title: In vitro splicing deficiency induced by a C to T mutation at position -3 in the intron 10 acceptor site of the phenylalanine hydroxylase gene in a patient with phenylketonuria.
abstract: A previous study has identified a C-->U mutation at position -3 in the 3' splice site of intron 10 of the phenylalanine hydroxylase pre-mRNA in a patient with phenylketonuria. In vivo, this mutation induces the skipping of the downstream exon. This result is puzzling because both CAG and UAG have been reported to function equally as 3' splice sites. In this report, we show that the C-->U mutation affects predominantly the first step of the splicing reaction and that it blocks spliceosome assembly at an early stage. The 3' region of the phenylalanine hydroxylase intron 10 has two unusual characteristic features: multiple potential branch sites and a series of four guanosine residues, which interrupt the polypyrimidine tract at positions -8 to -11 from the 3' splice site. We show that the mutation precludes the use of the proximal branch site, while having no effect on the remote one. We also show that in the UAG transcript, the four guanosine residues inhibit the splicing of intron 10. The substitution of these purine residues by one cytosine residue, regardless of the position, increases the splicing efficiency of the mutant UAG precursor while having no effect on the wild-type CAG precursor. Substituting the four purine residues by four pyrimidines relieves the inhibition and rescues the use of the proximal branch site. These results demonstrate that according to the context, the C and U nucleotides preceding the AG are not equivalent for the splicing reaction.
authors.affiliation: Unité 12 INSERM, Hôpital des Enfants Malades, Paris, France.
authors.forename: J
authors.initials: J
authors.lastname: Jaruzelska
authors.forename: V
authors.initials: V
authors.lastname: Abadie
authors.forename: Y
authors.initials: Y
authors.lastname: d'Aubenton-Carafa
authors.forename: E
authors.initials: E
authors.lastname: Brody
authors.forename: A
authors.initials: A
authors.lastname: Munnich
authors.forename: J
authors.initials: J
authors.lastname: Marie
mesh_terms: D001483:Base Sequence; D003062:Codon / Q000235:genetics; D003596:Cytosine; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D010651:Phenylalanine Hydroxylase / Q000096:biosynthesis / Q000235:genetics*; D010661:Phenylketonurias / Q000201:enzymology / Q000235:genetics*; D017354:Point Mutation; D012322:RNA Precursors / Q000096:biosynthesis / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics*; D017381:Spliceosomes / Q000378:metabolism; D013941:Thymine; D014158:Transcription, Genetic
chemical_list: D003062:Codon; D012322:RNA Precursors; D003596:Cytosine; D010651:Phenylalanine Hydroxylase; D013941:Thymine