Abstract
Antisense oligomers (AOs) are increasingly being used to modulate RNA splicing in live cells, both for research and for the development of therapeutics. While the most common intended effect of these AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven AO-induced cryptic splice sites in six genes. Five of these cryptic splice sites were discovered through our own experiments, and two originated from other published reports. We modelled the predicted effects of AO binding on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was disruption of the exon definition signal within the exon’s excluded segment.
Highlights
Abbreviations Antisense oligonucleotides (AOs) Antisense oligonucleotide SnRNP Small nuclear ribonucleoproteins 5′ss 5′ Splice site 3′ss 3′ Splice site Nt Nucleotide nonsense mediated decay (NMD) Nonsense mediated decay NC Negative control serine/arginine-rich splicing factor (SRSF) Serine/arginine-rich splicing factor ESE Exonic splicing enhancer exon splicing silencer (ESS) Exonic splicing silencer long non-coding RNA (lncRNA) Long non-coding RNA 2′-OMe PS 2′-O-Methyl modified bases on a phosphorothioate backbone FBS Fetal bovine serum Basic Local Alignment Search Tool (BLAST) Basic local alignment search tool
The major form of the spliceosome is composed of five small nuclear ribonucleoproteins, as well as numerous non-snRNP proteins[4,5]
A new donor splice site was activated by treatment with an AO targeting COL7A1 exon 15, H15A(+91+115), that resulted in cryptic splice site activation in 30% of the transcripts after transfection of the AO at both 100 nM and 50 nM
Summary
Abbreviations AO Antisense oligonucleotide SnRNP Small nuclear ribonucleoproteins 5′ss 5′ Splice site 3′ss 3′ Splice site Nt Nucleotide NMD Nonsense mediated decay NC Negative control SRSF Serine/arginine-rich splicing factor ESE Exonic splicing enhancer ESS Exonic splicing silencer lncRNA Long non-coding RNA 2′-OMe PS 2′-O-Methyl modified bases on a phosphorothioate backbone FBS Fetal bovine serum BLAST Basic local alignment search tool. In order to achieve alternative splicing, the spliceosome must recognize and select a splice site amid a variety of alternative splice sites and branchpoints within the transcript These splice sites are well defined and have evolutionarily conserved functions. According to DBASS, the mutations most commonly causative of cryptic splice site activation are those that weaken canonical exon splice sites, redirecting the spliceosome to utilize a viable cryptic site n earby[13]. This is a relatively rare outcome of such mutations, which are generally far more likely to induce whole exon s kipping[14]. Recent data has shown that cryptic splice sites can be activated by synthetic molecules such as antisense oligonucleotides
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