RNA Editing In Plant Organelles Research Articles

C-to-U and U-to-C: RNA editing in plant organelles and beyond.

The genomes in the two energy-converting organelles of plant cells, chloroplasts and mitochondria, contain numerous 'errors' that are corrected at the level of RNA transcript copies. The genes encoded in the two endosymbiotic organelles would not function properly if their transcripts were not altered by site-specific cytidine-to-uridine (C-to-U) exchanges and by additional reverse U-to-C exchanges in hornworts, lycophytes, and ferns. These peculiar processes of plant RNA editing, re-establishing genetic information that could alternatively be present at the organelle genome level, has spurred much research over >30 years. Lately new studies have revealed numerous interesting insights, notably on the biochemical machinery identifying specific pyrimidine nucleobases for conversion from C to U and vice versa. Here, I will summarize prominent research findings that lately have contributed to our better understanding of these phenomena introducing an added layer of information processing in plant cells. Some of this recent progress is based on the successful functional expression of plant RNA editing factors in bacteria and mammalian cells. These research approaches have recapitulated natural processes of horizontal gene transfer through which some protist lineages seem to have acquired plant RNA editing factors and adapted them functionally for their own purposes.

SlRIP1b is a global organellar RNA editing factor, required for normal fruit development in tomato plants.

RNA editing in plant organelles involves numerous C-U conversions, which often restore evolutionarily conserved codons and may generate new translation initiation and termination codons. These RNA maturation events rely on a subset of nuclear-encoded protein cofactors. Here, we provide evidence of the role of SlRIP1b on RNA editing of mitochondrial transcripts in tomato (Solanum lycopersicum) plants. SlRIP1b is a RIP/MORF protein that was originally identified as an interacting partner of the organellar editing factor SlORRM4. Mutants of SlRIP1b, obtained by CRISPR/Cas9 strategy, exhibited abnormal carpel development and grew into fruit with more locules. RNA-sequencing revealed that SlRIP1b affects the C-U editing of numerous mitochondrial pre-RNA transcripts and in particular altered RNA editing of various cytochrome c maturation (CCM)-related genes. The slrip1b mutants display increased H2 O2 and aberrant mitochondrial morphologies, which are associated with defects in cytochrome c biosynthesis and assembly of respiratory complex III. Taken together, our results indicate that SlRIP1b is a global editing factor that plays a key role in CCM and oxidative phosphorylation system biogenesis during fruit development in tomato plants. These data provide important insights into the molecular roles of organellar RNA editing factors during fruit development.

GRP23 plays a core role in E-type editosomes via interacting with MORFs and atypical PPR-DYWs in Arabidopsis mitochondria

Identifying the PPR-E+-NUWA-DYW2 editosome improves our understanding of the C-to-U RNA editing in plant organelles. However, the mechanism of RNA editing remains to be elucidated. Here, we report that GLUTAMINE-RICH PROTEIN23 (GRP23), a previously identified nuclear transcription regulator, plays an essential role in mitochondrial RNA editing through interacting with MORF (multiple organellar RNA-editing factor) proteins and atypical DYW-type pentatricopeptide repeat (PPR) proteins. GRP23 is targeted to mitochondria, plastids, and nuclei. Analysis of the grp23 mutants rescued by embryo-specific complementation shows decreased editing efficiency at 352 sites in mitochondria and 6 sites in plastids, with a predominant specificity for sites edited by the PPR-E and PPR-DYW proteins. GRP23 interacts with atypical PPR-DYW proteins (MEF8, MEF8S, DYW2, and DYW4) and MORF proteins (MORF1 and MORF8), whereas the four PPR-DYWs interact with the two MORFs. These interactions may increase the stability of the GRP23-MORF-atypical PPR-DYW complex. Furthermore, analysis of mef8N△64aamef8s double mutants shows that MEF8/MEF8S are required for the editing of the PPR-E protein-targeted sites in mitochondria. GRP23 could enhance the interaction between PPR-E and MEF8/MEF8S and form a homodimer or heterodimer with NUWA. Genetic complementation analysis shows that the C-terminal domains of GRP23 and NUWA possess a similar function, probably in the interaction with the MORFs. NUWA also interacts with atypical PPR-DYWs in yeast. Both GRP23 and NUWA interact with the atypical PPR-DYWs, suggesting that the PPR-E proteins recruit MEF8/MEF8S, whereas the PPR-E+ proteins specifically recruit DYW2 as the trans deaminase, and then GRP23, NUWA, and MORFs facilitate and/or stabilize the E or E+-type editosome formation.

