Human Molecular Genetics, 2002, Vol. 11, No. 9 1017-1027
© 2002 Oxford University Press
A novel association of the SMN protein with two major non-ribosomal nucleolar proteins and its implication in spinal muscular atrophy
INSERM U393, IRNEM Institute, Hôpital Necker-Enfants Malades, 149 Rue de Sèvres, 75743 Paris Cédex 15, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Spinal muscular atrophy (SMA) is caused by the loss of functional survival motor neuron 1 (SMN1) protein. This ubiquitously expressed protein is a component of a novel complex immunodetected in both the cytoplasm and the nucleus, which is associated with complexes involved in mRNA splicing, ribosome biogenesis and transcription. Here, we study a mutant protein corresponding to the N-terminal half of the protein that is encoded by the SMA frameshift mutation SMN 472del5. We show by confocal microscopy that the resulting mutant protein exhibits various distribution patterns in different transiently transfected COS cells. The mutant distributes into the nucleoplasm and/or the nucleolus, whereas the normal SMN protein accumulates at discrete nucleocytoplasmic dot-like structures previously named gems/Cajal bodies. The cell population with the nucleolar distribution is enriched upon treatment with mimosine, a synchronizing drug in late G1 phase. Co-immunoprecipitation studies carried out on nuclear extracts reveal that both the endogenous SMN and mutant proteins are associated with complexes containing two major non-ribosomal nucleolar proteins, namely nucleolin and protein B23, and that the association is mediated, by among other things, RNA moieties. Both the association of the SMN protein with nucleolin-containing complexes and the nucleolin/B23 complex are disrupted in fibroblasts derived from a type I SMA patient harboring a homozygous SMN1 gene deletion. These findings suggest that altered assembly and/or stability of ribonucleoprotein complexes may contribute to the pathophysiological processes in SMA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Proximal spinal muscular atrophy (SMA) is a frequent inherited neuromuscular disease of childhood characterized by degeneration of spinal motor neurons, leading to progressive muscular atrophy. SMA has been divided into three forms based on age of onset, clinical severity and lifespan (for a review, see 1). The disease is caused by mutations (deletions in 96% of patients) of the survival motor neuron 1 gene (SMN1) located in a duplication on chromosome 5q13 (2). SMN1 and its copy, SMN2, produce multiple mRNA isoforms, and the alternatively spliced form lacking exon 7 is specific to SMN2 and results from a C/T transition in exon 7 (2–4). Importantly, the reduced level of SMN protein produced from the SMN2 gene correlates with disease severity (5–7). Recently, the creation of mouse models showed the essential role of the mouse single-copy smn gene and the dose-dependent modulation of the phenotype with a human SMN2 transgene (for a review, see 8). The SMA causative function of SMN protein is still unknown, but recent findings show its involvement in small nuclear ribonucleoprotein (snRNP) assembly and recycling, ribosome biogenesis and transcription (9–14).
The SMN protein (294 amino acids) contains distinct functional domains that mediate various interactions, including interactions with itself, the Sm proteins, the tumor suppressor protein p53, fibrillarin, GAR1 and nucleic acids. These interactions are likely to be relevant to the disease, since SMN mutants identified in SMA patients were shown to be deficient in these activities (15–19). Furthermore, we have analyzed the RNA-binding capacity of the SMN protein from orthologuous genes and found that a deletion of the zebrafish SMN protein mimicking the SMA frameshift mutation SMN 472del5 (20) altered the RNA-binding properties of the protein in vitro (21).
The SMN protein is detected in the cytoplasm and in nuclear gems (gemini of Cajal bodies) colocalized with Cajal (coiled) bodies (G/CB) (22) in cell culture and also in nucleoli in tissues (5,7,23–25). Cajal bodies are dynamic nuclear suborganelles highly enriched in snRNPs and involved in pre-mRNA splicing, preribosomal RNA processing and histone pre-mRNA 3'-end processing (for a review, see 26). The zinc finger protein ZPR1, a mitogenic signal protein, has been shown to be involved in the nuclear localization of the SMN protein (27). More recently, the CB-specific protein p80 coilin was demonstrated to be essential for the recruitment of SMN to gems/Cajal bodies (28).
