A role for protein phosphatase PP1γ in SMN complex formation and subnuclear localization to Cajal bodies Benoît Renvoisé, Gwendoline Quérol, Eloi Rémi Verrier, Philippe Burlet, Suzie Lefebvre

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Benoît Renvoisé, Gwendoline Quérol, Eloi Rémi Verrier, Philippe Burlet, Suzie Lefebvre. A role for protein phosphatase PP1γ in SMN complex formation and subnuclear localization to Cajal bodies. Journal of Cell Science, Company of Biologists, 2012, 125 (12), pp.2862-2874. ￿10.1242/jcs.096255￿. ￿hal-00776457￿

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Journal of Cell Science iesoa rti e lcrpoei eeldta M shprhshrltdi ula xrcso PP1 of extracts nuclear in PP1 hyperphosphorylated of is extracts SMN nuclear and that cytoplasmic revealed in electrophoresis increases Gemin8 gel and protein SMN dimensional between interaction the Consequently, o:10.1242/jcs.096255 doi: 2862–2874 125, Science Cell of Journal 2012 February 14 Accepted auainadpoesn tp eutn ntepouto of production the in additional for resulting Following CBs steps to snRNPs. processing targeted core and are into maturation snRNPs proteins core snRNAs import, core the nuclear Sm assembles cytoplasm, the complex common In (SMN) with 2010). neuron the al., motor with et survival associate (Suzuki to complex nuclear targeting export small CB nuclear a and of precursor transcription (snRNA) the RNA with starts Synthesis andphases. al., factors et Matera processing 2000; Gall, 2005; mRNA 2007). Lamond, and histone (Cioce telomerase polymerases, (snoRNPs), RNPs nucleolar RNA small spliceosomal (snRNPs), RNPs as al., nuclear such in et small enriched (RNPs), (Lafarga ribonucleoproteins domains neurons subnuclear different dynamic and many highly cells are cancer They in 2009). prominent 2010). al., whereas are et mice, (Strzelecka and CBs embryos fly zebrafish fruit developing in in essential are not they are CBs distinct and suggest coilin studies various that and evolution, into in conserved (Ras been coilin has marker Coilin organized protein CBs one the four is by to identified two (CB) are contain is body that cells Most Cajal 2008). the (Morris, entities nucleus and these of 2011) al., cell et (Mao compartments mammalian The Introduction words: Key PP1 of role interact Gemin8 PP1 component and its PP1 and of complex expression SMN the that PP1 here show of we depletion is assays, complex Moreover, SMN binding the CBs. protein nuclear of to vitro localization PP1 small co-immunoprecipitation Subcellular phosphatase in Using spliceosomal protein cells. understood. and with SMA poorly of directly extracts in remain assembly of deficiency mechanisms cell concentration precise SMN the HeLa the The upon and (CBs). in for reduced manner bodies as is required phosphorylation-dependent Cajal known CBs a domain is (also in in subnuclear regulated snRNPs unrip which to and and localization complex, complex (Gemin2–8) their SMN 2–8 (SMN) and the import protein neuron nuclear gem-associated their motor with (snRNPs), forms survival ribonucleoproteins SMN ubiquitous product the gene (SMA) STRAP) atrophy muscular spinal The Summary " work § this to equally ` contributed authors *These 2 bodies 1 Cajal to Renvoise´Benoıˆt localization subnuclear PP1 and phosphatase formation protein for role A 2862 PP1 ieo,Sron ai ie,1 u He rue Cite´, 15 Paris Sorbonne Diderot, rsn drs:Clua erlg nt ergntc rnh ainlIsiueo erlgclDsresadSrk,Ntoa ntttso elh ehsa D282 USA 20892, MD Bethesda, Health, of Institutes National Stroke, and Disorders Neurological of Institute National Branch, Neurogenetics Unit, Neurology Cellular address: Present rsn drs:Isiu ainld aRceceArnmqe aoaor eGe de Laboratoire Agronomique, Recherche la de National Institut address: Present uhrfrcrepnec ( correspondence for Author aoaor eBooi elliedsMmrns rgam eBooi ellie ntttJcusMnd M 52CR,Universite´ Paris CNRS, 7592 UMR Jacques-Monod, Institut Ge Cellulaire, de Biologie Service de Programme Membranes, des Cellulaire Biologie de Laboratoire 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. h ignsso nNsivle yolsi n nuclear and cytoplasmic involves snRNPs of biogenesis The c satre o hrpui nevnini SMA. in intervention therapeutic for target a as c ntearpi kltlmsls ugsigta h ucino PP1 of function the that suggesting muscles, skeletal atrophic the in c pnlmsua toh,SNcmlx aa ois piesmlsRP,PoenpopaaePP1 