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Differences in tRNA expression have been implicated in a remarkable number of biological processes. There is growing evidence that tRNA genes can play dramatically different roles depending on both expression and post-transcriptional modification, yet sequencing tRNAs to measure abundance and detect modifications remains challenging. Their secondary structure and extensive post-transcriptional modifications interfere with RNA-seq library preparation methods and have limited the utility of high-throughput sequencing technologies. Here, we combine two modifications to standard RNA-seq methods by treating with the demethylating enzyme AlkB and ligating with tRNA-specific adapters in order to sequence tRNAs from four species of flowering plants, a group that has been shown to have some of the most extensive rates of post-transcriptional tRNA modifications. This protocol has the advantage of detecting full-length tRNAs and sequence variants that can be used to infer many post-transcriptional modifications. We used the resulting data to produce a modification index of almost all unique reference tRNAs inArabidopsis thaliana, which exhibited many anciently conserved similarities with humans but also positions that appear to be “hot spots” for modifications in angiosperm tRNAs. We also found evidence based on northern blot analysis and droplet digital PCR that, even after demethylation treatment, tRNA-seq can produce highly biased estimates of absolute expression levels most likely due to biased reverse transcription. Nevertheless, the generation of full-length tRNA sequences with modification data is still promising for assessing differences in relative tRNA expression across treatments, tissues or subcellular fractions and help elucidate the functional roles of tRNA modifications.

The unicellular photosynthetic organism, Chlamydomonas reinhardtii, represents a powerful model to study mitochondrial gene expression. Here, we show that the 5′- and 3′-extremities of the eight Chlamydomonas mitochondrial mRNAs present two unusual characteristics. First, all mRNAs start primarily at the AUG initiation codon of the coding sequence which is often marked by a cluster of small RNAs. Second, unusual tails are added post-transcriptionally at the 3′-extremity of all mRNAs. The nucleotide composition of the tails is distinct from that described in any other systems and can be partitioned between A/U-rich tails, predominantly composed of Adenosine and Uridine, and C-rich tails composed mostly of Cytidine. Based on 3′ RACE experiments, 22% of mRNAs present C-rich tails, some of them composed of up to 20 consecutive Cs. Polycytidylation is specific to mitochondria and occurs primarily on mRNAs. This unprecedented post-transcriptional modification seems to be a specific feature of the Chlorophyceae class of green algae and points out the existence of novel strategies in mitochondrial gene expression.

International audience; Genome packaging by nucleosomes is a hallmark of eukaryotes. Histones and the pathways that deposit, remove, and read histone modifications are deeply conserved. Yet, we lack information regarding chromatin landscapes in extant representatives of ancestors of the main groups of eukaryotes, and our knowledge of the evolution of chromatin-related processes is limited. We used the bryophyte Marchantia polymorpha, which diverged from vascular plants circa 400 mya, to obtain a whole chromosome genome assembly and explore the chromatin landscape and three-dimensional genome organization in an early diverging land plant lineage. Based on genomic profiles of ten chromatin marks, we conclude that the relationship between active marks and gene expression is conserved across land plants. In contrast, we observed distinctive features of transposons and other repetitive sequences in Marchantia compared with flowering plants. Silenced transposons and repeats did not accumulate around centromeres. Although a large fraction of constitutive heterochromatin was marked by H3K9 methylation as in flowering plants, a significant proportion of transposons were marked by H3K27me3, which is otherwise dedicated to the transcriptional repression of protein-coding genes in flowering plants. Chromatin compartmentalization analyses of Hi-C data revealed that repressed B compartments were densely decorated with H3K27me3 but not H3K9 or DNA methylation as reported in flowering plants. We conclude that, in early plants, H3K27me3 played an essential role in heterochromatin function, suggesting an ancestral role of this mark in transposon silencing.

Caenorhabditis elegans nematodes produce and keep imprints of attractive chemosensory cues to which they are exposed early in life. These imprints enhance adult chemo-attraction to the same cues. Depending on the number of odor-exposed generations, imprinting is transiently or stably inherited. Strinkingly, we have found that early odor-exposed C. elegans produce odor-specific forms of the transfer RNAAla (UGC). Naive animals fed on these tRNAs acquire transient or stable odor-specific imprinting. The tRNAAla (UGC) controls C. elegans chemo-attractive responses through the multifunctional Elongator complex. Mutations that affect the functions of Elongator sub-units 1 or 3, either impaired chemo-attraction, or definitely abolish responses to the odors nematodes were exposed. We hypothesize that early olfactory experiences translate into tRNAAla (UGC) bearing odor-specific signatures. These diffusible odor memory, together with Elongator, can stably reprogram the C. elegans chemo-attractive behavior.

In plants, the voltage-dependent anion-selective channel (VDAC) is a major component of a pathway involved in transfer RNA (tRNA) translocation through the mitochondrial outer membrane. However, the way in which VDAC proteins interact with tRNAs is still unknown. Potato mitochondria contain two major mitochondrial VDAC proteins, VDAC34 and VDAC36. These two proteins, composed of a N-terminal α-helix and of 19 β-strands forming a β-barrel structure, share 75% sequence identity. Here, using both northwestern and gel shift experiments, we report that these two proteins interact differentially with nucleic acids. VDAC34 binds more efficiently with tRNAs or other nucleic acids than VDAC36. To further identify specific features and critical amino acids required for tRNA binding, 21 VDAC34 mutants were constructed and analyzed by northwestern. This allowed us to show that the β-barrel structure of VDAC34 and the first 50 amino acids that contain the α-helix are essential for RNA binding. Altogether the work shows that during evolution, plant mitochondrial VDAC proteins have diverged so as to interact differentially with nucleic acids, and this may reflect their involvement in various specialized biological functions.

In the expanding repertoire of small noncoding RNAs (ncRNAs), tRNA-derived RNA fragments (tRFs) have been identified in all domains of life. Their existence in plants has been already proven but no detailed analysis has been performed. Here, short tRFs of 19–26 nucleotides were retrieved from Arabidopsis thaliana small RNA libraries obtained from various tissues, plants submitted to abiotic stress or fractions immunoprecipitated with ARGONAUTE 1 (AGO1). Large differences in the tRF populations of each extract were observed. Depending on the tRNA, either tRF-5D (due to a cleavage in the D region) or tRF-3T (via a cleavage in the T region) were found and hot spots of tRNA cleavages have been identified. Interestingly, up to 25% of the tRFs originate from plastid tRNAs and we provide evidence that mitochondrial tRNAs can also be a source of tRFs. Very specific tRF-5D deriving not only from nucleus-encoded but also from plastid-encoded tRNAs are strongly enriched in AGO1 immunoprecipitates. We demonstrate that the organellar tRFs are not found within chloroplasts or mitochondria but rather accumulate outside the organelles. These observations suggest that some organellar tRFs could play regulatory functions within the plant cell and may be part of a signaling pathway.

