title: Optimal antisense target reducing INS intron 1 retention is adjacent to a parallel G quadruplex.
abstract: Splice-switching oligonucleotides (SSOs) have been widely used to inhibit exon usage but antisense strategies that promote removal of entire introns to increase splicing-mediated gene expression have not been developed. Here we show reduction of INS intron 1 retention by SSOs that bind transcripts derived from a human haplotype expressing low levels of proinsulin. This haplotype is tagged by a polypyrimidine tract variant rs689 that decreases the efficiency of intron 1 splicing and increases the relative abundance of mRNAs with extended 5' untranslated region (5' UTR), which curtails translation. Co-expression of haplotype-specific reporter constructs with SSOs bound to splicing regulatory motifs and decoy splice sites in primary transcripts revealed a motif that significantly reduced intron 1-containing mRNAs. Using an antisense microwalk at a single nucleotide resolution, the optimal target was mapped to a splicing silencer containing two pseudoacceptor sites sandwiched between predicted RNA guanine (G) quadruplex structures. Circular dichroism spectroscopy and nuclear magnetic resonance of synthetic G-rich oligoribonucleotide tracts derived from this region showed formation of a stable parallel 2-quartet G-quadruplex on the 3' side of the antisense retention target and an equilibrium between quadruplexes and stable hairpin-loop structures bound by optimal SSOs. This region interacts with heterogeneous nuclear ribonucleoproteins F and H that may interfere with conformational transitions involving the antisense target. The SSO-assisted promotion of weak intron removal from the 5' UTR through competing noncanonical and canonical RNA structures may facilitate development of novel strategies to enhance gene expression.
authors.affiliation: University of Southampton, Faculty of Medicine, Southampton SO16 6YD, UK.
authors.forename: Jana
authors.initials: J
authors.lastname: Kralovicova
authors.affiliation: University of Southampton, Faculty of Medicine, Southampton SO16 6YD, UK.
authors.forename: Ana
authors.initials: A
authors.lastname: Lages
authors.affiliation: University of Nottingham, School of Chemistry, Centre for Biomolecular Sciences, Nottingham NG7 2RD, UK.
authors.forename: Alpa
authors.initials: A
authors.lastname: Patel
authors.affiliation: ICGEB, Padriciano 99, 34149 Trieste, Italy.
authors.forename: Ashish
authors.initials: A
authors.lastname: Dhir
authors.affiliation: ICGEB, Padriciano 99, 34149 Trieste, Italy.
authors.forename: Emanuele
authors.initials: E
authors.lastname: Buratti
authors.affiliation: University of Nottingham, School of Chemistry, Centre for Biomolecular Sciences, Nottingham NG7 2RD, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Searle
authors.affiliation: University of Southampton, Faculty of Medicine, Southampton SO16 6YD, UK igvo@soton.ac.uk.
authors.forename: Igor
authors.initials: I
authors.lastname: Vorechovsky
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D053487:DEAD-box RNA Helicases / Q000037:antagonists & inhibitors; D054856:G-Quadruplexes; D006801:Humans; D007438:Introns; D016376:Oligonucleotides, Antisense / Q000737:chemistry*; D011384:Proinsulin / Q000235:genetics*; D012313:RNA / Q000737:chemistry; D012322:RNA Precursors / Q000378:metabolism; D022821:RNA Splice Sites; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D016376:Oligonucleotides, Antisense; D012322:RNA Precursors; D022821:RNA Splice Sites; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012313:RNA; D011384:Proinsulin; D053487:DEAD-box RNA Helicases