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[This corrects the article DOI: 10.1371/journal.pgen.1002946.].
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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.
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Arabidopsis/enzimologia , Endorribonucleases/metabolismo , RNA de Transferência/metabolismo , Ribonuclease III/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Humanos , Mutação , Ribonucleases/genética , Ribonucleases/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
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.
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Arabidopsis/genética , Núcleo Celular/metabolismo , RNA de Transferência/metabolismo , RNA não Traduzido/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/metabolismo , Núcleo Celular/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plastídeos/metabolismo , RNA/metabolismo , RNA de Cloroplastos/metabolismo , RNA Mitocondrial , RNA de Transferência/química , RNA não Traduzido/química , Estresse FisiológicoRESUMO
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.
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Chlamydomonas reinhardtii/genética , Mitocôndrias/genética , RNA Mensageiro/genética , Transcrição Gênica , Sequência de Bases , Chlamydomonas reinhardtii/metabolismo , Clorófitas/classificação , Clorófitas/genética , Genoma Mitocondrial/genética , Mitocôndrias/metabolismo , Filogenia , Poli C/metabolismo , RNA Mensageiro/metabolismo , RNA Mitocondrial , Homologia de Sequência do Ácido NucleicoRESUMO
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.
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Proteínas Mitocondriais/metabolismo , RNA Mensageiro/metabolismo , Solanum tuberosum/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Núcleo Celular/metabolismo , Citosol/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Mutação , Transporte Proteico , RNA Mensageiro/genética , RNA de Plantas/genética , RNA de Plantas/metabolismo , Ribossomos/metabolismo , Solanum tuberosum/metabolismoRESUMO
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.
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Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Mitocôndrias/metabolismo , Transporte de RNA , RNA de Transferência/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Ligação Proteica , Biossíntese de Proteínas , Domínios Proteicos , RNA de Transferência/química , Proteínas de Ligação a RNA/metabolismo , Especificidade da Espécie , Transcriptoma/genéticaRESUMO
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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Proteínas de Arabidopsis/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Isoformas de RNA , Canais de Ânion Dependentes de Voltagem/genética , Regiões 3' não Traduzidas , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Genes de Plantas , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Mutação , Fenótipo , Porinas/metabolismo , Transporte Proteico , RNA Mensageiro/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
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.
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Proteínas Mitocondriais/química , Proteínas de Plantas/química , RNA de Transferência/metabolismo , Canais de Ânion Dependentes de Voltagem/química , DNA de Plantas/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Proteínas de Plantas/metabolismo , Ligação Proteica , Isoformas de Proteínas/metabolismo , RNA de Plantas/metabolismo , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
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.
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DNA Mitocondrial/genética , Genoma Mitocondrial , RNA de Transferência/genética , Animais , Crustáceos/genética , Regulação da Expressão GênicaRESUMO
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.
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Bases de Dados de Ácidos Nucleicos , Plantas/genética , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Estramenópilas/genética , Bryopsida/genética , Clorófitas/genética , Cyanophora/genética , Diatomáceas/genética , Enzimas/genética , Enzimas/metabolismo , Genoma Mitocondrial , Genoma de Planta , Genomas de Plastídeos , Internet , Magnoliopsida/genética , Phaeophyceae/genética , Fotossíntese/genética , RNA de Plantas/química , RNA de Transferência/química , Interface Usuário-ComputadorRESUMO
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.
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Chlamydomonas/genética , Mitocôndrias/genética , Biossíntese de Proteínas , RNA de Transferência/genética , Transporte Biológico , Respiração Celular/genética , Códon/genética , Evolução Molecular , Genoma Mitocondrial , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Mutação Puntual , RNA de Transferência/metabolismoRESUMO
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.
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Células Eucarióticas/metabolismo , RNA de Transferência/metabolismo , Endonucleases/metabolismo , Estabilidade de RNA , RNA não Traduzido/metabolismoRESUMO
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.
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Mitocôndrias/metabolismo , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/metabolismo , Transporte de RNA , Sequência de Bases , Larix/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Dados de Sequência Molecular , Edição de RNA , Precursores de RNA/química , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência de Histidina/química , RNA de Transferência de Histidina/metabolismo , Solanum tuberosum/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
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.
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Mitocôndrias/metabolismo , RNA de Transferência/metabolismo , Animais , Modelos Biológicos , Transporte de RNA/fisiologia , RNA de Plantas/metabolismoRESUMO
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.