Article7 May 2009free access Site-specific binding of a PPR protein defines and stabilizes 5′ and 3′ mRNA termini in chloroplasts Jeannette Pfalz Jeannette Pfalz Institute of Molecular Biology, University of Oregon, Eugene, OR, USAPresent address: Friedrich-Schiller-University, Jena, Germany Search for more papers by this author Omer Ali Bayraktar Omer Ali Bayraktar Institute of Molecular Biology, University of Oregon, Eugene, OR, USA Search for more papers by this author Jana Prikryl Jana Prikryl Institute of Molecular Biology, University of Oregon, Eugene, OR, USA Search for more papers by this author Alice Barkan Corresponding Author Alice Barkan Institute of Molecular Biology, University of Oregon, Eugene, OR, USA Search for more papers by this author Jeannette Pfalz Jeannette Pfalz Institute of Molecular Biology, University of Oregon, Eugene, OR, USAPresent address: Friedrich-Schiller-University, Jena, Germany Search for more papers by this author Omer Ali Bayraktar Omer Ali Bayraktar Institute of Molecular Biology, University of Oregon, Eugene, OR, USA Search for more papers by this author Jana Prikryl Jana Prikryl Institute of Molecular Biology, University of Oregon, Eugene, OR, USA Search for more papers by this author Alice Barkan Corresponding Author Alice Barkan Institute of Molecular Biology, University of Oregon, Eugene, OR, USA Search for more papers by this author Author Information Jeannette Pfalz1, Omer Ali Bayraktar1, Jana Prikryl1 and Alice Barkan 1 1Institute of Molecular Biology, University of Oregon, Eugene, OR, USA *Corresponding author. Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA. Tel.: +1 541 346 5145; Fax: +1 541 346 5891; E-mail: [email protected] The EMBO Journal (2009)28:2042-2052https://doi.org/10.1038/emboj.2009.121 There is a Have you seen ...? (July 2009) associated with this Article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Chloroplast mRNA populations are characterized by overlapping transcripts derived by processing from polycistronic precursors. The mechanisms and functional significance of these processing events are poorly understood. We describe a pentatricopeptide repeat (PPR) protein, PPR10, whose binding defines mRNA segments derived from two transcription units in maize chloroplasts. PPR10 interacts in vivo and in vitro with two intergenic RNA regions of similar sequence. The processed 5′ and 3′ RNA termini in these regions overlap by approximately 25 nucleotides. The PPR10-binding sites map precisely to these overlapping sequences, and PPR10 is required specifically for the accumulation of RNAs with these termini. These findings show that PPR10 serves as a barrier to RNA decay from either the 5′ or 3′ direction and that a bound protein provides an alternative to an RNA hairpin as a barrier to 3′ exonucleases. The results imply that protein ‘caps’ at both 5′ and 3′ ends can define the termini of chloroplast mRNA segments. These results, together with recent insights into bacterial RNA decay, suggest a unifying model for the biogenesis of chloroplast transcript populations and for the determinants of chloroplast mRNA stability. Introduction The chloroplast has retained many properties of its cyanobacterial ancestor, including operon-like polycistronic transcription units and an RNA polymerase that is closely akin to that in bacteria. However, the transfer of many endosymbiont genes encoding components of the photosynthetic apparatus to the nucleus, and the incorporation of the organelle into plants with distinct developmental stages and cell types necessitated the evolution of mechanisms to coordinate the activities of the nuclear and chloroplast genomes. Nucleus-encoded proteins that modulate chloroplast gene expression provide one means to this end. Several hundred such proteins are anticipated based on the current data, some of bacterial ancestry and others derived from the host genome. Prominent among the latter class are members of the pentatricopeptide repeat (PPR) family (Small and Peeters, 2000). PPR proteins are defined by degenerate 35 amino acid repeats that are related to the tetratricopeptide repeat (TPR). Genetic data have implicated PPR proteins in