Sam68 sequestration and partial loss of function are associated with splicing alterations in FXTAS patients

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1 The EMO Journl (21) 29, & 21 Europen Moleculr iology Orgniztion ll Rights Reserved /1 sequestrtion nd prtil loss of function re ssocited with splicing ltertions in FXTS ptients THE EMO JOURNL Chntl Sellier 1,Frédérique Ru 1, Yilei Liu 2, Flor Tssone 3,6, Rente K Hukem 4, Rent Gttoni 1, nne Schneider 1, Stéphne Richrd 5, Ro Willemsen 4, Dvid J Elliott 2, Pul J Hgermn 3,6 nd Nicols Chrlet-erguernd 1, * 1 Deprtment of Neuroiology nd Genetics, IGMC, INSERM U964, CNRS UMR714, University of Strsourg, Illkirch, Frnce, 2 Institute of Humn Genetics, Newcstle University, Newcstle, UK, 3 M.I.N.D. Institute, University of Cliforni, Dvis, Helth System, Scrmento, C, US, 4 CG-Deprtment of Clinicl Genetics, Ersmus MC, Rotterdm, The Netherlnds, 5 Ldy Dvis Institute for Medicl Reserch, Deprtments of Medicine nd Oncology, McGill University, Montrel, Queec, Cnd nd 6 Deprtment of iochemistry nd Moleculr Medicine, School of Medicine, University of Cliforni, Dvis, C, US Frgile X-ssocited Tremor/txi Syndrome (FXTS) is neurodegenertive disorder cused y expnsion of 55 2 repets in the 5 -UTR of the FMR1 gene. FXTS is chrcterized y ction tremor, git txi nd impired executive cognitive functioning. It hs een proposed tht FXTS is cused y titrtion of RN-inding proteins y the expnded repets. is n RNinding protein involved in lterntive splicing regultion nd its ltion in mouse leds to motor coordintion defects. Here, we report tht mrns contining expnded repets form lrge nd dynmic intrnucler RN ggregtes tht recruit severl RN-inding proteins sequentilly, first, then hnrnp-g nd MNL1. Importntly, is sequestered y expnded repets nd therey loses its splicing-regultory function. Consequently, -responsive splicing is ltered in FXTS ptients. Finlly, we found tht regultion of tyrosine phosphoryltion modultes its locliztion within ggregtes nd tht tutomycin prevents oth nd RN ggregte formtion. Overll, these dt support n RN gin-of-function mechnism for FXTS neuropthology, nd suggest possile trget routes for tretment options. The EMO Journl (21) 29, doi:1.138/ emoj.21.21; Pulished online 25 Ferury 21 Suject Ctegories: RN; moleculr iology of disese Keywords: FXTS; RN gin-of-function diseses; *Corresponding uthor. Deprtment of Neuroiology nd Genetics, IGMC, 1 rue Lurent Fries, Strsourg, Illkirch 674, Frnce. Tel.: þ , Fx: þ ; E-mil: nchrlet@igmc.fr Received: 29 My 29; ccepted: 2 Jnury 21; pulished online: 25 Ferury 21 Introduction Frgile X-ssocited Tremor/txi Syndrome (FXTS) is recently identified neurodegenertive disorder tht ffects principlly older dult mles who re crriers of pre-muttion expnsions (55 2 repets) in the 5 -untrnslted region (UTR) of the Frgile X Mentl Retrdtion-1 (FMR1) gene (Hgermn et l, 21; Hgermn nd Hgermn, 24; Ostr nd Willemesen, 29). The mjor clinicl fetures of FXTS re progressive intention tremor nd git txi, with more vrile ssocited fetures, including prkinsonism, dysutonomi, nxiety, peripherl neuropthy nd cognitive decline (Jcquemont et l, 23). The neuropthologicl hllmrk of FXTS is the presence of uiquitin-positive intrnucler inclusions in oth strocytes nd neurons throughout the rin (Greco et l, 22). It is estimted tht nerly 1 in 3 mles hs lifetime risk of developing FXTS, which would mke FXTS one of the most common single gene cuses of tremor, txi nd cognitive decline mong older dults (Jcquemont et l, 24). In Frgile X syndrome full muttions (42 repets) result in hypermethyltion nd silencing of the FMR1 gene. In contrst, crriers of the pre-muttion lleles (55 2 repets) hve incresed FMR1 mrn levels ut norml, or modertely low, FMR1 protein expression, especilly in the upper pre-muttion rnge (Tssone et l, 2, ; Kenneson et l, 21; Primerno et l, 22). These oservtions, coupled with the fct tht mrns contining expnded repets ccumulte in intrnucler ggregtes, suggest toxic RN gin-of-function model for FXTS (Tssone et l, 24). In support of this hypothesis, knock-in (KI) mouse model, in which the endogenous eight repets in the Fmr1 gene hs een replced with n expnsion contining 1 repets of humn origin, shows uiquitin-positive intrnucler inclusions nd mild neuromotor nd ehviourl disturnce (Willemsen et l, 23; Vn Dm et l, 25; rouwer et l, 28). Furthermore, sole expression of mrns contining 9 repets outside the context of Fmr1 in trnsgenic mouse model is sufficient to recpitulte the neuropthologicl nd moleculr fetures of FXTS (Hshem et l, 29). Similrly, heterologous expression of 9 repets in Drosophil is sufficient to cuse neurodegenertion long with formtion of neuronl inclusions (Jin et l, 23). These models show tht sole expression of expnded repets is necessry nd sufficient to cuse pthology similr to humn FXTS, nd thus indicte tht the expnded repets in RN re the likely cuse of the neurodegenertion in FXTS. The FXTS toxic RN gin-of-function model show similrities with Myotonic Dystrophies (DM), where expnded CUG or CCUG repets ccumulte in nucler RN ggregtes tht sequester the Musclelind-like (MNL1) splicing fctor. In DM, prtil depletion of the free pool of MNL1 leds to 1248 The EMO Journl VOL 29 NO 7 21 & 21 Europen Moleculr iology Orgniztion

2 sequestrtion in FXTS ptients C Sellier et l specific lterntive splicing chnges, which ultimtely result in the symptoms of DM (Rnum nd Cooper, 26; Wheeler nd Thornton, 27). Extending this model to FXTS, expnded repets re predicted to sequester specific proteins resulting in loss of their norml moleculr functions. Severl proteins, including numer of het-shock proteins, Pur, hnrnp-2/1, CUGP1, MNL1, lmin-/c nd MP were found to loclize with uiquitin-positive inclusions in -expressing Drosophil, KI mouse model nd FXTS ptients (Iwhshi et l, 26; Jin et l, 27; Sofol et l, 27). However, these proteins were not found to e sequestered y expnded repets nd consequently they re not expected to lose their functions in FXTS ptients. In this study, we found tht in contrst to CUG repets, expnded repets ccumulte in dynmic intrnucler RN structures tht expnd over time. Formtion of gint RN ggregtes ultimtely results in disorgniztion of the nucler lmin structure nd cell deth. MNL1 nd hnrnp-g proteins were found loclized within ggregtes ut only in the lrger inclusions nd t lte time points fter trnsfection, suggesting these re not the primry defects. In contrst, we identified the Src-ssocited sustrte during mitosis of 68-kD () protein s the only protein tht coloclizes with RN ggregtes t ech time point. is nucler RN-inding protein involved in lterntive splicing regultion (Stoss et l, 21; Pronetto et l, 27; Chwl et l, 29), nd its ltion in mouse knock-out model leds to motor coordintion defects (Lukong nd Richrd, 28). splicing ctivity, RNinding ility nd locliztion re regulted y phosphoryltion (Hegerth et l, 24; Lukong et l, 25), nd intercts with vrious RN-inding proteins through severl protein protein interction domins (Lukong nd Richrd, 23). We found tht is required for susequent ggregtion of MNL1 nd hnrnp G proteins within ggregtes. Most importntly, is sequestered y expnded -repet ggregtes nd therey loses its splicing-regultory function. s consequence, -regulted splicing is ltered in FXTS ptients. Finlly, we found tht regultion of phosphoryltion modultes its locliztion within ggregtes. Strikingly, mong the vrious kinse nd phosphtse inhiitors tested, we found one, tutomycin, which not only prevents colocliztion within ggregtes, ut lso olishes RN ggregte formtion. Results Expnded repets form dynmic nucler RN ggregtes tht expnd over time We first questioned whether pre-muttion-length repets cn form nucler RN ggregtes in cultured cells. We trnsfected plsmids expressing either 2, 4, 6 or 1 repets under the control of cytomeglovirus (CMV) promoter in vrious cell lines, nd tested the formtion of ggregtes y RN fluorescence in situ hyridtion (FISH) nlysis. We confirmed the specificity of the FISH conditions, nd the RN composition of ggregtes, s they were sensitive to RNse tretment (Supplementry Figure S1). Consistent with n RN gin-of-function model, expression of 6 or 1 repets within COS7 cells generted numerous intrnucler ggregtes, wheres expression of 2 repets did not (Figure 1). Expression of 4 repets resulted in n intermedite condition with formtion of rre smll intrnucler ggregtes. This is consistent with oservtions in FXTS ptients in whom it is estimted tht norml polymorphic repet lengths re 5 45 repets long, gry zone lleles contin 45 to 55 repets nd FXTS ptients re defined y pre-muttion llele contining 55 2 repets (Tssone et l, 27; Leehey et l, 28). Next, expression of ggregtes ws investigted in vrious cell types. Trnsfection of constructs contining 6 repets led to intrnucler RN ggregte formtion in primry cultures of hippocmpl emryonic mouse neurons, s well s in vrious immortlized cell lines such s neuronl (differentited PC12), ovrin (SKOV3 nd SW626) nd kidney (COS7)-derived cell lines (Figure 1). However, no ggregtes were oserved in 172, U-937, THP1, HeL, HEK293, NG18-15, IMR-32, Neuro-2, SH-SY5Y, SK-N-MC or SK-N-SH cell lines (dt not shown), confirming previous report tht not ll cell lines cn support -repet ggregte formtion (rocen et l, 25). Tests of colocliztion with vrious nucler structures indicted tht in trnsfected cells most of the ggregtes re ssocited with nucler speckles, ut not with other structures such s lmin, nucleoli, PML, Gems or Cjl odies (Supplementry Figure S2). Finlly, kinetic formtion of ggregtes ws investigted. COS7 cells were trnsfected with plsmid expressing 6 repets nd nlysed y RN FISH either 24, 48 or 72 h fter trnsfection. Surprisingly, expnded repets formed dynmic nucler structures tht expnded over time, resulting in gint inclusions, nucler lmin rchitecture disruption nd cell deth h fter trnsfection (Figure 1C). In contrst, expnded CUG (DM1 muttion) or UUCU (SC1 muttion) repets were less toxic nd formed stle nucler ggregtes, whose size did not evolve over time (Supplementry Figure S2). nnexin-v PE poptosis tests were negtive indicting tht the cytotoxicity of repets is not linked to poptotic cell deth (dt not shown), nd in greement with previous report (rocen et l, 25) no uiquitin-positive ggregtes were oserved in trnsfected cells. ggregtes recruit, then MNL1 nd hnrnp G To identify which proteins re ssocited with expnded repets, we first dopted n in vitro pproch. Proteins extrcted from mouse rin or COS7-cell nuclei were cptured on streptvidin resin coupled to iotinylted in vitrotrnscried RN composed of 6 repets, eluted, seprted on SDS PGE gels nd identified y MLDI-TOF nlysis. More thn 2 proteins were identified (Supplementry Tle 1), including het-shock protein nd severl RNinding proteins, including MNL1 nd hnrnp-g. To discrd non-specific inding proteins, we tested for colocliztion of these cndidtes with RN ggregtes in COS7 cells trnsfected with 6 repets. ggregtes expnd over time, suggesting tht these repets my recruit different proteins t different time points. Thus, we tested our cndidtes t 24 nd 72 h fter trnsfection (Figure 2 nd ). Colocliztion of MNL1 within ggregtes incresed from 14% t 24 h to 41% t 72 h fter trnsfection. Similrly, & 21 Europen Moleculr iology Orgniztion The EMO Journl VOL 29 NO

3 sequestrtion in FXTS ptients C Sellier et l x2 x4 x6 x1 c d e Mouse neurons primry culture Neuronl cells (PC12) Ovrin cells (172) Kidney cells (Cos7) d c C Lmin-GFP Cy3-CCG Merged 24 h 48 h c 72 h x6 Lmin Lmin Lmin Figure 1 Expnded repets form intrnucler RN ggregtes. () COS7 cells were trnsfected with plsmid expressing either no (), 2 (), 4 (c), 6 (d) or 1 (e) repets, trnsferred to.1% serum to lock cell divisions nd nlysed 24 h fter trnsfection y RN FISH using (CCG) 8x -Cy3 DN proe. () Primry cultures of hippocmpl emryonic mouse neurons (), differentited PC12 (), SKOV3 (c) nd COS7 (d) cells were trnsfected with plsmid expressing 6 repets nd nlysed 24 h fter trnsfection y FISH. (C) COS7 cells were cotrnsfected with plsmid expressing 6 repets nd plsmid expressing GFP-tgged lmin-, nd nlysed y RN FISH either 24 (), 48 () or 72 (c) hours fter trnsfection. In ll the figures, the mgnifiction is 63. The scle rs represent 1 mm; nuclei were counterstined with DPI nd one representtive experiment from t lest three seprte experiments is shown. colocliztion of hnrnp-g incresed from 26 to 73% (Figure 2D), suggesting tht ggregtes form dynmic structures, which constntly recruit proteins. y contrst, other in vitro-identified cndidtes such s SPNR, hnrnp-1, hnrnp-2/, hnrnp-c, hnrnp-d, hnrnp-e nd hnrnp-h showed no or very little colocliztion with RN ggregtes, nd ny colocliztion oserved ws only within the gint ggregtes tht form just efore cell deth (dt not shown). We lso oserved tht neither CUGP1, nor Pur, coloclized with ggregtes in COS7-trnsfected cells. These results suggest tht MNL1 nd hnrnp-g re not initilly recruited y repets, ut join the lrger ggregtes lter on, proly through protein protein interctions. On the sis of tht hypothesis, we serched for proteins tht would coloclize with expnded repets erly fter trnsfection, nd my cpture other RN-inding proteins through RN or protein interctions. We screened 5 cndidtes known to ind to RN or RN-inding proteins (Supplementry Tle 2 nd dt not shown) nd found one,, tht consistently co-loclized with RN ggregtes t ech time point, including the erliest (Figure 2C nd D). Next, we confirmed y FISH/IF tht endogenous coloclized with ggregtes in neuronl-differentited PC12 cells (Figure 2C). Similr to COS7 cells, endogenous MNL1 nd hnrnp-g were not recruited within ggregtes in PC12 cells 24 h fter trnsfection. We were not le to test, MNL1 or hnrnp-g colocliztion within ggregtes t lter trnsfection time points s PC12 cells re very sensitive to toxicity nd die fter less thn 48 h of expression. is nucler protein involved in vrious spects of mrn metolism nd intercts with severl RN-inding proteins, rising question its specificity towrds ggregtes versus other expnded RN repets. COS7 cells were trnsfected y plsmids expressing either expnded, CUG, CCUG or UUCU repets, which re the cuse of FXTS, DM1, DM2 nd SC1 diseses, respectively. We found tht 24 or 72 h fter trnsfection, endogenous 125 The EMO Journl VOL 29 NO 7 21 & 21 Europen Moleculr iology Orgniztion

