Abstract
SummaryThe 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.
Highlights
Pre-mRNA splicing has the potential to be a target of considerable importance for therapeutic intervention
One of the most successful techniques for redirecting the splicing patterns of specific genes is to use oligonucleotides complementary to splicing signals or auxiliary motifs in the premRNA (Eperon, 2012; Rigo et al, 2012; Singh and Cooper, 2012). These techniques were first designed to suppress the use of a particular pattern by blocking the binding of splicing factors to splice sites or exons (Dominski and Kole, 1993; Mayeda et al, 1990) and were subsequently developed as potential therapies for muscular dystrophy in cases where skipping of an exon carrying a nonsense mutation would be beneficial (Cirak et al, 2011; Dunckley et al, 1998; Goemans et al, 2011)
Previous we found that it augmented splicing effectively even in the absence of an exonic splicing enhancer (ESE) in exon 7 (Owen et al, 2011) that is otherwise essential (Hofmann et al, 2000; Martins de Araujo et al, 2009), the sites of annealing that enabled efficient activation of SMN2 exon 7 were very restricted, and stabilizing the annealing of the oligonucleotide with modified nucleotides reduced its activity (Owen et al, 2011). These findings suggest that the mechanistic models used as a basis for designing the oligonucleotides were inadequate, which compromises our ability to apply targeted oligonucleotide enhancers of splicing (TOES) oligonucleotides to rescue other exons
Summary
Pre-mRNA splicing has the potential to be a target of considerable importance for therapeutic intervention. The C-terminal domain of an ESE-bound SR protein was proposed to interact directly with the 30 splice site-recognition factor, U2AF, the recruitment of which is often a limiting step in splicing, thereby increasing the level of binding of U2AF (Graveley et al, 2001; Lavigueur et al, 1993; Staknis and Reed, 1994; Wang et al, 1995; Wu and Maniatis, 1993) This led to the development of two types of oligonucleotides to stimulate usage of an exon. Other sequences in or around exons have been found to act as silencers, and in such cases activation can be achieved by using oligonucleotides to block the binding of repressor proteins (Hua et al, 2007, 2008)
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