title: Molecular basis of two-exon skipping (exons 12 and 13) by c.1248+5g>a in OXCT1 gene: study on intermediates of OXCT1 transcripts in fibroblasts.
abstract: The molecular basis of simultaneous two-exon skipping induced by a splice-site mutation has yet to be completely explained. The splice donor site mutation c.1248+5g>a (IVS13) of the OXCT1 gene resulted predominantly in skipping of exons 12 and 13 in fibroblasts from a patient (GS23) with succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency. We compared heteronuclear RNA (hnRNA) intermediates between controls' and GS23's fibroblasts. Our strategy was to use RT-PCR of hnRNA to detect the presence or absence of spliced exon clusters in RNA intermediates (SECRIs) comprising sequential exons. Our initial hypothesis was that a SECRI comprising exons 12 and 13 was formed first followed by skipping of this SECRI in GS23 cells. However, such a pathway was revealed to be not a major one. Hence, we compared the intron removal of SCOT transcript between controls and GS23. In controls, intron 11 was the last intron to be spliced and the removal of intron 12 was also rather slow and occurred after the removal of intron 13 in a major pathway. However, the mutation in GS23 cells resulted in retention of intron 13, thus causing the retention of introns 12 and 11. This "splicing paralysis" may be solved by skipping the whole intron 11-exon 12-intron 12-exon 13-mutated intron 13, resulting in skipping of exons 12 and 13.
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Gifu, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Hori
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Fukao
authors.forename: Keiko
authors.initials: K
authors.lastname: Murase
authors.forename: Naomi
authors.initials: N
authors.lastname: Sakaguchi
authors.forename: Cary O
authors.initials: CO
authors.lastname: Harding
authors.forename: Naomi
authors.initials: N
authors.lastname: Kondo
mesh_terms: D000138:Acidosis / Q000235:genetics*; D015256:Coenzyme A-Transferases / Q000172:deficiency / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D005091:Exons; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007223:Infant; D007438:Introns; D007662:Ketosis / Q000235:genetics; D008297:Male; D009154:Mutation; D022821:RNA Splice Sites; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D018076:DNA, Complementary; D022821:RNA Splice Sites; D012333:RNA, Messenger; D015256:Coenzyme A-Transferases; C021460:3-ketoacid CoA-transferase