A novel nuclear structure containing the survival of motor neurons protein.

Spinal muscular atrophy (SMA) is a common, often fatal, autosomal recessive disease leading to progressive muscle wasting and paralysis as a result of degeneration of anterior horn cells of the spinal cord. A gene termed survival of motor neurons (SMN), at 5q13, has been identified as the determining gene of SMA (Lefebvre et al., 1995). The SMN gene is deleted in > 98% of SMA patients, but the function of the SMN protein is unknown. In searching for hnRNP-interacting proteins we found that SMN interacts with the RGG box region of hnRNP U, with itself, with fibrillarin and with several novel proteins. We have produced monoclonal antibodies to the SMN protein, and we report here on its striking cellular localization pattern. Immunolocalization studies using SMN monoclonal antibodies show several intense dots in HeLa cell nuclei. These structures are similar in number (2-6) and size (0.1-1.0 micron) to coiled bodies, and frequently are found near or associated with coiled bodies. We term these prominent nuclear structures gems, for Gemini of coiled bodies.     

Coupled in vitro import of U snRNPs and SMN, the spinal muscular atrophy protein

Cytoplasmic assembly of Sm-class small nuclear ribonucleoproteins (snRNPs) is a central process in eukaryotic gene expression. A large macromolecular complex containing the survival of motor neurons (SMN) protein is required for proper snRNP assembly in vivo. Defects in SMN function lead to a human neuromuscular disorder, spinal muscular atrophy (SMA). SMN protein localizes to both nuclear and cytoplasmic compartments, and a reduction in nuclear levels of SMN is correlated with the disease. The mechanism of SMN nuclear import, however, is unknown. Using digitonin-permeabilized cells, we show that SMN import depends on the presence of Sm snRNPs. Conversely, import of labeled U1 snRNPs was SMN complex dependent. Thus, import of SMN and U snRNPs are coupled in vitro. Furthermore, we identify nuclear import defects in SMA patient-derived SMN mutants, uncovering a potential mechanism for SMN dysfunction.     

A direct interaction between the survival motor neuron protein and p53 and its relationship to spinal muscular atrophy.

Mutations in the SMN1 (survival motor neuron 1) gene cause spinal muscular atrophy (SMA). We now show that SMN protein, the SMN1 gene product, interacts directly with the tumor suppressor protein, p53. Pathogenic missense mutations in SMN reduce both self-association and p53 binding by SMN, and the extent of the reductions correlate with disease severity. The inactive, truncated form of SMN produced by the SMN2 gene in SMA patients fails to bind p53 efficiently. SMN and p53 co-localize in nuclear Cajal bodies, but p53 redistributes to the nucleolus in fibroblasts from SMA patients. These results suggest a functional interaction between SMN and p53, and the potential for apoptosis when this interaction is impaired may explain motor neuron death in SMA.     

Direct interaction of the spinal muscular atrophy disease protein SMN with the small nucleolar RNA-associated protein fibrillarin

Disruption of the survival motor neuron (SMN) gene leads to selective loss of spinal motor neurons, resulting in the fatal human neurodegenerative disorder spinal muscular atrophy (SMA). SMN has been shown to function in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and pre-mRNA splicing. We have demonstrated that SMN also interacts with fibrillarin, a highly conserved nucleolar protein that is associated with all Box C/D small nucleolar RNAs and functions in processing and modification of rRNA. Fibrillarin and SMN co-immunoprecipitate from HeLa cell extracts indicating that the proteins exist as a complex in vivo. Furthermore, in vitro binding studies indicate that the interaction between SMN and fibrillarin is direct and salt-stable. We show that the glycine/arginine-rich domain of fibrillarin is necessary and sufficient for SMN binding and that the region of SMN encoded by exon 3, including the Tudor domain, mediates the binding of fibrillarin. Tudor domain missense mutations, including one found in an SMA patient, impair the interaction between SMN and fibrillarin (as well as the common snRNP protein SmB). Our results suggest a function for SMN in small nucleolar RNP biogenesis (akin to its known role as an snRNP assembly factor) and reveal a potential link between small nucleolar RNP biogenesis and SMA.     