RNA fragments deriving from tRNAs (tRFs) exist in all branches of life and the repertoire of their biological functions regularly increases. Paradoxically, their biogenesis remains unclear. The human RNase A, Angiogenin, and the yeast RNase T2, Rny1p, generate long tRFs after cleavage in the anticodon region. The production of short tRFs after cleavage in the D or T regions is still enigmatic. Here, we show that the Arabidopsis Dicer-like proteins, DCL1-4, do not play a major role in the production of tRFs. Rather, we demonstrate that the Arabidopsis RNases T2, called RNS, are key players of both long and short tRFs biogenesis. Arabidopsis RNS show specific expression profiles. In particular, RNS1 and RNS3 are mainly found in the outer tissues of senescing seeds where they are the main endoribonucleases responsible of tRNA cleavage activity for tRFs production. In plants grown under phosphate starvation conditions, the induction of RNS1 is correlated with the accumulation of specific tRFs. Beyond plants, we also provide evidence that short tRFs can be produced by the yeast Rny1p and that, in vitro, human RNase T2 is also able to generate long and short tRFs. Our data suggest an evolutionary conserved feature of these enzymes in eukaryotes.

Caenorhabditis elegansnematodes produce and maintain imprints of attractive chemosensory cues to which they are exposed early in life. Early odor-exposure increases adult chemo-attraction to the same cues. Imprinting is transiently or stably inherited, depending on the number of exposed generations.We show here that the Alanine tRNA (UGC) plays a central role in regulatingC. eleganschemo-attraction. Naive worms fed on tRNAAla(UGC) purified from odor-experienced worms, acquire odor-specific imprints.Chemo-attractive responses require the tRNA-modifying Elongator complex sub-units 1 (elpc-1) and 3 (elpc-3) genes.elpc-3deletions impair chemo-attraction, which is fully restored by wild-type tRNAAla(UGC) feeding. A stably inherited decrease of odor-specific responses ensues from early odor-exposition ofelpc-1deletion mutants.tRNAAla(UGC) may adopt various chemical forms to mediate the cross-talk between innately-programmed and environment-directed chemo-attractive behavior.

Beyond their central role in protein synthesis, transfer RNAs (tRNAs) have many other crucial functions. This includes various roles in the regulation of gene expression, stress responses, metabolic processes and priming reverse transcription. In the RNA world, tRNAs are, with ribosomal RNAs, among the most stable molecules. Nevertheless, they are not eternal. As key elements of cell function, tRNAs need to be continuously quality-controlled. Two tRNA surveillance pathways have been identified. They act on hypo-modified or mis-processed pre-tRNAs and on mature tRNAs lacking modifications. A short overview of these two pathways will be presented here. Furthermore, while the exoribonucleases acting in these pathways ultimately lead to complete tRNA degradation, numerous tRNA-derived fragments (tRFs) are present within a cell. These cleavage products of tRNAs now potentially emerge as a new class of small non-coding RNAs (sncRNAs) and are suspected to have important regulatory functions. The tRFs are evolutionarily widespread and created by cleavage at different positions by various endonucleases. Here, we review our present knowledge on the biogenesis and function of tRFs in various organisms.

Summary Intracellular sorting of mRNAs is an essential process for regulating gene expression and protein localization. Most mitochondrial proteins are nuclear-encoded and imported into the mitochondria through post-translational or co-translational processes. In the latter case, mRNAs are found to be enriched in the vicinity of mitochondria. A genome-scale analysis of mRNAs associated with mitochondria has been performed to determine plant cytosolic mRNAs targeted to the mitochondrial surface. Many messengers encoding mitochondrial proteins were found associated with mitochondria. These mRNAs correspond to particular functions and complexes, such as respiration or mitoribosomes, which indicates a coordinated control of mRNA localization within metabolic pathways. In addition, upstream AUGs in 5' untranslated regions (UTRs), which modulate the translation efficiency of downstream sequences, were found to negatively affect the association of mRNAs with mitochondria. A mutational approach coupled with in vivo mRNA visualization confirmed this observation. Moreover, this technique allowed the identification of 3'-UTRs as another essential element for mRNA localization at the mitochondrial surface. Therefore, this work offers new insights into the mechanism, function and regulation of the association of cytosolic mRNAs with plant mitochondria.

Chloroplast genetic engineering has long been recognised as a powerful technology to produce recombinant proteins. To date, however, little attention has been given to the causes of pleiotropic effects reported, in some cases, as consequence of the expression of foreign proteins in transgenic plastids. In this study, we investigated the phenotypic alterations observed in transplastomic tobacco plants accumulating the Pr55(gag) polyprotein of human immunodeficiency virus (HIV-1). The expression of Pr55(gag) at high levels in the tobacco plastome leads to a lethal phenotype of seedlings grown in soil, severe impairment of plastid development and photosynthetic activity, with chloroplasts largely resembling undeveloped proplastids. These alterations are associated to the binding of Pr55(gag) to thylakoids. During particle assembly in HIV-1 infected human cells, the binding of Pr55(gag) to a specific lipid [phosphatidylinositol-(4-5) bisphosphate] in the plasma membrane is mediated by myristoylation at the amino-terminus and the so-called highly basic region (HBR). Surprisingly, the non-myristoylated Pr55(gag) expressed in tobacco plastids was likely able, through the HBR motif, to bind to nonphosphorous glycerogalactolipids or other classes of lipids present in plastidial membranes. Although secondary consequences of disturbed chloroplast biogenesis on expression of nuclear-encoded plastid proteins cannot be ruled out, results of proteomic analyses suggest that their altered accumulation could be due to retrograde control in which chloroplasts relay their status to the nucleus for fine-tuning of gene expression.

