Mitochondrial DNA (mtDNA) mutations are an important cause of human disease for which there is no efficient treatment. Our aim was to determine whether the A8344G mitochondrial tRNA(Lys) mutation, which can cause the MERRF (myoclonic epilepsy with ragged-red fibers) syndrome, could be complemented by targeting tRNAs into mitochondria from the cytosol. Import of small RNAs into mitochondria has been demonstrated in many organisms, including protozoans, plants, fungi and animals. Although human mitochondria do not import tRNAs in vivo, we previously demonstrated that some yeast tRNA derivatives can be imported into isolated human mitochondria. We show here that yeast tRNALys derivatives expressed in immortalized human cells and in primary human fibroblasts are partially imported into mitochondria. Imported tRNAs are correctly aminoacylated and are able to participate in mitochondrial translation. In transmitochondrial cybrid cells and in patient-derived fibroblasts bearing the MERRF mutation, import of tRNALys is accompanied by a partial rescue of mitochondrial functions affected by the mutation such as mitochondrial translation, activity of respiratory complexes, electrochemical potential across the mitochondrial membrane and respiration rate. Import of a tRNALys with a mutation in the anticodon preventing recognition of the lysine codons does not lead to any rescue, whereas downregulation of the transgenic tRNAs by small interfering RNA (siRNA) transiently abolishes the functional rescue, showing that this rescue is due to the import. These findings prove for the first time the functionality of imported tRNAs in human mitochondria in vivo and highlight the potential for exploiting the RNA import pathway to treat patients with mtDNA diseases.

No abstract

From neurohaemal lobes of corpora cardiacu of Locusta migratoria a 5-kDa peptide has been isolated and its sequence established [see the accompanying paper, by Hietter et al. (1990) Eur. J . Biochem. 187, 241 -2471. We have designed oligonucleotide probes from the peptide sequence of this molecule and screened a library prepared from mRNA of the neurosecretory cell region of the brain of this insect. Several positive cDNAs were isolated, the combined nucleotide sequences of which predict a large precursor of 145 residues (15770 Da) containing the newly isolated 5-kDa peptide. The peptide is flanked by regions homologous to the A and B chains of the superfamily of insulins. The overall organization of the precursor is as follows: signal peptide/domain homologous to the B chain of insulins/C (connecting)-peptide (corresponding to the newly isolated 5-kDa peptide)/domain homologous to the A chain of insulins. The numbers and relative positions of the cysteines of the Locusta peptide are equivalent to those of the other members of the insulin superfamily and most of the hydrophobic core residues are conserved.A 5-kDa peptide has been isolated from neurohaemal lobes of the corpora cardiacu of adult females of the grasshopper Locustu migratoria and has been fully characterized, as reported in the accompanying paper [l]. Comparison with sequence data banks revealed no homology with other known peptides. We have synthesized oligonucleotide probes based on the peptide sequence and screened cDNA libraries prepared from the neurosecretory cell region of the brain of this insect. This region includes the established site of biosynthesis for neurohormones which are axonically transported to the neurohaemal lobes of the corpora cardiaca where they are stored prior to release into the blood. We report here the isolation of several cDNAs, the nucleotide sequences of which predict a large precursor molecule containing the newly isolated 5-kDa peptide. Interestingly, on either side of this peptide we find regions homologous to the A and B chains of the insulin superfamily. MATERIALS AND METHODS InsectsLocusta migratoria were reared in the gregarious phase at a day temperature of 28-30°C, falling to 25°C at night.Correspondence to M. Lagueux, Laboratoire de Biologie Genkrale, 12 rue de I'Universite, F-67000 Strasbourg, FranceAhbreviations. NSC, neurosecretory cells; LIRP, Locustu insulinrelated pcptide; p.f.u., plaque-forming unit; NaCl/Cit, 0.15 M NaCI/ 15 m M trisodium citrate (pH 7.0).Nate. The novel nucleotide sequence data published here has been deposited with the EM BL sequence data banks and is available under accession number X17024. The novel amino acid sequence data published here has been deposited with the EMBL sequence data bank .Electric bulbs inside the cages established temperature gradients up to 38 "C. The relative humidity was around 70%. Day conditions lasted from 11.00 h to 23.00 h. In these rearing conditions, the duration of the 5th larval instar is 8 days.Female adults initiate vitellogenesis around 10-12 ...

No abstract