SRp30c-dependent stimulation of survival motor neuron (SMN) exon 7 inclusion is facilitated by a direct interaction with hTra2 beta 1.
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Proximal spinal muscular atrophy (SMA) is caused by the homozygous loss of survival motor neuron (SMN1). SMN2, a nearly identical copy gene, is present in all SMA patients; however this gene cannot provide protection from disease due to the aberrant splicing of a critical exon. SMN1-derived transcripts are exclusively full-length, whereas SMN2-derived transcripts predominantly lack SMN exon 7. A single non-polymorphic nucleotide difference (C in SMN1; T in SMN2) is responsible for the alternative splicing patterns. We have previously shown that transient expression of an SR-like splicing factor, hTra2 beta 1, stimulates inclusion of exon 7 in SMN2-derived mini-gene transcripts through an interaction with the AG-rich exonic splice enhancer within exon 7. We now demonstrate that a second splicing factor, SRp30c, can stimulate SMN exon 7-inclusion and that this activity required the same AG-rich enhancer as hTra2 beta 1. SRp30c did not directly associate with SMN exon 7; rather its association with the exonic enhancer was mediated by a direct interaction with hTra2 beta 1. In the absence of the hTra2 beta 1 binding site, SRp30c failed to complex with SMN exon 7. Taken together, these results identify SRp30c as a modulator of SMN exon 7-inclusion and provide insight into the molecular regulation of this critical exon. (+info)
hnRNP-G promotes exon 7 inclusion of survival motor neuron (SMN) via direct interaction with Htra2-beta1.
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Proximal spinal muscular atrophy (SMA) is a common motor neuron disease caused by homozygous loss of the survival motor neuron gene (SMN1). SMN2, a nearly identical copy of the gene and present in all SMA patients, fails to provide protection from SMA, due to the disruption of an exonic splicing enhancer (ESE) by a single translationally silent nucleotide exchange, which causes alternative splicing of SMN2 exon 7. Identification of splicing factors that stimulate exon 7 inclusion and thereby produce sufficient amounts of full-length transcripts from the SMN2 gene is of great importance for therapy approaches. Here, by use of in vivo splicing assays, we identified the protein hnRNP-G and its paralogue RBM as two novel splicing factors that promote the inclusion of SMN2 exon 7. Moreover, hnRNP-G and RBM non-specifically bind RNA, but directly and specifically bind Htra2-beta1, an SR-like splicing factor which we have previously shown to stimulate inclusion of exon 7 through a direct interaction with the AG-rich ESE in SMN2 exon 7 pre-mRNA. By using deletion mutants of hnRNP-G, we show that the specific protein-protein interaction of hnRNP-G with Htra2-beta1 mediates the inclusion of SMN2 exon 7 rather than the non-specific interaction of hnRNP-G with SMN pre-mRNA. Additionally, we show for the first time that recombinant trans-acting splicing factors such as hnRNP-G and Htra2-beta1 are also effective on endogenous SMN2 transcripts and increase the endogenous SMN protein level. Finally, we suggest a model of how the exon 7 mRNA processing is regulated by the splicing factors identified so far. (+info)
Quantification of PCR bias caused by a single nucleotide polymorphism in SMN gene dosage analysis.
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Approximately 94% of patients with spinal muscular atrophy lack both copies of SMN1 exon 7, and most carriers have only one copy of SMN1 exon 7. We described previously the effect of SMN1/SMN2 heteroduplex formation on SMN gene dosage analysis, which is a multiplex quantitative PCR assay to determine the copy numbers of SMN1 and SMN2 using DraI digestion to differentiate SMN2 from SMN1. We describe herein the quantification of PCR bias between SMN1 exon 7 and SMN2 exon 7, which differ by only one nucleotide that is not present in either primer binding site. Using samples from 272 individuals with various SMN genotypes, we found that the amplification efficiency of SMN2 was consistent only approximately 80% that of SMN1. Thus, even a single nucleotide polymorphism, not in primer binding sites, can cause reproducible PCR bias. The precision and accuracy of our SMN gene dosage analysis are high because our assay design and controls take advantage of the consistency of the PCR bias. As additional clinically significant single nucleotide polymorphisms (SNPs) are discovered, assessment of PCR bias, and judicious selection of standards and controls, will be increasingly important for quantitative PCR assays. (+info)
A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy.
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5q spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and the leading genetic cause of infantile death. Patients lack a functional survival of motor neurons (SMN1) gene, but carry one or more copies of the highly homologous SMN2 gene. A homozygous knockout of the single murine Smn gene is embryonic lethal. Here we report that in the absence of the SMN2 gene, a mutant SMN A2G transgene is unable to rescue the embryonic lethality. In its presence, the A2G transgene delays the onset of motor neuron loss, resulting in mice with mild SMA. We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions. Mild SMA mice exhibit motor neuron degeneration, muscle atrophy, and abnormal EMGs. Animals homozygous for the mutant transgene are less severely affected than heterozygotes. This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele. Our mild SMA mice will be useful in (a) determining the effect of missense mutations in vivo and in motor neurons and (b) testing potential therapies in SMA. (+info)
Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts.
