A NYN domain protein directly interacts with DECAPPING1 and is required for phyllotactic pattern.

In eukaryotes, general mRNA decay requires the decapping complex. The activity of this complex depends on its catalytic subunit, DECAPPING2 (DCP2), and its interaction with decapping enhancers, including its main partner DECAPPING1 (DCP1). Here, we report that in Arabidopsis thaliana, DCP1 also interacts with a NYN domain endoribonuclease, hence named DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1). Interestingly, we found DNE1 predominantly associated with DCP1, but not with DCP2, and reciprocally, suggesting the existence of two distinct protein complexes. We also showed that the catalytic residues of DNE1 are required to repress the expression of mRNAs in planta upon transient expression. The overexpression of DNE1 in transgenic lines led to growth defects and a similar gene deregulation signature than inactivation of the decapping complex. Finally, the combination of dne1 and dcp2 mutations revealed a functional redundancy between DNE1 and DCP2 in controlling phyllotactic pattern formation. Our work identifies DNE1, a hitherto unknown DCP1 protein partner highly conserved in the plant kingdom and identifies its importance for developmental robustness.

How RNases Shape Mitochondrial Transcriptomes.

Mitochondria are the power houses of eukaryote cells. These endosymbiotic organelles of prokaryote origin are considered as semi-autonomous since they have retained a genome and fully functional gene expression mechanisms. These pathways are particularly interesting because they combine features inherited from the bacterial ancestor of mitochondria with characteristics that appeared during eukaryote evolution. RNA biology is thus particularly diverse in mitochondria. It involves an unexpectedly vast array of factors, some of which being universal to all mitochondria and others being specific from specific eukaryote clades. Among them, ribonucleases are particularly prominent. They play pivotal functions such as the maturation of transcript ends, RNA degradation and surveillance functions that are required to attain the pool of mature RNAs required to synthesize essential mitochondrial proteins such as respiratory chain proteins. Beyond these functions, mitochondrial ribonucleases are also involved in the maintenance and replication of mitochondrial DNA, and even possibly in the biogenesis of mitochondrial ribosomes. The diversity of mitochondrial RNases is reviewed here, showing for instance how in some cases a bacterial-type enzyme was kept in some eukaryotes, while in other clades, eukaryote specific enzymes were recruited for the same function.

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.

PRORP proteins support RNase P activity in both organelles and the nucleus in Arabidopsis.

RNase P is an essential enzyme that cleaves the 5' leader sequence of tRNA precursors. RNase Ps were believed until now to occur universally as ribonucleoproteins in organisms performing RNase P activity. Here we find that protein-only RNase P enzymes called PRORP (for proteinaceous RNase P) support RNase P activity in vivo in both organelles and the nucleus in Arabidopsis. Beyond tRNA, PRORP proteins are involved in the maturation of small nucleolar RNA (snoRNA) and mRNA. Finally, ribonucleoprotein RNase MRP is not involved in tRNA maturation in plants. Altogether, our results indicate that ribonucleoprotein enzymes have been entirely replaced by proteins for RNase P activity in plants.

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.

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.

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.

Distribution of Ribonucleoprotein and Protein-Only RNase P in Eukarya.

RNase P is the endonuclease that removes 5' leader sequences from tRNA precursors. In Eukarya, separate RNase P activities exist in the nucleus and mitochondria/plastids. Although all RNase P enzymes catalyze the same reaction, the different architectures found in Eukarya range from ribonucleoprotein (RNP) enzymes with a catalytic RNA and up to 10 protein subunits to single-subunit protein-only RNase P (PRORP) enzymes. Here, analysis of the phylogenetic distribution of RNP and PRORP enzymes in Eukarya revealed 1) a wealth of novel P RNAs in previously unexplored phylogenetic branches and 2) that PRORP enzymes are more widespread than previously appreciated, found in four of the five eukaryal supergroups, in the nuclei and/or organelles. Intriguingly, the occurrence of RNP RNase P and PRORP seems mutually exclusive in genetic compartments of modern Eukarya. Our comparative analysis provides a global picture of the evolution and diversification of RNase P throughout Eukarya.

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.

An Arabidopsis dual-localized pentatricopeptide repeat protein interacts with nuclear proteins involved in gene expression regulation.

