title: A new mechanism for mtDNA pathogenesis: impairment of post-transcriptional maturation leads to severe depletion of mitochondrial tRNASer(UCN) caused by T7512C and G7497A point mutations.
abstract: We have studied the consequences of two homoplasmic, pathogenic point mutations (T7512C and G7497A) in the tRNA(Ser(UCN)) gene of mitochondrial (mt) DNA using osteosarcoma cybrids. We identified a severe reduction of tRNA(Ser(UCN)) to levels below 10% of controls for both mutations, resulting in a 40% reduction in mitochondrial protein synthesis rate and in a respiratory chain deficiency resembling that in the patients muscle. Aminoacylation was apparently unaffected. On non-denaturating northern blots we detected an altered electrophoretic mobility for G7497A containing tRNA molecules suggesting a structural impact of this mutation, which was confirmed by structural probing. By comparing in vitro transcribed molecules with native RNA in such gels, we also identified tRNA(Ser(UCN)) being present in two isoforms in vivo, probably corresponding to the nascent, unmodified transcripts co-migrating with the in vitro transcripts and a second, faster moving isoform corresponding to the mature tRNA. In cybrids containing either mutations the unmodified isoforms were severely reduced. We hypothesize that both mutations lead to an impairment of post-transcriptional modification processes, ultimately leading to a preponderance of degradation by nucleases over maturation by modifying enzymes, resulting in severely reduced tRNA(Ser(UCN)) steady state levels. We infer that an increased degradation rate, caused by disturbance of tRNA maturation and, in the case of the G7497A mutant, alteration of tRNA structure, is a new pathogenic mechanism of mt tRNA point mutations.
authors.affiliation: Institute of Vegetative Physiology, University of Köln, Robert-Koch-Strasse 39, 50931 Köln, Germany.
authors.forename: Myriam
authors.initials: M
authors.lastname: Möllers
authors.forename: Katharina
authors.initials: K
authors.lastname: Maniura-Weber
authors.forename: Emina
authors.initials: E
authors.lastname: Kiseljakovic
authors.forename: Maria
authors.initials: M
authors.lastname: Bust
authors.forename: Armine
authors.initials: A
authors.lastname: Hayrapetyan
authors.forename: Michaela
authors.initials: M
authors.lastname: Jaksch
authors.forename: Mark
authors.initials: M
authors.lastname: Helm
authors.forename: Rudolf J
authors.initials: RJ
authors.lastname: Wiesner
authors.forename: Jürgen-Christoph
authors.initials: JC
authors.lastname: von Kleist-Retzow
mesh_terms: D049148:Aminoacylation; D001483:Base Sequence; D002460:Cell Line; D002648:Child; D002675:Child, Preschool; D004272:DNA, Mitochondrial / Q000235:genetics*; D042967:Electron Transport Complex I / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D006801:Humans; D008297:Male; D028361:Mitochondrial Diseases / Q000235:genetics* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D017354:Point Mutation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D020871:RNA Stability; D000077278:RNA, Mitochondrial; D012362:RNA, Transfer, Ser / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004272:DNA, Mitochondrial; D024101:Mitochondrial Proteins; D012322:RNA Precursors; D000077278:RNA, Mitochondrial; D012362:RNA, Transfer, Ser; D012313:RNA; D003576:Electron Transport Complex IV; D042967:Electron Transport Complex I