various aspects of organellar RNA metabolism in all eukaryotic lineages (reviewed in Schmitz-Linneweber and Small, 2008), but their mechanisms of action are poorly understood. The results presented here define precise RNA-binding sites for a chloroplast PPR protein, and link binding at those sites to RNA stabilization and the accumulation of specific processed mRNA isoforms. Chloroplast RNA turnover (reviewed in Bollenbach et al, 2007) is believed to be initiated by endonucleolytic cleavage, followed by 3′-polyadenylation of the cleavage products and 3′ → 5′ exonucleolytic degradation. RNA hairpins at 3′ termini impede the 3′ → 5′ exonucleases, thereby stabilizing upstream RNA. This scenario is similar to that proposed for Escherichia coli (reviewed in Condon, 2007). Superimposed on this model is the notion that site-specific intercistronic cleavages generate the complex RNA populations that are characteristic of polycistronic transcription units in land plant chloroplasts (reviewed in Bollenbach et al, 2007). In this context, several observations have been puzzling. First, the downstream products of endonucleolytic cleavage are rapidly degraded in vivo (reviewed in Bollenbach et al, 2007), suggesting that 5′ terminal features are important for stabilizing chloroplast mRNAs. This notion is supported by the analysis of chimeric genes in tobacco chloroplasts (Eibl et al, 1999) and by genetic data from Chlamydomonas reinhardtii (Boudreau et al, 2000; Vaistij et al, 2000a; Murakami et al, 2005; Loiselay et al, 2008). Second, in one of the few instances in which both the 5′ and 3′ termini of adjacent processed chloroplast RNAs have been mapped, the RNAs overlap in a manner that is not compatible with their biogenesis through a single cleavage event (Barkan et al, 1994). The results presented here provide evidence for a conceptually simple mechanism that can account for these and other disparate observations. These insights emerged from the analysis of a maize PPR protein dubbed PPR10. We present evidence that PPR10 stabilizes two sets of chloroplast transcripts: RNAs with a 5′- or 3′-end mapping in either the atpI-atpH or psaJ-rpl33 intergenic region. We show that bound PPR10 defines the positions of the 5′ and 3′ termini of processed transcripts derived from both regions, and that it substitutes for a 3′-terminal hairpin to protect upstream RNA from 3′ exonucleases. These and other findings suggest that protein ‘caps’ at 5′ and 3′ termini may be a major mechanism for defining chloroplast transcript populations. When considered in the context of recent advances in understanding RNA decay in bacteria, these results suggest revised models for the biogenesis of complex chloroplast transcript populations and for the determinants of chloroplast mRNA stability. Results PPR10 is required for chloroplast development PPR10 belongs to the ‘P’ subfamily of PPR proteins, whose members lack additional domains (Schmitz-Linneweber and Small, 2008). Rice and Arabidopsis PPR10 orthologs (Os05g19380 and At2g18940, respectively) were identified through phylogenetic analyses (Supplementary Figure 1). PPR10 orthologs are predicted to begin with a chloroplast transit peptide, followed by approximately 100 amino acids and then 18 or 19 PPR motifs (Supplementary Figure 2). However, the SCOP server (Tung and Yang, 2007) and structural modelling of PPR10 (Supplementary Figure 3) predict that the N-terminal region also adopts a TPR-like structure. Therefore, virtually the entire mature portion of PPR10 is likely to consist of helical hairpin units. We recovered two mutant alleles of ppr10 in a reverse-genetic screen of our collection of transposon-induced non-photosynthetic maize mutants. Both insertions disrupt the open reading frame (Figure 1A) and cosegregate with mutations conferring a yellow-green seedling phenotype (Figure 1B). The progeny of crosses between ppr10-1/+ and ppr10-2/+ segregate yellow-green seedlings, showing lack of complementation between the alleles. Heteroallelic progeny of complementation crosses (ppr10-1/-2) were used in all phenotypic assays to ensure that the phenotypes result from disruption of ppr10. The mutant seedlings die after the