eerhGopPstasrpinlCnrlo eeEpeso,DKFZ-ZMBH Germany. Expression, 69120, Heidelberg of Alliance, Control Posttranscriptional Group Research Spain. Biologı erlg,Wsigo nvriy t oi,M 31,USA. 63110, MO Louis, St. University, Washington Neurology, atne -90,Spain. E-39008, Santander h M uo I-iedmi skyt m1adcoilin and Tapia biogenesis Olga SmD1 body to Cajal key to is and domain interactions SIM-like Tudor SMN The REPORT SHORT ß eevd2 uy21;Acpe 4Dcme 2013 December 14 Accepted 2013; July 25 Received " Scripps USA. The 92037, Biology, CA Jolla, Molecular La and Institute, Cell Research of Department address: *Present to conjugates al., SMN et 1 Endogenous Nizami 2013). 2005; al., Lamond, et and Machyna 2010; (Cioce pre- in processing involved scaRNPs) mRNA Cajal and and ( nuclear small ribonucleoproteins and body (SMN) neuron motor survival 2007). Ramo (Kerscher, SUMO1 phosphorylated for and/or affinity the acidic al., ensure by that et (V/I- residues, that flanked core hydrophobic (Song generally a (SIMs) contain SUMO1 SIMs x-V/I-V/I), 2006). motifs al., with et Some Hecker interaction 2004; SUMO-interacting 2013). Melchior, non-covalent also and mediate Flotho have 2005; substrates (Hay, ( sequence protein consensus lysine target 2013). a a C-terminal and to the (SUMO) corresponding Melchior, modifier residue between ubiquitin-related small occurs and of glycine modification (Flotho SUMO and Covalent stability interactions localization, subcellular protein–protein that activity, modification protein post-translational regulates important an is Sumoylation INTRODUCTION complex Sm be SUMO1, SIM, can SMN, body, dysfunction Cajal WORDS: their KEY and biogenesis pathophysiology. CB SMA CB in interactions involved prevents for SIM-like-mediated important and SMN SNRPD1), are the interaction as Accordingly, known SMN– (also assembly. SmD1 decreases spliceosomal modifier the severely interaction the with ubiquitin-related abolishes SMN small constructs of The mutant a domain. SIM-like Tudor of has the expression in that motif (SIM)-like target motif SMN (SUMO)-interacting SUMO1 that survival demonstrate a occurs we the Here, assembly is the (SMA). CB atrophy and of muscular ribonucleoproteins in spinal Dysfunction snRNPs in (SMN). nuclear involved protein coilin, neuron organelles small concentrate motor They nuclear spliceosomal (snRNPs). are of (CBs) maturation bodies Cajal ABSTRACT *uhrfrcrepnec ([email protected]) correspondence for **Author Marı eateto ntm n elBooy nvriyo Cantabria-IFIMAV, of University Biology, Cell and Anatomy of Department hs uhr otiue qal oti work. this to equally contributed authors These 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,9996doi:10.1242/jcs.138537 939–946 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. aa ois(B)aencerognle icvrdby discovered organelles nuclear are (CBs) bodies Cajal aT Berciano T. ´a ´ ´ oeua eeoOha SC nvria Auto Universidad CSIC, Ochoa, Severo Molecular a aa Lfrae l,20) hyaeerce ncoilin, in enriched are They 2009). al., et (Lafarga Cajal y n 1, * , " aeaLafarga Vanesa , 1 2 eatmnod Biologı de Departamento § rsn drs:Dprmn of Department address: Present { rsn drs:HlhlzJunior Helmholtz address: Present 2, { , " oi Bengoechea Rocio , ´ oeua,Cnr de Centro Molecular, a ´ oa 84 Madrid, 28049 noma, y x/)i the in KxE/D) 1,§ n Palanca Ana , M sanwSM1tre.W ute hwta M a a has SMN that that show report further we We Here, target. SUMO1 2008). new al., a is et SMN (Navascues SUMO1 (Lefebvre active mortality 2011). infantile of al., 1997). cause et al., genetic et (Fischer major maturation a (SMA), their localize of the they culminate of where biogenesis Disruption produce to nucleus, complex to the CBs snRNAs the into SMN the to imported on the are in family that Sm , snRNPs involved the the of is In assembles snRNPs. which the form spliceosomal to complex, STRAP) as known SMN (also Unrip and Gemin2–Gemin8 Basml n ol