12 research outputs found
Caractérisation de la machinerie de traduction mitochondriale chez Arabidopsis thaliana
Ribosomes are the molecular machines translating the genetic information carried by mRNA into protein. Different translation machineries co-exist in eukaryote cells. While cytosolic translation is comparatively well characterized, it remains the most elusive step of gene expression in mitochondria. In plants, while numerous pentatricopeptide repeat (PPR) proteins are involved in all steps of gene expression, their function in translation remains unclear. My work focused on the biochemical characterisation of Arabidopsis mitochondrial ribosomes and the identification of its protein composition. Complementary biochemical approaches identified 19 plant specific mitoribosome proteins, among which 10 are PPR proteins. The knock out mutations of ribosomal PPR (rPPR) genes result in distinct macroscopic phenotypes including lethality or severe growth delays. The molecular analysis of rPPR1 mutants, using ribosome profiling, as well as the analysis of mitochondrial protein levels, revealed that rPPR1 is a generic translation factor, which is a novel function for PPR proteins. Finally, single particle cryo-electron microscopy was used and revealed the unique structural architecture of Arabidopsis mitoribosomes, characterised by a very large small ribosomal subunit, larger than the large subunit, with a novel head domain. Overall, my results showed that Arabidopsis mitoribosomes are completely distinct from bacterial and other eukaryote mitoribosomes, both in terms of structure and of protein content.Dans les cellules eucaryotes, diffĂ©rents types de ribosomes coexistent. Les ribosomes mitochondriaux synthĂ©tisent les quelques protĂ©ines codĂ©es par lâADN mitochondrial, qui sont essentielles au fonctionnement de lâorganisme. Ces ribosomes sont particuliĂšrement divergents des ribosomes procaryotes, mais sont Ă©galement trĂšs diffĂ©rents entre les eucaryotes. Mon travail de thĂšse s'est concentrĂ© sur la caractĂ©risation de la structure et de la composition en protĂ©ines du ribosome mitochondrial de la plante modĂšle Arabidopsis thaliana. Des approches biochimiques complĂ©mentaires ont permis dâidentifier 19 protĂ©ines uniquement trouvĂ©es dans le mitoribosome de plante, parmi lesquelles 10 sont des protĂ©ines PPR, des protĂ©ines particuliĂšrement abondantes chez les plantes. Les mutations des gĂšnes codant pour ces PPR ribosomales (rPPR) mĂšnent Ă lâapparition de phĂ©notypes macroscopiques distincts, notamment une lĂ©talitĂ© ou des retards de croissance importants. L'analyse molĂ©culaire du mutant rppr1 par profilage des ribosomes, ainsi que l'analyse du taux de protĂ©ines mitochondriales, rĂ©vĂšlent que la protĂ©ine rPPR1 est un facteur de traduction gĂ©nĂ©rique, ce qui constitue une nouvelle fonction des protĂ©ines PPR. De plus, la cryo-Ă©lectron microscopie a Ă©tĂ© utilisĂ©e pour dĂ©terminer lâarchitecture tridimensionnelle de ce mitoribosome. Cette approche a rĂ©vĂ©lĂ© la structure unique du mitoribosome de plante, caractĂ©risĂ©e par une trĂšs grande petite sous-unitĂ© ribosomale ayant un domaine additionnel jamais dĂ©crit jusquâĂ prĂ©sent. Globalement, mes rĂ©sultats ont montrĂ© que le mitoribosome dâArabidopsis est complĂštement diffĂ©rent des ribosomes bactĂ©riens et des autres mitoribosomes eucaryotes, Ă la fois en terme de structure mais aussi de composition, permettant ainsi de mieux comprendre lâĂ©volution de ce composant central de lâexpression gĂ©nĂ©tique
Striking Diversity of Mitochondria-Specific Translation Processes across Eukaryotes
Mitochondria are essential organelles that act as energy conversion powerhouses and metabolic hubs. Their gene expression machineries combine traits inherited from prokaryote ancestors and specific features acquired during eukaryote evolution. Mitochondrial research has wide implications ranging from human health to agronomy. We highlight recent advances in mitochondrial translation. Functional, biochemical, and structural data have revealed an unexpected diversity of mitochondrial translation systems, particularly of their key players, the mitochondrial ribosomes (mitoribosomes). Ribosome assembly and translation mechanisms, such as initiation, are discussed and put in perspective with the prevalence of eukaryote-specific families of mitochondrial translation factors such as pentatricopeptide repeat (PPR) proteins
Specific features and assembly of the plant mitochondrial complex I revealed by cryo-EM
Electrons enter the mitochondrial respiratory chain via complex I. Here, the authors report high-resolution structures of mature plant complex I and one of its assembly intermediates, highlighting plant-specific features including an ancestral carbonic anhydrase domain
Cryo-EM structure of the RNA-rich plant mitochondrial ribosome
