Overexpression of MLN51 Triggers P-Body Disassembly and Formation of a New Type of RNA Granules N

Overexpression of MLN51 triggers P-body disassembly and formation of a new type of RNA granules N. Cougot, E. Daguenet, A. Baguet, A. Cavalier, D. Thomas, P. Bellaud, A. Fautrel, F. Godey, Edouard Bertrand, C. Tomasetto, et al.

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N. Cougot, E. Daguenet, A. Baguet, A. Cavalier, D. Thomas, et al.. Overexpression of MLN51 triggers P-body disassembly and formation of a new type of RNA granules. Journal of Cell Science, Company of Biologists, 2014, 127 (21), pp.4692–701. 10.1242/jcs.154500. hal-02191565

HAL Id: hal-02191565 https://hal.archives-ouvertes.fr/hal-02191565 Submitted on 1 Jun 2021

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Euge em,Cmu eBale,302Rne ee,France. cedex, Rennes 35042 Beaulieu, de Campus Team», omto fanwtp fRAgranules RNA Cougot of Nicolas type new and a disassembly of P-body formation triggers MLN51 of Overexpression ARTICLE RESEARCH ß 4692 2014 August 17 Accepted 2014; April 3 Received 2005). al., et Ballut 2004; exon 1 al., the et into RNA-binding (Degot MLN51 core unique of 2004; complex incorporation a junction the al., localizer triggers 2005). (SELOR), speckle that al., et module the domain et harbors Tange Nott RNA-binding MLN51 2005; of 2004; and and al., region et al., N-terminal Gehring translation The 2004; et al., pre-mRNA localization, et (Degot regulate Palacios to EJC a of stability export, proposed fate junction. transcript been subsequent is cytoplasmic has the exon–exon splicing EJC determining EJC splicing, in the the the step The as crucial by at mRNA, a (EJC). deposited is mRNAs binding complex is onto that junction machinery association complex exon its macromolecular through a the metabolism is CASC3) mRNA with as in known also involved 51, protein node lymph (metastatic MLN51 INTRODUCTION Stress P-bodies, MLN51, granules mRNA, cancer, Breast WORDS: KEY P-body of in observed process (HER2 also is HER2-positive the count human P-body with in reduction correlates similar A foci disassembly. these directed The P-bodies. of in show and granules RNA, appearance stress localizes from contain distinct are These MLN51 and foci. movements overexpressed, cytoplasmic small stress processing When novel of of component new (P-bodies). assembly a is role the bodies MLN51 cytoplasmic that in the show We investigated role MLN51. further of key we study, the a this in In located plays granules. mainly it is MLN51 where RBM8A)], cytoplasm, Magoh as [eIF4AIII, known components (also core Y14 EJC and loaded their other determining is three in the which role Unlike essential (EJC), fate. an plays complex and mRNAs junction spliced onto exon core a the is CASC3) of as known component also (MLN51, 51 node lymph Metastatic ABSTRACT ltfrehsoahlgqe F 4 FS Universite France. GFAS, Rennes, 140 IFR histopathologique, Plateforme INSERM/Universite L5.Ti ugssta -oydssebyadsubsequent and disassembly P-body that suggests This MLN51. RAdrglto ih orlt ihcne progression. cancer with correlate might deregulation mRNA Atosfrcrepnec [email protected] [email protected]; [email protected];) ([email protected]; correspondence for *Authors France. 5, cedex Montpellier 34293 li Fautrel Alain Universite ´ 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,49–71doi:10.1242/jcs.154500 4692–4701 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. ne ` ´ eMrus u el aaleFade ukru 54 enscedex, Rennes 35042 – Dunkerque Flandres Bataille la de Rue Marquis, ne iu td ilgeMole Biologie de et tique 5 ntttd Ge de Institut ´ eRne ,URCR 20ID,«rnlto n Folding and «Translation IGDR, 6290 CNRS UMR 1, Rennes de 4 ´ eted esucsBooiusSante Biologiques Ressources de Centre 3 eSrsor,644Ilic,France. Illkirch, 67404 Strasbourg, de lrneGodey Florence , ´ ne 1 ´ ,E iu Mole tique ´ iaehDaguenet lisabeth ´ uar tCluar,UR70,CNRS/U964 7104, UMR Cellulaire, et culaire ´ + uar eMnplir NS M 5535, UMR CNRS, Montpellier, de culaire ratcne el overexpressing cells cancer breast ) 4 ,E ´ oadBertrand douard ´ 2 eRne ,35043 1, Rennes de Aure , 3 ´ Unite eRne,Centre Rennes, de 2 ntttde Institut ´ NEM991, INSERM leBaguet ´lie 5, ,CteieTomasetto Catherine *, 2 ni Cavalier Annie , oprmnsivle ntermdln fmessenger of remodeling cytoplasmic as the granules 2007), stress in Anderson, of and involved description (Kedersha the compartments granules to in stress to involved leading found been in factors have silencing Several localize mRNA or 2006). decay mRNA Kedersha, translation, arrest translational and general cause (Anderson can that stress stresses environmental cytoplasmic to MLN51 2007). al., of et is recruitment (Baguet that granules region the and export particular nucleus this for the nuclear is essential it between a interestingly, shuttle and, to contains cytoplasm protein the MLN51 the allows of that signal moiety C-terminal The n L5 oaiaini ml rnlsi loosre in observed also is granules HER2 count small P-body in human in localization reduction MLN51 similar and P-body a microtubule-dependent Interestingly, Moreover, to disassembly. (small movements. leads directed overexpression SMIGs mRNA- show MLN51 and cytoplasmic novel named RNAs described a contain we newly bodies in These that and granules). granules structure of MLN51-induced stress types granular in two containing in – accumulates foci of it cytoplasmic component novel overexpressed, a When is MLN51 and present P-bodies. endogenous the granules that In stress show cells. cancer in we breast study, protein malignant this al., and HeLa of et mRNA in role P-bodies the (Chazal the in explored polysomes MLN51 and of active cycle involvement in the investigated present we be 2013), to and activator granules stress and the 2009). P-bodies Parker, fits and mRNAs polysomes, (Balagopal substructures cytoplasmic active which Overall, cytoplasmic between to 2013). cycle two according translational al., a model, these et cycle’ and and Cougot ‘mRNA of 2012; polysomes factors presence al., the decay with et (Cougot mRNA enriched a activators 2007). – containing shell compartments Sheth, two core peripheral and into organized central Parker are P-bodies dense 2007; they of that organization al., Pillai showed structural 2004; and et the al., deciphered Eulalio et we Cougot Recently, 2005; 2003; Parker, al., and et Sheth (Eystathioy 2002; decay involved al., and cytoplasmic are repression et translational that non-membranous share storage, (P-bodies) mRNA They bodies in other processing 2008). called with al., structures components et (Mollet some particles ribonucleoprotein obte hrceietectpamcpteno MLN51 against of directed pattern antibodies cytoplasmic polyclonal raised the we characterize distribution, P-bodies better of component novel To a is MLN51 Endogenous RESULTS ugsigta -oydssebyadsbeun mRNA subsequent cancers. certain and to linked disassembly be might P-body deregulation that suggesting tesgaue efasml nrsos oawd ait of variety wide a to response in self-assemble granules Stress eas L5 a eetysont eatranslational a be to shown recently was MLN51 Because + 1 ratcne el vrxrsigMLN51, overexpressing cells cancer breast ailThomas Daniel , 2, n enl Gillet Reynald and * 1 acl Bellaud Pascale , 1, * 3 ,

