Discovery, structure, mechanisms, and evolution of protein-only RNase P enzymes.

The endoribonuclease RNase P is responsible for tRNA 5' maturation in all domains of life. A unique feature of RNase P is the variety of enzyme architectures, ranging from dual- to multi-subunit ribonucleoprotein forms with catalytic RNA subunits to protein-only enzymes, the latter occurring as single- or multi-subunit forms or homo-oligomeric assemblies. The protein-only enzymes evolved twice: a eukaryal protein-only RNase P termed PRORP and a bacterial/archaeal variant termed homolog of Aquifex RNase P (HARP); the latter replaced the RNA-based enzyme in a small group of thermophilic bacteria but otherwise coexists with the ribonucleoprotein enzyme in a few other bacteria as well as in those archaea that also encode a HARP. Here we summarize the history of the discovery of protein-only RNase P enzymes and review the state of knowledge on structure and function of bacterial HARPs and eukaryal PRORPs, including human mitochondrial RNase P as a paradigm of multi-subunit PRORPs. We also describe the phylogenetic distribution and evolution of PRORPs, as well as possible reasons for the spread of PRORPs in the eukaryal tree and for the recruitment of two additional protein subunits to metazoan mitochondrial PRORP. We outline potential applications of PRORPs in plant biotechnology and address diseases associated with mutations in human mitochondrial RNase P genes. Finally, we consider possible causes underlying the displacement of the ancient RNA enzyme by a protein-only enzyme in a small group of bacteria.

A tRNA-modifying enzyme facilitates RNase P activity in Arabidopsis nuclei.

RNase P is the essential activity that performs the 5' maturation of transfer RNA (tRNA) precursors. Beyond the ancestral form of RNase P containing a ribozyme, protein-only RNase P enzymes termed PRORP were identified in eukaryotes. In human mitochondria, PRORP forms a complex with two protein partners to become functional. In plants, although PRORP enzymes are active alone, we investigate their interaction network to identify potential tRNA maturation complexes. Here we investigate functional interactions involving the Arabidopsis nuclear RNase P PRORP2. We show, using an immuno-affinity strategy, that PRORP2 occurs in a complex with the tRNA methyl transferases TRM1A and TRM1B in vivo. Beyond RNase P, these enzymes can also interact with RNase Z. We show that TRM1A/TRM1B localize in the nucleus and find that their double knockout mutation results in a severe macroscopic phenotype. Using a combination of immuno-detections, mass spectrometry and a transcriptome-wide tRNA sequencing approach, we observe that TRM1A/TRM1B are responsible for the m22G26 modification of 70% of cytosolic tRNAs in vivo. We use the transcriptome wide tRNAseq approach as well as RNA blot hybridizations to show that RNase P activity is impaired in TRM1A/TRM1B mutants for specific tRNAs, in particular, tRNAs containing a m22G modification at position 26 that are strongly downregulated in TRM1A/TRM1B mutants. Altogether, results indicate that the m22G-adding enzymes TRM1A/TRM1B functionally cooperate with nuclear RNase P in vivo for the early steps of cytosolic tRNA biogenesis.

Types and Functions of Mitoribosome-Specific Ribosomal Proteins across Eukaryotes.

Mitochondria are key organelles that combine features inherited from their bacterial endosymbiotic ancestor with traits that arose during eukaryote evolution. These energy producing organelles have retained a genome and fully functional gene expression machineries including specific ribosomes. Recent advances in cryo-electron microscopy have enabled the characterization of a fast-growing number of the low abundant membrane-bound mitochondrial ribosomes. Surprisingly, mitoribosomes were found to be extremely diverse both in terms of structure and composition. Still, all of them drastically increased their number of ribosomal proteins. Interestingly, among the more than 130 novel ribosomal proteins identified to date in mitochondria, most of them are composed of a-helices. Many of them belong to the nuclear encoded super family of helical repeat proteins. Here we review the diversity of functions and the mode of action held by the novel mitoribosome proteins and discuss why these proteins that share similar helical folds were independently recruited by mitoribosomes during evolution in independent eukaryote clades.

