WHIRLY3 (WHY3) is a nuclear-encoded member of the plant-specific Whirly family of single-stranded DNA-binding proteins. It carries an N-terminal chloroplast transit peptide and is located in chloroplasts, where it associates with plastid nucleoids; in organello import shows it can also enter mitochondria. Like its plastid paralog WHY1, it folds into the Whirly domain and assembles into tetramers that bind ssDNA without sequence specificity. WHY1 and WHY3 act redundantly to maintain plastid genome stability: plants lacking both accumulate plastid DNA rearrangements generated by recombination between short direct repeats (microhomology-mediated break-induced replication), especially after double-strand breaks are induced, leading to maternally inherited leaf variegation. WHY3 is also captured in RNA-interactome studies, and a nuclear pool has been reported to bind, with WHY1, an upstream element of the kinesin gene AtKP1 and repress its transcription.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003677 DNA binding | IDA PMID:21911368 A conserved lysine residue of plant Whirly proteins is neces... | MODIFY | Summary: WHY3 binds DNA; in chloroplast extracts the WHY1/WHY3 pool is found in large DNA-containing complexes, and WHY3 alone (in why1 KO extracts) retains ssDNA-binding activity. Reason: DNA binding is correct but general. The specific, well-supported activity is single-stranded DNA binding: EMSA of why1 single-KO leaf extracts, in which WHY3 is the only plastid Whirly, still shows ssDNA binding, and the Whirly fold and ssDNA-binding residues are conserved. GO:0003697 is_a GO:0003677 and the gene already carries it (IBA, IEA). Refine to single-stranded DNA binding. Proposed replacements: single-stranded DNA binding Supporting Evidence: PMID:21911368 part of the WHY1/3 population is found in large complexes that contain DNA PMID:19666500 Only one Whirly is detected in the single KOs, and no Whirlies are detected in KO1/3 extracts. PMID:19666500 whereas less intense signals are detected with KO1 and KO3, respectively |
| GO:0003677 DNA binding | IEA GO_REF:0000120 | MODIFY | Summary: Electronic DNA binding annotation from the Whirly ssDNA-binding domain signature. Reason: The Whirly domain (IPR009044 ssDNA-binding transcriptional regulator) supports DNA binding, but the domain-level and experimental evidence specify single-stranded DNA binding, which the gene already carries. Refine to GO:0003697. Proposed replacements: single-stranded DNA binding Supporting Evidence: file:ARATH/WHY3/WHY3-uniprot.txt Pfam; PF08536; Whirly; 1. PMID:19666500 whereas less intense signals are detected with KO1 and KO3, respectively |
| GO:0003677 DNA binding | ISS PMID:15967440 DNA-binding proteins of the Whirly family in Arabidopsis tha... | MODIFY | Summary: ISS DNA binding transferred from potato p24/StWHY1 (AF233342), a characterised ssDNA-binding Whirly. Reason: The transfer is sound (WHY3 has the Whirly fold and the conserved ssDNA-binding residues), but the source protein and WHY3 both bind single-stranded DNA; refine to single-stranded DNA binding. Proposed replacements: single-stranded DNA binding Supporting Evidence: PMID:24192350 WHY1, WHY2 and WHY3 all exhibit the canonical Whirly fold. PMID:20551348 Importantly, ssDNA binding residues are also conserved in Arabidopsis proteins, suggesting that the mechanism of ssDNA binding of St-WHY2 is similar to the ones of Arabidopsis WHY1, WHY2, and WHY3 PMID:19666500 whereas less intense signals are detected with KO1 and KO3, respectively |