Journal of Cell Science ois( bodies is ebro h J oet edtce nPbde.T more To P-bodies. in the detected be fact, to in core is, EJC the that MLN51 of foci markers. member P- first P-bodies cytoplasmic of both in with detection accumulated colocalized allow observed MLN51 We that (also 1A,B). endogenous (Fig. Dcp1a that Ge1 2007) Anderson, human against and (Kedersha or bodies antibodies EDC4) which with as MLN51 cells, known immunolabelled 2002). HeLa al., in also et immunofluorescence were (Degot by protein detected first the was of part N-terminal the ARTICLE RESEARCH hnuigvrosPbd akr,sc sDp,G1 rck/p54 Ge1, Dcp1, as P-body confirmed such markers, 2B). was P-body (Fig. various overexpression using overexpression when MLN51 MLN51 of following by that Dcp1a disassembly with affected endogenous of compared not levels the P-bodies anti-Dcp1a are though of even an cells, number cells untransfected using reduced MLN51-overexpressing a P-bodies Surprisingly, showed 2A). of (Fig. and P- cells, co-labeling antibody on HeLa in overexpression performed expressed MLN51 was we CFP–MLN51 of so, do effects To the bodies. analyzed disassembly next P-body to We leads MLN51 of Overexpression – cytoplasm surrounding in the 48.47 of to MLN51- factor 1C). a compared (Fig. by 1990.54 P-bodies enriched antibodies were same the particles gold the immunoelectron labeling performed using we localization, microscopy its assess precisely eut hwta L5 sdsrbtdeulytruhu the throughout equally distributed is these MLN51 the granule. Collectively, that on of P-bodies). show counted gold-labeled 75% gold-particles results hDcp1a 806 About of different the respectively. periphery 11 of 75% the (604 area, to at P-bodies total accumulated mammalian correspond al., 50% particles the areas et gold a of (Cougot MLN51-labeling internal by body 25% and electron-dense calculated and peripheral entire The was the zone peripheral 2012). that and the to it area added contour between external P-body the of internal downscaling performed an was P-bodies the in in gold-labeling the of Quantification 6 n 5 341 odparticles/ gold 1354.16 1 n 41.07 and 11) 6 70 odparticles/ gold 17.04 m m 2 (mean 6 .. nieteP- the inside s.d.) m m 2 outside. oy hra hswstecs nol .%o untransfected of 5.7% P- only one in than case 2D). less after the (Fig. had fact, was cells In cells this one. in the whereas or of body, zero cell 88% having cells overexpression, strong per of a MLN51 number with cell P-bodies, the P-bodies per CFP– fewer in P-bodies having increase cells overexpressing of (2.66 towards number shift cells the 3.5 a of showed in distribution of the 0.76 and MLN51), versus factor cells a was one cell untransfected least per at by P-bodies with of and cells number decreased average transfected The analysis 54 at 3C,D). of containing Fig. quantitative out cells P-body, of (29 number P-body performed the one in least We decrease 1.9-fold 2C). a (Fig. observed Xrn1 or rnls(aute l,20) is,w ofre ywestern by confirmed we First, stress 2007). cytoplasmic al., to and protein et signal the of (Baguet export and recruitment nuclear granules 2005) the al., in a role et contains a through Ballut domain plays core 2004; C-terminal EJC MLN51 al., the the et (2) to (Degot of is domain MLN51 and SELOR of the the signal recruitment effect (1) in localization the 3A): MLN51 nuclear in (Fig. the involved a of domains contains two role of domain whether the composed N-terminal to is MLN51 linked explore EJC. is the P-bodies to on overexpression the wanted in MLN51 of We role the to linked EJC not is disassembly P-body rvosybe hw opeetMN1icroaininto has incorporation that MLN51 prevent MLN51HD) to (H220A/D221A, shown MLN51 been of overexpressed previously we this, mutant linked confirm results not To is EJC. a these number the with P-body association together, on its MLN51 the to of Taken effect of the that 3C,D). that MLN51 (Fig. to suggest the phenotype of protein similar [hDcp1 overexpression a full-length The examined induced 3D)]. fragment marker (Fig. C-terminal p54 P-body or the 3C) P-body (Fig. on of effect significant irrespective MLN51 a the Dcp1a have number, of not and did overexpression p54 SELOR that endogenous or observed N-terminus of we expression Then, 3B). of (Fig. level the no had on fragments MLN51 effect of overexpression that experiments blot ora fCl cec 21)17 6240 doi:10.1242/jcs.154500 4692–4701 127, (2014) Science Cell of Journal oun ihDP-tie uli(le.Saebr 20 bar: right Scale the (blue). in shown nuclei are DAPI-stained overlays with FITC column, and Cy3 P-body. colocalize the green), detected panel, in MLN51, central (A,B, endogenous anti- anti-MLN51 and rabbit with red) with detected panel, Ge1, left human (B, or Ge1 red) panel, left of (A, Dcp1a component novel a P-bodies. is mammalian MLN51 Endogenous 1. Fig. eacls ,nces ,ctpam cl as 0 nm panel). 500 (right bars: nm Scale 100 cytoplasm. panel), c, (left nucleus; n, chemical-embedded cells. and HeLa gold frozen (15-nm high-pressure anti-Dcp1a in the with particles) throughout labeled localized is is which particles) P-body, gold (6-nm MLN51 (C) ua c1,dtce ihanti- with detected Dcp1a, Human 4693 m m.