phosphatase Protein snRNPs, Spliceosomal bodies, Cajal complex, SMN atrophy, muscular Spinal ´ne ntefraino h M ope n h aneac fC nert.Fnly epooeGmn neato with interaction Gemin8 propose we Finally, integrity. CB of maintenance the and complex SMN the of formation the in tqeM´iae optlNce-nat aae,773Prscdx1,France 15, cedex Paris 75743 Malades, Me´dicale, Hoˆpital Necker-Enfants ´tique c etrsteeioom.Ntby M eiinyi M ed oteaern uclua oaiaino Gemin8 of localization subcellular aberrant the to leads SMA in deficiency SMN Notably, isoforms. these restores 1, [email protected] * ,` wnoieQue´rol Gwendoline , ´le c n ro,725Prscdx1,France 13, cedex Paris 75205 Brion, `ne c vrxrsino ei8i el nrae h ubro B n eut ntreigo PP1 of targeting in results and CBs of number the increases cells in Gemin8 of Overexpression . yRAitreec nacstelclzto fteSNcmlxadsRP oCBs. to snRNPs and complex SMN the of localization the enhances interference RNA by ˇ ae l,1991). al., et ka 1, ) ,Eo em Verrier Re´mi Eloi *, ´ne tqeAiaee ilgeItgaie oan eVlet 85 oye-oa,France Jouy-en-Josas, 78350 Vilvert, de Domaine Integrative, Biologie et Animale ´tique bnac fCs(rge ta. 00 eevee l,1997; al., et Lefebvre 2000; al., et (Frugier Renvoise Beattie, CBs and and SMN This of of levels abundance (Burghes (MNs). residual with neurons correlates elusive severity Disease motor 2009). remains spinal spinal vulnerability of in selective degeneration 1995). role genetic to al., human central leading et the (Lefebvre common a of infancy most Alterations plays of the disorder protein (SMA), neurodegenerative SMN atrophy Carvahlo 1999). 1996; nucleus,muscular Dreyfuss, and the al., (Liu and CBs et cytoplasm in concentrates STRAP) the as it to where known protein; localizes (also 2007), receptor-associated unrip (Pellizzoni, and kinase (Gemin2–8) serine-threonine 2–8 protein also associated 1998). could al., complex et SMN (Pellizzoni The processes 2008). Klingauf these al., 2004; in et al., operate Stanek et 2006; and (Nesic al., assembly splicing et increase after in to snRNPs storage reported of for been recycling or also nucleoplasm have the CBs in speckles. splicing for snRNPs mature htsRPpouto scuili M ies Wnlre al., et (Winkler disease SMA that in show crucial is models indicate production and animal snRNP defects, in that developmental rescue Studies DiDonato, could 2008). snRNPs and al., mature (Schmid et splicing Zhang in 2007; thus, and biogenesis snRNP h bqiosSNcmlxta nldsSN gem- SMN, includes that complex SMN ubiquitous The 1,§ c ´ hlpeBurlet Philippe , slkl ob fetdi ies.Orfnig eela reveal findings Our disease. in affected be to likely is ta. 2009). al., et c SMN1 nSNcomplex SMN in SMN1 eecueSNpoendeficiency protein SMN cause gene 2 n ui Lefebvre Suzie and sacnevdgn seta in essential gene conserved a is c dpee el.Two- cells. -depleted eerhArticle Research c dpee el and cells -depleted 1, " c Journal of Cell Science oi Miebc ta. 06 n h oaiainptenof pattern localization the ‘R/KVxF’ and targeting PP1-binding 2006) Most regulatory consensus al., 2010). et 180 primary al., (Meiselbach et a approximately motif (Bollen harbor of growing and proteins still list Bollen is 2002; The substrate (Cohen, subunits and subunits 2002). catalytic localization Beullens, targeting) the subcellular (or of regulatory the specificity a determines with association subunit The gene. different oln 04 orede l,20) nmmas hr are there mammals, and In ( (Ceulemans 2007). subunits survival, catalytic al., contraction PP1 et cell three muscle Moorhead 2004; splicing, and Bollen, plasticity pre-mRNA synaptic transcription, of PP1 including number How CB unknown. the 2008). Moreover, is al., increase localization temporal et 2003). SMN to (Novoyatleva regulate the shown al., fibroblasts could been SMA enhances et in has gems PP1 (Carnegie and of 1997; snRNPs complex inhibition al., associates of PP4 et SMN phosphatase (Lyon localization nuclear the snRNPs The of 1998). with the and al., affect et coilin coilin Sleeman of in localization mutation phosphoserine CB a as 2007). inhibiting and al., phosphorylation: reported et implicated phosphatases Petri have been CBs 2009; to on al., and has studies et coilin Other (Hearst CBs, and CBs in SMN SMN in accumulation of their present dephosphorylation and the not to phosphatase contributing is 1993) protein nuclear which The that 2002). al., PPM1G, al., coilin CBs et (Hebert