Aberrant splicing of spinal muscular atrophy gene is modulated by a RNA structure located at the exon-intron junction
Spinal Muscular Atrophy (SMA) is a severe autosomal recessive disorder caused by deletion or mutations within SMN1 (Survival of Motor Neuron 1) gene. Its nearly identical copy, SMN2, which is present in all SMA patients, fails to compensate for the loss of SMN1 due to aberrant splicing of exon 7. Using a novel in vivo selection method, we have determined that a weak 5'-splice site (5'ss) is the major cause of SMN2 exon 7 skipping (Singh et al., RNA 10, 1291-1305, 2004). Subsequently, we discovered that a unique intronic silencer located immediately downstream of the 5'ss of exon 7 has a strong inhibitory effect on the exon inclusion (Singh et al., Mol. Cell Biol., in press). Here we report the role of a local RNA structure we call Terminal Stem-Loop 2 (TSL2) in regulation of exon 7 splicing. TSL2 is formed by sequences at the junction of exon 7 and intron 7. Structure probing confirmed the existence of TSL2, formed by an eight-base-pair-long stem and a tri-loop. Using minigenes and transient transfection, we showed that point mutations that destabilized TSL2 restored SMN2 exon 7 inclusion, while strengthening of TSL2 promoted exon 7 skipping even in SMN1 mRNA. The above results highlight the inhibitory nature of TSL2. We demonstrate that the negative role of TSL2 is associated with the poor recruitment of U1 snRNP at the 5'ss of exon 7. TSL2 sequesters the first two intronic nucleotides, possibly interfering with the formation of the U1 snRNA:5'ss duplex. Consistently, intronic mutations that increased the length of this duplex fully overcame the inhibitory effect of TSL2. Our results highlight the surprising complexity of exon 7 splicing, where local RNA structure appears to be an integral part of the inhibitory network that sequesters the 5'ss and promotes exon skipping. We believe TSL2 presents an attractive target for the correction of SMN2 exon 7 splicing in SMA.