RNA Editing and Its Roles in Plant Organelles.

RNA editing, a vital supplement to the central dogma, yields genetic information on RNA products that are different from their DNA templates. The conversion of C-to-U in mitochondria and plastids is the main kind of RNA editing in plants. Various factors have been demonstrated to be involved in RNA editing. In this minireview, we summarized the factors and mechanisms involved in RNA editing in plant organelles. Recently, the rapid development of deep sequencing has revealed many RNA editing events in plant organelles, and we further reviewed these events identified through deep sequencing data. Numerous studies have shown that RNA editing plays essential roles in diverse processes, such as the biogenesis of chloroplasts and mitochondria, seed development, and stress and hormone responses. Finally, we discussed the functions of RNA editing in plant organelles.

Plant organellar RNA editing: what 30years of research has revealed.

The central dogma in biology defines the flow of genetic information from DNA to RNA to protein. Accordingly, RNA molecules generally accurately follow the sequences of the genes from which they are transcribed. This rule is transgressed by RNA editing, which creates RNA products that differ from their DNA templates. Analyses of the RNA landscapes of terrestrial plants have indicated that RNA editing (in the form of C-U base transitions) is highly prevalent within organelles (that is, mitochondria and chloroplasts). Numerous C→U conversions (and in some plants also U→C) alter the coding sequences of many of the organellar transcripts and can also produce translatable mRNAs by creating AUG start sites or eliminating premature stop codons, or affect the RNA structure, influence splicing and alter the stability of RNAs. RNA-binding proteins are at the heart of post-transcriptional RNA expression. The C-to-U RNA editing process in plant mitochondria involves numerous nuclear-encoded factors, many of which have been identified as pentatricopeptide repeat (PPR) proteins that target editing sites in a sequence-specific manner. In this review we report on major discoveries on RNA editing in plant organelles, since it was first documented 30years ago.

Multifarious Evolutionary Pathways of a Nuclear RNA Editing Factor: Disjunctions in Coevolution of DOT4 and Its Chloroplast Target rpoC1eU488SL.

Nuclear-encoded pentatricopeptide repeat (PPR) proteins are site-specific factors for C-to-U RNA editing in plant organelles coevolving with their targets. Losing an editing target by C-to-T conversion allows for eventual loss of its editing factor, as recently confirmed for editing factors CLB19, CRR28, and RARE1 targeting ancient chloroplast editing sites in flowering plants. Here, we report on alternative evolutionary pathways for DOT4 addressing rpoC1eU488SL, a chloroplast editing site in the RNA polymerase β′ subunit mRNA. Upon loss of rpoC1eU488SL by C-to-T conversion, DOT4 got lost multiple times independently in angiosperm evolution with intermediate states of DOT4 orthologs in various stages of degeneration. Surprisingly, we now also observe degeneration and loss of DOT4 despite retention of a C in the editing position (in Carica, Coffea, Vicia, and Spirodela). We find that the cytidine remains unedited, proving that DOT4 was not replaced by another editing factor. Yet another pathway of DOT4 evolution is observed among the Poaceae. Although the rpoC1eU488SL edit has been lost through C-to-T conversion, DOT4 orthologs not only remain conserved but also have their array of PPRs extended by six additional repeats. Here, the loss of the ancient target has likely allowed DOT4 to adapt for a new function. We suggest rps3 antisense transcripts as previously demonstrated in barley (Hordeum vulgare) arising from promotor sequences newly emerging in the rpl16 intron of Poaceae as a new candidate target for the extended PPR stretch of DOT4. Altogether, DOT4 and its target show more flexible pathways for evolution than the previously explored editing factors CLB19, CRR28, and RARE1. Certain plant clades (e.g., Amaranthus, Vaccinium, Carica, the Poaceae, Fabales, and Caryophyllales) show pronounced dynamics in the evolution of editing sites and corresponding factors.

Protein Complexes Implicated in RNA Editing in Plant Organelles

Even after 30 years of active research, RNA editing in plant organelles remains an enigma. Why do most plants post-transcriptionally alter hundreds or thousands of nucleotides in organellar transcripts? How do they achieve such an effective and targeted process? What is known is that in almost all plants, RNA editing is essential for correct synthesis of the photosynthetic and respiratory machinery in mitochondria and chloroplasts. The sites to be edited are defined by a large class of sequence-specific RNA binding proteins: pentatricopeptide repeat (PPR) proteins (Barkan and Small, 2014).

