Functional and Molecular Mitochondrial Abnormalities Associated with a C → T Transition at Position 3256 of the Human Mitochondrial Genome

Abstract

We have previously identified a mitochondrial DNA polymorphism (a C --> T transition at position 3256, within the mitochondrial tRNALeu(UUR) gene in a patient with a multisystem disorder. Although there were several indicators suggesting a pathogenetic role for this mtDNA polymorphism, its heteroplasmic nature made functional and molecular studies difficult to interpret. We have now fused enucleated fibroblasts from the patient with a mtDNA-less cell line to generate transmitochondrial cybrids harboring different proportions of mutated and wild-type mtDNA. Individual clones harboring essentially 100% wild-type or > 99% mutated mtDNAs were characterized and studied for respiratory capacity, respiratory chain enzymes activity, mitochondrial protein synthesis, and RNA steady-state levels and processing. Our results showed that cell lines containing exclusively mutated mtDNAs respire poorly, overproduce lactic acid, and have significantly impaired activity of respiratory complexes I and IV. Molecular studies showed that mutant clones have a decrease in steady-state levels of mitochondrial tRNALeu(UUR), and a partial impairment of mitochondrial protein synthesis and steady-state levels, suggesting that these molecular abnormalities are involved in the pathogenetic mechanism of the mtDNA 3256 mutation.

Impairment of tRNA processing by point mutations in mitochondrial tRNALeu(UUR) associated with mitochondrial diseases

“…Accumulation of this precursor, corresponding to 16S rRNA-tRNAL~"'(utm/-ND1 mRNA and termed RNA 19 [8], was somewhat more pronounced in 3271 cybrids than in 3243 [16], well in correspondence with the difference of the in vitro processing rate (this study). Yet, a similar analysis of C to T transition 3256, which has the most striking effect on processing in vitro, showed only a very slight increase in the abundance of RNA 19, but significantly reduced levels of (mt)tRNA L''''/t~uR) and NDI mRNA [14]. It must, however, be noted that although mutant cybrid clones may approach homoplasmy, they generally still show considerable phenotypic heterogeneity [6,8,14,16].…”

“…However, when comparing an in vitro analysis of mitochondrial tRNA processing to data obtained in vivo, one is inevitably faced with the complexities of the mitochondrial genetic system: a mixture of wild-type and mutant genomes (heteroplasmy) and variable nuclear genetic background. These problems can be partially avoided by the use of transmitochondrial cybrids [6 8], and this technology has helped to establish the causal relationship of certain (mt)tRNA L''"/cUR/ mutations (3243, 3256, 3260, 3271) and the pathological mitochondrial phenotype [8][9][10][11]14].…”

“…Steady-state levels of (mt)tRNA L'''l(UUR/, as well as of the flanking 16S rRNA and NDI mRNA, have been investigated in three of these mutant cell lines (3243, 3256, 3271) [8,14,16,37,38] and in tissue samples of eight MELAS-3243 patients [39]. While no significant alterations of size and amount of these RNAs were detected in cybrids harboring the 3243 or 3271 mutation, cell lines as well as patient tissues (3243) contained increased amounts of unprocessed (mt)tRNA r-''''(rU~).…”

“…3' pre-tRNase, precursor-tRNA 3'-endonuclease; MELAS, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes; (nat), mitochondrial; mtDNA, mitochondrial DNA; mtRNase P, mitochondrial RNase P; nRNase P, nuclear RNase P; PEG, polyethylene glycol tein synthesis and consequently a decrease in oxidative phosphorylation capacity [7][8][9][10][11][12][13][14]. More specifically, in one case a decreased aminoacylation of the affected tRNA was found [15], and in two other cases the specific accumulation of the tRNA precursor [8,16].…”

Titrating the Effects of Mitochondrial Complex I Impairment in the Cell Physiology

“…Culture conditions were modified as described below under "Growth Curves." SUB21 cell line (transmitochondrial cybrid clone containing wild type mtDNA) was previously characterized (24) and used in some experiments as a human control cell line.…”