The interactions of SMN protein with Sm proteins, heterogeneous nuclear ribonucleoprotein (hnRNP) K, hnRNP Q, fibrillarin, GAR1, RNA helicase A and coilin were shown to be mediated by their arginine and glycine (RG)-rich domains (12–14,22,28,29). Studies have established that RG-rich domains from nucleolin, fibrillarin, hnRNPs and Sm proteins are substrates for protein arginine methyltransferases (30 and references therein). Interestingly, it has been demonstrated that arginine dimethylation enhances the affinity of the SMN/Gemin2 complex for the RG-rich domain of the Sm proteins (31).
Nucleolin is a multifunctional protein that has been implicated in several cellular processes, including cell growth and proliferation control, programmed cell death, cell surface signal transduction, and differentiation and maintenance of neural tissues (for a review, see 32). There is a correlation between the level of nucleolin and the progression of the cell cycle. Indeed, the levels of nucleolin are low in non-dividing cell cultures and its expression is increased in mid to late G1 phase of the cell cycle. Nucleolin, protein B23 and fibrillarin have been shown to associate within large pre-ribosomal RNA particles (33). Also, the interaction between nucleolin and B23 has been studied in yeast two-hybrid experiments (34).
Here, we show that the human mutation SMN 472del5 impairs both the in vitro RNA-binding activity of the protein and the subcellular localization in transiently transfected COS cells. The mutant protein is immunodetected throughout the nucleoplasm and/or in the nucleolus in a cell-cycle-dependent manner. Co-immunoprecipitation experiments to test the possible association between SMN protein and nucleolin identify both nucleolin and protein B23 associated with SMN in a RNase-sensitive manner. These data indicate that SMN can associate with large particles of the ribosomal RNA metabolism. Importantly, we provide the first in vivo evidence that the composition of the nucleolin-containing particles is altered in association with a marked reduction of the SMN protein level in fibroblasts derived from a type I SMA patient.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The SMN 472del5 protein has lost RNA-binding preference in vitro
As predicted from our previous results with a truncated zebrafish SMN protein (21), we determined first whether the SMA frameshift mutation SMN 472del5 (20) can alter the RNA-binding properties of the protein in vitro. This SMA-linked mutation encoded a mutant protein with one nonsense aminoacid after C146, resulting in the loss of the C-terminal half of the protein (Fig. 1A). The corresponding recombinant protein (rSMNmut) was tested for its ability to bind to the ribonucleotide homopolymers in vitro and compared with that of the full-length SMN protein (rSMN, 294 amino acids; Fig. 1B). The wild-type SMN protein bound more strongly to poly(G) than to poly(C), poly(U) and poly(A), as previously reported (15). The mutant protein showed no preferential association with poly(G) RNA, despite the presence of the RNA-binding determinant, suggesting that the intramolecular modulatory properties of the SMN protein are conserved in evolution (21). Recently, these intramolecular interactions have been characterized using the BIAcore methodology (35).