Distinct role of Arabidopsis mitochondrial P-type pentatricopeptide repeat protein-modulating editing protein, PPME, in nad1 RNA editing

ABSTRACTThe mitochondrion is an important power generator in most eukaryotic cells. To preserve its function, many essential nuclear-encoded factors play specific roles in mitochondrial RNA metabolic processes, including RNA editing. RNA editing consists of post-transcriptional deamination, which alters specific nucleotides in transcripts to mediate gene expression. In plant cells, many pentatricopeptide repeat proteins (PPRs) participate in diverse organellar RNA metabolic processes, but only PLS-type PPRs are involved in RNA editing. Here, we report a P-type PPR protein from Arabidopsis thaliana, P-type PPR-Modulating Editing (PPME), which has a distinct role in mitochondrial nad1 RNA editing via RNA binding activity. In the homozygous ppme mutant, cytosine (C)-to-uracil (U) conversions at both the nad1-898 and 937 sites were abolished, disrupting Arg300-to-Trp300 and Pro313-to-Ser313 amino acid changes in the mitochondrial NAD1 protein. NAD1 is a critical component of mitochondrial respiration complex I; its activity is severely reduced in the homozygous ppme mutant, resulting in significantly altered growth and development. Both abolished RNA editing and defective complex I activity were completely rescued by CaMV 35S promoter- and PPME native promoter-driven PPME genomic fragments tagged with GFP in a homozygous ppme background. Our experimental results demonstrate a distinct role of a P-type PPR protein, PPME, in RNA editing in plant organelles.

A DYW‐protein knockout in Physcomitrella affects two closely spaced mitochondrial editing sites and causes a severe developmental phenotype

RNA-binding pentatricopeptide repeat (PPR) proteins carrying a carboxy-terminal DYW domain similar to cytidine deaminases have been characterized as site-specific factors for C-to-U RNA editing in plant organelles. Here we report that knockout of DYW-PPR_65 in Physcomitrella patens causes a severe developmental phenotype in the moss and specifically affects two editing sites located 18 nucleotides apart on the mitochondrial ccmFC mRNA. Intriguingly, PPR_71, another DYW-type PPR, had been identified previously as an editing factor specifically affecting only the downstream editing site, ccmFCeU122SF. The now characterized PPR_65 binds specifically only to the upstream target site, ccmFCeU103PS, in full agreement with a recent RNA-recognition code for PPR arrays. The functional interference between the two editing events may be caused by a combination of three factors: (i) the destabilization of an RNA secondary structure interfering with PPR_71 binding by prior binding of PPR_65; (ii) the resulting upstream C-U conversion; or (iii) a direct interaction between the two DYW proteins. Indeed, we find the Physcomitrella DYW-PPRs to interact in yeast-two-hybrid assays. The moss DYW-PPRs also interact yet more strongly with MORF (Multiple Organellar RNA editing Factor)/RIP (RNA editing factor interacting proteins) proteins of Arabidopsis known to be general editing factors in flowering plants, although MORF homologues are entirely absent in the moss. Finally, we demonstrate binding of Physcomitrella DYW-PPR_98, for which no KO lines could be raised, to its predicted target sequence upstream of editing site atp9eU92SL. Together with the functional characterization of DYW-PPR_65, this completes the assignment of RNA editing factors to all editing sites in the Physcomitrella mitochondrial transcriptome.

Improved Computational Target Site Prediction for Pentatricopeptide Repeat RNA Editing Factors

Pentatricopeptide repeat (PPR) proteins with an E domain have been identified as specific factors for C to U RNA editing in plant organelles. These PPR proteins bind to a unique sequence motif 5′ of their target editing sites. Recently, involvement of a combinatorial amino acid code in the P (normal length) and S type (short) PPR domains in sequence specific RNA binding was reported. PPR proteins involved in RNA editing, however, contain not only P and S motifs but also their long variants L (long) and L2 (long2) and the S2 (short2) motifs. We now find that inclusion of these motifs improves the prediction of RNA editing target sites. Previously overlooked RNA editing target sites are suggested from the PPR motif structures of known E-class PPR proteins and are experimentally verified. RNA editing target sites are assigned for the novel PPR protein MEF32 (mitochondrial editing factor 32) and are confirmed in the cDNA.

Contiguous RNA editing sites in the mitochondrial nad1 transcript of Arabidopsis thaliana are recognized by different proteins

Pentatricopeptide repeat (PPR) proteins have been identified as site-specific factors for RNA editing in plant organelles. These proteins recognize cis-elements near the editing site. It is unclear how contiguous sites are addressed, and whether one or two factors are required. We here show the PPR MEF25 to be essential for RNA editing at the nad1-308 site in Arabidopsis mitochondria. Another editing site just one nucleotide upstream, nad1-307, is edited normally in mef25 mutant lines. This finding shows that two independent factors recognizing similar cis-elements are involved at these contiguous sites without competing with each other in vivo.

