Enhanced expression of the central survival of motor neuron (SMN) protein during the pathogenesis of osteoarthritis

The identification of new components implicated in the pathogenesis of osteoarthritis (OA) might improve our understanding of the disease process. Here, we investigated the levels of the survival of motor neuron (SMN) expression in OA cartilage considering the fundamental role of the SMN protein in cell survival and its involvement in other stress‐associated pathologies. We report that SMN expression is up‐regulated in human OA compared with normal cartilage, showing a strong correlation with the disease severity, a result confirmed in vivo in an experimental model of the disease. We further show that the prominent inflammatory cytokines (IL‐1β, TNF‐α) are critical inducers of SMN expression. This is in marked contrast with the reported impaired levels of SMN in spinal muscular atrophy, a single inherited neuromuscular disorder characterized by mutations in the smn gene whereas OA is a complex disease with multiple aetiologies. While the precise functions of SMN during OA remain to be elucidated, the conclusions of this study shed light on a novel pathophysiological pathway involved in the progression of OA, potentially offering new targets for therapy.     

An intronic splicing enhancer element in survival motor neuron (SMN) pre-mRNA

Spinal muscular atrophy is caused by the homozygous loss of survival motor neuron 1 (SMN1). SMN2, a nearly identical copy gene, differs from SMN1 only by a single nonpolymorphic C to T transition in exon 7, which leads to alteration of exon 7 splicing; SMN2 leads to exon 7 skipping and expression of a nonfunctional gene product and fails to compensate for the loss of SMN1. The exclusion of SMN exon 7 is critical for the onset of this disease. Regulation of SMN exon 7 splicing was determined by analyzing the roles of the cis-acting element in intron 7 (element 2), which we previously identified as a splicing enhancer element of SMN exon 7 containing the C to T transition. The minimum sequence essential for activation of the splicing was determined to be 24 nucleotides, and RNA structural analyses showed a stem-loop structure. Deletion of this element or disruption of the stem-loop structure resulted in a decrease in exon 7 inclusion. A gel shift assay using element 2 revealed formation of RNA-protein complexes, suggesting that the binding of the trans-acting proteins to element 2 plays a crucial role in the splicing of SMN exon 7 containing the C to T transition.     

A comprehensive interaction map of the human survival of motor neuron (SMN) complex

Assembly of the Sm-class of U-rich small nuclear ribonucleoprotein particles (U snRNPs) is a process facilitated by the macromolecular survival of motor neuron (SMN) complex. This entity promotes the binding of a set of factors, termed LSm/Sm proteins, onto snRNA to form the core structure of these particles. Nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and unrip have been identified as the major components of the SMN complex. So far, however, only little is known about the architecture of this complex and the contribution of individual components to its function. Here, we present a comprehensive interaction map of all core components of the SMN complex based upon in vivo and in vitro methods. Our studies reveal a modular composition of the SMN complex with the three proteins SMN, Gemin8, and Gemin7 in its center. Onto this central building block the other components are bound via multiple interactions. Furthermore, by employing a novel assay, we were able to reconstitute the SMN complex from individual components and confirm the interaction map. Interestingly, SMN protein carrying an SMA-causing mutation was severely impaired in formation of the SMN complex. Finally, we show that the peripheral component Gemin5 contributes an essential activity to the SMN complex, most likely the transfer of Sm proteins onto the U snRNA. Collectively, the data presented here provide a basis for the detailed mechanistic and structural analysis of the assembly machinery of U snRNPs.     

Comparative FISH-mapping of the survival of motor neuron gene (SMN) in domestic bovids

A comparative fluorescence in situ mapping of the SMN gene was performed on R-banded chromosome preparations of cattle (Bos taurus, BTA, 2n = 60), river buffalo (Bubalus bubalis, BBU, 2n = 50), sheep (Ovis aries, OAR, 2n = 54) and goat (Capra hircus, CHI, 2n = 60), as well as on those of a calf from Piedmont breed affected by arthrogryposis. SMN was located on BTA20q13.1, OAR16q13.1, CHI20q13.1 and BBU19q13. These chromosomes and chromosome bands are believed to be homeologous, confirming the high degree of chromosome homeologies among bovids. The position of SMN was refined in cattle, compared to the two previous localizations, while it is a new gene assignment in the other three bovids. A comparative fiber-FISH performed on extended chromatin of both normal cattle and calf affected by arthrogryposis revealed more extended FITC signals in the calf, compared to the normal cattle (control), suggesting a possible duplication of the SMN gene in the calf affected by arthrogryposis. .     

Allele-specific amplification of exon 7 in the survival motor neuron (SMN) genes for molecular diagnosis of spinal muscular atrophy

There are two highly homologous survival motor neuron (SMN) genes in humans but molecular defects in the SMN1 gene cause spinal muscular atrophy (SMA). More than 90% of SMA patients are shown to have a homozygous deletion of exon 7 in the SMN1 gene. Therefore, a simple test for exon 7 deletion would be very useful in the molecular diagnosis of SMA. However, limited methods are available, and most of these methods utilize expensive instruments and consumables. Here, we describe a simple allele-specific PCR test, which can be performed using standard equipment in DNA laboratories. The principle of the test is based on a single nucleotide difference (C versus T) between the exon 7 of SMN1 and SMN2 genes. Using allele-specific primers, two PCR amplifications are performed for each sample to amplify a 404-bp diagnostic fragment, and consequent electrophoresis of PCR products on agarose gel provides definitive information concerning the exon 7 deletion To rule out false negatives, a 500-bp fragment from the N-acetyltransferase gene was coamplified as an internal control in each test. We have, so far, analyzed 41 SMA samples with our method, and tested the validity of results using an independent restriction fragment length polymorphism (RFLP) method. Genotyping results obtained by both methods were in complete agreement for all of the samples analyzed. Our method can also be used to detect heterozygous deletion of exon 7 in SMN genes, if the relative intensities of the diagnostic and internal control bands are determined.     