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The mutation SMN 472del5 alters the distribution of the encoded protein
To gain further insight into the cellular consequences of the SMA frameshift mutation SMN 472del5, we compared the distribution of the mutant protein in COS cells transiently transfected with constructs designed to produce either the wild-type or mutant SMN proteins fused to an N-terminal HA-epitope (Fig. 2A). Immunoblot analyses with mouse monoclonal anti-HA and anti-SMN antibodies revealed that both the HA-SMN and HA-SMNmut proteins were produced at their expected sizes of 38 and 21.5 kDa, respectively, suggesting that they were not subject to degradation (Fig. 2B). The subcellular distributions of both overexpressed proteins were investigated by indirect immunofluorescence analyses using confocal microscopy (Fig. 2C–F). The monoclonal anti-HA antibody detected the HA-SMN protein in the nucleoplasmic dot-like structures termed gems/Cajal bodies (G/CB) and in the cytoplasm, as previously reported (22) (Fig. 2C). Transient transfection of the HA-SMNmut protein resulted in various distribution patterns in different transfected cells: (i) the immunodetection throughout the nucleoplasm and the nucleolus, (ii) the immunolabeling of the nucleoplasm and (iii) the accumulation of the protein into the nucleolus (Fig. 2D–F). We estimated the fraction of transfected cells harbouring each specific distribution and found that the simultaneous immuno-reactivity of the nucleoplasm and the nucleolus was detected in 82% of cells and the accumulation into the nucleolus was observed in 11% of the cells (Fig. 3B and C). Interestingly, the mutant SMN 472del5 displayed a unique distribution pattern as compared with that of the normal protein and other SMA-linked SMN mutants recently reported in transiently transfected cell cultures (36,37). Our data suggest that this SMA-linked mutation might reflect a defect in the regulation of SMN subnuclear transport.
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The transfected cell population in late G1 phase displays a nucleolar distribution of the SMN 472del5 protein
Because the mutant protein showed various nuclear distribution patterns, we investigated whether there might be a relationship between the cell cycle and the spatial distribution of the fusion protein using synchronizing drug treatments to block at distinct cell cycle points (Fig. 3). We used three well-characterized drugs commonly employed in cell synchronization experiments (38). Mimosine, a non-protein amino acid, inhibits DNA initiation and arrests cell cycle progression at late G1 phase. Cells treated with thymidine to prevent DNA synthesis are blocked in S phase. Nocodazole is a microtubule-depolymerizing drug that results in a cell population synchronized at the G2-to-M phase transition. We determined by flow cytometric measurement of DNA content that the drugs employed in our experimental conditions gave partly synchronized cell populations in G1 (64%), S (68%) and G2 (82%) phases with mimosine, thymidine and nocodazole, respectively (data not shown). We compared the effects of each drug for the localization of the mutant protein in transiently transfected cells (Fig. 3). Treatment with mimosine enriched a cell population with the mutant protein almost exclusively immunodetected in the nucleolus (57%). Following a single thymidine block, the mutant showed strong immunostaining in both the nucleoplasm and the nucleolus in an enriched cell population corresponding to 88% of the transfected cells. In the presence of nocodazole, a significant increase was observed in the fraction of cells with the mutant protein accumulated in the nucleoplasm and excluded from the nucleolus (68%) compared with the untreated cells (7%). Those treatments also appeared to enrich specific populations of the cells transfected with the full-length SMN protein (Fig. 3A). Most of the HA-SMN protein was in the cytoplasm and in perinucleolar G/CB of mimosine-treated cells. Under thymidine block, the HA-SMN protein immunostaining concentrated in 2–8 large nuclear dot-like structures (G/CB) and was diffuse in the cytoplasm. The full-length protein did not concentrate at discrete nucleoplasmic foci in cells from the nocodazole-induced block – probably because of a low nuclear concentration of the protein.
In order to confirm the nucleolar distribution of the mutant protein, double immunolabeling experiments were carried out by confocal microscopy with a mouse monoclonal antibody to nucleolin, a major nucleolar RNA-binding protein, and a rabbit polyclonal anti-HA antibody (Fig. 4). The strong immunodetection signal of the nucleolin allowed visualization of the nucleolus. An overlay of both the nucleolin and the mutant protein specific signals demonstrated a complete superimposition of both nucleolar images. A view of the merge of the three-dimensional projection obtained using 16 focal planes for both proteins also confirmed that the mutant protein was detected within the nucleolus. Our results demonstrated that loss of the C-terminal half of the SMN protein resulted in the redistribution of the mutant protein to the nucleolus and the nucleoplasm, and agreed with a cell-cycle-dependent nucleolar phase of the SMN protein.