development of three leaves, as is typical of non-photosynthetic maize mutants. The mutants have reduced levels of subunits of several photosynthetic enzyme complexes (Figure 1C); the AtpF subunit of CF0 (the membrane-intrinsic portion of the ATP synthase) is the most severely reduced of the proteins assayed. Figure 1.ppr10 mutants. (A) Transposon insertions in ppr10. The PPR10 coding region is indicated by a rectangle. Sequences flanking the insertions are shown below with the target-site duplications underlined. Polymorphisms in the terminal inverted repeats identify the insertions as related to the MuDR member of the Mu family. (B) Phenotypes of ppr10 mutants. Seedlings were grown for 8 days in soil. ppr10-1/-2 is the progeny of a complementation cross. (C) Immunoblot analysis of photosynthetic complex subunits. Immunoblots of leaf extracts were probed with antibodies to proteins indicated to the right; the Ponceau S-stained blot below illustrates sample loading and the abundance of RbcL, the large subunit of Rubisco. AtpA and AtpF are subunits of the CF1 and CF0 portions of the ATP synthase, respectively. D1, PsaD and PetD are subunits of photosystem II, photosystem I and the cytochrome b6f complex, respectively. hcf7 illustrates protein losses resulting from a global decrease in plastid translation (Barkan, 1993). (D) Immunoblot detection of PPR10 in leaf extract, showing antibody specificity and loss of PPR10 in ppr10 mutants. A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint Polyclonal antibodies were raised against a PPR10 segment that lacks similarity to non-orthologous proteins. This antibody detects a protein of the size expected for mature PPR10 (∼80 kDa) in wild-type leaf; this protein is absent in ppr10 mutants (Figure 1D), verifying that it is PPR10. PPR10 is associated with atpH and psaJ RNAs in chloroplast extract Immunoblot analysis of leaf and subcellular fractions showed PPR10 to be enriched in isolated chloroplasts, where it was found solely in the stroma (Figure 2A). When stromal extract was fractioned by sedimentation through a sucrose gradient, PPR10 was found in a broad peak representing particles between ∼100 and 600 kDa (Figure 2B). Treatment of the extract with ribonuclease shifted the PPR10 distribution toward smaller particles, suggesting that a fraction of PPR10 is associated with RNA. Figure 2.PPR10 is localized to the chloroplast stroma. (A) Immunoblots of leaf and subcellular fractions. Chloroplast (Cp) subfractions were loaded on the basis of equal chloroplast number. Replicate blots were probed with antibodies to the proteins indicated at left. Cpn60, PetD, IM35 and PDH were used as markers for stroma (Str), thylakoid (Thy), envelope (Env) and mitochondria (Mito), respectively. (B) Size distribution of PPR10-containing particles. Stroma was incubated with ribonuclease A (RNAse) or mock-treated and fractionated by sedimentation through sucrose gradients. An equal volume of each gradient fraction was analysed by probing immunoblots with PPR10 antibody. The Ponceau S-stained blots illustrate the position of Rubisco (∼550 kDa). P, pelleted material. Download figure Download PowerPoint We used a ‘RIP-Chip’ assay as an initial screen to identify the RNAs that associate with PPR10 in chloroplast extract: RNAs purified from the pellets and supernatants of PPR10 immunoprecipitations were labelled with a red-fluorescing or green-fluorescing dye, respectively, combined and hybridized to a tiling microarray of the maize chloroplast genome (Schmitz-Linneweber et al, 2005). Two replicate experiments were performed, using PPR10 antibodies from different immunized rabbits. An immunoprecipitation with antiserum to OE16 served as a negative control. Figure 3A shows the median enrichment ratio for each array element plotted as a function of chromosomal position, after subtracting the corresponding values for the control assay. The most highly significant peaks represent sequences near the atpH and psaJ loci. Within these peaks, the strongest enrichment was detected near the 5′ end of atpH and the 3′ end of psaJ. Slot-blot hybridization of immunoprecipitated RNAs validated the specific enrichment of atpH 