Kenji Okuda

A Pentatricopeptide Repeat Protein Is a Site Recognition Factor in Chloroplast RNA Editing

In higher plants, RNA editing is a post-transcriptional process that converts C to U in organelle mRNAs. We have previously shown that an Arabidopsis thaliana crr4 mutant is defective with respect to RNA editing for creating the translational initial codon of the plastid ndhD gene (the ndhD-1 site). CRR4 contains 11 pentatricopeptide repeat motifs but does not contain any domains that are likely to be involved in the editing activity. The green fluorescent protein fused to the putative transit peptide of CRR4 targeted the plastid. The recombinant CRR4 expressed in Escherichia coli specifically bound to the 25 nucleotides of the upstream and the 10 nucleotides of the downstream sequences surrounding the editing site of ndhD-1. The target C nucleotide of this editing is not essential for the binding of CRR4. Taken together with the genetic evidence, we conclude that the pentatricopeptide repeat protein CRR4 is a sequence-specific RNA-binding protein that acts as a site recognition factor in plastid RNA editing.

Pentatricopeptide Repeat Proteins with the DYW Motif Have Distinct Molecular Functions in RNA Editing and RNA Cleavage in Arabidopsis Chloroplasts

The plant-specific DYW subclass of pentatricopeptide repeat proteins has been postulated to be involved in RNA editing of organelle transcripts. We discovered that the DYW proteins CHLORORESPIRATORY REDUCTION22 (CRR22) and CRR28 are required for editing of multiple plastid transcripts but that their DYW motifs are dispensable for editing activity in vivo. Replacement of the DYW motifs of CRR22 and CRR28 by that of CRR2, which has been shown to be capable of endonucleolytic cleavage, blocks the editing activity of both proteins. In return, the DYW motifs of neither CRR22 nor CRR28 can functionally replace that of CRR2. We propose that different DYW family members have acquired distinct functions in the divergent processes of RNA maturation, including RNA cleavage and RNA editing.

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

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.

The pentatricopeptide repeat protein OTP82 is required for RNA editing of plastid ndhB and ndhG transcripts

Several hundred nucleus-encoded factors are required for regulating gene expression in plant organelles. Among them, the most numerous are the members of the pentatricopeptide repeat (PPR) protein family. We found that PPR protein OTP82 is essential for RNA editing of the ndhB-9 and ndhG-1 sites within transcripts encoding subunits of chloroplast NAD(P)H dehydrogenase. Despite the defects in RNA editing, otp82 did not show any phenotypes in NDH activity, stability or interaction with photosystem I, suggesting that the RNA editing events mediated by OTP82 are functionally silent even though they induce amino acid alterations. In agreement with this result, both sites are partially edited even in the wild type, implying the possibility that a single gene produces heterogeneous proteins that are functionally equivalent. Although only five nucleotides separate the ndhB-8 and ndhB-9 sites, the ndhB-8 site is normally edited in otp82 mutants, suggesting that both sites are recognized by different PPR proteins. OTP82 falls into the DYW subclass containing conserved C-terminal E and DYW motifs. As in CRR22 and CRR28, the DYW motif present in OTP82 is not essential for RNA editing in vivo.

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

Complementary, reverse genetic, protein–protein interaction and fusion approaches reveal the requirement of at least two interacting PPR proteins for the editing of a specific site in Arabidopsis plastids. 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 sequence-specific 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.

A eukaryotic factor required for accumulation of the chloroplast NAD(P)H dehydrogenase complex in Arabidopsis.

The NAD(P)H dehydrogenase (NDH) complex in chloroplasts mediates photosystem I cyclic and chlororespiratory electron transport. Eleven chloroplast genes and three nuclear genes have been identified as encoding Ndh subunits, but the entire subunit composition is still unknown. An Arabidopsis (Arabidopsis thaliana) chlororespiratory reduction (crr3) mutant was isolated based on its lack of transient increase in chlorophyll fluorescence after actinic light illumination; this was due to a specific defect in accumulation of the NDH complex. The CRR3 gene (At2g01590) encodes a novel protein containing a putative plastid-targeting signal and a transmembrane domain. Consistent with the gene structure, CRR3 localized to the membrane fraction of chloroplasts. In addition to the essential function of CRR3 in stabilizing the NDH complex, the NDH complex is also required for the accumulation of CRR3. These results suggest that CRR3 interacts with the NDH complex in the thylakoid membrane. In contrast to other subunits in the chloroplast NDH complex, CRR3 is not conserved in cyanobacteria from which the chloroplast NDH complex is believed to have originated. We propose that CRR3 is a subunit of the NDH complex, which is specific to the chloroplast.