| GO:0003697 single-stranded DNA binding | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of single-stranded DNA binding for the plant Whirly family node. Reason: Single-stranded DNA binding is the core activity of the Whirly family and is supported directly for WHY3 by EMSA of why1 KO extracts. WHY3 sits inside the Whirly clade with an intact Whirly fold, so the inherited claim holds. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN008423852 SUPPORTS TRANSFER Whirly family node; WHY3 retains the Whirly fold and ssDNA-binding residues. Supporting Evidence: PMID:19666500 whereas less intense signals are detected with KO1 and KO3, respectively PMID:24192350 WHY1, WHY2 and WHY3 all exhibit the canonical Whirly fold. PMID:20551348 Importantly, ssDNA binding residues are also conserved in Arabidopsis proteins, suggesting that the mechanism of ssDNA binding of St-WHY2 is similar to the ones of Arabidopsis WHY1, WHY2, and WHY3 |
| GO:0003697 single-stranded DNA binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO single-stranded DNA binding from the Whirly domain (IPR013742). Reason: Whirly domain membership is a reliable indicator of ssDNA binding, and this is confirmed experimentally for WHY3. Supporting Evidence: file:ARATH/WHY3/WHY3-uniprot.txt Pfam; PF08536; Whirly; 1. PMID:19666500 whereas less intense signals are detected with KO1 and KO3, respectively |
| GO:0003729 mRNA binding | HDA PMID:27729395 In Planta Determination of the mRNA-Binding Proteome of Arab... | KEEP AS NON CORE | Summary: WHY3 was captured in the mRNA-binding proteome of etiolated seedlings (interactome capture). Reason: RNA binding by WHY3 is supported by two independent in planta RNA-interactome capture studies, but the RNA targets and the biological role of RNA binding by WHY3 are unknown; the core activity is ssDNA binding. Retained as non-core. The paper is cached as abstract only; the abstract names WHIRLY among captured protein families. Supporting Evidence: PMID:27729395 including WHIRLY, LIM, ALBA, DUF1296, and |
| GO:0003729 mRNA binding | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic inference of mRNA binding for the Whirly family. Reason: RNA binding by WHY3 is supported by two independent in planta RNA-interactome capture studies, but the RNA targets and the biological role of RNA binding by WHY3 are unknown; the core activity is ssDNA binding. Retained as non-core. The family node is seeded by Arabidopsis Whirlies captured in RIC data, including WHY3 itself. Supporting Evidence: PMID:32344669 ptRIC also assigned RNA-binding activity to all the members of the Alba- and the Whirly (WHY)-domain-containing protein families. PMID:27729395 including WHIRLY, LIM, ALBA, DUF1296, and |
| GO:0003729 mRNA binding | IDA PMID:32344669 Discovering the RNA-Binding Proteome of Plant Leaves with an... | KEEP AS NON CORE | Summary: Leaf ptRIC assigned RNA-binding activity to all Whirly family members, including WHY3. Reason: RNA binding by WHY3 is supported by two independent in planta RNA-interactome capture studies, but the RNA targets and the biological role of RNA binding by WHY3 are unknown; the core activity is ssDNA binding. Retained as non-core. Supporting Evidence: PMID:32344669 ptRIC also assigned RNA-binding activity to all the members of the Alba- and the Whirly (WHY)-domain-containing protein families. |
| GO:0005634 nucleus | IDA NOT PMID:15967440 DNA-binding proteins of the Whirly family in Arabidopsis tha... | UNDECIDED | Summary: NOT nucleus: GFP fusion of WHY3 in protoplasts and onion cells showed plastid, not nuclear, signal. Reason: The negative result comes from a transient GFP-fusion assay (paper cached as abstract only) and is corroborated by later reporter work, but a nuclear pool is suggested by ChIP of WHY1/WHY3 at the AtKP1 upstream element and by a predicted NLS; UniProt lists nucleus by similarity. Absence of detectable GFP signal does not exclude a small or conditional nuclear pool, so the negation can neither be confirmed nor refuted with the available evidence. Supporting Evidence: PMID:15967440 Transient expression of GFP fusion proteins in protoplasts and onion epidermal cells confirmed the localisation of these proteins in plastids or mitochondria, respectively. PMID:32490348 WHIRLY3 was likewise found solely in chloroplasts after transient expression of a reporter construct PMID:19669906 A ChIP assay showed that AtWHY1 and AtWHY3 bind to the upstream region of AtKP1 gene in vivo. |