Journal of Cell Science EERHARTICLE RESEARCH nue rnls(MG) ti motn ont htthe that of note expression the 4694 to with important only associated is is SMIGs It of MLN51- (SMIGs). ‘small appearance named granules foci we granule small that induced the stress granules stress-granule-related; unknown these are to foci of correspond larger suggests all the this only S2A–C), with were that Fig. colocalize material foci (supplementary that did Given MLN51 components foci 4). (Fig. small larger markers PABP the the 2009). and granule FMRP Parker, that TIA1, and stress for observed negative (Buchan three PABP we and against Interestingly, FMRP CFP- TIA1, antibodies series a – a performed using markers then expressed cells, co-stainings HeLa identify transiently in of better MLN51 we To of version granules. structures, tagged whether stress to wondered cytoplasmic 2; related and (Fig. we the be component assembly, could (3.7%) granule foci granule stress foci these a stress larger is in MLN51 and participates that Given (77.8%) 4C). foci Fig. diffuse of – distribution small fractions previously the cytoplasmic (18.5%), confirmed As three we into P-bodies. 2007), MLN51 we in overexpressed al., and present et cytoplasm, is (Baguet the protein reported in Magoh the found (eIF4AIII, that primarily components observed is EJC MLN51 core Y14), other and SMIGs three in the accumulation its Unlike to leads MLN51 of Overexpression both EJCs w te oeECcmoet,eFA n 1,adno P- (supplementary other and with noticed Xrn1). made Y14, (Ge1, were markers was observations and body Similar P-bodies eIF4A3 S1). Fig. on components, material EJC effect core significant overexpressed we other Finally, PB). two one least led at mutant contained this ( MLN51HD P-bodies of of overexpression disassembly 3C,D, the Fig. to in shown As 2012). nvitro in and nvivo in Blu ta. 05 aunte al., et Daguenet 2005; al., et (Ballut , 0 fteclsoverexpressing cells the of 40% olwn h vrxrsino h L5 -emnldomain. C-terminal MLN51 observed the be of also can overexpression foci the larger following the whereas MLN51, full-length xeiet eete efre nodrt eemn the determine to order in poly(A) performed MLN51, of then colocalization labeling were fluorescence Triple experiments CFP–MLN51. overexpressing cells situ tesgaue.W on htpoly(A) that found We granules. stress ofrhrcaatrz MG,w nlzdwehrthese whether analyzed we Poly(A) SMIGs, RNA. contained characterize granules further granules To RNA mobile are SMIGs MG hwdrce oeet iha vrg pe of granule speed stress for required average are microtubules an that mobile, previously shown with when that, movements directed showed 3.7 directed showed this show and was SMIGs SMIGs, displacement SMIGs move, 119 squared on mean not the performed of did MLN51-containing material Calculation the that movements. supplementary to granules contrary 6A; that, (Fig. stress observed We minutes every 1). 5 picture Movie over one taking seconds these CFP–MLN51, 0.5 of imaging overexpressing dynamics live-cell cells time-lapse the performed on studied We granules. first of we types two this, address To granule. been actually have S3). markers Fig. both SMIGs material these for (supplementary in negative were mRNAs SMIGs However, cap-binding order Co- translated. whether in nuclear performed, 5B). the determine were (Fig. NCBP1) against to granules as and known SMIGs (also PABP against stress CBP80 in of protein directed form antibodies MLN51 in nuclear with the with SMIGs ATXN2 of only experiments and staining and MLN51 5A) (Fig. with colocalized hs n osblt a htaSI sapeusrt stress a to precursor a is SMIG a that was possibility one Thus, m yrdzto sn y-aee lg ()poe nHeLa in probes d(T) oligo Cy5-labeled using hybridization / vra vrg itneo 4 of distance average an over m/s ora fCl cec 21)17 6240 doi:10.1242/jcs.154500 4692–4701 127, (2014) Science Cell of Journal nioisaantDpa e,p54 Ge1, Dcp1a, against antibodies with immunodetection following counted were P-bodies markers. P-body various with observed is MLN51 of overexpression following disassembly P-body CFP–MLN51. of overexpression following (PB) P-body one least at having cells of percentage the showing Graph signal.(C) non-specific a represents asterisk tubulin.The and Dcp1a p54, GFP, against antibodies using blotting western by analyzed were (+). populations YFP Sorted for positive other the and niDpa.N,utasetdcells. untransfected NT, anti-Dcp1a). with immunodetection by determined (as CFP–MLN51 of following overexpression cell per P-bodies of the number of Distribution (D) hXrn1. and ouain,oengtv o F ( YFP for negative one populations, distinct two into sorting cell fluorescence-activated by separated were 3,4) (lanes YFP–MLN51 and 1,2) (lanes alone YFP with transfected cells of HeLa level Dcp1a. the endogenous on effect no has MLN51 20 bar: Scale red). panel, (central anti-Dcp1a with detected P-bodies no have panel, green) right and panel, (left MLN51 CFP– overexpressing cells HeLa (A) disassembly. P-body MLN51 induces of Overexpression 2. Fig. + + n TN,acmoetof component a ATXN2, and RA eedtce by detected were mRNAs m m .()Oeepeso of Overexpression (B) m. Fg B.I a been has It 6B). (Fig. m + RA actually mRNAs 2 in )

