Moss PPR-SMR protein PpPPR_64 influences the expression of a psaA-psaB-rps14 gene cluster and processing of the 23S-4.5S rRNA precursor in chloroplasts.

KEY MESSAGE: Moss PPR-SMR protein PpPPR_64 is a pTAC2 homolog but is functionally distinct from pTAC2. PpPPR_64 is required for psaA gene expression and its function may have evolved in mosses. The pentatricopeptide repeat (PPR) proteins are key regulatory factors responsible for the control of plant organellar gene expression. A small subset of PPR proteins possess a C-terminal small MutS-related (SMR) domain and have diverse roles in plant organellar biogenesis. However, the function of PPR-SMR proteins is not fully understood. Here, we report the function of PPR-SMR protein PpPPR_64 in the moss Physcomitrium patens. Phylogenetic analysis indicated that PpPPR_64 belongs to the same clade as the Arabidopsis PPR-SMR protein pTAC2. PpPPR_64 knockout (KO) mutants grew autotrophically but with reduced protonemata growth and the poor formation of photosystems' antenna complexes. Quantitative reverse transcription-polymerase chain reaction and RNA gel blot hybridization analyses revealed a significant reduction in transcript levels of the psaA-psaB-rps14 gene cluster but no alteration to transcript levels of most photosynthesis- and non-photosynthesis-related genes. In addition, RNA processing of 23S-4.5S rRNA precursor was impaired in the PpPPR_64 KO mutants. This suggests that PpPPR_64 is specifically involved in the expression level of the psaA-psaB-rps14 gene and in processing of the 23S-4.5S rRNA precursor. Our results indicate that PpPPR_64 is functionally distinct from pTAC2 and is a novel PPR-SMR protein required for proper chloroplast biogenesis in P. patens.

The P-class pentatricopeptide repeat protein PpPPR_21 is needed for accumulation of the psbI-ycf12 dicistronic mRNA in Physcomitrella chloroplasts.

Chloroplast gene expression is controlled by numerous nuclear-encoded RNA-binding proteins. Among these, pentatricopeptide repeat (PPR) proteins are known to be key players in post-transcriptional regulation in chloroplasts. However, the functions of many PPR proteins remain unknown. In this study, we characterized the function of a chloroplast-localized P-class PPR protein PpPPR_21 in Physcomitrella patens. Knockout (KO) mutants of PpPPR_21 exhibited reduced protonemata growth and lower photosynthetic activity. Immunoblot analysis and blue-native gel analysis showed a remarkable reduction of the photosystem II (PSII) reaction center protein and poor formation of the PSII supercomplexes in the KO mutants. To assess whether PpPPR_21 is involved in chloroplast gene expression, chloroplast genome-wide microarray analysis and Northern blot hybridization were performed. These analyses indicated that the psbI-ycf12 transcript encoding the low molecular weight subunits of PSII did not accumulate in the KO mutants while other psb transcripts accumulated at similar levels in wild-type and KO mutants. A complemented PpPPR_21KO moss transformed with the cognate full-length PpPPR_21cDNA rescued the level of accumulation of psbI-ycf12 transcript. RNA-binding experiments showed that the recombinant PpPPR_21 bound efficiently to the 5' untranslated and translated regions of psbImRNA. The present study suggests that PpPPR_21 may be essential for the accumulation of a stable psbI-ycf12mRNA.

Two Novel PLS-Class Pentatricopeptide Repeat Proteins Are Involved in the Group II Intron Splicing of Mitochondrial Transcripts in the Moss Physcomitrella patens.

Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that function in posttranscriptional regulation as gene-specific regulators of RNA metabolism in plant organelles. Plant PPR proteins are divided into four classes: P, PLS, E and DYW. The E- and DYW-class proteins are mainly implicated in RNA editing, whereas most of the P-class proteins predominantly participate in RNA cleavage, splicing and stabilization. In contrast, the functions of PLS-class proteins still remain obscure. Here, we report the function of PLS-class PpPPR_31 and PpPPR_9 in Physcomitrella patens. The knockout (KO) mutants of PpPPR_31 and PpPPR_9 exhibited slower protonema growth compared to the wild type. The PpPPR_31 KO mutants showed a considerable reduction in the splicing of nad5 intron 3 and atp9 intron 1. The PpPPR_9 KO mutants displayed severely reduced splicing of cox1 intron 3. An RNA electrophoresis mobility shift assay showed that the recombinant PpPPR_31 protein bound to the 5' region of nad5 exon 4 and the bulged A region in domain VI of atp9 group II intron 1 while the recombinant PpPPR_9 bound to the translated region of ORF622 in cox1 intron 3. These results suggest that a certain set of PLS-class PPR proteins may influence the splicing efficiency of mitochondrial group II introns.