4 sequestrtion in FXTS ptients C Sellier et l C hnrnp G MNL1 COS7 COS7 c Protein Cy3-CCG Merged MNL1 hnrnp G MS2CP hnrnp G MNL1 Protein Cy3-CCG Merged MNL1 hnrnp G MS2CP 24 h fter trnsfection 72 h fter trnsfection Cy3-CCG Merged D 72 h 24 h % of co-locliztion 1 24 h 72 h MNL1 hnrnp G PC12 24 h Figure 2 coloclizes with RN ggregtes. () COS7 cells were trnsfected with plsmid expressing 6 repets nd nlysed y FISH/IF using n ntiody ginst MNL1 () 24 h fter trnsfection. None of the ntiodies ginst hnrnp-g tht we tested supported FISH conditions. Consequently, endogenous hnrnp-g () ws nlysed y co-trnsfection of COS7 cells with plsmid expressing 6 repets fused to three MS2 tgs nd plsmid expressing the GFP-MS2 Cot Protein. The MS2 Cot Protein (MS2CP) possesses very high nd specific ffinity for MS2 RN tgs. Endogenous hnrnp-g () ws detected y IF nd the ggregtes y locliztion of the GFP- MS2CP protein, which is ound to the MS2-() 6x RN. In the sence of MS2-() 6x, GFP-MS2CP ws diffuse in the nucleoplsm (dt not shown). () Similr to pnel ut nlysed 72 h fter trnsfection. (C) COS7 cells (, ) or differentited PC12 neuronl cells (c) were trnsfected with plsmid expressing 6 repets nd nlysed y FISH/IF using n ntiody ginst, 24 h ( nd c) or 72 h fter trnsfection (). (D) The percentge of endogenous, MNL1 nd hnrnp-g coloclized within RN ggregtes in trnsfected COS7 cells 24 or 72 h fter trnsfection. In ll the experiments, three independent trnsfections totlling hundred cells were counted, nd results re presented s men±s.d. coloclized only with RN ggregtes, ut not with other expnded RN repets (Figure 3). These results suggest tht is specific nd erly component of ggregtes in FXTS. is essentil for recruitment of MNL1 nd hnrnp-g within ggregtes is recruited erlier thn MNL1 nd hnrnp-g, suggesting sequentil recruitment of proteins within the ggregtes. Furthermore, protein contins severl protein protein interction domins, rising the question whether directly recruits hnrnp-g nd MNL1. In greement with previous report (Venles et l, 1999), we found y co-immunoprecipittion tht intercts with hnrnp-g. However, we found no roust interctions etween MNL1 nd or etween MNL1 nd hnrnp-g (Supplementry Figure S3). Next, we mpped the domin required for colocliztion with ggregtes. protein contins centrl KH RN-inding domin, nd N- nd C-terminl protein protein interction domins. Deletion of the N-terminl domin olished colocliztion with ggregtes, wheres deletion of the RN-inding domin did not (Supplementry Figure S3). Similrly, prlog proteins, Slm1 nd Slm2 (T-Str), which re devoid of the N-terminl extended region of the protein, did not coloclize with expnded ggregtes 24 h fter trnsfection. The N-terminl prt of the protein consists of numer of potentil protein protein interction domins, suggesting tht ssocition of with repets might not e direct ut medited through protein protein interctions. Finlly, we tested whether is required for colocliztion of MNL1 nd hnrnp-g within ggregtes. Trnsfection of COS7 cells with n shrn directed ginst gretly reduced (48%) the expression of endogenous (Figure 4C). Importntly, depletion of endogenous olished the colocliztion of MNL1 nd hnrnp-g within ggregtes (Figure 4 nd Supplementry Tle 3). These dt show tht protein is required for susequent recruitment of MNL1 nd hnrnp-g proteins within ggregtes. coloclizes with ggregtes in FXTS ptients Next, we tested whether protein coloclizes with endogenous ggregtes. We first nlysed the locliztion of endogenous nd repets in the rin sections of KI mouse model, in which endogenous repets hd een replced with n expnsion of 98 & 21 Europen Moleculr iology Orgniztion The EMO Journl VOL 29 NO

5 sequestrtion in FXTS ptients C Sellier et l UUCU x16 x6 Cy3-Oligo UUCU Merged deplete its moleculr ctivity. To test sequestrtion y repets, we first nlysed its moility y FRP (Fluorescence Recovery fter Photoleching) experiments. FRP of trnsfected GFP- ws mesured in nucler regions contining ggregtes nd compred to nucler regions contining diffuse either locted within the sme nucleus or locted in the nuclei of cells not trnsfected with repets (Figure 6). In oth cses, nucleoplsmic res without ggregtes recovered 95% of their initil fluorescence fter photoleching, wheres res contining ggregtes recovered only 6% of their initil fluorescence (Figure 6). This shows tht frction of is less moile in -trnsfected cells. C CUG x96 CCUG x3 D CUG CCUG Figure 3 colocliztion within nucler RN ggregtes is specific to expnded repets. COS7 cells were trnsfected with plsmid expressing either 6 repets (), 16 UUCU repets (), 96 CUG repets (C) or 3 CCUG repets (D). Endogenous nd repets were nlysed 24 h fter trnsfection y FISH/IF. repets (Willemsen et l, 23). FISH/IF experiments showed the presence of intrnucler ggregtes tht coloclized with in mice expressing 98 repets (Figure 5). y contrst, no RN ggregtes were detected nd ws diffuse throughout the nucleoplsm in control mice (Figure 5). Then, we investigted the presence of nd ggregtes in FXTS ptients. FISH/IF experiments show tht consistently coloclized with intrnucler RN ggregtes in the rin sections of FXTS ptients (Figure 5), ut neither nor ggregtes were found in control rin tissue (Figure 5). We noted tht in FXTS ptients, nd ggregtes were lrger nd more frequent thn in KI mice. This is consistent with the milder neuromotor nd ehviourl disturnces oserved in KI mice s compred with tht in FXTS ptients (Willemsen et l, 23; Vn Dm et l, 25). Finlly, presence of ggregtes in rin sections of FXTS ptients ws confirmed y immunohistochemistry. In rin sections of control ptients, ws diffuse within the nucleoplsm nd no detectle inclusions were found. In contrst, lrge intrnucler ggregtes of were oserved in FXTS ptients (Figure 5C), thus confirming tht expnded repets lter locliztion. protein is prtilly immoilized within expnded repets n RN gin-of-function model for FXTS predicts tht expnded repets should immoilize protein nd depletion y repets ffects lterntive splicing is nucler RN-inding protein with roles in lterntive splicing regultion (Stoss et l, 21; Pronetto et l, 27; Chwl et l, 29). ccording to the RN gin-offunction model, titrtion of free nucler into nucler ggregtes should deplete its functionl ctivity nd result in detectle pre-mrn splicing ltertions. To test this hypothesis, we co-trnsfected constructs encoding expnded repets with minigenes tht recpitulte splicing events directly regulted y. In greement with previous report (Pronetto et l, 27), overexpression of with cl-x minigene repressed the formtion of the long splicing form, cl-xl. We found