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

Abstract

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.

Figure 1.

Two models to explain the function of a single PPR protein that functions at multiple sites. Alignments of the nucleotide sequences in the region surrounding the editing sites affected in (A) otp82 and (B) crr22 are shown. The alignments include the sequences from −20 to +5 around the edited C (bold), with identical nucleotides shown in shaded boxes. Consensus sequences of the 15 nt immediately upstream of the edited C were identified by bioinformatics analysis (20) and are shown above the target sequences. In the consensus, full conservation of nucleotides (A, U, G and C), conservation of purines (A or G = R) or pyrimidines (U or C = Y), and conservation of the number of hydrogen bonding groups (A or U = W, G or C = S) are indicated. (C). In the upper model, each cis-acting element is recognized by the indicated PPR protein (medium and dark gray) and a second PPR protein (light gray) functions as a binding partner shared via the formation of a heterodimer at each site. The PPR protein may have additional functions other than RNA recognition, such as providing the DYW motif for the editing reaction. In the lower model, the PPR protein acts as a site-specificity factor at multiple sites.

Figure 2.

Purification of rOTP82 and rCRR22. (A) The domain organization of rOTP82 and rCRR22 fusion proteins. (B) Elution of rOTP82 and rCRR22 from a Superdex 200 gel filtration column. rOTP82 and rCRR22 were purified with Ni-NTA Agarose and then fractionated on a Superdex 200 gel filtration column. Equal proportions of contiguous column fractions were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue. Size standards BSA (67 kDa) and aldolase (160 kDa) are indicated. (C) The fractions indicated by the arrowheads in Figure 2 (B), and purified rTrx, were analyzed by SDS-PAGE. Six hundred nanograms of rOTP82, rCRR22 and rTrx was loaded onto the gel, and the gel was stained with Coomassie Brilliant Blue. The positions of molecular mass standards are indicated to the left of the gel. Lane 1 contains rCRR22, lane 2 contains rOTP82 and lane 3 contains rTRX.

Figure 3.

GMS assays with rOTP82 and target RNAs. (A) The RNA sequences used as probes are shown. Editing sites of ndhB-9 and ndhG-1 are indicated in bold and are marked with arrowheads. (B) GMS assays were performed with the indicated concentrations of rOTP82 and labeled RNAs (ndhB9L, ndhB9S, ndhB9S2, ndhG1L, ndhG1S and ndhG1S2), as described in the ‘Materials and Methods’ section. (C) Equilibrium K_d of rOTP82 for the ndhB9S and ndhB9S2 probes (left), and for the ndhG1S and ndhG1S2 probes (right). The rOTP82 concentrations and fraction of RNA bound in each lane are plotted. The K_d calculation assumed a 1:1 interaction between the RNA and protein. The K_d value and each data point are means ± SD of three experiments performed with the same rOTP82 preparation. All of the GMS assays were performed with the same preparation of rOTP82 and within 2 weeks after purification.

Figure 4.

Competition assays demonstrating sequence-specific interactions between rOTP82 and the target RNAs. (A) Two RNAs used as competitors (ndhD1 and ndhD2) are shown. Editing sites of ndhD-1 and ndhD-2 are indicated in bold and are marked with arrowheads. (B) Binding reactions included radioactive ndhB9S or ndhG1S RNA with a 1-, 10- or 100-fold molar excess of the non-radioactive RNA indicated above each panel. The concentration of rOTP82 was held constant at 56 nM. (C) GMS assays were performed with the indicated concentrations of rOTP82 and radioactive RNAs (ndhD1 and ndhD2). All of the competition and GMS assays were performed with the same preparation of rOTP82 as used in Figure 3 and within 2 weeks after purification.

Figure 5.

GMS assays with rCRR22 and target RNAs. (A) RNA sequences used as probes are shown. Editing sites of ndhB-7, ndhD-5 and rpoB-3 are indicated in bold and are marked with arrowheads. (B) GMS assays were performed with the indicated concentrations of rCRR22 and labeled RNAs (ndhB7L, ndhB7S-S3, ndhD5L, ndhD5S-S3, rpoB3L and rpoB3S-S3), as described in the ‘Materials and Methods’ section. (C) Equilibrium K_d of rCRR22 for the ndhB7S-S3 (left), ndhD5S-S3 (middle) and rpoB3S-S3 (right) probes. rCRR22 concentrations and fractions of RNA bound in each lane are plotted. The K_d calculation assumes a 1:1 interaction between the RNA and the protein. The K_d values and each data point are means ± SD of three experiments performed with the same rCRR22 preparation. All of the GMS assays were performed with the same preparation of rCRR22 and within 2 weeks after purification.

Figure 6.

Competition assays demonstrating sequence-specific interactions between rCRR22 and the target RNAs. (A) Binding reactions included radioactive ndhB7S, ndhD5S and rpoB3S RNAs, together with a 1-, 10-, or 100-fold molar excess of the non-radioactive RNAs indicated above each panel. The concentration of rCRR22 was held constant at 56 nM. (B) ycf1, the 20-nt competitor RNA used in these experiments, is shown. The 15-nt sequence, which shows similarity to the CRR22 binding region found by bioinformatics analysis (20) in the ycf1 RNA, is shown below the ycf1 sequence. In the sequence, full conservation of nucleotides (A, U, G and C), conservation of purines (A or G = R) or pyrimidines (U or C = Y), and conservation of the number of hydrogen bonding groups (A or U = W, G or C = S) are shown. (C) GMS assays were performed with the indicated concentrations of rCRR22 and radioactive RNAs (ndhD1 and ycf1). All of the competition and GMS assays were performed with the same preparation of rCRR22 as used in Figure 5 and within 2 weeks after purification.