5′ UTR and psaJ 3′ UTR RNA sequences in PPR10 immunoprecipitations (Figure 3B). RNA from near the petA locus appeared to be enriched by RIP-chip, but did not reproduce in the slot-blot assay. These results point to RNAs from the 5′ region of atpH and the 3′ region of psaJ as the primary and possibly the sole physiological ligands of PPR10. Figure 3.Coimmunoprecipitation assays identifying RNAs associated with PPR10. (A) Summary of RIP-chip data. The median log2-transformed enrichment ratios (F635/F532) for two replicate PPR10 immunoprecipitations are plotted according to chromosomal position after subtracting the corresponding values for a control immunoprecipitation with OE16 antibody. Data points that show significant differential enrichment between the PPR10 and control assays (P-value <1E-4) are marked with diamonds and are annotated with the locus name. The underlying data for the highest-ranking fragments are summarized in Supplementary Table 1. (B) Validation of RIP-chip data. Immunoprecipitations were performed as for RIP-chip assays except that ribonuclease inhibitor was not included. One-sixth of the RNA from each immunoprecipitation pellet (P) and one-twelfth of the RNA from the corresponding supernatant (S) were applied to replicate slot blots. The two PPR10 immunoprecipitations (a and b) used sera from different immunized rabbits. Blots were probed with oligonucleotides specific for the atpH 5′ UTR, the psaJ 3′ UTR, the petA 5′ UTR and the coding regions of the other genes indicated. Download figure Download PowerPoint Fine-mapping coimmunoprecipitated RNAs reveals a consensus sequence at sites of PPR10 association To precisely map the RNA sequences that are associated with PPR10, immunoprecipitations were performed without ribonuclease inhibitor so as to decrease the size of the coimmunoprecipitated RNA fragments. RNAs recovered from the immunoprecipitation pellets and supernatants were applied to slot-blots and hybridized to a series of 60-nt probes from the atpH and psaJ loci (Figure 4). The strongest enrichment at the atpH locus was detected with the atpH-2 60-mer (Figure 4A); the strongest enrichment at the psaJ locus was detected with the psaJ-5 60-mer (Figure 4B). An alignment of sequences in these regions (Figure 4C) revealed strong sequence similarity spanning approximately 30 nucleotides. Experiments described below showed that this shared sequence is directly recognized by PPR10. Figure 4.Fine-mapping RNAs that coimmunoprecipitate with PPR10. (A, B) Replicate slot blots were prepared from immunoprecipitation pellet (P) and supernatant (S) RNAs, as described in Figure 3B. The positions of the 60-mer oligonucleotide probes are diagrammed, using line weights that reflect the degree to which corresponding RNAs were enriched in PPR10 immunoprecipitations. The most strongly enriched sequences are shown below, with the consensus residues shared by the atpH and psaJ sites highlighted in bold. The data were quantified with a phosphorimager, and are graphed at right. (C) Alignment of sequences near atpH and psaJ that coimmunoprecipitate with PPR10, with the consensus highlighted. Download figure Download PowerPoint PPR10 is required for the accumulation of RNAs with a 5′- or 3′-end mapping to sites of PPR10 interaction To investigate the role of PPR10, chloroplast RNA in ppr10 mutants was initially profiled by hybridization to our chloroplast tiling microarray (Supplementary Figure 4). The results suggested that ppr10 mutants have reduced levels of atpH and psaJ RNAs, correlating with the finding that these are PPR10 ligands. RNA gel blots were used to validate and extend these findings (Figure 5). hcf7 mutants, which have a global decrease in chloroplast translation (Barkan, 1993), were analysed in parallel to control for indirect effects resulting from compromised photosynthesis. The results showed that ppr10 mutants lack specific transcripts from both the atpH and psaJ transcription units. Other transcripts suggested by either the RIP-chip data or microarray RNA profile to be PPR10 targets were also examined by RNA gel blot hybridization (Supplementary Figure 5); the abundance of petA RNA is reduced slightly in