RESUMO
Proximal spinal muscular atrophy (SMA) is a common neuromuscular disorder causing infant death in half of all patients. Homozygous absence of the survival motor neuron gene (SMN1) is the primary cause of SMA, while SMA severity is mainly determined by the number of SMN2 copies. One SMN2 copy produces only about 10% of full-length protein identical to SMN1, whereas the majority of SMN2 transcripts is aberrantly spliced due to a silent mutation within an exonic splicing enhancer in exon 7. However, correct splicing can be restored by over-expression of the SR-like splicing factor Htra2-beta 1. We show that in fibroblast cultures derived from SMA patients treated with therapeutic doses (0.5-500 microM) of valproic acid (VPA), the level of full-length SMN2 mRNA/protein increased 2- to 4-fold. Importantly, this up-regulation of SMN could be most likely attributed to increased levels of Htra2-beta 1 which facilitates the correct splicing of SMN2 RNA as well as to an SMN gene transcription activation. Especially at low VPA concentrations, the restored SMN level depended on the number of SMN2 copies. Moreover, VPA was able to increase SMN protein levels through transcription activation in organotypic hippocampal brain slices from rats. Finally, VPA also increased the expression of further SR proteins, which may have important implications for other disorders affected by alternative splicing. Since VPA is a drug highly successfully used in long-term epilepsy therapy, our findings open the exciting perspective for a first causal therapy of an inherited disease by elevating the SMN2 transcription level and restoring its correct splicing.
Assuntos
Fibroblastos/metabolismo , GABAérgicos/uso terapêutico , Atrofia Muscular Espinal/genética , Proteínas do Tecido Nervoso/genética , Ácido Valproico/uso terapêutico , Animais , Anticorpos Monoclonais/metabolismo , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Éxons , Fibroblastos/efeitos dos fármacos , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Terapia Genética , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/terapia , Proteínas do Tecido Nervoso/classificação , Técnicas de Cultura de Órgãos , Splicing de RNA , RNA Mensageiro/análise , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
Spinal muscular atrophy (SMA), a common motor neuron disease in humans, results from loss of functional survival motor neuron (SMN1) alleles. A nearly identical copy of the gene, SMN2, fails to provide protection from SMA because of a single translationally silent nucleotide difference in exon 7. This likely disrupts an exonic splicing enhancer and causes exon 7 skipping, leading to abundant production of a shorter isoform, SMN2Delta7. The truncated transcript encodes a less stable protein with reduced self-oligomerization activity that fails to compensate for the loss of SMN1. This report describes the identification of an in vivo regulator of SMN mRNA processing. Htra2-beta1, an SR-like splicing factor and ortholog of Drosophila melanogaster transformer-2, promoted the inclusion of SMN exon 7, which would stimulate full-length SMN2 expression. Htra2-beta1 specifically functioned through and bound an AG-rich exonic splicing enhancer in SMN exon 7. This effect is not species-specific as expression of Htra2-beta1 in human or mouse cells carrying an SMN2 minigene dramatically increased production of full-length SMN2. This demonstrates that SMN2 mRNA processing can be modulated in vivo. Because all SMA patients retain at least one SMN2 copy, these results show that an in vivo modulation of SMN RNA processing could serve as a therapeutic strategy to prevent SMA.
Assuntos
Éxons/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/metabolismo , Splicing de RNA/genética , Animais , Sequência de Bases , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Terapia Genética , Humanos , Camundongos , Dados de Sequência Molecular , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/terapia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas do Complexo SMN , Fatores de Processamento de Serina-Arginina , Proteína 1 de Sobrevivência do Neurônio Motor , Proteína 2 de Sobrevivência do Neurônio MotorRESUMO
The locus PKHD1 (polycystic kidney and hepatic disease 1) has been linked to all typical forms of the autosomal recessive polycystic kidney disease (ARPKD) and maps to chromosome 6p21.1-p12. We previously defined its genetic interval by the flanking markers D6S1714 and D6S1024. In our current work, we have fine-mapped the gene for the human P1 protein (MCM3), thought to be involved in the DNA replication process, to this critical region. We have also established its genomic structure. Mutation analyses using SSCP were performed in ARPKD patients' cDNA samples, leading to the exclusion of this gene as a candidate for this disorder. We also identified two intragenic polymorphisms that allowed families with critical recombination events to be evaluated. Although neither marker was informative in these individuals, they are the closest yet described for PKHD1 and may help to refine the candidate region.