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The multiplicity of proteins compared with genes in mammals owes much to alternative splicing. Splicing signals are so subtle and complex that small perturbations may allow the production of new mRNA variants. However, the flexibility of splicing can also be a liability, and several genetic diseases result from single-base changes that cause exons to be skipped during splicing. Conventional oligonucleotide strategies can block reactions but cannot restore splicing. We describe here a method by which the use of a defective exon was restored. Spinal muscular atrophy (SMA) results from mutations of the Survival Motor Neuron (SMN) gene. Mutations of SMN1 cause SMA, whereas SMN2 acts as a modifying gene. The two genes undergo alternative splicing with SMN1, producing an abundance of full-length mRNA transcripts, whereas SMN2 predominantly produces exon 7-deleted transcripts. This discrepancy is because of a single nucleotide difference in SMN2 exon 7, which disrupts an exonic splicing enhancer containing an SF2ASF binding site. We have designed oligoribonucleotides that are complementary to exon 7 and contain exonic splicing enhancer motifs to provide trans-acting enhancers. These tailed oligoribonucleotides increased SMN2 exon 7 splicing in vitro and rescued the incorporation of SMN2 exon 7 in SMA patient fibroblasts. This treatment also resulted in the partial restoration of gems, intranuclear structures containing SMN protein that are severely reduced in patients with SMA. The use of tailed antisense oligonucleotides to recruit positively acting factors to stimulate a splicing reaction may have therapeutic applications for genetic disorders, such as SMA, in which splicing patterns are altered. (+info)
Inverse correlation between SMN1 and SMN2 copy numbers: evidence for gene conversion from SMN2 to SMN1.
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Most carriers of autosomal recessive spinal muscular atrophy (SMA) have only one copy of SMN1 because of SMN1 gene deletions or gene conversions from SMN1 to SMN2, which has only one base difference in coding sequence from SMN1. Using SMN gene dosage analysis, we determined the copy numbers of SMN1 and SMN2 in the general population as well as in SMA patients and carriers. Increased SMN1 copy number is associated with decreased SMN2 copy number in the general population; that is, SMN2 copy number was decreased to one or zero copies in 11 of 13 individuals with three or four copies of SMN1, whereas only 71 of 164 individuals with two copies of SMN1 had one or zero copies of SMN2 (P<0.01). SMN2 copy number was increased to three or four in a subset of SMN1 deletion/conversion carriers, and in most SMA patients with a milder phenotype. In conclusion, our data provide evidence that gene conversion from SMN2 to SMN1 occurs, and that SMN1 converted from SMN2 is present in the general population. (+info)
Phenylbutyrate increases SMN expression in vitro: relevance for treatment of spinal muscular atrophy.
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Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease, characterized by degeneration of the anterior horn cells of the spinal cord. SMA presents with a highly variable phenotype ranging from very severe to mild (type I-III). No cure for SMA is available at present. All forms of SMA are caused by homozygous loss of the functional survival motor neuron (SMN1) gene. However, all patients have one or more copies of the SMN2 gene, nearly identical to SMN1. Both genes encode the SMN protein but the level produced by SMN2 is insufficient to protect from disease. Increasing SMN2 gene expression could be of considerable therapeutic importance. The aim of this study was to assess whether SMN2 gene expression can be increased by 4-phenylbutyrate (PBA). Fibroblast cell cultures from 16 SMA patients affected by different clinical severities were treated with PBA, and full-length SMN2 transcripts were measured by real-time PCR. In all cell cultures, except one, PBA treatment caused an increase in full-length SMN2 transcripts, ranging from 50 to 160% in type I and from 80 to 400% in type II and III cultures. PBA was found also effective in enhancing SMN protein levels and the number of SMN-containing nuclear structures (gems). These data show that SMN expression is considerably increased by PBA, and suggest that the compound, owing also to its favorable pharmacological properties, could be a good candidate for the treatment of SMA. (+info)
Identification of a novel cyclic AMP-response element (CRE-II) and the role of CREB-1 in the cAMP-induced expression of the survival motor neuron (SMN) gene.
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Spinal muscular atrophy, an autosomal recessive disorder, is caused by loss of the SMN1 (survival motor neuron) gene while retaining the SMN2 gene. SMN1 produces a majority of full-length SMN transcript, whereas SMN2 generates mostly an isoform lacking exon 7. Here, we demonstrate a novel cAMP-response element, CRE-II, in the SMN promoter that interacts with the cAMP-response element-binding (CREB) family of proteins. In vitro DNase I protection analysis and in vivo genomic footprinting of the SMN promoter using the brain and liver nuclei from SMN2 transgenic mice revealed footprinting at the CRE-II site. Site-directed mutation of the CRE-II element caused a marked reduction in the SMN promoter activity revealed by transient transfection assay. Activation of the cAMP pathway by dibutyryl cAMP (0.5 mm) alone or in combination with forskolin (20 microm) caused a 2-5-fold increase in the SMN promoter activity but had no effect on the CRE-II mutated promoter. Electrophoretic mobility shift assay and a UV-induced DNA-protein cross-linking experiment confirmed that CREB1 binds specifically to the CRE-II site. Transient overexpression of CREB1 protein resulted in a 4-fold increase of the SMN promoter activity. Intraperitoneal injection of epinephrine in mice expressing two copies of the human SMN2 gene resulted in a 2-fold increase in full-length SMN transcript in the liver. Combined treatment with dibutyryl cAMP and forskolin significantly increased the level of both the full-length and exon 7-deleted SMN (exonDelta7SMN) transcript in primary hepatocytes from mice expressing two copies of human SMN2 gene. Similar treatments of type I spinal muscular atrophy mouse and human fibroblasts as well as HeLa cells resulted in an augmented level of SMN transcript. These findings suggest that the CRE-II site in SMN promoter positively regulates the expression of the SMN gene, and treatment with cAMP-elevating agents increases expression of both the full-length and exonDelta7SMN transcript. (+info)