Genetic manipulation of the unicellular green alga Chlamydomonas reinhardtii is straightforward. Nuclear genes can be interrupted by insertional mutagenesis or targeted by RNA interference whereas random or site-directed mutagenesis allows the introduction of mutations in the mitochondrial genome. This, combined with a screen that easily allows discriminating respiratory-deficient mutants, makes Chlamydomonas a model system of choice to study mitochondria biology in photosynthetic organisms. Since the first description of Chlamydomonas respiratory-deficient mutants in 1977 by random mutagenesis, many other mutants affected in mitochondrial components have been characterized. These respiratory-deficient mutants increased our knowledge on function and assembly of the respiratory enzyme complexes. More recently some of these mutants allowed the study of mitochondrial gene expression processes poorly understood in Chlamydomonas. In this review, we update the data concerning the respiratory components with a special focus on the assembly factors identified on other organisms. In addition, we make an inventory of different mitochondrial respiratory mutants that are inactivated either on mitochondrial or nuclear genes.

A variety of eukaryotes, in particular plants, do not contain the required number of tRNAs to support the translation of mitochondria-encoded genes and thus need to import tRNAs from the cytosol. This study identified two Arabidopsis (Arabidopsis thaliana) proteins, Tric1 and Tric2 (for tRNA import component), which on simultaneous inactivation by T-DNA insertion lines displayed a severely delayed and chlorotic growth phenotype and significantly reduced tRNA import capacity into isolated mitochondria. The predicted tRNA-binding domain of Tric1 and Tric2, a sterile-α-motif at the C-terminal end of the protein, was required to restore tRNA uptake ability in mitochondria of complemented plants. The purified predicted tRNA-binding domain binds the T-arm of the tRNA for alanine with conserved lysine residues required for binding. T-DNA inactivation of both Tric proteins further resulted in an increase in the in vitro rate of in organello protein synthesis, which was mediated by a reorganization of the nuclear transcriptome, in particular of genes encoding a variety of proteins required for mitochondrial gene expression at both the transcriptional and translational levels. The characterization of Tric1/2 provides mechanistic insight into the process of tRNA import into mitochondria and supports the theory that the tRNA import pathway resulted from the repurposing of a preexisting protein import apparatus.

Although transfer RNA (tRNA) has a fundamental role in cell life, little is known about tRNA gene organization and expression on a genome-wide scale in eukaryotes, particularly plants. Here, we analyse the content and distribution of tRNA genes in five flowering plants and one green alga. The tRNA gene content is homogenous in plants, and is mostly correlated with genome size. The number of tRNA pseudogenes and organellar-like tRNA genes present in nuclear genomes varies greatly from one plant species to another. These pseudogenes or organellar-like genes appear to be generated or inserted randomly during evolution. Interestingly, we identified a new family of tRNA-related short interspersed nuclear elements (SINEs) in the Populus trichocarpa nuclear genome. In higher plants, intron-containing tRNA genes are rare, and correspond to genes coding for tRNA(Tyr) and tRNA(Mete) . By contrast, in green algae, more than half of the tRNA genes contain an intron. This suggests divergent means of intron acquisition and the splicing process between green algae and land plants. Numerous tRNAs are co-transcribed in Chlamydomonas, but they are mostly transcribed as a single unit in flowering plants. The only exceptions are tRNA(Gly) -snoRNA and tRNA(Mete) -snoRNA cotranscripts in dicots and monocots, respectively. The internal or external motifs required for efficient transcription of tRNA genes by RNA polymerase III are well conserved among angiosperms. A brief analysis of the mitochondrial and plastidial tRNA gene populations is also provided.

All tRNA(His) possess an essential extra G(-1) guanosine residue at their 5' end. In eukaryotes after standard processing by RNase P, G(-1) is added by a tRNA(His) guanylyl transferase. In prokaryotes, G(-1) is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G(-1) we find here that both maturation pathways can be used. Indeed, tRNA(His) with or without a G(-1) are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNA(His) precursors at both positions G(+1) and G(-1). The G(-1) is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNA(His) without G(-1) has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNA(His) with a G(-1) are recovered. This shows that a previously unreported tRNA(His) guanylyltransferase activity is present in plant mitochondria.

The mitochondrial genome of Chlamydomonas reinhardtii only encodes three expressed tRNA genes, thus most mitochondrial tRNAs are likely imported. The sharing of tRNAs between chloroplasts and mitochondria has been speculated in this organism. We first demonstrate that no plastidial tRNA is present in mitochondria and that the mitochondrial translation mainly relies on the import of nucleus-encoded tRNA species. Then, using northern analysis, we show that the extent of mitochondrial localization for the 49 tRNA isoacceptor families encoded by the C. reinhardtii nuclear genome is highly variable. Until now the reasons for such variability were unknown. By comparing cytosolic and mitochondrial codon usage with the sub-cellular distribution of tRNAs, we provide unprecedented evidence that the steady-state level of a mitochondrial tRNA is linked not only to the frequency of the cognate codon in mitochondria but also to its frequency in the cytosol, then allowing optimal mitochondrial translation.

Organellar nuclear-encoded proteins can be mitochondrial, chloroplastic or localized in both mitochondria and chloroplasts. Most of the determinants for organellar targeting are localized in the N-terminal part of the proteins, which were therefore analyzed in Arabidopsis thaliana. The mitochondrial, chloroplastic and dual N-terminal sequences have an overall similar composition. However, Arg is rare in the first 20 residues of chloroplastic and dual sequences, and Ala is more frequent at position 2 of these two types of sequence as compared to mitochondrial sequences. According to these observations, mutations were performed in three dual targeted proteins and analyzed by in vitro import into isolated mitochondria and chloroplasts. First, experiments performed with wild-type proteins suggest that the binding of precursor proteins to mitochondria is highly efficient, whereas the import and processing steps are more efficient in chloroplasts. Moreover, different processing sites are recognized by the mitochondrial and chloroplastic processing peptidases. Second, the mutagenesis approach shows the positive role of Arg residues for enhancing mitochondrial import or processing, as expected by the in silico analysis. By contrast, mutations at position 2 have dramatic and unpredicted effects, either enhancing or completely abolishing import. This suggests that the nature of the second amino acid residue of the N-terminal sequence is essential for the import of dual targeted sequences.

