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.
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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)