 clones (500 were and presented individual G418 selection, experiments containing cells G418 All medium of subcloned. transfection in weeks 2000 selection 2 after After clone Lipofectamine ml). individual hours allow using to Twenty-four diluted transfected instructions. were manufacturer’s cells HeLa 2666 ulu osuyternme n rai h ifrn iecn odtosusing conditions silencing LifeScience). different (GE the software in Analysis area Target and Multi number the their study to nucleus otae(ELfSine ysgetn h ulu fteclsacrigt the Poly(A) to according nuclear cells the The Analysis of staining. nucleus Target the Multi DAPI segmenting the by with LifeScience) pictures (GE 10 the a software been analyzed using has LifeSciences) We fluorescence For EGFP-PCID2 (GE objective. workstation S7, 2010). magnification Analyzer and Fig. al., INCell1000 bleach material et an laser EGFP-ENY2 with supplementary (Boulanger acquired milliseconds in in experiments (50 shown described experiments sample algorithm FRAP the using the For denoised in were spot point). mW) images bleached 50 per (BP a nm, set (491 create duration filter technology to Chroma laser used using MicroPoint confocal was filtered Instrument Photonic was Yokogawa nm). Emission CSU22 525/50 laser. argon-krypton a Nikon 60 mW a with a 100 on and based equipped was disk imaging microscope spinning video and inverted measurements FLIP TE2000-E and FRAP for used oor . hih . aia . aaj,W . ats . ok,M and M. Lohka, R., Bastos, H., W. Raharjo, D., Salina, S., Shaikh, K., Bodoor, ope erimn oitols n nrncnann es genes. yeast intron-containing and intronless 2620-2631. to recruitment complex hog h elcce euainadmmrn organization. membrane and regulation cycle: cell the through uk,B. Burke, 2016. h n fmitosis. of end the ora fCl cec 2 (12) 126 Science Cell of Journal 19) eunilrcuteto P rtist h ula eihr at periphery nuclear the to proteins NPC of recruitment Sequential (1999). ´ iae hswr a uddb rnsfo ANR from grants by funded was work This dicale. .Cl Sci. Cell J. 6 6 TM . es S2 ooaacnoa pnigds and disk spinning confocal Yokogawa CSU22 a lens, 1.4 lnAoN . es xiainue Melles-Griot a used Excitation lens. 1.4 NA Apo Plan hme Slide Chamber 112 2253-2264. , + RAfc aebe eetdi the in detected been have foci mRNA 20) ula oecmlxassembly complex pore Nuclear (2008). TM 20) iceia nlsso TREX of analysis Biochemical (2004). ytm(uc.Teotclset-up optical The (Nunc). System H ESLett. FEBS codn othe to according MOJ. EMBO 582 2004- , m 23 6 g/ , rb,A . emr,J,Kroia . hn,N . hn,A .adTr,L. Tora, and C. A. Chang, C., N. Chang, K., Karmodiya, J., Demmers, R., A. Krebs, Ko Ko Ko Rondo S., Jimeno, O. V. Wickramasinghe, and M. Stewart, A., R. Laskey, E., Hurt, S., Lutz, D., Jani, Ko T., Fischer, J., N. Marshall, S., Lutz, D., Jani, ura,P n giea A. Aguilera, and P. Huertas, L., Miguet, C., Weber, F., Robert, F., Klein, S., Sasorith, S., Hardy, D., Helmlinger, Gru ice,T,Stra T., Fischer, Gonza S., Jimeno, S., Kemmler, B., M. 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