The Gating Domain of MEF28 is Essential for Editing Two Contiguous Cytidines in nad2 mRNA in Arabidopsis thaliana.

In plant organelles, each C-to-U RNA editing site is specifically recognized by PLS class pentatricopeptide repeat (PPR) proteins with E1-E2, E1-E2-E+, or E1-E2-DYW domain extensions at the C-terminus. The distance between the PPR domain binding site and the RNA editing site is usually fixed at four bases, increasing the specificity of target site recognition in this system. We here report, in contrast to the general case, on MEF28, which edits two adjacent mitochondrial sites, nad2-89 and nad2-90. When the sDYW domain of MEF28 was replaced with one derived from MEF11 or CRR22, the ability to edit downstream sites was lost, suggesting that the DYW domain of MEF28 provides unique target flexibility for two continuous cytidines. By contrast, substitutions of the entire E1-E2-DYW domains by MEF19E1-E2, SLO2E1-E2-E+, or the CRR22E1-E2-E+ target both nad2 sites. In these cases, access to the contiguous sites in the chimeric PPR proteins is likely to be provided by the trans-associated DYW1-like proteins via the replaced E1-E2 or E1-E2-E+ domains. Furthermore, we demonstrated that the gating domain of MEF28 plays an important role in specific target site recognition of the DYW domain. This finding suggests that the DYW domain and its internal gating domain fine-tune the specificity of the target site, which is valuable information for designing specific synthetic RNA editing tools based on plant RNA editing factors.

DYW cytidine deaminase domains have a long-range impact on RNA recognition by the PPR array of chimeric plant C-to-U RNA editing factors and strongly affect target selection.

The protein factors for the specific C-to-U RNA editing events in plant mitochondria and chloroplasts possess unique arrays of RNA-binding pentatricopeptide repeats (PPRs) linked to carboxy-terminal cytidine deaminase DYW domains via the extension motifs E1 and E2. The E1 and E2 motifs have distant similarities to tetratricopeptide repeats known to mediate protein-protein interactions but their precise function is unclear. Here, we investigate the tolerance of PPR56 and PPR65, two functionally characterized RNA editing factors of the moss Physcomitrium patens, for the creation of chimeras by variably replacing their C-terminal protein regions. Making use of a heterologous RNA editing assay system in Escherichia coli we find that heterologous DYW domains can strongly restrict or widen the spectrum of off-targets in the bacterial transcriptome for PPR56. Surprisingly, our data suggest that these changes are not only caused by the preference of a given heterologous DYW domain for the immediate sequence environment of the cytidine to be edited but also by a long-range impact on the nucleotide selectivity of the upstream PPRs.

The MITOCHONDRIAL TRANSCRIPT STABILITY FACTOR 4 (MTSF4) is essential for the accumulation of dicistronic rpl5-cob mRNAs in Arabidopsis thaliana.

The Arabidopsis thaliana genome harbors more than 450 nuclear genes encoding pentatricopeptide repeat (PPR) proteins that operate in the RNA metabolism of mitochondria and/or plastids. To date, the molecular function of many PPR proteins is still unknown. Here we analyzed the nucleus-encoded gene At4g19440 coding for a P-type PPR protein. Knockout of this gene interferes with normal embryo development and seed maturation. Two experimental approaches were applied to overcome lethality and to investigate the outcome of At4g19440 knockout in adult plants. These studies revealed changes in the abundance of several mitochondria-encoded transcripts. In particular, steady-state levels of dicistronic rpl5-cob RNAs were markedly reduced, whereas levels of mature ccmC and rpl2-mttB transcripts were clearly increased. Predictions according to the one repeat to one nucleotide code for PPR proteins indicate binding of the At4g19440 protein to a previously detected small RNA at the 3' termini of the dicistronic rpl5-cob transcripts. This potential interaction indicates a function of this protein in 3' end formation and stabilization of these RNA species, whereas the increase in the levels of the ccmC mRNA along with other mitochondria-encoded RNAs seems to be a secondary effect of At4g19440 knockout. Since the inactivation of At4g19440 influences the stability of several mitochondrial RNAs we call this gene MITOCHONDRIAL TRANSCRIPT STABILITY FACTOR 4 (MTSF4). This factor will be an interesting subject to study opposing effects of a single nucleus-encoded protein on mitochondrial transcript levels.

Structural insight into the activation of an Arabidopsis organellar C-to-U RNA editing enzyme by active site complementation.

