Thalia Salinas

Recent advances in tRNA mitochondrial import

In many eukaryotes, tRNA import from the cytosol into mitochondria is essential for mitochondrial biogenesis and, consequently, for cell viability. Recent work has begun to unravel the molecular mechanisms involved in tRNA transport in yeast, trypanosomatids and plants. The mechanisms of tRNA targeting to, and translocation through, the double mitochondrial membrane in addition to how selectivity and regulation of these processes are achieved are the main questions that have been addressed. The characterization of both direct and co-import mechanisms involving distinct protein-import factors is in agreement with a polyphyletic origin of tRNA import. Moreover, our increased understanding of the tRNA-import pathway has been exploited recently to rescue dysfunctions associated with mitochondrial tRNA mutations.

The voltage-dependent anion channel, a major component of the tRNA import machinery in plant mitochondria

In plants, as in most eukaryotic cells, import of nuclear-encoded cytosolic tRNAs is an essential process for mitochondrial biogenesis. Despite its broad occurrence, the mechanisms governing RNA transport into mitochondria are far less understood than protein import. This article demonstrates by Northwestern and gel-shift experiments that the plant mitochondrial voltage-dependent anion channel (VDAC) protein interacts with tRNA in vitro. It shows also that this porin, known to play a key role in metabolite transport, is a major component of the channel involved in the tRNA translocation step through the plant mitochondrial outer membrane, as supported by inhibition of tRNA import into isolated mitochondria by VDAC antibodies and Ruthenium red. However VDAC is not a tRNA receptor on the outer membrane. Rather, two major components from the TOM (translocase of the outer mitochondrial membrane) complex, namely TOM20 and TOM40, are important for tRNA binding at the surface of mitochondria, suggesting that they are also involved in tRNA import. Finally, we show that proteins and tRNAs are translocated into plant mitochondria by different pathways. Together, these findings identify unexpected components of the tRNA import machinery and suggest that the plant tRNA import pathway has evolved by recruiting multifunctional proteins.

Helical repeats modular proteins are major players for organelle gene expression.

Mitochondria and chloroplasts are often described as semi-autonomous organelles because they have retained a genome. They thus require fully functional gene expression machineries. Many of the required processes going all the way from transcription to translation have specificities in organelles and arose during eukaryote history. Most factors involved in these RNA maturation steps have remained elusive for a long time. The recent identification of a number of novel protein families including pentatricopeptide repeat proteins, half-a-tetratricopeptide proteins, octotricopeptide repeat proteins and mitochondrial transcription termination factors has helped to settle long-standing questions regarding organelle gene expression. In particular, their functions have been related to replication, transcription, RNA processing, RNA editing, splicing, the control of RNA turnover and translation throughout eukaryotes. These families of proteins, although evolutionary independent, seem to share a common overall architecture. For all of them, proteins contain tandem arrays of repeated motifs. Each module is composed of two to three α-helices and their succession forms a super-helix. Here, we review the features characterising these protein families, in particular, their distribution, the identified functions and mode of action and propose that they might share similar substrate recognition mechanisms.

PlantRNA, a database for tRNAs of photosynthetic eukaryotes

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.

Steady-state levels of imported tRNAs in Chlamydomonas mitochondria are correlated with both cytosolic and mitochondrial codon usages.

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.

Co-evolution of mitochondrial tRNA import and codon usage determines translational efficiency in the green alga Chlamydomonas.

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.

On the Evolution and Expression of Chlamydomonas reinhardtii Nucleus-Encoded Transfer RNA Genes

In Chlamydomonas reinhardtii, 259 tRNA genes were identified and classified into 49 tRNA isoaccepting families. By constructing phylogenetic trees, we determined the evolutionary history for each tRNA gene family. The majority of the tRNA sequences are more closely related to their plant counterparts than to animals ones. Northern experiments also permitted us to show that at least one member of each tRNA isoacceptor family is transcribed and correctly processed in vivo. A short stretch of T residues known to be a signal for termination of polymerase III transcription was found downstream of most tRNA genes. It allowed us to propose that the vast majority of the tRNA genes are expressed and to confirm that numerous tRNA genes separated by short spacers are indeed cotranscribed. Interestingly, in silico analyses and hybridization experiments show that the cellular tRNA abundance is correlated with the number of tRNA genes and is adjusted to the codon usage to optimize translation efficiency. Finally, we studied the origin of SINEs, short interspersed elements related to tRNAs, whose presence in Chlamydomonas is exceptional. Phylogenetic analysis strongly suggests that tRNAAsp-related SINEs originate from a prokaryotic-type tRNA either horizontally transferred from a bacterium or originally present in mitochondria or chloroplasts.

The pea mitochondrial nucleoside diphosphate kinase cleaves DNA and RNA

Here, we present the characterization of a plant NDPK exhibiting nuclease activity. This is the first identification of a nuclease localised in the intermembrane space of plant mitochondria. The recombinant pea NDPK3 protein cleaves not only supercoiled plasmid DNA, but also highly structured RNA molecules such as tRNAs or the 3′UTR of the atp9 mRNA suggesting that the NDPK3 nuclease activity has a structural requirement. ATP inhibits this nuclease activity, while ADP has no effect. Furthermore, studies on NDPK mutant proteins indicate that the nuclease‐ and the kinase‐mechanisms are separate.

Molecular basis for the differential interaction of plant mitochondrial VDAC proteins with tRNAs

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.

Mitochondrial Translation in Green Algae and Higher Plants

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.