hypophosphorylated et of CBs 2004). the forms with lack al., (Carmo-Fonseca interacts with et preferentially mitosis coincides (Grimmler and coilin cytoplasm during 2009) the of al., et in Hyperphosphorylation SMN (Burnett complex of assembly the phosphorylation snRNP of The stability the phosphoproteins. SMN regulates 2010). are (Hebert, 2002; coilin phosphorylation protein–protein al., and et and 2009) Hebert in al., 2002; et al., et Clelland changes (Boisvert such methylation modifications, protein substantial These as post-translational 2010). by fully in al., influenced et interactions not result (Boulon number far and processes size so composition, are CB CBs in mechanisms accumulation understood. the at However, the present 2008). is al., assembly controlling SMN et non-linear when (Kaiser a levels components in as sufficient snRNP most reconstituted formation by been when CB triggered storage has novo process be SMN form de to of showing thought gems reaction cells, are is tethering and gems SMN The Thus, that sites. al., formation, disturbed. indicate et is CB data (Lemm metabolism These gems for 2008). whereas forms al., required gems, and et and Gems Whittom CBs CBs 1996). 2006; disrupts disrupts Dreyfuss, depletion and SMN coilin and coilin lack of and a (Liu complex Depletion SMN designated CBs snRNPs. the been body of of components nuclear also the of CB- Gemini contain has type small SMN for different of 2010). a ‘gem’ recruitment al., form CB to et in reported (Mahmoudi involved RNAs and RNA factor specific body encoding a 2004) Cajal SMN WD40 telomerase 1), as al., with known the protein also interaction et (WRAP53; of its p53 to (Narayanan on import antisense CBs snRNPs nuclear Nuclear to precise on unknown. targeting The depends is 2009). al., SMN complex et of (Turner nucleus function the in SMN 2011). al., et Hubers 2007; Bassell, disease and to (Rossoll contribute severity might functions SMN other However, 2005). P oonye euaeawd ag fclua functions, cellular of range wide a regulate holoenzymes PP1 on impact pathways signaling or stimuli stress, of forms Many of deficiency a with correlates diseases MN of severity The a , b /d and c ,ec noe ya by encoded each ), Results CBs. to localization and PP1 formation that complex conclusion SMN the regulate support results Our organization. P oonyecmlxs l he oaiet h cytoplasm PP1 the interphase. to during localize nucleus different three and many All of complexes. addition holoenzyme the PP1 from results subunit catalytic each muoltaaye eeldta PP1 that GFP–472D revealed 1D). (Fig. with analyses experiments immunoprecipitated co-immunoprecipitation Immunoblot PP1 of performed association the GFP–472D test To PP1c of Co-immunoprecipitation cells mammalian in localization subnuclear gain to its order (Renvoise in into mutants deletion insight SMN SMN of produced region previously N-terminal We the of study Proteomics h xrsinlvl fPP1 of levels expression PP1- a as the complex, PP1 SMN targeting the protein binding of component a Gemin8, identified kltlmsl eas ftevr pcfccytoplasmic PP1 with interaction specific new a very on focused the and with proteins co-immunopurified proteins of SMN the examined because We organization. the muscle chose we individuals skeletal SMA and control involving experiments immunofluorescence vivo and in interference For approaches. RNA immunofluorescence proteomic, biochemical, using determined rnltoa euaino h M ope yPP1 by complex SMN the of be. regulation could sites translational subcellular the specific (Moorhead transient different CBs and to dynamic in PP1 how occasionally of illustrate found targeting data These be 2007). can al., (Landsverk and et nucleoplasm 10 2005) the al., to subunit localizes et that regulatory p99] and 1 (PP1R10) as known phosphatase [also PNUTS serine/threonine-protein subunit targeting nuclear phosphatase the TikeMlaye l,20;TikeMlaye l,2003). PP1 of al., subunits targeting et nuclear major Trinkle-Mulcahy the localization proteins 2001; of targeting One subcellular al., the of their et levels expression (Trinkle-Mulcahy and the with mobile change could highly catalytic PP1 are The 1998). subunits al., et (Andreassen pattern accumulation PPP1b PP1 and nucleoplasm oaie otencepamadCs nrdcino mutations GFP–472D of of Introduction region lysine-rich CBs. the and in nucleoplasm the to protein localizes fusion a produced 