oetal eivle nSMA in involved be domain. Tudor potentially the Sm in mutations could the point controlling by of for and caused essential SNRPD1) pathophysiology is assembly as binding interaction known its This CB (also coilin. regulates and protein that complex domain SmD1 Tudor the the with in motif SIM-like cetrlsn ihnacnessst FK1--)wt a SUMO with a (F-K119-R-E) site of consensus presence a the within lysine (http://sumosp. revealed acceptor 2.0 servers SUMOsp biocuckoo.org) and the that www.abgent.com/tools) bodies. suggests of these CBs 2012) in localization al., occur in et can the (Schulz events However, detected CBs desumoylation in are sites. not USPL1 they sumoylation isopeptidase that was Interestingly, indicating the S1M–R), 1K). Ubc9 not Fig. (Fig. material enzyme with assay was (supplementary interaction conjugating sumoylation interact This a 1I,J). the GFP–SMNwt (Fig. with manner and covalent validated a endo- SMN in co- both SUMO1 by that monomeric Next, demonstrated 1A–H). genous of we (Fig. some assays, SUMO1 CBs, immunoprecipitation for canonical that to immunoreactive showed SUMO1 recruited them or always complex Sm was GFP- the co-immunostain- GFP–SMNwt either a (GFP–SMNwt), and Thus, coilin with experiments. construct for ing mutagenesis transfected SMN for CBs cells human here in used (wt) MCF7 SUMO1 of wild-type in localization tagged confirmed The whether also target. investigate SUMO1 to was in a us (supplementary not is prompted This SMN but bodies S1D–L). Fig. bodies, SMN-negative only material SUMO1 nuclear and active SMN-containing that coilin-positive investigate that observed in we cells Fig. to cells, concentrated UR61 material UR61 (supplementary and us In SUMO1 293T in SMN S1A–C). prompted enriched MCF7, and are coilin in endogenous CBs CBs in some confirmed of particularly of We CBs, presence in proteins. subset targets the SUMO a putative showing in results SUMO1 previous substrate SUMO1 Our novel a is SMN DISCUSSION AND RESULTS rvosy ehv hw htasbe fCsconcentrates CBs of subset a that shown have we Previously, iifrai nlsso ua M ihSMpo (http:// SUMOplot with SMN human of analysis Bioinformatic 1 iulLafarga Miguel , SMN1 eepoue pnlmsua atrophy muscular spinal produces gene 1, *and ** 939

Journal of Cell Science HR REPORT SHORT 940 15 bar: Scale CBs. substrate. SUMO1-immunoreactive SUMO1 showing a GFP–SMNwt is SMN 1. Fig. oimnpeiiainasy *M–UO ope) K uolto sa ftercmiatMcSNpoen L rti euneo h human the of sequence Protein (L) protein. Myc–SMN recombinant the of assay domain. Sumoylation Tudor (K) SMN complex). (*SMN–SUMO1 assays co-immunoprecipitation AH muotiigfrcii n nNs(–) rcii n UO EH nMF el expressing cells MCF7 in (E–H) SUMO1 and coilin or (A–D), snRNPs and coilin for Immunostaining (A–H) m .(,)Itrcin fbt noeosSNadGPSNtwt UO in SUMO1 with GFP–SMNwt and SMN endogenous both of Interactions (I,J) m. ora fCl cec 21)17 3–4 doi:10.1242/jcs.138537 939–946 127, (2014) Science Cell of Journal

Journal of Cell Science aoia Is(oge l,20) ecl tSIM-like. it call we proposed the 2004), to al., exactly et conform (Song not that Given SIMs does were 1L). sequence canonical (Fig. which species a for Y130), mammalian such residues and several putative Y127 in conserved and T128, (T122, E134-E135) phosphorylation (E121, by residues flanked (V124-V125-V126) acidic of residues GPS-SBM consisting valine SIM consecutive the three a Furthermore, identified sumoylation. server (http://sbm.biocuckoo.org) for probability high REPORT SHORT 15 n 14/15/16)adwt nices in the increase in an changes (V124A/ correlate with hydrophobicity to designing First, and of (V124I/V125I). by V124A/V125A/V126A) hydrophobicity loss analysis and a SIM-like bioinformatics V125A with SMN a mutations the performed theoretical of properties we biophysical motif, the protein– determine its capacity To reduces aggregation motif and SIM-like affinity SMN protein in