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Co-immunoprecipitation of SMN protein with nucleolin
Both the endogenous SMN protein on tissue sections (23–25) and the HA-SMN 472del5 protein in cell culture (Figs 2–4) were detected within the nucleolus. Taking this together with the observation that SMN protein binds to proteins via their RG-rich domain (for a review, see 39), we speculated about the possible association of the SMN protein with nucleolin, a nucleolar RG-containing protein. To determine whether nucleolin was able to associate with the HA-SMN and the HA-SMNmut proteins in transiently transfected cell cultures, we performed immunoprecipitation studies with an anti-nucleolin mouse monoclonal antibody and probed with an anti-SMN mouse monoclonal antibody (Fig. 5A). The co-immunoprecipitations were carried out under relatively non-denaturating conditions (150 mM KCl, 0.1% NP-40) using nuclear extracts from mimosine-treated cells, which corresponds to the enrichment of the nucleolar immunodetection of the mutant protein. We have shown earlier that treatment with the non-ionic detergent NP-40 (0.5%) did not dissociate SMN protein from RNP complexes (7). Both the HA-SMN and HA-SMNmut proteins were detectable in complexes associated with nucleolin (Fig. 5A). Interestingly, the immunoblot analysis showed that a fraction of the nuclear endogenous SMN protein was also immunoprecipitated with the anti-nucleolin mouse monoclonal antibody. To further establish this association, reverse immunoprecipitation was performed using a monoclonal antibody to SMN, and immunoblot analyses with anti-nucleolin monoclonal antibody revealed the presence of two major immunoreactive bands, 100 and 72 kDa (a proteolytic product of nucleolin; 32) (Fig. 5B). The phenotypic consequence of SMN mutation is primarily a disease of spinal motor neurons. Accordingly, we prepared nuclear fractions from mouse spinal cord and performed immunoprecipitations with an anti-SMN mouse monoclonal antibody and probed with anti-nucleolin mouse monoclonal antibody. These experiments showed that the association of SMN with nucleolin also occurs in spinal cord (Fig. 5C). Immunoprecipitations performed without an antibody or with a negative control mouse monoclonal antibody (DAKO) showed that no bands were significantly contributed by the Dynabeads M-280 sheep anti-mouse IgG alone nor by the negative control IgG, indicating that non-specific binding was not critical using the Dynabeads technique (data not shown). These data demonstrated that under our experimental conditions, the SMN protein and the nucleolin are associated in vivo.
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RNase treatment disrupts the SMN association with nucleolin-containing complex(es)
Because both SMN and nucleolin have characteristics of RNA-binding proteins, their association can be due to binding to common RNA moieties. To test this hypothesis, the co-immunoprecipitation with anti-SMN antibody was performed with nuclear extracts predigested at 30°C with either RNase A or RNase-free DNase RQ1 (Fig. 5D). The immunoblot analyses showed that nucleolin is dissociated from the SMN protein complex in RNase-treated nuclear extracts. Under DNase treatment, the association was similar to that of the untreated sample. The nucleolar protein B23 is associated with the nucleolin-containing complex in an RNA-dependent manner (33). The blot was probed with an anti-B23 antibody to characterize the association of SMN protein with nucleolin-containing RNP complexes. Interestingly, B23 was also immunoprecipitated with the SMN protein. These results demonstrated that RNA integrity should be preserved for SMN protein to associate with nucleolin/B23 complex(es).