Journal of Cell Science EERHARTICLE RESEARCH iasml nMN1lclzto n yais t2 hours 24 At dynamics. and these microtubule directed localization of that MLN51 effects the show on assembly. hypothesize investigated disassembly granule we SMIGs hypothesis, stress to this subsequent test As tempting To in involved be 2003). are would movements al., it et movements, (Ivanov assembly acrclsta ihrddo i o vrxrs MLN51 overexpress not did or did either that [HER2 cells cancer iecl mgn aldt hwaysr fSI uina a as fusion Taken SMIG shown). of not (data – sort formation 2003) any granule stress al., show for et to prerequisite (Ivanov failed granules imaging been stress have live-cell that for results to previously SMIGs similar – described containing 46.2% cells to found of 77.8% from we number decreased the Although antimitotic treatment, assembly. an following microtubule nocodazole, that, alter with to hours known 2 CFP–MLN51 agent for full-length treated the were expressing protein cells transfection, after atro .3Pbde e elfloignocodazole following cell per P-bodies 8.23 – ( 3 al., treatment et of by Aizer P-bodies factor of 2007; number anti- al., a for the increases with et treated treatment (Sweet detected nocodazole were 2008), established were already P-bodies cells As and expression, Dcp1a. nocodazole of with CFP– hours hours with 2 24 transfected After were cells MLN51. HeLa dependent. are microtubule SMIGs stress the nascent than that rather notion entities granules. the cytoplasmic distinct reinforce certainly results our together, ihteat-c1 nioy()o 5 nioy() aaso the show Data (D). antibody p54 or (C) detected mean are antibody P-bodies anti-Dcp1a mutant. the MLN51HD with the SELOR or fusions MLN51 C-terminus CFP N-terminus, MLN51 of MLN51 domain, overexpression (MLN51), following MLN51 of P-body full-length percentage one the of least of Graphs at against (C,D) having antibodies antibodies. cells using eIF4A3 and blotting Dcp1a western p54, by GFP, analyzed YFP–MLN51HD and or N- 9,10) 7,8) YFP–MLN51 (lanes (lanes 3,4), C-terminus transfected YFP–MLN51 (lanes 5,6), were YFP–MLN51 (lanes cells 1,2), terminus HeLa (lanes Dcp1a. alone level and YFP the p54 on with endogenous effect no of has expression fragments anti-Dcp1a of MLN51 the of with Overexpression immunofluorescence (B) or antibody. by YFP either detected to were fused P-bodies (Nt), MLN51HD, CFP. or N-terminus (Ct) MLN51 C-terminus MLN51, MLN51 with SELOR, transfected MLN51 were the cells in HeLa function study. this MLN51 of independent is EJC. disassembly P-body 3. Fig. ubro -oisi omlbes el ptetP22 821) the [HER2 P12/28 (patient with cells cells this breast HER2-negative normal compared and in P-bodies We of respectively]. number 20416E), (patient HER2 of 50% In HER2 in observed is disassembly P-body 08.W nlzdtenme fPbde nHER2 in P-bodies of number the analyzed We 2008). (Bie amplicon Erbb2 ihaHR muoitceity(H)soeo 0 (supplementary of IHC score by by and assessed (IHC) was 7) MLN51 (Fig. immunohistochemistry of immunofluorescence Overexpression HER2 207861)]. (patient a with htoeepesMLN51 overexpress that oehr hs eut ugs htoeepeso fMLN51 microtubule-dependent a of in overexpression P-bodies Taken manner. of that cells). disassembly in suggest the untreated induces contacts results in formed after these 7% (27% together, Interestingly, versus markers cells cells. P-body nocodazole-treated and untransfected contacts in SMIG P-bodies increase of fourfold between having a that observed cells we of treatment, to nocodazole percentage observed similar the reduction and 3.5-fold is the cells, reduced with was untreated compared cell 1.5 in per of P-bodies factor a of by number average nocodazole Following the P-bodies. treatment, MLN51 of of number effect the the on reduces overexpression strikingly also It cells. untransfected enx atdt e hte h -oycutwas count P-body the whether see to wanted next We A ceai ersnaino h ifrn L5 osrcsue in used constructs MLN51 different the of representation Schematic (A) 6 s.d. + tegn noigHR)adteohrgnso the of genes other the and HER2) encoding gene (the ora fCl cec 21)17 6240 doi:10.1242/jcs.154500 4692–4701 127, (2014) Science Cell of Journal /MLN51 n 5 ` h ta. 96 eo ta. 02 ril tal., et Arriola 2002; al., et Degot 1996; al., et che 5 ess26 -oisprcl ( cell per P-bodies 2.66 versus 55) + + ratcancers, breast ptet241)adHER2 and 204817) (patient MLN51 2 /MLN51 sc-mlfe with co-amplified is + ratcne cells cancer breast 2 rattumor breast a , + /MLN51 n 5 + 3 in 53) breast 4695 c- 2

Journal of Cell Science omlbes el 20 -oisprcl nHER2 in of cell that per P-bodies with [2.08 compared cells as breast MLN51 normal overexpressing cells cancer EERHARTICLE RESEARCH 4696 poly(A) contain SMIGs 5. Fig. HER2 in P-bodies of number the with compared ( when cell per P-bodies [3.7 MLN51 HER2 in number ( ( cells For 7). (Fig. by antibodies detection HER2 anti-Dcp1a following counted using were immunofluorescence P-bodies S4). Fig. material ubro -oisprcl a erae nHER2 average in the decreased was cells, in cell HeLa Noticeably, per in P-bodies S4D). observation of cells; Fig. our number 1380 material of with (128 agreement supplementary MLN51 of 7D; overexpression Fig. of levels higher TN n poly(A) and ATXN2 h niAX2atbd.DAi tie ihDP bu)()SIscnanpoly(A) contain SMIGs (A) (blue) DAPI with stained is DNA antibody. anti-ATXN2 fluorescence the by detected was white) panels, n 5 29] twsas erae hncmae ihthe with compared when decreased also was It 1299)]. + n /MLN51 5 2)vru .3Pbde e eli omlcells normal in cell per P-bodies 3.53 versus 128) + + + RA.Saebr:10 bars: Scale mRNAs. ape,w oue nclsta showed that cells on focused we samples, ratcne el htddntoverexpress not did that cells cancer breast + RNA. n 5 F–L5 lf aes re)wstasetdi eacls t2 or olwn rnfcin poly(A) transfection, following hours 24 At cells. HeLa in transfected was green) panels, (left CFP–MLN51 1).Adfnly tdecreased it finally, And 910)]. nsitu in m m. yrdzto ihaC5lbldoiodT rb,srs rnls(eodpnl,rd eedtce with detected were red) panels, (second granules stress probe, d(T) oligo Cy5-labeled a with hybridization + /MLN51 + 2 breast cells + fMN1i eacells. overexpression HeLa after observed in is that MLN51 P-bodies to of of manner similar number a the in MLN51, reduced overexpress that cells cancer hnsxPbde a erae nteHER2 the in more decreased with was cells P-bodies of percentage six the than whereas cells), breast normal in 39 -oisprcl nHER2 in cell per P-bodies [3.99 ecnaeo el otiigls hnoePbd sstrongly is P-body one than HER2 less in containing increased cells of percentage ratcls(i.7) hs eut hwta,i HER2 in normal a that, in show show found results MLN51 that These 7E). overexpress to inset). (Fig. number not cells 7D, P-body breast do (Fig. of that distribution SMIGs similar cells resemble small Finally, cancer into that 19%). Breast localization with its granules in compared cytoplasmic resulted (2% MLN51 of sample overexpression normal the versus + RAbtn tesgauemres B tesgaue contain granules Stress (B) markers. granule stress no but mRNA ora fCl cec 21)17 6240 doi:10.1242/jcs.154500 4692–4701 127, (2014) Science Cell of Journal + /MLN51 mgso h ih.Saebr:10 bars: Scale right. the the in on indicated images areas the granule from show enlargements panels Insets central nuclei. and DAPI-stained left with Right of column). overlay right shows in column red PABP column, and (B) (central FMRP (C) (A), TIA1 against with antibodies immunodetected column). were right granules in Stress green column, (left CFP–MLN51 are that granules. SMIGs stress in not accumulates MLN51 4. Fig. + ape 4%cmae ih18% with compared (43% samples 2 /MLN el eetasetdwith transfected were Cells 2 + /MLN51 ( n 5 + 13] The 1103)]. m RA(fourth mRNA m. + + sample breast

