RESUMO
Cockayne syndrome (CS) is a severe disorder with no effective treatment. The Cockayne syndrome group B (CSB) gene is one gene responsible for CS and also causes UV sensitive syndrome (UVSS), a disorder that causes mild symptoms. How the CSB gene determines a patient's fate is unknown, but one intriguing point is that in UVSS patient cell, there are nonsense mutations in both alleles at the same position in each upstream region of the PiggyBac transposable element derived 3 (PGBD3) inserted region. In contrast, in CS patient cells, there is at least one allele with several mutations downstream of the PGBD3 inserted region, or there are homozygous mutations in exon 1. Here, we designed and synthesized 24 splice switching oligonucleotides (SSOs) to skip exon 3 in CSB mRNA. Use of these SSOs induced a frame shift in order to generate an alternative stop codon at the upstream region of the PGBD3 invasion site. As a result, a reduction of mitochondrial membrane potential following H2O2 treatment in CS cell was recovered. It was demonstrated that up-regulation of several gene expression brought about by SSOs are related to mitochondrial dysfunction in CS cells.
Assuntos
Síndrome de Cockayne/genética , Síndrome de Cockayne/patologia , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Oligonucleotídeos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Splicing de RNA/genética , Linhagem Celular , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Éxons , Humanos , Peróxido de Hidrogênio/farmacologia , Potencial da Membrana Mitocondrial , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção , Raios UltravioletaRESUMO
Double-stranded DNA (dsDNA) templates can hybridize to and accelerate cleavage of oligonucleotides containing a P3'âN5' phosphoramidate (P-N) linkage. This dsDNA-templated cleavage of P-N linkages could be due to conformational strain placed on the linkage upon triplex formation. To determine whether duplex formation also induced conformational strain, we examined the reactivity of the oligonucleotides with a P-N linkage in the presence of single-stranded templates, and compared these reactions to those with dsDNA templates. P-N oligonucleotides that are cleaved upon duplex formation could be used as probes to detect single-stranded nucleic acids.
