A comparative genomics approach identifies a PPR-DYW protein that is essential for C-to-U editing of the Arabidopsis chloroplast accD transcript

Abstract

Several nuclear-encoded proteins containing pentatricopeptide repeat (PPR) motifs have previously been identified to be transfactors essential for particular chloroplast RNA editing events through analysis of mutants affected in chloroplast biogenesis or function. Other PPR genes are known to encode proteins involved in other aspects of organelle RNA metabolism. A function has not been assigned to most members of the large plant PPR gene family. Arabidopsis and rice each contain over 400 PPR genes, of which about a fifth exhibit a C-terminal DYW domain. We describe here a comparative genomics approach that will facilitate identification of the role of RNA-binding proteins in organelle RNA metabolism. We have implemented this strategy to identify an Arabidopsis nuclear-encoded gene RARE1 that is required for editing of the chloroplast accD transcript. RARE1 carries 15 PPR motifs, an E/E+ and a DYW domain, whereas previously reported editing factors CRR4, CRR21, and CLB19 lack a DYW domain. The accD gene encodes the b carboxyltransferase subunit of acetyl coA carboxylase, which catalyzes the first step in fatty acid biosynthesis in chloroplasts. Despite a lack of accD C794 editing and lack of restoration of an evolutionarily conserved leucine residue in the b carboxyltransferase protein, rare1 mutants are unexpectedly robust and reproduce under growth room conditions. Previously the serine-to-leucine alteration caused by editing was deemed essential in the light of the finding that a recombinantly expressed ''unedited'' form of the pea acetyl coA carboxylase was catalytically inactive.Keywords: chloroplast; RNA editing; accD; RARE1; pentatricopeptide; trans-factor INTRODUCTION Vascular plant organelle transcripts undergo C-to-U RNA editing (for review, see Hanson et al. 1996;Bock 2000;Tillich et al. 2005;Shikanai 2006). In Arabidopsis, 34 editing events are known to occur in chloroplast transcripts (Tillich et al. 2005;Chateigner-Boutin and Small 2007), while 508 Cs are known to be modified to Us in Arabidopsis mitochondria (Bentolila et al. 2008). The amino acid encoded by edited transcripts often differs from the one predicted from unedited transcripts, usually resulting in increased evolutionary conservation of the amino acid sequence from the one predicted from genomic sequence (Gualberto et al. 1989), although start and stop codons in organelle transcripts are also sometimes created by C-to-U editing (Wintz and Hanson 1991;Kudla et al. 1992). The residues modified by RNA editing are often important for the three-dimensional structure of the protein (Yura and Go 2008). RNA editing appears to be a mechanism to correct defective organelle genes at the transcript level.The suite of particular C-to-U editing events varies from one plant species to another, even though RNA editing probably arose in an ancestor common to the land plants (Tillich et al. 2006). Between divergent species, such as between dicots and monocots, RNA editing C targets vary considerably. In species that do not contain a particular C-...

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

“…Unlike the majority of the previously characterized editing mutants (Okuda et al, 2006(Okuda et al, , 2007(Okuda et al, , 2009Chateigner-Boutin et al, 2008;Kim et al, 2009;Yu et al, 2009;Zhou et al, 2009), which were identified via visible growth or fluorescence phenotypes, these mutants were identified directly via a screen for unedited RNAs. It is striking that of the eight mutants discovered in this way, including RARE1 (Robbins et al, 2009) and MEF1 (Zehrmann et al, 2009), none have growth or The curve shows a typical trace of chlorophyll fluorescence in the wild type and a mutant totally impaired in NDH activity (crr21) (Okuda et al, 2007) compared with traces from otp85 and two independent otp84 mutants (otp84-1 and otp84-2). Leaves were exposed to actinic light (AL) (50 mmol of photons m À2 s À1 ) for 5 min.…”

“…Investigation of Arabidopsis mutants with such phenotypes has identified a number of nuclear genes suspected to encode RNAediting specificity factors (Kotera et al, 2005;Okuda et al, 2007Okuda et al, , 2009Chateigner-Boutin et al, 2008;Cai et al, 2009;Kim et al, 2009;Robbins et al, 2009;Yu et al, 2009;Zehrmann et al, 2009;Zhou et al, 2009). In each case, loss of one of these factors leads to a specific loss of editing of one or at most a few sites.…”

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

“…RNA extraction and RT-PCR methods were as previously described (4) and chloroplast isolation as described in Hayes and Hanson (55). Primers to amplify the mitochondrial and plastid transcripts have been previously described (4,29,56). Analysis of RNA editing by PPE was done as in ref.…”

“…Analysis of RNA editing by PPE was done as in ref. 29. The editing extent in maize plastid genes was measured primarily by bulk-sequencing of RT-PCR products amplified with primers listed in Table S4.…”

“…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).…”