Journal of Cell Science EERHARTICLE RESEARCH ute tde ilb eesr oeuiaetemolecular the functions. extra elucidate these EJC. to that perform the to suggests necessary them of This allow be that independently EJC. will mechanisms the mRNAs mutated studies to on a bind Further assemble to overexpressing can unable when MLN51 its is reproduced on that in overexpression be MLN51 involved MLN51 of one can effect report). the the P-bodies this that 2004), as show al., et we same ((Degot Moreover, in complex the involved core is the not that to recruitment region is MLN51 the function to Y14, has non-EJC with it as However, that, metabolism. factors noted mRNA its be in of changes roles some additional allowing conformational play thus an core, to following had several EJC the that, cells within translation, occur suggest HeLa might of studies in rounds both overexpression numbers, pioneer Y14 P-body that on see Although effect MLN51. not for This here did 2013). reported al., that we Y14 et to recently (Chuang opposite core, P-bodies is action EJC of al. Y14 number to the leads the overexpression in in et an its increase role and an of functions, its Chuang important hypothesis other to has the addition localization. also favors in 2013) that, of demonstrated al., mode et Chuang core independent (Chan 2004; other P-bodies is al., from with of only Y14) question et and absence is associated Magoh or The (eiF4A3, interesting components EJC P-bodies. is EJC An the EJC-associated MLN51 of in 2004). independently present of been that localizes al., has part MLN51 time et whether It first small (Degot P-bodies. the a of for polysomes component show that a we suggested is study, in MLN51 mainly this localizes endogenous In that component cytoplasm. EJC core the only the is MLN51 DISCUSSION asteris and foci by the separated of is position the panel indicate Each arrowheads figure. insets, this the for In ( selected image. time main were of the cell in function one box of a black Photographs the video. by at indicated time-lapse positions interest using their of by region minutes a 5 show insets for The observed were cells movements. hours, directed 24 show SMIGs 6. Fig. t 5 6 .Saebr 10 bar: Scale 0. ...Teuboe ierpeet h ensurddslcmn o atce negigBona movements. Brownian undergoing particles for displacement squared mean the represents line unbroken The s.d.). m .()Ma qae ipaeetcluainfrSIs(os.Tegahrpeet h ensurddslcmn as displacement squared mean the represents graph The (dots). SMIGs for calculation displacement squared Mean (B) m. A L5 hw ietdmvmn ntectpam eaclswr rnfce ihCPMN1 After CFP–MLN51. with transfected were cells HeLa cytoplasm. the in movement directed shows MLN51 (A) ta. 01.Fnly thsbe eetysonta in that shown recently been has it (Carbonaro Finally, repression P- miRNA 2011). the translational in of al., to member accumulates a leading et for tumors, Ago2, recent thus with solid required a associates pathway, in then Furthermore, is It HIF-1 overexpressed 2005). bodies. treatment, mRNA is al., nocodazole after et which that Andrei that Ferraiuolo 2004; showed reported al., study 2005; et al., 2008). been (Cougot Shav-Tal, active et maintenance and the also P-body a (Aizer on it cytoplasmic P-body relying has reach than a to rather in P-body It way a localize own Staufen1 of to its movement in that find 2003). the must shown al., located target in et have (Macchi also microtubules studies neurons is Dynamics along hippocampal mouse mRNAs Barentsz of dendrites transport 2001). which al., for particles, et essential shown has is Eeden RNA- of screening third homolog of barentsz genetic Functional human type that the barentsz. the mouse new under is favor and now a fly MLN51 are SMIGs observations are Interestingly, of these investigation. components These Other in that granule. Ge1 SMIGs. suggest containing differ p54, Dcp1a, and in granules as hypothesis (such stress Xrn1) marker P-body and or common could a we SMIGs granule. detect immunofluorescence, of not Using that type kinetics. and new show composition entirely granule we an stress or be Here, might P-bodies SMIGs small movements. precursors, directed show and nue rnls SIs.SIscnanpoly(A) contain SMIGs uncharacterized MLN51- ‘small (SMIGs). currently named granules’ and have we induced new that granules in cytoplasmic small assembles mainly also hnoeepesd L5 oaie nsrs rnls but granules, stress in localizes MLN51 overexpressed, When ora fCl cec 21)17 6240 doi:10.1242/jcs.154500 4692–4701 127, (2014) Science Cell of Journal oskar RAlclzto (van localization mRNA a + sindicate ks mRNA, mRNA 4697 . s. 0.5

Journal of Cell Science EERHARTICLE RESEARCH 4698 ora fCl cec 21)17 6240 doi:10.1242/jcs.154500 4692–4701 127, (2014) Science Cell of Journal W)bes el ptetP22 821), HER2 P12/28 (B) (patient cells breast wild-type (WT) (A) in anti-MLN51 using immunofluorescence MLN51. 1 n 2 el,rsetvl.Dt show Data mean respectively. the 1103, cells, 1299, 128 on and were counted 910 fields were random P-bodies ten and each, captured For (white). cells breast i.7 h ubro -oisi eue in reduced is P-bodies of HER2 number The 7. Fig. ptet246)ad()HER2 (D) and 20416E) (patient 081,()HER2 (C) 207861), tie ihDP bu) cl as 20 bars: are Scale nuclei (blue). where DAPI overlays with as stained Dcp1a well and as left) right), Insets (upper (upper images. MLN51 main of the detection in show black in the represent outlined insets regions The 204817). (patient cells cancer MLN51 MLN51 E itiuino h ubro -ois(B e cell per (PB) P-bodies HER2 of in number the of Distribution (E) + 2 2 ratcne el htoverexpress that cells cancer breast + eeto fedgnu L5 by MLN51 endogenous of Detection ratcne el lgtga)o normal or gray) (light cells HER2 cancer gray), breast (dark cells cancer breast /MLN51 6 2 /MLN51 s.d. + ratcne el bak,HER2 (black), cells cancer breast 2 + /MLN51 ratcne el (patient cells cancer breast 2 ratcne cells cancer breast + /MLN51 + m breast m 2 / + /