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.

Purification and Cryo-electron Microscopy Analysis of Plant Mitochondrial Ribosomes.

Plants make up by far the largest part of biomass on Earth. They are the primary source of food and the basis of most drugs used for medicinal purposes. Similarly to all eukaryotes, plant cells also use mitochondria for energy production. Among mitochondrial gene expression processes, translation is the least understood; although, recent advances have revealed the specificities of its main component, the mitochondrial ribosome (mitoribosome). Here, we present a detailed protocol to extract highly pure cauliflower mitochondria by differential centrifugation for the purification of mitochondrial ribosomes using a sucrose gradient and the preparation of cryo-electron microscopy (cryo-EM) grids. Finally, the specific bioinformatics pipeline used for image acquisition, the processing steps, and the data analysis used for cryo-EM of the plant mitoribosome are described. This protocol will be used for further analysis of the critical steps of mitochondrial translation, such as its initiation and regulation.

Synthetic biological circuit tested in spaceflight.

Synthetic biology has potential spaceflight applications yet few if any studies have attempted to translate Earth-based synthetic biology tools into spaceflight. An exogenously inducible biological circuit for protein production in Arabidopsis thaliana, pX7-AtPDSi (Guo et al. 2003), was flown to ISS and functionally investigated. Seedlings were grown in a custom built 1.25 U plant greenhouse. Images recorded during the experiment show that leaves of pX7-AtPDSi seedlings photobleached as designed while wild type Col-0 leaves did not, which reveals that the synthetic circuit led to protein production during spaceflight. Polymerase chain reaction analysis post-flight also confirms that the Cre/LoxP (recombination system) portions of the circuit were functional in spaceflight. The subcomponents of the biological circuit, estrogen-responsive transcription factor XVE, Cre/LoxP DNA recombination system, and RNAi post-transcriptional gene silencing system now have flight heritage and can be incorporated in future designs for space applications. To facilitate future plant studies in space, the full payload design and manufacturing files are made available.

Towards plant resistance to viruses using protein-only RNase P.

Plant 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 5' 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.

La Société de Biologie de Strasbourg : 100 ans au service de la science et de la société.

Founded in 1919, the Society of Biology of Strasbourg (SBS) is a learned society whose purpose is the dissemination and promotion of scientific knowledge in biology. Subsidiary of the Society of Biology, the SBS celebrated its Centenary on Wednesday, the 16th of October 2019 on the Strasbourg University campus and at the Strasbourg City Hall. This day allowed retracing the various milestones of the SBS, through its main strengths, its difficulties and its permanent goal to meet scientific and societal challenges. The common thread of this day was the transmission of knowledge related to the past, the present, but also the future. At the start of the 21st century, the SBS must continue to reinvent itself to pursue its objective of transmitting scientific knowledge in biology and beyond. Scientific talks performed by senior scientists and former SBS thesis prizes awardees, a round table, and informal discussions reflected the history and the dynamism of the SBS association. All SBS Centennial participants have set the first milestone for the SBS Bicentennial.

TITLE: La Société de Biologie de Strasbourg : 100 ans au service de la science et de la société. ABSTRACT: Filiale de la Société de Biologie, la Société de Biologie de Strasbourg (SBS) est une société savante qui a pour objet la diffusion et la promotion du savoir scientifique en biologie et en médecine. Fondée en 1919, La SBS a célébré son Centenaire le mercredi 16 octobre 2019. Cette journée a permis de retracer les différents jalons de la SBS, à travers ses lignes de forces, ses difficultés et sa volonté permanente de mettre en exergue les défis scientifiques et sociétaux auxquels participent les recherches strasbourgeoises. Le fil rouge de cette journée a été la transmission d'un savoir en lien avec le passé, le présent, mais également le futur. En ce début du 21e siècle, la SBS se doit de continuer de se réinventer pour poursuivre son objectif de transmission des connaissances scientifiques en biologie et au-delà. L'ensemble des participants du Centenaire de la SBS a ainsi posé la première pierre du Bicentenaire de la SBS.