hydrophobicity of Loss biogenesis. CB SUMO in with involved SMN proteins of the target interaction of other inefficient structure or an the by it in or consider modifications domain by Tudor we explained context, SIM-like be SMN an in could this disruption mutants plays CB In the results K119, whether assembly. investigate the to CB these important not in but Collectively, sequence, role important SIM-like S1). the 2G,H; transfection that Table (Fig. identified all analysis material in quantitative which distribution by supplementary phenotype, CB confirmed in were normal SIM-like Changes experiments the 2E,F). the (Fig. rescue with of relocalization coilin intranucleolar and CBs to transfections of loss dramatic unable a contrast, displayed essential were In not is assembly. mutants residue phenotype K119 CB lysine nuclear the for that normal construct indicating GFP–SMNwt 2C,D), the the with (Fig. transfected nucleolar rescued cells MCF7 in cells and observed SMN-depleted loss in CB 2012). causes al., et patients (Tapia motor coilin human of SMA relocalization in depletion from SMN that neurons and observation 2011) recent Hebert, and studies our Gilder 2010; with previous al., experimental et (Tapia the with CBs upon of disruption coilin consistent of accumulations is nucleolar 2006). showing reorganization al., et coilin (Lemm cells HeLa This in reported Fig. previously in as and material S2B,C), (supplementary the microfoci within coilin nucleoplasmic of numerous relocalization and CB of including by expression, loss coilin confirmed in changes was dramatic induced SMN siRNA depletion S2A). Fig. the material supplementary 2B; of (Fig. efficiency immunoblotting The 3 (siRNA). a with RNA SMN endogenous from depleted alanine hydrophobic minor the 2A). arginine (Fig. for GFP–SMN-V124/125/126A) for or sequence (GFP–SMN-V124/125A residues 119 residues valine SIM-like three GFP–SMNwt lysine the or the replacing two of replacing assembly, by by or CB mutated (GFP-SMN-K119R) in was SMN–SUMO1 involved construct non-covalent or are covalent interactions whether determine and assembly To CB regulates that localization sequence coilin SIM-like a has SMN proteins CB crucial several Renvoise a 2001; with is 2011). al., which interactions et 1L), (Fig. multiple (Selenko are the domain for motif Tudor within SMN sequence SIM-like sequences the and consecutive of strand in residue located K119 strategically the both Interestingly, et eepesdGPSNti C7clspreviously cells MCF7 in GFP–SMNwt expressed we Next, h xrsino h uatK1Ri h UOacpo site acceptor SUMO the in K119R mutant the of expression The ´ ta. 06 rpinse al., et Tripsianes 2006; al., et 9 URsalinterfering small -UTR b 3- oiid(i.3) hs hsmtto rbbyaffects bodies expressing cells probably nuclear MCF7 in whereas mutation present were fact, SMN this the ectopic In affect containing was Thus, recognition. not hydrophobicity surface 3D). did protein–protein but (Fig. residues domain, Tudor alanine modified predicted the for the of the In residues structure of 1G5V). replacement valine mutant (PDB three V124A/V125A/V126A, structure had of its 124–126 motif structure residues in Tudor valine the localized 2001), al., that were et given (Selenko targets mutant Finally, crystallized binding hydrophobic 2008). been its for al., the SMN et of of affinity (Uversky the disorder the modify to might and proteins (Conchillo-Sole propensity wild-type 3C) The the (Fig. both 2007). proteins with V124I/V125I comparison mutant SMN in the within domain IUPred disorder to Tudor the SIM-like propensity the relative Second, in a hydrophobicity conveys in 3B). mutants decrease mutant (Fig. the V124I/V125I that hydrophobicity showed SMN server higher as the aggregation, has or R120– protein wild-type that for the the scores with of lower V124A/V125A/V126A compared cause hydrophobicity and and stretch reduce G129 V124A/V125A domain, dramatically the