title: 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.
abstract: 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.
authors: Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors: Bruno
authors: B
authors: Palhais
authors: Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors: Veronica S
authors: VS
authors: Præstegaard
authors: Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors: Rugivan
authors: R
authors: Sabaratnam
authors: Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors: Thomas Koed
authors: TK
authors: Doktor
authors: Division of Metabolism, University Children's Hospital, Zürich, Switzerland.
authors: Seraina
authors: S
authors: Lutz
authors: Division of Metabolism, University Children's Hospital, Zürich, Switzerland.
authors: Patricie
authors: P
authors: Burda
authors: Division of Metabolism, University Children's Hospital, Zürich, Switzerland.
authors: Terttu
authors: T
authors: Suormala
authors: Division of Metabolism, University Children's Hospital, Zürich, Switzerland.
authors: Matthias
authors: M
authors: Baumgartner
authors: Division of Metabolism, University Children's Hospital, Zürich, Switzerland.
authors: Brian
authors: B
authors: Fowler
authors: Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors: Gitte Hoffmann
authors: GH
authors: Bruun
authors: Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors: Henriette Skovgaard
authors: HS
authors: Andersen
authors: Institute of Inherited Metabolic Disorders, Charles University in Prague-First Faculty of Medicine and General University Hospital, Praha, Czech Republic.
authors: Viktor
authors: V
authors: Kožich
authors: Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark bragea@bmb.sdu.dk.
authors: Brage Storstein
authors: BS
authors: Andresen
mesh_terms: D000749:Anemia, Megaloblastic / Q000201:enzymology / Q000235:genetics*; D002460:Cell Line; D002478:Cells, Cultured; D005091:Exons; D005287:Ferredoxin-NADP Reductase / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006712:Homocystinuria / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D009154:Mutation; D009841:Oligonucleotides; D022821:RNA Splice Sites; D012326:RNA Splicing; D038621:Regulatory Sequences, Ribonucleic Acid
chemical_list: D009841:Oligonucleotides; D022821:RNA Splice Sites; D038621:Regulatory Sequences, Ribonucleic Acid; C111241:methionine synthase reductase; D005287:Ferredoxin-NADP Reductase