| GO:0005739 mitochondrion | HDA PMID:28887381 Global Analysis of Membrane-associated Protein Oligomerizati... | KEEP AS NON CORE | Summary: WHY3 was detected in a mitochondrial/membrane-associated proteome profiling dataset. Reason: The mitochondrion location is independently supported by in organello import showing protease-protected accumulation of WHY3 in mitochondria, competed by a mitochondrial precursor. The assay cannot resolve the sub-compartment, and the documented functional role of WHY3 is in plastids, so the location is kept as non-core. Supporting Evidence: PMID:32490348 Indeed, as proven here by in organello import as well as competition experiments, WHIRLY3 is dually targeted into both, mitochondria and chloroplasts |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic inference that Whirly proteins are active in mitochondria, seeded by the mitochondrial paralog WHY2. Reason: Checked the source: the node is seeded by WHY2 (AT1G71260), the mitochondrial paralog, so the transfer to the plastid paralog WHY3 is a paralog extrapolation. Nevertheless WHY3 is imported into mitochondria in organello and has been proposed to compensate for WHY2 loss, so the organelle claim holds for WHY3; activity there is not demonstrated, so it is kept as non-core. Propagation Review Root cause: NO FAILURE NON CORE Failure modes: WRONG ORTHOLOG OR PARALOG Sources checked: AGI_LocusCode:AT1G71260 SUPPORTS TRANSFER WHY2 is mitochondrial; WHY3 is plastid-targeted but also imported into mitochondria in organello (PMID:32490348). Supporting Evidence: PMID:32490348 Indeed, as proven here by in organello import as well as competition experiments, WHIRLY3 is dually targeted into both, mitochondria and chloroplasts |
| GO:0006281 DNA repair | IBA GO_REF:0000033 | MODIFY | Summary: Phylogenetic inference of DNA repair for the Whirly family. Reason: DNA repair is correct but general. The why1 why3 evidence comes from DSB-inducing treatments (ciprofloxacin, bleomycin), and the mechanism proposed is binding of resected ssDNA at DSBs to limit error-prone microhomology-mediated break-induced replication in favour of accurate repair. Refine to double-strand break repair (GO:0006302, is_a DNA repair). Note that the in vivo genetics are on the double mutant, so WHY3 acts redundantly with WHY1. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN008423852 SUPPORTS TRANSFER Whirly DSB-repair role is shown for plastid WHY1/WHY3 and mitochondrial WHY2 in Arabidopsis. Proposed replacements: double-strand break repair Supporting Evidence: PMID:20551348 Importantly, rearrangements in the plastid genome of why2-1 mutant plants and in the mitochondria of why1 why3 mutant plants were at wild-type levels PMID:21911368 Whirly proteins are believed to modulate the MMBIR pathway either by binding the resected DNA that forms during processing of DSBs, thus favoring error-free repair through homologous recombination |
| GO:0006281 DNA repair | IEA GO_REF:0000117 | MODIFY | Summary: ARBA machine-learning prediction of DNA repair. Reason: DNA repair is correct but general. The why1 why3 evidence comes from DSB-inducing treatments (ciprofloxacin, bleomycin), and the mechanism proposed is binding of resected ssDNA at DSBs to limit error-prone microhomology-mediated break-induced replication in favour of accurate repair. Refine to double-strand break repair (GO:0006302, is_a DNA repair). Note that the in vivo genetics are on the double mutant, so WHY3 acts redundantly with WHY1. Proposed replacements: double-strand break repair Supporting Evidence: PMID:20551348 Importantly, rearrangements in the plastid genome of why2-1 mutant plants and in the mitochondria of why1 why3 mutant plants were at wild-type levels PMID:21911368 Whirly proteins are believed to modulate the MMBIR pathway either by binding the resected DNA that forms during processing of DSBs, thus favoring error-free repair through homologous recombination |