An evolutionarily conserved P-subfamily pentatricopeptide repeat protein is required to splice the plastid ndhA transcript in the moss Physcomitrella patens and Arabidopsis thaliana.

Pentatricopeptide repeat (PPR) proteins are known to play important roles in post-transcriptional regulation in plant organelles. However, the function of the majority of PPR proteins remains unknown. To examine their functions, Physcomitrella patens PpPPR_66 knockout (KO) mutants were generated and characterized. The KO mosses exhibited a wild-type-like growth phenotype but showed aberrant chlorophyll fluorescence due to defects in chloroplast NADH dehydrogenase-like (NDH) activity. Immunoblot analysis suggested that disruption of PpPPR_66 led to a complete loss of the chloroplast NDH complex. To examine whether the loss of PpPPR_66 affects the expression of plastid ndh genes, the transcript levels of 11 plastid ndh genes were analyzed by reverse transcription PCR. This analysis indicated that splicing of the ndhA transcript was specifically impaired while mRNA accumulation levels as well as the processing patterns of other plastid ndh genes were not affected in the KO mutants. Complemented PpPPR_66 KO lines transformed with the PpPPR_66 full-length cDNA rescued splicing of the ndhA transcript. Arabidopsis thaliana T-DNA tagged lines of a PPR_66 homolog (At2 g35130) showed deficient splicing of the ndhA transcript. This indicates that the two proteins are functionally conserved between bryophytes and vascular plants. An in vitro RNA-binding assay demonstrated that the recombinant PpPPR_66 bound preferentially to the region encompassing a part of exon 1 to a 5' part of the ndhA group II intron. Taken together, these results indicate that PpPPR_66 acts as a specific factor to splice ndhA pre-mRNA.

The DYW Domains of Pentatricopeptide Repeat RNA Editing Factors Contribute to Discriminate Target and Non-Target Editing Sites.

In land plant organelles, many transcripts are modified by cytidine to uridine RNA editing. Target cytidines are specifically recognized by nuclear-encoded pentatricopeptide repeat (PPR) proteins via their sequence-specific RNA-binding motifs. In the moss Physcomitrella patens, all PPR editing factors have C-terminal E and DYW domains. To examine the contribution of E and DYW domains in RNA editing, we performed a complementation assay using mutated PpPPR_56 and PpPPR_71, which are responsible for mitochondrial editing sites. This assay showed that both E and DYW domains are required for RNA editing at the target sites, and that the conserved zinc-binding signature and the terminal triplet of the DYW domain are essential for editing. In addition, DYW domain-swapping experiments demonstrated that DYW domains are functionally different between PpPPR_56 and other mitochondrial PPR editing factors, and that residues 37-42 of the DYW domain are involved in site-specific editing. Our results suggest that PPR-DYW proteins specifically recognize their target editing sites via PPR motifs and the DYW domain.

Light-regulated PAS-containing histidine kinases delay gametophore formation in the moss Physcomitrella patens.

Two-component systems (TCSs) are signal transduction mechanisms for responding to various environmental stimuli. In angiosperms, TCSs involved in phytohormone signaling have been intensively studied, whereas there are only a few reports on TCSs in basal land plants. The moss Physcomitrella patens possesses several histidine kinases (HKs) that are lacking in seed plant genomes. Here, we studied two of these unique HKs, PAS-histidine kinase 1 (PHK1) and its paralog PHK2, both of which have PAS (Per-Arnt-Sim) domains, which are known to show versatile functions such as sensing light or molecular oxygen. We found homologs of PHK1 and PHK2 only in early diverged clades such as bryophytes and lycophytes, but not in seed plants. The PAS sequences of PHK1 and PHK2 are more similar to a subset of bacterial PAS sequences than to any angiosperm PAS sequences. Gene disruption lines that lack either PHK1 or PHK2 or both formed gametophores earlier than the wild-type, and consistently, more caulonema side branches were induced in response to light in the disruption lines. Therefore, PHK1 and PHK2 delay the timing of gametophore development, probably by suppressing light-induced caulonema branching. This study provides new insights into the evolution of TCSs in plants.

