Redefinition of exon 7 in the COL1A1 gene of type I collagen by an intron 8 splice-donor-site mutation in a form of osteogenesis imperfecta: influence of intron splice order on outcome of splice-site mutation.
Most splice-site mutations lead to a limited array of products, including exon skipping, use of cryptic splice-acceptor or -donor sites, and intron inclusion. At the intron 8 splice-donor site of the COL1A1 gene, we identified a G+1-->A transition that resulted in the production of several splice products from the mutant allele. These included one in which the upstream exon 7 was extended by 96 nt, others in which either intron 8 or introns 7 and 8 were retained, one in which exon 8 was skipped, and one that used a cryptic donor site in exon 8. To determine the mechanism by which exon-7 redefinition might occur, we examined the order of intron removal in the region of the mutation by using intron/exon primer pairs to amplify regions of the precursor nuclear mRNA between exon 5 and exon 10. Removal of introns 5, 6, and 9 was rapid. Removal of intron 8 usually preceded removal of intron 7 in the normal gene, although, in a small proportion of copies, the order was reversed. The proportion of abnormal products suggested that exon 7 redefinition, intron 7 plus intron 8 inclusion, and exon 8 skipping all represented products of the impaired rapid pathway, whereas the intron-8 inclusion product resulted from use of the slow intron 7-first pathway. The very low-abundance cryptic exon 8 donor site product could have arisen from either pathway. These results suggest that there is commitment of the pre-mRNA to the two pathways, independent of the presence of the mutation, and that the order and rate of intron removal are important determinants of the outcome of splice-site mutations and may explain some unusual alterations.
Departments of Pathology and Medicine, University of Washington, Seattle, WA 98195-7470, USA.
U
U
Schwarze
B J
BJ
Starman
P H
PH
Byers
D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007223:Infant; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010013:Osteogenesis Imperfecta / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D013997:Time Factors
D012333:RNA, Messenger; D003094:Collagen