Tudor algorithm mutations The that SMN R120–G129. and wild-type predicts A111–I116 residues the the two acid used in of amino localization we stretches the illustrates propensity hydrophobic 3A Fig. aggregation server. the AGGRESCAN with hydrophobicity ta. 01 ai ta. 2012). al., (Hebert neurons et of motor Tapia targeting SMA of the 2001; feature in al., cardinal defect et a a is import way, SMN CBs this nuclear to of In SMN defective 2004). a interaction al., than et SIM-like defective rather (Narayanan SMN a partners mutant CB-protein suggesting of with 3J), S2G–J). import did (Fig. nuclear hydrophobicity the proteins in Fig. with reduction few GFP– interfere the nuclear not the that material of revealed in analysis proteins intensity SMN colocalized (supplementary fluorescence Gemin2 a expressing Interestingly, and CBs cells In coilin 3F,G,I). construct in endogenous (Fig. cells, hydrophobicity disappear SIM-like latter of loss to these hydrophobic had that tended more proteins mutant these the 3E,H,I), or (Fig. wild-type the eut niaeta h I-iedmi sntrequired the that interactions. the not or molecular that SMN other to is discard mediate SUMO1 cannot bound sequence domain covalently we SIM-like the SMN, SIM-like of groove of free the sumoylation our covalent that Although for indicate 4C). (Fig. was results mutant co-immunoprecipitation GFP–SMN-V124/125A analysis. SIM-like by and Covalent confirmed the blot GFP–SMNwt 4B). (Fig. in of interaction western SMN–SUMO1 sumoylation hydrophobicity the to abrogate lower were not did the cells subjected that antibody 293T were anti-SUMO showed with issue, GFP–SMN-V124/125A GFP–SMN-SUMO lysates of this or Immunodetection total address GFP–SMNwt GFP, To and with 4A). (Fig. transfected of investigated sumoylation SMN covalent have in the involved we is covalent domain 2007), enhances SIM-like the (Kerscher, and whether SUMO precedes with often SUMO1 conjugation CBs and protein of target assembly molecular and the pathway, of 4A). biogenesis we in (Fig. interaction snRNP interactions SMN, the biogenesis the of of SMN–coilin CB in components sumoylation with involved for SMN processes: is three needed motif studied SIM-like interactions the protein–protein whether determine To spliceosomal with coilin interactions and SMN SmD1 regulates domain SIM-like The ie httehdohbcitrcinbtenteSMo the of SIM the between interaction hydrophobic the that Given ora fCl cec 21)17 3–4 doi:10.1242/jcs.138537 939–946 127, (2014) Science Cell of Journal ´ tal., et 941

Journal of Cell Science HR REPORT SHORT 942 Means cells proteins. SMN-depleted mutant in SIM-like immunostaining and *** Coilin 10 SMNwt assayed; (C–F) bar: expressing were here. Scale cells experiment used constructs. MCF7 constructs GFP–SMN-V124/125/126A SMN-depleted GFP–SMN and in the GFP–SMN-V124/125A CBs with GFP–SMN-K119R, transfected GFP–SMNwt, cells with MCF7 assembly. transfected CB SMN-depleted regulates in sequence levels SIM-like protein SMN The 2. Fig. M neato ihGmnpoen Zage l,21) (3) 2011), (2) al., 2012), et al., (Zhang et proteins SMN Gemin Martin (1) with 1998; 4A): interaction al., (Fig. SMN steps et following (Lorson the in oligomerization involved is SMN that eadn h nN ignssptwy ti elknown well is it pathway, biogenesis snRNP the Regarding P , .0 scmae ihcnrlsiRNA). control with compared as 0.001 A al fGPSNmtn ainsue nti td.()Rdcino noeosSMN endogenous of Reduction (B) study. this in used variants mutant GFP–SMN of Table (A) 4 M–olnitrcini B Hbr ta. 2001) al., et (Hebert CBs and in 2002), al., interaction 4A). et (Fig. are SMN–coilin (Pellizzoni which snRNPs, spliceosomes (4) of of proteins constituents complex Sm key the to binding SMN 6 ora fCl cec 21)17 3–4 doi:10.1242/jcs.138537 939–946 127, (2014) Science Cell of Journal ..fo he needn xeiet a es 0clsper cells 70 least (at experiments independent three from s.d. m .(,)Dsrbto of Distribution (G,H) m.

