RNA splicing: more clues from spinal muscular atrophy.
Spinal muscular atrophy is caused by mutations in the SMN1 gene, the product of which is part of a multi-component complex involved in the assembly of small nuclear ribonucleoproteins. A recent study indicates that SMN may also play a role in pre-mRNA splicing. (+info)
Quantitative analysis of survival motor neuron copies: identification of subtle SMN1 mutations in patients with spinal muscular atrophy, genotype-phenotype correlation, and implications for genetic counseling.
Problems with diagnosis and genetic counseling occur for patients with autosomal recessive proximal spinal muscular atrophy (SMA) who do not show the most common mutation: homozygous absence of at least exon 7 of the telomeric survival motor neuron gene (SMN1). Here we present molecular genetic data for 42 independent nondeleted SMA patients. A nonradioactive quantitative PCR test showed one SMN1 copy in 19 patients (45%). By sequencing cloned reverse-transcription (RT) PCR products or genomic fragments of SMN1, we identified nine different mutations in 18 of the 19 patients, six described for the first time: three missense mutations (Y272C, T274I, S262I), three frameshift mutations in exons 2a, 2b, and 4 (124insT, 241-242ins4, 591delA), one nonsense mutation in exon 1 (Q15X), one Alu-mediated deletion from intron 4 to intron 6, and one donor splice site mutation in intron 7 (c.922+6T-->G). The most frequent mutation, Y272C, was found in 6 (33%) of 18 patients. Each intragenic mutation found in at least two patients occurred on the same haplotype background, indicating founder mutations. Genotype-phenotype correlation allowed inference of the effect of each mutation on the function of the SMN1 protein and the role of the SMN2 copy number in modulating the SMA phenotype. In 14 of 23 SMA patients with two SMN1 copies, at least one intact SMN1 copy was sequenced, which excludes a 5q-SMA and suggests the existence of further gene(s) responsible for approximately 4%-5% of phenotypes indistinguishable from SMA. We determined the validity of the test, and we discuss its practical implications and limitations. (+info)
SMN protein analysis in fibroblast, amniocyte and CVS cultures from spinal muscular atrophy patients and its relevance for diagnosis.
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by the homozygous absence of the telomeric copy of the survival motor neuron (SMNt) gene, due to deletion, gene conversion or point mutation. SMNt and its homologous centromeric copy (SMNc) encode the SMN protein, which is diffusely present in the cytoplasm and in dot-like structures, called gems, in the nucleus. We have studied the SMN protein in different cell cultures, including fibroblasts, amniocytes and CVS cells from SMA individuals and controls. By immunofluorescence analysis we found a marked reduction in the number of gems in fibroblasts, amniocytes and chorionic villus cells of all SMA patients and foetuses, independent of the type of the genetic defect. We also show that immunolocalisation of the SMN protein may be a useful tool for the characterisation of particular patients of uncertain molecular diagnosis. (+info)
A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy.
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. (+info)
A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2.
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. (+info)
Detection of the survival motor neuron (SMN) genes by FISH: further evidence for a role for SMN2 in the modulation of disease severity in SMA patients.
Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder which presents with various clinical phenotypes ranging from severe to very mild. All forms are caused by the homozygous absence of the survival motor neuron ( SMN1 ) gene. SMN1 and a nearly identical copy ( SMN2 ) are located in a duplicated region at 5q13 and encode identical proteins. The genetic basis for the clinical variability of SMA remains unclear, but it has been suggested that the copy number of SMN2 could influence the disease severity. We have assessed the number of SMN2 genes in patients with different clinical phenotypes by fluorescence in situ hybridization (FISH) using as SMN probe a mixture of small specific DNA fragments. Gene copy number was established by FISH on interphase nuclei, but the presence of two SMN2 genes on the same chromosome could also be revealed by FISH on metaphase spreads. All patients had at least two SMN2 genes. We found two or three copies of SMN2 in severely affected type I patients, three copies in intermediately affected type II patients, generally four copies in mildly affected type III patients and four or eight copies in patients with very mild adult-onset SMA. No alterations of the genes were detected by Southern blot and sequence analysis, suggesting that all gene copies of SMN2 were intact. These data provide additional evidence that the SMN2 genes modulate the disease severity and suggest that knowledge of the gene copy number could be of some prognostic value. (+info)
Subcellular localization and axonal transport of the survival motor neuron (SMN) protein in the developing rat spinal cord.
The subcellular localization of the survival motor neuron (SMN) protein, encoded by the spinal muscular atrophy determining gene, was investigated in motor neurons of the developing and adult rat spinal cord by light and electron microscopy immunocytochemistry. The experiments were carried out with a panel of anti-SMN antibodies, all recognizing an SMN-specific protein band at 39 kDa in HeLa cells and rat spinal cord protein extracts. SMN protein expression decreased during postnatal spinal cord development, but it remained unchanged in distribution and intensity in motor neurons at all ages examined. SMN protein was mainly organized in immunoreactive aggregates sparse in the nucleoplasm and cytoplasm of both mature and developing motor neurons, and it was more rarely localized within the endoplasmic reticulum and in apposition to the external mitochondrial membrane. Most strikingly, the SMN protein was found in association with cytoskeletal elements in spinal dendrites and axons, where it was particularly evident during postnatal development. The present findings suggest that SMN protein may be transported via axoplasmic flow in maturing neurons. Given the RNA-binding activity of SMN, the SMN protein could be involved in the transport of specific mRNAs in axons and dendrites of motor neurons. The reduced transport of specific mRNAs within motor neurons during development could play a role in the motoneuronal degeneration and impaired axonal sprouting observed in spinal muscular atrophy. (+info)
An exonic enhancer is required for inclusion of an essential exon in the SMA-determining gene SMN.
The survival motor neuron genes, SMN1 and SMN2, encode identical proteins; however, only homo- zygous loss of SMN1 correlates with the development of spinal muscular atrophy (SMA). We have previously shown that a single non-polymorphic nucleotide difference in SMN exon 7 dramatically affects SMN mRNA processing. SMN1 primarily produces a full-length RNA whereas SMN2 expresses dramatically reduced full-length RNA and abundant levels of an aberrantly spliced transcript lacking exon 7. The importance of proper exon 7 processing has been underscored by the identification of several mutations within splice sites adjacent to exon 7. Here we show that an AG-rich exonic splice enhancer (ESE) in the center of SMN exon 7 is required for inclusion of exon 7. This region functioned as an ESE in a heterologous context, supporting efficient in vitro splicing of the Drosophila double-sex gene. Finally, the protein encoded by the exon-skipping event, Delta7, was less stable than full-length SMN, providing additional evidence of why SMN2 fails to compensate for the loss of SMN1 and leads to the development of SMA. (+info)