Mitochondria possess their own genome but critically depend on the import of many nuclear-encoded macromolecules to ensure its expression. Beside ~1,500 proteins, in apparently all eukaryotes, from protists to humans, some RNAs (tRNAs, 5S rRNA, miRNAs...) are partially redirected into mitochondria where they participate in gene expression processes(1). Therefore, the mitochondrial RNome represents an intricate mixture of transcriptome and importome. While studies of the mitochondrial transcriptome have now been facilitated by such techniques as RNA-seq(2,3), robust identification of nuclear-encoded transcripts imported into the organelles is still challenging since cytosolic contamination remains even after most thorough purification of mitochondria. Our laboratory is currently developing a conceptually novel experimental approach, Controlled Level of Contamination (CoLoC) which allows, by following RNase-mediated depletion dynamics of each transcript, to unequivocally distinguish between RNAs genuinely present inside mitochondria and mere contaminants. Coupled with deep sequencing (CoLoC-seq), this methodology will provide the first global view of the human mitochondrial RNA importome in diverse cell types and conditions. This knowledge will help a better understanding of nuclear-mitochondrial communication and will open ways to exploit RNA targeting pathways for manipulation of the mitochondrial genetic system and development of therapeutic approaches to currently incurable mitochondrial diseases(4).
The yeast tRNA CUU Lys is transcribed from a nuclear gene and then unequally redistributed between the cytosol (97–98%) and mitochondria (2–3%). We have optimized the conditions for its specific import into isolated mitochondria. However, only a minor fraction (about 0.5%) of the added tRNA was translocated into the organelles. An in vitro transcript, once aminoacylated, appeared to be a better import substrate than the natural tRNA which carries modified nucleosides. The tRNA is translocated across mitochondrial membranes in its aminoacylated form and remains relatively stable inside the organelle. Possible roles of aminoacylation, tRNA‐protein interactions and nucleoside modification in subcellular partitioning of the tRNA are discussed.
In the yeast Saccharomyces cerevisiae , nuclear DNA-encoded is partially imported into mitochondria. We previously found that the synthetic transcripts of yeast tRNA Lys and a number of their mutant versions could be specifically internalized by isolated yeast and human mitochondria. The mitochondrial targeting of tRNA Lys in yeast was shown to depend on the cytosolic precursor of mitochondrial lysyl-tRNA synthetase and the glycolytic enzyme enolase. Here we applied the approach of in vitro selection (SELEX) to broaden the spectrum of importable tRNA-derived molecules. We found that RNAs selected for their import into isolated yeast mitochondria have lost the potential to acquire a classical tRNA-shape. Analysis of conformational rearrangements in the importable RNAs by in-gel fluorescence resonance energy transfer (FRET) approach permitted us to suggest that protein factor binding and subsequent import require formation of an alternative structure, different from a classic L-form tRNA model. We show that in the complex with targeting protein factor, enolase 2, tRK1 adopts a particular conformation characterized by bringing together the 3′-end and the TΨC loop. This is a first evidence for implication of RNA secondary structure rearrangement in the mechanism of mitochondrial import selectivity. Based on these data, a set of small RNA molecules with significantly improved efficiency of import into yeast and human mitochondria was constructed, opening the possibility of creating a new mitochondrial vector system able to target therapeutic oligoribonucleotides into deficient human mitochondria.
In Saccharomyces cerevisiae , one of two cytosolic lysine‐tRNAs is partially imported into mitochondria. We demonstrate that three components of the ubiquitin/26S proteasome system (UPS), Rpn13p, Rpn8p and Doa1p interact with the imported tRNA and with the essential factor of its mitochondrial targeting, pre‐Msk1p. Genetic and biochemical assays demonstrate that UPS plays a dual regulatory role, since the overall inhibition of cellular proteasome activity reduces tRNA import, while specific depletion of Rpn13p or Doa1p increases it. This result suggests a functional link between UPS and tRNA mitochondrial import in yeast and indicates on the existence of negative and positive import regulators.
Defects in mitochondrial DNA often cause neuromuscular pathologies, for which no efficient therapy has yet been developed. MtDNA targeting nucleic acids might therefore be promising therapeutic candidates. Nevertheless, mitochondrial gene therapy has never been achieved because DNA molecules can not penetrate inside mitochondria in vivo. In contrast, some small non-coding RNAs are imported into mitochondrial matrix, and we recently designed mitochondrial RNA vectors that can be used to address therapeutic oligoribonucleotides into human mitochondria. Here we describe an approach of carrier-free targeting of the mitochondrially importable RNA into living human cells. For this purpose, we developed the protocol of chemical synthesis of oligoribonucleotides conjugated with cholesterol residue through cleavable covalent bonds. Conjugates containing pH-triggered hydrazone bond were stable during the cell transfection procedure and rapidly cleaved in acidic endosomal cellular compartments. RNAs conjugated to cholesterol through a hydrazone bond were characterized by efficient carrier-free cellular uptake and partial co-localization with mitochondrial network. Moreover, the imported oligoribonucleotide designed to target a pathogenic point mutation in mitochondrial DNA was able to induce a decrease in the proportion of mutant mitochondrial genomes. This newly developed approach can be useful for a carrier-free delivery of therapeutic RNA into mitochondria of living human cells.
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