title: A targeted oligonucleotide enhancer of SMN2 exon 7 splicing forms competing quadruplex and protein complexes in functional conditions.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester LE1 9HN, UK.
authors.forename: Lindsay D
authors.initials: LD
authors.lastname: Smith
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester LE1 9HN, UK; Department of Chemistry, University of Leicester, Leicester LE1 7RH, UK.
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Dickinson
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester LE1 9HN, UK.
authors.forename: Christian M
authors.initials: CM
authors.lastname: Lucas
authors.affiliation: School of Chemistry, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, UK.
authors.forename: Alex
authors.initials: A
authors.lastname: Cousins
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
authors.forename: Alexey A
authors.initials: AA
authors.lastname: Malygin
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester LE1 9HN, UK.
authors.forename: Carika
authors.initials: C
authors.lastname: Weldon
authors.affiliation: Department of Chemistry, University of Leicester, Leicester LE1 7RH, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Perrett
authors.affiliation: Protein and Nucleic Acid Chemistry Laboratory, University of Leicester, Leicester LE1 9HN, UK.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Bottrill
authors.affiliation: School of Chemistry, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, UK.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Searle
authors.affiliation: Department of Pure & Applied Chemistry, University of Strathclyde, Glasgow G1 1XL, UK. Electronic address: glenn.burley@strath.ac.uk.
authors.forename: Glenn A
authors.initials: GA
authors.lastname: Burley
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester LE1 9HN, UK. Electronic address: eci@leicester.ac.uk.
authors.forename: Ian C
authors.initials: IC
authors.lastname: Eperon
mesh_terms: D001483:Base Sequence; D005091:Exons; D006801:Humans; D007438:Introns; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics* / Q000378:metabolism; D022821:RNA Splice Sites; D012326:RNA Splicing; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D017381:Spliceosomes / Q000235:genetics* / Q000378:metabolism; D000072668:Splicing Factor U2AF; D055540:Survival of Motor Neuron 2 Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D009687:Nuclear Proteins; D009841:Oligonucleotides; D022821:RNA Splice Sites; D017413:Ribonucleoprotein, U2 Small Nuclear; D012261:Ribonucleoproteins; C526477:SMN2 protein, human; D000072668:Splicing Factor U2AF; D055540:Survival of Motor Neuron 2 Protein; C000607494:U2AF2 protein, human