During evolution, most of the ancestral genes from the endosymbiotic α-proteobacteria at the origin of mitochondria have been either lost or transferred to the nuclear genome. To allow the comeback of proteins and RNAs [in particular transfer RNA (tRNAs)] into the organelle, macromolecule import systems were universally established. While protein import processes have been studied into details, much less is known about tRNA mitochondrial import. In plants, part of the knowledge on the tRNA import process into mitochondria has been acquired thanks to in vitro import assays. Furthermore, the development of in vitro RNA import strategies allowed the study of plant mitochondrial gene expression. The purpose of this chapter is to provide detailed protocols to perform in vitro RNA uptake into potato (Solanum tuberosum) or Arabidopsis (Arabidopsis thaliana) mitochondria as well as approaches to analyze them.

During evolution, most of the ancestral genes from the endosymbiotic α-proteobacteria at the origin of mitochondria have been either lost or transferred to the nuclear genome. To allow the comeback of proteins and RNAs [in particular transfer RNA (tRNAs)] into the organelle, macromolecule import systems were universally established. While protein import processes have been studied into details, much less is known about tRNA mitochondrial import. In plants, part of the knowledge on the tRNA import process into mitochondria has been acquired thanks to in vitro import assays. Furthermore, the development of in vitro RNA import strategies allowed the study of plant mitochondrial gene expression. The purpose of this chapter is to provide detailed protocols to perform in vitro RNA uptake into potato (Solanum tuberosum) or Arabidopsis (Arabidopsis thaliana) mitochondria as well as approaches to analyze them.

International audience; A faithful expression of the mitochondrial DNA is crucial for cell survival. Animal mitochondrial DNA (mtDNA) presents a highly compact gene organization. The typical 16.5 kbp animal mtDNA encodes 13 proteins, 2 rRNAs and 22 tRNAs. In the backyard pillbug Armadillidium vulgare, the rather small 13.9 kbp mtDNA encodes the same set of proteins and rRNAs as compared to animal kingdom mtDNA, but seems to harbor an incomplete set of tRNA genes. Here, we first confirm the expression of 13 tRNA genes in this mtDNA. Then we show the extensive repair of a truncated tRNA, the expression of tRNA involved in large gene overlaps and of tRNA genes partially or fully integrated within protein-coding genes in either direct or opposite orientation. Under selective pressure, overlaps between genes have been likely favored for strong genome size reduction. Our study underlines the existence of unknown biochemical mechanisms for the complete gene expression of A. vulgare mtDNA, and of co-evolutionary processes to keep overlapping genes functional in a compacted mitochondrial genome.

In higher plant mitochondria, post-transcriptional C to U conversion known as editing mostly affects mRNAs. However, three tRNAs were also shown to be edited. Among them, three editing sites were identified in larch mitochondrial tRNAHis. We have previously shown that only the edited version can undergo maturation in vitro. In this paper, we introduced via direct DNA uptake the edited or unedited version of larch mitochondrial trnH into isolated potato mitochondria and expressed them under the control of potato mitochondrial 18 S rRNA promoter. As expected, the edited form of larch mitochondrial tRNAHis precursor was processed in the isolated organelles. By contrast, no mature tRNAHis was detected when using the unedited version of trnH. However, precursor molecules could be characterized by reverse transcription-PCR. These data demonstrate that the potato mitochondrial editing machinery is not able to recognize these “foreign” editing sites and confirm that these unedited tRNA precursor molecules are not correctly processed in organello. As a consequence, the fate of these RNA precursor molecules is likely to be degradation. Indeed, we detected by PCR two 3′-end truncated precursor RNAs. Interestingly, both RNA species exhibit poly(A) tails, a hallmark of degradation in plant mitochondria. Taken together, these data suggest that, in plant mitochondria, a defective unedited RNA precursor that cannot be processed to give a mature stable tRNA, is degraded through a polyadenylation-dependent pathway.

Import of tRNAs into plant mitochondria appears to be highly specific. We recently showed that the anticodon and the D-domain sequences are essential determinants for tRNAVal import into tobacco cell mitochondria. To determine the minimal set of elements required to direct import of a cytosol-specific tRNA species, tobacco cells were transformed with an Arabidopsis thaliana intron-containing tRNAMet-e gene carrying the D-domain and the anticodon of a valine tRNA. Although well expressed and processed into tobacco cells, this mutated tRNA was shown to remain in the cytosol. Furthermore, a mutant tRNAVal carrying the T-domain of the tRNAMet-e, although still efficiently recognized by the valyl-tRNA synthetase, is not imported into mitochondria. Altogether these results suggest that mutations affecting the core of a tRNA molecule also alter its import ability into plant mitochondria.

In higher plants, one-third to one-half of the mitochondrial tRNAs are encoded in the nucleus and are imported into mitochondria. This process appears to be highly specific for some tRNAs, but the factors that interact with tRNAs before and/or during import, as well as the signals present on the tRNAs, still need to be identified. The rare experiments performed so far suggest that, besides the probable implication of aminoacyl-tRNA synthetases, at least one additional import factor and/or structural features shared by imported tRNAs must be involved in plant mitochondrial tRNA import. To look for determinants that direct tRNA import into higher plant mitochondria, we have transformed BY2 tobacco cells with Arabidopsis thaliana cytosolic tRNA(Val)(AAC) carrying various mutations. The nucleotide replacements introduced in this naturally imported tRNA correspond to the anticodon and/or D-domain of the non-imported cytosolic tRNA(Met-e). Unlike the wild-type tRNA(Val)(AAC), a mutant tRNA(Val) carrying a methionine CAU anticodon that switches the aminoacylation of this tRNA from valine to methionine is not present in the mitochondrial fraction. Furthermore, mutant tRNAs(Val) carrying the D-domain of the tRNA(Met-e), although still efficiently recognized by the valyl-tRNA synthetase, are not imported any more into mitochondria. These data demonstrate that in plants, besides identity elements required for the recognition by the cognate aminoacyl-tRNA synthetase, tRNA molecules contain other determinants that are essential for mitochondrial import selectivity. Indeed, this suggests that the tRNA import mechanism occurring in plant mitochondria may be different from what has been described so far in yeast or in protozoa.