ppr10 mutants, but no other differences were detected between ppr10 and the control hcf7 mutants. Figure 5.RNA gel blot analysis of atpH (A) and psaJ (B) RNAs in ppr10 mutants. Probes are diagrammed above the maps as black lines. Numbered probes correspond to the same 60-mer oligonucleotides used for slot blot hybridizations in Figure 4. Transcript maps were generated based on transcript size, the probes with which they hybridized and by the cRT–PCR data in Supplementary Table 2. RNAs missing in ppr10 mutants are in grey. Dashed lines indicate the spliced atpF intron. The band marked with an asterisk on the psaJ-6 blot derives from a prior probing with the petG probe. These transcript maps are consistent with and expand on those reported earlier (Stahl et al, 1993; Miyagi et al, 1998; Yamazaki et al, 2004); remaining ambiguities are indicated with question marks. All lanes in each panel come from the same gel. Download figure Download PowerPoint The atpH and psaJ transcript data revealed a striking correspondence between those RNAs that are absent in ppr10 mutants and those with termini mapping near PPR10's in vivo interaction sites. Thus, all transcripts with a 5′- or a 3′-end mapping in either the atpH -atpI (Figure 5A) or the psaJ-rpl33 (Figure 5B) intergenic region fail to accumulate in ppr10 mutants (transcripts in grey). The positions of the termini of PPR10-dependent transcripts were estimated by probing blots with the 60-mers used to fine-map the coimmunoprecipitating RNAs. The atpH-2 60-mer hybridized to the PPR10-dependent atpH transcripts, whereas the adjacent atpH-3 60-mer did not (Figure 5A). A primer extension assay placed this 5′ end approximately 46 nucleotides upstream of the atpH start codon (Figure 6A), consistent with the RNA gel blot data and with an earlier report of a 5′ end near this position (Stahl et al, 1993). The psaJ-5 60-mer hybridized to PPR10-dependent psaJ transcripts, whereas the adjacent psaJ-6 60-mer did not (Figure 5B); this placed the 3′ end of PPR10-dependent psaJ RNAs near the ‘downstream’ end of the psaJ-5 sequence. Together, the genetic and coimmunoprecipitation data indicate that PPR10 is bound very near the termini of the transcripts that fail to accumulate in its absence. The loss of processed RNAs in ppr10 mutants was not accompanied by an increased abundance of precursor transcripts, arguing that transcript loss is caused by transcript instability rather than by a defect in precursor cleavage. Figure 6.Positions of PPR10-dependent RNA termini. (A) Primer extension mapping of the PPR10-dependent atpH-5′ end. A sequencing ladder generated with the same primer on an in vitro transcript is shown to the right. The U-tract disrupts the fidelity of reverse transcriptase, making the distal sequence ambiguous. (B) cRT–PCR procedure used to map transcript termini. Primer RP1 was used for reverse transcription. PCR was performed initially with RP1 and FP1 primers; nested PCR was then performed with RP1 and FP2. (C) Overlapping transcript termini in the atpI/atpH and psaJ/rpl33 intergenic regions. Residue numbers refer to position relative to the start codons of atpH and rpl33 or the stop codons of atpI and psaJ. Triangle size reflects relative transcript frequency, based on the data in Supplementary Table 2. Residues that are most highly conserved between the two sites are highlighted. Download figure Download PowerPoint Phosphorimager analysis of the RNA gel blot data showed that the total abundance of atpH transcripts is reduced by approximately three-fold in ppr10 mutants (data not shown). This is insufficient to account for the >10-fold decrease in the abundance of the CF0 complex (as monitored by its AtpF subunit in Figure 1C), suggesting that PPR10 might enhance the translation of the atpH ORF. To address this possibility, we examined the association of atpH mRNAs with polysomes (Supplementary Figure 6). The residual atpH transcripts in the ppr10 mutant were shifted toward polysomes of smaller size, consistent with a translation-enhancing role for PPR10. However, the polycistronic nature of these RNAs precludes firm conclusions. In fact, these data suggested a small global decrease in