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.
Bottom Line: 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.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.
Affiliation: Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.Show MeSH
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Mentions: Because exon recognition is usually determined by a finely tuned balance between positive and negative splicing regulatory sequences, we hypothesized that additional SREs may contribute to the regulation of MTRR pseudoexon splicing. Therefore we inspected the sequences flanking the ESE created by the c.903+469T>C mutation. A potential SRSF1 binding motif (CAGCCTG; Figure 2A) similar to the SRSF1-binding ESE (CAGACTG) present in ACADM exon 5 (33) is present 11 bp downstream from the created ESE and a hnRNP A1 binding motif (38) recently demonstrated to cause exon skipping and disease when it is created by a mutation in the weak exon 2 in ETFDH (13), is present 9 bp upstream (Figure 2A). To investigate the potential role of these motifs in regulation of pseudoexon inclusion, we designed a set of MTRR minigenes where the ACADM-like motif is disrupted in a mutant MTRR background (MUT-362T and MUT-361T) and the hnRNP A1 motif is disrupted in the wild-type MTRR background (WT-TCGGGA) (Figure 2A). Furthermore, we generated the original ACADM ESE (WT-361A/365C) and its corresponding ESE-inactivating c.362C>T mutant version in the wild-type MTRR minigene (WT-361A/362T/365C). Disruption of the ACADM-like ESE by introducing the c.362C>T mutation or another ESE-inactivating mutation, c.361A>T, causes decreased pseudoexon inclusion from the minigenes with the activating MTRR c.903+469T mutation (Figure 2B).
Affiliation: Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.