The vast majority of eukaryotic cells contain mitochondria, essential powerhouses and metabolic hubs; 1; . These organelles have a bacterial origin and were acquired during an early endosymbiosis event; 2; . Mitochondria possess specialized gene expression systems composed of various molecular machines, including the mitochondrial ribosomes (mitoribosomes). Mitoribosomes are in charge of translating the few essential mRNAs still encoded by mitochondrial genomes; 3; . While chloroplast ribosomes strongly resemble those of bacteria; 4,5; , mitoribosomes have diverged significantly during evolution and present strikingly different structures across eukaryotic species; 6-10; . In contrast to animals and trypanosomatids, plant mitoribosomes have unusually expanded ribosomal RNAs and have conserved the short 5S rRNA, which is usually missing in mitoribosomes; 11; . We have previously characterized the composition of the plant mitoribosome; 6; , revealing a dozen plant-specific proteins in addition to the common conserved mitoribosomal proteins. In spite of the tremendous recent advances in the field, plant mitoribosomes remained elusive to high-resolution structural investigations and the plant-specific ribosomal features of unknown structures. Here, we present a cryo-electron microscopy study of the plant 78S mitoribosome from cauliflower at near-atomic resolution. We show that most of the plant-specific ribosomal proteins are pentatricopeptide repeat proteins (PPRs) that deeply interact with the plant-specific rRNA expansion segments. These additional rRNA segments and proteins reshape the overall structure of the plant mitochondrial ribosome, and we discuss their involvement in the membrane association and mRNA recruitment prior to translation initiation. Finally, our structure unveils an rRNA-constructive phase of mitoribosome evolution across eukaryotes
Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis
International audienc
Towards plant resistance to viruses using protein-only RNase P
International audiencePlant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5âČ maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases
Determination of proteinâonly RNase P interactome in Arabidopsis mitochondria and chloroplasts identifies a complex between PRORP1 and another NYN domain nuclease
International audienc
Small is big in Arabidopsis mitochondrial ribosome
Mitochondria are responsible for energy production through aerobic respiration and represent the powerhouse of eukaryotic cells. Their metabolism and gene expression processes combine bacterial-like features and traits that evolved in eukaryotes. Among mitochondrial gene expression processes, translation remains the most elusive. In plants, while numerous pentatricopeptide repeat (PPR) proteins are involved in all steps of gene expression, their function in translation remained unclear. Here we present the biochemical characterisation of Arabidopsis mitochondrial ribosomes and identify their protein subunit composition. Complementary biochemical approaches identify 19 plant specific mitoribosome proteins, among which 10 are PPR proteins. The knock out mutations of ribosomal PPR genes result in distinct macroscopic phenotypes including lethality or severe growth delays. The molecular analysis of rPPR1 mutants using ribosome profiling as well as the analysis of mitochondrial protein levels reveal that rPPR1 is a generic translation factor, which is a novel function for PPR proteins. Finally, single particle cryo-electron microscopy reveals the unique structural architecture of Arabidopsis mitoribosomes, characterised by a very large small ribosomal subunit, larger than the large ribosomal subunit, with a huge head expansion. Overall, our results show that Arabidopsis mitoribosomes are completely distinct from bacterial and other eukaryote mitoribosomes, both in terms of structure and of protein content
Towards the Visual Proteomics of C. reinhardtii using High-throughput Collaborative in situ Cryo-ET
No abstract availabl
How to build a ribosome from RNA fragments in Chlamydomonas mitochondria
Mitochondria are the powerhouse of eukaryotic cells. They possess their own gene expression machineries where highly divergent and specialized ribosomes, named hereafter mitoribosomes, translate the few essential messenger RNAs still encoded by mitochondrial genomes. Here, we present a biochemical and structural characterization of the mitoribosome in the model green alga Chlamydomonas reinhardtii, as well as a functional study of some of its specific components. Single particle cryo-electron microscopy resolves how the Chlamydomonas mitoribosome is assembled from 13 rRNA fragments encoded by separate non-contiguous gene pieces. Additional proteins, mainly OPR, PPR and mTERF helical repeat proteins, are found in Chlamydomonas mitoribosome, revealing the structure of an OPR protein in complex with its RNA binding partner. Targeted amiRNA silencing indicates that these ribosomal proteins are required for mitoribosome integrity. Finally, we use cryo-electron tomography to show that Chlamydomonas mitoribosomes are attached to the inner mitochondrial membrane via two contact points mediated by Chlamydomonas-specific proteins. Our study expands our understanding of mitoribosome diversity and the various strategies these specialized molecular machines adopt for membrane tethering