Cryo-EM structure of the RNA-rich plant mitochondrial ribosome.

The vast majority of eukaryotic cells contain mitochondria, essential powerhouses and metabolic hubs1. These organelles have a bacterial origin and were acquired during an early endosymbiosis event2. 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 genomes3. While chloroplast ribosomes strongly resemble those of bacteria4,5, mitoribosomes have diverged significantly during evolution and present strikingly different structures across eukaryotic species6-10. 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 mitoribosomes11. We have previously characterized the composition of the plant mitoribosome6, 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.

Specificities of the plant mitochondrial translation apparatus.

Mitochondria are endosymbiotic organelles responsible for energy production in most eukaryotic cells. They host a genome and a fully functional gene expression machinery. In plants this machinery involves hundreds of pentatricopeptide repeat (PPR) proteins. Translation, the final step of mitochondrial gene expression is performed by mitochondrial ribosomes (mitoribosomes). The nature of these molecular machines remained elusive for a very long time. Because of their bacterial origin, it was expected that mitoribosomes would closely resemble bacterial ribosomes. However, recent advances in cryo-electron microscopy have revealed the extraordinary diversity of mitoribosome structure and composition. The plant mitoribosome was characterized for Arabidopsis. In plants, in contrast to other species such as mammals and kinetoplastids where rRNA has been largely reduced, the mitoribosome could be described as a protein/RNA-augmented bacterial ribosome. It has an oversized small subunit formed by expanded ribosomal RNAs and additional protein components when compared to bacterial ribosomes. The same holds true for the large subunit. The small subunit is characterized by a new elongated domain on the head. Among its additional proteins, several PPR proteins are core mitoribosome proteins. They mainly act at the structural level to stabilize and maintain the plant-specific ribosomal RNA expansions but could also be involved in translation initiation. Recent advances in plant mitoribosome composition and structure, its specialization for membrane protein synthesis, translation initiation, the regulation and dynamics of mitochondrial translation are reviewed here and put in perspective with the diversity of mitochondrial translation processes in the green lineage and in the wider context of eukaryote evolution.

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.

Determination of protein-only RNase P interactome in Arabidopsis mitochondria and chloroplasts identifies a complex between PRORP1 and another NYN domain nuclease.

The essential type of endonuclease that removes 5' leader sequences from transfer RNA precursors is called RNase P. While ribonucleoprotein RNase P enzymes containing a ribozyme are found in all domains of life, another type of RNase P called 'PRORP', for 'PROtein-only RNase P', is composed of protein that occurs only in a wide variety of eukaryotes, in organelles and in the nucleus. Here, to find how PRORP functions integrate with other cell processes, we explored the protein interaction network of PRORP1 in Arabidopsis mitochondria and chloroplasts. Although PRORP proteins function as single subunit enzymes in vitro, we found that PRORP1 occurs in protein complexes and is present in high-molecular-weight fractions that contain mitochondrial ribosomes. The analysis of immunoprecipitated protein complexes identified proteins involved in organellar gene expression processes. In particular, direct interaction was established between PRORP1 and MNU2 a mitochondrial nuclease. A specific domain of MNU2 and a conserved signature of PRORP1 were found to be directly accountable for this protein interaction. Altogether, results revealed the existence of an RNA maturation complex in Arabidopsis mitochondria and suggested that PRORP proteins cooperated with other gene expression factors for RNA maturation in vivo.

Involvement of PIN-like domain nucleases in tRNA processing and translation regulation.

Transfer RNAs require essential maturation steps to become functional. Among them, RNase P removes 5' leader sequences of pre-tRNAs. Although RNase P was long thought to occur universally as ribonucleoproteins, different types of protein-only RNase P enzymes were discovered in both eukaryotes and prokaryotes. Interestingly, all these enzymes belong to the super-group of PilT N-terminal-like nucleases (PIN)-like ribonucleases. This wide family of enzymes can be subdivided into major subgroups. Here, we review recent studies at both functional and mechanistic levels on three PIN-like ribonucleases groups containing enzymes connected to tRNA maturation and/or translation regulation. The evolutive distribution of these proteins containing PIN-like domains as well as their organization and fusion with various functional domains is discussed and put in perspective with the diversity of functions they acquired during evolution, for the maturation and homeostasis of tRNA and a wider array of RNA substrates. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1117-1125, 2019.

