Pentatricopeptide repeat proteins with the DYW motif have distinct molecular functions in RNA editing and RNA cleavage in Arabidopsis chloroplasts

Abstract

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.

Figure 1.

Monitoring NDH Activity Using Chlorophyll Fluorescence Analysis. The curve shows a typical trace of chlorophyll fluorescence in the wild type. Leaves were exposed to actinic light (50 μmol photons m⁻² s⁻¹) for 5 min. Actinic light was turned off, and the subsequent change in chlorophyll fluorescence level was monitored. The transient increase in chlorophyll fluorescence is due to the plastoquinone reduction based on NDH activity. Insets are magnified traces from the boxed area. The fluorescence levels were normalized by the maximum fluorescence at closed photosystem II centers in the dark (Fm) levels. ML, measuring light; SP, saturating pulse of white light; crr22-1+CRR22, crr22-1 complemented by introduction of the wild-type genomic CRR22; crr28-1+CRR28, crr28-1 complemented by introduction of the wild-type genomic CRR28.

Figure 2.

Predicted Motif Structure of CRR22 and CRR28. (A) PPR (or PPR-like), E, and DYW motifs are depicted as boxes with letters. The designation of the P, L, and S corresponds to the PPR motif, PPR-like S (for short) motif, and PPR-like L (for long) motif, respectively, proposed by Lurin et al. (2004). The putative plastid transit peptides are underlined. Sites of Ds or T-DNA insertions in mutant alleles are indicated. (B) Comparison of the E and DYW motifs among CLB19, CRR4, CRR21, YS1, and CRR2. Alignment was performed using the ClustalW program. The consensus sequence of the E and DYW motifs according to Lurin et al. (2004) is shown at the top. Amino acids that are fully or semiconserved are shaded black and gray, respectively. The invariant Cys and His residues in the DYW motif (Salone et al., 2007) are indicated above the sequences. Amino acids that are conserved among CRR22, CRR28, and YS1 but not in CRR2 are indicated by arrows. The point at which the sequences were truncated in the CRR2ΔDYW, CRR22ΔDYW, CRR28ΔDYW, CRR22ΔEDYW, and CRR28ΔEDYW constructs is specified.

Figure 3.

Editing Defects in crr28 and crr22 Mutants. Poisoned primer extension assays were conducted on the editing sites ndhD-3 (116290) and ndhB-2 (96698) for crr28 (A) and ndhD-5 (116281), ndhB-7 (96419), and rpoB-3 (25779) for crr22 (B). The editing sites are specified relative to the nucleotide sequence of the complete Arabidopsis chloroplast genome (Genbank Accession number AP000423). RT-PCR products were obtained with primers surrounding the editing sites and serve as templates for the extension reaction from a 5′-labeled 6-carboxyfluorescein primer that anneals next to the target editing site (a forward poisoned primer extension primer was used for all sites except for ndhD-5, for which we used a reverse primer). The extension is stopped by the incorporation of ddCTP at the location of the editing site for unedited molecules producing a short unedited product. The extension is stopped at the next C/G for the edited molecules producing a longer edited product. For ndhD-5, the extension is stopped by the incorporation of ddATP giving a longer product for the unedited ndhD-5.

Figure 4.

Protein Blot Analysis of the NDH Complex and the Major Photosynthetic Complexes. Immunodetection of NDH subunits, NdhH and NdhL; a subunit of the Cytb₆f complex, Cytf; a subunit of photosystem I, PsaB; a subunit of photosystem II, PsbO; and the γ-subunit of the chloroplast F₀F₁-ATPase, CF₁-γ. The proteins were extracted from thylakoid membrane fractions. Lanes were loaded with protein samples corresponding to 0.5 μg chlorophyll for Cytf, PsaB, PsbO, and CF₁-γ, 1 μg chlorophyll for NdhL, and 5 μg chlorophyll for NdhH (100%) and the series of dilutions indicated.

Figure 5.

The Effects on RNA Editing of Deleting or Swapping the DYW Motifs in CRR22 and CRR28. Nucleotide sequences including the RNA editing sites of ndhB-2, ndhB-7, ndhD-3, ndhD-5, and rpoB-3 are shown as sequencing chromatograms. Editing sites are indicated by arrows pointing to the corresponding peaks. crr22-1+CRR22ΔDYW, crr22-1 transformed with CRR22 lacking the DYW motif; crr28-1+CRR28ΔDYW, crr28-1 transformed with CRR28 lacking the DYW motif; crr22-1+CRR22DYW2, crr22-1 transformed with CRR22, in which the DYW motif was replaced by that of CRR2; crr28-1+CRR28DYW2, crr28-1 transformed with CRR28, in which the DYW motif was replaced by that of CRR2; crr22-1+CRR22DYW28, crr22-1 transformed with CRR22, in which the DYW motif was replaced by that of CRR28; crr28-1+CRR28DYW22, crr28-1 transformed with CRR28, in which the DYW motif was replaced by that of CRR22; crr22-1+CRR22ΔE/DYW, crr22-1 transformed with CRR22 lacking the E and DYW motifs; crr28-1+CRR28ΔE/DYW, crr28-1 transformed with CRR28 lacking the E and DYW motifs.

Figure 6.

The Effects of Deleting or Swapping the DYW Motif in CRR2. The rps7-ndhB region is shown schematically. The arrowhead indicates the site that is not cleaved in crr2; this site is located at position −12 with respect to the ndhB translation initiation codon. Total RNA (5 μg) isolated from leaves of 4-week-old wild-type and transgenic plants was analyzed by RNA gel blot and hybridization. The probe used for the experiments is indicated by a bar beneath the 3′ exon of ndhB. The signal identities (I and II) were based on previous analysis (Hashimoto et al., 2003). The migration of RNA size markers is indicated at the left. crr2-1+CRR2ΔDYW, crr2-1 transformed with CRR2 lacking the DYW motif; crr2-1+CRR2DYW22, crr2-1 transformed with CRR2, in which the DYW motif was replaced by that of CRR22; crr2-1+CRR2DYW28, crr2-1 transformed with CRR2, in which the DYW motif was replaced by that of CRR28; crr2-1+CRR2, crr2-1 complemented by introduction of the wild-type genomic CRR2.

Figure 7.

Activity Assay of the DYW Motif in CRR22. (A) The in vitro RNA editing assay. The [α-³²P]CTP–labeled NB2 RNA (0.2 nM) was incubated with the indicated proteins (100 nM) or without proteins (−protein) in the presence or absence of zinc ions (Zn; 0 to 2 mM). The RNA was digested into mononucleotides and separated by thin layer chromatography. The position of the U spot was confirmed using [α-³²P]UTP–labeled RNA. The spots indicated by an asterisk are probably pCp residues resulting from RNA cleavage by DYW/CRR2. (B) The in vitro RNA cleavage assay. The indicated protein (100 nM) was incubated with 0.2 nM ³²P-labeled NB2 RNA in buffer containing 10 mM MgCl₂. The reaction was performed in the absence or presence of metal ion chelator, EDTA (0 to 75 mM). The reaction was also performed with recombinant thioredoxin-Hisx6 tag protein (Trx-His₆) or without any protein (−protein). The ³²P-labeled RNAs were extracted and then separated by denaturing PAGE.