Expression of higher plant mitochondrial (mt) genes is regulated at the transcriptional, posttranscriptional, and translational levels, but the vast majority of the mtDNA and RNA-binding proteins involved remain to be identified. Plant mt single-stranded nucleic acid-binding proteins were purified by affinity chromatography, and corresponding genes have been identified. A majority of these proteins belong to a family of RNA-binding proteins characterized by the presence of an N-terminal RNA-recognition motif (RRM) sequence. They diverge in their C-terminal sequences, suggesting that they can be involved in different plant mt regulation processes. Mitochondrial localization of the proteins was confirmed both in vitro and in vivo and by immunolocalization. Binding experiments showed that several proteins have a preference for poly(U)-rich sequences. This mt protein family contains the ubiquitous RRM motif and has no known mt counterpart in non-plant species. Phylogenetic and functional analysis suggest a common ancestor with RNA-binding glycine-rich proteins (GRP), a family of developmentally regulated proteins of unknown function. As with several plant, cyanobacteria, and animal proteins that have similar structures, the expression of one of the Arabidopsis thaliana mt RNA-binding protein genes is induced by low temperatures.

International audience; Intracellular targeting of mRNAs has recently emerged as a prevalent mechanism to control protein localization. For mitochondria, a cotranslational model of protein import is now proposed in parallel to the conventional posttranslational model, and mitochondrial targeting of mRNAs has been demonstrated in various organisms. Voltage-dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane and the major transport pathway for numerous metabolites. Four nucleus-encoded VDACs have been identified in Arabidopsis thaliana. Alternative cleavage and polyadenylation generate two VDAC3 mRNA isoforms differing by their 3' UTR. By using quantitative RT-PCR and in vivo mRNA visualization approaches, the two mRNA variants were shown differentially associated with mitochondria. The longest mRNA presents a 3' extension named alternative UTR (aUTR) that is necessary and sufficient to target VDAC3 mRNA to the mitochondrial surface. Moreover, aUTR is sufficient for the mitochondrial targeting of a reporter transcript, and can be used as a tool to target an unrelated mRNA to the mitochondrial surface. Finally, VDAC3-aUTR mRNA variant impacts mitochondria morphology and size, demonstrating the role of mRNA targeting in mitochondria biogenesis.

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Mitochondria contain hundreds of proteins but only a few are encoded by the mitochondrial genome. The other proteins are nuclear-encoded and imported into mitochondria. These proteins can be translated on free cytosolic polysomes, then targeted and imported into mitochondria. Nonetheless, numerous cytosolic mRNAs encoding mitochondrial proteins are detected at the surface of mitochondria in yeast, plants and animals. The localization of mRNAs to the vicinity of mitochondria would be a way for mitochondrial protein sorting. The mechanisms responsible for mRNA targeting to mitochondria are not clearly identified. Sequences within the mRNA molecules (cis-elements), as well as a few trans-acting factors, have been shown to be essential for targeting of some mRNAs. In order to identify receptors involved in mRNA docking to the mitochondrial surface, we have developed an in vitro mRNA binding assay with isolated plant mitochondria. We show that naked mRNAs are able to bind to isolated mitochondria, and our results strongly suggest that mRNA docking to the plant mitochondrial outer membrane requires at least one component of TOM complex.

Editing in plant mitochondria consists in C to U changes and mainly affects messenger RNAs, thus providing the correct genetic information for the biosynthesis of mitochondrial (mt) proteins. But editing can also affect some of the plant mt tRNAs encoded by the mt genome. In dicots, a C to U editing event corrects a C:A mismatch into a U:A base-pair in the acceptor stem of mt tRNAPhe (GAA). In larch mitochondria, three C to U editing events restore U:A base-pairs in the acceptor stem, D stem and anticodon stem, respectively, of mt tRNAHis (GUG). For both these mt tRNAs editing of the precursors is a prerequisite for their processing into mature tRNAs. In potato mt tRNACys (GCA), editing converts a C28:U42 mismatch in the anticodon stem into a U28:U42 non-canonical base-pair, and reverse transcriptase minisequencing has shown that the mature mt tRNACys is fully edited. In the bryophyte Marchantia polymorpha this U residue is encoded in the mt genome and evolutionary studies suggest that restoration of the U28 residue is necessary when it is not encoded in the gene. However, in vitro studies have shown that neither processing of the precursor nor aminoacylation of tRNACys requires C to U editing at this position. But sequencing of the purified mt tRNACys has shown that psi is present at position 28, indicating that C to U editing is a prerequisite for the subsequent isomerization of U into psi at position 28.

In plant mitochondria, editing of messenger RNA by C-to-U conversions is essential for correct gene expression as it usually improves the protein-sequence conservation between different species or sometimes affects the reading frames (for a review, see Maier et al+, 1996)+ Editing sites have been identified in mitochondrial (mt) RNA of all major groups of land plants, including Bryophytes, Pteridophytes, Prespermaphytes, and Spermaphytes (Hiesel et al+, 1994a,b;Malek et al+, 1996)+ Editing mainly affects messenger RNA, but editing sites have also been identified in three transfer RNAs+ In dicot mitochondria a C-to-U editing event corrects a C:A mismatch into a U:A base pair in the acceptor stem of tRNAPhe(GAA) (Marechal-Drouard et al+, 1993; Binder et al+, 1994)+ In the gymnosperm Larix leptoeuropaea, three C-to-U conversions restore a U:A base pair in the acceptor stem, D stem, and anticodon stem of tRNAHis(GUG), respectively (Marechal-Drouard et al+, 1996b)+ The third example described is the Oenothera berteriana mt tRNACys(GCA), where a C28:U42 mismatch is converted into a U28:U42 noncanonical base pair (Binder et al+, 1994)+ In the case of both tRNAPhe and tRNAHis, editing of precursors is a prerequisite for 59 and 39 processing to generate a mature tRNA (Marchfelder et al+, 1996; Marechal-Drouard et al+, 1996a, 1996b; Kunzmann et al+, 1998)+ The role of editing in the case of tRNACys has not been studied so far, although it has been shown that it occurs at the precursor level (Binder et al+, 1994)+ In this letter, we report an evolutionary and functional study of mt tRNACys(GCA) editing in plant mitochondria+ The cloverleaf structure of the mt tRNACys(GCA) deduced from the sequence of the single Solanum tuberosum mt trnC gene (EMBL Accession Number X93575) is identical to its counterpart in O. berteriana and reveals a weak anticodon stem with a U27:G43 noncanonical interaction, and a C28:U42 mismatch+ By analyzing RT-PCR amplified cDNAs of S. tuberosum mt tRNACys precursors (362 nt in length), we found that 7 out of 11 independent clones contained a T at position 28+ The ratio of edited versus nonedited mature tRNACys was determined by RT-mini-sequencing+ When total S. tuberosum mt tRNAs were used as template, only dATP was incorporated, demonstrating that the mature tRNACys is fully edited in vivo (Fig+ 1B)+ From an evolutionary point of view, the comparison of the S. tuberosum mt trnC gene with its counterpart in Marchantia polymorpha shows in particular two differences in the anticodon stem (Fig+ 1A)+ In M. polymorpha, an A residue at position 43 allows a T27:A43 base pairing, and a T residue is present at position 28+ Considering that this sequence is more closely related to the ancestral sequence, we postulated that the C-to-U editing site found in dicot mitochondria restores this ancestral sequence+ To confirm this hypothesis, we first tried to determine when, during the evolution of land plants, the mt trnC gene acquired a C at position 28 and when the C28-to-U28 editing event occurred+ To do so, the internal sequence of trnC (from position 25 to 52) was PCR-amplified, cloned, and sequenced in several species that belong to different groups of land plants+ A single difference could be observed in this region between the different plants tested: a T residue was present at position 28 of mt trnC in the Pteridophyte Pteris nephrolepis (Filicales order) and in the Reprint requests to: Laurence Marechal-Drouard, Institut de Biologie Moleculaire des Plantes, Centre National de la Recherche Scientifique, Universite Louis Pasteur, 12 rue du General Zimmer, F-67084 Strasbourg Cedex, France; e-mail: laurence+drouard@ibmpulp+u-strasbg+fr 2Present address: Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853-1801, USA 3Present address: Department of Molecular Biophysics and Biochemistry, Yale University, School of Medicine, New Haven, Connecticut 06520-8024, USA RNA (2000), 6:470–474+ Cambridge University Press+ Printed in the USA+ Copyright © 2000 RNA Society+

