title: The deep intronic c.903+469T>C mutation in the MTRR gene creates an SF2/ASF binding exonic splicing enhancer, which leads to pseudoexon activation and causes the cblE type of homocystinuria.
abstract: Deep intronic mutations are often ignored as possible causes of human diseases. A deep intronic mutation in the MTRR gene, c.903+469T>C, is the most frequent mutation causing the cblE type of homocystinuria. It is well known to be associated with pre-mRNA mis-splicing, resulting in pseudoexon inclusion; however, the pathological mechanism remains unknown. We used minigenes to demonstrate that this mutation is the direct cause of MTRR pseudoexon inclusion, and that the pseudoexon is normally not recognized due to a suboptimal 5' splice site. Within the pseudoexon we identified an exonic splicing enhancer (ESE), which is activated by the mutation. Cotransfection and siRNA experiments showed that pseudoexon inclusion depends on the cellular amounts of SF2/ASF and in vitro RNA-binding assays showed dramatically increased SF2/ASF binding to the mutant MTRR ESE. The mutant MTRR ESE sequence is identical to an ESE of the alternatively spliced MST1R proto-oncogene, which suggests that this ESE could be frequently involved in splicing regulation. Our study conclusively demonstrates that an intronic single nucleotide change is sufficient to cause pseudoexon activation via creation of a functional ESE, which binds a specific splicing factor. We suggest that this mechanism may cause genetic disease much more frequently than previously reported.
authors.affiliation: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague 2, Czech Republic.
authors.forename: Katerina
authors.initials: K
authors.lastname: Homolova
authors.forename: Petra
authors.initials: P
authors.lastname: Zavadakova
authors.forename: Thomas Koed
authors.initials: TK
authors.lastname: Doktor
authors.forename: Lisbeth Dahl
authors.initials: LD
authors.lastname: Schroeder
authors.forename: Viktor
authors.initials: V
authors.lastname: Kozich
authors.forename: Brage S
authors.initials: BS
authors.lastname: Andresen
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D019295:Computational Biology; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D005287:Ferredoxin-NADP Reductase / Q000235:genetics*; D006712:Homocystinuria / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D000090063:Proto-Oncogene Mas; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D014805:Vitamin B 12 / Q000378:metabolism
chemical_list: C000715470:MAS1 protein, human; D050505:Mutant Proteins; D009687:Nuclear Proteins; D000090063:Proto-Oncogene Mas; D022821:RNA Splice Sites; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; C111241:methionine synthase reductase; D005287:Ferredoxin-NADP Reductase; D014805:Vitamin B 12