The nucleocytoplasmic conflict, a driving force for the emergence of plant organellar RNA editing

RNA editing challenges the central dogma of molecular biology by changing the genetic information at the transcript level. In plant organelles, RNAs are modified by deamination of some specific cytosine residues, but the origin of this process remains puzzling. Different from the generally accepted neutral model to explain the emergence of RNA editing in plant organelles, we propose a new hypothesis based on the nucleocytoplasmic conflict theory. We assume that mutations in organellar genomes arose first and spread into the population provided they increased the transmission of their own maternally inherited genome. RNA editing appeared subsequently as a nuclear-encoded correction mechanism to restore the transmission of the nuclear genome. In plants, a well-known consequence of the nucleocytoplasmic conflict is cytoplasmic male sterility (CMS) which is counteracted by the emergence of fertility restorer genes (Rf) belonging to the pentatricopeptide repeat (PPR) protein family. Interestingly, RNA-editing deficiency can lead to CMS, and it now clearly appears that PPR proteins are major players in RNA editing. This striking similarity between the mechanisms of fertility restoration and RNA editing can be explained if both reactions are the consequence of the same driving force, the nucleocytoplasmic conflict. Similarly, the prevalence of RNA editing in eukaryotic organellar genomes could also be a consequence of the genetic antagonism between organellar and nuclear genomes.

RNA editing in plant organelles. Why make it easy?

Gene expression in plant organelles involves a number of distinct co- or posttranscriptional nucleic acid modifications: 5' and 3' RNA processing, cis- and trans-splicing, RNA stability, and RNA editing. All contribute to the steady-state RNA levels available for the translation of the reduced but essential organellar genetic information. Different from other maturation processes, RNA editing at the transcript level modifies the information encoded by organellar genes and is an essential step for the production of functional proteins. Editing changes are extensive in mitochondria from flowering plants with more than 400 cytidine-to-uridine changes that involve most transcripts, while in chloroplasts they are limited to some RNAs. An additional U-to-C RNA editing reaction is observed with the C-to-U transitions in fern and moss organelles. While RNA editing targets mostly concern coding regions, some events occur in untranslated regions. Whereas RNA editing is genetically and biochemically distinct from other RNA modification activities, evidence is growing for a tight connection between the different processing events. Although the understanding of this astonishing mechanism has increased since its discovery in 1989, some important questions remain unanswered. In this review we discuss the current knowledge on the different aspects of C-to-U, and to a lesser extent U-to-C, and look at RNA editing in plants with a particular emphasis on recent developments involving the role of pentatricopeptide repeat (PPR) proteins in this process.

The moss Physcomitrella patens, a model plant for the study of RNA editing in plant organelles

RNA editing is an enigmatic phenomenon in which specific cytidines (C) in the transcripts are changed to uridines (U). In flowering plants, over 500 editing sites have been identified in the mitochondria and plastids. By contrast, in a moss Physcomitrella patens, only 12 editing sites are found in both organelles. Recent extensive genetics studies have revealed involvement of the pentatricopeptide repeat (PPR) proteins with a C-terminal DYW domain (PPR-DYW) in site-specific RNA editing events. Flowering plants have ~100 PPR-DYW genes while P. patens has only 10 PPR-DYW genes. Thus, the number of PPR-DYW genes is somewhat related to the number of RNA editing sites. The P. patens gametophyte, the haploid phase of the life cycle, is dominant, making it possible to study the phenotype of knockouts directly after transformation for gene-targeting. This makes it easier to identify the PPR-DYW proteins required for RNA editing. Recently, we have shown that one of 10 PPR-DYW proteins, PpPPR_71, was responsible for RNA editing in the mitochondrial ccmFc mRNA. In this study, we propose a working model of PpPPR_71 function on RNA editing.

RNA editing in plant mitochondria: 20 years later

In 1989, three laboratories (in Canada, France and Germany) independently and simultaneously reported the discovery of C-to-U RNA editing in plant mitochondria (1-3). To mark the 20th anniversary of this finding, the leaders of the three research teams have written personal essays describing the events leading up to the discovery in each of their laboratories. These essays are intended not only to capture historical facts but also to illustrate unexpected convergence in the process of scientific discovery, with different groups coming to the same conclusion, often very close together in time, drawing on different types of evidence and via sometimes quite different hypotheses and approaches. Essential background information pertaining to RNA editing in general and RNA editing in plant organelles in particular is provided in this overview.

