Characterization of parallel G-quadrplex formation by highly conserved G-rich motifs in INS intron 1

Abstract

Individuals predisposed to type 1 diabetes (T1DM) carry an adenine allele at rs689, a T-to-A genetic variant located 6 nucleotides upstream of the 3’ splice site of INS intron 1. The A allele disrupts the polypyrimidine tract (Py-tract) and impairs splicing, increasing intron retention (IR) levels in mature transcripts. Intron 1-containing messenger RNAs have an extended 5’ untranslated region (5’UTR), introducing an upstream open reading frame (uORF) and curtailing translation. IR can be reduced using oligonucleotides complementary to an intronic segment flanked by G-rich sequences, mitigating the splicing defect. To investigate their potential to form G-quadruplex (G4), a G4-specific fluorescent probe, thioflavin T, was used to examine DNA and RNA G-tracts flanking the antisense target region, in vitro. Fluorescence intensity was shown to be specific for G4 structures in DNA and RNA. G4 formation was influenced by the adjacent target region in each direction and by concentrations of K+ and Mg2+. G4s were also detected in RNA transcribed in real time and their formation was influenced by mutations that affected intron 1 removal. To identify proteins binding to this intronic region, in vitro RNA transcripts containing the antisense target region were used in RNA pull-down assays with HeLa nuclear extracts, revealing heterogeneous nuclear ribonucleoproteins (hnRNPs) F and H1 binding to G-rich segments. The G-rich 5’ UTRs of representative primate species were also cloned into a dual-luciferase-reporter system to establish primate-specific translation rates. Elimination of the Homoninae-specific uORF significantly increased luciferase translation, demonstrating its importance for INS gene expression. Overall, data obtained in this project has improved the understanding of the molecular mechanisms underlying the allele-specific expression of preproinsulin expression. These results may facilitate development of future preventative strategies for T1DM.

More information

Identifiers

ORCID for John Holloway: orcid.org/0000-0001-9998-0464

ORCID for Igor Vorechovsky: orcid.org/0000-0002-6740-6502

Catalogue record

Export record