plastid translation in ppr10 mutants, consistent with their moderate reduction in all photosynthetic enzyme complexes harbouring plastid-encoded subunits (Figure 1C). Overlapping transcript termini in the atpI-atpH and psaJ-rpl33 intergenic regions highlight PPR10 interaction sites To obtain more comprehensive information about the termini of PPR10-dependent transcripts, transcript termini in the atpI-atpH and psaJ-rpl33 intergenic regions were determined by circularization RT–PCR (cRT–PCR) (Figure 6B; Supplementary Table 2). The results showed that the adjacent processed RNAs in both regions overlap in a manner that is not compatible with their biogenesis through a single endonucleolytic cleavage (Figure 6C). Strikingly, the length of the overlap between the most abundant transcript isoforms is almost identical in the two regions (24–25 nts) (Figure 6C), and this overlap lies entirely within the consensus sequence shared by the RNAs that most strongly coimmunoprecipitate with PPR10. These results together with the genetic and RNA gel blot data strongly suggest that PPR10 is associated in vivo with the termini of the overlapping transcripts in both intergenic regions, and that this interaction is necessary to stabilize the processed transcripts. Recombinant PPR10 binds with specificity to the consensus sequence in the atpH 5′-UTR and psaJ-3′ UTR The above results highlight the ‘consensus’ sequences shared by the overlapping PPR10-dependent RNA termini in the atpI-atpH and psaJ-rpl33 regions as potential PPR10-binding sites. To test whether PPR10 directly binds these sequences, in vitro assays were performed with purified recombinant PPR10 (rPPR10). rPPR10 has a monomeric molecular weight of 79 kDa, but eluted from a size-exclusion column at a position corresponding to a globular protein of ∼160 kDa, suggesting that it forms a homodimer (Figure 7A). Preliminary analytical ultracentrifugation data support this interpretation (data not shown), but we cannot eliminate the possibility that rPPR10 is a highly elongated monomer. Figure 7.rPPR10 binds with specificity to the consensus sequences shared by the atpH 5′ UTR and psaJ 3′ UTR. (A) Elution of rPPR10 from a size-exclusion column. Column fractions were fractionated by SDS–PAGE and stained with Coomassie blue. The elution positions of β-amylase (200 kDa) and BSA (67 kDa) are shown. Fractions 10, 11 and 12 were pooled for binding assays. (B) RNA oligonucleotides used for binding assays. The atpH-a and psaJ-a sequences are shown below, aligned according to their consensus sequence. The positions of mapped RNA termini (see Figure 6) are marked with arrows. (C) Gel mobility shift assays. Binding reactions contained RNA at100 pM and the indicated concentration of rPPR10. The control RNAs (atpH-b and psaJ-b) were matched to the corresponding RNAs in length but migrated differently due to distinct structures. Download figure Download PowerPoint Gel mobility shift assays were performed with rPPR10 and synthetic RNAs of ∼30 nts (Figure 7B and C). rPPR10 bound with high affinity to RNAs containing the sequences shared by the overlapping termini in the atpI-atpH and psaJ-rpl33 intergenic regions, but did not bind to RNAs of the same length containing sequences from nearby regions. Under the assay conditions used here (RNA concentration well below the Kd and protein in excess), the Kd can be estimated as the protein concentration at which half of the RNA is bound. Thus, the Kd for the interaction between rPPR10 and the atpH 30-mer is <10 nM. These results indicate that PPR10 is an RNA-binding protein that binds with high specificity and affinity to sequences shared at the termini of transcripts whose accumulation it promotes. Discussion The results presented here elucidate how a PPR protein modulates gene expression and the factors that determine the termini and stability of chloroplast mRNAs. We show that PPR10 is a sequence-specific RNA-binding protein that binds in vivo and in vitro to RNAs from two genomic regions of similar sequence. PPR10 binds the immediate 5′ or 3′ termini of RNAs that accumulate in a PPR10-dependent manner, indicating that PPR10 defines the positions of these termini