RNA-binding pentatricopeptide repeat (PPR) proteins catalyze hundreds of cytidine to uridine RNA editing events in plant organelles; these editing events are essential for proper gene expression. More than half of the PPR-type RNA editing factors, however, lack the DYW cytidine deaminase domain. Genetic analyses have suggested that their cytidine deaminase activity arises by association with a family of DYW1-like proteins that contain an N-terminally truncated DYW domain, but their molecular mechanism has been unclear. Here, we report the crystal structure of the Arabidopsis thaliana DYW1 deaminase domain at 1.8 Å resolution. DYW1 has a cytidine deaminase fold lacking the PG box. The internal insertion within the deaminase fold shows an α-helical fold instead of the ß-finger reported for the gating domain of the A. thaliana ORGANELLE TRANSCRIPT PROCESSING 86. The substrate-binding pocket is incompletely formed and appears to be complemented in the complex by the E2 domain and the PG box of the interacting PPR protein. In vivo RNA editing assays corroborate the activation model for DYW1 deaminase. Our study demonstrates the common activation mechanism of the DYW1-like proteins by molecular complementation of the DYW domain and reconstitution of the substrate-binding pocket.

DYW deaminase domain has a distinct preference for neighboring nucleotides of the target RNA editing sites.

C-to-U RNA editing sites in plant organelles show a strong bias for neighboring nucleotides. The nucleotide upstream of the target cytidine is typically C or U, whereas A and G are less common and rare, respectively. In pentatricopeptide repeat (PPR)-type RNA editing factors, the PPR domain specifically binds to the 5' sequence of target cytidines, whereas the DYW domain catalyzes the C-to-U deamination. We comprehensively analyzed the effects of neighboring nucleotides of the target cytidines using an Escherichia coli orthogonal system. Physcomitrium PPR56 efficiently edited target cytidines when the nucleotide upstream was U or C, whereas it barely edited when the position was G or the nucleotide downstream was C. This preference pattern, which corresponds well with the observed nucleotide bias for neighboring nucleotides in plant organelles, was altered when the DYW domain of OTP86 or DYW1 was adopted. The PPR56 chimeric proteins edited the target sites even when the -1 position was G. Our results suggest that the DYW domain possesses a distinct preference for the neighboring nucleotides of the target sites, thus contributing to target selection in addition to the existing selection determined by the PPR domain.

Targeted base editing in the mitochondrial genome of Arabidopsis thaliana.

Beyond their well-known role in respiration, mitochondria of land plants contain biologically essential and/or agriculturally important genes whose function and regulation are not fully understood. Until recently, it has been difficult to analyze these genes or, in the case of crops, to improve their functions, due to a lack of methods for stably modifying plant mitochondrial genomes. In rice, rapeseed, and Arabidopsis thaliana, mitochondria-targeting transcription activator-like effector nucleases (mitoTALENs) have recently been used to disrupt targeted genes in an inheritable and stable manner. However, this technique can also induce large deletions around the targeted sites, as well as cause ectopic homologous recombinations, which can change the sequences and gene order of mitochondrial genomes. Here, we used mitochondria-targeting TALEN-based cytidine deaminase to successfully substitute targeted C:G pairs with T:A pairs in the mitochondrial genomes of plantlets of A. thaliana without causing deletions or changes in genome structure. Expression vectors of the base editor genes were stably introduced into the nuclear genome by the easy-to-use floral dipping method. Some T1 plants had apparent homoplasmic substitutions that were stably inherited by seed progenies, independently of the inheritance of nuclear-introduced genes. As a demonstration of the method, we used it to restore the growth of an organelle transcript processing 87 (otp87) mutant that is defective in the editing of RNA transcripts of the mitochondrial atp1 gene and to identify bases in atp1 that affect the efficiency of RNA editing by OTP87.

Quantification of Mitochondrial RNA Editing Efficiency Using Sanger Sequencing Data.

C-to-U RNA editing in mitochondria and plastids is widespread in almost all terrestrial plants, where it mainly changes codons to encode conserved amino acids in organelle mRNAs. In flowering plants, the number of RNA editing sites reaches 400-600 in mitochondria and about 40 in plastids, respectively. To date, more than 100 factors involved in RNA editing have been identified. Since target cytidines of each factor are often distributed across multiple transcripts, comprehensive monitoring of all RNA editing sites is necessary for their characterization. Comparing the signals of C and T in the Sanger sequencing chromatogram of RT-PCR products is the most frequently employed method for quantification of RNA editing efficiency, although several methods based on next-generation sequencing have been developed. I here describe a quick and easy method for quantification of RNA editing efficiency at several hundred sites using the Sanger sequencing chromatogram data.