Journal of Cell Science rgoi Kapre l,20;Dgte l,20) spr of patient part As poor 2002). al., and et Degot overproduction 2000; chemo-resistance al., its et tumor because (Klapper pathogenesis, prognosis with in role correlates major is a the HER2 to play Importantly, belonging family. kinase in receptor tyrosine overexpressed factor a growth HER2, protein epidermal the for codes h E2o rb mlcn L5 sc-vrxrse in co-overexpressed is MLN51 amplicon, Erbb2 , or HER2 the netgtn -oysau ncne el tvrossae of currently stages various are at We cells P- malignancy. cancer is bodies, in cell nuclear tumorigenesis. It status for mirror P-bodies. P-body case could the investigating of is status component as that, body identified speculate newly to tempting a overexpression following MLN51, disassembly of P-body discuss we paper, ta. 08.Smlrt hti bevdi eaclsafter also cells are HeLa P-bodies HER2 in that MLN51-overexpressing observed show in is we what disassembled here, to overexpression, Similar MLN51 2008). al., et trciemdlwudb htoeepeso fMN1in MLN51 of One overexpression 2007). that al., be processing et HER2 would miRNA (Kumar model the tumorigenesis impairing attractive cells and promotes tumor 2006), in pathway impaired al., is et subset miRNA (Thomson an of Maturation cells. aesm erse RA u fPbde,adta this that and P-bodies, to of able out is P- mRNAs MLN51 MLN51 repressed overexpressed, mammalian by when some of that, attractive induced take one be miRNA-mediated disassembly Thus, would al., SMIGs manner. model the microtubule-dependent et that in a to in (Fritzsche show bodies lead granules involved we translational overexpression RNA Here, mRNAs, factors in 2013). with repression and associates translational barentsz regulators brains, rat ARTICLE RESEARCH h ogamo hoooe1,argo locnann h c- the containing also region a 17, chromosome Arriola Erbb2 of 2002; arm al., long et the Degot better 1995; 2008). al., al., to et et (Tomasetto us cells cancer help more will A SMIGs function. preventing transcripts. these their of understand P-bodies, SMIG-associated of characterization in Nocodazole the SMIGs complete out the manner. of trap RNA overexpression microtubule-dependent thus model, would P-body a cytoplasmic a treatment induce certain such in that titrating In for granules structures of by network. transport factor out RNA microtubule disassembly recycling transport be the would their a via SMIGs in as P-body involved the act being mRNAs, also therefore, repressed function, would EJC its MLN51 Besides disassembly. P-body causes ugsigta h rsneo bec fncerbodies nuclear of The absence thus (de reassembly, or malignancy body cell presence PML–RAR nuclear Interestingly, with the induces correlates to the acid that leukemogenesis. leads of retinoic suggesting also because to or but cancer arsenic degradation contributes to with link treatment and a have bodies PML–RAR to the that thought observation changes. cancer-associated are show to They found been has that organelle following P-bodies from released MLN51. which and of identify in mRNAs to overexpression is trapped be miRNA-repressed will be effect then can this step of mRNAs first whether The subclasses P-bodies. or in certain localized cells, repression remaining cancer translation to in miRNA restricted A perturbed general is the repression. pathway whether translational is question miRNA-mediated of 0 fHER2 of 50% neetnl,MN1i vrxrse nmlgatbreast malignant in overexpressed is MLN51 Interestingly, M ula oisaea xml fanon-membranous a of example an are bodies nuclear PML + ee(oaet ta. 95.Tec- The 1995). al., et (Tomasetto gene ratcne el ed odrglto ftemechanism the of deregulation to leads cells cancer breast MLN51 + , ratcne el Dgte l,20;Arriola 2002; al., et (Degot cells cancer breast 0 fhmnbes acr,weei must it where cancers, breast human of 30% a enmpe oteq1q13rgo on region q11–q21.3 the to mapped been has a uinpoendsut nuclear disrupts protein fusion ´ ta. 02.I this In 2012). al., et Erbb2 + ratcancer breast oncogene a npstvl hre lds hndida 58 at dried then slides, charged positively on 5mntsa omtmeaue AIsann a efre in performed for was staining Invitrogen) DAPI from temperature. room (both at anti-mouse-IgG using minutes Alexa-Fluor- 45 performed goat the was and Detection 488-conjugated anti-rabbit-IgG anti-Dcp1a. goat mouse Alexa-Fluor-555-conjugated 1:50 and MLN51 ujce osadr muoloecne one n nlzdas analyzed and mounted immunofluorescence, above. described standard to subjected 2 with washes g nioiswr ucae rmJcsnImmunoResearch Jackson from purchased PA). were Grove, (West (Montluc anti-rabbit- Laboratories antibodies or INTERCHIM anti-mouse-IgG goat from TX) IgG FITC-conjugated (Dallas, were and Biotechnology Anti- Cy3- anti-ATXN2 Cruz 2012). France). Santa al., anti-DDX6, from et were Daguenet and anti-Ge1 2004; and al., and et TIA1 Taiwan) were (Degot City, antibodies previously -eIF4A3 (Taipei described and Abnova Anti-MLN51 respectively. from Zealand Sigma-Aldrich, were New antibodies Bertrand into by anti-tubulin supplied injected kindly was and antibody Se protein ovalbumin anti-Dcp1a MLN51 rabbit to human The synthetic rabbits. the coupled the of was 1–283 antibody, sequence residues polyclonal to rabbit corresponding anti-MLN51 peptide the generate To Antibodies 1 and (FCS) serum calf fetal Paisley, 10% (Invitrogen, GlutaMAX containing DMEM in UK) maintained were cells HeLa transfections and culture Cell METHODS AND MATERIALS uoae H tiigsse Vnaa usn Z.Following 75 at AZ). solution Tucson, Prep XT EZ (Ventana, Discovery Ventana system the with staining deparaffination on IHC performed Automated was staining 4- Immunohistochemical into cut was tissue Paraffin-embedded HER2 on Immunofluorescence upright Z1 AxioImager Poly(A) an Fluorescence with Germany). Oberkochen, (Zeiss, made software Axiovision were using microscope Observations Burlingame, Laboratories, Medium (Vector CA). 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Ebel FACSVantage C. a using by was on provided Purity was performed Biosciences). transfected (BD was DiVa sorting option were Cell 30 with transfection. treated after MO), DNA Louis, were St cells (Sigma-Aldrich, manufacturer’s the nocodazole plasmid the with following treatment For (Invitrogen) of protocol. Reagent Plus six-well ng Lipofectamine in 100–200 transfection transient plates, For Technologies). (Life streptomycin ´ ahn(NSIBC lkrh rne.Temueat-c1 and anti-Dcp1a mouse The France). Illkirch, (CNRS/IGBMC, raphin + ora fCl cec 21)17 6240 doi:10.1242/jcs.154500 4692–4701 127, (2014) Science Cell of Journal Nswr eetdby detected were RNAs 6 6 nsitu in ˚ o 0mntswt :0dlto frbi anti- rabbit of dilution 1:50 a with minutes 60 for C S otiig3%fraie h oesiswere coverslips the formamide, 30% containing SSC B,clswr nuae o orwt the with hour 1 for incubated were cells PBS, 6 hybridization B,clswr nuae o ora room at hour 1 for incubated were cells PBS, 6 B o 0mnts hnstrtdfor saturated then minutes, 10 for PBS m 6 ooaoefr2husa 4hours 24 at hours 2 for nocodazole M 6 + S otiig5%fraie and formamide, 50% containing SSC n omlbes cells breast normal and nsitu in B,clswr emaiie with permeabilized were cells PBS, ˚ o 8mntsuigC1 a CC1, using minutes 48 for C m yrdzto (Bu hybridization . -hc etos mounted sections, m-thick 8.Hl o elsorting cell for Help 98%. ˚ o or.Atrthree After hours. 3 for C 6 ˚ ih7%ethanol. 70% with C B n ie for fixed and PBS ˚ o 0minutes. 60 for C ˚ o minutes, 8 for C 6 6 he tal., et ¨hler B.After PBS. penicillin- 4699 ¸on, 6

