Arabidopsis tRNA-derived fragments as potential modulators of translation

International audienceTransfer RNA-derived fragments (tRFs) exist in all branches of life. They are involved in RNA degradation, regulation of gene expression, ribosome biogenesis. In archaebacteria, kinetoplastid, yeast and human cells, they were also shown to regulate translation. In Arabidopsis, the tRFs population fluctuates under developmental or environmental conditions but their functions are yet poorly understood. Here, we show that populations of long (30-35 nt) or short (19-25 nt) tRFs produced from Arabidopsis tRNAs can inhibit in vitro translation of a reporter gene. Analyzing a series of oligoribonucleotides mimicking natural tRFs, we demonstrate that only a limited set of tRFs possess the ability to affect protein synthesis. Out of a dozen of tRFs, only two deriving from tRNA Ala (AGC) and tRNA Asn (GUU) strongly attenuate translation in vitro. Contrary to human tRF(Ala), the 4 Gs present at the 5' extremity of Arabidopsis tRF(Ala) are not implicated in this inhibition while the G18 and G19 residues are essential. Protein synthesis inhibition by tRFs does not require complementarity with the translated mRNA but, having the capability to be associated with polyribosomes, tRFs likely act as general modulation factors of the translation process in plants

PlantRNA, a database for tRNAs of photosynthetic eukaryotes.

International audiencePlantRNA 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

Transfer RNA maturation in Chlamydomonas mitochondria, chloroplast and the nucleus by a single RNase P protein.

The maturation of tRNA precursors involves the 5' cleavage of leader sequences by an essential endonuclease called RNase P. Beyond the ancestral ribonucleoprotein (RNP) RNase P, a second type of RNase P called PRORP (protein-only RNase P) evolved in eukaryotes. The current view on the distribution of RNase P in cells is that multiple RNPs, multiple PRORPs or a combination of both, perform specialised RNase P activities in the different compartments where gene expression occurs. Here, we identify a single gene encoding PRORP in the green alga Chlamydomonas reinhardtii while no RNP is found. We show that its product, CrPRORP, is triple-localised to mitochondria, the chloroplast and the nucleus. Its downregulation results in impaired tRNA biogenesis in both organelles and the nucleus. CrPRORP, as a single-subunit RNase P for an entire organism, makes up the most compact and versatile RNase P machinery described in either prokaryotes or eukaryotes

Isolation of Cytosolic Ribosomes Associated with Plant Mitochondria and Chloroplasts

International audienceExcluding 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

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

How to build a ribosome from RNA fragments in Chlamydomonas mitochondria

International audienceMitochondria 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

The Chlamydomonas mitochondrial ribosome:how to build a ribosome from RNA fragments

International audienceMitochondria 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. Novel proteins, mainly helical repeat proteins, including OPR, PPR and mTERF proteins are found in Chlamydomonas mitoribosome, revealing the first structure of an OPR protein in complex with its RNA target. Targeted amiRNA silencing indicated that the novel ribosomal proteins are required for mitoribosome integrity. Finally, we use cryo-electron tomography to show that Chlamydomonas mitoribosomes are attached to the mitochondrial inner membrane via two contact points mediated by Chlamydomonas-specific proteins. Our study expands our understanding of the mitoribosome diversity and the various strategies they adopt for membrane tethering