Restoration of SMN expression in mesenchymal stem cells derived from gene-targeted patient-specific iPSCs

Spinal muscular atrophy (SMA) is primarily a neurodegenerative disease caused by the homozygous deletion of the survival motor neuron 1 (SMN1) gene, thereby reducing SMN protein expression. Mesenchymal stem cells (MSCs) have been implicated in the treatment of SMA. In the present study, we overexpressed exogenous SMN1 at the ribosomal DNA (rDNA) locus of induced pluripotent stem cells (iPSCs) generated from a SMA patient using an rDNA-targeting vector. The gene-targeted patient iPSCs differentiated into MSCs (SMN1-MSCs). A 2.1-fold higher expression level of SMN protein was detected in SMN1-MSCs than that detected in MSCs derived from patient iPSCs, and the results of the immunofluorescence analysis showed no difference in the quantity of SMN nuclear structures (gems) between SMN1-MSCs and MSCs derived from normal human iPSCs (h-MSCs). These findings provide a novel strategy for obtaining gene-targeted MSCs for potential clinical applications in autologous cell-based therapy.     

Ultrastructural characterisation of a nuclear domain highly enriched in survival of motor neuron (SMN) protein

Mutations in the survival of motor neuron (SMN) gene are the major cause of spinal muscular atrophy (SMA). The SMN gene encodes a 38-kDa protein that localises in the cytoplasm and in nuclear bodies termed Gemini of coiled bodies (gems). When visualised by immunofluorescence microscopy, gems often appeared either in close proximity to, or entirely overlapping with coiled (Cajal) bodies (CBs) implying a possible functional relationship between these nuclear domains. With the aim of identifying subnuclear compartments corresponding to gems, we have investigated the intranuclear localisation of SMN and of its interacting protein Gemin2 by immunoelectron microscopy in cultured cells and in liver cells of hibernating dormouse. These antigens are highly enriched in round-shaped electron-dense fibro-granular clusters (EFGCs), which also display a biochemical composition similar to gems visualised by immunofluorescence microscopy. Our data reveal a novel SMN/Gemin2 containing nuclear domain and support the idea that it represents the structural counterpart of gems seen in the light microscope.     

Purification of native survival of motor neurons complexes and identification of Gemin6 as a novel component.

The survival of motor neurons (SMN) protein, the product of the gene responsible for the motor neuron degenerative disease spinal muscular atrophy (SMA), is part of a large macromolecular complex. The SMN complex is localized in both the cytoplasm and the nucleus and contains SMN, Gemin2, Gemin3, Gemin4, Gemin5, and a few not yet identified proteins. The SMN complex plays a key role in the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and other ribonucleoprotein particles. As a step toward the complete characterization of the components of the SMN complex, we generated stable cell lines that express FLAG-tagged SMN or Gemin2 under the control of a tetracycline-inducible promoter. Native SMN complexes of identical protein composition to those isolated by immunoprecipitation with anti-SMN antibodies were purified by affinity chromatography from extracts of both cell lines. Here we report the identification by mass spectrometry of a novel protein component of the SMN complex termed Gemin6. Co-immunoprecipitation, immunolocalization, and in vitro binding experiments demonstrate that Gemin6 is a component of the SMN complex that localizes to gems and interacts with several Sm proteins of the spliceosomal snRNPs.     

Overexpressed human survival motor neurone isoforms, SMNDeltaexon7 and SMN+exon7, both form intranuclear gems but differ in cytoplasmic distribution

Homozygous mutations of the telomeric survival motor neurone gene (SMN1) cause spinal muscular atrophy (SMA). The centromeric copy gene (SMN2) generally skips exon 7 during splicing and fails to compensate for SMN1 deficits, so SMA cells have reduced SMN protein and few nuclear gems. To investigate the role of exon 7 in SMN localisation, cDNAs for full-length SMN and SMNDeltaexon 7 were overexpressed in COS cells, neurones and SMA fibroblasts. Both constructs formed discrete intranuclear bodies colocalising with p80-coilin, but produced more cytoplasmic aggregates in cells overexpressing exon 7. Hence, the exon 7 domain enhances SMN aggregation but is not critical for gem formation.     