| GO:0006281 DNA repair | IMP PMID:21911368 A conserved lysine residue of plant Whirly proteins is neces... | MODIFY | Summary: why1 why3 double mutants are hypersensitive to ciprofloxacin (DSB inducer) and complementation requires the conserved KGKAAL lysine. Reason: DNA repair is correct but general. The why1 why3 evidence comes from DSB-inducing treatments (ciprofloxacin, bleomycin), and the mechanism proposed is binding of resected ssDNA at DSBs to limit error-prone microhomology-mediated break-induced replication in favour of accurate repair. Refine to double-strand break repair (GO:0006302, is_a DNA repair). Note that the in vivo genetics are on the double mutant, so WHY3 acts redundantly with WHY1. Proposed replacements: double-strand break repair Supporting Evidence: PMID:21911368 the absence of Whirly proteins in the plastids of these plants makes them sensitive to low doses of ciprofloxacin PMID:21911368 Whirly proteins are believed to modulate the MMBIR pathway either by binding the resected DNA that forms during processing of DSBs, thus favoring error-free repair through homologous recombination |
| GO:0006281 DNA repair | NAS PMID:20551348 Crystal structures of DNA-Whirly complexes and their role in... | MODIFY | Summary: Author statement that Whirlies function in organelle genome repair, from the DNA-Whirly crystal structure paper. Reason: DNA repair is correct but general. The why1 why3 evidence comes from DSB-inducing treatments (ciprofloxacin, bleomycin), and the mechanism proposed is binding of resected ssDNA at DSBs to limit error-prone microhomology-mediated break-induced replication in favour of accurate repair. Refine to double-strand break repair (GO:0006302, is_a DNA repair). Note that the in vivo genetics are on the double mutant, so WHY3 acts redundantly with WHY1. Proposed replacements: double-strand break repair Supporting Evidence: PMID:20551348 Importantly, rearrangements in the plastid genome of why2-1 mutant plants and in the mitochondria of why1 why3 mutant plants were at wild-type levels PMID:21911368 Whirly proteins are believed to modulate the MMBIR pathway either by binding the resected DNA that forms during processing of DSBs, thus favoring error-free repair through homologous recombination |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO regulation of transcription from the Whirly "ssDNA-binding transcriptional regulator" domain. Reason: Whirly proteins were first described as nuclear transcriptional regulators (potato p24/WHY1). For WHY3 the only support is a single report that WHY1 and WHY3 are components of KBF1, which binds an upstream element of AtKP1 (ChIP) and represses it on overexpression. The role is plausible for WHY3 but peripheral to its established organellar genome-maintenance function. Supporting Evidence: PMID:19669906 A ChIP assay showed that AtWHY1 and AtWHY3 bind to the upstream region of AtKP1 gene in vivo. PMID:19669906 KBF1 is a single and double-stranded DNA-binding factor. |
| GO:0006952 defense response | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro2GO defense response from the Whirly domain. Reason: Defense-gene regulation by Whirlies is established for Arabidopsis WHY1 and potato WHY1 (nuclear pool), not for WHY3. No defense phenotype or defense-gene binding has been shown for WHY3; the annotation is a family-level extrapolation of a function not demonstrated for this paralog. Supporting Evidence: PMID:15708347 the Arabidopsis Whirly protein AtWhy1 contributes to both basal and specific defense responses |