Two copies of the chloroplast-like tRNA(Asn) gene, trnN1 and trnN2, are expressed in potato mitochondria. While Northern-blot analysis revealed only mature tRNA(Asn), RT-PCR indicated that trnN1 is co-transcribed with trnY and nad2 (exons c, d and e). Using primer-extension and capping experiments, four transcription initiation sites have been mapped in the vicinity of these genes. The first site, responsible for the co-transcription of trnN1, trnY and nad2 (exons c, d and e), gives rise to a primary transcript of at least 7000 nt. A second site, 58 nt downstream from trnY, corresponds to an alternative promoter specific for nad2. In both cases, only the CRTA core motif of the consensus CRTAaGaGA of dicot mitochondrial promoters was found. Finally, two transcription initiation sites were identified 135 and 128 nt upstream of trnN2 in a region which shows no sequence homology with this consensus motif.

The translation systems of plant mitochondria differ from those of other mitochondria in that they incorporate tRNAs of three different origins: native mitochondrial tRNAs, plastid tRNAs transcribed from plastid DNA insertions in mitochondrial DNA, and nuclearly encoded imported tRNAs. The complicated evolutionary history of the tRNA replacement events leading up to this situation is slowly being unraveled. Recent research on plant aminoacyl-tRNA synthetases is starting to reveal how the mitochondrial compartment can cope with this unusual mix of tRNAs and has uncovered an unprecedented degree of sharing of isoforms between compartments. Many plant aminoacyl-tRNA synthetases are dual targeted to two compartments, either cytosol/mitochondria or plastids/mitochondria. The molecular basis for some of these cases of dual targeting are described.

In potato ( Solanum tuberosum ) mitochondria, about two-thirds of the tRNAs are encoded by the mitochondrial genome and one-third is imported from the cytosol. In the case of tRNAGly isoacceptors, a mitochondrial-encoded tRNAGly(GCC) was found in potato mitochondria, but this is likely to be insufficient to decode the four GGN glycine codons. In this work, we identified a cytosolic tRNAGly(UCC), which was found to be present in S.tuberosum mitochondria. The cytosolic tRNAGly(CCC) was also present in mitochondria, but to a lesser extent. By contrast, the cytosolic tRNAGly(GCC) could not be detected in mitochondria. This selective import of tRNAGly isoacceptors into S. tuberosum mitochondria raises further questions about the mechanism under-lying the specificity of the import process.

We have analyzed 67 sequences surrounding transcription initiation sites identified in higher plant mitochondria. The sequences were classified, independently for monocots and dicots, according to the presence of the CRTA core element found upstream of the first transcribed nucleotide and previously reported as an essential element of plant mitochondrial consensus promoters. This compilation provides new elements concerning the structure of consensus promoters and the relative importance of non-conserved promoters in plant mitochondria. It can be emphasized that promoter regions exhibit several differences between monocot and dicot mitochondria, presumably reflecting a divergent evolution: The sequences classified among consensus promoters as well as the distance between the first transcribed nucleotide and the core element are highly conserved in dicots while more plasticity is observed in monocots. It also appears that the proportion of promoters with neither the conserved promoter sequence nor any conserved motif is far greater in dicots than in monocots.

Bean nuclear genes for tRNAPro, tRNAThrand tRNALeu were isolated. Expression of the tRNAPro genes was demonstrated in vivo and sequence analysis suggested amplification of the tRNAPro gene copy number through duplication of a gene cluster at the same locus of the bean genome. The two tRNAThr genes isolated were actively transcribed and their transcripts processed in a HeLa cell system. In vivo expression tests of these genes and aminoacylation assays of the corresponding in vitro transcripts showed the presence of identity determinants in the anticodon of plant tRNAThr. The tRNALeu gene was not expressed due to deviation from the consensus in the internal B-box promoter. The same sequence deviation also prevented aminoacylation of the corresponding in vitro transcript. This tRNALeu however exists in plants and is synthesized from another gene with a consensus B-box promoter. Plant mitochondria import from the cytosol a number of nucleus-encoded tRNAs, including tRNALeu and tRNAThr. From the available sequence data, we could not identify any conserved structural motif characteristic for the nucleus-encoded tRNAs imported into plant mitochondria, either in the tRNAs, or in the gene flanking sequences. These results suggest that recognition of tRNAs for import is idiosyncratic and likely to depend on protein/RNA interactions that are specific to each tRNA or each isoacceptor group.