tht overexpression of expnded repets reproduced depletion of nd stimulted the expression of cl-xl (Figure 7). Next, prlogues SLM1 nd SLM2 re known to regulte the lterntive splicing of exon-7 of the survivl motor neuron-2 (SMN2) pre-mrn (Stoss et l, 24); thus we tested whether lso regultes SMN2 splicing. We found tht overexpression of modestly repressed the inclusion of the exon-7 of n SMN2 minigene, while expression of repets reproduced depletion of nd stimulted the inclusion of tht exon (Figure 7). Finlly, in ioinformtic nlysis, we identified novel exon in intron-28 of the humn TP11 gene, which ws predicted to e specificlly included in the centrl neurl system (Clrk et l 27; Liu et l, 29). We constructed minigene in which TP11 exon-28 nd 3 p of its flnking introns were ordered y -gloin exons nd cotrnsfection experiments showed tht ctivted its inclusion. In contrst, overexpression of repets reproduced depletion of endogenous y shrn nd repressed exon-28 inclusion (Figure 7C). hnrnp-g nd MNL1 re lso recruited within ggregtes, rising the question whether the splicing defects oserved in expressing cells re only due to sequestrtion. To test tht hypothesis, we used mutnt of deleted of its N-terminl domin (DNter), which ws no longer recruited within ggregtes (Supplementry Figure S3), ut ws still le to regulte lterntive splicing (Supplementry Figure S4). Co-trnsfection of DNter rescued the splicing defects cused y expression of repets (Supplementry Figure S4), suggesting tht the splicing ltertions oserved in -expressing cells were mostly due to sequestrtion of The EMO Journl VOL 29 NO 7 21 & 21 Europen Moleculr iology Orgniztion

6 sequestrtion in FXTS ptients C Sellier et l MNL1 Cy3-CCG Merged MNL1 shrn MNL1 GFP-hnRNP G Cy3-CCG Merged GFP-hnRNP G shrn GFP-hnRNP G C sh GPDH D % of co-locliztion 8 CTL sh MNL1 hnrnp G Figure 4 is essentil for recruitment of MNL1 nd hnrnp-g within ggregtes. () COS7 cells were co-trnsfected with plsmid expressing 6 repets nd plsmid expressing either control LcZ shrn (), or shrn (), nd nlysed 72 h fter trnsfection y FISH/IF. Endogenous MNL1 ws detected y IF using n lex-488-lelled secondry ntiody. Simultneous detection of y IF using Cy5-lelled secondry ntiody confirmed shrn-medited depletion of endogenous. () Endogenous hnrnp-g cnnot e detected y FISH/IF. Thus, COS7 cells were co-trnsfected with plsmid expressing 6 repets, plsmid expressing GFP-hnRNP-G nd either control () or () shrn nd nlysed 72 h fter trnsfection y FISH. (C) shrn-medited depletion of endogenous ws confirmed y western lotting ginst. (D) The percentge of endogenous, MNL1 nd GFP-hnRNP-G coloclized within RN ggregtes 72 h fter trnsfection in COS7 cells trnsfected with plsmid expressing either control or shrn. lterntive splicing is ltered in FXTS ptients hs reduced nucler sptil moility nd splicing regultory function in -expressing cells; so we tested whether lterntive pre-mrn splicing is ltered in humn FXTS ptients. RT PCR nlysis of TP11 lterntive splicing in rin smples of control nd FXTS ptients showed significnt decrese of exon-28 inclusion from 47±2% in control to 31±8% in FXTS ptients (Po.5). We confirmed these results y qrt PCR nd found tht expression of TP11 exon-28 is downregulted in FXTS ptients s compred with tht in control (Figure 8). Similrly, we tested y qrt PCR the expression of exon-7 of the SMN2 pre-mrn nd found it under-expressed in FXTS ptients (Figure 8). y contrst, we found no significnt differences in the expression of SMN1 exon-7, which is consistent with no lterntive splicing regultion of tht exon. These results re consistent with lterntive splicing eing ltered in FXTS ptients. Tyrosine phosphoryltion of regultes its recruitment within ggregtes is sustrte of the SIK/RK-tyrosine kinse, which regultes the RN-inding ility nd locliztion of the protein (Derry et l, 2; Hegerth et l, 24; & 21 Europen Moleculr iology Orgniztion The EMO Journl VOL 29 NO

7 sequestrtion in FXTS ptients C Sellier et l C x98 FXTS Cy3-CCG Cy3-CCG FXTS Merged Merged Figure 5 coloclizes with endogenous ggregtes. () rin sections of mouse expressing either control eight repets () or expnded 98 repets () were nlysed y FISH/IF. () Similr to pnel. rin sections (hippocmpl re) of ge-mtched control () or FXTS () ptients were nlysed y FISH/IF. Mgnifiction: 63. Scle r, 1 mm. (C) rin (hippocmpl re) sections of ge-mtched control () or FXTS () ptients were nlysed y immunohistochemistry directed ginst. Mgnifiction: 35. Reltive fluorescence intensity x Prelech s 1 s 4 s -GFP within nucleoplsm () -GFP within ggregtes () Time (s) Figure 6 FRP nlysis uncovers n immoile frction of GFP- within ggregtes. () Photoleching ws performed 24 h fter trnsfection of COS7 cells with GFP- nd plsmid expressing either no repets () or 6 repets (). The white circles denote the photoleched regions in the ggregtes nd the nucleoplsm. Representtive imges show single z-section otined efore photoleching (pre-lech) nd t the indicted time points fter photoleching. () Recovery curves of photoleched ggregtes nd nucleoplsm in cells expressing or 6 repets re shown s reltive fluorescence intensity versus time. In -expressing cells, recovery reched plteu t 6% round 3 s. Ech dt point is the verge of 1 nuclei. The error rs indicte the s.e.m. s. Lukong et l, 25). We thus tested the effect of phosphoryltion on the formtion of ggregte. We found tht expression of SIK/RK disrupted protein locliztion within ggregtes nd returned to free nucleoplsmic locliztion (Figure 9 nd ). is tyrosine-phosphorylted in response to EGF tretment (Lukong et l, 25). We found tht inhiition of the EGFR tyrosine kinse pthwys through tyrphostin/g49 tretment stimulted the recruitment of within ggregtes, leding to formtion of lrge ggregtes t erly time points. y contrst, tretment of trnsfected cells with dephosttin, tyrosine phosphtse inhiitor, reduced the recruitment of within ggregtes (Figure 9 nd ). These dt suggest model in which tyrosine phosphoryltion of, medited y the EGFR-SIK/RK tyrosine kinse pthwy, reduces the recruitment of within ggregtes (Supplementry Figure S6). We confirmed tht phosphoryltion ws stimulted y trnsfection of the SIK/RK kinse or y inhiition of tyrosine phosphtse pthwys through dephosttin tretment. y contrst, inhiition of the EGFR pthwy (tyrphostin/g49 tretment) reduced phosphoryltion (Figure 9C). Tyrosine phosphoryltion of inhiits its splicing ctivity (Pronetto et l, 27). We found tht trnsfection of SIK/RK or dephosttin tretment, oth of which induced tyrosine phosphoryltion (Figure 9C), modified the splicing of TP11 nd mimicked depletion of y or shrn expression (Figure 9D nd dt not shown). Interestingly, we noted no cumultive effects on splicing when -expressing cells were co-trnsfected with SIK/RK or treted with dephosttin. These dt suggest tht either TP11 splicing regultion ws sturted or tht repets cted similrly to SIK/RK or dephosttin, proly through inhiition of ctivity. We oserved similr sence of cumultive effects when repets nd shrn were coexpressed (dt not shown), suggesting tht repets cted on splicing mostly through depletion. Tutomycin reduces the formtion of ggregtes mong the vrious inhiitors of kinses nd phosphtses tht we tested, we found one, tutomycin, which not only reduced colocliztion ut lso reduced ggregte formtion (Figure 1 nd Supplementry Tle 3). y contrst, tutomycin tretment did not impir the formtion 1254 The EMO Journl VOL 29 NO 7 21 & 21 Europen Moleculr iology Orgniztion