91–151) (aa) GFP–SMN472 (aa acids domain [amino Tudor 1A. region and Fig. lysine-rich 71–83] in the shown containing is fragment proteins The fusion fluorescent for study present M ope oaiainadfnto r euae yprotein by regulated are function phosphorylation. PP1 and localization between complex relationship SMN the sought We from determine S1). Fig. to and material (supplementary GFP–N86 detected were mass, from PP1 Peptides phosphatase by of analysed protein (MS). and mass of digested spectrometry cut, apparent band was the mass band performed The co- major to 1C). In (Fig. a corresponding and GFP–N86 1B,C). kDa observed (Fig. 40 aa) SMN we approximately to of proteins, first partners antibody immunoprecipitated interaction anti-GFP 86 new GFP with a identify as (GFP–N86; experiments SMN of immunoprecipitation region protein N-terminal the fusion expressed We CBs. in erpr eeapeiul nnw ehns fpost- of mechanism unknown previously a here report We /d ´ sdtce hogottenceswt oparticular no with nucleus the throughout detected is ta. 06.Adarmo h oan sdi the in used domains the of diagram A 2006). al., et ,GPN6adGPN62fso rtisand proteins fusion GFP–N86M2 and GFP–N86 5, P euaino M ope 2863 complex SMN of regulation PP1 D hratrrfre oa GFP–472 as to referred (hereafter 5 c locnetae nncel,whereas nucleoli, in concentrates also c sacniaeprnro expected of partner candidate a as , c oCs eso htmodulating that show We CBs. to c c a mato h subnuclear the on impact ihSN eexpressed we SMN, with ihteSNcomplex SMN the with blse h localization the abolishes 5 n PP1 and n F–8.We GFP–N86. and 5 c n M because SMN and c r on nthe in found are c a n PP1 and c a co- was D htwas that c )that 5) c could .We c is Journal of Cell Science h y-ihdmi fSN ghadIG r h muolblnheavy immunoglobulin the are IgGl respectively. and chains, IgGh light SMN. and of domain Lys-rich PP1 of the association The SMN. endogenous ( 8M,rsetvl.Budpoen eeaaye yimnbotn with immunoblotting by analysed anti-PP1 were proteins Bound 472 respectively. eGFP-tagged N86M2, the expressing stably of cells position COS expected ( from the Gemin8. indicates component arrow asterisk complex An The SMN staining. band. silver 40-kDa were with a proteins visualized indicates Bound and N86. SDS-PAGE eGFP-tagged by with expressing separated and cells antibody COS (IP-GFP) from anti-GFP extracts or Control) (IP immunoglobulins ( eitdwt h y-ih uo,Porc,Y-o n exon7-encoded and YG-box Pro-rich, ( Tudor, domains. Lys-rich, the with depicted ugsigta ihrteat-M nioymssthe interaction masks the antibody mediates component anti-SMN third them. a the between or site either PP1 interaction and that between GFP–SMN between Interaction of suggesting than 2007). shown) localization not al., CB (data et and PP1 and (Morse stability SMN endogenous complex regulate SMN could the that motif self-interacting SMN minor a disrupts of (Renvoise mutation mutated This are 2006). residues al., lysine et the which PP1 in between protein interaction fusion any if little found (Renvoise N-terminus the at eGFP to fused mutants PP1 and SMN immunoprecipitation. of Interaction 1. Fig. 2864 E muorcpttos(P)wr efre ihngtv oto mouse control negative with performed were (IPs) Immunoprecipitations C) on rtisatrI fGPSNwr nlsdfrPP1 for analysed were GFP–SMN of IP after proteins Bound ) c niois h niGPicbto evda odn control. loading a as served incubation anti-GFP The antibodies. B ora fCl cec 2 (12) 125 Science Cell of Journal lwcato h muouiiainprocedure. immunopurification the of Flow-chart ) ( ceai ersnaino M a –9)and 1–294) (aa SMN of representation Schematic A) c rtisapae ob esefficient less be to appeared proteins P eepromdwt extracts with performed were IPs D) c sae yco- by assayed c ihtefso rtisrequires proteins fusion the with c D ´ ,N6o obemutant double or N86 5, n F–8M,a GFP–N86M2, and ta. 06.SNis SMN 2006). al., et c Fg 1E), (Fig. c and ´ M niois h seik niaeteimmunoglobulins. the indicate asterisks anti-PP1 The with antibodies. immunoblotting by SMN analysed were extracts cell HeLa niois sacnrl n ftepiayatbde a mte.Saebar: Scale omitted. was antibodies primary the anti-PP1 3 of and one anti-Gemin8 control, a with As cells antibodies. HeLa in proteins endogenous uinpoen hw nCwr nuae ihprfe i-agdPP1 PP1 ( His-tagged immunoblotting. Gemin8. purified by with with analysed incubated were were proteins ( C Bound staining. in Coomassie shown by proteins analysed fusion and SDS-PAGE by separated ( immunoblotting. opnn Gemin8 component ltd eovdb D-AEadaaye ysle staining. silver by analysed were Proteins and ( SMN). SDS-PAGE (IP by antibody resolved anti-SMN eluted, or Control) (IP control negative .coli E. nioy43alwdu oprf h M ope from complex remove to SMN order the in conditions purify stringent under to lysates us cell interaction HeLa allowed direct 4B3 demonstrate antibody to PP1 used was between assay pull-down A PP1c of interaction Direct i.2 ietitrcino PP1c Gemin8. of component interaction Direct 2. Fig. B m muorcpttdpoen hw nAwr nuae ihpurified with incubated were A in shown proteins Immunoprecipitated ) .