Molecular determinants of survival motor neuron (SMN) protein cleavage by the calcium-activated protease, calpain

Spinal muscular atrophy (SMA) is a leading genetic cause of childhood mortality, caused by reduced levels of survival motor neuron (SMN) protein. SMN functions as part of a large complex in the biogenesis of small nuclear ribonucleoproteins (snRNPs). It is not clear if defects in snRNP biogenesis cause SMA or if loss of some tissue-specific function causes disease. We recently demonstrated that the SMN complex localizes to the Z-discs of skeletal and cardiac muscle sarcomeres, and that SMN is a proteolytic target of calpain. Calpains are implicated in muscle and neurodegenerative disorders, although their relationship to SMA is unclear. Using mass spectrometry, we identified two adjacent calpain cleavage sites in SMN, S192 and F193. Deletion of small motifs in the region surrounding these sites inhibited cleavage. Patient-derived SMA mutations within SMN reduced calpain cleavage. SMN(D44V), reported to impair Gemin2 binding and amino-terminal SMN association, drastically inhibited cleavage, suggesting a role for these interactions in regulating calpain cleavage. Deletion of A188, a residue mutated in SMA type I (A188S), abrogated calpain cleavage, highlighting the importance of this region. Conversely, SMA mutations that interfere with self-oligomerization of SMN, Y272C and SMNΔ7, had no effect on cleavage. Removal of the recently-identified SMN degron (Δ268-294) resulted in increased calpain sensitivity, suggesting that the C-terminus of SMN is important in dictating availability of the cleavage site. Investigation into the spatial determinants of SMN cleavage revealed that endogenous calpains can cleave cytosolic, but not nuclear, SMN. Collectively, the results provide insight into a novel aspect of the post-translation regulation of SMN.     

Degradation of survival motor neuron (SMN) protein is mediated via the ubiquitin/proteasome pathway

Homozygous deletion or mutation in the survival motor neuron (SMN)1 gene causes proximal spinal muscular atrophy (SMA), whereas SMN2 acts as a modifying gene that can influence the severity of SMA. It has been suggested that restoration of the SMN protein level in neuronal cells may prevent cell loss and may be helpful for treatment of SMA. Recent studies indicate that the ubiquitin/proteasome pathway is a major system for proteolysis of intracellular proteins. In this study, we investigate whether SMN protein is degraded via the ubiquitin/proteasome pathway. Primary fibroblasts were established from the skin biopsies of SMA patients and the effect of a proteasome inhibitor MG132 and lysosome inhibitor NH(4)Cl on SMN protein level was examined. We found that MG132, but not NH(4)Cl, significantly increased the amount and nuclear accumulation of SMN protein in SMA patient's fibroblasts. Immunoprecipitation/western blot analysis indicated that SMN protein was ubiquitinated in cells. In vitro protein ubiquitination assay also demonstrated that SMN protein could be conjugated with ubiquitin. Taken together, we have provided clear evidences that degradation of SMN protein is mediated via the ubiquitin/proteasome pathway and suggest that proteasome inhibitors may up-regulate SMN protein level and may be useful for the treatment of SMA.     

SMN, the product of the spinal muscular atrophy gene, binds preferentially to dimethylarginine-containing protein targets

The survival of motor neurons protein (SMN), the product of the neurodegenerative disease spinal muscular atrophy (SMA) gene, functions as an assembly factor for snRNPs and likely other RNPs. SMN binds the arginine- and glycine-rich (RG) domains of the snRNP proteins SmD1 and SmD3. Specific arginines in these domains are modified to dimethylarginines, a common modification of unknown function. We show that SMN binds preferentially to the dimethylarginine-modified RG domains of SmD1 and SmD3. The binding of other SMN-interacting proteins is also strongly enhanced by methylation. Thus, methylation of arginines is a novel mechanism to promote specific protein-protein interactions and appears to be key to generating high-affinity SMN substrates. It is reasonable to expect that protein hypomethylation may contribute to the severity of SMA.