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formation and subnuclear localization to Cajal bodies
Benoît Renvoisé, Gwendoline Quérol, Eloi Rémi Verrier, Philippe Burlet,
Suzie Lefebvre
To cite this version:
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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A role for protein phosphatase PP1c in SMN complex
formation and subnuclear localization to Cajal bodies
Benoıˆt Renvoise´1,*,`, Gwendoline Que´rol1,*, Eloi Re´mi Verrier1,§, Philippe Burlet2and Suzie Lefebvre1,"1Laboratoire de Biologie Cellulaire des Membranes, Programme de Biologie Cellulaire, Institut Jacques-Monod, UMR 7592 CNRS, Universite´ Paris
Diderot, Sorbonne Paris Cite´, 15 rue He´le`ne Brion, 75205 Paris cedex 13, France
2
Service de Ge´ne´tique Me´dicale, Hoˆpital Necker-Enfants Malades, 75743 Paris cedex 15, France
*These authors contributed equally to this work
`Present address: Cellular Neurology Unit, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA §Present address: Institut National de la Recherche Agronomique, Laboratoire de Ge´ne´tique Animale et Biologie Integrative, Domaine de Vilvert, 78350 Jouy-en-Josas, France "
Author for correspondence (lefebvre.suzie@ijm.univ-paris-diderot.fr) Accepted 14 February 2012
Journal of Cell Science 125, 2862–2874
2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.096255
Summary
The spinal muscular atrophy (SMA) gene product SMN forms with gem-associated protein 2–8 (Gemin2–8) and unrip (also known as STRAP) the ubiquitous survival motor neuron (SMN) complex, which is required for the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), their nuclear import and their localization to subnuclear domain Cajal bodies (CBs). The concentration of the SMN complex and snRNPs in CBs is reduced upon SMN deficiency in SMA cells. Subcellular localization of the SMN complex is regulated in a phosphorylation-dependent manner and the precise mechanisms remain poorly understood. Using co-immunoprecipitation in HeLa cell extracts and in vitro protein binding assays, we show here that the SMN complex and its component Gemin8 interact directly with protein phosphatase PP1c. Overexpression of Gemin8 in cells increases the number of CBs and results in targeting of PP1c to CBs. Moreover, depletion of PP1c by RNA interference enhances the localization of the SMN complex and snRNPs to CBs. Consequently, the interaction between SMN and Gemin8 increases in cytoplasmic and nuclear extracts of PP1c-depleted cells. Two-dimensional protein gel electrophoresis revealed that SMN is hyperphosphorylated in nuclear extracts of PP1c-depleted cells and expression of PP1c restores these isoforms. Notably, SMN deficiency in SMA leads to the aberrant subcellular localization of Gemin8 and PP1c in the atrophic skeletal muscles, suggesting that the function of PP1c is likely to be affected in disease. Our findings reveal a role of PP1c in the formation of the SMN complex and the maintenance of CB integrity. Finally, we propose Gemin8 interaction with PP1c as a target for therapeutic intervention in SMA.
Key words: Spinal muscular atrophy, SMN complex, Cajal bodies, Spliceosomal snRNPs, Protein phosphatase PP1
Introduction
The mammalian cell nucleus is organized into distinct compartments (Mao et al., 2011) and the Cajal body (CB) is one of these entities (Morris, 2008). Most cells contain two to four CBs that are identified by the protein marker coilin (Rasˇka et al., 1991). Coilin has been conserved in evolution, and various studies suggest that coilin and CBs are not essential in fruit fly and mice, whereas they are in developing zebrafish embryos (Strzelecka et al., 2010). CBs are prominent in cancer cells and neurons (Lafarga et al., 2009). They are highly dynamic subnuclear domains enriched in many different ribonucleoproteins (RNPs), such as spliceosomal small nuclear RNPs (snRNPs), small nucleolar RNPs (snoRNPs), RNA polymerases, histone mRNA processing factors and telomerase (Cioce and Lamond, 2005; Gall, 2000; Matera et al., 2007).
The biogenesis of snRNPs involves cytoplasmic and nuclear phases. Synthesis starts with the transcription of a small nuclear RNA (snRNA) precursor and CB targeting to associate with the nuclear export complex (Suzuki et al., 2010). In the cytoplasm, survival motor neuron (SMN) complex assembles the snRNAs with common Sm core proteins into core snRNPs. Following nuclear import, core snRNPs are targeted to CBs for additional maturation and processing steps resulting in the production of
mature snRNPs for splicing in the nucleoplasm or for storage in speckles. CBs have also been reported to increase assembly and recycling of snRNPs after splicing (Nesic et al., 2004; Klingauf et al., 2006; Stanek et al., 2008). The SMN complex could also operate in these processes (Pellizzoni et al., 1998).
The ubiquitous SMN complex that includes SMN, gem-associated protein 2–8 (Gemin2–8) and unrip (also known as serine-threonine kinase receptor-associated protein; STRAP) (Pellizzoni, 2007), localizes to the cytoplasm and the nucleus, where it concentrates in CBs (Liu and Dreyfuss, 1996; Carvahlo et al., 1999). SMN protein plays a central role in spinal muscular atrophy (SMA), the most common human genetic neurodegenerative disorder of infancy (Lefebvre et al., 1995). Alterations of the SMN1 gene cause SMN protein deficiency leading to degeneration of spinal motor neurons (MNs). This selective vulnerability remains elusive (Burghes and Beattie, 2009). Disease severity correlates with residual levels of SMN and abundance of CBs (Frugier et al., 2000; Lefebvre et al., 1997; Renvoise´ et al., 2009). SMN1 is a conserved gene essential in snRNP biogenesis and thus, in splicing (Schmid and DiDonato, 2007; Zhang et al., 2008). Studies in animal models show that mature snRNPs could rescue developmental defects, and indicate that snRNP production is crucial in SMA disease (Winkler et al.,
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2005). However, other SMN functions might contribute to disease severity (Rossoll and Bassell, 2007; Hubers et al., 2011).
The severity of MN diseases correlates with a deficiency of SMN in the nucleus (Turner et al., 2009). The precise nuclear function of SMN is unknown. Nuclear import of the SMN complex depends on snRNPs (Narayanan et al., 2004) and targeting to CBs on its interaction with WD40 encoding RNA antisense to p53 (WRAP53; also known as telomerase Cajal body protein 1), a factor involved in CB recruitment of small CB-specific RNAs (Mahmoudi et al., 2010). SMN has also been reported to form a different type of nuclear body designated a ‘gem’ for Gemini of CBs (Liu and Dreyfuss, 1996). Gems contain the components of the SMN complex and lack coilin and snRNPs. Depletion of SMN disrupts CBs and gems, whereas coilin depletion disrupts CBs and forms gems (Lemm et al., 2006; Whittom et al., 2008). These data indicate that SMN is required for CB formation, and gems form when snRNP metabolism is disturbed. Thus, gems are thought to be storage sites. The tethering reaction of SMN has been reconstituted in cells, showing de novo CB formation as a non-linear assembly process triggered by most components when SMN is present at sufficient levels (Kaiser et al., 2008). However, the mechanisms controlling the accumulation in CBs are so far not fully understood.