The E domains of pentatricopeptide repeat proteins from different organelles are not functionally equivalent for RNA editing

RNA editing in plants is an essential post-transcriptional process that modifies the genetic information encoded in organelle genomes. Forward and reverse genetics approaches have revealed the prevalent role of pentatricopeptide repeat (PPR) proteins in editing in both plastids and mitochondria, confirming the shared origin of this process in both organelles. The E domain at or near the Cxa0terminus of these proteins has been shown to be essential for editing, and is presumed to recruit the enzyme that deaminates the target cytidine residue. Here, we describe two mutants, otp71 and otp72, disrupted in genes encoding mitochondrial E-type PPR proteins with single editing defects in ccmFN 2 and rpl16 transcripts, respectively. Comparisons between the E domains of these proteins and previously reported editing factors from chloroplasts suggested that there are characteristic differences in the proteins between the two organelles. To test this, we swapped E domains between two mitochondrial and two chloroplast editing factors. In all cases investigated, E domains from the same organelle (chloroplast or mitochondria) were found to be exchangeable; however, swapping the E domain between organelles led to non-functional editing factors. We conclude that the E domains of mitochondrial and plastid PPR proteins are not functionally equivalent, and therefore that an important component of the putative editing complexes in the two organelles may be different.

PROTON GRADIENT REGULATION 3 recognizes multiple targets with limited similarity and mediates translation and RNA stabilization in plastids

PROTON GRADIENT REGULATION 3 (PGR3) contains 27 pentatricopeptide repeat (PPR) motifs and belongs to the P-subfamily. Previous studies have suggested that PGR3 functions in the stabilization of petL operon RNA and also in the translation of petL and one, or some, of the 11 plastid ndh genes encoding subunits of chloroplast NADH dehydrogenase-like complex (NDH). The pgr3-3 allele has been suggested to be specifically defective in the putative PGR3 function of translation. Herein, we show that the polysome association of the monocistronic petL transcript is impaired in pgr3-3. We detected sequences weakly conserved in the 5 untranslated regions (UTRs) of petL and ndhA, and these putative elements were recognized by recombinant PGR3 in vitro. Previously, pgr3-2 was shown to be specifically defective in stabilizing petL operon RNA and to accumulate NDH at wild-type levels. Consistent with this pgr3-2 phenotype, we show here that a recombinant protein carrying the pgr3-2 mutation in the 12th PPR motif bound to the 5 UTR of ndhA but not of petL. This indicates that a single amino acid alteration changes the binding specificity of PGR3. In contrast, the recombinant protein carrying the pgr3-3 mutation in the final, 27th, incomplete PPR motif can bind to both petL and ndhA 5 UTRs, suggesting that the C-terminal end of PGR3 is not required for binding to targets but is essential for translation of petL and probably also ndhA. Our results fully support the model in which PGR3 recognizes two target sequences and is involved in multiple functions, i.e. stabilizing RNA and activating translation.

A pentatricopeptide repeat protein acts as a site-specificity factor at multiple RNA editing sites with unrelated cis-acting elements in plastids

In plant organelles, RNA editing alters specific cytidine residues to uridine in transcripts. All of the site-specificity factors of RNA editing identified so far are pentatricopeptide repeat (PPR) proteins. A defect in a specific PPR protein often impairs RNA editing at multiple sites, at which the cis-acting elements are not highly conserved. The molecular mechanism for sharing a single PPR protein over multiple sites is still unclear. We focused here on the PPR proteins OTP82 and CRR22, the putative target elements of which are, respectively, partially and barely conserved. Recombinant OTP82 specifically bound to the −15 to 0 regions of its target sites. Recombinant CRR22 specifically bound to the −20 to 0 regions of the ndhB-7 and ndhD-5 sites and to the −17 to 0 region of the rpoB-3 site. Taking this information together with the genetic data, we conclude that OTP82 and CRR22 act as site-specificity factors at multiple sites in plastids. In addition, the high-affinity binding of CRR22 to unrelated cis-acting elements suggests that only certain specific nucleotides in a cis-acting element are sufficient for high-affinity binding of a PPR protein. The cis-acting elements can therefore be rather divergent and still be recognized by a single PPR protein.

Quantitative analysis of motifs contributing to the interaction between PLS-subfamily members and their target RNA sequences in plastid RNA editing

In plant organelles, RNA editing alters specific cytidine residues to uridine in transcripts. Target cytidines are specifically recognized by pentatricopeptide repeat (PPR) proteins of the PLS subfamily, which have additional C-terminal E or E-DYW motifs. Recent in silico analysis proposed a model for site recognition by PLS-subfamily PPR proteins, with a correspondence of one PPR motif to one nucleotide, and with the C-terminal last S motif aligning with the nucleotide at position -4 with respect to the editing site. Here, we present quantitative biochemical data on site recognition by four PLS-subfamily proteins: CRR28 and OTP85 are DYW-class members, whereas CRR21 and OTP80 are E-class members. The minimal RNA segments required for high-affinity binding by these PPR proteins were experimentally determined. The results were generally consistent with the in silico-based model; however, we clarified that several PPR motifs, including the C-terminal L2 and S motifs of CRR21 and OTP80, are dispensable for the RNA binding, suggesting distinct contributions of each PPR motif to site recognition. We also demonstrate that the DYW motif interacts with the target C and its 5 proximal region (from -3 to 0), whereas the E motif is not involved in binding.