P-class pentatricopeptide repeat protein PTSF1 is required for splicing of the plastid pre-tRNA(I) (le) in Physcomitrella patens.

Pentatricopeptide repeat (PPR) proteins are widely distributed in eukaryotes and are mostly localized in mitochondria or plastids. PPR proteins play essential roles in various RNA processing steps in organelles; however, the function of the majority of PPR proteins remains unknown. To examine the function of plastid PPR proteins, PpPPR_4 gene knock-out mutants were characterized in Physcomitrella patens. The knock-out mosses displayed severe growth retardation and reduced effective quantum yield of photosystem II. Immunoblot analysis showed that knock-out of PpPPR_4 resulted in a strongly reduced level of plastid-encoded proteins, such as photosystem II reaction center protein D1, the ß subunit of ATP synthase, and the stromal enzyme, Rubisco. To further investigate whether knock-out of the PpPPR_4 gene affects plastid gene expression, we analyzed steady-state transcript levels of protein- and rRNA-coding genes by quantitative RT-PCR. This analysis showed that the level of many protein-coding transcripts increased in the mutants. In contrast, splicing of a spacer tRNA(I) (le) precursor encoded by the rrn operon was specifically impaired in the mutants, whereas the accumulation of other plastid tRNAs and rRNAs was not largely affected. Thus, the defect in tRNA(I) (le) splicing leads to a considerable reduction of mature tRNA(I) (le) , which may be accountable for the reduced protein level. An RNA mobility shift assay showed that the recombinant PpPPR_4 bound preferentially to domain III of the tRNA(I) (le) group-II intron. These results provide evidence that PpPPR_4 functions in RNA splicing of the tRNA(I) (le) intron, and hence PpPPR_4 was named plastid tRNA splicing factor 1 (PTSF1).

A PPR-DYW protein is required for splicing of a group II intron of cox1 pre-mRNA in Physcomitrella patens.

The pentatricopeptide repeat (PPR) protein family is involved in various steps of RNA metabolism in plastids and mitochondria. To investigate the function of a DYW sub-class PPR protein in the moss Physcomitrella patens, we constructed and characterized knockout mutants of the PpPPR_43 gene, which encodes a mitochondrial localized PPR protein with a C-terminal DYW domain. The disruptants showed poor growth of moss protonemata. To investigate whether mitochondrial transcripts were affected by disruption of PpPPR_43, we sequenced the cDNA to detect RNA editing events and performed RT-PCR analyses to measure steady-state mitochondrial transcript levels. Disruption of PpPPR_43 did not result in defective RNA editing, but a substantial reduction in the level of mature cox1 transcript was observed in the disruptants. RT-PCR analysis showed that the 3rd intron of cox1 pre-mRNA was not spliced out in the disruptants, but the 1st, 2nd and 4th introns were efficiently spliced out. This suggests that PpPPR_43 is an intron 3-specific splicing factor. The role of the C-terminal domains of PpPPR_43 in intron 3 splicing was analyzed by complementation experiments with truncated constructs lacking the DYW domain or both the E and DYW domains. Both truncated genes completely restored splicing in the PpPPR_43 knockout mutant. This indicates that the E and DYW domains of PpPPR_43 are not required for splicing, and can be deleted without loss of cox1 intron 3 splicing.

Two DYW subclass PPR proteins are involved in RNA editing of ccmFc and atp9 transcripts in the moss Physcomitrella patens: first complete set of PPR editing factors in plant mitochondria.

The moss Physcomitrella patens has 11 RNA editing sites in mitochondrial transcripts. We previously identified six DYW subclass pentatricopeptide repeat (PPR) proteins as RNA editing factors for nine out of 11 sites. In this study, we identified two novel DYW subclass PPR proteins, PpPPR_65 and PpPPR_98, as RNA editing factors. Disruption of the PpPPR_65 gene resulted in a complete loss of RNA editing at two neighboring sites, ccmFc-C103 and ccmFc-C122, in the mitochondrial ccmFc transcript. To confirm this result, we further generated PpPPR_65 knockdown (KD) mutants by an inducible RNA interference (RNAi) system. The generated RNAi lines displayed reduced levels of RNA editing at both ccmFc-C103 and ccmFc-C122 sites. Next, we characterized the function of PpPPR_98 by constructing a KD mutant of PpPPR_98 expression. The KD mutant showed a 30% reduction in the level of atp9-C92 editing. When PpPPR_98 cDNA was introduced into the KD mutant, RNA editing levels were restored to the wild-type level. This indicates that PpPPR_98 is an editing factor for the atp9-C92 site. The recombinant PpPPR_98 protein bound to the upstream sequence of the editing site that was created by splicing of atp9 transcript. This suggests that atp9 RNA editing occurs after splicing of atp9 transcript. Our present and previous data provide the first evidence that all 11 known editing events require at least eight DYW subclass PPR proteins in the moss mitochondria.

Architecture of the PPR gene family in the moss Physcomitrella patens.

Pentatricopeptide repeat (PPR) proteins are widespread in eukaryotes and in particular, include several hundred members in land plants. The majority of PPR proteins are localized in mitochondria and plastids, where they play a crucial role in various aspects of RNA metabolism at the post-transcriptional level in gene expression. However, many of their functions remain to be characterized. In contrast to vascular plants, the moss Physcomitrella patens has only 105 PPR genes. This number may represent a minimum set of PPR proteins required for post-transcriptional regulation in plant organelles. Here, we review the overall structure of the P. patens PPR gene family and the current status of the functional characterization of moss PPR proteins.

U-to-C RNA editing by synthetic PPR-DYW proteins in bacteria and human culture cells.

Programmable RNA editing offers significant therapeutic potential for a wide range of genetic diseases. Currently, several deaminase enzymes, including ADAR and APOBEC, can perform programmable adenosine-to-inosine or cytidine-to-uridine RNA correction. However, enzymes to perform guanosine-to-adenosine and uridine-to-cytidine (U-to-C) editing are still lacking to complete the set of transition reactions. It is believed that the DYW:KP proteins, specific to seedless plants, catalyze the U-to-C reactions in mitochondria and chloroplasts. In this study, we designed seven DYW:KP domains based on consensus sequences and fused them to a designer RNA-binding pentatricopeptide repeat (PPR) domain. We show that three of these PPR-DYW:KP proteins edit targeted uridine to cytidine in bacteria and human cells. In addition, we show that these proteins have a 5' but not apparent 3' preference for neighboring nucleotides. Our results establish the DYW:KP aminase domain as a potential candidate for the development of a U-to-C editing tool in human cells.

Substitutional RNA Editing in Plant Organelles.

RNA editing by cytidine (C) to uridine (U) conversions frequently occurs in land plant mitochondria and plastids. Target cytidines are specifically recognized by nuclear-encoded pentatricopeptide repeat (PPR) proteins in a sequence-specific manner. In the moss Physcomitrella patens, all PPR editing factors possess the DYW-deaminase domain at the C-terminus. Here, we describe methods for the direct sequencing of cDNA to detect RNA editing events and the RNA electrophoresis mobility shift assay (REMSA) to analyze the specific binding of PPR editing factors to their target RNA.

Targeted gene disruption identifies three PPR-DYW proteins involved in RNA editing for five editing sites of the moss mitochondrial transcripts.

In plant organelles, RNA editing frequently occurs in many transcripts, but little is known about its molecular mechanism. Eleven RNA editing sites are present in the moss Physcomitrella patens mitochondria. Recently PpPPR_71, one member of 10 DYW-subclass pentatricopeptide repeat (PPR-DYW) proteins, has been identified as a site-specific recognition factor for RNA editing in the mitochondrial transcript. In this study, we disrupted three genes encoding a PPR-DYW protein-PpPPR_56, PpPPR_77, and PpPPR_91-to investigate whether they are involved in RNA editing. Transient expression of an N-terminal amino acid sequence fused to the green fluorescent protein (GFP) suggests that the three PPR-DYW proteins are targeted to mitochondria. Disruption of each gene by homologous recombination revealed that PpPPR_56 was involved in RNA editing at the nad3 and nad4 sites, PpPPR_77 at the cox2 and cox3 sites, and PpPPR_91 at the nad5-2 site in the mitochondrial transcripts. The nucleotide sequences surrounding the two editing sites targeted by a single PPR-DYW protein share 42 to 56% of their identities. Thus, moss PPR-DYW proteins seem to be site-specific factors for RNA editing in mitochondrial transcripts.

The L motifs of two moss pentatricopeptide repeat proteins are involved in RNA editing but predominantly not in RNA recognition.

Pentatricopeptide repeat (PPR) proteins, composed of PPR motifs repeated in tandem, are sequence-specific RNA binding proteins. Recent bioinformatic studies have shown that the combination of polar amino acids at positions 5 and last in each PPR motif recognizes RNA bases, and an RNA recognition code for PPR proteins has been proposed. Subsequent studies confirmed that the P (canonical length) and S (short) motifs bind to specific nucleotides according to this code. However, the contribution of L (long) motifs to RNA recognition is mostly controversial, owing to the presence of a nonpolar amino acid at position 5. The PLS-class PPR protein PpPPR_56 is a mitochondrial RNA editing factor in the moss Physcomitrella patens. Here, we performed in vitro RNA binding and in vivo complementation assays with PpPPR_56 and its variants containing mutated L motifs to investigate their contributions to RNA recognition. In vitro RNA binding assay showed that the original combination of amino acids at positions 5 and last in the L motifs of PpPPR_56 is not required for RNA recognition. In addition, an in vivo complementation assay with RNA editing factors PpPPR_56 and PpPPR_78 revealed the importance of nonpolar amino acids at position 5 of C-terminal L motifs for efficient RNA editing. Our findings suggest that L motifs function as non-binding spacers, not as RNA-binding motifs, to facilitate the formation of a complex between PLS-class PPR protein and RNA. As a result, the DYW domain, a putative catalytic deaminase responsible for C-to-U RNA editing, is correctly placed in proximity to C, which is to be edited.

RNA Editing and Its Molecular Mechanism in Plant Organelles.

RNA editing by cytidine (C) to uridine (U) conversions is widespread in plant mitochondria and chloroplasts. In some plant taxa, "reverse" U-to-C editing also occurs. However, to date, no instance of RNA editing has yet been reported in green algae and the complex thalloid liverworts. RNA editing may have evolved in early land plants 450 million years ago. However, in some plant species, including the liverwort, Marchantia polymorpha, editing may have been lost during evolution. Most RNA editing events can restore the evolutionarily conserved amino acid residues in mRNAs or create translation start and stop codons. Therefore, RNA editing is an essential process to maintain genetic information at the RNA level. Individual RNA editing sites are recognized by plant-specific pentatricopeptide repeat (PPR) proteins that are encoded in the nuclear genome. These PPR proteins are characterized by repeat elements that bind specifically to RNA sequences upstream of target editing sites. In flowering plants, non-PPR proteins also participate in multiple RNA editing events as auxiliary factors. C-to-U editing can be explained by cytidine deamination. The proteins discovered to date are important factors for RNA editing but a bona fide RNA editing enzyme has yet to be identified.

Diversity of plant circadian clocks: Insights from studies of Chlamydomonas reinhardtii and Physcomitrella patens.

Arabidopsis thaliana has long been the model plant of choice for elucidating the mechanisms of the circadian clock. Recently, relevant results have accumulated in other species of green plant lineages, including green algae. This mini-review describes a comparison of the mechanism of the A. thaliana clock to those of the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens, focusing on commonalities and divergences of subsystems of the clock. The potential of such an approach from an evolutionary viewpoint is discussed.

Identification of a pentatricopeptide repeat RNA editing factor in Physcomitrella patens chloroplasts.

The moss Physcomitrella patens has two RNA editing sites in the chloroplasts. Here we identified a novel DYW-subclass pentatricopeptide repeat (PPR) protein, PpPPR_45, as a chloroplast RNA editing factor in P. patens. Knockdown of the PpPPR_45 gene reduced the extent of RNA editing at the chloroplast rps14-C2 site, whereas over-expression of PpPPR_45 increased the levels of RNA editing at both the rps14-C2 site and its neighboring C site. This indicates that the expression level of PpPPR_45 affects the extent of RNA editing at the two neighboring sites.

The PPR-DYW proteins are required for RNA editing of rps14, cox1 and nad5 transcripts in Physcomitrella patens mitochondria.

We identified two DYW subclass pentatricopeptide repeat (PPR) proteins, PpPPR_78 and PpPPR_79, as RNA editing factors in the moss Physcomitrella patens. Disruption of each gene by homologous recombination revealed that PpPPR_78 was involved in RNA editing at the rps14 (rps14-C137) and cox1 (cox1-C755) sites and PpPPR_79 at the nad5-1 (nad5-C598) site in the mitochondrial transcripts. RNA editing defects did not affect transcript patterns of the target genes. Thus, DYW subclass PPR proteins seem to be site-specific trans-acting factors for RNA editing.