Many forms of stress, stimuli or signaling pathways impact on CB composition, size and number (Boulon et al., 2010). These processes result in substantial changes in protein–protein interactions influenced by post-translational modifications, such as protein methylation (Boisvert et al., 2002; Hebert et al., 2002; Clelland et al., 2009) and phosphorylation (Hebert, 2010). SMN and coilin are phosphoproteins. The phosphorylation of SMN regulates the stability of the complex (Burnett et al., 2009) and snRNP assembly in the cytoplasm (Grimmler et al., 2004). Hyperphosphorylation of coilin coincides with lack of CBs during mitosis (Carmo-Fonseca et al., 1993) and SMN preferentially interacts with the hypophosphorylated coilin that forms CBs (Hebert et al., 2002). The nuclear protein phosphatase PPM1G, which is not present in CBs, has been reported as contributing to the dephosphorylation of SMN and coilin and to their accumulation in CBs (Hearst et al., 2009; Petri et al., 2007). Other studies on CBs have implicated phosphorylation: inhibiting phosphatases and a phosphoserine mutation in coilin affect the CB localization of coilin and of snRNPs (Lyon et al., 1997; Sleeman et al., 1998). The nuclear phosphatase PP4 associates with the SMN complex and enhances the temporal CB localization of snRNPs (Carnegie et al., 2003). Moreover, inhibition of PP1 has been shown to increase the number of gems in SMA fibroblasts (Novoyatleva et al., 2008). How PP1 could regulate SMN localization is unknown.
PP1 holoenzymes regulate a wide range of cellular functions, including transcription, pre-mRNA splicing, cell survival, synaptic plasticity and muscle contraction (Ceulemans and Bollen, 2004; Moorhead et al., 2007). In mammals, there are three PP1 catalytic subunits (a, b/d and c), each encoded by a different gene. The association with a regulatory (or targeting) subunit determines the subcellular localization and substrate specificity of the catalytic subunits (Cohen, 2002; Bollen and Beullens, 2002). The list of approximately 180 regulatory subunits is still growing (Bollen et al., 2010). Most targeting proteins harbor a primary consensus PP1-binding ‘R/KVxF’ motif (Meiselbach et al., 2006) and the localization pattern of
each catalytic subunit results from the addition of many different PP1 holoenzyme complexes. All three localize to the cytoplasm and nucleus during interphase. PP1a and PP1c are found in the nucleoplasm and PP1c also concentrates in nucleoli, whereas PPP1b/d is detected throughout the nucleus with no particular accumulation pattern (Andreassen et al., 1998). The PP1 catalytic subunits are highly mobile and their subcellular localization could change with the expression levels of the targeting proteins (Trinkle-Mulcahy et al., 2001; Trinkle-Mulcahy et al., 2003). One of the major nuclear targeting subunits of PP1a and PP1c is the phosphatase nuclear targeting subunit PNUTS [also known as serine/threonine-protein phosphatase 1 regulatory subunit 10 (PP1R10) and p99] that localizes to the nucleoplasm (Landsverk et al., 2005) and can be found occasionally in CBs (Moorhead et al., 2007). These data illustrate how dynamic and transient the targeting of PP1 to different specific subcellular sites could be.
We report here a previously unknown mechanism of post-translational regulation of the SMN complex by PP1c that was determined using biochemical, proteomic, RNA interference and immunofluorescence approaches. For in vivo immunofluorescence experiments involving control and SMA individuals we chose the skeletal muscle because of the very specific cytoplasmic organization. We examined the proteins co-immunopurified with SMN proteins and focused on a new interaction with PP1c. We identified Gemin8, a component of the SMN complex, as a PP1-binding protein targeting PP1c to CBs. We show that modulating the expression levels of PP1c impact on the subnuclear organization. Our results support the conclusion that PP1c could regulate SMN complex formation and localization to CBs. Results
Proteomics study of the N-terminal region of SMN
We previously produced SMN deletion mutants in order to gain insight into its subnuclear localization in mammalian cells (Renvoise´ et al., 2006). A diagram of the domains used in the present study for fluorescent fusion proteins is shown in Fig. 1A. The fragment containing the lysine-rich region [amino acids (aa) 71–83] and Tudor domain (aa 91–151) produced a fusion protein GFP–SMN472D5 (hereafter referred to as GFP–472D5) that localizes to the nucleoplasm and CBs. Introduction of mutations in the lysine-rich region of GFP–472D5 abolishes the localization in CBs. We expressed the N-terminal region of SMN as a GFP fusion protein (GFP–N86; 86 first aa) and performed immunoprecipitation experiments with anti-GFP antibody to identify new interaction partners of SMN (Fig. 1B,C). In co-immunoprecipitated proteins, we observed a major band of approximately 40 kDa corresponding to the apparent mass of GFP–N86 (Fig. 1C). The band was cut, digested and analysed by mass spectrometry (MS). Peptides from GFP–N86 and from protein phosphatase PP1c, as a candidate partner of expected mass, were detected (supplementary material Fig. S1). We sought to determine the relationship between PP1c and SMN because SMN complex localization and function are regulated by protein phosphorylation.
Co-immunoprecipitation of PP1c with the SMN complex
To test the association of PP1c with SMN, we expressed GFP–472D5, GFP–N86 and GFP–N86M2 fusion proteins and performed co-immunoprecipitation experiments (Fig. 1D). Immunoblot analyses revealed that PP1c was co-immunoprecipitated with GFP–472D5 and GFP–N86. We
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found little if any interaction between PP1c and GFP–N86M2, a fusion protein in which the lysine residues are mutated (Renvoise´ et al., 2006). This mutation disrupts a minor self-interacting motif of SMN that could regulate stability and CB localization of the SMN complex (Morse et al., 2007). Interaction between endogenous SMN and PP1c proteins appeared to be less efficient (data not shown) than between GFP–SMN and PP1c (Fig. 1E), suggesting that either the anti-SMN antibody masks the interaction site or a third component mediates the interaction between them.
Direct interaction of PP1c with SMN complex and its component Gemin8
A pull-down assay was used to demonstrate direct interaction between PP1c and the SMN complex (Fig. 2). Anti-SMN antibody 4B3 allowed us to purify the SMN complex from HeLa cell lysates under stringent conditions in order to remove
Fig. 1. Interaction of SMN and PP1c assayed by
co-immunoprecipitation. (A) Schematic representation of SMN (aa 1–294) and mutants fused to eGFP at the N-terminus (Renvoise´ et al., 2006). SMN is depicted with the Lys-rich, Tudor, Pro-rich, YG-box and exon7-encoded domains. (B) Flow-chart of the immunopurification procedure.