Following the endosymbiotic acquisition of mitochondria by eukaryotic cells, most of the genes in this organelle were transferred to the nucleus. To maintain mitochondrial biogenesis and function, nuclear and mitochondrial genomes require regulated and coordinated expression. In plant organelles, nuclear-encoded proteins targeted to the organelles control posttranscriptional and posttranslational mechanisms. Pentatricopeptide repeat (PPR) proteins are good candidates to play such regulatory roles. Here, we identify PNM1 (for PPR protein localized to the nucleus and mitochondria 1), a novel PPR protein that is dual localized to mitochondria and nuclei in Arabidopsis thaliana, as observed by green fluorescent protein fusions and immunodetection on subcellular fractions and on histological sections. Genetic complementation showed that loss of PNM1 function in mitochondria, but not in nuclei, is lethal for the embryo. In mitochondria, it is associated with polysomes and may play a role in translation. A genetic screen in yeast identified protein partners of PNM1. These partners, the nucleosome assembly protein NAP1, and the transcription factor TCP8 interact with PNM1 in the nucleus in planta. Furthermore, TCP8 can bind the promoter of PNM1. This suggests that PNM1 might be involved in the regulation of its own gene expression in the nucleus and could thus play a role in gene expression adjustments between mitochondria and the nucleus.

PPR proteins shed a new light on RNase P biology.

A fast growing number of studies identify pentatricopeptide repeat (PPR) proteins as major players in gene expression processes. Among them, a subset of PPR proteins called PRORP possesses RNase P activity in several eukaryotes, both in nuclei and organelles. RNase P is the endonucleolytic activity that removes 5' leader sequences from tRNA precursors and is thus essential for translation. Before the characterization of PRORP, RNase P enzymes were thought to occur universally as ribonucleoproteins, although some evidence implied that some eukaryotes or cellular compartments did not use RNA for RNase P activity. The characterization of PRORP reveals a two-domain enzyme, with an N-terminal domain containing multiple PPR motifs and assumed to achieve target specificity and a C-terminal domain holding catalytic activity. The nature of PRORP interactions with tRNAs suggests that ribonucleoprotein and protein-only RNase P enzymes share a similar substrate binding process.

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.

Plant mitochondria use two pathways for the biogenesis of tRNAHis.

All tRNA(His) possess an essential extra G(-1) guanosine residue at their 5' end. In eukaryotes after standard processing by RNase P, G(-1) is added by a tRNA(His) guanylyl transferase. In prokaryotes, G(-1) is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G(-1) we find here that both maturation pathways can be used. Indeed, tRNA(His) with or without a G(-1) are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNA(His) precursors at both positions G(+1) and G(-1). The G(-1) is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNA(His) without G(-1) has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNA(His) with a G(-1) are recovered. This shows that a previously unreported tRNA(His) guanylyltransferase activity is present in plant mitochondria.

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.

RNA uridylation and decay in plants.

RNA uridylation consists of the untemplated addition of uridines at the 3' extremity of an RNA molecule. RNA uridylation is catalysed by terminal uridylyltransferases (TUTases), which form a subgroup of the terminal nucleotidyltransferase family, to which poly(A) polymerases also belong. The key role of RNA uridylation is to regulate RNA degradation in a variety of eukaryotes, including fission yeast, plants and animals. In plants, RNA uridylation has been mostly studied in two model species, the green algae Chlamydomonas reinhardtii and the flowering plant Arabidopsis thaliana Plant TUTases target a variety of RNA substrates, differing in size and function. These RNA substrates include microRNAs (miRNAs), small interfering silencing RNAs (siRNAs), ribosomal RNAs (rRNAs), messenger RNAs (mRNAs) and mRNA fragments generated during post-transcriptional gene silencing. Viral RNAs can also get uridylated during plant infection. We describe here the evolutionary history of plant TUTases and we summarize the diverse molecular functions of uridylation during RNA degradation processes in plants. We also outline key points of future research.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'.

RNA degradation by the plant RNA exosome involves both phosphorolytic and hydrolytic activities.

