PROTON GRADIENT REGULATION 3 (PGR3, At4g31850) is a nucleus-encoded, chloroplast-targeted P-class pentatricopeptide repeat (PPR) protein of about 1100 residues with an N-terminal transit peptide and 27 tandem PPR motifs. It is a sequence-specific RNA-binding protein that acts on plastid mRNAs in the chloroplast stroma. PGR3 binds a site in the petL 5' untranslated region, where it protects the processed 5' terminus of petL operon transcripts from exonucleolytic decay (leaving a small-RNA footprint) and activates petL translation, which is needed for accumulation of the cytochrome b6f complex. It also binds the ndhG 5' UTR and activates ndhG translation, contributing to accumulation of the chloroplast NADH dehydrogenase-like (NDH) complex, and binds the rpl14-rps8 intercistronic region, where, together with the PPR-SMR protein ATP4/SVR7, it stabilizes a processed rpl14 3' end and stimulates rps8 translation; reduced expression of these ribosomal protein genes contributes to the loss of plastid ribosomes in pgr3 mutants. The N-terminal PPR motifs mediate RNA stabilization through sequence-specific binding, whereas the C-terminal motifs are required for translational activation, apparently by counteracting inhibitory RNA secondary structure at the ribosome-binding region. Mutants show high chlorophyll fluorescence, reduced cytochrome b6f and NDH activity, slight chlorophyll deficiency and slower growth; functions are conserved in the maize ortholog.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003729 mRNA binding | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of mRNA binding for a clade of organellar P-class PPR proteins that includes PGR3 itself among the descendant evidence. Reason: PGR3 is a long-tract P-class PPR protein shown to bind specific plastid mRNA sites in vitro and in vivo (petL 5' UTR, ndhG 5' UTR, rpl14-rps8 intercistronic region). mRNA binding is the correct family-level claim for this PAINT node; the more specific 5'-UTR binding activity is recorded as a separate NEW row with direct evidence. PGR3 appearing in its own With/From list reflects its own experimental grounding, not circularity. Supporting Evidence: PMID:30125002 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. PMID:21457370 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. |
| GO:0003729 mRNA binding | IDA PMID:32344669 Discovering the RNA-Binding Proteome of Plant Leaves with an... | ACCEPT | Summary: Leaf RNA interactome capture (UV crosslinking and oligo(dT) capture) identified PGR3 among in vivo RNA-binding proteins. Reason: Proteome-scale in vivo RNA-binding evidence, consistent with the targeted binding studies. The assay cannot resolve which RNA region is bound, so mRNA binding is as precise as this evidence allows. PGR3 is not named in the main text (supplementary data), but the function is independently established for this gene. Supporting Evidence: PMID:32344669 Using this improved RIC method in Arabidopsis leaves, we identified 717 RBPs, generating a deep RNA-binding proteome for leaf tissues. PMID:33439244 Here, we show that PGR3 facilitates the translation from ndhG, in addition to petL, through binding to their 5' untranslated regions (UTRs). |
| GO:0005575 cellular_component | ND GO_REF:0000015 | MODIFY | Summary: TAIR 'no data' placeholder for cellular component. Reason: The location is now inferable. PGR3 has a predicted chloroplast transit peptide, acts exclusively on chloroplast-encoded RNAs, and its maize ortholog was immunoprecipitated from chloroplast stroma. The placeholder should be replaced by chloroplast (with the stroma as the likely sub-compartment, added as a separate row). Proposed replacements: chloroplast Supporting Evidence: file:ARATH/PGR3/PGR3-uniprot.txt TRANSIT 1..62 FT /note="Chloroplast" PMID:30125002 Antibody to Zm-PGR3 was used for immunoprecipitation from maize chloroplast stroma. |
| GO:0006412 translation | IMP PMID:30125002 Unexpected functional versatility of the pentatricopeptide r... | MODIFY | Summary: Ribosome profiling of the pgr3-4 null mutant showed reduced ribosome footprints on rpl14 and rps8 (and petL), and reduced plastid rRNA, which the authors attribute to PGR3 binding in the rpl14-rps8 intergenic region and stimulation of rps8 translation. Reason: Under acts_upstream_of_or_within the row is not wrong, but it is uninformative. PGR3 is not a component of the translation machinery; it activates translation of specific plastid mRNAs by binding near their translation initiation regions, and the broader loss of plastid ribosomes is a secondary consequence of reduced rpl14/rps8 expression. Positive regulation of translation captures what PGR3 does. GO has no plastid-specific regulation of translation term. Proposed replacements: positive regulation of translation Supporting Evidence: PMID:30125002 We conclude, therefore, that the binding of PGR3 in the rpl14-rps8 intergenic region simultaneously stabilizes a processed 3′-RNA terminus and stimulates rps8 translation. PMID:30125002 PGR3’s stimulatory effect on the expression of two genes that encode ribosomal proteins (rpl14 and rps8) likely contributes to the loss of plastid ribosomes in maize and Arabidopsis pgr3 mutants. |