Multiplex single-base extension typing to identify nuclear genes required for RNA editing in plant organelles

We developed a multiplex single-base extension single-nucleotide polymorphism-typing procedure for screening large numbers of plants for mutations in mitochondrial RNA editing. The high sensitivity of the approach detects changes in the RNA editing status generated in total cellular cDNA from pooled RNA preparations of up to 50 green plants. The method has been employed to tag several nuclear encoded genes required for RNA editing at specific sites in mitochondria of Arabidopsis thaliana. This approach will allow large-scale screening for mutations in genes encoding trans-factors for many types of RNA editing as well as for other RNA modifications.

A conserved DYW domain of the pentatricopeptide repeat protein possesses a novel endoribonuclease activity

Many plant pentatricopeptide repeat (PPR) proteins are known to contain a highly conserved C-terminal DYW domain whose function is unknown. Recently, the DYW domain has been proposed to play a role in RNA editing in plant organelles. To address this possibility, we prepared recombinant DYW proteins and tested their cytidine deaminase activity. However, we could not detect any activity in the assays we used. Instead, we found that the recombinant DYW domains possessed endoribonuclease activity and cleaved before adenosine residues in the RNA molecule. Some DYW-containing PPR proteins may catalyze site-specific cleavage of target RNA species.

Correlation between amino acid residues converted by RNA editing and functional residues in protein three-dimensional structures in plant organelles

BackgroundIn plant organelles, specific messenger RNAs (mRNAs) are subjected to conversion editing, a process that often converts the first or second nucleotide of a codon and hence the encoded amino acid. No systematic patterns in converted sites were found on mRNAs, and the converted sites rarely encoded residues located at the active sites of proteins. The role and origin of RNA editing in plant organelles remain to be elucidated.ResultsHere we study the relationship between amino acid residues encoded by edited codons and the structural characteristics of these residues within proteins, e.g., in protein-protein interfaces, elements of secondary structure, or protein structural cores. We find that the residues encoded by edited codons are significantly biased toward involvement in helices and protein structural cores. RNA editing can convert codons for hydrophilic to hydrophobic amino acids. Hence, only the edited form of an mRNA can be translated into a polypeptide with helix-preferring and core-forming residues at the appropriate positions, which is often required for a protein to form a functional three-dimensional (3D) structure.ConclusionWe have performed a novel analysis of the location of residues affected by RNA editing in proteins in plant organelles. This study documents that RNA editing sites are often found in positions important for 3D structure formation. Without RNA editing, protein folding will not occur properly, thus affecting gene expression. We suggest that RNA editing may have conferring evolutionary advantage by acting as a mechanism to reduce susceptibility to DNA damage by allowing the increase in GC content in DNA while maintaining RNA codons essential to encode residues required for protein folding and activity.

A hypothesis on the identification of the editing enzyme in plant organelles

RNA editing in plant organelles is an enigmatic process leading to conversion of cytidines into uridines. Editing specificity is determined by proteins; both those known so far are pentatricopeptide repeat (PPR) proteins. The enzyme catalysing RNA editing in plants is still totally unknown. We propose that the DYW domain found in many higher plant PPR proteins is the missing catalytic domain. This hypothesis is based on two compelling observations: (i) the DYW domain contains invariant residues that match the active site of cytidine deaminases; (ii) the phylogenetic distribution of the DYW domain is strictly correlated with RNA editing.

RNA editing in plant organelles: machinery, physiological function and evolution

In plants, RNA editing is a process for converting a specific nucleotide of RNA from C to U and less frequently from U to C in mitochondria and plastids. To specify the site of editing, the cis-element adjacent to the editing site functions as a binding site for the trans-acting factor. Genetic approaches using Arabidopsis thaliana have clarified that a member of the protein family with pentatricopeptide repeat (PPR) motifs is essential for RNA editing to generate a translational initiation codon of the chloroplast ndhD gene. The PPR motif is a highly degenerate unit of 35 amino acids and appears as tandem repeats in proteins that are involved in RNA maturation steps in mitochondria and plastids. The Arabidopsis genome encodes approximately 450 members of the PPR family, some of which possibly function as trans-acting factors binding the cis-elements of the RNA editing sites to facilitate access of an unidentified RNA editing enzyme. Based on this breakthrough in the research on plant RNA editing, I would like to discuss the possible steps of co-evolution of RNA editing events and PPR proteins.