[40 Years of Studying RNA Import into Mitochondria: From Basic Mechanisms to Gene Therapy Strategies].

Mitochondria of many living species internalize nuclear DNA-encoded ribonucleic acids. The pools of imported RNA molecules, as well as fine mechanisms of these processes, are highly species-specific. To date, baker's yeast Saccharomyces cerevisiae are the best studied in this regard. Moreover, the processes of yeast RNA mitochondrial import have been the basis of modeling several gene therapy strategies aimed to palliate negative effects of pathogenic mutations in human mitochondrial DNA. In this review, we summarize our current knowledge about the molecular events taking place in course of yeast RNA import into mitochondria. Also, we describe how this process can be used for compensation of pathogenic mutations in mitochondrial genomes of humans.

Factors beyond Enolase 2 and Mitochondrial Lysyl-tRNA Synthetase Precursor Are Required for tRNA Import into Yeast Mitochondria.

In yeast, the import of tRNALys with CUU anticodon (tRK1) relies on a complex mechanism where interaction with enolase 2 (Eno2p) dictates a deep conformational change of the tRNA. This event is believed to mask the tRNA from the cytosolic translational machinery to re-direct it towards the mitochondria. Once near the mitochondrial outer membrane, the precursor of the mitochondrial lysyl-tRNA synthetase (preMsk1p) takes over enolase to carry the tRNA within the mitochondrial matrix, where it is supposed to participate in translation following correct refolding. Biochemical data presented in this report focus on the role of enolase. They show that despite the inability of Eno2p alone to form a complex with tRK1, mitochondrial import can be recapitulated in vitro using fractions of yeast extracts sharing either recombinant or endogenous yeast Eno2p as one of the main components. Taken together, our data suggest the existence of a protein complex containing Eno2p that is involved in RNA mitochondrial import.

Procedure for Purification of Recombinant preMsk1p from E. coli Determines Its Properties as a Factor of tRNA Import into Yeast Mitochondria.

Mitochondrial genomes of many eukaryotic organisms do not code for the full tRNA set necessary for organellar translation. Missing tRNA species are imported from the cytosol. In particular, one out of two cytosolic lysine tRNAs of the yeast Saccharomyces cerevisiae is partially internalized by mitochondria. The key protein factor of this process is the precursor of mitochondrial lysyl-tRNA synthetase, preMsk1p. In this work, we show that recombinant preMsk1p purified from E. coli in native conditions, when used in an in vitro tRNA import system, demonstrates some properties different from those shown by the renatured protein purified from E. coli in the denatured state. We also discuss the possible mechanistic reasons for this phenomenon.

Noncanonical functions of aminoacyl-tRNA synthetases.

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.

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 (approximately 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.

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.

Targeting of tRNA into yeast and human mitochondria: the role of anticodon nucleotides.

In vivo, yeast mitochondria import a single cytoplasmic tRNA, tRNA(CUU)Lys, while human mitochondria do not import any cytoplasmic tRNA. We have previously demonstrated that both yeast and human isolated mitochondria can specifically internalize tRNA(CUU)Lys, several of its mutant versions and some mutant versions of yeast cytosolic tRNA(UUU)Lys (not imported in vivo). Aminoacylation of tRNA(CUU)Lys by the cytoplasmic lysyl-tRNA synthetase was a prerequisite for its import. Here we are studying the influence of one-base replacements in the anticodon of tRNAs(Lys) on their aminoacylation, on binding to the precursor of the mitochondrial lysyl-tRNA synthetase (carrier protein directing the import), and on the efficiency of import into isolated yeast and human mitochondria. We show that the base U35 is the main identity element for the yeast cytoplasmic lysyl-tRNA synthetase. The single replacement that abolished import was C34G, while all the others only modulated the import efficiency. The need of aminoacylation for import and for interaction with the carrier protein was shown only for a subset of mutant versions, while the others could be recognized and internalized without aminoacylation or in misacylated forms.

5 S rRNA and tRNA import into human mitochondria. Comparison of in vitro requirements.

In vivo, human mitochondria import 5 S rRNA and do not import tRNAs from the cytoplasm. We demonstrated previously that isolated human mitochondria are able to internalize a yeast tRNA(Lys) in the presence of yeast soluble factors. Here, we describe an assay for specific uptake of 5 S rRNA by isolated human mitochondria and compare its requirements with the artificial tRNA import. The efficiency of 5 S rRNA uptake by isolated mitochondria was comparable with that found in vivo. The import was shown to depend on ATP and the transmembrane electrochemical potential and was directed by soluble proteins. Blocking the pre-protein import channel inhibited internalization of both 5 S rRNA and tRNA, which suggests this apparatus be involved in RNA uptake by the mitochondria. We show that human mitochondria can also selectively internalize several in vitro synthesized versions of yeast tRNA(Lys) as well as a transcript of the human mitochondrial tRNA(Lys). Either yeast or human soluble proteins can direct this import, suggesting that human cells possess all factors needed for such an artificial translocation. On the other hand, the efficiency of import directed by yeast or human protein factors varies significantly, depending on the tRNA version. Similarly to the yeast system, tRNA(Lys) import into human mitochondria depended on aminoacylation and on the precursor of the mitochondrial lysyl-tRNA synthetase. 5 S rRNA import was also dependent upon soluble protein(s), which were distinct from the factors providing tRNA internalization.