DYW domain structures imply an unusual regulation principle in plant organellar RNA editing catalysis.

RNA editosomes selectively deaminate cytidines to uridines in plant organellar transcripts-mostly to restore protein functionality and consequently facilitate mitochondrial and chloroplast function. The RNA editosomal pentatricopeptide repeat proteins serve target RNA recognition, whereas the intensively studied DYW domain elicits catalysis. Here we present structures and functional data of a DYW domain in an inactive ground state and activated. DYW domains harbour a cytidine deaminase fold and a C-terminal DYW motif, with catalytic and structural zinc atoms, respectively. A conserved gating domain within the deaminase fold regulates the active site sterically and mechanistically in a process that we termed gated zinc shutter. Based on the structures, an autoinhibited ground state and its activation are cross-validated by RNA editing assays and differential scanning fluorimetry. We anticipate that, in vivo, the framework of an active plant RNA editosome triggers the release of DYW autoinhibition to ensure a controlled and coordinated cytidine deamination playing a key role in mitochondrial and chloroplast homeostasis.

A single nucleotide substitution in the coding region of Ogura male sterile gene, orf138, determines effectiveness of a fertility restorer gene, Rfo, in radish.

Cytoplasmic male sterility (CMS) observed in many plants leads defect in the production of functional pollen, while the expression of CMS is suppressed by a fertility restorer gene in the nuclear genome. Ogura CMS of radish is induced by a mitochondrial orf138, and a fertility restorer gene, Rfo, encodes a P-type PPR protein, ORF687, acting at the translational level. But, the exact function of ORF687 is still unclear. We found a Japanese variety showing male sterility even in the presence of Rfo. We examined the pollen fertility, Rfo expression, and orf138 mRNA in progenies of this variety. The progeny with Type H orf138 and Rfo showed male sterility when their orf138 mRNA was unprocessed within the coding region. By contrast, all progeny with Type A orf138 were fertile though orf138 mRNA remained unprocessed in the coding region, demonstrating that ORF687 functions on Type A but not on Type H. In silico analysis suggested a specific binding site of ORF687 in the coding region, not the 5' untranslated region estimated previously, of Type A. A single nucleotide substitution in the putative binding site diminishes affinity of ORF687 in Type H and is most likely the cause of the ineffectiveness of ORF687. Furthermore, fertility restoration by RNA processing at a novel site in some progeny plants indicated a new and the third fertility restorer gene, Rfs, for orf138. This study clarified that direct ORF687 binding to the coding region of orf138 is essential for fertility restoration by Rfo.

The Pentatricopeptide Repeat Protein PGR3 Is Required for the Translation of petL and ndhG by Binding Their 5' UTRs.

PGR3 is a P-class pentatricopeptide repeat (PPR) protein required for the stabilization of petL operon RNA and the translation of the petL gene in plastids. Irrespective of its important roles in plastids, key questions have remained unanswered, including how PGR3 protein promotes translation and which plastid mRNA PGR3 activates the translation. Here, we show that PGR3 facilitates the translation from ndhG, in addition to petL, through binding to their 5' untranslated regions (UTRs). Ribosome profiling and RNA sequencing in pgr3 mutants revealed that translation from petL and ndhG was specifically suppressed. Harnessing small RNA fragments protected by PPR proteins in vivo, we probed the PGR3 recruitment to the 5' UTRs of petL and ndhG. The putative PGR3-bound RNA segments per se repress the translation possibly with a strong secondary structure and thereby block ribosomes' access. However, the PGR3 binding antagonizes the effects and facilitates the protein synthesis from petL and ndhG in vitro. The prediction of the 3-dimensional structure of PGR3 suggests that the 26th PPR motif plays important roles in target RNA binding. Our data show the specificity of a plastidic RNA-binding protein and provide a mechanistic insight into translational control.

One C-to-U RNA Editing Site and Two Independently Evolved Editing Factors: Testing Reciprocal Complementation with DYW-Type PPR Proteins from the Moss Physcomitrium (Physcomitrella) patens and the Flowering Plants Macadamia integrifolia and Arabidopsis.