( m. eobnn i-agdPP1 His-tagged recombinant F oI fteedgnu rtiswt niGmn nioyand antibody anti-Gemin8 with proteins endogenous the of Co-IP ) E c nst L eeto fteitrcinbetween interaction the of detection PLA situ In ) C S n S–ei8fso rtiswr purified, were proteins fusion GST–Gemin8 and GST ) n h M ope Fg ) Anti-SMN 2). (Fig. complex SMN the and ( eacl yaewsimnpeiiae with immunoprecipitated was lysate cell HeLa A) ihSNcmlxadits and complex SMN with c ihteSNcmlxadits and complex SMN the with on rtiswr nlsdby analysed were proteins Bound . c neat directly interacts D c GST ) c n anti- and c . Journal of Cell Science markers Fg F.Orrslsspottecnlso htGmn sa is Gemin8 that conclusion the support protein. protein results PP1-binding Our Gemin8– each 2F). (Fig. the PP1 interacted for PLA, Gemin8 with endogenous situ Moreover, specific CBs. in and nucleoplasm Using antibodies 2002). This PP1 al., 2E). in using Fig. et interactions PLA; (Fredriksson cells protein–protein situ (in endogenous fixed between assay detects interaction ligation Direct approach proximity 2D). situ (Fig. in not PP1 did and Gemin8 alone His-PP1 GST retained the Gemin8 glutathione immobilized (GST)-tagged between The transferase 2C). interaction PP1 (Fig. Direct proteins recombinant motif. and to PP1-binding appeared Gemin8 potential 80–83) (aa a were Gemin8 8, in be and motif 3 HVAW Gemin2, namely The SMN, examined. PP1 to SMN directly for that bind hypothesis that substrate gemins the a on Based be proline). might except any {P} and on oteSNcmlx ete ogtt dniythe identify PP1 to to sought binding mediates then that We complex PP1 complex. mouse the recombinant of SMN unrelated component the to The to bound (control). bound the that antibody for with tested monoclonal compared and were eluted proteins were His–PP1 complex of the presence complex to bound the purified proteins for a PP1 complex) with affinity histidine-tagged (SMN incubated weak complex was antibody-immobilized of The 2A). proteins (Fig. interacting any ooaiainwt M n PP1 was its and and Gemin8 3A) SMN Fig. holoenzymes, (FP–Gemin8; with fusion PP1 colocalization fluorescent to a as localization specificity expressed subcellular substrate confer proteins and PP1-binding that Given PP1c colocalizes Gemin8 of Overexpression otcmo P-idn VFmtfhsteconsensus the has motif RVxF [R/K]x PP1-binding sequence common most oyfrainadw nrdcdmttosi h uaiePP1- (FP–G8 putative the motif in mutations binding introduced we and the formation (FP–G8 including body PP1-binding Gemin8 domain other of coil region of C-terminal coiled presence The the with CBs. to Colocalization in FP– proteins due 3C). either (Fig. be alone could expressing in FP FP–Gemin8 cells FP– concentrated or and was In myc–Gemin4 cells it SMN, 2006). untransfected whereas fusion, of al., the nucleoli et with bodies (Carissimi nuclear PP1 reported nuclear cells, as and Gemin8-expressing cytoplasm 3B), the (Fig. to CBs SMN with colocalized completely G8 FP–G8 eurdfrtelclzto fPP1 of is localization Gemin8 of the motif for HVAW the required that revealed experiments These eut upr h ocuinta ei8cnrbtst CB to contributes Gemin8 that PP1 of conclusion localization the support results Fg AE.AlF–ei8ncerbde eeCsas were CBs They the as coilin. such were markers, snRNP-specific with and bodies Gemin3 colocalization in enriched nuclear also complete FP–Gemin8 by examined (a indicated was All snRNPs coilin of 4A–E). with components (Fig. and colocalization Gemin3 constituents, marker), CB CB other contained odtriewehrteF–ei8ncerbodies nuclear FP–Gemin8 the whether determine To D c erie PP1 recruited C neato perda rgtdt ntecytoplasm, the in dots bright as appeared interaction D 3 oaie otencepamadt ee B.FP– CBs. fewer to and nucleoplasm the to localized C3A c n M nc-muorcptto experiments co-immunoprecipitation in SMN and c 0–1 a ute etdi ie eaclsuigthe using cells HeLa fixed in tested further was c VI{}FW xrpeet n mn acid, amino any represents (x [V/I]{P}[F/W] c c c c nmmaincells. mammalian in D yimnbotn Fg B.Tebound The 2B). (Fig. immunoblotting by noCs hra FP–G8 whereas CBs, into (His–PP1 a etduigprfe bacterial purified using tested was 3;Fg AC.Bt FP–G8 Both 3A,C). 