Spinal Muscular Atrophy (SMA) is caused by degeneration of α-motor neurons, as a consequence of null mutations in the Survival of Motor Neuron-1 (SMN1) gene. SMA is the leading genetic cause of death in infancy, urging the development of novel therapeutic approaches. Humans carry a highly homologous duplicated copy of the gene, named SMN2, that potentially encodes for the same protein. However, the majority of the SMN2 transcripts lack exon 7 because a C-to-T transition determines the skipping of the exon during splicing, thereby leading to production of a non functional SMN protein. For this reason, much of the research efforts have been oriented at correcting this splicing event. Many splicing factors that modulate exon 7 inclusion or skipping have been described. Among them, we have identified Sam68 as a crucial splicing inhibitor, whose binding to SMN2 pre-mRNA triggers exon 7 skipping, potentially concurring to the SMA phenotype (Pedrotti et al., EMBO J. 2010). RNA pull-down, UV-crosslinking and EMSA experiments demonstrated that Sam68 binds to a consensus site created by the C-to-T transition in SMN2 exon 7 (UUUUA), whose integrity is absolutely required for Sam68-dependent exon 7 skipping. Dominant-negative mutants of Sam68 that interfere with its RNA-binding activity, or suppress its association with the splicing repressor hnRNP A1, abrogate Sam68-induced SMN2 exon 7 skipping. Retroviral infection of SMA fibroblast with these Sam68 mutants rescued SMN protein levels and its functional assembly in nuclear gems. Moreover, since the splicing activity of Sam68 is regulated by post-translational modification, like phosphorylation by Src-family and ERK kinases, the contribution of different signaling pathways on Sam68-dependent SMN2 exon 7 splicing in SMA fibroblast was investigated. Our results demonstrate that Sam68 is a novel, crucial regulator of SMN2 splicing and provide new tools that may correct this aberrant splicing and ameliorate the SMA phenotype.
The RNA-binding protein Sam68 regulates SMN2 exon 7 alternative splicing in spinal muscular atrophy
Paronetto MP;
2010-01-01
Abstract
Spinal Muscular Atrophy (SMA) is caused by degeneration of α-motor neurons, as a consequence of null mutations in the Survival of Motor Neuron-1 (SMN1) gene. SMA is the leading genetic cause of death in infancy, urging the development of novel therapeutic approaches. Humans carry a highly homologous duplicated copy of the gene, named SMN2, that potentially encodes for the same protein. However, the majority of the SMN2 transcripts lack exon 7 because a C-to-T transition determines the skipping of the exon during splicing, thereby leading to production of a non functional SMN protein. For this reason, much of the research efforts have been oriented at correcting this splicing event. Many splicing factors that modulate exon 7 inclusion or skipping have been described. Among them, we have identified Sam68 as a crucial splicing inhibitor, whose binding to SMN2 pre-mRNA triggers exon 7 skipping, potentially concurring to the SMA phenotype (Pedrotti et al., EMBO J. 2010). RNA pull-down, UV-crosslinking and EMSA experiments demonstrated that Sam68 binds to a consensus site created by the C-to-T transition in SMN2 exon 7 (UUUUA), whose integrity is absolutely required for Sam68-dependent exon 7 skipping. Dominant-negative mutants of Sam68 that interfere with its RNA-binding activity, or suppress its association with the splicing repressor hnRNP A1, abrogate Sam68-induced SMN2 exon 7 skipping. Retroviral infection of SMA fibroblast with these Sam68 mutants rescued SMN protein levels and its functional assembly in nuclear gems. Moreover, since the splicing activity of Sam68 is regulated by post-translational modification, like phosphorylation by Src-family and ERK kinases, the contribution of different signaling pathways on Sam68-dependent SMN2 exon 7 splicing in SMA fibroblast was investigated. Our results demonstrate that Sam68 is a novel, crucial regulator of SMN2 splicing and provide new tools that may correct this aberrant splicing and ameliorate the SMA phenotype.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
