Mizuho Ichinose

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.

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 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.

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.

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.

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.

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.

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

ABSTRACT 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.

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

In the moss Physcomitrella patens, PAS-containing histidine kinases regulate the timing of developmental processes of gametophyte generation in response to light, suggesting their functional significance unique to basal land plants.