A Targeted Oligonucleotide Enhancer of SMN2 Exon 7 Splicing Forms Competing Quadruplex and Protein Complexes in Functional Conditions

Abstract

Summary: The use of oligonucleotides to activate the splicing of selected exons is limited by a poor understanding of the mechanisms affected. A targeted bifunctional oligonucleotide enhancer of splicing (TOES) anneals to SMN2 exon 7 and carries an exonic splicing enhancer (ESE) sequence. We show that it stimulates splicing specifically of intron 6 in the presence of repressing sequences in intron 7. Complementarity to the 5′ end of exon 7 increases U2AF65 binding, but the ESE sequence is required for efficient recruitment of U2 snRNP. The ESE forms at least three coexisting discrete states: a quadruplex, a complex containing only hnRNP F/H, and a complex enriched in the activator SRSF1. Neither hnRNP H nor quadruplex formation contributes to ESE activity. The results suggest that splicing limited by weak signals can be rescued by rapid exchange of TOES oligonucleotides in various complexes and raise the possibility that SR proteins associate transiently with ESEs. : The use of oligonucleotides to activate selected exons is hampered by a poor understanding of the mechanisms of splicing enhancers. In this study, Smith et al. show that a bifunctional oligonucleotide enhancer of splicing of SMN2 exon 7 stimulates recruitment of U2 snRNP to the upstream intron. Surprisingly, the GGA-rich enhancer forms a quadruplex and complexes enriched in hnRNP H and other proteins. Nonetheless, these complexes do not prevent enhancer activity, suggesting that SR proteins function here via dynamic exchange.