RESUMEN
Programmable RNA editing offers significant therapeutic potential for a wide range of genetic diseases. Currently, several deaminase enzymes, including ADAR and APOBEC, can perform programmable adenosine-to-inosine or cytidine-to-uridine RNA correction. However, enzymes to perform guanosine-to-adenosine and uridine-to-cytidine (U-to-C) editing are still lacking to complete the set of transition reactions. It is believed that the DYW:KP proteins, specific to seedless plants, catalyze the U-to-C reactions in mitochondria and chloroplasts. In this study, we designed seven DYW:KP domains based on consensus sequences and fused them to a designer RNA-binding pentatricopeptide repeat (PPR) domain. We show that three of these PPR-DYW:KP proteins edit targeted uridine to cytidine in bacteria and human cells. In addition, we show that these proteins have a 5' but not apparent 3' preference for neighboring nucleotides. Our results establish the DYW:KP aminase domain as a potential candidate for the development of a U-to-C editing tool in human cells.
Asunto(s)
Citidina , Edición de ARN , Adenosina/metabolismo , Bacterias/genética , Bacterias/metabolismo , Citidina/genética , Citidina/metabolismo , Guanosina/metabolismo , Humanos , Inosina , Nucleótidos/metabolismo , Proteínas de Plantas/genética , ARN/metabolismo , Uridina/metabolismoRESUMEN
Members of the pentatricopeptide repeat (PPR) protein family act as specificity factors in C-to-U RNA editing. The expansion of the PPR superfamily in plants provides the sequence variation required for design of consensus-based RNA-binding proteins. We used this approach to design a synthetic RNA editing factor to target one of the sites in the Arabidopsis chloroplast transcriptome recognised by the natural editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that our synthetic editing factor specifically recognises the target sequence in in vitro binding assays. The designed factor is equally specific for the target rpoA site when expressed in chloroplasts and in the bacterium E. coli. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Bacterianas/metabolismo , Cloroplastos/genética , Escherichia coli/genética , Edición de ARN , Proteínas de Unión al ARN/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/genética , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Cloroplastos/metabolismo , Escherichia coli/metabolismo , ARN Bacteriano/genética , ARN de Planta/genética , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genéticaRESUMEN
In Arabidopsis thaliana there are more than 600 C-to-U RNA editing events in the mitochondria and at least 44 in the chloroplasts. Pentatricopeptide repeat (PPR) proteins provide the specificity for these reactions. They recognize RNA sequences in a partially predictable fashion via key amino acids at the fifth and last position in each PPR motif that bind to individual ribonucleotides. A combined approach of RNA-Seq, mutant complementation, electrophoresis of mitochondrial protein complexes and Western blotting allowed us to show that MEF100, a PPR protein identified in a genetic screen for mutants resistant to an inhibitor of γ -glutamylcysteine synthetase, is required for the editing of nad1-493, nad4-403, nad7-698 and ccmFN2-356 sites in Arabidopsis mitochondria. The absence of editing in mef100 leads to a decrease in mitochondrial Complex I activity, which probably explains the physiological phenotype. Some plants have lost the requirement for MEF100 at one or more of these sites through mutations in the mitochondrial genome. We show that loss of the requirement for MEF100 editing leads to divergence in the MEF100 binding site.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Proteínas Mitocondriales/metabolismo , Proteínas de Plantas/metabolismo , Mitocondrias/metabolismoRESUMEN
Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are attractive tools for RNA processing in synthetic biology applications given their modular structure and ease of design. Several distinct types of motifs have been described from natural PPR proteins, but almost all work so far with synthetic PPR proteins has focused on the most widespread P-type motifs. We have investigated synthetic PPR proteins based on tandem repeats of the more compact S-type PPR motif found in plant organellar RNA editing factors and particularly prevalent in the lycophyte Selaginella. With the aid of a novel plate-based screening method, we show that synthetic S-type PPR proteins are easy to design and bind with high affinity and specificity and are functional in a wide range of pH, salt and temperature conditions. We find that they outperform a synthetic P-type PPR scaffold in many situations. We designed an S-type editing factor to edit an RNA target in E. coli and demonstrate that it edits effectively without requiring any additional cofactors to be added to the system. These qualities make S-type PPR scaffolds ideal for developing new RNA processing tools.
