Conservation of the moss RNA editing factor PPR78 despite the loss of its known cytidine-to-uridine editing sites is explained by a hidden extra target.

Cytidine (C)-to-uridine (U) RNA editing in plant organelles relies on specific RNA-binding pentatricopeptide repeat (PPR) proteins. In the moss Physcomitrium patens, all such RNA editing factors feature a C-terminal DYW domain that acts as the cytidine deaminase for C-to-U conversion. PPR78 of Physcomitrium targets 2 mitochondrial editing sites, cox1eU755SL and rps14eU137SL. Remarkably, the latter is edited to highly variable degrees in different mosses. Here, we aimed to unravel the coevolution of PPR78 and its 2 target sites in mosses. Heterologous complementation in a Physcomitrium knockout line revealed that the variable editing of rps14eU137SL depends on the PPR arrays of different PPR78 orthologues but not their C-terminal domains. Intriguingly, PPR78 has remained conserved despite the simultaneous loss of editing at both known targets among Hypnales (feather mosses), suggesting it serves an additional function. Using a recently established RNA editing assay in Escherichia coli, we confirmed site-specific RNA editing by PPR78 in the bacterium and identified 4 additional off-targets in the bacterial transcriptome. Based on conservation profiles, we predicted ccmFNeU1465RC as a candidate editing target of PPR78 in moss mitochondrial transcriptomes. We confirmed editing at this site in several mosses and verified that PPR78 targets ccmFNeU1465RC in the bacterial editing system, explaining the conservation and functional adaptation of PPR78 during moss evolution.

Beyond a PPR-RNA recognition code: Many aspects matter for the multi-targeting properties of RNA editing factor PPR56.

The mitochondrial C-to-U RNA editing factor PPR56 of the moss Physcomitrium patens is an RNA-binding pentatricopeptide repeat protein equipped with a terminal DYW-type cytidine deaminase domain. Transferred into Escherichia coli, PPR56 works faithfully on its two native RNA editing targets, nad3eU230SL and nad4eU272SL, and also converts cytidines into uridines at over 100 off-targets in the bacterial transcriptome. Accordingly, PPR56 is attractive for detailed mechanistic studies in the heterologous bacterial setup, allowing for scoring differential RNA editing activities of many target and protein variants in reasonable time. Here, we report (i) on the effects of numerous individual and combined PPR56 protein and target modifications, (ii) on the spectrum of off-target C-to-U editing in the bacterial background transcriptome for PPR56 and two variants engineered for target re-direction and (iii) on combinations of targets in tandem or separately at the 5'- and 3'-ends of large mRNAs. The latter experimentation finds enhancement of RNA editing at weak targets in many cases, including cox3eU290SF as a new candidate mitogenome target. We conclude that C-to-U RNA editing can be much enhanced by transcript features also outside the region ultimately targeted by PPRs of a plant editing factor, possibly facilitated by its enrichment or scanning along transcripts.

Conquering new grounds: plant organellar C-to-U RNA editing factors can be functional in the plant cytosol.

Plant mitochondrial and chloroplast transcripts are subject to numerous events of specific cytidine-to-uridine (C-to-U) RNA editing to correct genetic information. Key protein factors for this process are specific RNA-binding pentatricopeptide repeat (PPR) proteins, which are encoded in the nucleus and post-translationally imported into the two endosymbiotic organelles. Despite hundreds of C-to-U editing sites in the plant organelles, no comparable editing has been found for nucleo-cytosolic mRNAs raising the question why plant RNA editing is restricted to chloroplasts and mitochondria. Here, we addressed this issue in the model moss Physcomitrium patens, where all PPR-type RNA editing factors comprise specific RNA-binding and cytidine deamination functionalities in single proteins. To explore whether organelle-type RNA editing can principally also take place in the plant cytosol, we expressed PPR56, PPR65 and PPR78, three editing factors recently shown to also function in a bacterial setup, together with cytosolic co-transcribed native targets in Physcomitrium. While we obtained unsatisfying results upon their constitutive expression, we found strong cytosolic RNA editing under hormone-inducible expression. Moreover, RNA-Seq analyses revealed varying numbers of up to more than 900 off-targets in other cytosolic transcripts. We conclude that PPR-mediated C-to-U RNA editing is not per se incompatible with the plant cytosol but that its limited target specificity has restricted its occurrence to the much less complex transcriptomes of mitochondria and chloroplast in the course of evolution.

