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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.
C-to-U RNA editing frequently occurs in many transcripts of most plant organelles.1,2 For example, Arabidopsis thaliana has 508 RNA editing sites in the mitochondria3 and 34 sites in the plastids.4 In P. patens, RNA editing in the translated regions is a rarely occurring event as only one site in the plastids5 and 11 sites in the mitochondria.6,7 Since RNA editing was discovered in plant organelles 20 year ago,8,9 the molecular mechanism of RNA editing was unknown for a long time. In 2005, a nucleus-encoded and plastid-targeted PPR protein was first identified as an RNA editing factor for plastid ndhD transcript from A. thaliana.10 Last year several PPR-DYW proteins were found to act as site recognition factors for RNA editing.11–18 Among 450 A. thaliana PPR genes, 87 encode PPR-DYW proteins.19,20 The C-terminal DYW domain (95 amino acids) contains invariant residues that match the active site of cytidine deaminases from bacteria, plants, animals and yeast.21 Interestingly, neither RNA editing nor DYW domains could be identified in algae and one clade of liverworts. There is a correlation between the presence of nuclear DYW genes and organelle RNA editing among embryophytes.21,22 Therefore, the DYW domains are proposed to be responsible for RNA editing in plant organelles and catalyze RNA editing. The moss P. patens has only 10 PPR-DYW genes,20 which raises the question whether PPR-DYW proteins are responsible for RNA editing in mosses.
One of the 10 PPR-DYW proteins, PpPPR_71, is a mitochondrial-targeted protein that consists of 833 amino acids with 17 PPR motifs.20 Targeted-disruption of the PpPPR_71 gene resulted in an RNA editing defect for the ccmF-2 site of the ccmFc transcript while the 10 editing sites, including the ccmF-1 site, were not affected.7 The PpPPR_71 disruptants displayed severe growth retardation of the moss protonemata. RNA editing at this site (the 122nd nucleotide from the AUG of ccmFc mRNA) results in a serine (uCu) to phenylalanine (uUu) change at the 41st residue of CcmFc. This suggests that phenylalanine at this position is critical for the functioning of CcmFc.23
The ccmF-1 and ccmF-2 editing sites are separated only by 18 nt and therefore RNA editing at both sites could be influenced by each other. To investigate this possibility the RNA editing status of ccmFc transcripts was examined. We cloned the RT-PCR products from the wild type protonemata RNA sample and randomly sequenced 108 independent cDNA clones. Among them, 89 cDNAs (82.4%) were fully edited and 10 (9.3%) were unedited for both sites, and 9 were edited at the ccmF-1 site but not at the ccmF-2 site (Fig. 1). However, only cDNA edited at ccmF-2 could not be isolated. This strongly suggests that RNA editing for ccmF-2 depends on RNA editing of ccmF-1.
To investigate the RNA binding properties of PpPPR_71, we performed an electrophoretic mobility shift assay (EMSA) using the recombinant (r) PPR-DYW or rDYW proteins and 46-nt RNA probes, RNA1 to RNA3.7 The sequences of the three RNA probes differed by the C or U residue at the ccmF-1 and ccmF-2 editing sites (Fig. 1). This assay indicated that PpPPR_71 binds strongly to the RNA2 probe, which carries edited U at ccmF-1 and unedited C at ccmF-2. In contrast, PpPPR_71 binds weakly to RNA1 (unedited) and RNA3 (fully edited). Thus, the binding activity of PpPPR_71 likely depends on the RNA editing status of the target RNA. It is interesting to observe that the rDYW domain also binds to the same extent to the three RNA probes but its binding affinity is much weaker than that of rPPR-DYW.
Furthermore, EMSA with excess amounts of cold RNA1 to 3 probes as a competitor indicated that rDYW binds somewhat stronger to RNA1 and RNA2, which carry unedited C at ccmF-2, than to RNA3 carrying an edited ccmF-2 site. This suggests that the DYW domain itself recognizes and binds to the surrounding unedited ccmF-2 site of ccmFc RNA. Taken together with the in vivo RNA editing status of ccmFc transcripts, we propose a model of how PpPPR_71 protein works for RNA editing of the ccmFc transcript (Fig. 2). When RNA editing at the ccmF-1 site is completed, then PpPPR_71 recognizes and binds to the cis-acting element carrying U at ccmF-1 and acts as a trans-acting factor for ccmF-2 RNA editing. PPR motifs and the DYW domain cooperatively bind to the cis-acting element for RNA editing, then recruit unidentified RNA editing enzyme. After the ccmF-2 site is edited, PpPPR_71 dissociates from the target RNA.
Alternatively, the result of cDNA sequence analysis can be explained as follows. Editing at the ccmF-1 site is simply faster or more efficient than editing at the ccmF-2 site.2 If a defect of the ccmF-1 recognition factor also abolishes or strongly inhibits the editing of ccmF-2, our model will be supported. To answer this question, trans-acting factors for ccmF-1 RNA editing remain to be identified. One of the remaining 9 PPR-DYW proteins of P. patens is possibly involved in RNA editing at the ccmF-1 site. We are currently screening the knockout mutants of other PPR-DYW genes and have identified five PPR-DYW proteins required for RNA editing in the mitochondrial transcripts (Ohtani S, Tasaki E, Koumura Y, Aoki Y and Sugita M, unpublished results).
Addendum to: Tasaki E, Hattori M, Sugita M. The moss pentatricopeptide repeat protein with a DYW domain is responsible for RNA editing of mitochondrial ccmFc transcriptPlant J2010 doi: 10.1111/j.1365-313X.2010.04175.x.
Previously published online: www.landesbioscience.com/journals/psb/article/11664