FRIENDLY (FMT) is an RNA binding protein associated with cytosolic ribosomes at the mitochondrial surface.

The spatial organization of protein synthesis in the eukaryotic cell is essential for maintaining the integrity of the proteome and the functioning of the cell. Translation on free polysomes or on ribosomes associated with the endoplasmic reticulum has been studied for a long time. More recent data have revealed selective translation of mRNAs in other compartments, in particular at the surface of mitochondria. Although these processes have been described in many organisms, particularky in plants, the mRNA targeting and localized translation mechanisms remain poorly understood. Here, the Arabidopsis thaliana Friendly (FMT) protein is shown to be a cytosolic RNA binding protein that associates with cytosolic ribosomes at the surface of mitochondria. FMT knockout delays seedling development and causes mitochondrial clustering. The mutation also disrupts the mitochondrial proteome, as well as the localization of nuclear transcripts encoding mitochondrial proteins at the surface of mitochondria. These data indicate that FMT participates in the localization of mRNAs and their translation at the surface of mitochondria.

The interactome of CLUH reveals its association to SPAG5 and its co-translational proximity to mitochondrial proteins.

BACKGROUND: Mitochondria require thousands of proteins to fulfill their essential function in energy production and other fundamental biological processes. These proteins are mostly encoded by the nuclear genome, translated in the cytoplasm before being imported into the organelle. RNA binding proteins (RBPs) are central players in the regulation of this process by affecting mRNA translation, stability, or localization. CLUH is an RBP recognizing specifically mRNAs coding for mitochondrial proteins, but its precise molecular function and interacting partners remain undiscovered in mammals. RESULTS: Here we reveal for the first time CLUH interactome in mammalian cells. Using both co-IP and BioID proximity-labeling approaches, we identify novel molecular partners interacting stably or transiently with CLUH in HCT116 cells and mouse embryonic stem cells. We reveal stable RNA-independent interactions of CLUH with itself and with SPAG5 in cytosolic granular structures. More importantly, we uncover an unexpected proximity of CLUH to mitochondrial proteins and their cognate mRNAs in the cytosol. We show that this interaction occurs during the process of active translation and is dependent on CLUH TPR domain. CONCLUSIONS: Overall, through the analysis of CLUH interactome, our study sheds a new light on CLUH molecular function by revealing new partners and by highlighting its link to the translation and subcellular localization of some mRNAs coding for mitochondrial proteins.

Plant RNases T2, but not Dicer-like proteins, are major players of tRNA-derived fragments biogenesis.

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.

Polycytidylation of mitochondrial mRNAs in Chlamydomonas reinhardtii.

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.

The nuclear and organellar tRNA-derived RNA fragment population in Arabidopsis thaliana is highly dynamic.

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.

A genome-scale analysis of mRNAs targeting to plant mitochondria: upstream AUGs in 5' untranslated regions reduce mitochondrial association.

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.

Differential targeting of VDAC3 mRNA isoforms influences mitochondria morphology.

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.

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 ß-strands forming a ß-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 ß-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.

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.

Surveillance and cleavage of eukaryotic tRNAs.

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.

Isolation of Cytosolic Ribosomes Associated with Plant Mitochondria and Chloroplasts.

Excluding the few dozen proteins encoded by the chloroplast and mitochondrial genomes, the majority of plant cell proteins are synthesized by cytosolic ribosomes. Most of these nuclear-encoded proteins are then targeted to specific cell compartments thanks to localization signals present in their amino acid sequence. These signals can be specific amino acid sequences known as transit peptides, or post-translational modifications, ability to interact with specific proteins or other more complex regulatory processes. Furthermore, in eukaryotic cells, protein synthesis can be regulated so that certain proteins are synthesized close to their destination site, thus enabling local protein synthesis in specific compartments of the cell. Previous studies have revealed that such locally translating cytosolic ribosomes are present in the vicinity of mitochondria and emerging views suggest that localized translation near chloroplasts could also occur. However, in higher plants, very little information is available on molecular mechanisms controlling these processes and there is a need to characterize cytosolic ribosomes associated with organelles membranes. To this goal, this protocol describes the purification of higher plant chloroplast and mitochondria and the organelle-associated cytosolic ribosomes.

A protein shuttle system to target RNA into mitochondria.

Mitochondria play a key role in essential cellular functions. A deeper understanding of mitochondrial molecular processes is hampered by the difficulty of incorporating foreign nucleic acids into organelles. Mitochondria of most eukaryotic species import cytosolic tRNAs. Based on this natural process, we describe here a powerful shuttle system to internalize several types of RNAs into isolated mitochondria. We demonstrate that this tool is useful to investigate tRNA processing or mRNA editing in plant mitochondria. Furthermore, we show that the same strategy can be used to address both tRNA and mRNA to isolated mammalian mitochondria. We anticipate our novel approach to be the starting point for various studies on mitochondrial processes. Finally, our study provides new insights into the mechanism of RNA import into mitochondria.

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 E3 ubiquitin ligase FBXL6 controls the quality of newly synthesized mitochondrial ribosomal proteins.

In mammals, about 99% of mitochondrial proteins are synthesized in the cytosol as precursors that are subsequently imported into the organelle. The mitochondrial health and functions rely on an accurate quality control of these imported proteins. Here, we show that the E3 ubiquitin ligase F box/leucine-rich-repeat protein 6 (FBXL6) regulates the quality of cytosolically translated mitochondrial proteins. Indeed, we found that FBXL6 substrates are newly synthesized mitochondrial ribosomal proteins. This E3 binds to chaperones involved in the folding and trafficking of newly synthesized peptide and to ribosomal-associated quality control proteins. Deletion of these interacting partners is sufficient to hamper interactions between FBXL6 and its substrate. Furthermore, we show that cells lacking FBXL6 fail to degrade specifically mistranslated mitochondrial ribosomal proteins. Finally, showing the role of FBXL6-dependent mechanism, FBXL6-knockout (KO) cells display mitochondrial ribosomal protein aggregations, altered mitochondrial metabolism, and inhibited cell cycle in oxidative conditions.

A global picture of tRNA genes in plant genomes.

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.

Voltage-dependent-anion-channels (VDACs) in Arabidopsis have a dual localization in the cell but show a distinct role in mitochondria.

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.

Dual-targeted tRNA-dependent amidotransferase ensures both mitochondrial and chloroplastic Gln-tRNAGln synthesis in plants.

Aminoacyl-tRNAs are generally formed by direct attachment of an amino acid to tRNAs by aminoacyl-tRNA synthetases, but Gln-tRNA is an exception to this rule. Gln-tRNA(Gln) is formed by this direct pathway in the eukaryotic cytosol and in protists or fungi mitochondria but is formed by an indirect transamidation pathway in most of bacteria, archaea, and chloroplasts. We show here that the formation of Gln-tRNA(Gln) is also achieved by the indirect pathway in plant mitochondria. The mitochondrial-encoded tRNA(Gln), which is the only tRNA(Gln) present in mitochondria, is first charged with glutamate by a nondiscriminating GluRS, then is converted into Gln-tRNA(Gln) by a tRNA-dependent amidotransferase (AdT). The three subunits GatA, GatB, and GatC are imported into mitochondria and assemble into a functional GatCAB AdT. Moreover, the mitochondrial pathway of Gln-tRNA(Gln) formation is shared with chloroplasts as both the GluRS, and the three AdT subunits are dual-imported into mitochondria and chloroplasts.