Journal of Cell Science HR REPORT SHORT ntelf,hdohbcrsde fteSMlk oanaehglgtdb tc ersnaini rne ntergtpnl 14 15adV2 we V126 and V125 V124, G assemble panel, to right fail the V124A/V125A/V126A In or orange. V124A/V125A in ( mutants representation SIM-like Tudor (E–H) stick SMN 15 model. means a the bar: mutant condition; for by the server Scale representation highlighted create IUpred bodies. ribbon are to PyMOL The nuclear sticks) domain A (C) SMN-positive (purple (D) SIM-like domain. hydrophobicity. residues the decreased SIM-like alanine of with by the mutants residues replaced of SIM-like hydrophobic of propensity left, disorder aggregation the for and propensity. tendency On aggregation hydrophobicity a the is the there with reduce that correlates mutations predicts motif V124A/V125A/V126A SIM-like the SMN and in V125A hydrophobicity The 3. Fig. novdi M oooioeiain epromdco- with performed transfected cells We 293T homo-oligomerization. in experiments SMN immunoprecipitation in involved n 5 is,w att eemn hte h I-iedmi is domain SIM-like the whether determine to want we First, 0 e odto;means condition; per 100 6 .. *** s.d.; P , 6 .0 scmae ihcnrl) J loecneitniyaayi fncerGPSNtadmtn ainsi C7cells. MCF7 in variants mutant and GFP–SMNwt nuclear of analysis intensity Fluorescence (J) controls). with compared as 0.001 .. ** s.d.; P , .1 *** 0.01, m .()Dsrbto ftencerbde nMF el rnfce ihGPSNcntut ( constructs GFP–SMN with transfected cells MCF7 in bodies nuclear the of Distribution (I) m. P , .0 scmae ihwl-yeo h 14/14 mutant). V124I/V124I the or wild-type with compared as 0.001 o F muorcpttswt h niSNantibody proteins GFP–SMN anti-SMN mutant the and with wild-type that immunoprecipitates demonstrated GFP Immunoblotting GFP–SMN-V124/125A. for or GFP–SMNwt GFP, ora fCl cec 21)17 3–4 doi:10.1242/jcs.138537 939–946 127, (2014) Science Cell of Journal AB h GRSA evrpeit htbt V124A/ both that predicts server AGGRESCAN The (A,B) n 5 0 el per cells 200 domain. 943 FP– re

Journal of Cell Science HR REPORT SHORT 944 by detected were levels GFP, *** with protein experiments. transfected coilin independent were or three cells SmD1 of 293T Gemin2, average (E) endogenous GFP the analysis. lysates, After show blot cell GFP–SMN-V124/125A. western Graphs from or in (F–H) western immunoprecipitation anti-SMN GFP–SMNwt by blotting. GFP, GFP by detected western with After was monitored transfected SUMO1 was GFP–SMN-V124/125A. were with enrichment or GFP–SMN-V124/125A cells SMN SMNwt 293T and lysates, (D) GFP–SMNwt cell of (C). from interaction coimmunoprecipitation immunoprecipitation Covalent or coilin. (B,C) asterisk) and assembly. (B, SmD1 CB with blotting and interactions biogenesis SMN snRNP regulates in domain domain SIM-like The 4. Fig. A ceai ersnaino h uaiefntoso h M SIM-like SMN the of functions putative the of representation Schematic (A) P , 0.001. ora fCl cec 21)17 3–4 doi:10.1242/jcs.138537 939–946 127, (2014) Science Cell of Journal GFP–