(C) Immunoprecipitations (IPs) were performed with negative control mouse immunoglobulins (IP Control) or anti-GFP (IP-GFP) antibody and with extracts from COS cells expressing eGFP-tagged N86. Bound proteins were separated by SDS-PAGE and visualized with silver staining. An arrow indicates a 40-kDa band. The asterisk indicates the expected position of the SMN complex component Gemin8. (D) IPs were performed with extracts from COS cells stably expressing eGFP-tagged 472D5, N86 or double mutant N86M2, respectively. Bound proteins were analysed by immunoblotting with anti-PP1c antibodies. The anti-GFP incubation served as a loading control. (E) Bound proteins after IP of GFP–SMN were analysed for PP1c and endogenous SMN. The association of PP1c with the fusion proteins requires the Lys-rich domain of SMN. IgGh and IgGl are the immunoglobulin heavy and light chains, respectively.
Fig. 2. Direct interaction of PP1c with the SMN complex and its component Gemin8. (A) HeLa cell lysate was immunoprecipitated with negative control (IP Control) or anti-SMN antibody (IP SMN). Proteins were eluted, resolved by SDS-PAGE and analysed by silver staining.
(B) Immunoprecipitated proteins shown in A were incubated with purified E. coli recombinant His-tagged PP1c. Bound proteins were analysed by immunoblotting. (C) GST and GST–Gemin8 fusion proteins were purified, separated by SDS-PAGE and analysed by Coomassie staining. (D) GST fusion proteins shown in C were incubated with purified His-tagged PP1c. Bound proteins were analysed by immunoblotting. PP1c interacts directly with Gemin8. (E) In situ PLA detection of the interaction between endogenous proteins in HeLa cells with anti-Gemin8 and anti-PP1c antibodies. As a control, one of the primary antibodies was omitted. Scale bar: 3mm. (F) Co-IP of the endogenous proteins with anti-Gemin8 antibody and HeLa cell extracts were analysed by immunoblotting with PP1c and anti-SMN antibodies. The asterisks indicate the immunoglobulins.
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any interacting proteins of weak affinity for the complex (Fig. 2A). The antibody-immobilized complex (SMN complex) was incubated with a purified Escherichia coli recombinant histidine-tagged PP1c (His–PP1c). After extensive washes, the proteins bound to the complex were eluted and tested for the presence of His–PP1c by immunoblotting (Fig. 2B). The bound proteins were compared with that bound to unrelated mouse monoclonal antibody (control). The recombinant PP1c was bound to the SMN complex. We then sought to identify the component of the complex that mediates binding to PP1c. The most common PP1-binding RVxF motif has the consensus sequence [R/K]x0–1[V/I]{P}[F/W] (x represents any amino acid,
and {P} any except proline). Based on the hypothesis that SMN might be a substrate for PP1c, the sequences of SMN and of gemins that bind directly to SMN, namely Gemin2, 3 and 8, were examined. The HVAW motif in Gemin8 (aa 80–83) appeared to be a potential PP1-binding motif. Direct interaction between Gemin8 and PP1c was tested using purified bacterial recombinant proteins (Fig. 2C). The immobilized glutathione S-transferase (GST)-tagged Gemin8 retained His-PP1c, whereas the GST alone did not (Fig. 2D). Direct interaction between Gemin8 and PP1c was further tested in fixed HeLa cells using the in situ proximity ligation assay (in situ PLA; Fig. 2E). This approach detects endogenous protein–protein interactions in fixed cells using antibodies specific for each protein (Fredriksson et al., 2002). Using in situ PLA, the Gemin8– PP1c interaction appeared as bright dots in the cytoplasm, nucleoplasm and CBs. Moreover, endogenous Gemin8 interacted with PP1c and SMN in co-immunoprecipitation experiments (Fig. 2F). Our results support the conclusion that Gemin8 is a PP1-binding protein.
Overexpression of Gemin8 colocalizes PP1c with CB markers
Given that PP1-binding proteins confer subcellular localization and substrate specificity to PP1 holoenzymes, Gemin8 was expressed as a fluorescent fusion (FP–Gemin8; Fig. 3A) and its colocalization with SMN and PP1c was tested. FP–Gemin8 completely colocalized with SMN to the cytoplasm and nuclear CBs (Fig. 3B), as reported (Carissimi et al., 2006). In FP– Gemin8-expressing cells, PP1c was concentrated in numerous nuclear bodies with the fusion, whereas it was concentrated in nucleoli of untransfected cells and cells expressing either FP– SMN, myc–Gemin4 or FP alone (Fig. 3C). Colocalization with FP–Gemin8 could be due to the presence of other PP1-binding proteins in CBs. The C-terminal region of Gemin8 including the coiled coil domain (FP–G8DC) was deleted to reduce nuclear body formation and we introduced mutations in the putative PP1-binding motif (FP–G8DC3A; Fig. 3A,C). Both FP–G8DC and FP–G8DC3A localized to the nucleoplasm and to fewer CBs. FP– G8DC recruited PP1c into CBs, whereas FP–G8DC3A did not. These experiments revealed that the HVAW motif of Gemin8 is required for the localization of PP1c to CBs. Altogether, our results support the conclusion that Gemin8 contributes to CB localization of PP1c in mammalian cells.
To determine whether the FP–Gemin8 nuclear bodies contained other CB constituents, colocalization with coilin (a CB marker), Gemin3 and components of snRNPs was examined (Fig. 4A–E). All FP–Gemin8 nuclear bodies were CBs as indicated by complete colocalization with coilin. They were also enriched in Gemin3 and snRNP-specific markers, such as the
Fig. 3. Targeting of PP1c to Cajal bodies in cells overexpressing Gemin8. (A) Schematic representation of Gemin8 (aa 1–242) and mutants fused to destabilized YFP (FP) at the N-terminus. Gemin8 is depicted with the putative PP1-binding motif HVAW and a self-association coiled-coil domain (accession number Q9NWZ8). The expressed fusion proteins were assayed in total cell lysates by immunoblotting with anti-FP antibody. The asterisk indicates an unspecific protein band from HeLa cells. (B) Immunofluorescence experiments were performed in HeLa cells expressing FP–Gemin8 fusion protein with anti-SMN antibody. FP–Gemin8 colocalizes with anti-SMN in the cytoplasm and nuclear bodies as shown in yellow (merged images) in low- and high-expressing cells. (C) FP–Gemin8, FP–G8DC, FP–G8DC3A, FP–SMN, myc– Gemin4 and FP alone were expressed and analysed by immunofluorescence with anti-PP1c antibody. Overexpression of FP–Gemin8 changes the localization of PP1c from nucleoli to Gemin8-expressing nuclear bodies in a manner dependent on the HVAW motif of Gemin8. Arrowheads point to untransfected cells and arrows to cells expressing fusions. Scale bar: 3mm.