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FP–Gemin8 SMN expressing cells HeLa in performed were ( an cells. indicates HeLa asterisk from band The protein cell antibody. unspecific total anti-FP in with assayed immunoblotting were by proteins lysates fusion expressed The Q9NWZ8). (accession putative number domain the coiled-coil self-association with a depicted and is HVAW motif Gemin8 PP1-binding N-terminus. the at (FP) YFP destabilized ( i.3 agtn fPP1 of Targeting 3. Fig. A m ceai ersnaino ei8(a122 n uat ue to fused mutants and 1–242) (aa Gemin8 of representation Schematic ) m. c nioy vrxrsino PGmn hne the changes FP–Gemin8 of Overexpression antibody. P euaino M ope 2865 complex SMN of regulation PP1 C c PGmn,FP–G8 FP–Gemin8, ) rmncel oGmn-xrsigncerbde na in bodies nuclear Gemin8-expressing to nucleoli from c oCjlbde nclsoeepesn Gemin8. overexpressing cells in bodies Cajal to B D muoloecneexperiments Immunofluorescence ) ,FP–G8 C, D 3,F–M,myc– FP–SMN, C3A, Journal of Cell Science sseii.Orrslsdmntae htGmn ol form PP1 could and Gemin8 snRNPs complex, that SMN demonstrated contain which results CBs, Our specific. is efrttse h rti eeso oa yae fHL cells HeLa of lysates total of levels protein the tested first We ncdw xeiet eepromduigtredistinct three using performed PP1 were between experiments link functional knockdown a be might PP1 there whether determine CBs To in snRNPs and complex PP1c of Depletion cells, FP–Gemin8-expressing PP1 of of CBs accumulation the in that a conclusion not occurs the is staining supporting The and SC35 in that that concentrate speckles protein. showed not SC35 4F in does Fig. kDa factor and investigated. was 1993) 70 splicing CBs (Spector, snRNPs speckles abundant all U1-specific of to protein the marker and common core of proteins snRNP) Sm localization Sm snRNP U6 seven snRNAs), the except of of (one import SmD3 nuclear the for signature (molecular snRNAs (TMG)-capped trimethylguanosine 2866 c c seii ml nefrn N sRA ulxs(i.5). (Fig. duplexes (siRNA) RNA interfering small -specific n M agtn oCs N nefrne(RNAi) interference RNA CBs, to targeting SMN and ora fCl cec 2 (12) 125 Science Cell of Journal nacsteacmlto fSMN of accumulation the enhances c . c nCBs in oto,teoealpopoyainpoieo oa protein PP1 between were total There differences panel). of right major furthest profile 5A, no (Fig. phosphorylation PP1 examined was reduce overall lysates to indicating used the same, siRNAs the control, the remained of levels specificity complex the SMN 5A). (Fig. rnfce ihete eaiecnrlfrfylcfrs (Gl2) PP1 luciferase firefly control PP1 negative or a either with transfected h omto fCsuo elto fPP1 components, had of all CB markers, depletion snRNP-specific and and coilin Gemin8 upon Gemin3, with including CBs experiments with of cells Colocalization of PP1 formation proportion three All the the bodies. in There nuclear increase five 5B,C). than fourfold (Fig. more to cells three- control a than was bodies nuclear SMN more ( significant 5B). (Fig. a experiments was PP1 immunofluorescence of overlapping cells Depletion siRNA-treated by have in examined SMN holoenzymes then of localization PP1 The that substrates. indicating controls, c seii iNsrdcdPP1 reduced siRNAs -specific c nncerfc.Saebr 3 bar: Scale focused was foci. microscope nuclear overlapping The on above. the observed of yellow specificity in the localizations indicating red), (in images). speckles merged the (in signals ( yellow in results Colocalization rti n ( and protein ( akr,( marker), el n nlsdb muoloecnewt ( with immunofluorescence by analysed and cells snRNPs. and complex SMN i.4 vrxrse ei8clclzsPP1 colocalizes Gemin8 Overexpressed 4. 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SMN multiprotein i.8 elto fPP1 ( of Depletion 8. Fig. 2870 eaclswr rnfce ihcnrlG2o he ifrn PP1 different three or Gl2 control with transfected were cells HeLa A) ei8bnsdrcl oPP1 to directly binds Gemin8 c iN 5wr muorcpttdwt oto mouse control with immunoprecipitated were #5 siRNA P ora fCl cec 2 (12) 125 Science Cell of Journal , B .4 Wloo indrn et.