Nina Entelis

Mitochondrial import of a cytoplasmic lysine-tRNA in yeast is mediated by cooperation of cytoplasmic and mitochondrial lysyl-tRNA synthetases.

Cytoplasmic tRNA(Lys)CUU is the only nuclear‐encoded tRNA of Saccharomyces cerevisiae found to be associated with mitochondria. Selective import of this tRNA into isolated organelles requires cytoplasmic factors. Here we identify two of these factors as the cytoplasmic and mitochondrial lysyl‐tRNA synthetases. The cytoplasmic enzyme is obligatory for in vitro import of the deacylated, but not of the aminoacylated tRNA. We thus infer that it is needed for aminoacylation of the tRNA, which is a prerequisite for its import. The mitochondrial synthetase, which cannot aminoacylate tRN(Lys)CUU, is required for import of both aminoacylated and deacylated forms. Its depletion leads to a total arrest of tRNA import, in vitro and in vivo. The mitochondrial lysyl‐tRNA synthetase is able to form specific and stable RNP complexes with the amino‐acylated tRNA. Furthermore, an N‐terminal truncated form of the synthetase which cannot be targeted into mitochondria is unable to direct the import of the tRNA. We therefore hypothesize that the cytosolic precursor form of the mitochondrial synthetase has a carrier function for translocation of the tRNA across the mitochondrial membranes. However, cooperation of the two synthetases is not sufficient to direct tRNA import, suggesting the need of additional factor(s).

Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18

5S rRNA is an essential component of ribosomes of all living organisms, the only known exceptions being mitochondrial ribosomes of fungi, animals, and some protists. An intriguing situation distinguishes mammalian cells: Although the mitochondrial genome contains no 5S rRNA genes, abundant import of the nuclear DNA-encoded 5S rRNA into mitochondria was reported. Neither the detailed mechanism of this pathway nor its rationale was clarified to date. In this study, we describe an elegant molecular conveyor composed of a previously identified human 5S rRNA import factor, rhodanese, and mitochondrial ribosomal protein L18, thanks to which 5S rRNA molecules can be specifically withdrawn from the cytosolic pool and redirected to mitochondria, bypassing the classic nucleolar reimport pathway. Inside mitochondria, the cytosolic 5S rRNA is shown to be associated with mitochondrial ribosomes.

RNA delivery into mitochondria

Mitochondria, though containing their own genome, import the vast majority of their macromolecular components from the cytoplasm. If the mechanisms of pre-protein import are well understood, the import of nuclear-coded RNAs into mitochondria was investigated to a much lesser extent. This targeting, if not universal, is widely spread among species. The origin and the mechanisms of RNA import seem to differ from one system to another and striking differences are observed even in closely related species. We describe data concerning the various experimental systems of studying RNA import with emphasis on the model of the yeast Saccharomyces cerevisiae, which was studied in our laboratory. We compare various requirements of RNA import into mitochondria in different species and demonstrate that this pathway can be transferred from yeast to human cells, in which tRNAs normally are not imported. We speculate on the possibility to use RNA import for biomedical purposes.

Mitochondrial Enzyme Rhodanese Is Essential for 5 S Ribosomal RNA Import into Human Mitochondria

5 S rRNA is an essential component of ribosomes. In eukaryotic cells, it is distinguished by particularly complex intracellular traffic, including nuclear export and re-import. The finding that in mammalian cells 5 S rRNA can eventually escape its usual circuit toward nascent ribosomes to get imported into mitochondria has made the scheme more complex, and it has raised questions about both the mechanism of 5 S rRNA mitochondrial targeting and its function inside the organelle. Previously, we showed that import of 5 S rRNA into mitochondria requires unknown cytosolic proteins. Here, one of them was identified as mitochondrial thiosulfate sulfurtransferase, rhodanese. Rhodanese in its misfolded form was found to possess a strong and specific 5 S rRNA binding activity, exploiting sites found earlier to function as signals of 5 S rRNA mitochondrial localization. The interaction with 5 S rRNA occurs cotranslationally and results in formation of a stable complex in which rhodanese is preserved in a compact enzymatically inactive conformation. Human 5 S rRNA in a branched Mg2+-free form, upon its interaction with misfolded rhodanese, demonstrates characteristic functional traits of Hsp40 cochaperones implicated in mitochondrial precursor protein targeting, suggesting that it may use this mechanism to ensure its own mitochondrial localization. Finally, silencing of the rhodanese gene caused not only a proportional decrease of 5 S rRNA import but also a general inhibition of mitochondrial translation, indicating the functional importance of the imported 5 S rRNA inside the organelle.

Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria

RNA import into mitochondria is a widespread phenomenon. Studied in details for yeast, protists, and plants, it still awaits thorough investigation for human cells, in which the nuclear DNA-encoded 5S rRNA is imported. Only the general requirements for this pathway have been described, whereas specific protein factors needed for 5S rRNA delivery into mitochondria and its structural determinants of import remain unknown. In this study, a systematic analysis of the possible role of human 5S rRNA structural elements in import was performed. Our experiments in vitro and in vivo show that two distinct regions of the human 5S rRNA molecule are needed for its mitochondrial targeting. One of them is located in the proximal part of the helix I and contains a conserved uncompensated G:U pair. The second and most important one is associated with the loop E-helix IV region with several noncanonical structural features. Destruction or even destabilization of these sites leads to a significant decrease of the 5S rRNA import efficiency. On the contrary, the beta-domain of the 5S rRNA was proven to be dispensable for import, and thus it can be deleted or substituted without affecting the 5S rRNA importability. This finding was used to demonstrate that the 5S rRNA can function as a vector for delivering heterologous RNA sequences into human mitochondria. 5S rRNA-based vectors containing a substitution of a part of the beta-domain by a foreign RNA sequence were shown to be much more efficiently imported in vivo than the wild-type 5S rRNA.

Correction of the consequences of mitochondrial 3243A>G mutation in the MT-TL1 gene causing the MELAS syndrome by tRNA import into mitochondria

Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, an important number have been identified in tRNA genes, including 29 in the gene MT-TL1 coding for the tRNALeu(UUR). The m.3243A>G mutation was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation was reported to reduce tRNALeu(UUR) aminoacylation and modification of its anti-codon wobble position, which results in a defective mitochondrial protein synthesis and reduced activities of respiratory chain complexes. In the present study, we have tested whether the mitochondrial targeting of recombinant tRNAs bearing the identity elements for human mitochondrial leucyl-tRNA synthetase can rescue the phenotype caused by MELAS mutation in human transmitochondrial cybrid cells. We demonstrate that nuclear expression and mitochondrial targeting of specifically designed transgenic tRNAs results in an improvement of mitochondrial translation, increased levels of mitochondrial DNA-encoded respiratory complexes subunits, and significant rescue of respiration. These findings prove the possibility to direct tRNAs with changed aminoacylation specificities into mitochondria, thus extending the potential therapeutic strategy of allotopic expression to address mitochondrial disorders.

Mitochondrial targeting of recombinant RNAs modulates the level of a heteroplasmic mutation in human mitochondrial DNA associated with Kearns Sayre Syndrome

Mitochondrial mutations, an important cause of incurable human neuromuscular diseases, are mostly heteroplasmic: mutated mitochondrial DNA is present in cells simultaneously with wild-type genomes, the pathogenic threshold being generally >70% of mutant mtDNA. We studied whether heteroplasmy level could be decreased by specifically designed oligoribonucleotides, targeted into mitochondria by the pathway delivering RNA molecules in vivo. Using mitochondrially imported RNAs as vectors, we demonstrated that oligoribonucleotides complementary to mutant mtDNA region can specifically reduce the proportion of mtDNA bearing a large deletion associated with the Kearns Sayre Syndrome in cultured transmitochondrial cybrid cells. These findings may be relevant to developing of a new tool for therapy of mtDNA associated diseases.

Specific features of 5S rRNA structure — Its interactions with macromolecules and possible functions

Small non-coding RNAs are today a topic of great interest for molecular biologists because they can be regarded as relicts of a hypothetical “RNA world” which, apparently, preceded the modern stage of organic evolution on Earth. The small molecule of 5S rRNA (∼120 nucleotides) is a component of large ribosomal subunits of all living beings (5S rRNAs are not found only in mitoribosomes of fungi and metazoans). This molecule interacts with various protein factors and 23S (28S) rRNA. This review contains the accumulated data to date concerning 5S rRNA structure, interactions with other biological macromolecules, intracellular traffic, and functions in the cell.

Mitochondrial import of a yeast cytoplasmic tRNALys: possible roles of aminoacylation and modified nucleosides in subcellular partitioning

The yeast tRNACUU 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.

Import of Nuclear Encoded RNAs into Yeast and Human Mitochondria: Experimental Approaches and Possible Biomedical Applications

Mitochondria import from the cytoplasm the vast majority of proteins and some RNAs. Although there exists extended knowledge concerning the mechanisms of protein import, the import of RNA is poorly understood. It was almost exclusively studied on the model of tRNA import, in several protozoans, plants and yeast. Mammalian mitochondria, which do not import tRNAs naturally, are hypothesized to import other small RNA molecules from the cytoplasm. We studied tRNA import in the yeast system, both in vitro and in vivo, and applied similar approaches to study 5S rRNA import into human mitochondria. Despite the obvious divergence of RNA import systems suggested for different species, we find that in yeast and human cells this pathway involves similar mechanisms exploiting cytosolic proteins to target the RNA to the organelle and requiring the integrity of pre-protein import apparatus. The import pathway might be of interest from a biomedical point of view, to target into mitochondria RNAs that could suppress pathological mutations in mitochondrial DNA. Yeast represents a good model to elaborate such a gene therapy approach. We have described here the various approaches and protocols to study RNA import into mitochondria of yeast and human cells in vitro and in vivo.