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trimethylguanosine (TMG)-capped snRNAs (molecular signature for the nuclear import of snRNAs), snRNP Sm core protein SmD3 (one of the seven Sm proteins common to all snRNPs except U6 snRNP) and U1-specific 70 kDa protein. The localization of the abundant splicing factor SC35 that is a marker of speckles (Spector, 1993) and does not concentrate in CBs was investigated. Fig. 4F showed that SC35 staining occurs in speckles and not in CBs of FP–Gemin8-expressing cells, supporting the conclusion that the accumulation of PP1c in CBs is specific. Our results demonstrated that Gemin8 could form CBs, which contain SMN complex, snRNPs and PP1c.
Depletion of PP1c enhances the accumulation of SMN complex and snRNPs in CBs
To determine whether there might be a functional link between PP1c and SMN targeting to CBs, RNA interference (RNAi) knockdown experiments were performed using three distinct PP1c-specific small interfering RNA (siRNA) duplexes (Fig. 5). We first tested the protein levels of total lysates of HeLa cells
transfected with either a negative control firefly luciferase (Gl2) or PP1c siRNAs (si#5, si#6 and si#8) by immunoblotting. The PP1c-specific siRNAs reduced PP1c protein levels by ,65% (Fig. 5A). SMN complex levels remained the same, indicating the specificity of the siRNAs used to reduce PP1c levels. As a control, the overall phosphorylation profile of total protein lysates was examined (Fig. 5A, furthest right panel). There were no major differences between PP1c-depleted cell lysates and controls, indicating that PP1 holoenzymes have overlapping substrates. The localization of SMN in siRNA-treated cells was then examined by immunofluorescence experiments (Fig. 5B). Depletion of PP1c (as shown by the reduced PP1c staining) led to a significant (P,0.001) increase in the proportion of cells with more SMN nuclear bodies than control cells (Fig. 5B,C). There was a three- to fourfold increase in the proportion of cells with more than five nuclear bodies. All three PP1c siRNAs confirmed the formation of CBs upon depletion of PP1c (Fig. 5C). Colocalization experiments with coilin and CB components, including Gemin3, Gemin8 and snRNP-specific markers, all had
Fig. 4. Overexpressed Gemin8 colocalizes PP1c in CBs with the SMN complex and snRNPs. FP–Gemin8 was expressed in HeLa cells and analysed by immunofluorescence with (A) anti-coilin (CB marker), (B) anti-Gemin3, a component of the SMN complex, (C) anti-TMG-capped snRNA, (D) anti-SmD3 common core snRNP protein and (E) anti-U1 snRNP-specific 70 kDa protein antibodies. Colocalization results in yellow signals (in the merged images). (F) The distribution of the splicing factor SC35 remains in the speckles (in red), indicating the specificity of the overlapping localizations in yellow observed above. The microscope was focused on nuclear foci. Scale bar: 3mm.
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similar results (Fig. 6A–F). These data indicate that depletion of PP1c increases CB numbers and has no adverse effect on their composition.
In the nucleus, SMN is localized with coilin in CBs and in gems, which lack coilin and snRNPs (Liu and Dreyfuss, 1996).
To examine whether SMN localizes to either type of nuclear body in PP1c-depleted cells, triple immunolabelling experiments were performed with PP1c, SMN and coilin antibodies (Fig. 7A– D). In control Gl2 siRNA-treated cells SMN localized to both gems and CBs (Fig. 7A), whereas depletion of PP1c led to complete localization of SMN to CBs (Fig. 7B). To demonstrate the specificity of the effects observed above, FP–PP1c was expressed using a cDNA resistant to siRNA#5 (Fig. 7C). FP– PP1c expression restored the localization of SMN to gems and CBs, whereas FP alone did not (Fig. 7D). Statistical analyses confirmed these results (Fig. 7E). The increased number of CBs observed in PP1c-depleted cells was significantly (P,0.001) reduced in rescue experiments, indicating that the siRNA-mediated effects are specific of PP1c. Our results support the conclusion that localization of SMN complex in either CBs or gems involves PP1c.
Depletion of PP1c changes the post-translational modification pattern of SMN
To attempt to understand how PP1c is involved in CB localization of the SMN complex as demonstrated above, we sought to determine whether depletion of PP1c might influence the phosphorylation status of SMN. To test the hyperphosphorylation hypothesis, SMN immunoblotting of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) gels was performed. As a control, protein levels in siRNA-transfected cell lysates were examined (Fig. 7F). The PP1c siRNA led to PP1c reduction when compared with Gl2 siRNA (control RNAi). Transfection of PP1c siRNA#5 alone or with a FP–PP1c expression plasmid resistant to the siRNA#5 did not reduce either PP1a (PP1 catalytic subunit alpha) or SMN levels, again indicating the specificity of this approach. The post-translational modification patterns of SMN from nuclear extracts of transfected cells were compared and relative signal intensities were measured (Fig. 7G,H). A complex pattern of numerous SMN isoforms was detected in control conditions as reported previously (Grimmler et al., 2004). Examination of SMN isoforms from PP1c-depleted cell extracts revealed striking differences. First, SMN phosphorylation increased upon depletion of PP1c as indicated by the accumulation of more acidic isoforms (section 1), whereas expression of FP–PP1c reduced them. Second, the spots at pH 6.8 (section 2) and pH 8.5 (section 3) that were reduced upon PP1c depletion (Fig. 7G, arrows) were also rescued by FP–PP1c. Third, a basic isoform at pH 9.2 (section 4, arrowhead) accumulated in PP1c-depleted extracts, whereas FP–PP1c caused it to disappear, and a slightly less basic spot to form. Finally, overall populations of spots from section 2–4 were partially rescued by FP–PP1c. Our results support the conclusion that PP1c regulates changes in the post-translational modification pattern of SMN.
