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 moss Physcomitrella patens, a model plant for the study of RNA editing in plant organelles.

RNA editing is an enigmatic phenomenon in which specific cytidines (C) in the transcripts are changed to uridines (U). In flowering plants, over 500 editing sites have been identified in the mitochondria and plastids. By contrast, in a moss Physcomitrella patens, only 12 editing sites are found in both organelles. Recent extensive genetics studies have revealed involvement of the pentatricopeptide repeat (PPR) proteins with a C-terminal DYW domain (PPR-DYW) in site-specific RNA editing events. Flowering plants have ~100 PPR-DYW genes while P. patens has only 10 PPR-DYW genes. Thus, the number of PPR-DYW genes is somewhat related to the number of RNA editing sites. The P. patens gametophyte, the haploid phase of the life cycle, is dominant, making it possible to study the phenotype of knockouts directly after transformation for gene-targeting. This makes it easier to identify the PPR-DYW proteins required for RNA editing. Recently, we have shown that one of 10 PPR-DYW proteins, PpPPR_71, was responsible for RNA editing in the mitochondrial ccmFc mRNA. In this study, we propose a working model of PpPPR_71 function on RNA editing.

The moss pentatricopeptide repeat protein with a DYW domain is responsible for RNA editing of mitochondrial ccmFc transcript.

In most land plants RNA editing frequently occurs in many organelle transcripts, but little is known about the molecular mechanisms of the organelle RNA editing process. In this study, we have characterized the Physcomitrella patens PpPPR_71 gene that is required for RNA editing of the ccmFc transcript. This transcript harbors two RNA editing sites, ccmF-1 and ccmF-2, that are separated by 18 nucleotides. Complementary DNA sequence analysis of ccmFc suggested that RNA editing at the ccmF-1 site occurred before ccmF-2 editing. RNA editing of the ccmF-2 downstream site was specifically impaired by disruption of the PpPPR_71 gene that encodes a polypeptide with 17 pentatricopeptide repeat motifs and a C-terminal DYW domain. The recombinant PpPPR_71 protein expressed in Escherichia coli specifically bound to the 46-nucleotide sequence containing the ccmF-2 editing site. The binding affinity of the recombinant PpPPR_71 was strongest when using the edited RNA at ccmF-1. In addition, the DYW domain also binds to the surrounding ccmF-2 editing site. We conclude that PpPPR_71 is an RNA-binding protein that acts as a site recognition factor in mitochondrial RNA editing.