Journal of Cell Science h ape eete ahdoc ihaeoeadtretmswith times three and acetone at with once ethanol washed 100% then were samples The oirlH2 sflos or t2% or t50%, at Finally, hours 2 100%. at 25%, times) (four at of hour at 1 hours proportion out 2 carried finally, was volume-to-volume and, follows: polymerization 75% the at as raising overnight HM20 by Lowicryl mixtures ethanol npstvl hre lds hndida 58 at dried then slides, 4- charged into positively cut on was tissue Ni-E Paraffin-embedded Nikon a Immunohistochemistry on software. 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Sunnyvale, ˚ e or n ape eekp tti eprtr o 4hours. 24 for temperature this at kept were samples and hour, per C + )wsue stescnayatbd Vco) o HER2 For (Vector). antibody secondary the as used was L) 2 O 2 Vnaa o iue t37 at minutes 4 for (Ventana) m rpo h apewslae noapehae,0.5- pre-heated, a onto loaded was sample the of drop l 2 50 ˚ .Smlswr nitae ihresin/100% with infiltrated were Samples C. 2 90 2 ˚ usiuinslto.Clswere Cells solution. substitution C 50 ˚ o 0mntswt 1:1000 a with minutes 20 for C ˚ o 8husada 20 at and hours 48 for C ˚ o 0mntswt 1:100 a with minutes 60 for C ˚ m .FrMN1sann,the staining, MLN51 For C. -hc etos mounted sections, m-thick 6 ˚ A14 objective) 1.45 NA o 0minutes. 60 for C Z 2 ˚ sak eeused were -stacks o 8minutes 48 for C 50 ˚ tart of rate a at C 2 ˚ ˚ Cfor for C 90 ˚ Z C - eeto sn os niDparvae ihga nimueIgG anti-mouse goat particles. with gold revealed 15-nm second to a anti-Dcp1a revealed and conjugated anti-MLN51 mouse particles, gold rabbit using 6-nm using to detection conjugated detection IgG first immuno- anti-rabbit a goat single with row: two a repeating in of detections consisted immuno-detections Double uhrcontributions Author interests. competing no declare authors The interests Competing de (Centre Dugast Mireille and Godey Florence Sante Turlin, Biologiques Bruno Ressources to grateful are We Acknowledgements non- of samples two HER2 included study three and The patients cells, ten above. breast of described up malignant as made analyzed was were cohort samples patient Human samples patient Human alt . acair . aut . oaet,C,Se C., Tomasetto, A., Baguet, B., Marchadier, L., Ballut, R. Parker, and V. Balagopal, C., Wendling, P., M. Chenard, E., Bertrand, N., Cougot, S., Degot, A., Baguet, R., Natrajan, B., M. Lambros, C., S. Drury, S., D. Tan, C., Marchio, R. E., Rivera-Pomar, Arriola, T., Achsel, R., Heintzmann, D., Ingelfinger, A., M. Andrei, N. Kedersha, Y. and Shav-Tal, P. and Anderson, H. R. Singer, N., Sonenberg, W., L. Ler, Y., Brody, A., Aizer, Y. Shav-Tal, and A. Aizer, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.154500/-/DC1 online available material Supplementary material Supplementary (CCIR-GE). pour Fondation Cancer the Le from Contre fellowship a Me had the Recherche E.D. and la Cancer. ANR- France; le numbers de Contre [grant Universitaire Nationale Agency Institut Ligue National the French ANR-09-MIE]; the and by 08JCJC-0027-01 supported was work This and Funding design the to contributed authors the experiments. and All of advice authors. interpretation with other manuscript the the wrote from N.C. R.G. comments data. process; analyzed and revision project the the during directed experiments W.B. performed and experiments, performed immunofluorescence conceived and C.T. experiments analysis; conceived data and E.B. designed videomicroscopy F.G. cohort; samples; study patient the human selected with P.B. experiments microscopy; performed freeze immunoelectron A.F. and and with freezing experiments pressure performed high D.T. with substitution; revision experiments the performed during A.C. and process; before immunofluorescence, the A.B. with wrote immunofluorescence; experiments and with performed data experiments analyzed performed experiments; E.D. the manuscript. performed and conceived N.C. (De ape.Ti td a prvdb h ntttoa orso the of boards institutional the by Sante Biologiques approved Ressources was de Centre study This samples. lcrnmcocp xeiet eepoesda h irsoyRennes (http://microscopie.univ-rennes1.fr/). Microscopy facilities the (MRic) at Center manuscript. processed Imaging the were on experiments comments insightful microscopy for Electron Berland Juliana to and antibody present Dcp1a the Se of Bertrand part thank Mole clinical to Biologie the like with would help We invaluable investigation. for France) Rennes, de ttsadftso uaytcmRNAs. eukaryotic of fates stress- and C. in states Tomasetto, functions and 51 C. M. node assembly. Rio, lymph granule H., metastatic Hir, junction-complex-component Le breast P., and Kessler, cancer breast in al. amplicon et lines. HER2/TOP2A K. cell the Fenwick, cancer N., of Tamber, analysis A., Genomic Mackay, M., S. Rodriguez-Pinilla, bodies. processing mammalian to mRNPs and Lu and assembly, transport, body P 808. mammalian of vivo. in dynamics disassembly The (2008). bodies. I4IIAPs activity. ATPase eIF4AIII H. preetd Gyne de ´partement 20) h xnjnto oecmlxi okdot N yihbto of inhibition by RNA onto locked is complex core junction exon The (2005). ran R. hrmann, ¨ ora fCl cec 21)17 6240 doi:10.1242/jcs.154500 4692–4701 127, (2014) Science Cell of Journal Prion clieCluar,Srsor,Fac)frpoiigu ihteanti- the with us providing for France) Strasbourg, Cellulaire, ćulaire dcl FM.Fnst ..wr rmTeLgeNationale Ligue The from were C.T. to Funds (FRM). ´dicale 2 131-134. , a.Invest. Lab. .Cl Sci. Cell J. clgee d’Obste et ćologie 20) oefreFEadeFEtasotri targeting in eIF4E-transporter and eIF4E for role A (2005). o.Bo.Cell Biol. Mol. a.Src.Ml Biol. Mol. Struct. Nat. 20) nrclua rfikn n yaiso P of dynamics and trafficking Intracellular (2008). eRne,Fac)adJa Leve Jean and France) Rennes, de ´ 20) oyoe,Pbde n tesgranules: stress and bodies P Polysomes, (2009). 88 120 20) N granules. RNA (2006). 491-503. , 2774-2784. , 19 tiu,Cnr optle Universitaire Hospitalier Centre ´trique, 4154-4166. , 2 ur pn elBiol. Cell Opin. Curr. ´ n ieHER2 five and RNA eRennes. de rpi Isiu Ge (Institut ´raphin 12 11 861-869. , 717-727. , rpi,B n eHir, Le and B. ´raphin, .Cl Biol. Cell J. 20) h exon- The (2007). + 21 ratcancer breast 403-408. , ˆque ńe ´tique 172 (2008). 803- ,