Mitochondria originate from the α-proteobacterial domain of life. Since this unique event occurred, mitochondrial genomes of protozoans, fungi, plants and metazoans have highly derived and diverged away from the common ancestral DNA. These resulting genomes highly differ from one another, but all present-day mitochondrial DNAs have a very reduced coding capacity. Strikingly however, ATP production coupled to electron transport and translation of mitochondrial proteins are the two common functions retained in all mitochondrial DNAs. Paradoxically, most components essential for these two functions are now expressed from nuclear genes. Understanding how mitochondrial translation evolved in various eukaryotic models is essential to acquire new knowledge of mitochondrial genome expression. In this review, we provide a thorough analysis of the idiosyncrasies of mitochondrial translation as they occur between organisms. We address this by looking at mitochondrial codon usage and tRNA content. Then, we look at the aminoacyl-tRNA-forming enzymes in terms of peculiarities, dual origin, and alternate function(s). Finally we give examples of the atypical structural properties of mitochondrial tRNAs found in some organisms and the resulting adaptive tRNA-protein partnership.

International audience; PlantRNA database (http://plantrna.ibmp.cnrs.fr/) compiles transfer RNA (tRNA) gene sequences retrieved from fully annotated plant nuclear, plastidial and mitochondrial genomes. The set of annotated tRNA gene sequences has been manually curated for maximum quality and confidence. The novelty of this database resides in the inclusion of biological information relevant to the function of all the tRNAs entered in the library. This includes 5'- and 3'-flanking sequences, A and B box sequences, region of transcription initiation and poly(T) transcription termination stretches, tRNA intron sequences, aminoacyl-tRNA synthetases and enzymes responsible for tRNA maturation and modification. Finally, data on mitochondrial import of nuclear-encoded tRNAs as well as the bibliome for the respective tRNAs and tRNA-binding proteins are also included. The current annotation concerns complete genomes from 11 organisms: five flowering plants (Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Medicago truncatula and Brachypodium distachyon), a moss (Physcomitrella patens), two green algae (Chlamydomonas reinhardtii and Ostreococcus tauri), one glaucophyte (Cyanophora paradoxa), one brown alga (Ectocarpus siliculosus) and a pennate diatom (Phaeodactylum tricornutum). The database will be regularly updated and implemented with new plant genome annotations so as to provide extensive information on tRNA biology to the research community.

This review focuses on the mitochondrial translation system of higher plants and algae. A few mitochondrial mRNAs have to be expressed in the mitochondrion, and a functional translational machinery is required. With the exception of some ribosomal proteins, ribosomal RNAs and part of the transfer RNA population, all the other components are nucleus-encoded and depend on numerous macromolecular trafficking processes. The presence of a second endosymbiotic organelle within the plant cell, i.e., the chloroplast increases the complexity of the mitochondrial translation machinery by having several important repercussions on the origin as well as on the targeting of the mitochondrial translation components. As an illustration of this complexity, our present knowledge on the mitochondrial aminoacyl-tRNA synthetase (aaRS) population and the mitochondrial transfer RNA (tRNA) population in both higher plants and in green algae, are summarized. Concerning the translation process by itself little is known. The existence of cis- and trans-acting factors and the emergence of novel family proteins such as the PentatricoPeptide Repeat (PPR) proteins either as direct components of the ribosome or implicated in the regulation of mitochondrial translation is also tackled.

Prasinoviruses are among the largest viruses (>200 kbp) and encode several hundreds of protein coding genes, including most genes of the DNA replication machinery and several genes involved in transcription and translation, as well as tRNAs. They can infect and lyse small eukaryotic planktonic marine green algae, thereby affecting global algal population dynamics. Here we investigate the causes of codon usage bias in one prasinovirus, OtV5, and its host Ostreococcus tauri, during a viral infection using microarray expression data. We show that (i) codon usage bias in the host and in the viral genes increases with expression levels and (ii) optimal codons use those tRNAs encoded by the most abundant host tRNA genes, supporting the notion of translational optimization by natural selection. We find evidence that viral tRNA genes complement the host tRNA pool for those viral amino acids whose host tRNAs are in short supply. We further discuss the coevolution of Codon usage bias in hosts and prasinoviruses by comparing optimal codons in 3 evolutionary Diverged host--‐virus specific pairs whose complete genome sequences are known.

The expression of two mitochondrial gene clusters (orf87-nad3-nad1/A and orf87-nad3-rps12) was studied in Nicotiana sylvestris. 5' and 3' termini of transcripts were mapped by primer extension and nuclease S1 protection. Processing and transcription initiation sites were differentiated by in vitro phosphorylation and capping experiments. A transcription initiation site, present in both gene clusters, was found 213 nucleotides upstream of orf87. This promoter element matches the consensus motif for dicotyledonous mitochondrial promoters and initiates run-off transcription in a pea mitochondrial purified protein fraction. Processing sites were identified 5' of nad3, nad1/A and rps12 respectively. These results suggest that (i) the expression of the two cistrons is only controlled by one duplicated promoter element, and (ii) multiple processing events are required to produce monocistronic nad3, nad1/A and rps12 transcripts.

Larch mitochondria contain a'native'tRNAHis which is absent from angiosperms. Sequence comparisons of genomic DNA and cDNA obtained from unprocessed primary transcripts of the larch mitochondrial gene trnH encoding this tRNA revealed three nucleotide discrepancies. These three nucleotide alterations, in the acceptor stem, D stem and anticodon stem respectively, are conversions of genomic cytidines to thymidines in the cDNA (uridines in the tRNA) and thus resemble the RNA editing events observed in nearly all plant mitochondrial mRNAs. Two cases of editing affecting mitochondrial tRNAs from angiosperms have already been described, but we present here the first example of such events in a gymnosperm mitochondrial tRNA. All three editing events correct mismatched C x A base pairs which appear when folding the gene sequence into the standard cloverleaf structure, thereby improving the secondary structure of the tRNA. When incubated with a heterologous potato mitochondrial processing extract, only the edited form of the larch mitochondrial tRNAHis precursor was efficiently processed in vitro. These data strongly suggest that editing of larch mitochondrial tRNAHis is a prerequisite for its processing.