The RNA exosome provides eukaryotic cells with an essential 3'-5' exoribonucleolytic activity, which processes or eliminates many classes of RNAs. Its nine-subunit core (Exo9) is structurally related to prokaryotic phosphorolytic exoribonucleases. Yet, yeast and animal Exo9s have lost the primordial phosphorolytic capacity and rely instead on associated hydrolytic ribonucleases for catalytic activity. Here, we demonstrate that Arabidopsis Exo9 has retained a distributive phosphorolytic activity, which contributes to rRNA maturation processes, the hallmark of exosome function. High-density mapping of 3' extremities of rRNA maturation intermediates reveals the intricate interplay between three exoribonucleolytic activities coordinated by the plant exosome. Interestingly, the analysis of RRP41 protein diversity across eukaryotes suggests that Exo9's intrinsic activity operates throughout the green lineage, and possibly in some earlier-branching non-plant eukaryotes. Our results reveal a remarkable evolutionary variation of this essential RNA degradation machine in eukaryotes.

Differential NO3 - dependent patterns of NO3 - uptake in Pinus pinaster, Rhizopogon roseolus and their ectomycorrhizal association.

• A different NO3 - dependent pattern of NO3 - uptake at low [NO3 - ] (0-0.1 mM) is shown in Pinus pinaster and in the ectomycorrhizal fungus Rhizopogon roseolus. In ectomycorrhizal symbiosis, the fungal pattern is pre-eminent. • Net NO3 - uptake rates were deduced in plant and fungus from solution depletion measurements. Net NO3 - fluxes were estimated at the surface of mycorrhizal and nonmycorrhizal short roots, using NO3 - selective microelectrodes. • In NO3 - starved seedlings, maximum NO3 - uptake rates were reached after 3 d of incubation in 0.05 mM NO3 - . In R. roseolus mycelia, NO3 - uptake rates did not change after withdrawing NO3 - for up to 7 d, or after adding NO3 - for 3 d. Net NO3 - fluxes in nonmycorrhizal short roots were increased twofold by a 3-d exposure to NO3 - whereas in ectomycorrhiza they were similar irrespective of the NO3 - pretreatment, but always higher than the fluxes measured in nonmycorrhizal roots. • Ectomycorrhiza have a greater capacity to use NO3 - than nonmycorrhizal short roots, whatever the NO3 - concentration in the solution. This may give mycorrhizal plants a greater ability to use fluctuating concentrations of NO3 - in the soil solution.

Helical repeats modular proteins are major players for organelle gene expression.

Mitochondria and chloroplasts are often described as semi-autonomous organelles because they have retained a genome. They thus require fully functional gene expression machineries. Many of the required processes going all the way from transcription to translation have specificities in organelles and arose during eukaryote history. Most factors involved in these RNA maturation steps have remained elusive for a long time. The recent identification of a number of novel protein families including pentatricopeptide repeat proteins, half-a-tetratricopeptide proteins, octotricopeptide repeat proteins and mitochondrial transcription termination factors has helped to settle long-standing questions regarding organelle gene expression. In particular, their functions have been related to replication, transcription, RNA processing, RNA editing, splicing, the control of RNA turnover and translation throughout eukaryotes. These families of proteins, although evolutionary independent, seem to share a common overall architecture. For all of them, proteins contain tandem arrays of repeated motifs. Each module is composed of two to three α-helices and their succession forms a super-helix. Here, we review the features characterising these protein families, in particular, their distribution, the identified functions and mode of action and propose that they might share similar substrate recognition mechanisms.

Structural insights into protein-only RNase P complexed with tRNA.

RNase P is the essential activity removing 5'-leader sequences from transfer RNA precursors. RNase P was always associated with ribonucleoprotein complexes before the discovery of protein-only RNase P enzymes called PRORPs (PROteinaceous RNase P) in eukaryotes. Here we provide biophysical and functional data to understand the mode of action of PRORP enzymes. Activity assays and footprinting experiments show that the anticodon domain of transfer RNA is dispensable, whereas individual residues in D and TψC loops are essential for PRORP function. PRORP proteins are characterized in solution and a molecular envelope is derived from small-angle X-ray scattering. Conserved residues are shown to be involved in the binding of one zinc atom to PRORP. These results facilitate the elaboration of a model of the PRORP/transfer RNA interaction. The comparison with the ribonucleoprotein RNase P/transfer RNA complex suggests that transfer RNA recognition by PRORP proteins is similar to that by ribonucleoprotein RNase P.