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.

Suppression of mutations in mitochondrial DNA by tRNAs imported from the cytoplasm.

Mitochondrial import of a cytoplasmic transfer RNA (tRNA) in yeast requires the preprotein import machinery and cytosolic factors. We investigated whether the tRNA import pathway can be used to correct respiratory deficiencies due to mutations in the mitochondrial DNA and whether this system can be transferred into human cells. We show that cytoplasmic tRNAs with altered aminoacylation identity can be specifically targeted to the mitochondria and participate in mitochondrial translation. We also show that human mitochondria, which do not normally import tRNAs, are able to internalize yeast tRNA derivatives in vitro and that this import requires an essential yeast import factor.

Functional analysis of six genes from chromosomes XIV and XV of Saccharomyces cerevisiae reveals YOR145c as an essential gene and YNL059c/ARP5 as a strain-dependent essential gene encoding nuclear proteins.

We report here basic functional analysis of strains deleted for six open reading frames (ORFs), YNL059c and YNL148c from chromosome XIV and YOR145c, YOR152c, YOR161c and YOR162c from chromosome XV of Saccharomyces cerevisiae. ORFs were replaced with the KanMX4 resistance marker using a long flanking homology PCR strategy in FY1679 and W303 diploid strains. Replacement cassettes were constructed in plasmid pUG7 and the cognate wild-type genes were recovered by gap repair. Sporulation and tetrad analysis revealed that deletion of YNL059c/ARP5 was lethal for vegetative growth in strain W303 and caused severe growth defects in strain FY1679 while YOR145c was essential for growth in both strains. Fusion of the green fluorescent protein (GFP) gene to the 3' ends of the YNL059c/ARP5 and YOR145c coding sequences created functional chimeric genes at the respective chromosomal loci. Both Arp5-GFP and Yor145-GFP localized to the nucleus, Yor145-GFP concentrating in the nucleolus. The vectors containing the deletion cassettes and the cognate wild-type genes, the oligonucleotides, and the deletant strains are available from the EUROFAN resource centre EUROSCARF (Frankfurt).

The aminoacceptor stem of the yeast tRNA(Lys) contains determinants of mitochondrial import selectivity.

Yeast tRNA(Lys)CUU is nucleus-encoded and is partially imported into the mitochondria. Another lysine isoacceptor, tRNA(Lys)SUU, is also nucleus-encoded but is not imported. These two tRNAs differ in 21 bases. We have previously localised import selectivity determinants in the anticodon arm. By in vitro import of mutant transcripts and by expression of mutant tRNA genes in vivo we show here that the first base pair (1:72) and the discriminator base 73 are also relevant to import selectivity. Replacement of bases 1:72 in tRNA(Lys)SUU by those of tRNA(Lys)CUU makes it importable with a transport efficiency similar to natural.

Structural requirements of tRNALys for its import into yeast mitochondria.

In the yeast Saccharomyces cerevisiae, one of the two cytoplasmic lysine tRNAs, tRNACUULys, is partially associated with the mitochondrial matrix. Mitochondrial import of this tRNA requires binding to the precursor of the mitochondrial lysyl-tRNA synthetase, pre-MSK, and aminoacylation by the cytoplasmic lysyl-tRNA synthetase, KRS, appears to be a prerequisite for this binding. The second lysine isoacceptor tRNAmnmLys5s2UUU [where 5-[(methylamino)-methyl]-2-thiouridine is mnm5s2U] is exclusively localized in the cytoplasm. To study import determinants within the tRNACUULys molecule, we introduced a panel of replacements in the original sequences of the imported and nonimported lysine tRNAs that correspond to domains or individual residues that differ between these two isoacceptors. The mutant transcripts were tested for import, aminoacylation, and binding to pre-MSK. Import and aminoacylation efficiencies correlate well for the majority of mutant transcripts. However, some poorly aminoacylated transcripts were rather efficiently imported. Surprisingly, these transcripts retained binding capacity to pre-MSK. In fact, all imported transcripts retained pre-MSK binding capacity but nonimported versions did not, suggesting that this binding, rather than aminoacylation, is essential for import. Substitution of the anticodon arm of tRNACUULys with that of tRNAmnmLys5s2UUU abolished import without affecting aminoacylation. A version of tRNAmnmLys5s2UUU with an anticodon CUU was efficiently imported in vitro and was also found to be imported in vivo. This implies that the anticodon arm, especially position 34, is important for recognition by the import machinery. A nicked tRNACUULys transcript is still imported but its import requires reannealing of the two tRNA moieties, which implies that tRNACUULys is imported as a folded molecule.