8 sequestrtion in FXTS ptients C Sellier et l C x6 sh- x6 sh- x6 sh- xl + ex7 + ex28 xs ex7 ex28 % of cl-xl cl-x % of exon 7 inclusion SMN2 exon 7 % of exon 28 inclusion TP11 exon 28 Figure 7 lterntive splicing is ltered in -expressing cells. COS7 cells were co-trnsfected with cl-x minigene (), n SMN2 exon-7 minigene (), or n TP11 exon-28 minigene (C) nd plsmid expressing either no repet, or 6 repets, or shrn. cl-xl/s, SMN2 nd TP11 splicing isoforms were identified 24 h fter trnsfection y RN extrction followed y RT PCR. Rtio of exon 28/TP TP11 exon 28 FXTS Rtio of exon 7/PO mrn SMN2 exon 7 Figure 8 lterntive splicing is ltered in FXTS ptients. () qrt PCR nlysis of TP11 exon-28 in rin RN smples of three controls nd four FXTS ptients. Results re expressed s the rtio etween lterntive TP11 exon-28 nd constitutive TP11 exons () qrt PCR nlysis of SMN2 exon-7 in rin RN smples from three controls nd four FXTS ptients. We were not le to discriminte etween SMN1 nd SMN2 constitutive exons; therefore, results re expressed s the rtio of the lterntive SMN2 exon-7/po mrn. FXTS of expnded CUG- or UUCU-repet RN ggregtes (dt not shown). We confirmed y qrt PCR tht the quntity of RN contining the expnded repets ws not ltered y tutomycin tretment (Figure 1C), suggesting tht tutomycin disrupts ggregtion without ltering their expression. Finlly, we oserved tht in the presence of tutomycin, TP11 splicing ws no longer ltered y expression of expnded repets (Figure 1D), suggesting tht tutomycin reduced the deleterious effects of the repets on splicing. However nd s noted previously (Mermoud et l, 1992; Novoytlev et l, 28), tretment with tutomycin lone resulted in splicing chnges (Figure 1D), rendering difficult to pinpoint the precise role of tutomycin on the deleterious effects of repets on splicing. Discussion ggregtes re dynmic structures tht recruit vrious RN-inding proteins sequentilly striking chrcteristic of expnded repets is tht they form dynmic intrnucler RN ggregtes tht enlrge with time, resulting in the formtion of gint inclusions, n oservtion tht stnds in contrst to the sence of growth over time of expnded CUG (DM1 muttion), CCUG (DM2) or UUCU (SC1) repets. Continuous enlrgement of RN ggregtes suggests tht these repets my constntly recruit proteins. Consistent with tht hypothesis, we nd others hve found vrious proteins, minly RN-inding proteins, to e cptured y repets in vitro (Iwhshi et l, 26; Jin et l, 27; Sofol et l, 27), suggesting tht repets cn sequester lrge numer of proteins through direct RN protein interctions, ut proly lso through indirect protein protein interctions. When tested in cells, we found tht endogenous, MNL1 nd hnrnp-g coloclized with ggregtes. In contrst, other cndidte proteins such s Pur, FMRP nd CUGP1 did not coloclize with expnded RN, wheres hnrnp-1, hnrnp-2/, hnrnp-c, hnrnp-d, hnrnp-e nd hnrnp-h presented n intermediry sitution with some colocliztion, ut only t very lte time points nd within the gint ggregtes tht form in dying cells. This questions whether these other cndidte proteins re recruited specificlly t the very lst step of ggregte formtion, or tht they form non-specific ggregtes in dying cells. We tested the locliztion of some of these cndidtes y FISH/IF in rin sections of control nd FXTS ptients. & 21 Europen Moleculr iology Orgniztion The EMO Journl VOL 29 NO

9 sequestrtion in FXTS ptients C Sellier et l Cy3-CCG Merged Dephosttin G 49 SIK-YF 447 c % of co-loc SIK G49 Dephosttin C SIK Dephosttin G49 P- D SIK G49 Dephosttin CTL CTL CTL CTL + Ex 28 Ex 28 6 % of exon 28 inclusion SIK G49 Dephosttin Figure 9 Tyrosine phosphoryltion reduces colocliztion within ggregtes. () COS7 cells were trnsfected with plsmid expressing 6 repets nd either co-trnsfected with plsmid expressing constitutively ctive YF447 mutnt of the SIK/rk kinse (), or treted with 1 mm of tyrphostin/g49 () or 2 mm dephosttin (c) nd nlysed y FISH/IF 24 h fter trnsfection. () The percentge of endogenous coloclized within RN ggregtes in trnsfected COS7 cells 24 h fter trnsfection. (C) Similr to pnel ut phosphoryltion of ws ssyed y immunoprecipittion followed y western lotting using -Y44 phospho-specific ntiody. (D) Similr to pnel ut COS7 cells were lso co-trnsfected with n TP11 minigene. Inclusion of exon-28 ws quntified 24 h fter trnsfection y RN extrction followed y RT PCR. First, we tested Pur, ut could only detect smll quntities of Pur within the neuronl intrnucler inclusions in sections from humn FXTS rin s compred with undnt cytoplsmic lelling in the sme neurons, which suggests tht Pur is still ville for its cellulr function. This is in greement with recent report showing sence of Purpositive inclusions in FXTS mouse model expressing 9 repets (Hshem et l, 29). However, Pur ws found 1256 The EMO Journl VOL 29 NO 7 21 & 21 Europen Moleculr iology Orgniztion

10 sequestrtion in FXTS ptients C Sellier et l Cy3-CCG Merged Tutomycin CCG CCG % of ggregtes C Rtio of /Neo µm 1 µm Tutomycin Tutomycin D % of exon 28 inclusion Tutomycin CTL CTL Tutomycin + Ex 28 Ex 28 Figure 1 Tutomycin olishes nd ggregte formtion. () COS7 cells were co-trnsfected with plsmid expressing 6 repets nd either treted with solvent () or 1 mm of tutomycin (), then nlysed y FISH/IF 24 h fter trnsfection. () The percentge of cells contining RN ggregtes in trnsfected COS7 cells treted with solvent,.3 or 1 mm of tutomycin. (C) Similr to pnel ut followed y RN extrction 24 h fter trnsfection nd qrt PCR quntifiction of the repets RN nd of the neomycin mrn. nd neomycin cssettes re expressed from the sme pcdn3.1 plsmid, ut under different promoters. (D) Similr to pnel or C ut COS7 cells were lso co-trnsfected with n TP11 minigene. Inclusion of exon-28 ws identified 24 h fter trnsfection y RN extrction followed y RT PCR. to coloclize with HSP7 inclusions in -expressing Drosophil nd in single reported humn inclusion (Jin et l, 27). possile explntion is tht Pur, which is cytoplmic, is not found for the most prt in ggregtes, which re strictly intrnucler in the mmmlin cells nd FXTS ptients tht we nlysed (Greco et l, 26). In contrst, in -repet-expressing Drosophil, sustntil frction of the HSP7 inclusions were cytoplsmic nd found to coloclize with Pur (Jin et l, 23, 27). Next, we found tht MNL1, which is recruited trdily within ggregtes in trnsfected cells, lso coloclizes with inclusions in FXTS ptients (Supplementry Figure S5, Iwhshi et l, 26). However, we found tht the splicing events regulted y MNL1 re