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

Nucleic Acids Research 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 Bruno Palhais 2 Veronica S. Praestegaard 2 Rugivan Sabaratnam 2 Thomas Koed Doktor 2 Seraina Lutz 1 Patricie Burda 1 Terttu Suormala 1 Matthias Baumgartner 1 Brian Fowler 1 Gitte Hoffmann Bruun 2 Henriette Skovgaard Andersen 2 Viktor Koz ich 0 Brage Storstein Andresen 2 0 Institute of Inherited Metabolic Disorders, Charles University in Prague-First Faculty of Medicine and General University Hospital , Praha , Czech Republic 1 Division of Metabolism, University Children's Hospital , Z u rich , Switzerland 2 Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark , Odense M , Denmark C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. - 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. Expression of protein coding genes in eukaryotes relies on correct splicing of pre-mRNA transcripts. During this process the spliceosome removes intronic sequences from the initial transcripts and joins together the exons to produce a mature mRNA. It is thus crucial for the cell to identify and process exons with high fidelity. Splice site sequences are the major splicing signals recognized by the spliceosomal machinery but due to their degeneracy (1,2) they are not by themselves sufficient for efficient recognition of exons, and in silico analysis shows that non-functional copies of splice site sequences are highly abundant in intronic regions (3). Therefore, other cis-acting splicing regulatory elements (SRE) are necessary to direct the spliceosomal proteins to the correct splice sites for efficient splicing. Exonic splicing enhancers (ESE) and intronic splicing enhancers (ISE) are sequences commonly bound by proteins of the serine/arginine-rich (SR) family, which stimulate exon inclusion (4). Conversely, members from the heterogeneous nuclear ribonucleoprotein (hnRNP) family bind to exonic splicing silencers (ESS) and intronic splicing silencers (ISS) to repress exon inclusion (5). Aberrant splicing often causes human diseases and according to the Human Gene Mutation Database (HGMD R Professional Release 2014.4) 14 849 of 163 670 (i.e. about *To whom correspondence should be addressed. Tel: +45 65502413; Fax: +45 65502467; Email: These authors contributed equally to the paper as first authors. 9.1%) of all reported mutations affect the splicing process. Furthermore, an estimated 25% of the mutations presumed to be missense and nonsense mutations are in fact splicing mutations (6,7). The majority of the reported splicing mutations alter the conserved splice sites at exonintron junctions. However, in a growing number of cases aberrant splicing results from mutations in positive or negative SREs (3,813). Sequences resembling functional splice sites (pseudosplice-sites) are highly frequent in introns but are rarely used during splicing (3,14). It is believed that this may be due to intrinsic defects in the sequences themselves (14) and the enrichment of splicing silencer motifs (15). When two matching pseudosplice-sites are located close in an intron they define a pseudoexon. Activation of a pseudoexon, so that it is spliced into an mRNA, disrupts gene expression and will often cause disease. All genes harbor pseudoexons, but the number of pseudoexons in our genome is not known and has so far only been loosely estimated based on computational approaches (15,16). Pseudoexon activation has traditionally been regarded as a rare disease mechanism, which requires multiple changes (mutations) to occur (14), but in recent years it has become clear that apparently benign single nucleotide variations can be sufficient to activate pseudoexons and cause disease and the number of reported cases has been increasing (3,17). In most of the reported cases, pseudoexon activation results from single nucleotide changes creating new splice sites or increasing the strength of existing suboptimal splice sites. This is most likely due to the fact that changes involving the splice site sequences are easier to recognize since the consensus motifs are well established. We have recently reported that the most frequent mutation in the methionine synthase reductase (MTRR) gene, a deep intronic mutation (c.903+469T>C), creates an SRSF1 binding ESE, which leads to pseudoexon inclusion and causes the cblE type of homocystinuria (18). Similarly, other groups have also reported in other genes (PCCA, GLA, FGB, CFTR, ATM, Col4A5 and MFGE8) that single nucleotide changes in introns located outside splice site sequences can cause pseudoexon activation and human disease (1925). These and other studies convincingly show that an intronic single nucleotide change by affecting splicing regulatory elements outside of the splice sites is sufficient to activate a pseudoexon and cause disease. It can therefore be hypothesized that pseudoexons, like constitutive exons, are regulated by a finely tuned balance between positive and negative splicing regulatory elements and any single nucleotide substitution that changes this balance may lead to pseudoexon activation. Because intronic sequences are typically not examined during routine diagnostic procedures it is likely that the prev (...truncated)