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Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

Palhais, Bruno ; Præstegaard, Veronica S. ; Sabaratnam, Rugivan ; Doktor, Thomas Koed ; Lutz, Seraina ; Burda, Patricie ; Suormala, Terttu ; Baumgartner, Matthias ; Fowler, Brian ; Bruun, Gitte Hoffmann ; Andersen, Henriette Skovgaard ; Kožich, Viktor ; Andresen, Brage Storstein

In: Nucleic Acids Research, 2015, vol. 43, no. 9, p. 4627-4639

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    Titel
    • Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Autor
    • Palhais, Bruno. Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark
    • Præstegaard, Veronica S.. Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark
    • Sabaratnam, Rugivan. Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark
    • Doktor, Thomas Koed. Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark
    • Lutz, Seraina. Division of Metabolism, University Children's Hospital, Zürich, Switzerland
    • Burda, Patricie. Division of Metabolism, University Children's Hospital, Zürich, Switzerland
    • Suormala, Terttu. Division of Metabolism, University Children's Hospital, Zürich, Switzerland
    • Baumgartner, Matthias. Division of Metabolism, University Children's Hospital, Zürich, Switzerland
    • Fowler, Brian. Division of Metabolism, University Children's Hospital, Zürich, Switzerland
    • Bruun, Gitte Hoffmann. Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark
    • Andersen, Henriette Skovgaard. Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark
    • Kožich, Viktor. Institute of Inherited Metabolic Disorders, Charles University in Prague-First Faculty of Medicine and General University Hospital, Praha, Czech Republic
    • Andresen, Brage Storstein. Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark
    Dokumententyp
    • Postprint
    Sprache
    • Englisch
    Veröffentlicht in
    • Nucleic Acids Research, 2015, vol. 43, no. 9, p. 4627-4639. Oxford University Press
    Andere elektronische Ausgabe
    Klassifikation
    • Gesundheit
    OAI-PMH-ID
    • oai:doc.rero.ch:292780
    Summary
    The prevalent c.903+469T>C mutation in MTRR causes the cblE type of homocystinuria by strengthening an SRSF1 binding site in an ESE leading to activation of a pseudoexon. We hypothesized that other splicing regulatory elements (SREs) are also critical for MTRR pseudoexon inclusion. We demonstrate that the MTRR pseudoexon is on the verge of being recognized and is therefore vulnerable to several point mutations that disrupt a fine-tuned balance between the different SREs. Normally, pseudoexon inclusion is suppressed by a hnRNP A1 binding exonic splicing silencer (ESS). When the c.903+469T>C mutation is present two ESEs abrogate the activity of the ESS and promote pseudoexon inclusion. Blocking the 3′splice site or the ESEs by SSOs is effective in restoring normal splicing of minigenes and endogenous MTRR transcripts in patient cells. By employing an SSO complementary to both ESEs, we were able to rescue MTRR enzymatic activity in patient cells to approximately 50% of that in controls. We show that several point mutations, individually, can activate a pseudoexon, illustrating that this mechanism can occur more frequently than previously expected. Moreover, we demonstrate that SSO blocking of critical ESEs is a promising strategy to treat the increasing number of activated pseudoexons