Cytidine-to-uridine RNA editing is a posttranscriptional process in plant organelles, mediated by specific pentatricopeptide repeat (PPR) proteins. In angiosperms, hundreds of sites undergo RNA editing. By contrast, only 13 sites are edited in the moss Physcomitrium (Physcomitrella) patens Some are conserved between the two species, like the mitochondrial editing site nad5eU598RC. The PPR proteins assigned to this editing site are known in both species: the DYW-type PPR protein PPR79 in P. patens and the E+-type PPR protein CWM1 in Arabidopsis (Arabidopsis thaliana). CWM1 also edits sites ccmCeU463RC, ccmBeU428SL, and nad5eU609VV. Here, we reciprocally expressed the P. patens and Arabidopsis editing factors in the respective other genetic environment. Surprisingly, the P. patens editing factor edited all target sites when expressed in the Arabidopsis cwm1 mutant background, even when carboxy-terminally truncated. Conversely, neither Arabidopsis CWM1 nor CWM1-PPR79 chimeras restored editing in P. patens ppr79 knockout plants. A CWM1-like PPR protein from the early diverging angiosperm macadamia (Macadamia integrifolia) features a complete DYW domain and fully rescued editing of nad5eU598RC when expressed in P. patens. We conclude that (1) the independently evolved P. patens editing factor PPR79 faithfully operates in the more complex Arabidopsis editing system, (2) truncated PPR79 recruits catalytic DYW domains in trans when expressed in Arabidopsis, and (3) the macadamia CWM1-like protein retains the capacity to work in the less complex P. patens editing environment.

MEF46 and MEF47 are novel specificity factors for RNA editing sites in mitochondrial nad transcripts.

Terrestrial plants have C-to-U RNA editing in the transcripts of plastids and mitochondria. Target specificity for more than several hundred editing sites are governed by PLS (PPR, Long and Short) class Pentatricopeptide repeat (PPR) proteins with additional C-terminal domains. Half of these PPR proteins have DYW (Aspartate (D), Tyrosine (Y) and Tryptophan (W)) domains, which most likely harbour cytidine deaminase activity. The other half of them, E subclass and E+ subclass proteins, contain no or only a part of the DYW domain. Missing DYW domains in the E and E+ subclass PPR proteins are likely to be complemented by other DYW containing proteins. All target sites of so far characterized E+ subclass PPR proteins show defects in dyw2 mutants, suggesting that the DYW2 protein complements the missing DYW domains in the E+ subclass PPR proteins. Here we report two novel RNA editing factors, MEF46 and MEF47, which belong to E+ and E subclass, respectively. The defective editing site in mef46, nad5-1958, overlaps with the affected sites in dyw2 mutants, while that in mef47, nad3-64 and ccmC-614 do not, further supporting the specific functional connection between E+ subclass PPR proteins and DYW2.

Mitochondrial Pentatricopeptide Repeat Protein, EMB2794, Plays a Pivotal Role in NADH Dehydrogenase Subunit nad2 mRNA Maturation in Arabidopsis thaliana.

The Arabidopsis genome encodes >450 proteins containing the pentatricopeptide repeat (PPR) motif. The PPR proteins are classified into two groups, termed as P and P Long-Short (PLS) classes. Typically, the PLS subclass proteins are mainly involved in the RNA editing of mitochondrial and chloroplast transcripts, whereas most of the analyzed P subclass proteins have been mainly implicated in RNA metabolism, such as 5' or 3' transcript stabilization and processing, splicing and translation. Mutations of PPR genes often result in embryogenesis and altered seedling developmental defect phenotypes, but only a limited number of ppr mutants have been characterized in detail. In this report, we show that null mutations in the EMB2794 gene result in embryo arrest, due to altered splicing of nad2 transcripts in the Arabidopsis mitochondria. In angiosperms, nad2 has five exons that are transcribed individually from two mitochondrial DNA regions. Biochemical and in vivo analyses further indicate that recombinant or transgenic EMB2794 proteins bind to the nad2 pre-mRNAs in vitro as well as in vivo, suggesting a role for this protein in trans-splicing of nad2 intron 2 and possibly in the stability of the second pre-mRNA of nad2. Homozygous emb2794 lines, showing embryo-defective phenotypes, can be partially rescued by the addition of sucrose to the growth medium. Mitochondria of rescued homozygous mutant plants contain only traces of respiratory complex I, which lack the NADH-dehydrogenase activity.

The Analysis of the Editing Defects in the dyw2 Mutant Provides New Clues for the Prediction of RNA Targets of Arabidopsis E+-Class PPR Proteins.