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.

Plant mitochondrial RNA editing factors can perform targeted C-to-U editing of nuclear transcripts in human cells.

RNA editing processes are strikingly different in animals and plants. Up to thousands of specific cytidines are converted into uridines in plant chloroplasts and mitochondria whereas up to millions of adenosines are converted into inosines in animal nucleo-cytosolic RNAs. It is unknown whether these two different RNA editing machineries are mutually incompatible. RNA-binding pentatricopeptide repeat (PPR) proteins are the key factors of plant organelle cytidine-to-uridine RNA editing. The complete absence of PPR mediated editing of cytosolic RNAs might be due to a yet unknown barrier that prevents its activity in the cytosol. Here, we transferred two plant mitochondrial PPR-type editing factors into human cell lines to explore whether they could operate in the nucleo-cytosolic environment. PPR56 and PPR65 not only faithfully edited their native, co-transcribed targets but also different sets of off-targets in the human background transcriptome. More than 900 of such off-targets with editing efficiencies up to 91%, largely explained by known PPR-RNA binding properties, were identified for PPR56. Engineering two crucial amino acid positions in its PPR array led to predictable shifts in target recognition. We conclude that plant PPR editing factors can operate in the entirely different genetic environment of the human nucleo-cytosol and can be intentionally re-engineered towards new targets.

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.

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.

A functional twintron, 'zombie' twintrons and a hypermobile group II intron invading itself in plant mitochondria.

The occurrence of group II introns in plant mitochondrial genomes is strikingly different between the six major land plant clades, contrasting their highly conserved counterparts in chloroplast DNA. Their present distribution likely reflects numerous ancient intron gains and losses during early plant evolution before the emergence of seed plants. As a novelty for plant organelles, we here report on five cases of twintrons, introns-within-introns, in the mitogenomes of lycophytes and hornworts. An internal group II intron interrupts an intron-borne maturase of an atp9 intron in Lycopodiaceae, whose splicing precedes splicing of the external intron. An invasive, hypermobile group II intron in cox1, has conquered nine further locations including a previously overlooked sdh3 intron and, most surprisingly, also itself. In those cases, splicing of the external introns does not depend on splicing of the internal introns. Similar cases are identified in the mtDNAs of hornworts. Although disrupting a group I intron-encoded protein in one case, we could not detect splicing of the internal group II intron in this 'mixed' group I/II twintron. We suggest the name 'zombie' twintrons (half-dead, half-alive) for such cases where splicing of external introns does not depend any more on prior splicing of fossilized internal introns.

The dual-targeted RNA editing factor AEF1 is universally conserved among angiosperms and reveals only minor adaptations upon loss of its chloroplast or its mitochondrial target.