The marked reduction of SMN protein in type I SMA-derived fibroblasts disrupts nucleolin/B23 complex(es)
A consequence of SMN gene mutation is the reduction of the functional SMN protein level in tissues derived from SMA patients (5–7). Here, we derived SV40-immortalized fibroblasts from a type I SMA patient harboring a homozygous SMN1 gene deletion. Western blot analysis of these cells revealed a residual level of SMN protein of about 10% compared to control SV40 immortalized fibroblasts derived from a normal individual (Fig. 6A). The association of SMN protein with nucleolin/B23 complex(es) was also demonstrated by using the nuclear extract from SV40-immortalized human fibroblast cell culture (Fig. 6B). We performed co-immunoprecipitation experiments with anti-nucleolin using the nuclear extract from type I SMA-derived fibroblasts. Neither SMN nor B23 were detected in the nucleolin-containing complexes isolated from the SMA fibroblast cell culture under our experimental conditions, showing a close correlation with defects in nucleolin/B23 complex assembly and/or stability.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Ever since the identification of the SMA-determining gene, SMN, the efforts have aimed to elucidate the function of the SMN protein and unravel the mechanism responsible for spinal muscular atrophy. The nuclear fraction of the SMN protein has been shown to contribute to the recycling of the splicing machinery (10,11), small nucleolar ribonucleoprotein (snoRNP) biogenesis (12,13) and regulation of RNA pol II-dependent transcription (14,40–42). These candidate functions as possible causes of SMA have been identified in control cell systems. SMN mutants derived from SMA patients were shown to be deficient in a number of protein–protein interactions in vitro, including self-oligomerization and interactions with the Sm proteins, p53, fibrillarin and GAR1 (15–19). However, these mutants failed to reveal any abnormal distribution pattern in transiently transfected cells (36,37). To characterize the cellular consequences associated with the disease, one approach will be to identify SMN mutants derived from patients that show aberrant spatial distribution.
In the present study, we first compared the RNA-binding capacity in vitro and the subcellular distribution of the SMN mutant encoded by the frameshift mutation SMN 472del5 derived from a type I SMA patient (20) and the normal full-length protein (294 amino acids). The resulting mutant protein (147 amino acids), corresponding to the N-terminal half of the SMN protein, loses its preference for the poly(G) homopolymer in an in vitro RNA-binding assay. The full-length SMN protein was shown to bind more strongly to the poly(G) homopolymer than to the poly(U), poly(A) and poly(C) homopolymers (19,20). The SMA-derived SMN mutants Y272C, S262I and SMN
ex7 also have reduced RNA-binding activities in vitro (19). Together, these findings show that the integrity of the SMN protein must be preserved to allow proper RNA-binding activity in vitro.
Immunodetection studies revealed that the SMN protein is located in both cell lines and tissues in the cytoplasm and nuclear gems/Cajal bodies (G/CB) (5,7,22). SMN immunolabeling within the nucleolus and perinucleolar G/CB were reported in postnatal tissues (23–25). Also, in cell culture upon inhibition of protein synthesis, the SMN protein was transiently detected at the nucleolar periphery (13). Here, the truncated protein SMN 472del5 transiently expressed in COS cells was immunodetected throughout the nucleoplasm and within the nucleolus, whereas the full-length SMN protein was concentrated in nucleoplasmic dot-like structures previously identified as G/CB (22). Our results suggest that altered dynamics of the SMN distribution between the G/CB and the nucleolus is involved in the pathogenesis of SMA.
The SMN protein is part of in a novel complex including several new components: Gemin2/SIP1, Gemin3/dp103, Gemin4/GIP1 and Gemin5 (43 and references therein). Interestingly, both the SMN protein and Gemin4/GIP1 are the only components of this novel complex thus far detected in both G/CB and nucleolus. The SMN protein interacts directly with Gemin2/SIP1 and Gemin3/dp103, and Gemin4/GIP1 interacts directly with Gemin3/dp103. The SMN 472del5 mutant lacks the self-association site and interacting domains for Gemin4–Gemin3. The lack of these functional domains impairs the accumulation of the protein into G/CB and accounts for the nucleoplasmic and nucleolar accumulation in cell culture. This observation further underlines the close link between the G/CB and the nucleolus (for a review, see 26).
In previous studies, the dynamics of CB have been examined during the cell cycle using p80 coilin as a CB marker (44). During mitosis, the CB disassemble and distribute throughout the cytoplasm. CB were observed to be larger and fewer (one or two) in S and G2 phases of the cell cycle, and smaller and numerous (three to eight) during G1 phase. Furthermore, cyclin-dependent kinase 2 (CDK2) and cyclin E were shown to accumulate in CB during the G1-to-S phase transition, which coincide with the expression of cyclin E and its role in the regulation of the G1-to-S transition (45 and references therein). Treatment with a CDK inhibitor redistributed CDK2 to the nucleolus, but cyclin E localization remained similar to that of the untreated cells. Interestingly, among the CB proteins phosphorylated by the CDK2–cyclin E complex, there is a subset of proteins interacting with SMN protein, including p80 coilin, p53 and the Sm B/B' proteins. These data indicate that several CB components are associated with mechanisms regulating cell cycle progression during the G1-to-S phase transition.