C to U editing is one of the post-transcriptional steps which are required for the proper expression of chloroplast and mitochondrial genes in plants. It depends on several proteins acting together which include the PLS-class pentatricopeptide repeat proteins (PPR). DYW2 was recently shown to be required for the editing of many sites in both organelles. In particular almost all the sites associated with the E+ subfamily of PPR proteins are depending on DYW2, suggesting that DYW2 is required for the function of E+-type PPR proteins. Here we strengthened this link by identifying 16 major editing sites controlled by 3 PPR proteins: OTP90, a DYW-type PPR and PGN and MEF37, 2 E+-type PPR proteins. A re-analysis of the DYW2 editotype showed that the 49 sites known to be associated with the 18 characterized E+-type PPR proteins all depend on DYW2. Considering only the 288 DYW2-dependent editing sites as potential E+-type PPR sites, instead of the 795 known editing sites, improves the performances of binding predictions systems based on the PPR code for E+-type PPR proteins. However, it does not compensate for poor binding predictions.

Plant organellar RNA editing: what 30 years 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 30 years ago.

RNA editing mutants as surrogates for mitochondrial SNP mutants.

In terrestrial plants, RNA editing converts specific cytidines to uridines in mitochondrial and plastidic transcripts. Most of these events appear to be important for proper function of organellar encoded genes, since translated proteins from edited mRNAs show higher similarity with evolutionary conserved polypeptide sequences. So far about 100 nuclear encoded proteins have been characterized as RNA editing factors in plant organelles. Respective RNA editing mutants reduce or lose editing activity at different sites and display various macroscopic phenotypes from pale or albino in the case of chloroplasts to growth retardation or even embryonic lethality. Therefore, RNA editing mutants can be a useful resource of surrogate mutants for organellar encoded genes, especially for mitochondrially encoded genes that it is so far unfeasible to manipulate. However, connections between RNA editing defects and observed phenotypes in the mutants are often hard to elucidate, since RNA editing factors often target multiple RNA sites in different genes simultaneously. In this review article, we summarize the physiological aspects of respective RNA editing mutants and discuss them as surrogate mutants for functional analysis of mitochondrially encoded genes.

The Yeast Three-Hybrid System for Protein Interactions.

Proteins rarely act alone as their functions tend to be regulated in vivo. Therefore, protein-protein interaction analyses provide key clues for understanding the complex biological processes in the living cell. Several techniques have been developed to elucidate the conformation of large protein complexes, dynamic protein complex rearrangement and transient protein interactions. Yeast two-hybrid system is a well-established method to analyze binary protein interactions. Here we describe a basic yeast three-hybrid method, which represents an additional refinement of the classical yeast two-hybrid system for analyzing further complex interactions among three proteins.

The Pentatricopeptide Repeat Protein MEF31 is Required for Editing at Site 581 of the Mitochondrial tatC Transcript and Indirectly Influences Editing at Site 586 of the Same Transcript.

Pentatricopeptide repeat (PPR) proteins constitute the largest family of proteins in angiosperms, and most members are predicted to play roles in the maturation of organellar RNAs. Here we describe the novel mitochondrial editing factor 31 (MEF31), an E-PPR protein involved in editing at two close sites in the same transcript encoding subunit C of the twin-arginine translocation (tat) pathway. MEF31 is essential for editing at site tatC-581 and application of the recently proposed amino acid code for RNA recognition by PPR proteins supports the view that MEF31 directly targets this site by recognizing its cis sequence. In contrast, editing at site tatC-586 five nucleotides downstream is only partially affected in plants lacking MEF31, being restored to wild-type levels in complemented plants. Application of the amino acid code and analysis of individual RNA molecules for editing at sites 581 and 586 suggest that MEF31 does not directly target site tatC-586, and only indirectly influences editing at this site. It is likely that editing at site tatC-581 improves recognition of the site tatC-586 cis sequence by a second unknown PPR protein.

Two interacting PPR proteins are major Arabidopsis editing factors in plastid and mitochondria.

RNA editing is converting hundreds of cytosines into uridines during organelle gene expression of land plants. The pentatricopeptide repeat (PPR) proteins are at the core of this posttranscriptional RNA modification. Even if a PPR protein defines the editing site, a DYW domain of the same or another PPR protein is believed to catalyze the deamination. To give insight into the organelle RNA editosome, we performed tandem affinity purification of the plastidial CHLOROPLAST BIOGENESIS 19 (CLB19) PPR editing factor. Two PPR proteins, dually targeted to mitochondria and chloroplasts, were identified as potential partners of CLB19. These two proteins, a P-type PPR and a member of a small PPR-DYW subfamily, were shown to interact in yeast. Insertional mutations resulted in embryo lethality that could be rescued by embryo-specific complementation. A transcriptome analysis of these complemented plants showed major editing defects in both organelles with a very high PPR type specificity, indicating that the two proteins are core members of E+-type PPR editosomes.