KEY MESSAGE: Upon loss of either its chloroplast or mitochondrial target, a uniquely dual-targeted factor for C-to-U RNA editing in angiosperms reveals low evidence for improved molecular adaptation to its remaining target. RNA-binding pentatricopeptide repeat (PPR) proteins specifically recognize target sites for C-to-U RNA editing in the transcriptomes of plant chloroplasts and mitochondria. Among more than 80 PPR-type editing factors that have meantime been characterized, AEF1 (or MPR25) is a special case given its dual targeting to both organelles and addressing an essential mitochondrial (nad5eU1580SL) and an essential chloroplast (atpFeU92SL) RNA editing site in parallel in Arabidopsis. Here, we explored the angiosperm-wide conservation of AEF1 and its two organelle targets. Despite numerous independent losses of the chloroplast editing site by C-to-T conversion and at least four such conversions at the mitochondrial target site in other taxa, AEF1 remains consistently conserved in more than 120 sampled angiosperm genomes. Not a single case of simultaneous loss of the chloroplast and mitochondrial editing target or of AEF1 disintegration or loss could be identified, contrasting previous findings for editing factors targeted to only one organelle. Like in most RNA editing factors, the PPR array of AEF1 reveals potential for conceptually "improved fits" to its targets according to the current PPR-RNA binding code. Surprisingly, we observe only minor evidence for adaptation to the mitochondrial target also after deep losses of the chloroplast target among Asterales, Caryophyllales and Poales or, vice versa, for the remaining chloroplast target after a deep loss of the mitochondrial target among Malvales. The evolutionary observations support the notion that PPR-RNA mismatches may be essential for proper function of editing factors.

Towards a plant model for enigmatic U-to-C RNA editing: the organelle genomes, transcriptomes, editomes and candidate RNA editing factors in the hornwort Anthoceros agrestis.

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.

Plant organelle RNA editing and its specificity factors: enhancements of analyses and new database features in PREPACT 3.0.

BACKGROUND: Gene expression in plant chloroplasts and mitochondria is affected by RNA editing. Numerous C-to-U conversions, accompanied by reverse U-to-C exchanges in some plant clades, alter the genetic information encoded in the organelle genomes. Predicting and analyzing RNA editing, which ranges from only few sites in some species to thousands in other taxa, is bioinformatically demanding. RESULTS: Here, we present major enhancements and extensions of PREPACT, a WWW-based service for analysing, predicting and cataloguing plant-type RNA editing. New features in PREPACT's core include direct GenBank accession query input and options to restrict searches to candidate U-to-C editing or to sites where editing has been documented previously in the references. The reference database has been extended by 20 new organelle editomes. PREPACT 3.0 features new modules "EdiFacts" and "TargetScan". EdiFacts integrates information on pentatricopeptide repeat (PPR) proteins characterized as site-specific RNA editing factors. PREPACT's editome references connect into EdiFacts, linking editing events to specific co-factors where known. TargetScan allows position-weighted querying for sequence motifs in the organelle references, optionally restricted to coding regions or sequences around editing sites, or in queries uploaded by the user. TargetScan is mainly intended to evaluate and further refine the proposed PPR-RNA recognition code but may be handy for other tasks as well. We present an analysis for the immediate sequence environment of more than 15,000 documented editing sites finding strong and different bias in the editome data sets. CONCLUSIONS: We exemplarily present the novel features of PREPACT 3.0 aimed to enhance the analyses of plant-type RNA editing, including its new modules EdiFacts integrating information on characterized editing factors and TargetScan aimed to analyse RNA editing site recognition specificities.

Expected and unexpected evolution of plant RNA editing factors CLB19, CRR28 and RARE1: retention of CLB19 despite a phylogenetically deep loss of its two known editing targets in Poaceae.

BACKGROUND: C-to-U RNA editing in mitochondria and chloroplasts and the nuclear-encoded, RNA-binding PPR proteins acting as editing factors present a wide field of co-evolution between the different genetic systems in a plant cell. Recent studies on chloroplast editing factors RARE1 and CRR28 addressing one or two chloroplast editing sites, respectively, found them strictly conserved among 65 flowering plants as long as one of their RNA editing targets remained present. RESULTS: Extending the earlier sampling to 117 angiosperms with high-quality genome or transcriptome data, we find more evidence confirming previous conclusions but now also identify cases for expected evolutionary transition states such as retention of RARE1 despite loss of its editing target or the degeneration of CRR28 truncating its carboxyterminal DYW domain. The extended angiosperm set was now used to explore CLB19, an "E+"-type PPR editing factor targeting two chloroplast editing sites, rpoAeU200SF and clpPeU559HY, in Arabidopsis thaliana. We found CLB19 consistently conserved if one of the two targets was retained and three independent losses of CLB19 after elimination of both targets. The Ericales show independent regains of the ancestrally lost clpPeU559HY editing, further explaining why multiple-target editing factors are lost much more rarely than single target factors like RARE1. The retention of CLB19 despite loss of both editing targets in some Ericaceae, Apocynaceae and in Camptotheca (Nyssaceae) likely represents evolutionary transitions. However, the retention of CLB19 after a phylogenetic deep loss in the Poaceae rather suggests a yet unrecognized further editing target, for which we suggest editing event ndhAeU473SL. CONCLUSION: Extending the scope of studies on plant organelle RNA editing to further taxa and additional nuclear cofactors reveals expected evolutionary transitions, strikingly different evolutionary dynamics for multiple-target editing factors like CLB19 and CRR28 and suggests additional functions for editing factor CLB19 among the Poaceae.

Monilophyte mitochondrial rps1 genes carry a unique group II intron that likely originated from an ancient paralog in rpl2.

Intron patterns in plant mitochondrial genomes differ significantly between the major land plant clades. We here report on a new, clade-specific group II intron in the rps1 gene of monilophytes (ferns). This intron, rps1i25g2, is strikingly similar to rpl2i846g2 previously identified in the mitochondrial rpl2 gene of seed plants, ferns, and the lycophyte Phlegmariurus squarrosus Although mitochondrial ribosomal protein genes are frequently subject to endosymbiotic gene transfer among plants, we could retrieve the mitochondrial rps1 gene in a taxonomically wide sampling of 44 monilophyte taxa including basal lineages such as the Ophioglossales, Psilotales, and Marattiales with the only exception being the Equisetales (horsetails). Introns rps1i25g2 and rpl2i846g2 were likewise consistently present with only two exceptions: Intron rps1i25g2 is lost in the genus Ophioglossum and intron rpl2i846g2 is lost in Equisetum bogotense Both intron sequences are moderately affected by RNA editing. The unprecedented primary and secondary structure similarity of rps1i25g2 and rpl2i846g2 suggests an ancient retrotransposition event copying rpl2i846g2 into rps1, for which we suggest a model. Our phylogenetic analysis adding the new rps1 locus to a previous data set is fully congruent with recent insights on monilophyte phylogeny and further supports a sister relationship of Gleicheniales and Hymenophyllales.

Ginkgo and Welwitschia Mitogenomes Reveal Extreme Contrasts in Gymnosperm Mitochondrial Evolution.

Mitochondrial genomes (mitogenomes) of flowering plants are well known for their extreme diversity in size, structure, gene content, and rates of sequence evolution and recombination. In contrast, little is known about mitogenomic diversity and evolution within gymnosperms. Only a single complete genome sequence is available, from the cycad Cycas taitungensis, while limited information is available for the one draft sequence, from Norway spruce (Picea abies). To examine mitogenomic evolution in gymnosperms, we generated complete genome sequences for the ginkgo tree (Ginkgo biloba) and a gnetophyte (Welwitschia mirabilis). There is great disparity in size, sequence conservation, levels of shared DNA, and functional content among gymnosperm mitogenomes. The Cycas and Ginkgo mitogenomes are relatively small, have low substitution rates, and possess numerous genes, introns, and edit sites; we infer that these properties were present in the ancestral seed plant. By contrast, the Welwitschia mitogenome has an expanded size coupled with accelerated substitution rates and extensive loss of these functional features. The Picea genome has expanded further, to more than 4 Mb. With regard to structural evolution, the Cycas and Ginkgo mitogenomes share a remarkable amount of intergenic DNA, which may be related to the limited recombinational activity detected at repeats in Ginkgo Conversely, the Welwitschia mitogenome shares almost no intergenic DNA with any other seed plant. By conducting the first measurements of rates of DNA turnover in seed plant mitogenomes, we discovered that turnover rates vary by orders of magnitude among species.