Depletion of PP1c results in an increased association of SMN and Gemins
We tested the possibility that the increased number of CBs demonstrated above in PP1c-depleted cells might correlate with accumulation of the SMN complex in the nucleus. Cytosolic and nuclear fractions were prepared from HeLa cells transfected with either Gl2 or PP1c siRNAs and the distribution of the SMN complex was monitored by immunoblotting (Fig. 8A). It was not possible to probe for Gemin5, -6 and -7 because of the limited affinities of commercial antibodies. Gemin8 and unrip were
Fig. 5. Depletion of PP1c enhances the accumulation of SMN nuclear bodies. HeLa cells were transfected with negative control Gl2 (GL2; firefly luciferase) or three different PP1c siRNAs (#5, #6 and #8). (A) Cell lysates were analysed by immunoblotting using antibodies against components of the SMN complex. Incubation of a-tubulin served as a loading control. siRNA #5, #6 and #8 reduced the levels of PP1c by 69, 64 and 64%, respectively (n55 experiments). The immunoblot in the right panel shows that lowering PP1c levels (siRNA#5) had no major effect on the overall phosphoserine profile. (B) Double-labeling immunofluorescence experiments were performed using anti-PP1c (red) and anti-SMN antibodies (green). The arrows point to silenced cells. The asterisk indicates an unsilenced cell. The microscope was focused on the nuclear foci. Scale bar: 3mm. (C) Statistical analyses of SMN nuclear bodies in silenced cells (n.1000 cells). Error bars indicate the standard error of the mean (s.e.m.); ***P,0.001 (Student’s t-test).
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predominantly cytosolic as reported (Carissimi et al., 2005; Carissimi et al., 2006; Grimmler et al., 2005). There was no major change in the nucleocytoplasmic distribution of the SMN complex, indicating that PP1c is not involved in this process.
Given the role of phosphorylation in protein–protein interactions, we tested whether PP1c might regulate the association of the Gemins with SMN. To this effect, cytosolic and nuclear fractions were immunoprecipitated using anti-SMN antibodies, and co-immunoprecipitated proteins were analysed by immunoblotting (Fig. 8B–D). In SMN complexes from the cytosol of PP1c-depleted cells, Gemin4 and -8 and unrip were significantly (P,0.042) increased relative to SMN, whereas Gemin2 and -3 were not (Fig. 8C). Similar changes were found when the nuclear SMN complexes were examined (Fig. 8D). This indicated no major change in the association of SMN– Gemin2–Gemin3 subcomplex, whereas the association of the peripheral Gemins changed. As described previously (Carissimi et al., 2006; Charroux et al., 2000; Otter et al., 2007), SMN interacts directly with Gemin8, and Gemin3 with Gemin4. Our results support the conclusion that PP1c regulates these interactions.
PP1c and Gemin8 colocalize in the cytoplasm of skeletal muscles
As a first approach to understand the in vivo physiological relevance of the interaction between Gemin8 and PP1c demonstrated here, we sought to determine their colocalization in mammalian tissue sections. The skeletal muscle was chosen for its specific subcellular organization and localization of the SMN complex at the Z-discs of mouse myofibrils (Walker et al., 2008). Using the Prestige anti-Gemin8 antibodies validated by the Human Protein Atlas project in numerous tissues including the skeletal muscle (www.proteinatlas.org), the expected striated pattern of Z-discs was found on 14-day post-natal mouse and human foetal skeletal muscles (Fig. 9A,B). Our colocalization experiments revealed substantial, although not complete, overlap of PP1c with Gemin8. These results support the conclusion that Gemin8 and PP1c could colocalize in vivo.
Our previous studies showed a marked reduction of SMN levels in skeletal muscles from foetuses with severe SMA (Burlet et al., 1998) (Fig. 9D). Morphological defects consistent with a Z-disc deficiency have been reported in a mouse model with severe SMA (Walker et al., 2008; Kariya et al., 2008). To address
Fig. 6. Depletion of PP1c enhances localization of components of the SMN complex and snRNPs in CBs. Double-immunofluorescence analyses of HeLa cells transfected with control scrambled or PP1c siRNA using antibodies against (A) PP1c, (B) SMN-complex component Gemin3, (C) Gemin8, (D) TMG-capped snRNA, (E) SmD3 core protein and (F) U1-specific 70 kDa protein (green) and co-stained for DNA (DAPI) or the CB marker coilin (red). The yellow in merge indicates overlapping signals. Scale bars: 3mm.
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the question of whether similar defects occur in the human disease, colocalization experiments were carried out on muscle sections from SMA foetuses (Fig. 9C). An altered organization of the striated pattern was observed as indicated by aberrant Gemin8 and PP1c localization and a severe atrophy, reflecting denervated fibers. To provide molecular insights into pathobiology, control and severe SMA fibroblast cell extracts were immunoprecipitated using anti-Gemin8 antibodies and co-immunoprecipitated proteins were analysed by immunoblotting (Fig. 9E). As reported previously (Lefebvre et al., 2002), a reduction of SMN levels of ,75% was detected in SMA fibroblast cell cultures compared with control fibroblasts. There was a significant (P,0.01) 60% reduction in interaction between SMN and Gemin8 or PP1c in SMA fibroblasts, whereas no significant difference of the Gemin8 interaction with PP1c was detected (Fig. 9F). This is consistent with the 50–70% reduction of in vitro snRNP assembly activity previously reported for SMA fibroblast cell extracts (Gabanella et al., 2007; Wan et al., 2005). Our
results support the conclusion that the production of SMN complex is less efficient in SMA conditions.
Discussion
The SMN complex plays a central role in snRNPs biogenesis (Fischer et al., 1997) and in SMA, the second most frequent autosomal recessive disease of children (Lefebvre et al., 1995). snRNP biogenesis is fundamental for splicing and transcriptome integrity (Kaida et al., 2010). One key consequence of splicing activity in cells is the accumulation of SMN and snRNPs in CBs (Liu and Dreyfuss, 1996; Carvahlo et al., 1999), which is disrupted in SMA motor neurones (Lefebvre et al., 1997). Understanding the mechanisms underlying SMN interaction is important for cell survival in general and particularly for the development of SMA therapy. Using co-immunoprecipitation experiments we identified a previously unknown regulator of the SMN complex. Protein phosphatase PP1c interacts with the complex and regulates its formation and localization to CBs.
Fig. 7. Depletion of PP1c enhances CB accumulation and the post-translational modification status of SMN. Triple-labelling immunofluorescence experiments were performed with anti-PP1c, anti-SMN and anti-coilin antibodies on HeLa cells transfected with (A) negative control Gl2 (GL2) or (B) PP1c siRNA#5. (C) The PP1c-silenced cells were transfected with FP–PP1c or (D) FP alone. The arrowheads and arrows point to one of the nuclear body gem (SMN alone) and to one of the CBs (SMN and coilin), respectively. The microscope was focused on the nuclear foci. Scale bar: 3mm. (E) Statistical analyses of SMN nuclear bodies in silenced cells. Error bars indicate the s.e.m.; ***P,0.001 (Student’s t-test). (F) HeLa cells were transfected with negative control (control RNAi) or PP1c siRNA (PP1c RNAi). Cell lysates were analysed by immunoblotting with PP1c, PP1a and SMN antibodies. The anti-a-tubulin incubation served as a loading control. (G) Nuclei preparations from cells analysed in F were separated by conventional 2D gel electrophoresis (IF; followed by second dimension, SDS-PAGE). Immunoblots were revealed with anti-SMN antibody. Brackets indicate four sections with major changes upon PP1c knockdown and rescue. The highest to the lowest phosphorylated forms of SMN are from section 1 to 4. The arrowheads point to isoforms accumulated in PP1c-depleted nuclei that disappeared when FP–PP1c was expressed. The arrows point to isoforms absent in PP1c-depleted nuclei and restored in rescue experiments. (H) The relative intensities of signals detected in sections of the SMN immunoblots shown in G.