RESUMEN
The RNA-binding pentatricopeptide repeat (PPR) family comprises hundreds to thousands of genes in most plants, but only a few dozen in algae, indicating massive gene expansions during land plant evolution. The nature and timing of these expansions has not been well defined due to the sparse sequence data available from early-diverging land plant lineages. In this study, we exploit the comprehensive OneKP datasets of over 1000 transcriptomes from diverse plants and algae toward establishing a clear picture of the evolution of this massive gene family, focusing on the proteins typically associated with RNA editing, which show the most spectacular variation in numbers and domain composition across the plant kingdom. We characterize over 2 250 000 PPR motifs in over 400 000 proteins. In lycophytes, polypod ferns, and hornworts, nearly 10% of expressed protein-coding genes encode putative PPR editing factors, whereas they are absent from algae and complex-thalloid liverworts. We show that rather than a single expansion, most land plant lineages with high numbers of editing factors have continued to generate novel sequence diversity. We identify sequence variations that imply functional differences between PPR proteins in seed plants versus non-seed plants and variations we propose to be linked to seed-plant-specific editing co-factors. Finally, using the sequence variations across the datasets, we develop a structural model of the catalytic DYW domain associated with C-to-U editing and identify a clade of unique DYW variants that are strong candidates as U-to-C RNA-editing factors, given their phylogenetic distribution and sequence characteristics.
Asunto(s)
Embryophyta/genética , Proteínas de Plantas/genética , Edición de ARN/genética , Proteínas de Unión al ARN/genética , Secuencias de Aminoácidos , Bases de Datos Genéticas , Embryophyta/clasificación , Evolución Molecular , Duplicación de Gen , Variación Genética , Modelos Moleculares , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas/clasificación , Plantas/genética , Dominios Proteicos , ARN de Planta/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Secuencias Repetitivas de AminoácidoRESUMEN
Hornworts are crucial to understand the phylogeny of early land plants. The emergence of 'reverse' U-to-C RNA editing accompanying the widespread C-to-U RNA editing in plant chloroplasts and mitochondria may be a molecular synapomorphy of a hornwort-tracheophyte clade. C-to-U RNA editing is well understood after identification of many editing factors in models like Arabidopsis thaliana and Physcomitrella patens, but there is no plant model yet to investigate U-to-C RNA editing. The hornwort Anthoceros agrestis is now emerging as such a model system. We report on the assembly and analyses of the A. agrestis chloroplast and mitochondrial genomes, their transcriptomes and editomes, and a large nuclear gene family encoding pentatricopeptide repeat (PPR) proteins likely acting as RNA editing factors. Both organelles in A. agrestis feature high amounts of RNA editing, with altogether > 1100 sites of C-to-U and 1300 sites of U-to-C editing. The nuclear genome reveals > 1400 genes for PPR proteins with variable carboxyterminal DYW domains. We observe significant variants of the 'classic' DYW domain, in the meantime confirmed as the cytidine deaminase for C-to-U editing, and discuss the first attractive candidates for reverse editing factors given their excellent matches to U-to-C editing targets according to the PPR-RNA binding code.
Asunto(s)
Anthocerotophyta , Bryopsida , Anthocerotophyta/metabolismo , Bryopsida/genética , Orgánulos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Edición de ARN/genética , ARN de Planta/genética , ARN de Planta/metabolismo , Transcriptoma/genéticaRESUMEN
Pentatricopeptide repeat (PPR) proteins are modular RNA-binding proteins involved in different aspects of RNA metabolism in organelles. PPR proteins of the PLS subclass often contain C-terminal domains that are important for their function, but the role of one of these domains, the E domain, is far from resolved. Here, we elucidate the role of the E domain in CRR2 in plastids. We identified a surprisingly large number of small RNAs that represent in vivo footprints of the Arabidopsis PLS-class PPR protein CRR2. An unexpectedly strong base conservation was found in the nucleotides aligned to the E domain. We used both in vitro and in vivo experiments to reveal the role of the E domain of CRR2. The E domain of CRR2 can be predictably altered to prefer different nucleotides in its RNA ligand, and position 5 of the E1-motif is biologically important for the PPR-RNA interaction. The 'code' of the E domain PPR motifs is different from that of P- and S-motifs. The findings presented here show that the E domain of CRR2 is involved in sequence-specific interaction with its RNA ligand and have implications for our ability to predict RNA targets for PLS-PPRs and their use as biotechnological tools to manipulate specific RNAs in vivo.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/química , Proteínas de Cloroplastos/metabolismo , Plastidios/metabolismo , ARN de Planta/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia Conservada , Glicina/metabolismo , Mutación/genética , Dominios ProteicosRESUMEN
RNA editing in plastids is known to be required for embryogenesis, but no single editing event had been shown to be essential. We show that the emb2261-2 mutation is lethal through a failure to express an editing factor that specifically recognizes the rps14-2 site. EMB2261 was predicted to bind the cis-element upstream of the rps14-2 site and genetic complementation with promoters of different strength followed by RNA-seq analysis was conducted to test the correlation between rps14-2 editing and EMB2261 expression. Rps14-2 is the only editing event in Arabidopsis chloroplasts that correlates with EMB2261 expression. Sequence divergence between the cis-element and the EMB2261 protein sequence in plants where rps14-2 editing is not required adds support to the association between them. We conclude that EMB2261 is the specificity factor for rps14-2 editing. This editing event converts P51 in Rps14 to L51, which is conserved among species lacking RNA editing, implying the importance of the editing event to Rps14 function. Rps14 is an essential ribosomal subunit for plastid translation, which, in turn, is essential for Arabidopsis embryogenesis.