Journal of Cell Science hn .C,Dsi,J,De,M . eg . an . apibr .and J. Rappsilber, M., Mann, W., Feng, D., M. Diem, J., Dostie, C., C. Chan, P. Giannakakou, and A. O’Brate, M., Carbonaro, Bu Bie ARTICLE RESEARCH ogt . oz,A . idc,E,Cvle,A,Toa,D n ilt R. Gillet, and D. Thomas, A., Cavalier, E., Giudice, E., A. Molza, N., Cougot, R. Gillet, and D. Thomas, A., Cavalier, N., Cougot, Se Y. and W. S. Tarn, Babajko, and N., Cougot, M. K. Lee, L., W. Chang, W., T. Chuang, Sargueil, C., Tomasetto, N., Ulryck, C., Wendling, E., Daguenet, E., P. Chazal, rtsh,R,Kra . ent,K . n,F . eadFro,J . Tolino, E., J. Heraud-Farlow, Y., F. Ang, L., K. Bennett, and D., Karra, R. R., D. Fritzsche, Schoenberg, L., E. Murray, J., Dostie, S., Basak, A., M. and Ferraiuolo, K. Griffith, D., J. Keene, A., S. Tenenbaum, K., E. Chan, T., Eystathioy, eo,S,Re S., Degot, C., Wendling, The F., de Alpy, U., Schmidt, S., Degot, A., Baguet, E., Daguenet, R. Parker, and R. J. Buchan, uai,A,Bh-nmn,I,Shezr .adIarad,E. 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Journal of Cell Science J. Cell Science Research Article Running Title: MLN51 and P-bodies disassembly

SUPPLEMENTARY FIGURES

Supplementary Figure 1: Overexpression of other EJC core components do not lead to disassembly of PB. Graph of the percentage of cells having tat one leas P-body following overexpression of CFP fusion of eIF4A3 or Y14. Each experiment corresponds to at least 40 cells captured on 11 different fields. B and C: Distribution of the number of PBs per cell following overexpression of CFP-eIF4A3 (B) or CFP-Y14 (C). NT, untransfected cells. Data represent the mean of 3 independent experiments, the error bar representing standard deviation.

Journal of Cell Science | Supplementary Material J. Cell Science Research Article Running Title: MLN51 and P-bodies disassembly

Supplementary Figure 2: The large cytoplasmic granules observed upon expression of the CFP-MLN51 C- terminal fragment (CFP-MLN51Ct) are stress granules. Cells were transfected with CFP-MLN51Ct (left panels, green in right panels). SGs (central panels, red in right panels) were then immuno-detected with the following antibodies: anti-TIA1 (A), anti-FMRP (B), or anti-PABP (C). Right panels show overlays with DAPI-stained nuclei (blue). Scale bar: 10µm. Insets show granule enlargements.

Journal of Cell Science | Supplementary Material J. Cell Science Research Article Running Title: MLN51 and P-bodies disassembly

SMIG

Supplementary Figure 3: co-staining experiments of MLN51 with the nuclear cap-binding protein CBP80 and with the nuclear form of PABP. CFP-MLN51 (left panels, green on right panels) was transfected in HeLa cells. 24 hours following transfection, poly(A)+ mRNA (fourth panels, white on right panels) were detected by fluorescence in situ hybridization with a Cy3-labelled oligod(T) probe. CBP80 or PABPN1 (second panels, red on right panels) were detected with anti-CBP80 (top) or anti-PABPN1 (bottom) antibodies, respectively. (Top) SG contain poly(A)+ mRNA and CBP80; SG contain PABPN1 and poly(A)+ mRNAs. SMIG contain poly(A)+ mRNAs but not CBP80. (Bottom) SMIG contain poly(A)+ mRNA but no PABPN1. Scale bar: 10µm.

Journal of Cell Science | Supplementary Material J. Cell Science Research Article Running Title: MLN51 and P-bodies disassembly

Supplementary Figure 4: Immunohistochemical analysis of MLN51 subcellular localization in breast cells.(A) breast cells (patient P12/28 821); (B) HER2-negative breast cancer cells (patient 207861); (C) HER2- positive cancer cells not overexpressing MLN51 (patient 20416E); and (D) HER2-positive cancer cells overexpressing MLN51 (patient 204817).

Journal of Cell Science | Supplementary Material Movie 1. CFP-MLN51 foci in U2OS transfected cells. Duration: 5 minutes. Interval: 1 second.

Journal of Cell Science | Supplementary Material