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

Nucleic Acids Research, Jan 2005

We have studied the consequences of two homoplasmic, pathogenic point mutations (T7512C and G7497A) in the tRNASer(UCN) gene of mitochondrial (mt) DNA using osteosarcoma cybrids. We identified a severe reduction of tRNASer(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 tRNASer(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 tRNASer(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.

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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

Myriam Mo llers 2 Katharina Maniura-Weber 2 Emina Kiseljakovic 1 2 Maria Bust 2 Armine Hayrapetyan 0 Michaela Jaksch 5 Mark Helm 0 Rudolf J. Wiesner 2 4 Ju rgen-Christoph von Kleist-Retzow 3 4 0 Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg , Im Neuenheimer Feld 364, 69120 Heidelberg, Germany 1 Department of Biochemistry, Medical Faculty , Sarajevo, Cekalusa 90, Bosnia and Herzegovina 2 Institute of Vegetative Physiology, University of Ko ln , Robert-Koch-Strasse 39, 50931 Ko ln, Germany 3 Department of Pediatrics, University of Ko ln , Kerpener Strasse 62, 50924 Ko ln, Germany 4 Center for Molecular Medicine Cologne (CMMC), University of Ko ln , Joseph-Stelzmann-Strasse 52, 50931 Ko ln, Germany 5 Institute of Clinical Chemistry and Mitochondrial Genetics , Ko lner Platz 1, 80804 Mu nchen, Germany We have studied the consequences of two homoplasmic, pathogenic point mutations (T7512C and G7497A) in the tRNASer(UCN) gene of mitochondrial (mt) DNA using osteosarcoma cybrids. We identified a severe reduction of tRNASer(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 tRNASer(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 posttranscriptional modification processes, ultimately leading to a preponderance of degradation by nucleases over maturation by modifying enzymes, resulting in severely reduced tRNASer(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. - Mitochondrial DNA (mtDNA) codes for a total of 37 genes and different human mtDNA alterations, including rearrangements as well as mutations in most of them have been identified as underlying various clinical diseases. In fact, point mutations are responsible for a tremendous number of different clinical phenotypes. This variability has been related to the peculiarities of mitochondrial genetics, e.g. (i) random segregation of a given mutation within the human body, (ii) heteroplasmy, i.e. the coexistence of wild-type and mutated mtDNA molecules within mitochondria, cells and tissues and (iii) threshold effects, meaning that a given mutation becomes The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors functionally relevant only if it exceeds a certain level of heteroplasmy. However, it has become clear that beyond those characteristics mtDNA point-mutations may have quite variable consequences on the genuine function of the corresponding protein, tRNA or rRNA, and several patterns of those consequences have been identified in the last years (1,2). It is tempting to speculate that these different pathomechanisms not only discriminate, whether a given base pair substitution results in either a functionally irrelevant silent polymorphism or in a potentially disease causing pointmutation, they most likely contribute as well to the quite variable clinical phenotypes of point mutations in any mitochondrial gene. This is particularly true for the 22 tRNA genes where mutations are causing more complex processes than the simple alternative between an either fully functional tRNA or a molecule which is not participating at all in the translational process. Several different pathomechanisms at different levels of cellular function have been identified in the last years: point mutations may reduce the steady state levels of the corresponding tRNA (3), may interfere with the level of aminoacylation with the corresponding amino acid (4), induce an atypical base-modification pattern (5) and may result in quantitative and/or qualitative alterations of protein synthesis and respiratory chain (RC) function (6,7). Structure, as the basis for all biochemical function, represents a common factor that potentially can be affected by any mutation, and which may have detrimental effects to any number of biochemical interactions. Thus, structural perturbation of tRNAs by pathogenic point mutations represents a potentially significant pathomechanism. Consequently, structural influence of pathogen (...truncated)


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Myriam Möllers, Katharina Maniura-Weber, Emina Kiseljakovic, Maria Bust, Armine Hayrapetyan, Michaela Jaksch, Mark Helm, Rudolf J. Wiesner, Jürgen-Christoph von Kleist-Retzow. 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, Nucleic Acids Research, 2005, pp. 5647-5658, 33/17, DOI: 10.1093/nar/gki876