Asunto(s)
Orgánulos/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Edición de ARN/fisiología , ARN de Planta/metabolismo , Proteínas de Unión al ARN/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Orgánulos/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas/genética , Dominios Proteicos , ARN de Planta/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
The pentatricopeptide repeat (PPR) proteins form one of the largest protein families in land plants. They are characterised by tandem 30-40 amino acid motifs that form an extended binding surface capable of sequence-specific recognition of RNA strands. Almost all of them are post-translationally targeted to plastids and mitochondria, where they play important roles in post-transcriptional processes including splicing, RNA editing and the initiation of translation. A code describing how PPR proteins recognise their RNA targets promises to accelerate research on these proteins, but making use of this code requires accurate definition and annotation of all of the various nucleotide-binding motifs in each protein. We have used a structural modelling approach to define 10 different variants of the PPR motif found in plant proteins, in addition to the putative deaminase motif that is found at the C-terminus of many RNA-editing factors. We show that the super-helical RNA-binding surface of RNA-editing factors is potentially longer than previously recognised. We used the redefined motifs to develop accurate and consistent annotations of PPR sequences from 109 genomes. We report a high error rate in PPR gene models in many public plant proteomes, due to gene fusions and insertions of spurious introns. These consistently annotated datasets across a wide range of species are valuable resources for future comparative genomics studies, and an essential pre-requisite for accurate large-scale computational predictions of PPR targets. We have created a web portal (http://www.plantppr.com) that provides open access to these resources for the community.
Asunto(s)
Embryophyta/genética , Modelos Estructurales , Proteínas de Plantas/química , Edición de ARN/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Embryophyta/metabolismo , Mitocondrias/metabolismo , Modelos Moleculares , Anotación de Secuencia Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plastidios/metabolismo , Transporte de Proteínas , Proteínas con Motivos de Reconocimiento de ARN/química , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , ARN de Planta/genética , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Eucariontes/metabolismo , Ribonucleasa P/metabolismo , Ribonucleoproteínas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia Conservada , Eucariontes/enzimología , Eucariontes/genética , Datos de Secuencia Molecular , Filogenia , Estructura Secundaria de Proteína , ARN/genética , ARN/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Ribonucleasa P/genética , Ribonucleoproteínas/genética , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , Ribonucleasa P/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Células Eucariotas/metabolismo , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Ribonucleasa P/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , ARN de Transferencia/química , Ribonucleasa P/química , Ribonucleasa P/metabolismo , Modelos Biológicos , Modelos Moleculares , Unión Proteica , ARN/química , Precursores del ARN/química , ARN Mitocondrial , ARN de Planta/química , ARN de Planta/metabolismo , ARN de Transferencia/metabolismo , Recombinación Genética/genética , Secuencias Reguladoras de Ácido Ribonucleico , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Dispersión del Ángulo Pequeño , Soluciones , Espectrofotometría Atómica , Difracción de Rayos X , Zinc/metabolismoRESUMEN
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.
Asunto(s)
Bases de Datos de Ácidos Nucleicos , Plantas/genética , ARN de Planta/genética , ARN de Planta/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Estramenopilos/genética , Bryopsida/genética , Chlorophyta/genética , Cyanophora/genética , Diatomeas/genética , Enzimas/genética , Enzimas/metabolismo , Genoma Mitocondrial , Genoma de Planta , Genoma de Plastidios , Internet , Magnoliopsida/genética , Phaeophyceae/genética , Fotosíntesis/genética , ARN de Planta/química , ARN de Transferencia/química , Interfaz Usuario-ComputadorRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , ARN Mensajero/metabolismo , ARN Nucleolar Pequeño/metabolismo , ARN de Transferencia/metabolismo , Ribonucleasa P/metabolismo , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/genética , Núcleo Celular/enzimología , Mitocondrias/enzimología , Procesamiento Postranscripcional del ARN , Ribonucleasa P/genética , Ribonucleoproteínas/metabolismoRESUMEN
The ubiquitous endonuclease RNase P is responsible for the 5' maturation of tRNA precursors. Until the discovery of human mitochondrial RNase P, these enzymes had typically been found to be ribonucleoproteins, the catalytic activity of which is associated with the RNA component. Here we show that, in Arabidopsis thaliana mitochondria and plastids, a single protein called 'proteinaceous RNase P' (PRORP1) can perform the endonucleolytic maturation of tRNA precursors that defines RNase P activity. In addition, PRORP1 is able to cleave tRNA-like structures involved in the maturation of plant mitochondrial mRNAs. Finally, we show that Arabidopsis PRORP1 can replace the bacterial ribonucleoprotein RNase P in Escherichia coli cells. PRORP2 and PRORP3, two paralogs of PRORP1, are both localized in the nucleus.