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. 2018 Nov 2;46(19):10448-10459.
doi: 10.1093/nar/gky737.

Unexpected Functional Versatility of the Pentatricopeptide Repeat Proteins PGR3, PPR5 and PPR10

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Free PMC article

Unexpected Functional Versatility of the Pentatricopeptide Repeat Proteins PGR3, PPR5 and PPR10

Margarita Rojas et al. Nucleic Acids Res. .
Free PMC article

Abstract

Pentatricopeptide repeat (PPR) proteins are a large family of helical repeat proteins that bind RNA in mitochondria and chloroplasts. Sites of PPR action have been inferred primarily from genetic data, which have led to the view that most PPR proteins act at a very small number of sites in vivo. Here, we report new functions for three chloroplast PPR proteins that had already been studied in depth. Maize PPR5, previously shown to promote trnG splicing, is also required for rpl16 splicing. Maize PPR10, previously shown to bind the atpI-atpH and psaJ-rpl33 intercistronic regions, also stabilizes a 3'-end downstream from psaI. Arabidopsis PGR3, shown previously to bind upstream of petL, also binds the rpl14-rps8 intercistronic region where it stabilizes a 3'-end and stimulates rps8 translation. These functions of PGR3 are conserved in maize. The discovery of new functions for three proteins that were already among the best characterized members of the PPR family implies that functional repertoires of PPR proteins are more complex than have been appreciated. The diversity of sequences bound by PPR10 and PGR3 in vivo highlights challenges of predicting binding sites of native PPR proteins based on the amino acid code for nucleotide recognition by PPR motifs.

