SAM 68 is a physiological regulator of SMN 2 splicing in spinal muscular atrophy

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality, with an incidence of approximately one in 6,000–10,000 newborns (Arnold and Burghes, 2013; Nurputra et al., 2013). SMA is an autosomal recessive neuromuscular disorder primarily characterized by degeneration of spinal cord motor neurons and consequent atrophy of skeletal muscles. The disease is most commonly caused by homozygous deletion of the Survival Motor Neuron 1 (SMN1) gene, leading to reduced levels of the ubiquitously expressed SMN protein (Arnold and Burghes, 2013; Nurputra et al., 2013). SMN plays key roles in several canonical cellular pathways, including biogenesis of the small nuclear RNPs, which are core components of the spliceosome (Wahl et al., 2009), pre-mRNA splicing, and RNA transport (Li et al., 2014). Nevertheless, attempts to directly link aberrant RNA processing with SMA pathogenesis have proven controversial (Zhang et al., 2008; Bäumer et al., 2009). SMA patients retain at least one copy of the almost identical SMN2 gene. However, although it encodes a virtually identical protein, the expression levels of SMN2 are not sufficient to restore full SMN activity (Arnold and Burghes, 2013; Nurputra et al., 2013). The coding regions of SMN1 and SMN2 differ only for a silent C to T transition at position 6 in exon 7, leading to the skipping of exon 7 in most SMN2 transcripts and to production of an unstable SMNΔ7 protein that is rapidly degraded (Burnett et al., 2009; Cho and Dreyfuss, 2010). The residual low levels of SMN2 transcripts that include exon 7 produce small amounts of fully functional SMN protein, supporting early development and viability in patients and mouse models of SMA (Arnold and Burghes, 2013; Nurputra et al., 2013). Currently, there is no cure for SMA patients (Lorson and Lorson, 2012; Arnold and Burghes, 2013). Because rescue of exon 7 splicing represents a suitable therapeutic approach for SMA, understanding the molecular mechanisms involved in the regulation of this splicing event is of fundamental importance. Many RNA-binding proteins (RBPs) have been proposed to play a role in the regulation of SMN2 splicing in vitro (Hofmann et al., 2000; Hofmann and Wirth, 2002; Kashima and Manley, 2003; Kashima et al., 2007; Bose et al., 2008; Chen et al., 2008; Pedrotti et al., 2010; Singh et al., 2011), including hnRNP A1 (Kashima and Manley, 2003; Kashima et al., 2007) and SAM68 (Pedrotti et al., 2010), suggesting that the relative expression levels or activity of specific splicing factors can modulate SMN2 splicing (Pedrotti and Sette, 2010). However, Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. The almost identical SMN2 gene is unable to compensate for this deficiency because of the skipping of exon 7 during pre–messenger RNA (mRNA) processing. Although several splicing factors can modulate SMN2 splicing in vitro, the physiological regulators of this disease-causing event are unknown. We found that knockout of the splicing factor SAM68 partially rescued body weight and viability of SMAΔ7 mice. Ablation of SAM68 function promoted SMN2 splicing and expression in SMAΔ7 mice, correlating with amelioration of SMA-related defects in motor neurons and skeletal muscles. Mechanistically, SAM68 binds to SMN2 pre-mRNA, favoring recruitment of the splicing repressor hnRNP A1 and interfering with that of U2AF65 at the 3′ splice site of exon 7. These findings identify SAM68 as the first physiological regulator of SMN2 splicing in an SMA mouse model. SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy