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.

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.

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.

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.

PCIS1, Encoded by a Pentatricopeptide Protein Co-expressed Gene, Is Required for Splicing of Three Mitochondrial nad Transcripts in Angiosperms.

Group II introns are large catalytic RNAs, which reside mainly within genes encoding respiratory complex I (CI) subunits in angiosperms' mitochondria. Genetic and biochemical analyses led to the identification of many nuclear-encoded factors that facilitate the splicing of the degenerated organellar introns in plants. Here, we describe the analysis of the pentatricopeptide repeat (PPR) co-expressed intron splicing-1 (PCIS1) factor, which was identified in silico by its co-expression pattern with many PPR proteins. PCIS1 is well conserved in land plants but has no sequence similarity with any known protein motifs. PCIS1 mutant lines are arrested in embryogenesis and can be maintained by the temporal expression of the gene under the embryo-specific ABI3 promoter. The pABI3::PCIS1 mutant plants display low germination and stunted growth phenotypes. RNA-sequencing and quantitative RT-PCR analyses of wild-type and mutant plants indicated that PCIS1 is a novel splicing cofactor that is pivotal for the maturation of several nad transcripts in Arabidopsis mitochondria. These phenotypes are tightly associated with respiratory CI defects and altered plant growth. Our data further emphasize the key roles of nuclear-encoded cofactors that regulate the maturation and expression of mitochondrial transcripts for the biogenesis of the oxidative phosphorylation system, and hence for plant physiology. The discovery of novel splicing factors other than typical RNA-binding proteins suggests further complexity of splicing mechanisms in plant mitochondria.

Identification and variation of a new restorer of fertility gene that induces cleavage in orf138 mRNA of Ogura male sterility in radish.

KEY MESSAGE: A new restorer of fertility gene, Rfs, of Ogura cytoplasmic male sterility (CMS) in radish encodes a pentatricopeptide repeat protein that binds to 15 nucleotides in mRNA of the CMS gene, orf138. Nucleotide substitutions in both Rfs and orf138 determine effectiveness and specificity of restoration. Cytoplasmic male sterility (CMS) in plants caused by the expression of abnormal mitochondrial genes results from impaired pollen production. The manifestation of CMS is suppressed by the restorer of fertility (Rf) genes in the nuclear genome. Thus, the CMS-Rf system is a suitable model for studying the direct interactions of mitochondrial and nuclear genes. At least nine haplotypes, of which Type B is ancestry, have been reported for the Ogura CMS gene, orf138, in radish (Raphanus sativus). We previously observed that Rfo encoding a pentatricopeptide repeat (PPR) protein, ORF687, which inhibits the translation of orf138 is ineffective in one haplotype (i.e., Type H). Here, we carried out map-based cloning of another Rf gene (Rfs) that cleaves the orf138 mRNA of Type H. Rfs produces a PPR protein consisting of 15 PPR motifs that binds to the mRNA, cleaving the mRNA at about 50nt downstream of the binding site. However, Rfs was ineffective for Type A because of a single nucleotide substitution in the binding site. Both Rfo and Rfs suppress orf138 expression in ancestral Type B, but they are rendered ineffective in Type H and Type A, respectively, by a single nucleotide substitution in orf138.

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.

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.

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.

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.

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.

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.

RNA editing in plants and its evolution.

RNA editing alters the identity of nucleotides in RNA molecules such that the information for a protein in the mRNA differs from the prediction of the genomic DNA. In chloroplasts and mitochondria of flowering plants, RNA editing changes C nucleotides to U nucleotides; in ferns and mosses, it also changes U to C. The approximately 500 editing sites in mitochondria and 40 editing sites in plastids of flowering plants are individually addressed by specific proteins, genes for which are amplified in plant species with organellar RNA editing. These proteins contain repeat elements that bind to cognate RNA sequence motifs just 5' to the edited nucleotide. In flowering plants, the site-specific proteins interact selectively with individual members of a different, smaller family of proteins. These latter proteins may be connectors between the site-specific proteins and the as yet unknown deaminating enzymatic activity.