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.     

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.     

Molecular analysis of survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes of spinal muscular atrophy patients and their parents

We have assayed deletions of two candidate genes for spinal muscular atrophy (SMA), the survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes, in 101 patients from 86 Chinese SMA families. Deletions of exons 7 and 8 of the telomeric SMN gene were detected in 100%, 78.6%, 96.6%, and 16.7%, in type I, II, III, and adult-onset SMA patients, respectively. Deletion of exon 7 only was found in eight type II and one type III patient. One type II patient did not have a deletion of either exon 7 or 8. The prevalence of deletions of exons 5 and 6 of the NAIP gene were 22.5% and 2.4% in type I and II SMA patients, respectively. We also examined four polymorphisms of SMN genes and found that there were only two, SMN-2 and ^CBCD541-2, in Chinese subjects. In our study, analysis of the ratio of the telomeric to centromeric portion (T/C ratio) of the SMN gene after enzyme digestion was performed to differentiate carriers, normals, and SMA patients. We found the T/C ratio of exon 7 of the SMN gene differed significantly among the three groups, and may be used for carrier analysis. An asymptomatic individual with homozygous deletion of exons 7 and 8 of the SMN gene showed no difference in microsatellite markers in the SMA-related 5q11.2–5q13.3. In conclusion, SMN deletion in clinically presumed child-onset SMA should be considered as confirmation of the diagnosis. However, adult-onset SMA, a heterogeneous disease with phenotypical similarities to child-onset SMA, may be caused by SMN or other gene(s). Received: 13 November 1996 / Accepted: 13 May 1997     

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.     

Dephosphorylation of survival motor neurons (SMN) by PPM1G/PP2Cgamma governs Cajal body localization and stability of the SMN complex

The survival motor neuron (SMN) complex functions in maturation of uridine-rich small nuclear ribonucleoprotein (RNP) particles. SMN mediates the cytoplasmic assembly of Sm proteins onto uridine-rich small RNAs, and then participates in targeting RNPs to nuclear Cajal bodies (CBs). Recent studies have suggested that phosphorylation might control localization and function of the SMN complex. Here, we show that the nuclear phosphatase PPM1G/PP2Cgamma interacts with and dephosphorylates the SMN complex. Small interfering RNA knockdown of PPM1G leads to an altered phosphorylation pattern of SMN and Gemin3, loss of SMN from CBs, and reduced stability of SMN. Accumulation in CBs is restored upon overexpression of catalytically active, but not that of inactive, PPM1G. This demonstrates that PPM1G's phosphatase activity is necessary to maintain SMN subcellular distribution. Concomitant knockdown of unr interacting protein (unrip), a component implicated in cytoplasmic retention of the SMN complex, also rescues the localization defects. Our data suggest that an interplay between PPM1G and unrip determine compartment-specific phosphorylation patterns, localization, and function of the SMN complex.     

ZPR1 is essential for survival and is required for localization of the survival motor neurons (SMN) protein to Cajal bodies

Mutation of the survival motor neurons 1 (SMN1) gene causes motor neuron apoptosis and represents the major cause of spinal muscular atrophy in humans. Biochemical studies have established that the SMN protein plays an important role in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and that the SMN complex can interact with the zinc finger protein ZPR1. Here we report that targeted ablation of the Zpr1 gene in mice disrupts the subcellular localization of both SMN and spliceosomal snRNPs. Specifically, SMN localization to Cajal bodies and gems was not observed in cells derived from Zpr1-/- embryos and the amount of cytoplasmic snRNP detected in Zpr1-/- embryos was reduced compared with that in wild-type embryos. We found that Zpr1-/- mice die during early embryonic development, with reduced proliferation and increased apoptosis. These effects of Zpr1 gene disruption were confirmed and extended in studies of cultured motor neuron-like cells using small interfering RNA-mediated Zpr1 gene suppression; ZPR1 deficiency caused growth cone retraction, axonal defects, and apoptosis. Together, these data indicate that ZPR1 contributes to the regulation of SMN complexes and that it is essential for cell survival.     

Promoter analysis of the human centromeric and telomeric survival motor neuron genes (SMNC and SMNT).

Proximal spinal muscular atrophy (SMA) is caused by mutations in the telomeric (SMNT), but not centromeric (SMNC), survival motor neuron gene. Here we have identified and analyzed the two SMN promoters. We show that a 750-bp 5'-flanking fragment from each is capable of driving expression from a reporter construct. Within this fragment, we define a approximately 200-bp element that results in high expression in a motor neuron cell line. Sequence comparison of a 3. 4-kb upstream fragment from each gene shows minimal differences. Although these differences produce a 2-fold difference in reporter activity between the two promoters, this is not sufficiently high to explain why SMNT, but not SMNC, is the disease determining gene. Our data thus demonstrate, for the first time, almost complete equivalence between the SMN promoters and rule out the important possibility that differences in them might explain why mutations in only the telomeric SMN gene cause SMA.