Optimal antisense target reducing INS intron 1 retention is adjacent to a parallel G quadruplex

Jana Kralovicova, Ana Lages, Alpa Patel, Ashish Dhir, Emanuele Buratti, Mark Searle, Igor Vorechovsky

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Pages (from-to)8161-8173
Number of pages13
JournalNucleic Acids Research
Issue number12
Early online date16 Jun 2014
Publication statusPublished - 8 Jul 2014


  • 5' Untranslated Regions
  • Animals
  • COS Cells
  • Cell Line
  • Cercopithecus aethiops
  • DEAD-box RNA Helicases/antagonists & inhibitors
  • G-Quadruplexes
  • Humans
  • Introns
  • Oligonucleotides, Antisense/chemistry
  • Proinsulin/genetics
  • RNA/chemistry
  • RNA Precursors/metabolism
  • RNA Splice Sites
  • RNA Splicing
  • RNA, Messenger/metabolism
  • RNA-Binding Proteins/metabolism


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