SMN1 and SMN2 (survival motor neuron) encode identical proteins. A critical question is why only the homozygous loss of SMN1, and not SMN2, results in spinal muscular atrophy (SMA). Analysis of transcripts from SMN1/SMN2 hybrid genes and a new SMN1 mutation showed a direct relationship between presence of disease and exon 7 skipping. We have reported previously that the exon-skipped product SMNDelta7 is partially defective for self-association and SMN self-oligomerization correlated with clinical severity. To evaluate systematically which of the five nucleotides that differ between SMN1 and SMN2 effect alternative splicing of exon 7, a series of SMN minigenes was engineered and transfected into cultured cells, and their transcripts were characterized. Of these nucleotide differences, the exon 7 C-to-T transition at codon 280, a translationally silent variance, was necessary and sufficient to dictate exon 7 alternative splicing. Thus, the failure of SMN2 to fully compensate for SMN1 and protect from SMA is due to a nucleotide exchange (C/T) that attenuates activity of an exonic enhancer. These findings demonstrate the molecular genetic basis for the nature and pathogenesis of SMA and illustrate a novel disease mechanism. Because individuals with SMA retain the SMN2 allele, therapy targeted at preventing exon 7 skipping could modify clinical outcome.

Spinal muscular atrophy (SMA) is a recessive disorder characterized by loss of motor neurons in the spinal cord. It is caused by mutations in the telomeric survival motor neuron 1 ( SMN1 ) gene. Alterations within an almost identical copy gene, the centromeric survival motor neuron 2 ( SMN2 ) gene produce no known phenotypic effect. The exons of the two genes differ by just two nucleotides, neither of which alters the encoded amino acids. At the genomic level, only five nucleotides that differentiate the two genes from one another have been reported. The entire genomic sequence of the two genes has not been determined. Thus, differences which might explain why SMN1 is the SMA gene are not readily apparent. In this study, we have completely sequenced and compared genomic clones containing the SMN genes. The two genes show striking similarity, with the homology being unprecedented between two different yet functional genes. The only critical difference in an approximately 32 kb region between the two SMN genes is the C->T base change 6 bp inside exon 7. This alteration but not other variations in the SMN genes affects the splicing pattern of the genes. The majority of the transcript from the SMN1 locus is full length, whereas the majority of the transcript produced by the SMN2 locus lacks exon 7. We suggest that the exon 7 nucleotide change affects the activity of an exon splice enhancer. In SMA patients, the loss of SMN1 but the presence of SMN2 results in low levels of full-length SMN transcript and therefore low SMN protein levels which causes SMA.

The 38 kDa survival motor neuron (SMN) protein is encoded by two ubiquitously expressed genes: telomeric SMN (SMN(T)) and centromeric SMN (SMN(C)). Mutations in SMN(T), but not SMN(C), cause proximal spinal muscular atrophy (SMA), an autosomal recessive disorder that results in loss of motor neurons. SMN is found in the cytoplasm and nucleus. The nuclear form is located in structures termed gems. Using a panel of anti-SMN antibodies, we demonstrate that the SMN protein is expressed from both the SMN(T) and SMN(C) genes. Western blot analysis of fibroblasts from SMA patients with various clinical severities of SMA showed a moderate reduction in the amount of SMN protein, particularly in type I (most severe) patients. Immunocytochemical analysis of SMA patient fibroblasts indicates a significant reduction in the number of gems in type I SMA patients and a correlation of the number of gems with clinical severity. This correlation to phenotype using primary fibroblasts may serve as a useful diagnostic tool in an easily accessible tissue. SMN is expressed at high levels in brain, kidney and liver, moderate levels in skeletal and cardiac muscle, and low levels in fibroblasts and lymphocytes. In SMA patients, the SMN level was moderately reduced in muscle and lymphoblasts. In contrast, SMN was expressed at high levels in spinal cord from normals and non-SMA disease controls, but was reduced 100-fold in spinal cord from type I patients. The marked reduction of SMN in type I SMA spinal cords is consistent with the features of this motor neuron disease. We suggest that disruption of SMN(T) in type I patients results in loss of SMN from motor neurons, resulting in the degeneration of these neurons.

