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

Characterization of parallel G-quadrplex formation by highly conserved G-rich motifs in INS intron 1
Characterization of parallel G-quadrplex formation by highly conserved G-rich motifs in INS intron 1
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.
University of Southampton
das Lages, Ana Luísa Gonçalves
88542ce9-2e63-4900-8d1f-653cbea0d003
das Lages, Ana Luísa Gonçalves
88542ce9-2e63-4900-8d1f-653cbea0d003
Holloway, John
4bbd77e6-c095-445d-a36b-a50a72f6fe1a
Proud, Christopher G
5832db56-9069-4617-a80d-08b069093dba
Vorechovsky, Igor
7245de2f-8c9b-4034-8935-9a451d9b682e

das Lages, Ana Luísa Gonçalves (2018) Characterization of parallel G-quadrplex formation by highly conserved G-rich motifs in INS intron 1. University of Southampton, Doctoral Thesis, 290pp.

Record type: Thesis (Doctoral)

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.

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Ana Lages eThesis Characterization of parallel G-quadruplex formation by highly conserved G-rich motifs in INS intron 1 - Version of Record
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Published date: June 2018

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Local EPrints ID: 435487
URI: http://eprints.soton.ac.uk/id/eprint/435487
PURE UUID: 083de908-d34e-4904-9909-a28a9a020f91
ORCID for John Holloway: ORCID iD orcid.org/0000-0001-9998-0464

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Date deposited: 07 Nov 2019 17:30
Last modified: 08 Nov 2019 01:37

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Contributors

Author: Ana Luísa Gonçalves das Lages
Thesis advisor: John Holloway ORCID iD
Thesis advisor: Christopher G Proud
Thesis advisor: Igor Vorechovsky

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