( test). signed-rank (Wilcoxon 0.042 h yooi rcin fHL el rnfce ihG2or Gl2 with transfected cells HeLa of fractions cytosolic The ) c rmtsfraino h M complex. SMN the of formation promotes c a tblnadanti-HDAC and -tubulin n rnsi nothe into it brings and iia nlsso the of analyses Similar D) h relative The C) c PM ciiyi rbbyntatrdi PP1 in altered findings, complex not our with probably SMN Consistent is 2007). activity regulates al., et PP1MG (Petri PP1MG CBs Gemin8 in that stability with showed interaction nucleoplasm, its and complex 2009) al., 2006). 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R. Margolis, and E. Villa-Moruzzi, B., F. Lacroix, R., P. Andreassen, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.096255/-/DC1 online (to available material Myopathies Supplementary (to les Technologie Ministe Contre la Francaise the de Association from et B.R.). the Recherche fellowship and la B.R.); undergraduate de Nationale, an l’Education (grant S.L.); Myopathies Sante les to la Franc¸aise contre Association de the National scientifique; Institut by Me supported Recherche was the work of This facility Funding Gemin4 core proteomics myc-tagged the the Monod. Jacques and acknowledge provided Institut manuscript We are generously the We plasmid. of Massenet Nancy) support. S. reading constant antibodies. (CNRS, and for critical controls Cartaud protocols, for providing J. generously colleagues and to A. thank grateful to wish We Acknowledgements Protein using facility. performed was proteomics S1 Jacques-Monod (www.expasy.org/tools/). Fig. tools Institut material proteomics supplementary at expasy in nm) YAG- presented (355 a identification with laser equipped extracted Biosystems) and Hz (Applied 200 spectrometer trypsin, mass with TOF-TOF The 4800 digested a Invitrogen). extensively using by analysed excised, Quest, were visualized were peptides (Silver interest and of SDS-PAGE staining bands by silver separated mass-spectrometry-compatible were proteins spectroscopy Co-immunopurified mass by proteins Candidate drystrips IPG facility. linear proteomics mM 3–10 Monod 23 pH Jacques CHAPS, using Institut IPG first- 2.5% the The with at thiourea, needle. Healthcare) strips 27-gauge M (GE in a 1 using performed urea, (IPG) was gradient M dimension pH Santa and 8 NE-PER immobilized the 0.5% in or Abcam) and using solubilized 1:5000; DTT Healthcare prepared directly pellets at GE nuclear were 1:200; rabbit the kit 1:10,000; at electrophoresis, or 2D-gel (at (rabbit For Roche; phosphoserine Cruz). 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Richard, and et sfrsdrn ohitrhs n ioi nmmaincells. mammalian and in gamma1, mitosis and alpha, interphase phosphatase-1 141 both protein during isoforms of delta localization subcellular Differential 2 1207-1215. , Mdtitrio DT n .5 P0 h on rtiswere proteins bound The NP40. 0.05% and (DTT) dithiothreitol mM 1 , c 6 ga t11,0;SnaCruz), Santa 1:10,000; at (goat D apebuffer. sample SDS ˚ ih2.5 with C ´ iae(aayt ..;Cnr ainld arecherche la de national Centre S.L.); to (salary dicale ˆte 20) ymtia iehlriiemtyaini eurdfor required is methylation dimethylarginine Symmetrical (2002). ,J,Buagr .C,Ceox . ahn,F,Atxe,C. Autexier, F., Bachand, P., Cleroux, C., M. Boulanger, J., ´, m fprfe eobnn i-agdPP1 His-tagged recombinant purified of g a cao4hdoyinmcai CC)mti on matrix (CHCA) acid -cyano-4-hydroxycinnamic m fiecl idn ufradeue by eluted and buffer binding ice-cold of l m fmgei ed lry eepre- were slurry) beads magnetic of l a tbln(os A t1:10,000; at mAb (mouse -tubulin a rbi t11,0;Santa 1:10,000; at (rabbit .Cl Biol. Cell J. ´ ta. 2006), al., et c ´ .Cl Biol. Cell J. n25 in td la de et ` ede re (1998). m 159 lof , Journal of Cell Science rmlr . te,S,Ptr . ule,F,Cai .adFshr U. Fischer, and A. Chari, F., Mu¨ller, C., Peter, S., Otter, M., U. Grimmler, Fischer, and G. Meister, Ko¨rner, R., M., Nousiainen, L., Bauer, M., Grimmler, agai . irt . hru,S,Sam,M n ai,R J. R. Davis, and M. Sharma, S., Theroux, M., Mikrut, L., Gangwani, al .G. J. Gall, hn,H . ilc,D . ooua . uhre,A,Kne,M . e,A., Sen, W., M. Kankel, A., Mukherjee, T., Yokokura, N., D. Dimlich, C., H. Chang, aca . al,X,Curn . eeu,E,Rna,F n eol,A. Rebollo, and F. 