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Gemin8 binds directly to PP1c and brings it into the multiprotein SMN complex, which is essential for snRNP biogenesis. SMN complex subunits associate in a stepwise manner in the cytoplasm at various stages of the snRNP biogenesis pathway (Carissimi et al., 2006; Massenet et al., 2002; Yong et al., 2010). Direct Gemin8 binding to SMN is at the heart of the association of SMN–Gemin2 and Gemin6–Gemin7– Gemin8–unrip subunits to form an intermediate complex that is competent to associate the Sm proteins of snRNPs. Thereafter, Gemin5, bound to the pre-snRNA, and Gemin3 and -4 are
considered to separately join the intermediate complex. We observed here that the reduction of PP1c levels enhances SMN interaction with Gemin8 and the number of CBs. As suggested by a mathematical model predicting that CBs accelerate snRNP biogenesis (Klingauf et al., 2006), our data indicate that PP1c depletion enhances the cytoplasmic assembly of snRNPs and thus, their nuclear import and accumulation in CBs.
Our observations suggest that the formation of an intermediate complex made of SMN–Gemin2 and Gemin6–Gemin7–Gemin8– unrip is regulated by phosphorylation. We propose that PP1c regulates the phosphorylation state of SMN to prevent premature formation of the intermediate complex. Although the exact PP1c target sites of SMN are unknown, 2D-gel analyses reveal that lowering PP1c levels lead to accumulation of hyperphosphorylated SMN (Fig. 7). Moreover, a reduction in the extent of SMN phosphorylation was observed by expressing PP1c in PP1c-depleted cells, which correlates with a lower CB number, reflecting downregulation of snRNP biogenesis. This provides a link between previous studies showing that SMN complex formation depends on SMN phosphorylation (Grimmler et al., 2004; Burnett et al., 2009) and its interaction with Gemin8 (Carissimi et al., 2006).
Other studies involving dephosphorylation of SMN in the nucleoplasm, showed that PP1MG regulates SMN complex stability in CBs (Petri et al., 2007). Consistent with our findings, PP1MG activity is probably not altered in PP1c-depleted cells. The Gemin8 interaction with PP1c in CBs (Fig. 2E) suggests that the two phosphatases function in different subnuclear compartments. Although SMN is the best candidate for a PP1c substrate, other proteins interacting with Gemin8 or located in the vicinity could also be candidates. There are sixteen potential phosphoserines in SMN. It will be interesting to determine whether PP1c targets the same residues as PP1MG and if not, this could explain why depletion of PP1MG decreases SMN localization to CBs (Petri et al., 2007), whereas PP1c depletion increases it.
Regulation of SMN interactions could stabilize the complex (Burnett et al., 2009; Ogawa et al., 2009). SMA mutations in the C-terminal region prevent self-oligomerization (Lorson et al., 1998) that disrupts Gemin8 binding to SMN and hinder SMN complex formation (Otter et al., 2007). There is a second minor self-oligomerization domain lying in the Lys-rich domain of SMN (Morse et al., 2007). Indeed, PP1c interaction with SMN complex appears to require two elements, the self-oligomerization Lys-rich domain of SMN (Fig. 1) and Gemin8 (Fig. 2). Dependence on the production of a functional SMN complex would provide a rationale for the failure of PP1c to associate with a mutant lacking the self-oligomerization domains. Another possibility is that the phosphoserine S80-P (MS information, PhosphositePlus; http://www.phosphosite.org/) located within the Lys-rich domain might be sensitive to PP1c. This aspect awaits further investigation.
It is worth noting that SMA modelling in Drosophila melanogaster and Caenorhabditis elegans has identified PP1 regulatory (inhibitory) subunit 13B (PP1R13B) as a modifier of SMN-deficiency phenotypes (Chang et al., 2008; Dimitriadi et al., 2010). This is consistent with our model in which ablation of the PP1 regulatory subunit exacerbates SMN-related defects by upregulating PP1. Only SMN and Gemins2, -3 and -5 has been identified in Drosophila (Kroiss et al., 2009). It is therefore conceivable that another SMN-associated protein binds PP1c. In
Fig. 8. Depletion of PP1c promotes formation of the SMN complex. (A) HeLa cells were transfected with control Gl2 or three different PP1c siRNAs (#5, #6 and #8) and separated into cytosolic and nuclear fractions. The fractions were analysed by immunoblotting with antibodies against components of the SMN complex. The anti-a-tubulin and anti-HDAC incubations served as loading controls for the cytosolic and nuclear fractions, respectively. (B) The cytosolic fractions of HeLa cells transfected with Gl2 or PP1c siRNA #5 were immunoprecipitated with control mouse
immunoglobulins (Ctrl) and anti-SMN antibody (SMN). The
immunoprecipitates were analysed by immunoblotting. (C) The relative amounts of the proteins associated with SMN were estimated using the relative band intensities detected by immunoblotting, normalized to the SMN band. The histograms show the results of five experiments. Error bars indicate the s.e.m. *P,0.042 (Wilcoxon signed-rank test). (D) Similar analyses of the SMN complex in the nucleus.
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the human SMN complex, Gemin5 has a putative PP1-binding RVxF motif, but Gemin5 does not form CBs (Hao et al., 2007). Other PP1-binding motifs are Fxx[R/K]x[R/K] (Garcia et al., 2004) and DxxDxxxD (Neduva et al., 2005). The former motif occurs in Gemin4 and the latter in SMN. We have shown here that neither Gemin4 nor SMN recruits PP1c to CBs (Fig. 3). Collectively, our results are most consistent with Gemin8 targeting PP1c to CBs. The similarity of effects upon Gemin8 overexpression (Fig. 4) and PP1c depletion (Fig. 6) indicate that Gemin8 is a previously unknown regulatory subunit of PP1.
Loss of SMN leads to motor neuron degeneration and muscle atrophy in SMA. The importance of SMN in muscles is beginning to be appreciated, given the varing degrees of disease severity (Bosch-Marce´ et al., 2011; Kariya et al., 2008; Mutsaers et al., 2011). Although the mechanisms are still elusive, several observations can be considered. Lowering SMN in cultured cells alters fusion and morphology of differentiated myoblasts (Shafey et al., 2005). Ablation of the muscular SMN causes loss of regeneration by satellite cells (Nicole et al., 2003). Moreover, localization of the SMN complex with a-actinin at the level of Z-discs suggests a function outside snRNP biogenesis (Walker et al., 2008). Our observations that PP1c and Gemin8 colocalize at Z-discs indicate that PP1c could regulate a function of the SMN complex in vivo (Fig. 9). Lack of subcellular organization and aberrant localization of Gemin8 and PP1c, as observed in SMA muscles, compares well with the Z-disc phenotype in SMA mouse models (Walker et al., 2008). Another interesting observation is that the interaction of PP1c with SMN is reduced in SMA cells. These defects might impair the regulation of SMN complex in SMA.