and is required for the accumulation of RNAs harbouring them. The simplest explanation of our results is that bound PPR10 serves as a protective ‘cap’ to stabilize upstream and downstream RNA by blocking exonucleases from both the 5′ and 3′ directions. The loss of processed transcripts in ppr10 mutants with no accompanying increase in their precursors argues for a defect in RNA stabilization rather than a defect in precursor cleavage. Furthermore, the results are not consistent with a role for PPR10 in promoting simple endonucleolytic cleavages because the overlapping 5′ and 3′ PPR10-dependent termini in each region are not products of a single cleavage event. An RNA stabilization function for PPR10 is compatible with evidence that two other PPR proteins directly protect RNA from nucleases. Genetic data support a model in which MCA1 in C. reinhardtii binds to the 5′ terminal ∼21 nucleotides of chloroplast petA mRNA and protects the downstream RNA from degradation (Loiselay et al, 2008). Biochemical and genetic experiments showed that maize PPR5 stabilizes a chloroplast tRNA precursor by binding to an endonuclease-sensitive site within its intron (Beick et al, 2008; Williams-Carrier et al, 2008). The results with PPR10 add to this picture by providing evidence that a bound PPR protein (1) can block 5′ → 3′ degradation of chloroplast RNAs in a land plant and (2) can protect upstream RNA from 3′ → 5′ exonucleases. Taken together, these findings provide strong evidence that a bound PPR protein can directly block the cleavage of chloroplast RNAs from the 5′ direction, from the 3′ direction and from internal endonucleolytic attack. The conclusion that a bound PPR protein can protect both upstream and downstream RNA from degradation was foreshadowed by prior studies. The PPR protein CRP1 is required for the accumulation of processed transcripts with a 5′- or 3′-end mapping between the petB and petD loci in maize chloroplasts (Barkan et al, 1994; Fisk et al, 1999). The CRP1-dependent termini overlap by approximately 30 nucleotides, and the loss of the processed isoforms in crp1 mutants is not accompanied by an increased level of precursors (Barkan et al, 1994). In light of the current findings, a reasonable hypothesis is that CRP1 (or a CRP1-dependent partner) stabilizes both the upstream and downstream processed RNAs by binding their overlapping sequence. Although interactions between CRP1 and the petB/petD intergenic RNA were not unambiguously detected in RNA coimmunoprecipitation assays (Schmitz-Linneweber et al, 2005), we suspect that heterodimerization of CRP1 may mask the CRP1 epitope in petB/D ribonucleoprotein particles (unpublished results). The PPR protein HCF152 may also function analogously to PPR10. Arabidopsis hcf152 mutants lack chloroplast transcripts with either a 5′- or 3′-end mapping between the psbH and petB loci, again with no increase in uncleaved precursors (Meierhoff et al, 2003). To explore this possibility, we mapped RNA termini in the psbH/petB intergenic region in maize (Supplementary Figure 8); indeed, the processed psbH and petB termini overlap by approximately 20 nucleotides. In light of the other results presented here, it seems likely that this overlap represents the HCF152-binding site and that the hcf152 phenotype results from the ‘uncapping’ of the upstream and downstream RNA sequences. Interlinked RNA processing, RNA stabilization and translation in chloroplasts A dual role in RNA stabilization and translational enhancement has been proposed for several PPR-like proteins in chloroplasts (Barkan et al, 1994; Felder et al, 2001; Yamazaki et al, 2004; Loiselay et al, 2008). The magnitude of the CF0 deficiency in ppr10 mutants (Figure 1) and polysome data (Supplementary Figure 6) hint that PPR10, in addition to stabilizing atpH mRNA, also stimulates atpH translation. The position of the PPR10-binding site suggests a mechanism for this link. It seems likely that PPR10 bound to the atpH 5′ UTR spans the overlap between the upstream and downstream processed transcripts, which we believe represents an in vivo PPR10 ‘footprint’. Thus, the downstream edge of PPR10 is likely to be very near position −20 with r