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Cell protein Probl. neuron motor of 1189. h oso h nRPcaeoeNp10fo aa oiso ain fibroblasts atrophy. patient muscular of bodies spinal Cajal of from severity Nopp140 the chaperone with snoRNP correlates the of loss cells. The mammalian in bodies Cajal to targeting for S. ihatimn antibodies. autoimmune with M. E. ehshrlto fsria oo ern SN yPPM1G/PP2C complex. SMN by the (SMN) of stability neurons and localization motor body Cajal survival of splicing. pre-mRNA Dephosphorylation in product, gene disease atrophy muscular Cell spinal the SMN, for neuron motor of Rep. survival EMBO human the of map complex. interaction (SMN) comprehensive A (2007). ik m oFFsignaling. FGF to Smn links S. Artavanis-Tsakonas, uvvlmtrnuo ope opnnsi ml ula ribonucleoprotein nuclear small in components complex neuron assembly. motor survival .CidNeurol. Child J. rti hshts id oteRArcgiinmtfo eea piigfactors splicing several of processing. motif al. pre-mRNA recognition et RNA alternative L. the regulates Bracco, to P., and Fehlbaum, binds C., 1 Ben-Dov, phosphatase A., Protein M. Lorson, L., C. Lorson, el edt eakbepoeto gis m eedfc ndfeetae skeletal differentiated in al. defect et gene N. Smn Roblot, against L., protection muscle. Languille, remarkable to F., lead Backer, cells De L., M. Cao, biogenesis. snRNP U2 of steps networks. interaction B. protein R. mediating Russell, peptides and L. protein. Serrano, atrophy J., muscular spinal the SMN, and snRNPs 223-234. U of import 20) itntdmiso h pnlmsua toh rti M r required are SMN protein atrophy muscular spinal the of domains Distinct (2006). 615-624. 95, 19) muooia n lrsrcua tde ftencercie body coiled nuclear the of studies ultrastructural and Immunological (1991). .Cl Biol. Cell J. ora fCl cec 2 (12) 125 Science Cell of Journal .Bo.Chem. Biol. 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Lam, A., 4219-4228. 20) M eiinycue iseseii etrain nterpror of repertoire the in splicing. perturbations in tissue-specific defects widespread causes and snRNAs deficiency SMN (2008). 551-562. ybat eelitiscdfcsi ybatfso n ytb morphology. myotube and fusion myoblast Res. in Cell defects intrinsic reveal myoblasts Biol. 304. rti hshts ihntence flvn amla cells. mammalian living of nuclei the within 1 phosphatase protein assembly. complex export eiessRApeusr oteSNcmlxfrsRPbiogenesis. snRNP for complex SMN the to precursors snRNA delivers 2320-2330. xndgnrto na nmlmdlfrsia uclratrophy. muscular spinal for model animal an in degeneration axon U. Fischer, and B. Laggerbauer, sn nN rti uin otegenfursetprotein. revealed fluorescent nucleoli green the and to bodies fusions coiled protein snRNP snRNPs, using splicing between interactions Dynamic . ar,A .adNueae,K M. K. bodies. Neugebauer, Cajal through and cycle repeatedly K. particles ribonucleoprotein A. Sapra, X., erfs embryogenesis. M. zebrafish K. Neugebauer, 2534-2543. 5 20) M ope oaie otesroei -icadi proteolytic a is and Z-disc sarcomeric the to localizes complex SMN (2008). o.Bo.Cell Biol. Mol. 442-447. , erbo.Dis. Neurobiol. 311 ˆte 19) ula raiaino r-RAprocessing. pre-mRNA of organization Nuclear (1993). ,P .adKtay R. Kothary, and D. P. ´, 49-61. , u.Ml Genet. Mol. Hum. 107-117. 14, 21) olndpnetsRPasml sesnilfor essential is assembly snRNP Coilin-dependent (2010). 511-517. 34, a.Src.Ml Biol. Mol. Struct. Nat. .Cl Biol. Cell J. 20) eue nN sebycue motor causes assembly snRNP U Reduced (2005). 3399-3410. 17, el o.Lf Sci. Life Mol. Cell. 21) aa oysrelac fUsnRNA U of surveillance body Cajal (2010). 20) yoopi m ncdw C2C12 knockdown Smn Hypomorphic (2005). 190 603-612. , 20) olnlvl n modifications and levels Coilin (2008). Cell 20) piesmlsalnuclear small Spliceosomal (2008). 17 133 403-409. , 585-600. , 20) yai agtn of targeting Dynamic (2001). 1256-1271. 65, x.Cl Res. Cell Exp. 20) uvvlmotor Survival (2009). o.Cl.Biol. Cell. Mol. 20) Time-lapse (2003). o.Bo.Cell Biol. Mol. ur pn Cell Opin. Curr. .Cl Sci. Cell J. 21) Gemin5 (2010). ee Dev. Genes o.Cell Mol. 243 (1998). 290- , Exp. 114 25 38 19 19 , , , , , Fig. S1. Identi cation of the 40 kDa protein associated with the N-terminal region of SMN. (A) Protein iden- ti cation was made with the Mascot search soware using the peptides derived from a trypsin digestion of the 40 kDa band. Peptide coverage map for the protein phosphatase catalytic subunit PP1γ revealed 73% coverage, including the N- and C-terminal ends. (B) Profound search also showed the candidate to be PP1γ.