A plausible strategy for the development of therapeutics for SMA is to increase SMN protein levels (Lorson et al., 2010). The physiological relevance of our data is that a specific manipulation
of PP1c might impact on the SMN complex independently of an increase in SMN levels (Figs 5, 8). However, pharmacological ablation of all three PP1 catalytic subunits causes formation of SMN nuclear gems in SMA cells (Novoyatleva et al., 2008; our unpublished data). Because gems are storage sites formed when pre-mRNA splicing is inhibited, more specific molecules will be required. Modeling the interaction shown here between PP1c and the SMN complex could be a promising target in SMA therapy. Future in vivo experiments will need to address whether this has any beneficial effects.
Remarkable pleiotropy is seen for PP1 holoenzymes in distinct cellular pathways, including the EGF, TGF-b and FGF signaling pathways that have been linked to SMN (Bruns et al., 2009; Chang et al., 2008; Gangwani et al., 2001; Sen et al., 2011). Our studies reveal a regulation of the SMN complex and of nuclear body formation by PP1c, identify Gemin8 as directly interacting with PP1c, show mislocalization of PP1c and Gemin8 in SMA muscles in vivo and altered interaction of PP1c with SMN in SMA cells. A tight regulation of SMN complex assembly is revealed. It is therefore possible that imbalance of SMN sub-complexes produced by the reduction of SMN contributes to SMA severity.
Materials and Methods
Cell culture and transfections
COS7 and HeLa cell cultures were maintained in DMEM supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100mg/ml) in a humidified CO2
incubator at 37˚C. HeLa cells were plated in 60 mm dishes or eight-chamber culture slides (Becton Dickinson) for transfection using HiPerFect transfection reagent (Quiagen) with PP1c-validated siRNAs (Hs_PPP1CC_5, _6 and _8) and negative controls Gl2 or scrambled duplexes (25 nM). For the rescue experiments, HeLa cells were first transfected with siRNA (Hs_PPP1CC_5) and 16 hours later the cells were transfected with pdEYFP-PP1c plasmid (IOH14587-pdEYFPC1amp, imagines, Source BioScience, Nottingham, UK) using Transmessenger reagent (Qiagen). After a 5-hour incubation, the medium was Fig. 9. Localization of Gemin8 and PP1c in skeletal muscles. (A,B) Subcellular localization of Gemin8 and PP1c in longitudinal mouse and human foetal skeletal muscle sections as shown by immunofluorescence. Gemin8 has the striated appearance of the Z-discs as previously described for the components of the SMN complex (Walker et al., 2008). PP1c colocalizes with Gemin8 at the Z-discs as shown in merged images (yellow). ‘2nd only’ are control images of muscle sections incubated with the two fluorescent secondary antibodies alone. (C) SMN deficiency altered the muscle structure organization and localization of Gemin8 and PP1c in human Type I SMA foetuses. (D) Immunoblot analyses of muscle lysates from human control and type I SMA foetuses with Gemin8, SMN and PP1c. The anti-a-tubulin incubation served as loading control. (E) Immunoprecipitation of Gemin8 from control and type I SMA patient fibroblasts were analysed by immunoblotting. (F) Statistical analyses of the relative levels of SMN and PP1c co-precipitated with anti-Gemin8 antibody, and normalized to the levels of immunoprecipitated Gemin8 and to the levels of SMN input in samples (n53). Error bars indicate s.e.m.; **P,0.01 (Student’s t-test).
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replaced with fresh medium and incubated for a further 21 hours. Using FuGENE (Roche), HeLa cells were transfected with expression plasmids for pEGFP-SMN (Renvoise´ et al., 2006), myc-Gemin4 (a gift from S. Massenet, CNRS, Nancy) and Gemin8 (IOH3877-C1amp; imaGenes), and pdEYFP-Gemin8DC or pdEYFP-pdEYFP-Gemin8DC3A.
Indirect immunofluorescence
Forty-two hours post-transfection with siRNAs or 21 hours with DNA plasmids, HeLa cells were prepared for indirect immunofluorescence microscopy as described previously (Renvoise´ et al., 2006). Briefly, transfected cells were fixed in 4% formaldehyde, permeabilized with 0.5% Triton X-100 and incubated with the following antibodies (polyclonal unless otherwise designated as monoclonal: mAb): anti-SMN at 1:1000, mouse mAb 4B3 (Burlet et al., 1998) or 2B1 from Transduction Laboratories, anti-coilin [mouse mAb at 1:1000 (Transduction Laboratories) or rabbit at 1:1000 (Santa Cruz)], anti-PP1c (goat at 1:1000, SC), anti-Gemin3 (mouse mAb at 1:250; Abcam), anti-Gemin8 (rabbit at 1:30; Sigma), anti-TMG (mouse mAb at 1:4000; Calbiochem), anti-SmD3 protein (rabbit at 1:100; Sigma), anti-U1-specific 70 kDa protein (rabbit at 1:500; Aviva), anti-SC35 (mouse mAb at 1:1000; Sigma), anti-myc (mouse mAb at 1:1000; Covance) and either Cy3 or Cy5 (1:400; Jackson Laboratories) or Alexa-Fluor-488-conjugated secondary antibodies (1:400; Invitrogen). The final wash contained 0.1mg/ml 4, 6-diamidino-2-phenylindole (DAPI; Molecular probes) to stain DNA. Cryosections (7–10mm) of muscle tissues from mouse (provided by J. Cartaud) and human foetuses frozen at –80˚C in isopentane were mounted for immunostaining as described previously (Lefebvre et al., 1997; Burlet et al., 1998).
In situ proximity ligation assay
In situ proximity ligation assay (PLA) was performed as recommended by the manufacturer (DuolinkII kit, Olink Bioscience AB). Briefly, HeLa cells were fixed and permeabilized as above. Primary antibodies were diluted at 1.75 ng/ml rabbit anti-Gemin8 (Atlas antibodies, Sigma) and 2 ng/ml goat anti-PP1c (C19, SC) in 16 antibody diluent and incubated for 30 minutes at room temperature. The negative control consisted of using only one primary antibody. The cells were washed twice for 5 minutes in Tris-buffered saline with 0.05% Tween. The PLA probes (Rabbit-MINUS and Goat-PLUS; Olink BioScience AB) were incubated for 90 minutes at 37˚C. Subsequent steps were performed using the detection reagent Orange according to the DuolinkII kit protocol. In situ PLA signals were visible as dots with the RITC filter on the microscope. Nuclei were counterstained with DAPI to be able to select image position.