Here, we also showed that the subnuclear distribution of the mutant SMN 472del5 protein is cell-cycle-dependent using synchronizing drugs. The mutant accumulated in the nucleolus at late G1 phase (mimosine treatment) and redistributed from the nucleolus to the nucleoplasm during S phase (thymidine block), being exclusively nucleoplasmic at the G2-to-M phase transition (nocodazole treatment). These results strongly suggest that the SMN protein exhibits transient association with the nucleolus in a cell-cycle-dependent fashion.
An important question in view of our findings is whether the SMN mutant is recruited to the nucleolus by interacting with the endogenous SMN complex and/or with nucleolar components. One can speculate that SMN protein has a nucleolar localization signal (NoLS). Indeed, SMN exon 2B encodes a lysine-rich region that resembles the NoLS of p80 coilin (46). None of the SMN expression constructs reported so far has allowed the identification of an NoLS. In view of the localization studies reported by Thanh et al. (37), SMN exon 4- and/or exon 6-encoded domains might be responsible for masking the NoLS. This idea is further supported by the finding that the SMN exon 2A–2B region of the protein interacts in vitro with that of exon 4 (35). Similarly, intramolecular interactions are important for the subnuclear localization of the CB-specific p80 coilin. It will be interesting to verify whether the interaction between SMN exons 2A–2B and 4 might serve to release the SMN protein into the nucleolus.
On the other hand, the nucleolar targeting of the SMN protein may result from its association with nucleolar components. Here, we demonstrated that the anti-SMN antibody is able to immunoprecipitate RNP complexes containing two major non-ribosomal nucleolar proteins, and we established that the association of nucleolin and protein B23 with the SMN protein is due to, among other things, binding to common RNA moieties. Nucleolin is a multifunctional protein that plays a role in transcription/processing of pre-rRNA, nucleocytoplasmic shuttling, mRNA stability and mRNP assembly (for a review, see 32). These functions are regulated by proteolysis, autodegradation, phosphorylation, methylation and ADP-ribosylation. For example, the cleavage of the phosphorylated nucleolin generates 30 and 72 kDa proteins, being involved in the activation of rDNA transcription. Nucleolin is required for the first step of pre-rRNA processing and associates in an RNA-dependent manner with protein B23 (33). Protein B23 is a multifunctional protein involved in the processing of 5.8S rRNA (47), and assembly and transport of the pre-ribosomal particles (48). The localization of these proteins is not limited to the nucleolus, and they can be detected in the nucleoplasm. The rDNA genes are transcribed as a large pre-rRNA of 47S, which is extensively processed to give mature 18S, 5.8S and 28S rRNAs. The characterization of proteins associated with pre-ribosomal complexes using monoclonal antibody to nucleolin revealed the existence of intermediate complexes containing both B23 and fibrillarin (33). Interestingly, SMN protein interacts directly with fibrillarin, a nucleolar protein involved in pre-rRNA processing (12,13). Therefore, the association described here between SMN and the nucleolin-containing complexes might be mediated by the interaction with fibrillarin and small nucleolar (sno) RNAs. Our results suggest that SMN protein associates in complexes with precursor and processing rRNA intermediates, and one can speculate that SMN may be implicated in different aspects of ribosome biogenesis. Our finding that the association between SMN and nucleolin is sensitive to RNase treatment does not rule out the possibility that protein–protein interaction might also participate in the biogenesis of those complexes. Indeed, SMN protein interacts directly with RG-rich domains (12–14,22,28,29) and it remains to be established whether it interacts directly with the RG-rich domain of the nucleolin.
