Mitochondrial tRNA cleavage by tRNA-targeting ribonuclease causes mitochondrial dysfunction observed in mitochondrial disease

Mitochondrial DNA (mtDNA) is a genome possessed by mitochondria. Since reactive oxygen species (ROS) are generated during aerobic respiration in mitochondria, mtDNA is commonly exposed to the risk of DNA damage. Mitochondrial disease is caused by mitochondrial dysfunction, and mutations or deletions on mitochondrial tRNA (mt tRNA) genes are often observed in mtDNA of patients with the disease. Hence, the correlation between mt tRNA activity and mitochondrial dysfunction has been assessed. Then, cybrid cells, which are constructed by the fusion of an enucleated cell harboring altered mtDNA with a ρ⁰ cell, have long been used for the analysis due to difficulty in mtDNA manipulation. Here, we propose a new method that involves mt tRNA cleavage by a bacterial tRNA-specific ribonuclease. The ribonuclease tagged with a mitochondrial-targeting sequence (MTS) was successfully translocated to the mitochondrial matrix. Additionally, mt tRNA cleavage, which resulted in the decrease of cytochrome c oxidase (COX) activity, was observed.     

Complete Mitochondrial DNA Sequence of the Scallop <Emphasis Type="Italic">Placopecten magellanicus</Emphasis>: Evidence of Transposition Leading to an Uncharacteristically Large Mitochondrial Genome

Complete sequence determination of the mitochondrial (mt) genome of the sea scallop Placopecten magellanicus reveals a molecule radically different from that of the standard metazoan. With a minimum length of 30,680 nucleotides (nt; with one copy of a 1.4 kilobase (kb) repeat) and a maximum of 40,725 nt, it is the longest reported metazoan mitochondrial DNA (mtDNA). More than 50% of the genome is noncoding (NC), consisting of dispersed, imperfectly repeated sequences that are associated with tRNAs or tRNA-like structures. Although the genes for atp8 and two tRNAs were not discovered, the genome still has the potential for encoding 46 genes (the additional genes are all tRNAs), 9 of which encode tRNAs for methionine. The coding portions appear to be evolving at a rate consistent with other members of the pectinid clade. When the NC regions containing “dispersed repeat families” are examined in detail, we reach the conclusion that transposition involving tRNAs or tRNA-like structures is occurring and is responsible for the large size and abundance of noncoding DNA in the molecule. The rarity of enlarged mt genomes in the face of a demonstration that they can exist suggests that a small, compact organization is an actively maintained feature of metazoan mtDNA. Reviewing Editor: Gail Simmons     

Dual mode recognition of two isoacceptor tRNAs by mammalian mitochondrial seryl-tRNA synthetase

Animal mitochondrial translation systems contain two serine tRNAs, corresponding to the codons AGY (Y = U and C) and UCN (N = U, C, A, and G), each possessing an unusual secondary structure; tRNA(GCU)(Ser) (for AGY) lacks the entire D arm, whereas tRNA(UGA)(Ser) (for UCN) has an unusual cloverleaf configuration. We previously demonstrated that a single bovine mitochondrial seryl-tRNA synthetase (mt SerRS) recognizes these topologically distinct isoacceptors having no common sequence or structure. Recombinant mt SerRS clearly footprinted at the TPsiC loop of each isoacceptor, and kinetic studies revealed that mt SerRS specifically recognized the TPsiC loop sequence in each isoacceptor. However, in the case of tRNA(UGA)(Ser), TPsiC loop-D loop interaction was further required for recognition, suggesting that mt SerRS recognizes the two substrates by distinct mechanisms. mt SerRS could slightly but significantly misacylate mitochondrial tRNA(Gln), which has the same TPsiC loop sequence as tRNA(UGA)(Ser), implying that the fidelity of mitochondrial translation is maintained by kinetic discrimination of tRNAs in the network of aminoacyl-tRNA synthetases.     

Assembly of the mitochondrial membrane system. Sequences of yeast mitochondrial tRNA genes

Two cytoplasmic "petite" (rho-) clones of Saccharomyces cerevisiae have been selected for the retention of the aspartic acid tRNA gene. The two clones, designated DS200/A102 and DS200/A5, have tandemly repeated segments of mitochondrial DNA (mtDNA) with unit lengths of 1,000 and 6,400 base pairs, respectively. The DS200/A102 genome has a single tRNA gene with a 3'-CUG-5' anticodon capable of recognizing the 5'-GAC-3' and 5'-GAU-3' codons for aspartic acid. The mtDNA segment of DS200/A102 has been determined to represent the wild type sequence from 5.3 to 6.8 map units. The genome of DS200/A5 is more complex encompassing the region of wild type mtDNA from 3.5 to 12.7 units. A continuous sequence has been obtained from 3.5 to 8.6 units. In addition to the aspartic acid tRNA, this region codes for the tRNAUGCAla,tRNAUCUArg, tRNAACGArg, tRNAGCUSer,tRNAUCCGly and tRNAUUULys. The DNA sequence of the DS200/A5 genome has allowed us to deduce the secondary structures of the seven tRNAs and to assign precise map positions for their genes. All the tRNAs except tRNA GUCAsp exhibit most of the invariant features of prokaryotic and eukaryotic tRNAs. The aspartic acid tRNA has unusual D and T psi C loops. The structure of this tRNA is similar to the mitochondrial initiator tRNA of Neurospora crassa (Heckman, J.E., Hecker, L.I., Shwartzbach, S.D., Barnett, W.E., Baumstark, B., and RajBhandary, U.L. Cell 13, 83-95).     

Nucleotide sequence of a wheat mitochondrial lysine tRNA gene

We present the sequence of a wheat mitochondrial (mt) lysine tRNA gene (trnK-UUU). This gene more closely resembles its E. coli counterpart than it does the corresponding gene in fungal or mammalian mtDNA. Hybridization experiments with a trnK-specific probe suggest that at least two copies of this tRNALys gene are present in the wheat mitochondrial genome.