Humans have two nearly identical copies of the Survival Motor Neuron (SMN) gene, SMN1 and SMN2. In spinal muscular atrophy (SMA), SMN2 is not able to compensate for the loss of SMN1 due to exclusion of exon 7. Here we describe a novel inhibitory element located immediately downstream of the 5 splice site in intron 7. We call this element intronic splicing silencer N1 (ISS-N1). Deletion of ISS-N1 promoted exon 7 inclusion in mRNAs derived from the SMN2 minigene. Underlining the dominant role of ISS-N1 in exon 7 skipping, abrogation of a number of positive cis elements was tolerated when ISS-N1 was deleted. Confirming the silencer function of ISS-N1, an antisense oligonucleotide against ISS-N1 restored exon 7 inclusion in mRNAs derived from the SMN2 minigene or from endogenous SMN2. Consistently, this oligonucleotide increased the levels of SMN protein in SMA patient-derived cells that carry only the SMN2 gene. Our findings underscore for the first time the profound impact of an evolutionarily nonconserved intronic element on SMN2 exon 7 splicing. Considering that oligonucleotides annealing to intronic sequences do not interfere with exon-junction complex formation or mRNA transport and translation, ISS-N1 provides a very specific and efficient therapeutic target for antisense oligonucleotide-mediated correction of SMN2 splicing in SMA.Alternative splicing increases the coding potential of the human genome by producing multiple proteins from a single gene (2). It is also associated with a growing number of human diseases (13,15,47). Regulation of alternative splicing is dependent upon the relative concentrations of spliceosomal and nonspliceosomal proteins, namely, serine-arginine-rich proteins (SR proteins), SR-like proteins, and heterogeneous nuclear ribonucleoproteins (hnRNPs) (16,35,43). Some of these proteins bind to pre-mRNA sequences called exonic splicing enhancers (ESEs), intronic splicing enhancers, exonic splicing silencers, and intronic splicing silencers (ISSs). Enhancers and silencers promote or suppress splice site (ss) selection, respectively. Over the last several years, methods have been developed to predict exonic cis elements (7,12,58). Analogous methods to predict intronic cis elements do not exist. Local RNA structure presents an additional level of splicing regulation. Several studies have focused on RNA structures that facilitate specific interactions during pre-mRNA splicing (3). However, the role of critical RNA structures in pre-mRNA splicing remains largely unpredictable.Spinal muscular atrophy (SMA), the second most common autosomal recessive disorder, is caused by the absence of the Survival of Motor Neuron 1 (SMN1) gene (27). SMN1 encodes a ubiquitously expressed 38-kDa SMN protein that is necessary for snRNP assembly, an essential process for cell survival (57).A nearly identical copy of the gene, SMN2, fails to compensate for the loss of SMN1 because of exon 7 skipping, producing an unstable truncated protein, SMN⌬7 (30). SMN1 and SMN2 differ by a critical C-to-T substitution at po...

Spinal muscular atrophy (SMA) is a motor-neuron disorder resulting from anterior-horn-cell death. The autosomal recessive form has a carrier frequency of 1 in 50 and is the most common genetic cause of infant death. SMA is categorized as types I-III, ranging from severe to mild, based upon age of onset and clinical course. Two closely flanking copies of the survival motor neuron (SMN) gene are on chromosome 5q13 (ref. 1). The telomeric SMN (SMN1) copy is homozygously deleted or converted in >95% of SMA patients, while a small number of SMA disease alleles contain missense mutations within the carboxy terminus. We have identified a modular oligomerization domain within exon 6 of SMN1. All previously identified missense mutations map within or immediately adjacent to this domain. Comparison of wild-type to mutant SMN proteins of type I, II and III SMA patients showed a direct correlation between oligomerization and clinical type. Moreover, the most abundant centromeric SMN product, which encodes exons 1-6 but not 7, demonstrated reduced self-association. These findings identify decreased SMN self-association as a biochemical defect in SMA, and imply that disease severity is proportional to the intracellular concentration of oligomerization-competent SMN proteins.