Figures

Figure 1.
Figure 1.
PPR10 defines and stabilizes the 3′-end of monocistronic psaI RNA. (A) The psaI-ycf4-cemA-petA transcription unit. The three transcripts detected by RNA gel blot hybridization are diagrammed at top. The oligonucleotide probes used in panel (C) are diagrammed below in relation to the expansion of the psaI-ycf4 intergenic region. Probe lengths are not drawn to scale, but their overlap with one another is precisely shown by the nucleotide sequence above. The sequence underlined in red resembles known PPR10-binding sites (see panel (D)), and ends 430 nt downstream from the psaI transcription start site (53). (B) RNA gel blot hybridization showing the loss of monocistronic psaI RNA in ppr10 mutants. The two panels come from the same gel and were hybridized with gene-specific probes for either psaI or ycf4 RNA. Excerpts of the same blots stained with methylene blue are shown below to illustrate rRNA abundance. The transcripts are numbered to the right, according to the scheme shown in panel (A). (C) Mapping the 3′-end of monocistronic psaI mRNA by high-resolution RNA gel blot hybridization. Replicate panels from the same gel were hybridized to the oligonucleotide probes diagrammed in panel (A). (D) Gel mobility shift assay demonstrating that PPR10 can bind to the sequence at the 3′-end of the PPR10-dependent psaI transcript. A comparison of the psaI site with the known atpH and psaJ sites is shown at top; the red line marks the minimal binding site established for atpH (14). PPR10 was used at 10, 5, 2.5, 1.25 or 0 nM.
Figure 2.
Figure 2.
Analysis of chloroplast gene expression in Arabidopsis and maize pgr3 mutants by ribosome profiling. (A) Ribosome footprints from seedling leaves of the Arabidopsis pgr3 mutants and their normal siblings were detected by hybridization to high-resolution microarrays spanning the whole chloroplast genome. The values shown are the median ratio (wild-type:mutant) of the median normalized signal intensities among all 50-nt array probes mapping within each ORF. Error bars represent the standard deviation calculated from all probes covering the ORF. Each ORF is annotated with the number of probes whose signal was above background as a fraction of the total number of probes. Genes for which fewer than half of the probes were above background are marked with an asterisk; their values could not be assessed with confidence. (B) Ribosome footprints from seedling leaves of the maize pgr3 mutant (Zm-pgr3) were mapped by deep sequencing. The values shown are the ratio of normalized read counts for each gene in cps1-1/2, a mutant with a plastid ribosome deficiency similar in magnitude to that of Zm-pgr3. Read counts were normalized to the total number of reads mapping to chloroplast ORFs. A comparison to the wild-type is shown in Supplementary Figure S2A.
Figure 3.
Figure 3.
RNA gel blot hybridizations demonstrating loss of dicistronic rpl16-rpl14 transcripts in maize and Arabidopsis pgr3 mutants. Seedling leaf RNA from maize (A) or Arabidopsis (B) plants of the indicated genotype was analyzed by RNA gel blot hybridization, using probes specific for the indicated regions. The maize atp1 mutant lacks the chloroplast ATP synthase (54) and was included to control for effects resulting from the loss of this complex. The first three lanes of the blots shown in (A) (WT, atp4 and atp1) were published previously (15) and are reproduced here with permission from: Zoschke, R., Watkins, K.P. and Barkan, A. (2013) A rapid ribosome profiling method elucidates chloroplast ribosome behavior in vivo. Plant Cell,25, 22652275; DOI:10.1105/tpc.113.111567, www.plantcell.org, Copyright American Society of Plant Biologists (2013). Arabidopsis svr7 is orthologous to maize atp4. The rpl16 and rpl14 blots in panel (B) came from the same gel, whereas the rps8 blot came from a different gel. The probe for rpl14 in Arabidopsis cross-hybridized with the 16S rRNA, as marked in (B).
Figure 4.
Figure 4.
RIP-seq analysis showing that Zm-PGR3 associates with RNA from the rpl14-rps8 intergenic region in vivo. Antibody to Zm-PGR3 was used for immunoprecipitation from maize chloroplast stroma. RNA purified from the immunoprecipitate was analyzed by deep sequencing. An immunoprecipitation with antibody to AtpB (a subunit of the chloroplast ATP synthase) served as a negative control. (A) Sequence read counts in the rpl14 region. Read counts were normalized to the total number of reads mapping to the chloroplast genome. A genome-wide view of the data is shown in Supplementary Figure S3. (B) Position of the PGR3-binding site and PGR3-dependent 3′-ends in the rpl14-rps8 intergenic region. A screen capture of an excerpt of the PGR3 RIP-seq data displayed with the Geneious browser is shown at top. The sequence is expanded below, where the positions of the PGR3-dependent 3′-ends as mapped by circular RT-PCR (maize) or 3′-RACE (Arabidopsis) are marked. The number of clones representing each 3′-end is indicated. The asterisk marks a difference in the sequences we obtained from that in the reference maize chloroplast sequence (GENBANK Accession NC_001666). (C) Comparison of PGR3’s binding sites near petL and rpl14. Alignments between the orthologous sites in maize (Zm) and Arabidopsis (At) are shown at left. An alignment between the maize petL and rpl14 sites and the Zm-PGR3 binding site as predicted by the PPR code (ZmPred) is shown to the right. The code prediction was based on the nucleotide specificities according to the published PPR code (8). PPR motifs that lacked canonical amino acids at the specificity-determining positions are marked with question marks. The binding site prediction for Arabidopsis PGR3 is similar, with the exception of the position in bold underline near the center of the protein, which is predicted to be a pyrimidine in Arabidopsis.
Figure 5.
Figure 5.
RNA gel blot hybridization demonstrating a defect in rpl16 splicing in ppr5 mutants. Seedling leaf RNA from ppr5-1 mutants was compared to that in ppr103-2/-3 and ppr4-1 mutants. The ppr103 mutants lack both spliced and unspliced rpl16 RNAs due to a defect in stabilizing the processed 5′-end in the rpl16 5′-UTR (24). The ppr4-1 mutants are deficient for plastid ribosomes due to a defect in rps12 splicing (23). rRNA abundance is shown on the methylene blue stained blots below. The blot was probed to detect the second exon of rpl16. Transcripts were identified based on the data in Figure 3 and in (15).
Figure 6.
Figure 6.
Summary of the known functions of PPR10 (A), PGR3 (B) and PPR5 (C). See text for details. Arrows indicate an enhancement of translational efficiency (PPR10, PGR3) or RNA splicing (PPR5). RIP-seq data suggest that Zm-PGR3 may also interact weakly in the atpF-atpA intergenic region and possibly with a transcript related to the 23S rRNA. The diagrammed binding sites for PPR10, PGR3 and PPR5 are supported by RNA coimmunoprecipitation and/or in vitro RNA-binding assays. However, the effects of ATP4/SVR7 have not been shown to result from direct interaction with RNA. The fact that Zm-PGR3 mutants have a much more severe plastid ribosome deficiency than does Arabidopsis pgr3 implies that Zm-PGR3 has an as yet unidentified target involved in ribosome biogenesis. The RIP-seq data for Zm-PGR3 (Supplementary Figure S3) suggest that this might involve an interaction with 23S rRNA or a precursor thereof.

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