Taken together, the co-immunoprecipitation with anti-nucleolin antibody of both mutant and endogeneous SMN proteins and the nucleolar immunodetection of the mutant suggest that both proteins may travel through the nucleolus in association with the nucleolin-containing RNP complexes and that the mutant may interrupt its journey owing to the absence of functional assocations with other SMN-interacting proteins. Further, the observation is that nucleolin and B23 associate with SMN protein and that the nucleolin/B23 complex is disrupted in the fibroblasts derived from an SMA patient shed light on novel nuclear SMN functions, and further characterization of the SMN-associated RNPs in SMA patients should help elucidate the physiopathology of this neuromuscular disease. Although the exact function of CB is not known, they are postulated to play a role in the preassembly of the transcription and processing factors of the three eukaryotic RNA polymerases pol I, pol II and pol III (for a review, see 26). The efficiency with which RNP assembly occurs in SMA patients would influence the cellular activity of both motor neurons and muscle cells. An overall picture emerges of the SMN protein as an assembly factor (39), enhancing different aspects of RNA metabolism, and thus facilitating cell proliferation and viability.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Engineered fusion proteins
Full-length 1.6 kbp human SMN cDNA (2) was used to prepare DNA fragments by restriction enzyme digestion or by PCR amplification with Taq polymerase (Life Technologies). The DNA fragment for the SMN 472del5 mutant was generated by PCR amplification using sense, 5'-CATCCGCGGAATTCCCATGG-3', and antisense 5'-TTATTAGCTACAGATTGGGGAAAG-3', oligonucleotides. The digested PCR fragment was subcloned in pET30 (Novagen, Madison, WI) for protein expression in E. coli BL21(DE3). For the eukaryotic expression construct, the PCR product was subcloned into pT7linkHA and inserted as a HindIII–XbaI fragment into the pcDNA3 vector (Invitrogen). The hemagglutinin epitope (HA) is fused at the N-terminus of the protein. A natural PstI restriction site in exon 2B was used to insert the 3' end of the SMN cDNA into the SMN 472del5 constructs in order to generate the normal SMN protein. The constructs were analyzed by DNA sequencing (Perkin-Elmer/ABI, Foster City, USA).
Cell culture, transfection and drug treatments
COS-7 cells and SV40-immortalized human fibroblasts were maintained in complete modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptamycin (100 µg/ml). For the immunofluorescence analyses, cells were plated in an 8-chamber CultureSlide (Becton Dickinson Laboratories) some 12–24 h before transfection. Cells were transfected with Fugene 6 (Roche Diagnostics) and plasmid DNA was prepared using a purification kit (Quiagen). Cells were partly synchronized during the cell cycle by drug treatment for 16 h at 37°C with 400 µM mimosine (Sigma), 2 mM thymidine (Sigma) or 400 ng/ml nocodazole (Sigma) to enrich cell populations in late G1, S or at the G2-to-M phase transition, respectively (38). For cellular DNA content analyses, the trypsined cells were fixed in 70% ethanol at 4°C overnight. The cells were stained for 1 h at room temperature in PBS solution containing 100 µg/ml RNase A, 50 µg/ml propidium iodide and 1 mg/ml glucose. The samples were analyzed using FACSort, and the percentages of the different phases of the cell cycle were determined using FACScan/CellFit System (Becton-Dickinson, Mountain View, CA) as described previously (49).
Indirect immunofluorescence confocal microscopy
Cells were washed in PBS, incubated for 30 min in 4% paraformaldehyde with 0.5% Triton and blocked with 10% horse serum (HS)–3% BSA for 1 h. The cells were incubated with the primary antibody diluted in 1% HS–0.3% BSA for 1 h, washed in PBS and incubated with the secondary antibodies for 30 min. Double immunostaining experiments were performed by sequential incubations of each primary antibody and its appropriate secondary antibody. The cells were washed and mounted with AF1 (Cityfluor, UK). The following primary antibodies were used: anti-HA (rabbit serum at 1 : 700, Santa Cruz; mouse mAb 12CA5 at 1 : 200, Roche Diagnostics) and anti-nucleolin (mouse mAb at 1 : 500, Santa Cruz). Secondary anti-rabbit Cy3 (1 : 500) and anti-mouse Cy3 (1 : 500) antibodies were purchased from Jackson Lab and anti-mouse fluorescein (1 : 100) from DAKO. Laser confocal fluorescence images were generated using a Zeiss LSM 510 confocal microscope.
