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

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.

Do we know whether potential G-quadruplexes actually form in long functional RNA molecules?

“…However, the requirement of comparing G4 formation in different buffers prevents the use of these methods directly in physiological environments such as nuclear or cell extracts. Pausing of reverse transcriptase at G4s has been detected in nuclear extracts, although in such cases other evidence is still required to show that the pause was caused by a G4 [31]. …”

“…An alternative to site-directed mutagenesis of guanines therefore consists of evaluating the effect of such ligands in functional assays [25,26,29–31]. It is generally believed that all G4 binders act as stabilizers of G4s; therefore, the effect observed upon the addition of a ligand in functional assay can be attributed to the effect of the G4.…”

“…This is also the case for the G4 antibody [17], as addition of the antibody may shift the equilibrium of the RNA population to the G4-containing form. The possibility of this is emphasized by our previous work, showing that a short oligonucleotide that acts as a trans -acting enhancer of RNA splicing exists as a diverse population of molecules under functional conditions, bound as a linear sequence by various sets of mutually incompatible proteins and also a G4 [31]. The possibility that RNA structures are not static but in protein-augmented dynamic equilibria adds to the complexity of the challenge in studying G4s.…”

Pharmacology of Modulators of Alternative Splicing

“…In this case, transfection of cells with this targeted oligonucleotide enhancer of splicing results in stimulation of alternative splicing to a specific exon. This has been demonstrated in vitro in cell free systems and in intact cells, resulting in the extra inclusion of exon 7 in the SMN2 gene (Smith et al, 2014). The equivalent has also been undertaken with antisense oligonucleotides directed against SMN2 that contains the sequence for the ISS in exon 7, which then sequesters the splicing machinery that causes exon skipping, resulting in exon inclusion and rescue of the disease phenotype (Hua et al, 2007).…”

“…One example is targeted oligonucleotide enhancers of splicing (Smith et al, 2014) and another is to use alternative intronic splicing silencers [ISS (Singh et al, 2015)], which can then be used to stimulate or repress splicing to a specific exon, respectively.…”