Journal of Cell Science eas ohpoen omahtrdmri h oeo h SMN the of core the in heterodimer a importance form particular proteins of both the is because which interaction SMN–Gemin2 in and The Gemin2–Gemin8 complex, proteins, Unrip. SMN not eight to the conjugates is of oligomer SMN assembly domain the in SIM-like participates 4D), the (Fig. of SMN self-oligomerization. integrity SMN endogenous for the to necessary that affinity similar indicating with interact REPORT SHORT xeiet,hmnGPSNtadmtn osrcswere constructs mutant and rescue For GFP–SMNwt transfection. the after h human against 48 directed assayed experiments, were siRNA cells with and (Invitrogen), Transfections previously described 2012). 3 as human al., cultured were et lines cell (Tapia 293T and MCF7 assays UR61, transfection and culture Cell METHODS AND MATERIALS for spot hot a important the a an of is pa be add SMA might in data mutations splicing domain point our screening SIM-like 2011), aberrant the on al., Further that studies et investigation. pathophysiology SMA (Zhang Given to component SMA significant proteins. in defect SmD1 biochemical and not S coilin do of localization V124A/V125A/V126A) mutations nuclear the SMN and affect new (V124A/V125A involved the is here that it demonstrate reported that We suggest also biogenesis. and snRNP assembly to in CB for essential contributes is SMN results also biochemical sequence our SIM-like interaction. 2001), this the regulating al., SMN that et the with demonstrate (Hebert interacts coilin domain of assays C-terminal Tudor binding the previous that compared Although shown 4E,H). 2-fold have (Fig. than SMNwt) (more the coilin with endogenous coilin the its co- with impaired with the severely binding SIM-like GFP–SMN-V124/125A SMN Interestingly, the in 2001). present that of al., mutation revealed et also interaction experiment Tucker direct immunoprecipitation 2001; to al., snRNPs the et and on SMN (Hebert Previous of 4A). depends recruitment (Fig. the CBs SMN that to shown coilin have of studies binding the with interferes with the compared 5-fold with than GFP–SMN-V124/125A 4E,G). more (Fig. of reduced GFP–SMN-wt is binding SmD1 experiments the endogenous the co-immunoprecipitation that for our context, showed essential this is In 2001). E134 (Bu Particularly, interaction the flanking point SMN–SmD1 SMA. residues the in cause that are (Grimm which demonstrated SIM-like protein E134K, have SmD1 and studies the Y130C assembly Previous mutations to snRNP 2013). SMN al., Interestingly, of et binding 2011). direct al., of a domain requires et single-stranded (Fischer a complex in This snRNA encircled complex. Sm proteins the seven of contains protein Tudor SmD1 the the with domain from SMN far 2012). for are al., responsible et binding (Martin motifs domain Gemin2 SMN and the oligomerization previous statistically that with not consistent suggesting are is results reports change These 4E,F). this (Fig. Gemin2, significant endogenous of interaction with the reduced SMN that slightly mutation GFP– demonstrated SIM-like the with proteins although GFP–SMN-V124/125A experiments and Co-immunoprecipitation SMNwt (Paushkin 2002). proteins al., Sm et spliceosomal recognizes which complex, et eivsiae h neato fteSNSIM-like SMN the of interaction the investigated we Next, domain SIM-like the whether investigated we Second, ncnlso,ordt eosrt htteSMlk euneof sequence SIM-like the that demonstrate data our conclusion, In mutation SIM-like SMN