Analysis of a 35.6 kb region on the right arm of Saccharomyces cerevisiae chromosome XV.

We report the sequence of a 35,600 bp fragment covering the PET123 region on the right arm of chromosome XV from Saccharomyces cerevisiae. This region contains 19 possible open reading frames (ORFs) of which 16 are non-overlapping ORFs. Eight ORFs correspond to the SPP2, SMP3, PDR5, NFI1, PUP1, PET123 and MTR10 loci, described previously. Two ORFs correspond to yeast homologues of genes from other organisms: O3530 is a member of the large ribosomal subunit protein L13 family and O3560 (SME1 gene) is a 94-codon ORF and is a homologue of the mammalian SmE spliceosomal core protein. Three ORFs (O3513, O3521, O3548) present significant similarities to proteins of unknown function and three ORFs (O3510, O3536, O3545) lack homology to sequences within the databases screened.

Analysis of a 22,956 bp region on the right arm of Saccharomyces cerevisiae chromosome XV.

We present here the sequence analysis of a DNA fragment (cosmid pUOA1258) located on the right arm of chromosome XV. The 22,956 bp sequence reveals 14 open reading frames (ORFs) longer than 300 bp and the 201 bp RPS33 gene. Among the 14 large ORFs, two overlapping frames are likely to be non-expressed and one corresponds to the known GLN4 gene encoding glutaminyl-tRNA synthetase. Two ORFs, O3571 and O3620, encode putative transcriptional regulators with a Zn(2)-Cys(6) DNA binding domain characteristic of members of the GAL4 family. Among the nine remaining ORFs, five (O3568, O3575, O3590, O3615 and O3625) present significant similarity to proteins of unknown function and four (O3580, O3595, O3630 and O3635) lack homology to sequences present in the databases screened.

The yeast SME1 gene encodes the homologue of the human E core protein.

Removal of introns from pre-messenger RNA (pre-mRNA) requires small nuclear RNAs (snRNAs) packaged into stable small ribonucleoprotein particles (snRNP). These snRNPs contain specific and common proteins also called Sm proteins. Correct assembly of the snRNAs with the common proteins is an essential step for the biogenesis of snRNP particles. We have identified a new Saccharomyces serevisiae gene, SME1 whose product shows 45% identity with the E core protein of human snRNP. The Sme1p contains the evolutionary conserved residues found in all Sm proteins. Combining genetic and biochemical experiments, we show that SME1 is an essential gene required for pre-mRNA splicing, cap modification and U1, U2, U4 and U5 snRNA stability. We show also that the human E core protein complements a yeast SME1 disruption demonstrating the functional equivalence of Sme1p and the human E core protein.

Mitochondrial import of a yeast cytoplasmic tRNA (Lys): possible roles of aminoacylation and modified nucleosides in subcellular partitioning.

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.

Mechanisms of tRNA import into yeast mitochondria: an overview.

Mitochondrial import of tRNA is now considered as a quasi-universal phenomenon. In the yeast Saccharomyces cerevisiae, one of the three lysine isoacceptors, the tRNA(Lys)1 with the anticodon CUU (tRNA-K1), is encoded by the nuclear genome and distributed between the cytoplasmic (> 95%) and mitochondrial (< 5%) compartments. In vivo and in vitro import assays were developed to study the mechanisms of tRNA-K1 mitochondrial import. Transmembrane translocation of the tRNA requires the intactness of at least two of the components of the mitochondrial import machinery of pre-proteins, MOM19 and MIM44, as well as energy of ATP hydrolysis and an electrochemical potential across the inner membrane. The import of tRNA-K1 involves formation of an RNP complex on the mitochondrial outer membrane. tRNA-K1 import is also dependent upon cytosolic protein factors, one of which was identified as the precursor of the mitochondrial lysyl-tRNA synthetase (MSK). Although essential for tRNA-K1 import in vitro and in vivo, pre-MSK is however not sufficient to direct the import in vitro, which suggests the need of additional cytosolic factor(s). The tRNA can be imported in its mature form and nucleoside modification is not essential. Aminoacylation of the imported tRNA by the cytoplasmic lysyl-tRNA synthetase is a prerequisite for import. Possible mechanisms of intracellular partitioning and mitochondrial membrane translocation of tRNA-K1 are discussed.

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).