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

A previous study has identified a C --> U mutation at position -3 in t he 3' splice site of intron 10 of the phenylalanine hydroxylase pre-mR NA in a patient with phenylketonuria. In vivo, this mutation induces t he skipping of the downstream exon. This result is puzzling because bo th CAG and UAG have been reported to function equally as 3' splice sit es. In this report, we show that the C --> U mutation affects predomin antly the first step of the splicing reaction and that it blocks splic eosome assembly at an early stage. The 3' region of the phenylalanine hydroxylase intron 10 has two unusual characteristic features: multipl e potential branch sites and a series of four guanosine residues, whic h interrupt the polypyrimidine tract at positions -8 to -11 from the 3 ' splice site. We show that the mutation precludes the use of the prox imal branch site, while having no effect on the remote one. We also sh ow that in the UAG transcript, the four guanosine residues inhibit the splicing of intron 10. The substitution of these purine residues by o ne cytosine residue, regardless of the position, increases the splicin g efficiency of the mutant UAG precursor while having no effect on the wild type CAG precursor. Substituting the four purine residues by fou r pyrimidines relieves the inhibition and rescues the use of the proxi mal branch site. These results demonstrate that according to the conte xt, the C and U nucleotides preceding the AG are not equivalent for th e splicing reaction.