Two Interacting Proteins Are Necessary for the Editing of the NdhD-1 Site in Arabidopsis Plastids  

Abstract

After transcription, mRNA editing in angiosperm chloroplasts and mitochondria results in the conversion of cytidine to uridine by deamination. Analysis of Arabidopsis thaliana mutants affected in RNA editing have shown that many pentatricopeptide repeat proteins (PPRs) are required for specific cytidine deamination events. PPR proteins have been shown to be sequencespecific RNA binding proteins allowing the recognition of the C to be edited. The C-terminal DYW domain present in many editing factors has been proposed to catalyze C deamination, as it shows sequence similarities with cytidine deaminases in other organisms. However, many editing factors, such as the first to be discovered, CHLORORESPIRATORY REDUCTION4 (CRR4), lack this domain, so its importance has been unclear. Using a reverse genetic approach, we identified DYW1, an RNA editing factor acting specifically on the plastid ndhD-1 editing site recognized by CRR4. Unlike other known editing factors, DYW1 contains no identifiable PPR motifs but does contain a clear DYW domain. We were able to show interaction between CRR4 and DYW1 by bimolecular fluorescence complementation and to reconstitute a functional chimeric CRR4-DYW1 protein complementing the crr4 dyw1double mutant. We propose that CRR4 and DYW1 act together to edit the ndhD-1 site.

Identification of Two Pentatricopeptide Repeat Genes Required for RNA Editing and Zinc Binding by C-terminal Cytidine Deaminase-like Domains

“…These observations support a model that a PPR protein serves a RNA recognition function and that the editing deaminase activity can be provided in trans (14,16,55). A recent study demonstrated that two genes are responsible for editing ndhD C2 (38). CRR4 is a PLS-type PPR protein with a truncated DYW deaminase domain and provides editing site recognition, and DYW1 contains an intact DYW deaminase domain and could serve a catalytic function (38).…”

“…In at least one case, editing has been shown to require a PPR that provides site specificity, and a separate protein has been shown to supply a portion of the DYW deaminase domain (38). Both PPR proteins CRR4 and DYW1 are required for editing ndhD C2 (38). CRR4 lacks an intact DYW domain and is apparently required as a site recognition factor, whereas DYW1 has an intact DYW domain and may contribute the catalytic activity.…”

“…Thus, although the entire DYW deaminase domain is under strong selection (37), most of the DYW deaminase domain is dispensable. In at least one case, editing has been shown to require a PPR that provides site specificity, and a separate protein has been shown to supply a portion of the DYW deaminase domain (38). Both PPR proteins CRR4 and DYW1 are required for editing ndhD C2 (38).…”

“…3 The DYW Deaminase Domain Binds Two Zinc Atoms-All of the known PPR genes that are required for RNA editing include an intact or truncated DYW deaminase domain. DYW1 is a small PPR protein with a modified repeat region and an intact C-terminal DYW deaminase domain, and was recently reported to be required for editing ndhD C2 (38). Because the DYW deaminase domains from ELI1 and DYW1 could potentially serve a catalytic function in the editing mechanism, and zinc is a critical cofactor for most nucleotide deaminases, zinc binding by the DYW deaminase domain was investigated.…”

Tetrapyrrole biosynthetic enzyme protoporphyrinogen IX oxidase 1 is required for plastid RNA editing

“…A number of PPR factors, including CRR4, CRR28, OTP80, and OTP82, and a DYW domain-containing protein, DYW1, are known to be involved in the editing of ndhB and ndhD transcripts (22)(23)(24)(25)(26), which we show in this study to contain editing sites severely affected in ppo1. To examine whether PPO1 promotes RNA editing by associating with these PPR proteins and DYW1, we carried out a yeast two-hybrid assay.…”