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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 B, Præstegaard VS, Sabaratnam R, Doktor TK, Lutz S, Burda P, Suormala T, Baumgartner M, Fowler B, Bruun GH, Andersen HS, Kožich V, Andresen BS - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.

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The balanced interplay between an hnRNP A1 binding ESS and two SRSF1 binding ESEs dictate MTRR pseudoexon activation. (A) Schematic representation of the sequences from MTRR-minigenes used and the nucleotide changes introduced. A pictogram of hnRNP A1 position weight matrix (38) is shown above the putative ESS and the invariant position 2 of this motif is underscored. A pictogram of the SRSF1 position weight matrix (36) is shown above the previously described ESE1 (18) and the ACADM like ESE. Indicated positions 361, 362 and 365 are relative to the ACADM coding sequence. Nucleotide positions that were changed are underscored. (B) Splicing minigene assay. MTRR-minigenes were transiently transfected into HEK293. After RNA isolation the splicing products were analyzed by RT-PCR. The upper panel shows the average of pseudoexon inclusion from two measurements of each duplicate. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument. The lower panel shows a sample agarose gel electrophoresis displaying pseudoexon inclusion levels in the different cell lines. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inserted between minigene exons. Ψ marks the pseudoexon. (C) Binding of hnRNPA1 and SRSF1 proteins. Biotinylated RNA oligonucleotides were used in a pull-down experiment with HeLa nuclear extract followed by SDS PAGE and western blot analysis using antibodies against SRSF1 and hnRNP A1. Blank indicates a control lane from pull down without RNA oligonucleotides. NE is nuclear extract. The displayed blots are representative result from at least three pull-down experiments.
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Figure 2: The balanced interplay between an hnRNP A1 binding ESS and two SRSF1 binding ESEs dictate MTRR pseudoexon activation. (A) Schematic representation of the sequences from MTRR-minigenes used and the nucleotide changes introduced. A pictogram of hnRNP A1 position weight matrix (38) is shown above the putative ESS and the invariant position 2 of this motif is underscored. A pictogram of the SRSF1 position weight matrix (36) is shown above the previously described ESE1 (18) and the ACADM like ESE. Indicated positions 361, 362 and 365 are relative to the ACADM coding sequence. Nucleotide positions that were changed are underscored. (B) Splicing minigene assay. MTRR-minigenes were transiently transfected into HEK293. After RNA isolation the splicing products were analyzed by RT-PCR. The upper panel shows the average of pseudoexon inclusion from two measurements of each duplicate. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument. The lower panel shows a sample agarose gel electrophoresis displaying pseudoexon inclusion levels in the different cell lines. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inserted between minigene exons. Ψ marks the pseudoexon. (C) Binding of hnRNPA1 and SRSF1 proteins. Biotinylated RNA oligonucleotides were used in a pull-down experiment with HeLa nuclear extract followed by SDS PAGE and western blot analysis using antibodies against SRSF1 and hnRNP A1. Blank indicates a control lane from pull down without RNA oligonucleotides. NE is nuclear extract. The displayed blots are representative result from at least three pull-down experiments.

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).


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 B, Præstegaard VS, Sabaratnam R, Doktor TK, Lutz S, Burda P, Suormala T, Baumgartner M, Fowler B, Bruun GH, Andersen HS, Kožich V, Andresen BS - Nucleic Acids Res. (2015)

The balanced interplay between an hnRNP A1 binding ESS and two SRSF1 binding ESEs dictate MTRR pseudoexon activation. (A) Schematic representation of the sequences from MTRR-minigenes used and the nucleotide changes introduced. A pictogram of hnRNP A1 position weight matrix (38) is shown above the putative ESS and the invariant position 2 of this motif is underscored. A pictogram of the SRSF1 position weight matrix (36) is shown above the previously described ESE1 (18) and the ACADM like ESE. Indicated positions 361, 362 and 365 are relative to the ACADM coding sequence. Nucleotide positions that were changed are underscored. (B) Splicing minigene assay. MTRR-minigenes were transiently transfected into HEK293. After RNA isolation the splicing products were analyzed by RT-PCR. The upper panel shows the average of pseudoexon inclusion from two measurements of each duplicate. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument. The lower panel shows a sample agarose gel electrophoresis displaying pseudoexon inclusion levels in the different cell lines. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inserted between minigene exons. Ψ marks the pseudoexon. (C) Binding of hnRNPA1 and SRSF1 proteins. Biotinylated RNA oligonucleotides were used in a pull-down experiment with HeLa nuclear extract followed by SDS PAGE and western blot analysis using antibodies against SRSF1 and hnRNP A1. Blank indicates a control lane from pull down without RNA oligonucleotides. NE is nuclear extract. The displayed blots are representative result from at least three pull-down experiments.
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Figure 2: The balanced interplay between an hnRNP A1 binding ESS and two SRSF1 binding ESEs dictate MTRR pseudoexon activation. (A) Schematic representation of the sequences from MTRR-minigenes used and the nucleotide changes introduced. A pictogram of hnRNP A1 position weight matrix (38) is shown above the putative ESS and the invariant position 2 of this motif is underscored. A pictogram of the SRSF1 position weight matrix (36) is shown above the previously described ESE1 (18) and the ACADM like ESE. Indicated positions 361, 362 and 365 are relative to the ACADM coding sequence. Nucleotide positions that were changed are underscored. (B) Splicing minigene assay. MTRR-minigenes were transiently transfected into HEK293. After RNA isolation the splicing products were analyzed by RT-PCR. The upper panel shows the average of pseudoexon inclusion from two measurements of each duplicate. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument. The lower panel shows a sample agarose gel electrophoresis displaying pseudoexon inclusion levels in the different cell lines. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inserted between minigene exons. Ψ marks the pseudoexon. (C) Binding of hnRNPA1 and SRSF1 proteins. Biotinylated RNA oligonucleotides were used in a pull-down experiment with HeLa nuclear extract followed by SDS PAGE and western blot analysis using antibodies against SRSF1 and hnRNP A1. Blank indicates a control lane from pull down without RNA oligonucleotides. NE is nuclear extract. The displayed blots are representative result from at least three pull-down experiments.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.

Show MeSH
Related in: MedlinePlus