not ltered in -expressing cells or in FXTS ptients (Supplementry Figure S5). These results suggest tht, while MNL1 is present within ggregtes, it is not immoilized nd does not lose its splicing function. We lso tested the presence of hnrnp-g within ggregtes in FXTS ptients, ut none of the nti-hnrnp-g ntiodies tht we tested resisted the FISH conditions. Thus, whether hnrnp G coloclizes with ggregtes in FXTS ptients, remins to e determined. Finlly, we found tht, while very lte recruited within ggregtes, nd only in dying cells, oth hnrnp-1 nd hnrnp-2/1 were present in the ggregtes of FXTS ptients (dt not shown; Iwhshi et l, 26). y contrst, little or no hnrnp-2/1 ggregtes were oserved in KI mice (Hshem et l, 29 nd R Willemsen, personl communiction), which is consistent with smller ggregtes in mice s compred with tht in humns. hnrnp-1 regultes the lterntive splicing of exon-7 of the PP pre-mrn (Donev et l, 27), ut we found no splicing ltertions of PP in FXTS ptients (Supplementry Figure S5), suggesting tht, similr to MNL1, recruitment of hnrnp-1 within ggregtes my not impir its splicing-regultory function. The presence of hnrnp-1 nd hnrnp-2/1 ggregtes only in FXTS ptients is reminiscent of the uiquitin sitution, where no uiquitin ggregtes re found in -trnsfected cells, while uiquitin-positive inclusions re hllmrk of FXTS ptients (Greco et l, 22; rocen & 21 Europen Moleculr iology Orgniztion The EMO Journl VOL 29 NO

11 sequestrtion in FXTS ptients C Sellier et l et l, 25). Overll, these dt suggest tht ccumultion of some proteins, such s MNL1, hnrnp-1, hnrnp-2 nd uiquitinylted proteins, in ggregtes of rin from FXTS ptients is very lte-onset event, which my not result in sequestrtion nd loss of function of these proteins. nucletion model In contrst to lte recruited proteins, we found tht is erly nd constntly ssocited with ggregtes. Furthermore, we found tht depletion of olished the recruitment of MNL1 nd hnrnp-g within ggregtes nd reduced the formtion of gint ggregtes. These results suggest tht is essentil for recruitment of novel proteins nd the continuous enlrgement of the ggregtes. is involved in vrious spects of mrn metolism nd contins severl domins enling protein protein interctions, llowing to interct with vrious RN-inding proteins, including hnrnp-g, hnrnp-1, Tr2 (SFRS1), Slm1 nd Slm2 (T-Str) (Venles et l, 1999, 2; Pronetto et l, 27). Co-trnsfection of these cndidtes with expnded repets showed tht Slm1, Slm2 nd Tr2 coloclized with lrge ggregtes 48 h fter trnsfection. We propose tht these proteins cn in turn ssocite with other proteins such s MNL1 nd proly mny others, ultimtely resulting in mssive protein ggregtion disstrous for norml cell function nd viility (Supplementry Figure S6). This sequentil recruitment model is consistent with the lte colocliztion of hnrnp-, hnrnp-g nd MNL1 in trnsfected cells, nd with the lrge intrnucler inclusions, cell deth nd glol cererl nd cereellr trophy oserved in FXTS ptients (Greco et l, 22, 26; Tssone et l, 24). Such model of sequentil protein recruitment within ggregtes implies founding nd nucleting RN protein interction event tht would susequently trp other proteins through indirect RN protein or protein protein interctions. We propose tht is prt of this founding event, s its depletion y shrn inhiits susequent protein ggregtion nd suppresses the formtion of gint ggregtes. However, does not ind directly to RN (dt not shown), nd its locliztion within ggregtes requires its N-terminl domin (Supplementry Figure S3), which contins numer of protein protein interction domins. These dt suggest tht ssocition of with repets it not direct ut requires n intermediry RN-inding protein (Supplementry Figure S6). Chrcteriztion of tht protein is ongoing (C Sellier, in preprtion), ut eyond the scope of this pper. protein is prtilly sequestered nd functionlly inctivted in FXTS ptients The RN gin-of-function model for FXTS predicts tht protein should e sequestered y RN repets nd consequently lose its ctivity. FRP nlysis showed tht there is significnt decrese in the moile frction of in -trnsfected cells, suggesting tht frction of nucler protein is immoilized within ggregtes. Interestingly, the proportion of immoilized in trnsfected cells is identicl to the frction of MNL1 immoilized in CUG-trnsfected cells (Ho et l, 25). Through nlogy with DM, this shows prtil depletion of free in -expressing cells, nd hence confirms n RN gin-offunction model for FXTS. is involved in vrious spects of mrn metolism, such s lterntive splicing regultion, nucler export, somtodendritic trnsport, polydenyltion nd trnsltion (reviewed y Lukong nd Richrd, 23). sirn depletion of impirs neuronl differentition in cell cultures (Chwl et l, 29), nd its ltion in mouse leds to motor coordintion defects (Lukong nd Richrd, 28), suggesting tht prtil sequestrtion nd loss of function might e involved in the progressive intention tremor nd git txi oserved in FXTS ptients. One of the importnt functions of is in lterntive splicing regultion (Stoss et l, 21; Pronetto et l, 27; Chwl et l, 29). We found mis-regultion of pre-mrn lterntive splicing controlled y in -trnsfected cells nd in FXTS ptients. Notly, nlysis of lterntive splicing of TP11 pre-mrn showed splicing mis-regultion similr in FXTS ptients nd in -depleted cells y shrn trnsfection. These results re consistent with prtil loss of function of in -trnsfected cells nd in FXTS ptients. Phospho-type-4 TPse-11 (TP11) is puttive flippse predicted to ctlyse minophospholipid trnsport nd crete lipid symmetry in lte secretory nd endocytic comprtments. Exclusion of exon-28 results in protein isoform with distinct C-terminl prt. The physiologicl functions of this splice form remin to e determined. Next, we tested other splicing events regulted y (Chwl et l, 29). However, we found no significnt ltertions of lterntive splicing of the KTN1, IN1, DNCIC2, CLSP2 nd SGCE2 pre-mrns in rin smples of FXTS ptients, possily due to differences etween humn ptients nd murine cells where they were identified s trgets (Chwl et l, 29). Furthermore, we found no significnt ltertions of TP11, IN1, DNCIC2 nd CLSP2 lterntive splicing in rin smples of - knockout mice, proly due to the compenstory effects of prlogues, Slm1 nd Slm2, which re highly expressed in rin (Stoss et l, 24). We lso tested the lterntive splicing of cl-x nd CD44 pre-mrns in FXTS ptients (Mtter et l, 22; Pronetto et l, 27). However, trnscripts contining the exon-v5 of the CD44, or corresponding to the cl-x Short isoform, were not expressed in the rin smples tht we tested. Finlly, we tested the lterntive splicing of the SMN1 nd SMN2 pre-mrns. Loss of the SMN1 gene is responsile for spinl musculr trophy (SM), the most common inherited motor neuron disese. The ner-identicl SMN2 gene does not compenste the deficiency of SMN1 due to skipping of SMN2 exon-7 through lterntive splicing. We found tht wekly regultes the lterntive splicing of n SMN2 minigene nd inhiits the inclusion of exon-7. However, we found in FXTS ptients decresed expression of SMN2 exon- 7 ws, which is contrdictory with depletion of function in FXTS. SMN2 exon-7 inclusion is roustly stimulted y hnrnp G nd Tr2 (Hofmnn nd Wirth, 22; Heinrich et l, 29), which re oth lte recruited within ggregtes in COS7-trnsfected cells. Whether hnrnp-g nd Tr2 re sequestered within ggregtes nd lose their splicing functions in FXTS ptients remin to e determined. Furthermore, whether decrese of SMN2 exon- 7 in FXTS ptients results in lower quntity of SMN protein 1258 The EMO Journl VOL 29 NO 7 21 & 21 Europen Moleculr iology Orgniztion

12 sequestrtion in FXTS ptients C Sellier et l nd is involved in the neuronl deth oserved in these ptients remins lso to e determined. Tutomycin inhiits the formtion of ggregtes ctivity is regulted y phosphoryltion (Derry et l, 2; Hegerth et l, 24; Lukong et l, 25), chrcteristic tht mde the prototype of the Signl Trnsducers nd ctivtors of RN (STR) fmily, which trnsduces informtion from signlling pthwys to mrn metolism. Consistent with such function, we found tht tyrosine phosphoryltion of reduces its recruitment within ggregtes nd, consequently, the deleterious effects of the repets on splicing regultion (Supplementry Figure S6). y contrst, we found tht ctivtion of the MP serine/threonine kinse pthwy stimultes recruitment within ggregtes. However, we filed to detect direct phosphoryltion of due to the poor qulity of the serine nd threonine ntiodies tht we tested (Supplementry Figure S7). Strikingly, mong the vrious drugs tested, we found one, tutomycin, which not only prevents colocliztion with ggregtes, ut lso reduces ggregte formtion. We found tht tutomycin prevents the deleterious effects of the repets on lterntive splicing regultion, ut due to the inherent toxicity of tutomycin, we were not le to ssess whether tutomycin lso reduces the toxicity of the repets. y contrst to tutomycin, depletion of y shrn does not lter the formtion of ggregtes, suggesting tht tutomycin my ct upstrem from, mye on the unchrcterized protein tht ridges repets nd (Supplementry Figure S6). Tutomycin ws first descried s n inhiitor of the serine/threonine PP1 phosphtse, nd more recently s n inhiitor of the glycogen synthse kinse-3 (GSK-3) (dler et l, 29) nd of the Rf1 pthwy (Pinchot et l, 29). Whether formtion of ggregtes requires the PP1, GSK- 3 or Rf pthwy remins to e determined. In conclusion, our results support n RN gin-of-function mechnism in which is prtilly sequestered within ggregtes nd consequently loses its regultory function in neurons from FXTS ptients. Furthermore, our dt suggest tht ggregtes cn e dispersed, thus, ringing hope of drug tretments le to reduce ggregte formtion in FXTS ptients. Mterils nd methods Plsmids nd constructions Plsmids expressing 2, 4 or 6 repets were constructed y ligtion of oligonucleotides contining 2 repets in pcdn3.1. The plsmid expressing 98 repets ws descried previously (rocen et l, 25). TP11 exon-28 ws mplified using primers Fwd: 5 -CTTGCCCTTCTTGGTGG CTG nd Rev: 5 -CTTGGTTGTGGTTCTT CCGC, using C RP11-36G17 s templte, nd cloned within the vector pxj41. WT nd mutnts, Slm1, Slm2 nd hnrnp-g expression plsmids were descried previously (Venles et l, 24). Cell cultures, trnsfections nd tretments COS7 cells were cultured in Dulecco s modified Egle s Medium (DMEM), 1% foetl ovine serum nd gentmicin t 371C in 5% CO 2. PC12 were cultured in DMEM, 1% horse serum, 5% foetl clf serum nd penicillin t 371C, 5% CO 2. Cells were plted on glss coverslips in 24-well plte for immunofluorescence nd trnsfected 24 h fter plting in DMEM þ.1% foetl ovine serum to lock cell divisions, using either FugenHD (Roche) for COS7 cells or Lipofectmine 2 (Invitrogen) for PC12 cells. PC12 cells were differentited 6 h fter trnsfection y growing cells in 1% horse serum, 1% foetl clf serum plus 5 ng/ml of NGF (Clinisciences). Primry cultures of hippocmpl neurons were otined from WT E18 mouse nd grown into 24-wells pltes in 5 ml neurosl medium (Gico), 1 27 (Gico),.5 mm L-glutmine nd penicillin t 371C, 5% CO 2, nd were trnsfected fter 4 DIV with Lipofectmine 2 (Invitrogen). Cell tretments Cells were incuted 6 h fter trnsfection in 5 mm PD9859, 4 nm okdic cid, 4 ng/ml TP, 1 mm G49, 2 mm dephosttin or 1 mm tutomycin (Cliochem). Ptients nd rin sections FXTS ptients hve een descried previously (cse 6, 7 9 nd 1 of Greco et l, 26). KI mouse (72 weeks old, 98 repets) or humn rin sections were deprffinized two times for 2 min in Histosol Plus (Shndon) nd dehydrted s follows: twice in ethnol 1% (5 min), twice in ethnol 95% (5 min), once in ethnol 8% (5 min), once in ethnol 7% (5 min) nd rinsed in TS-Tween 1% efore FISH. FISH comined with immunofluorescence Glss coverslips contining plted cells or rin sections on slides treted s descried ove were fixed in 4% prformldehyde in PS (ph 7.4) for 15 min nd wshed three times with PS. The coverslips were incuted for 5 min in PS/.5% Triton X-1 nd wshed three times with PS efore pre-hyridiztion in 4% DMSO, 4% formmide, 1% S (1 mg/ml), 2 SCC for 3 min. The coverslips were hyridized for 2 h in 4% formmide, 1% DMSO, 2 SCC, 2 mm vndyl rionucleoside, 6 mg/ml trn, 3 mg/ml S plus.75 mg (CCG) 8x -Cy3 DN oligonucleotide proe (Sigm). The coverslips were wshed twice in 2 SCC/5% formmide nd twice in 2 SCC. Following FISH, the coverslips were wshed twice successively in 2 SCC/5% formmide, in 2 SCC nd in PS. The coverslips were incuted overnight with primry nti-hnrnp-g (1/2 dilution; Heinrich et l, 29), hnrnp-2/1 (1/2 dilution, clone DP33; Teu), hnrnp-1 (1/2 dilution, clone 9H1; cm), MNL1 (1/2 dilution, clone HL E9; Sigm) nd (1/4 dilution, C2 SC-333; Snt Cruz iotechnology) t 41C. The coverslips were wshed twice with PS efore incution with got nti-rit secondry ntiody conjugted with lex-fluor 488 (1/5 dilution; Fisher) or Cy5 (1/5 dilution; Interchim) for 6 min. Then, the coverslips were incuted for 1 min in 2 SCC/DPI (1/1 dilution) nd rinsed twice in 2 SSC efore mounting in Pro-Long medi (Moleculr Proes). Slides were exmined using either simple fluorescence microscope (Leic) or Leic DM4 confocl microscope, equipped with Leic 1 HCX Pln po CS 1.4 ojective, in 1-mm opticl sections. FRP nlysis COS7 cells were plted in 35-mm glss se dish (Iwki) nd trnsfected with Fugene HD 24 h fter plting. Twenty-four hours fter trnsfection, FRP experiments were performed using Leic DM4 confocl microscope comined to heted stge. The cells were mintined for 15 min in growth medi t 371C with no CO 2 or humidifier systems; five single scns were otined followed y five lech pulses. fter photoleching, imges were tken every 1 s for 15 s (post-lech 1) followed y 4 cquisitions every 2 s (post-lech 2). Fluorescence intensities were clculted using the Imge J softwre nd normlized y totl cellulr fluorescence intensity. s foci re moile, n re round the foci ws determined for the durtion of the experiment. MLDI nlysis Nucler extrcts were prepred from COS7 cells or mouse rin extrct s descried y Dignm et l (1983). Nucler extrct ws pssed over 6x in vitro T7 trnscried nd iotinylted RN (mion) ound to streptvidin grose column (Invitrogen) in the presence of KCl (1 mm), HEPES (1 mm) nd MgCl 2 (1 mm). The column ws wshed with 33 mm MgCl 2 nd glcil cetic cid. The eluted protein ws seprted y gel electrophoresis nd detected y silver stining. The protein nds were excised, & 21 Europen Moleculr iology Orgniztion The EMO Journl VOL 29 NO