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 mitochondrial translation remains unclear. Here we present the biochemical characterization of Arabidopsis mitochondrial ribosomes and identify their protein subunit composition. Complementary biochemical approaches identified 19 plant-specific mitoribosome proteins, of which ten are PPR proteins. The knockout mutations of ribosomal PPR (rPPR) genes result in distinct macroscopic phenotypes, including lethality and severe growth delay. The molecular analysis of rppr1 mutants using ribosome profiling, as well as the analysis of mitochondrial protein levels, demonstrate rPPR1 to be a generic translation factor that is a novel function for PPR proteins. Finally, single-particle cryo-electron microscopy (cryo-EM) reveals the unique structural architecture of Arabidopsis mitoribosomes, characterized by a very large small ribosomal subunit, larger than the large subunit, bearing an additional RNA domain grafted onto the head. Overall, our results show that Arabidopsis mitoribosomes are substantially divergent from bacterial and other eukaryote mitoribosomes, in terms of both structure and protein content.

Biophysical analysis of Arabidopsis protein-only RNase P alone and in complex with tRNA provides a refined model of tRNA binding.

RNase P is a universal enzyme that removes 5' leader sequences from tRNA precursors. The enzyme is therefore essential for maturation of functional tRNAs and mRNA translation. RNase P represents a unique example of an enzyme that can occur either as ribonucleoprotein or as protein alone. The latter form of the enzyme, called protein-only RNase P (PRORP), is widespread in eukaryotes in which it can provide organellar or nuclear RNase P activities. Here, we have focused on Arabidopsis nuclear PRORP2 and its interaction with tRNA substrates. Affinity measurements helped assess the respective importance of individual pentatricopeptide repeat motifs in PRORP2 for RNA binding. We characterized the PRORP2 structure by X-ray crystallography and by small-angle X-ray scattering in solution as well as that of its complex with a tRNA precursor by small-angle X-ray scattering. Of note, our study reports the first structural data of a PRORP-tRNA complex. Combined with complementary biochemical and biophysical analyses, our structural data suggest that PRORP2 undergoes conformational changes to accommodate its substrate. In particular, the catalytic domain and the RNA-binding domain can move around a central hinge. Altogether, this work provides a refined model of the PRORP-tRNA complex that illustrates how protein-only RNase P enzymes specifically bind tRNA and highlights the contribution of protein dynamics to achieve this specific interaction.

Mechanistic and Structural Studies of Protein-Only RNase P Compared to Ribonucleoproteins Reveal the Two Faces of the Same Enzymatic Activity.

RNase P, the essential activity that performs the 5' maturation of tRNA precursors, can be achieved either by ribonucleoproteins containing a ribozyme present in the three domains of life or by protein-only enzymes called protein-only RNase P (PRORP) that occur in eukaryote nuclei and organelles. A fast growing list of studies has investigated three-dimensional structures and mode of action of PRORP proteins. Results suggest that similar to ribozymes, PRORP proteins have two main domains. A clear functional analogy can be drawn between the specificity domain of the RNase P ribozyme and PRORP pentatricopeptide repeat domain, and between the ribozyme catalytic domain and PRORP N4BP1, YacP-like Nuclease domain. Moreover, both types of enzymes appear to dock with the acceptor arm of tRNA precursors and make specific contacts with the corner of pre-tRNAs. While some clear differences can still be delineated between PRORP and ribonucleoprotein (RNP) RNase P, the two types of enzymes seem to use, fundamentally, the same catalytic mechanism involving two metal ions. The occurrence of PRORP and RNP RNase P represents a remarkable example of convergent evolution. It might be the unique witness of an ongoing replacement of catalytic RNAs by proteins for enzymatic activities.

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.