| GO:0009507 chloroplast | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping to chloroplast, based on the predicted transit peptide. Reason: Root organelle term; the claim holds. PGR3 has a predicted 62-residue chloroplast transit peptide, every characterized target is a chloroplast-encoded RNA, and the maize ortholog was immunoprecipitated from chloroplast stroma. The stroma sub-compartment is added as a separate NEW row rather than substituted. Supporting Evidence: file:ARATH/PGR3/PGR3-uniprot.txt TRANSIT 1..62 FT /note="Chloroplast" PMID:30125002 Antibody to Zm-PGR3 was used for immunoprecipitation from maize chloroplast stroma. |
| GO:0032544 plastid translation | IMP PMID:33439244 The Pentatricopeptide Repeat Protein PGR3 Is Required for th... | MODIFY | Summary: Ribosome profiling in pgr3 mutants showed specific suppression of petL and ndhG translation; PGR3 binding to their 5' UTRs relieves translational repression by the RNA segment in vitro. Reason: The study shows a gene-specific translational activator rather than a general participant in plastid translation; the PGR3-bound RNA segment itself represses translation and PGR3 binding antagonizes that effect. Positive regulation of translation describes the role. The plastid context is captured by the chloroplast location. Proposed replacements: positive regulation of translation Supporting Evidence: PMID:33439244 Ribosome profiling and RNA sequencing in pgr3 mutants revealed that translation from petL and ndhG was specifically suppressed. PMID:33439244 However, the PGR3 binding antagonizes the effects and facilitates the protein synthesis from petL and ndhG in vitro. |
| GO:0043489 RNA stabilization | IMP PMID:21457370 PROTON GRADIENT REGULATION 3 recognizes multiple targets wit... | MODIFY | Summary: pgr3 alleles (notably pgr3-2) fail to stabilize petL operon RNA; the recombinant pgr3-2 protein no longer binds the petL 5' UTR. Reason: Correct but general. The stabilized RNAs are mRNAs (petL operon transcripts protected at their processed 5' end; the rpl16-rpl14 dicistronic mRNA protected at its 3' end), so mRNA stabilization (an is_a descendant of RNA stabilization) is the precise term. Proposed replacements: mRNA stabilization Supporting Evidence: PMID:21457370 Previously, pgr3-2 was shown to be specifically defective in stabilizing petL operon RNA and to accumulate NDH at wild-type levels. PMID:22156165 the binding site of PGR3, a PPR protein that stabilizes the petL 5′-terminus in Arabidopsis (14,29), is represented by an sRNA in these same datasets PMID:30125002 Taken together, these data provide strong evidence that PGR3 in Arabidopsis and maize binds within the rpl14-rps8 intergenic region and, in so doing, stabilizes the RNA upstream of its binding site. |
| GO:0048027 mRNA 5'-UTR binding | IDA PMID:21457370 PROTON GRADIENT REGULATION 3 recognizes multiple targets wit... | NEW | Summary: Recombinant PGR3 binds conserved elements in the petL 5' UTR in vitro, and in vivo sRNA footprints and translation assays place PGR3 on the 5' UTRs of petL and ndhG. Reason: This is the specific molecular activity behind PGR3's stabilization and translation functions and is directly shown for the Arabidopsis protein with recombinant protein; a single amino-acid change in PPR motif 12 (pgr3-2) abolishes binding to the petL 5' UTR. Existing rows stop at mRNA binding. Supporting Evidence: PMID:21457370 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. PMID:21457370 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. PMID:33439244 Here, we show that PGR3 facilitates the translation from ndhG, in addition to petL, through binding to their 5' untranslated regions (UTRs). |
| GO:0009570 chloroplast stroma | IC PMID:30125002 Unexpected functional versatility of the pentatricopeptide r... | NEW | Summary: PGR3 is inferred to reside in the chloroplast stroma. Reason: Two independent lines agree: the maize ortholog Zm-PGR3, whose functions are conserved, was immunoprecipitated with its target RNAs from chloroplast stromal extract, and PGR3 acts on stromal processes (binding nascent plastid mRNAs, activating their translation on stromal 70S ribosomes). PGR3 has a transit peptide but no transmembrane segment. Added beside, not in place of, the chloroplast row. Supporting Evidence: PMID:30125002 Antibody to Zm-PGR3 was used for immunoprecipitation from maize chloroplast stroma. PMID:30125002 These functions of PGR3 are conserved in maize. |
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Download this section (compressed HTML)Q: How do the C-terminal PPR motifs of PGR3 activate translation once bound - by remodelling inhibitory 5' UTR structure alone, or by recruiting ribosomes or other factors?
Q: Does maize PGR3 have an additional target involved in ribosome biogenesis (for example 23S rRNA precursors), and is that target shared by Arabidopsis PGR3?
Q: Which ndh mRNA (ndhG, ndhE or ndhA) accounts for the NDH deficiency of pgr3 mutants, given the conflicting reports?
Experiment: In vivo crosslinking and immunoprecipitation (CLIP/RIP-seq) of epitope-tagged Arabidopsis PGR3 to map all binding sites, including any rRNA or ndh targets.
Type: RIP-seq
Experiment: Toeprinting or in vitro translation with structure-probing (SHAPE) of petL and ndhG 5' UTRs with wild-type PGR3 versus C-terminally truncated (pgr3-3-like) protein to separate binding from translational activation.
Type: in vitro translation and RNA structure probing
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