Activation of a cryptic 5′ splice site reverses the impact of pathogenic splice site mutations in the spinal muscular atrophy gene
AbstractSpinal muscular atrophy (SMA) is caused by deletions or mutations of the Survival Motor Neuron 1 (SMN1) gene coupled with predominant skipping of SMN2 exon 7. The only approved SMA treatment is an antisense oligonucleotide that targets the intronic splicing silencer N1 (ISS-N1), located downstream of the 5′ splice site (5′ss) of exon 7. Here, we describe a novel approach to exon 7 splicing modulation through activation of a cryptic 5′ss (Cr1). We discovered the activation of Cr1 in transcripts derived from SMN1 that carries a pathogenic G-to-C mutation at the first position (G1C) of intron 7. We show that Cr1-activating engineered U1 snRNAs (eU1s) have the unique ability to reprogram pre-mRNA splicing and restore exon 7 inclusion in SMN1 carrying a broad spectrum of pathogenic mutations at both the 3′ss and 5′ss of the exon 7. Employing a splicing-coupled translation reporter, we demonstrate that mRNAs generated by an eU1-induced activation of Cr1 produce full-length SMN. Our findings underscore a wider role for U1 snRNP in splicing regulation and reveal a novel approach for the restoration of SMN exon 7 inclusion for a potential therapy of SMA.
[…] The strength of the 5′ and 3′ splice sites was determined using MaxEntScan scoring algorithm (http://genes.mit.edu/cgi-bin/Xmaxentscan_scoreseq.pl) (). The 5′ splice sites were also scored using the HBond score web interface, version 3.4 (http://www2.hhu.de/rna/html/hbond_score.php). CRYP-SKIP prediction algorithm was used to determine whether a splicing mutation of interest would result in exon skipping or activation of cryptic/di novo splice sites (). […]
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