13 sequestrtion in FXTS ptients C Sellier et l digested nd identified using Reflex IV MLDI-TOF spectrometer (ruker Dltonics) nd the Profound serch engine, s descried y rgentini et l, 28. RN isoltion nd PCR COS cells were cultured s descried ove in six-well plte. RN ws isolted 24 h fter trnsfection using GenElute kit (Sigm). Endogenous TP11 splicing nlysis ws performed on totl RN extrcted (Trizol regent) from control or FXTS ptient rin smples. cdn synthesis rections were performed with Superscript II (Invitrogen). The primers re descried in Supplementry Tle 4. PCRs were performed with Tq polymerse (Roche). The conditions for SMN2 nd cl-x minigenes were descried previously (Pronetto et l, 27; Heinrich et l, 29). The conditions for TP11 minigene were 4 min t 941C; 3 cycles of 4 s t 941C, 45 s t 61C nd 1 min t 721C; nd finl extension t 721C for 4 min. The PCR rection products were nlysed on 8% polycrylmide gels. Sttisticl nlysis The percentge of endogenous, MNL1 nd hnrnp-g coloclized within RN is expressed s the numer of nuclei presenting colocliztion/totl numer of nuclei contining ggregtes. Three independent trnsfections totlling hundred cells were counted. Results re presented s men±s.d. The lnce etween the mrn levels of the SMN2, cl-x nd TP11 splicing vrints is clculted s ((mrn þ exon)/ (mrn exon þ mrn þ exon)) 1. The results re derived from t lest three independent experiments. The error rs in the figures indicte the s.e.m. s. Sttisticl differences were clculted using t-test. Supplementry dt Supplementry dt re ville t The EMO Journl Online ( cknowledgements We thnk the imgery, cell culture nd MLDI pltforms of the IGMC; Thoms Cooper for the gift of the DT96 nd INSR minigene plsmid; Lur Rnum for the gift of the CCTG3 expression plsmid; Chrles Thornton for the gift of the polyclonl MNL1 ntiody; ngel Tyner for the gift of the SIK/rk constructs; Cludio Sette for the gift of the cl-x, sh nd WT nd mutnt constructs; Stefn Stmm for the gift of the SMN2 minigene; Stefn Huner for the gift of the GFP-lmin- construct; nd ll memers of the French DM Network for fruitful discussion. This work ws supported y INSERM VENIR funding (NC), NR GENOPT grnt P7942 (NC), NIH-NINDS grnt NS62411 (RW), NIH Rodmp Inititive DE19583 nd G32119 (PJH), SRC grnt /D13917/1 (DJE), Wellcome Trust grnt numer WT8368M (DJE) nd n ORSS interntionl studentship (YL). Conflict of interest The uthors declre tht they hve no conflict of interest. References dler JT, Cook M, Luo Y, Pitt SC, Ju J, Li W, Shen, Kunnimliyn M, Chen H (29) Tutomycetin nd tutomycin suppress the growth of medullry thyroid cncer cells vi inhiition of glycogen synthse kinse-3et. Mol Cncer Ther 8: rgentini M, Stru JM, Crpito C, Snglier S, Vn-Dorsseler (28) n optimized MLDI mss spectrometry method for improved detection of lysine/rginine/histidine free peptides. J Proteome Res 7: rocen DG, Iwhshi CK, Won N, eilin, Ludwig L, Tssone F, Schwrtz PH, Hgermn PJ (25) Induction of inclusion formtion nd disruption of lmin /C structure y premuttion repet RN in humn cultured neurl cells. Humn Mol Genet 14: rouwer JR, Huizer K, Severijnen L, Hukem RK, ermn RF, Oostr, Willemsen R (28) repet length nd neuropthologicl nd moleculr correltes in mouse model for frgile X-ssocited tremor/txi syndrome. J Neurochem 17: Chwl G, Lin CH, Hn, Shiue L, res Jr M, lck DL (29) regultes set of lterntively spliced exons during neurogenesis. Mol Cell iol 29: Clrk T, Schweitzer C, Chen TX, Stples MK, Lu G, Wng H, Willims, lume JE (27) Discovery of tissue-specific exons using comprehensive humn exon microrrys. Genome iol 8: 64 Derry JJ, Richrd S, Vlderrm Crvjl H, Ye X, Vsioukhin V, Cochrne W, Chen T, Tyner L (2) SIK (RK) phosphoryltes in the nucleus nd negtively regultes its RN inding ility. Mol Cell iol 2: Dignm JD, Leovitz RM, Roeder RG (1983) ccurte trnscription initition y RN polymerse II in solule extrct from isolted mmmlin nuclei. Nucleic cids Res 11: Donev R, Newll, Thome J, Sheer D (27) role for SC35 nd hnrnp1 in the determintion of myloid precursor protein isoforms. Mol Psychitry 12: Greco CM, ermn RF, Mrtin RM, Tssone F, Schwrtz PH, Chng, Trpp D, Iwhshi C, runerg J, Grigsy J, Hessl D, ecker EJ, Ppzin J, Leehey M, Hgermn RJ, Hgermn PJ (26) Neuropthology of frgile X-ssocited tremor/txi syndrome (FXTS). rin 1: Greco CM, Hgermn RJ, Tssone F, Chudley E, Del igio MR, Jcquemont S, Leehey M, Hgermn PJ (22) Neuronl intrnucler inclusions in new cereellr tremor/txi syndrome mong frgile X crriers. rin 125: Hegerth, Hep D, ie W, Derry JJ, Richrd S, Tyner L (24) The nucler tyrosine kinse RK/SIK phosphoryltes nd inhiits the RN-inding ctivities of the -like mmmlin proteins SLM-1 nd SLM-2. J iol Chem 279: Hgermn PJ, Hgermn RJ (24) The frgile-x premuttion: mturing perspective. m J Hum Genet 74: Hgermn RJ, Leehey M, Heinrichs W, Tssone F, Wilson R, Hills J, Grigsy J, Gge, Hgermn PJ (21) Intention tremor, prkinsonism, nd generlized rin trophy in mle crriers of frgile X. Neurology 1: Hshem V, Gllowy JN, Mori M, Willemsen R, Oostr, Pylor R, Nelson DL (29) Ectopic expression of contining mrn is neurotoxic in mmmls. Hum Mol Genet 18: Heinrich, Zhng Z, Ritskin O, Hiller M, endersk N, Hrtmnn M, rcco L, Elliott D, en-ri S, Soreq H, Sperling J, Sperling R, Stmm S (29) Heterogeneous nucler rionucleoprotein G regultes splice site selection y inding to CC(/C)-rich regions in pre-mrn. J iol Chem 284: Ho TH, Svkur RS, Poulos MG, Mncini M, Swnson MS, Cooper T (25) Colocliztion of musclelind with RN foci is seprle from mis-regultion of lterntive splicing in myotonic dystrophy. J Cell Sci 118 (Prt 13): Hofmnn Y, Wirth (22) hnrnp-g promotes exon 7 inclusion of survivl motor neuron (SMN) vi direct interction with Htr2- et1. Hum Mol Genet 11: Iwhshi CK, Ysui DH, n HJ, Greco CM, Tssone F, Nnnen K, ineu, Lerill C, Hgermn RJ, Hgermn PJ (26) Protein composition of the intrnucler inclusions of FXTS. rin 129: Jcquemont S, Hgermn RJ, Leehey M, Grigsy J, Zhng L, runerg J, Greco C, Des Portes V, Jrdini T, Levine R, erry- Krvis E, rown WT, Scheffer S, Kissel J, Tssone F, Hgermn PJ (23) Frgile X premuttion tremor/txi syndrome: moleculr, clinicl, nd neuroimging correltes. m J Hum Genet 4: Jcquemont S, Hgermn RJ, Leehey M, Hll D, Levine R, runerg J, Zhng L, Jrdini T, Gne LW, Hrris SW, Hermn K, Grigsy J, Greco CM, erry-krvis E, Tssone F, Hgermn PJ (24) Penetrnce of the frgile X-ssocited tremor/txi syndrome in premuttion crrier popultion. JM 4: The EMO Journl VOL 29 NO 7 21 & 21 Europen Moleculr iology Orgniztion