the whether investigated we Finally, SMN1 9 T M eepromdwt Lipofectamine-RNAiMAX with performed were SMN UTR gene. he ta. 99 eek tal., et Selenko 1999; al., et ¨hler Nbtdsutteitrcinwith interaction the disrupt but MN inswt eeoyosdeletion heterozygous with tients M osrcsue nti td aebe ecie previously described been 1998). have al., study et Pellizzoni this 2003; Myc– al., in and et GFP–SMNwt (Sleeman used The transfection. constructs siRNA SMN after h 48 transfected ebae eevsaie ihteOysysse (LI-COR system Odyssey the with visualized difluoride polyvinylidene Biotechnology). to were gels SDS-PAGE membranes from transferred Proteins blotting Western 4 rabbit at or IgG h as rabbit 2 with for mixed was antibody first immunoprecipitation anti-SMN were For binder lysates 2008). protein, al., endogenous GFP of et (Rothbauer Sepharose-coupled described previously with Immunoprecipitation Immunoprecipitation S1. and Fig. 63 material system markers supplementary microscopy a CB confocal LSM-510 with other Zeiss equipped a SMN, with imaged expressed were SUMO ectopically and Endogenous quantification and microscopy Confocal (Stratagene). kit mutagenesis II QuikChange the using PCR by mutagenesis site-directed by constructed were mutants GFP–SMN-binding Mutagenesis Bu References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.138537/-/DC1 online available material Supplementary material Supplementary ‘‘Direccio BFU2011-23983]. the number by [grant supported was work This Funding manuscript. the wrote and M.L. experiments experiments, designed transfection M.T.B. and and co-immunoprecipitation mutagenesis performed performed A.P. and and R.B. experiments assays, of conceived V.L. and O.T. contributions Author interests. competing no declare authors The interests Competing of University (IMM, reagents. USA), Desterro Joana for Carolina, and Portugal) North UK) (CLS, Lisbon, of Dundee, Lamond of (University I. University Matera (CLS, crystal Angus Gregory Hay the to Ron of indebted UK), studies are Dundee, simulation authors of the The University for motifs. Moreno Tudor Maria of thank structure to wish authors The Acknowledgements Student’s two-tailed unpaired, t by determined was significance Statistical analysis Statistical antibodies. (Ab1014) Zymed) anti-Myc C7, using (21 blotting anti-SUMO1 was western and sumoylation and Protein SDS-PAGE protocol. by manufacturer’s analyzed beads the the to For anti-Myc according SDS-PAGE. used using by verified purified and vitro MO) immunoaffinity Louis, St. were (Sigma-Aldrich, vitro system proteins in Myc– transcription/translation were Recombinant SMN instructions. coupled proteins manufacturer’s the quick SMN following (Promega) TnT Myc-tagged using assay, synthesized sumoylation the For assay Sumoylation o t4 at h 2 for ic,M n aod .I. A. Lamond, and M. Cioce, -tests. lr . ae,V,Lu V., Raker, D., hler, ¨ uo oano M nslcooa nN seby mlctosfor implications assembly: snRNP U spliceosomal in atrophy. SMN muscular spinal of domain tudor nu e.Cl e.Biol. Dev. Cell Rev. Annu. uolto sa,teK0 i LABoehItrainl was International) Biotech (LEA kit K007 the assay, sumoylation ora fCl cec 21)17 3–4 doi:10.1242/jcs.138537 939–946 127, (2014) Science Cell of Journal ˚ C. 6 ran .adFshr U. Fischer, and R. hrmann, ¨ i betv 14A.Seatbde sdin used antibodies See (1.4NA). objective oil u.Ml Genet. Mol. Hum. 21 20) aa ois oghsoyo discovery. of history long a bodies: Cajal (2005). 105-131. , ˚ eoeadto fprotein-A–agarose of addition before C nGnrld Investigacio de General ´n 8 2351-2357. , 19) seta oefrthe for role Essential (1999). n’o Spain of ´n’’ 945 in