Image acquisition and manipulation
Immunofluorescence signals were observed by nonconfocal microscopy (Leica DMR, objective 636/1.32). Black and white images were acquired with a cooled CCD camera (Micromax, Princetown Instruments, Inc.) using the MetaView Imaging System. Figures were processed and assembled with Photoshop (Adobe).
Protein extract preparation and immunoprecipitation
Cytosolic and nuclear fractionation was performed with a NE-PER kit (Pierce). For immunoprecipitations, the cytosolic fractions were used directly and isolated nuclei or frozen cell pellets were resuspended in immunoprecipitation (or RIPA) buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, EDTA-free protease inhibitors cocktail (Roche)], passed through a 27-gauge needle and clarified by centrifugation at 15,000 g for 15 minutes at 4˚C. Protein G dynabeads (25ml; Invitrogen) were covalently bound to 10mg of the following antibodies according to the manufacturer’s protocol: mouse monoclonal anti-GFP (Roche), mouse monoclonal anti-SMN 4B3 or mouse negative control antibodies (DAKO). The coated dynabeads were incubated with protein extracts for 1 hour at 4˚C. After five to seven washes in 1 ml immunoprecipitation buffer, the bound proteins were eluted by boiling in 26 SDS sample buffer and resolved by SDS-PAGE. Cultured COS cells expressing either GFP–SMN or GFP–N86M2 were crosslinked with dithiobis[succinimidyl propionate] (Pierce) to facilitate the biochemical analysis for Fig. 1E only. The anti-Gemin8 immunoprecipitations were performed in IP buffer containing 0.01% SDS using rabbit anti-Gemin8 antibodies (Santa Cruz) and negative control rabbit antibodies (DAKO).
Protein expression and purification
His-tagged PP1c was expressed from IOH14587-pDEST17-D18 (imaGenes) in E. coli BL21 (DE3) pLysS (Stratagene) at 30˚C for 5 hours and purified on Ni-NTA agarose (Qiagen) as previously described for His-tagged proteins (Lefebvre et al., 2002). Purified His–PP1c was either used immediately or snap-frozen and kept at 280˚C. GST vector (Pharmacia) and GST–Gemin8 (IOH3877-pDEST15, imaGenes) were expressed in E. coli Rosetta2 (DE3) pLysS (Novagen, Merck) at room temperature for 5 hours. Recombinant GST proteins were purified and retained on magnetic beads (magneGST protein purification system, Promega) for protein-binding assays, or eluted by boiling in 26 SDS sample buffer and resolved by SDS-PAGE.
In vitro protein-binding assay
SMN complex was immunopurified from HeLa whole-cell extracts with monoclonal antibody 4B3 under stringent conditions (500 mM NaCl and 0.1% NP40) on magnetic dynabeads as described above. The immobilized proteins (SMN complex or GST proteins, 12.5ml of magnetic beads slurry) were pre-incubated for 5 minutes with 1 mg/ml BSA, washed and pre-incubated for 30– 60 minutes at 4˚C with 2.5mg of purified recombinant His-tagged PP1c in 25 ml of binding buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5 mM MnCl2, 1 mM dithiothreitol (DTT) and 0.05% NP40. The bound proteins were
washed five to seven times with 500ml of ice-cold binding buffer and eluted by boiling in 26 SDS sample buffer.
Protein separation and immunoblot analysis
Protein samples were separated by 11% SDS-PAGE (Prosieve acrylamide) or a 4– 12% gradient (Invitrogen) and electrotransferred to PVDF membranes (Millipore). Immunoblotting was performed as described previously (Renvoise´ et al., 2006), revealed by chemiluminescence (ECL; GE Healthcare) and exposed to Fuji high-speed X-ray films or captured with a Fujifilm LSA-3000 camera system. The following antibodies were used: SMN (mouse mAb 4B3 at 1:5000), anti-Gemin2 and -3 (mouse mAb at 1:500; Abcam), Gemin4 (goat at 1:4000; Santa Cruz), Gemin8 (mouse mAb at 1:1000; Abcam), PP1a (rabbit at 1:10,000; Santa Cruz), PP1c (goat at 1:10,000; Santa Cruz), a-tubulin (mouse mAb at 1:10,000; Sigma), HDAC (rabbit at 1:500; Santa Cruz), phosphoserine (rabbit at 1:200; Invitrogen), GFP (mouse at 1:1000; Roche; or rabbit at 1:5000; Abcam) and horseradish peroxidase-conjugated IgG (at 1:10,000; GE Healthcare or Santa Cruz). For 2D-gel electrophoresis, the nuclear pellets prepared using the NE-PER kit were directly solubilized in 8 M urea, 1 M thiourea, 2.5% CHAPS, 23 mM DTT and 0.5% immobilized pH gradient (IPG) using a 27-gauge needle. The first-dimension was performed in strips with IPG using pH 3–10 linear IPG drystrips (GE Healthcare) at the Institut Jacques Monod proteomics facility.
Statistical analyses of ECL signals were performed with ImageJ using grayscale images generated with an Epson Perfection 4990 PHOTO transparency scanner (1000 d.p.i. images, as shown in the figures assembled with Photoshop) or with a luminescent analyser LAS-3000 camera system (Fujifilm).
Candidate proteins by mass spectroscopy
Co-immunopurified proteins were separated by SDS-PAGE and visualized by mass-spectrometry-compatible silver staining (Silver Quest, Invitrogen). The bands of interest were excised, extensively digested with trypsin, and extracted peptides were analysed using a-cyano-4-hydroxycinnamic acid (CHCA) matrix on a 4800 TOF-TOF mass spectrometer (Applied Biosystems) equipped with a YAG-200 Hz laser (355 nm) at Institut Jacques-Monod proteomics facility. Protein identification presented in supplementary material Fig. S1 was performed using expasy proteomics tools (www.expasy.org/tools/).
Acknowledgements
We wish to thank A. and J. Cartaud for constant support. We are grateful to colleagues for critical reading of the manuscript and generously providing protocols, controls and antibodies. S. Massenet (CNRS, Nancy) generously provided the myc-tagged Gemin4 plasmid. We acknowledge the proteomics core facility of the Institut Jacques Monod.
Funding
This work was supported by Institut National de la Sante´ et de la Recherche Me´dicale (salary to S.L.); Centre national de la recherche scientifique; the Association Franc¸aise contre les Myopathies (grant to S.L.); an undergraduate fellowship from the Ministe`re de l’Education Nationale, de la Recherche et de la Technologie (to B.R.); and the Association Francaise Contre les Myopathies (to B.R.).
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.096255/-/DC1
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