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.
Highlights
Expression of protein coding genes in eukaryotes relies on correct splicing of pre-mRNA transcripts
MTRR pseudoexon inclusion depends on the balanced interplay between exonic splicing silencers (ESS) and Exonic splicing enhancers (ESE) elements We have previously shown that the c.903+469T>C mutation in the MTRR gene strengthens a preexisting SRSF1binding motif and that pseudoexon activation is most likely caused by creation of an ESE and not loss of an ESS [18]
Pseudoexon activation was originally thought to be a rare and rather exotic event, which in order to occur requires that the function of more than one splicing regulatory element is altered by mutations [14]
Summary
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 crucial for the cell to identify and process exons with high fidelity. 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]. Members from the heterogeneous nuclear ribonucleoprotein (hnRNP) family bind to exonic splicing silencers (ESS) and intronic splicing silencers (ISS) to repress exon inclusion [5]
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