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.

Multiple PPR protein interactions are involved in the RNA editing system in Arabidopsis mitochondria and plastids.

Recent identification of several different types of RNA editing factors in plant organelles suggests complex RNA editosomes within which each factor has a different task. However, the precise protein interactions between the different editing factors are still poorly understood. In this paper, we show that the E+-type pentatricopeptide repeat (PPR) protein SLO2, which lacks a C-terminal cytidine deaminase-like DYW domain, interacts in vivo with the DYW-type PPR protein DYW2 and the P-type PPR protein NUWA in mitochondria, and that the latter enhances the interaction of the former ones. These results may reflect a protein scaffold or complex stabilization role of NUWA between E+-type PPR and DYW2 proteins. Interestingly, DYW2 and NUWA also interact in chloroplasts, and DYW2-GFP overexpressing lines show broad editing defects in both organelles, with predominant specificity for sites edited by E+-type PPR proteins. The latter suggests a coordinated regulation of organellar multiple site editing through DYW2, which probably provides the deaminase activity to E+ editosomes.

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.

Crystal structures of the Arabidopsis thaliana organellar RNA editing factors MORF1 and MORF9.

In flowering plant plastids and mitochondria, multiple organellar RNA editing factor (MORF/RIP) proteins are required at most sites for efficient C to U RNA editing catalyzed by the RNA editosome. MORF proteins harbor a conserved stretch of residues (MORF-box), form homo- and heteromers and interact with selected PPR (pentatricopeptide repeat) proteins, which recognize each editing site. The molecular function of the MORF-box remains elusive since it shares no sequence similarity with known domains. We determined structures of the A. thaliana mitochondrial MORF1 and chloroplast MORF9 MORF-boxes which both adopt a novel globular fold (MORF domain). Our structures state a paradigmatic model for MORF domains and their specific dimerization via a hydrophobic interface. We cross-validate the interface by yeast two-hybrid studies and pulldown assays employing structure-based mutants. We find a structural similarity of the MORF domain to an N-terminal ferredoxin-like domain (NFLD), which confers RNA substrate positioning in bacterial 4-thio-uracil tRNA synthetases, implying direct RNA contacts of MORF proteins during RNA editing. With the MORF1 and MORF9 structures we elucidate a yet unknown fold, corroborate MORF interaction studies, validate the mechanism of MORF multimerization by structure-based mutants and pave the way towards a complete structural characterization of the plant RNA editosome.

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.

A PPR protein in the PLS subfamily stabilizes the 5'-end of processed rpl16 mRNAs in maize chloroplasts.

Pentatricopeptide repeat (PPR) proteins are a large family of helical-repeat proteins that bind RNA in mitochondria and chloroplasts. Precise RNA targets and functions have been assigned to only a small fraction of the >400 members of the PPR family in plants. We used the amino acid code governing the specificity of RNA binding by PPR repeats to infer candidate-binding sites for the maize protein PPR103 and its ortholog Arabidopsis EMB175. Genetic and biochemical data confirmed a predicted binding site in the chloroplast rpl16 5'UTR to be a site of PPR103 action. This site maps to the 5' end of transcripts that fail to accumulate in ppr103 mutants. A small RNA corresponding to the predicted PPR103 binding site accumulates in a PPR103-dependent fashion, as expected of PPR103's in vivo footprint. Recombinant PPR103 bound specifically to this sequence in vitro These observations imply that PPR103 stabilizes rpl16 mRNA by impeding 5'→3' RNA degradation. Previously described PPR proteins with this type of function consist of canonical PPR motifs. By contrast, PPR103 is a PLS-type protein, an architecture typically associated with proteins that specify sites of RNA editing. However, PPR103 is not required to specify editing sites in chloroplasts.