A Study of New Arabidopsis Chloroplast RNA Editing Mutants Reveals General Features of Editing Factors and Their Target Sites  

Abstract

RNA editing in higher plant organelles results in the conversion of specific cytidine residues to uridine residues in RNA. The recognition of a specific target C site by the editing machinery involves trans-acting factors that bind to the RNA upstream of the C to be edited. In the last few years, analysis of mutants affected in chloroplast biogenesis has identified several pentatricopeptide repeat (PPR) proteins from the PLS subfamily that are essential for the editing of particular RNA transcripts. We selected other genes from the same subfamily and used a reverse genetics approach to identify six new chloroplast editing factors in Arabidopsis thaliana (OTP80, OTP81, OTP82, OTP84, OTP85, and OTP86). These six factors account for nine editing sites not previously assigned to an editing factor and, together with the nine PPR editing proteins previously described, explain more than half of the 34 editing events in Arabidopsis chloroplasts. OTP80, OTP81, OTP85, and OTP86 target only one editing site each, OTP82 two sites, and OTP84 three sites in different transcripts. An analysis of the target sites requiring the five editing factors involved in editing of multiple sites (CRR22, CRR28, CLB19, OTP82, and OTP84) suggests that editing factors can generally distinguish pyrimidines from purines and, at some positions, must be able to recognize specific bases.

Predictable Alteration of Sequence Recognition by RNA Editing Factors from Arabidopsis

“…A drop in clpP editing of 70% was found with a single amino acid change in CLB19 that led to a relatively minor decrease in affinity for the clpP ligand in vitro. Nevertheless, we have shown that binding of CLB19 to its targets is relatively robust to single changes in either the RNA sequences or the protein itself, consistent with the common ability of editing factors to recognize more than one editing site, often with quite divergent sequences (Hammani et al, 2009;Okuda and Shikanai, 2012). Overall, the results suggest that individual PPR motifs contribute to different extents to RNA recognition.…”

“…The process requires PPR proteins to convey the necessary RNA specificity by binding in close proximity to the cytidine that will be edited (Okuda and Shikanai, 2012). Each PPR editing factor displays a high degree of specificity, directing editing at a very limited number of RNA target sites (Hammani et al, 2009;Okuda and Shikanai, 2012).…”

To Thy Proteins Be True: RNA Editing in Plants

“…Using a reverse genetic screen of T-DNA mutants, Hammani et al (2009) searched for unedited transcripts of 34 known plastid RNAs that undergo editing, leading to the identification of six PPR proteins that together are responsible for editing nine sites in Arabidopsis plastid RNA. Interestingly, lack of editing in eight of these sites did not lead to an obvious difference in phenotype or growth habit between the mutant and wild-type plants grown under normal growth conditions.…”

“…Further analysis demonstrated that five of these PPR proteins edit multiple sites. Hammani et al (2009) examined the RNA sequences around the PPR target site to elucidate common RNA sequences. They were able of identify a 15-nucleotide stretch for four out of the five.…”

An RNA recognition motif-containing protein is required for plastid RNA editing in Arabidopsis and maize

“…The editing extent of these sites is severely reduced to more than 90% in the orrm1 mutant (Table S1). In the Y2H analysis we also included RARE1 and OTP84, which are required for the editing of accD-794, ndhF-290, ndhB-1481, and psbZ-50, respectively (29,30). The editing extent of accD-794, ndhF-290, ndhB-1481, and psbZ-50 in the orrm1 mutant is identical to the wild-type plant (Table S1).…”