Ribonucleotide homopolymer-binding assays
The His-tagged SMN and mutant 472del5 proteins were produced by IPTG (1 mM) induction of E. coli BL21(DE3) transformants. The proteins were purified in urea and eluted under native conditions (0.05 M sodium phosphate, 0.15 M NaCl, 0.01 M Tris–HCl, 0.01 M ß-mercaptoethanol, 10% glycerol and 0.25 M imidazole, pH 8.0). Binding experiments to RNA homopolymers of the recombinant proteins were performed as described elsewhere (21).
Co-immunoprecipitations
Nuclear fractions from cell cultures were prepared in the presence of protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 3 µg/ml pepstatin, 3 µg/ml anti-papain, 15 µg/ml benzaminidine and 40 µg/ml leupeptin) in extraction reagents as recommended by the manufacturer (Pierce, Rockford, II). Immunoprecipitations were performed using mouse monoclonal antibodies to SMN or nucleolin. Monoclonal antibodies (2 µg) were bound to the Dynabeads M-280 sheep anti-mouse IgG as indicated (Dynal, Norway). After overnight incubation at 4°C with the nuclear extract (25–50 µg), the immunoprecipitates were washed four times (10 min) with IPP150 buffer (0.05 M Tris–HCl, pH 7.4, 0.15 M KCl, 0.1% NP40), and bound proteins were eluted from the magnetic beads in SDS loading buffer. Control immunoprecipitations in which the primary antibody was omitted or replaced by a negative control mouse IgG (DAKO) were included. When indicated, RNase A (Sigma) and RNase-free DNase RQ1 (Promega) were added to the extracts for 10 min at 30°C prior to co-immunoprecipitation.
Protein gel electrophoresis and immunoblot analysis
The proteins were resolved on 10% ProSieve 50 polyacrylamide gel (FMC Bioproducts, Rockland, ME) and transferred to Immobilon membranes (Millipore), and immunoblotting experiments were performed as described previously (5). The immunoblots were incubated with antibodies directed against HA epitope (mouse mAb at 1 : 1000, Roche Diagnostics), SMN (mouse mAb at 1 : 1000, Transduction Laboratories), nucleolin (mouse mAb at 1 : 1000, Santa Cruz) and protein B23 (goat polyclonal IgG at 1 : 1000, Santa Cruz).
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ACKNOWLEDGEMENTS |
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We thank Y. Zermati and E. Scheinder for assistance with FACS analyses. We also thank J. Cartaud for critical reading of the manuscript. Yann Goureau (IRNEM) for technical assistance with the confocal microscopy and Y. Deris and B. Delamain (IRNEM) for skilful photography assistance are gratefully acknowledged. We thank the International SMA Consortium for stimulating workshops. This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Assitance Publique-Hôpitaux de Paris (AP-HP), Association Française contre les Myopathies (AFM), Families of SMA and Andrew's Buddies.
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FOOTNOTES |
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* To whom correspondence should be addressed at: INSERM U393, IRNEM Institute, 149 Rue de Sèvres, 75743 Paris Cédex 15, France. Tel: +33 1 44 49 40 00 ext 97831; Fax: +33 1 47 34 85 14; Email: lefebvre{at}necker.fr
Present address: Laboratoire de Biologie Cellulaire des Membranes, Département de Biologie Cellulaire, Institut Jacques Monod, UMR 7592, CNRS/Universités Paris 6 et Paris 7, 2 Place Jussieu, 75251 Paris Cedex 5, France.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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3
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