A Single-Target Mitochondrial RNA Editing Factor of Funaria hygrometrica Can Fully Reconstitute RNA Editing at Two Sites in Physcomitrella patens.

Nuclear-encoded pentatricopeptide repeat (PPR) proteins are key factors for site-specific RNA editing, converting cytidines into uridines in plant mitochondria and chloroplasts. All editing factors in the model moss Physcomitrella patens have a C-terminal DYW domain with similarity to cytidine deaminase. However, numerous editing factors in flowering plants lack such a terminal DYW domain, questioning its immediate role in the pyrimidine base conversion process. Here we further investigate the Physcomitrella DYW-type PPR protein PPR_78, responsible for mitochondrial editing sites cox1eU755SL and rps14eU137SL. Complementation assays with truncated proteins demonstrate that the DYW domain is essential for full PPR_78 editing functionality. The DYW domain can be replaced, however, with its counterpart from another editing factor, PPR_79. The PPR_78 ortholog of the related moss Funaria hygrometrica fully complements the Physcomitrella mutant for editing at both sites, although the editing site in rps14 is lacking in Funaria. Editing factor orthologs in different taxa may thus retain editing capacity for multiple sites despite the absence of editing requirement.

Frequent chloroplast RNA editing in early-branching flowering plants: pilot studies on angiosperm-wide coexistence of editing sites and their nuclear specificity factors.

BACKGROUND: RNA editing by cytidine-to-uridine conversions is an essential step of RNA maturation in plant organelles. Some 30-50 sites of C-to-U RNA editing exist in chloroplasts of flowering plant models like Arabidopsis, rice or tobacco. We now predicted significantly more RNA editing in chloroplasts of early-branching angiosperm genera like Amborella, Calycanthus, Ceratophyllum, Chloranthus, Illicium, Liriodendron, Magnolia, Nuphar and Zingiber. Nuclear-encoded RNA-binding pentatricopeptide repeat (PPR) proteins are key editing factors expected to coevolve with their cognate RNA editing sites in the organelles. RESULTS: With an extensive chloroplast transcriptome study we identified 138 sites of RNA editing in Amborella trichopoda, approximately the 3- to 4-fold of cp editing in Arabidopsis thaliana or Oryza sativa. Selected cDNA studies in the other early-branching flowering plant taxa furthermore reveal a high diversity of early angiosperm RNA editomes. Many of the now identified editing sites in Amborella have orthologues in ferns, lycophytes or hornworts. We investigated the evolution of CRR28 and RARE1, two known Arabidopsis RNA editing factors responsible for cp editing events ndhBeU467PL, ndhDeU878SL and accDeU794SL, respectively, all of which we now found conserved in Amborella. In a phylogenetically wide sampling of 65 angiosperm genomes we find evidence for only one single loss of CRR28 in chickpea but several independent losses of RARE1, perfectly congruent with the presence of their cognate editing sites in the respective cpDNAs. CONCLUSION: Chloroplast RNA editing is much more abundant in early-branching than in widely investigated model flowering plants. RNA editing specificity factors can be traced back for more than 120 million years of angiosperm evolution and show highly divergent patterns of evolutionary losses, matching the presence of their target editing events.

Reverse U-to-C editing exceeds C-to-U RNA editing in some ferns - a monilophyte-wide comparison of chloroplast and mitochondrial RNA editing suggests independent evolution of the two processes in both organelles.

