Published online 30 September 2005
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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
1Institute of Vegetative Physiology, University of Köln Robert-Koch-Strasse 39, 50931 Köln, Germany 2Department of Biochemistry, Medical Faculty Sarajevo, Cekalusa 90, Bosnia and Herzegovina 3Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg Im Neuenheimer Feld 364, 69120 Heidelberg, Germany 4Institute of Clinical Chemistry and Mitochondrial Genetics Kölner Platz 1, 80804 München, Germany 5Center for Molecular Medicine Cologne (CMMC), University of Köln Joseph-Stelzmann-Strasse 52, 50931 Köln, Germany 6Department of Pediatrics, University of Köln Kerpener Strasse 62, 50924 Köln, Germany
*To whom correspondence should be addressed. Tel: +49 221 478 3610; Fax: +49 221 478 3538; Email: rudolf.wiesner{at}uni-koeln.de
Received July 21, 2005. Revised September 14, 2005. Accepted September 14, 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.
Present address: Katharina Maniura-Weber, Empa, Materials Testing and Research, Lerchenfeldstrasse 5, 9014 St Gallen, Switzerland
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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