In order to identify the sequences promoting the expression of plant mitochondrial tRNA genes, we have characterized the trnS (GCU), trnF (GAA) and trnP (UGG) transcription unit of the potato mitochondrial genome. These three tRNA genes were shown to be co-transcribed as a 1800 nt long primary transcript. The transcription initiation site located 305 to 312 nt upstream of trnS is surrounded by a purine-rich region but does not contain the consensus motif proposed as a promoter element in dicotyledonous plants. Differential labelling of potato mitochondrial RNA with either guanylyltransferase or T4 polynucleotide kinase suggests that this site corresponds to the unique functional region responsible for the transcription of these three tRNA genes. The initiation site recently found upstream of Oenothera mitochondrial trnF does not seem to be used in potato mitochondria, although a very similar sequence is present 317 nt upstream of the corresponding potato gene. Major processing sites were identified at the 3′ end of each tRNA gene. Another processing site, surrounded by a double hairpin structure, is located 498 nt downstream of trnP in stretch of 10 A residues. As judged from northern experiments, this region is close to the determination site of this transcription unit.

A protocol for the rapid isolation of polysaccharide-free DNA without phenol extraction is described. Total nucleic acids are first extracted according to Guillemaut and Marechal-Drouard (1992) and then bound to an ion exchanger while contaminants not bound are washed away. The entire procedure can be carried out in Eppendorf tubes. The highly purified DNA obtained is suitable for restriction by the common endonucleases and serves as a substrate for PCR.

Mitochondria from diverse phyla, including protozoa, fungi, higher plants, and humans, import tRNAs from the cytosol in order to ensure proper mitochondrial translation. Despite the broad occurrence of this process, our understanding of tRNA import mechanisms is fragmentary, and crucial questions about their regulation remain unanswered. In the unicellular green alga Chlamydomonas, a precise correlation was found between the mitochondrial codon usage and the nature and amount of imported tRNAs. This led to the hypothesis that tRNA import might be a dynamic process able to adapt to the mitochondrial genome content. By manipulating the Chlamydomonas mitochondrial genome, we introduced point mutations in order to modify its codon usage. We find that the codon usage modification results in reduced levels of mitochondrial translation as well as in subsequent decreased levels and activities of respiratory complexes. These effects are linked to the consequential limitations of the pool of tRNAs in mitochondria. This indicates that tRNA mitochondrial import cannot be rapidly regulated in response to a novel genetic context and thus does not appear to be a dynamic process. It rather suggests that the steady-state levels of imported tRNAs in mitochondria result from a co-evolutive adaptation between the tRNA import mechanism and the requirements of the mitochondrial translation machinery., Author Summary Mitochondria are endosymbiotic organelles involved in diverse fundamental cellular processes. They contain their own genome that encodes a few but essential proteins (e.g. proteins of the respiratory chain complexes). Their synthesis requires functional mitochondrial translational machinery that necessitates a full set of transfer RNAs (tRNAs). As mitochondrial genomes of various organisms do not code for the complete set of tRNA genes, nucleus-encoded tRNAs have to be imported into mitochondria to compensate. Mitochondrial import of tRNAs is highly specific and tailored to the mitochondrial needs. Because transformation of the mitochondrial genome is possible in Chlamydomonas, we used this green alga as model to know if a fine regulation of the tRNA import process is possible so that the tRNA population can rapidly adapt to codon usage changes in mitochondria. Here we provide evidence that the regulation of tRNA mitochondrial import process is not dynamic but is rather the result of a co-evolutive process between the import and the mitochondrial codon bias in order to optimize the mitochondrial translation efficiency.

International audience; In mammals, the Voltage-dependent anion channels (VDACs) are predominant proteins of the outer mitochondrial membrane (OMM) where they contribute to the exchange of small metabolites essential for respiration. They were shown to be as well associated with the plasma membrane (PM) and act as redox enzyme or are involved in ATP release for example. In Arabidopsis, we show that four out of six genomic sequences encode AtVDAC proteins. All four AtVDACs are ubiquitously expressed in the plant but each of them displays a specific expression pattern in root cell types. Using two complementary approaches, we demonstrate conclusively that the four expressed AtVDACs are targeted to both mitochondria and plasma membrane but in differential abundance, AtVDAC3 being the most abundant in PM, and conversely, AtVDAC4 almost exclusively associated with mitochondria. These are the first plant proteins to be shown to reside in both these two membranes. To investigate a putative function of AtVDACs, we analyzed T-DNA insertion lines in each of the corresponding genes. Knock-out mutants for AtVDAC1, AtVDAC2 and AtVDAC4 present slow growth, reduced fertility and yellow spots in leaves when atvdac3 does not show any visible difference compared to wildtype plants. Analyses of atvdac1 and atvdac4 reveal that yellow areas correspond to necrosis and the mitochondria are swollen in these two mutants. All these results suggest that, in spite of a localization in plasma membrane for three of them, AtVDAC1, AtVDAC2 and AtVDAC4 have a main function in mitochondria.

The Strasbourg University PhD school in Life and Health Sciences launched an initiative called "OpenLAB." This project was developed in an effort to help high school teenagers understand theoretical and abstract concepts in genetics. A second objective of this program is to help students in defining their future orientation and to attract them to biology. The general idea is a 2-hour PCR-based practical that is developed around a fictitious criminal investigation. The practical is taught by PhD graduate students who bring all the required reagents and modern equipment into the classroom. Running the PCR provides free time dedicated to discussions with students about their future plans after the high school diploma. A specific website and a powerpoint presentation were developed to provide appropriate scientific information. Starting on a modest scale in Strasbourg in December 2008, "OpenLAB" was rapidly and well received all around, visiting 53 classes spread over a 200 km area in Alsace until May 2009. It permitted interactions with almost one thousand students in their last year of high school, with the prospect to visit 20% more classes this school year. Our experience, along with feedback from students and their teachers, suggests that it is possible to reach out to many students and have a strong impact with a rather limited budget.

Mitochondria, owing to their bacterial origin, still contain their own DNA. However, the majority of bacterial genes were lost or transferred to the nuclear genome and the biogenesis of the "present-day" mitochondria mainly depends on the expression of the nuclear genome. Thus, most mitochondrial proteins and a small number of mitochondrial RNAs (mostly tRNAs) expressed from nuclear genes need to be imported into the organelle. During evolution, macromolecule import systems were universally established. The processes of protein mitochondrial import are very well described in the literature. By contrast, deciphering the mitochondrial RNA import phenomenon is still a real challenge. The purpose of this review is to present a general survey of our present knowledge in this field in different model organisms, protozoa, plants, yeast, and mammals. Questions still under debate and major challenges are discussed. Mitochondria are involved in numerous human diseases. The targeting of macromolecule to mitochondria represents a promising way to fight mitochondrial disorders and recent developments in this area of research are presented.