BACKGROUND: RNA editing by C-to-U conversions is nearly omnipresent in land plant chloroplasts and mitochondria, where it mainly serves to reconstitute conserved codon identities in the organelle mRNAs. Reverse U-to-C RNA editing in contrast appears to be restricted to hornworts, some lycophytes, and ferns (monilophytes). A well-resolved monilophyte phylogeny has recently emerged and now allows to trace the side-by-side evolution of both types of pyrimidine exchange editing in the two endosymbiotic organelles. RESULTS: Our study of RNA editing in four selected mitochondrial genes show a wide spectrum of divergent RNA editing frequencies including a dominance of U-to-C over the canonical C-to-U editing in some taxa like the order Schizaeales. We find that silent RNA editing leaving encoded amino acids unchanged is highly biased with more than ten-fold amounts of silent C-to-U over U-to-C edits. In full contrast to flowering plants, RNA editing frequencies are low in early-branching monilophyte lineages but increase in later emerging clades. Moreover, while editing rates in the two organelles are usually correlated, we observe uncoupled evolution of editing frequencies in fern mitochondria and chloroplasts. Most mitochondrial RNA editing sites are shared between the recently emerging fern orders whereas chloroplast editing sites are mostly clade-specific. Finally, we observe that chloroplast RNA editing appears to be completely absent in horsetails (Equisetales), the sister clade of all other monilophytes. CONCLUSIONS: C-to-U and U-to-C RNA editing in fern chloroplasts and mitochondria follow disinct evolutionary pathways that are surprisingly different from what has previously been found in flowering plants. The results call for careful differentiation of the two types of RNA editing in the two endosymbiotic organelles in comparative evolutionary studies.

Horizontal gene transfer of chlamydial-like tRNA genes into early vascular plant mitochondria.

Mitochondrial genomes of lycophytes are surprisingly diverse, including strikingly different transfer RNA (tRNA) gene complements: No mitochondrial tRNA genes are present in the spikemoss Selaginella moellendorffii, whereas 26 tRNAs are encoded in the chondrome of the clubmoss Huperzia squarrosa. Reinvestigating the latter we found that trnL(gag) and trnS(gga) had never before been identified in any other land plant mitochondrial DNA. Sensitive sequence comparisons showed these two tRNAs as well as trnN(guu) and trnS(gcu) to be very similar to their respective counterparts in chlamydial bacteria. We identified homologs of these chlamydial-type tRNAs also in other lycophyte, fern, and gymnosperm DNAs, suggesting horizontal gene transfer (HGT) into mitochondria in the early vascular plant stem lineages. These findings extend plant mitochondrial HGT to affect individual tRNA genes, to include bacterial donors, and suggest that Chlamydiae on top of their recently proposed key role in primary chloroplast establishment may also have participated in early tracheophyte genome evolution.

Chloroplast RNA editing going extreme: more than 3400 events of C-to-U editing in the chloroplast transcriptome of the lycophyte Selaginella uncinata.

RNA editing in chloroplasts and mitochondria of land plants differs significantly in abundance. For example, some 200-500 sites of cytidine-to-uridine RNA editing exist in flowering plant mitochondria as opposed to only 30-50 such C-to-U editing events in their chloroplasts. In contrast, we predicted significantly more chloroplast RNA editing for the protein-coding genes in the available complete plastome sequences of two species of the spike moss genus Selaginella (Lycopodiophyta). To evaluate these predictions we investigated the Selaginella uncinata chloroplast transcriptome. Our exhaustive cDNA studies identified the extraordinary number of 3415 RNA-editing events, exclusively of the C-to-U type, in the 74 mRNAs encoding intact reading frames in the S. uncinata chloroplast. We find the overwhelming majority (61%) of the 428 silent editing events leaving codon meanings unaltered directly neighboring other editing events, possibly suggesting a sterically more flexible RNA-editing deaminase activity in Selaginella. No evidence of RNA editing was found for tRNAs or rRNAs but we identified a total of 74 editing sites in cDNA sequences of four group II introns (petBi6g2, petDi8g2, ycf3i124g2, and ycf3i354g2) retained in partially matured transcripts, which strongly contribute to improved base-pairing in the intron secondary structures as a likely prerequisite for their splicing.