In many organisms, mitochondria import nuclear DNA-encoded small RNAs. In yeast Saccharomyces cerevisiae, one out of two cytoplasmic isoacceptor tRNAs^Lys is partially addressed into the organelle. Mitochondrial targeting of this tRNA was shown to depend on interaction with the precursor of mitochondrial lysyl–tRNA synthetase, preMsk1p. However, preMsk1p alone was unable to direct tRNA targeting, suggesting the existence of additional protein factor(s). Here, we identify the glycolytic enzyme, enolase, as such a factor. We demonstrate that recombinant enolase and preMSK1p are sufficient to direct tRNA import in vitro and that depletion of enolase inhibits tRNA import in vivo. Enzymatic and tRNA targeting functions of enolase appear to be independent. Three newly characterized properties of the enolase can be related to its novel function: (1) specific affinity to the imported tRNA, (2) the ability to facilitate formation of the complex between preMsk1p and the imported tRNA, and (3) partial targeting toward the mitochondrial outer membrane. We propose a model suggesting that the cell exploits mitochondrial targeting of the enolase in order to address the tRNA toward peri-mitochondrially synthesized preMsk1p. Our results indicate an alternative molecular chaperone function of glycolytic enzyme enolase in tRNA mitochondrial targeting.

Although mitochondrial import of nuclear DNA-encoded RNAs is widely occurring, their functions in the organelles are not always understood. Mitochondrial function(s) of tRNA^Lys_CUU, tRK1, targeted into Saccharomyces cerevisiae mitochondria was mysterious, since mitochondrial DNA-encoded tRNA^Lys_UUU, tRK3, was hypothesized to decode both lysine codons, AAA and AAG. Mitochondrial targeting of tRK1 depends on the precursor of mitochondrial lysyl-tRNA synthetase, pre-Msk1p. Here we show that substitution of pre-Msk1p by its Ashbya gossypii ortholog results in a strain in which tRK3 is aminoacylated, while tRK1 is not imported. At elevated temperature, drop of tRK1 import inhibits mitochondrial translation of mRNAs containing AAG codons, which coincides with the impaired 2-thiolation of tRK3 anticodon wobble nucleotide. Restoration of tRK1 import cures the translational defect, suggesting the role of tRK1 in conditional adaptation of mitochondrial protein synthesis. In contrast with the known ways of organellar translation control, this mechanism exploits the RNA import pathway.

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.

The highly streamlined mitochondrial genome encodes almost exclusively a handful of transmembrane components of the respiratory chain complex. In order to ensure the correct assembly of the respiratory chain, the products of these genes must be produced in the correct stoichiometry and inserted into the membrane, posing a unique challenge to the mitochondrial translational system. In this review we describe the proteins orchestrating mitochondrial translation initiation: bacterial-like general initiation factors mIF2 and mIF3, as well as mitochondria-specific components - mRNA-specific translational activators and mRNA-nonspecific accessory initiation factors. We consider how the fast rate of evolution in these organelles has not only created a system that is divergent from that of its bacterial ancestors, but has led to a huge diversity in lineage specific mechanistic features of mitochondrial translation initiation among eukaryotes.

Targeting nuclear DNA-encoded tRNA is a quasi-ubiquitous process, found in a variety of species, although the mechanisms of this pathway seem to differ from one system to another. In all cases reported, this import concerns small non-coding RNAs and the vast majority of imported RNAs are transfer RNAs. If was commonly assumed that the main criterion to presume a tRNA to be imported is the absence of the corresponding gene in mitochondrial genome, in some cases the imported species seemed redundant in the organelle. By studying one of such "abnormal" situation in yeast S. cerevisiae, we discovered an original mechanism of conditional regulation of mitochondrial translation exploiting the RNA import pathway. Here, we provide an outline of the current state of RNA import in yeast and discuss the possible impact of the newly described mechanism of translational adaptation.

Aminoacyl-tRNA synthetases, together with their main function of covalent binding of an amino acid to a corresponding tRNA, also perform many other functions. They take part in regulation of gene transcription, apoptosis, translation, and RNA splicing. Some of them function as cytokines or catalyze different reactions in living cells. Noncanonical functions can be mediated by additional domains of these proteins. On the other hand, some of the noncanonical functions are directly associated with the active center of the aminoacylation reaction. In this review we summarize recent data on the noncanonical functions of aminoacyl-tRNA synthetases and on the mechanisms of their action.