Journal of Cell Science aaaa,U,Ahe,T,Lu T., Achsel, D. U., G. Narayanan, Duyne, Van and K. Perry, S., N. Ninan, K., Gupta, R., Martin, ahn,M,Hy,P n egbur .M. K. Neugebauer, and P. Heyn, M., Machyna, B., Wirth, E., Hahnen, D., J. Baleja, M., Bordonne J. Yao, M., J., Strasswimmer, Schneider, L., C. J., Lorson, N. Watkins, N., A. A., Kuhn, Munnich, C., Girard, O., I., Clermont, Lemm, S., Bertrandy, T. Q., M. Liu, P., Berciano, Burlet, and S., O. Lefebvre, Tapia, R., Bengoechea, I., Casafont, M., Lafarga, I. O. Dikic, Kerscher, and P. Bayer, K., Haglund, M., Rabiller, M., C. Hecker, G. A. Matera, and B. K. Shpargel, W., P. Szymczyk, D., M. Hebert, a,R T. R. Hay, rm,C,Cai . ez .P,Kpr . ikr . idrcs . tr,H., Stark, K., Diederichs, C., Kisker, J., Kuper, P., J. Pelz, A., Chari, C., Grimm, Gilder,A.S.andHebert,M. F. Melchior, and A. A. Flotho, Chari, and C. Englbrecht, U., Fischer, Conchillo-Sole REPORT SHORT 946 askn . uiz .K,Msee,S n ryus G. Dreyfuss, and S. Massenet, K., A. Gubitz, S., G. Paushkin, J. Gall, and S. and Deryusheva, T. Z., M. Nizami, Berciano, I., Casafont, O., Tapia, R., Bengoechea, J., Navascues, ir moto nNsadSN h pnlmsua toh protein. atrophy muscular spinal the SMN, and snRNPs U of import vitro oligomers. zipper glycine soluble forms protein neuron motor et function. meets J. severity. E. atrophy Androphy, muscular and spinal M. with correlates H. defect A. Burghes, T., Le, bodies. Cajal of integrity Lu and R. atrophy. muscular spinal nucleus. J. in Melki, cell level protein and neuronal G. the Dreyfuss, of knowledge the to Chromosoma contribution Cajal’s (2009). motifs. SUMO-interacting motifs. SUMO2-interacting and atrophy SUMO1- muscular of spinal the SMN, and bodies Cajal protein. between bridge the forms eitdsRPformation. snRNP mediated U. Fischer, and H. Schindelin, Nucleolus The disease. and health in ribonucleoproteins. nuclear small polypeptides. in aggregation of spots’’ ‘‘hot S. Ventura, nasmlooeo ribonucleoproteins. of assemblyosome an body. locus neurons. M. Lafarga, Cell 16 223-234. , ee Dev. 20) UO itr fmodification. of history a SUMO: (2005). ran R. hrmann, ¨ .Src.Biol. Struct. J. 20) UOjnto-htsyu ucin e nihsthrough insights New function? your junction-what’s SUMO (2007). odSrn ab eset Biol. Perspect. Harb. Spring Cold 20) GRSA:asre o h rdcinadeauto of evaluation and prediction the for server a AGGRESCAN: (2007). 20) UO1tasetylclzst aa oisi mammalian in bodies Cajal to localizes transiently SUMO-1 (2008). ,O,d ro,N . Avile S., N. Groot, de O., ´, d.O akadJ lo.p 6-8.Bri:Springer. Berlin: 361-380. pp Olson. J. and Mark O. Edt. . 118 ie nedsi.Rv RNA Rev. Interdiscip. Wiley 437-443. , 15 20) non nN ignssi eurdfrthe for required is biogenesis snRNP U Ongoing (2006). nu e.Biochem. Rev. Annu. 2720-2729. , 163 o.Bo.Cell Biol. 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