| GO:0006952 defense response | TAS PMID:15708347 Whirly transcription factors: defense gene regulation and be... | MARK AS OVER ANNOTATED | Summary: Traceable author statement from a Whirly review that Whirly transcription factors regulate defense genes. Reason: Defense-gene regulation by Whirlies is established for Arabidopsis WHY1 and potato WHY1 (nuclear pool), not for WHY3. No defense phenotype or defense-gene binding has been shown for WHY3; the annotation is a family-level extrapolation of a function not demonstrated for this paralog. The cited review attributes defense roles to AtWhy1 and potato Whirly orthologs. Supporting Evidence: PMID:15708347 the Arabidopsis Whirly protein AtWhy1 contributes to both basal and specific defense responses |
| GO:0009507 chloroplast | HDA PMID:18431481 Sorting signals, N-terminal modifications and abundance of t... | ACCEPT | Summary: WHY3 identified in large-scale MS proteome of purified chloroplasts. Reason: Chloroplast location is supported by a predicted chloroplast transit peptide, in vitro import and GFP fusion, and multiple chloroplast proteomes; it is where WHY3 maintains plastid genome stability. Supporting Evidence: PMID:21911368 WHY1 and WHY3 are directed to the chloroplasts whereas WHY2 is directed to the mitochondria PMID:15967440 Transient expression of GFP fusion proteins in protoplasts and onion epidermal cells confirmed the localisation of these proteins in plastids or mitochondria, respectively. |
| GO:0009507 chloroplast | IDA PMID:15967440 DNA-binding proteins of the Whirly family in Arabidopsis tha... | ACCEPT | Summary: In vitro import and GFP fusion show WHY3 is targeted to plastids. Reason: Chloroplast location is supported by a predicted chloroplast transit peptide, in vitro import and GFP fusion, and multiple chloroplast proteomes; it is where WHY3 maintains plastid genome stability. Supporting Evidence: PMID:15967440 Transient expression of GFP fusion proteins in protoplasts and onion epidermal cells confirmed the localisation of these proteins in plastids or mitochondria, respectively. file:ARATH/WHY3/WHY3-uniprot.txt SUBCELLULAR LOCATION: Plastid, chloroplast {ECO:0000269|PubMed:15967440}. |
| GO:0009507 chloroplast | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic mapping from UniProt subcellular location (Plastid, chloroplast). Reason: Chloroplast location is supported by a predicted chloroplast transit peptide, in vitro import and GFP fusion, and multiple chloroplast proteomes; it is where WHY3 maintains plastid genome stability. The UniProt location is experimentally grounded (PubMed:15967440). Supporting Evidence: file:ARATH/WHY3/WHY3-uniprot.txt SUBCELLULAR LOCATION: Plastid, chloroplast {ECO:0000269|PubMed:15967440}. PMID:15967440 Transient expression of GFP fusion proteins in protoplasts and onion epidermal cells confirmed the localisation of these proteins in plastids or mitochondria, respectively. |
| GO:0009507 chloroplast | ISM GO_REF:0000122 | ACCEPT | Summary: AtSubP prediction of chloroplast localization. Reason: Chloroplast location is supported by a predicted chloroplast transit peptide, in vitro import and GFP fusion, and multiple chloroplast proteomes; it is where WHY3 maintains plastid genome stability. Prediction agrees with the experimental data. Supporting Evidence: file:ARATH/WHY3/WHY3-uniprot.txt TRANSIT 1..75 /note="Chloroplast" PMID:15967440 Transient expression of GFP fusion proteins in protoplasts and onion epidermal cells confirmed the localisation of these proteins in plastids or mitochondria, respectively. |
| GO:0009570 chloroplast stroma | HDA PMID:20061580 AT_CHLORO, a comprehensive chloroplast proteome database wit... | ACCEPT | Summary: WHY3 identified in the stromal fraction of the AT_CHLORO chloroplast proteome. Reason: Stromal location is consistent with a soluble nucleoid-associated ssDNA-binding protein; chloroplast nucleoids are part of the stroma. Direct proteomic evidence. Supporting Evidence: PMID:21911368 part of the WHY1/3 population is found in large complexes that contain DNA |
| GO:0009863 salicylic acid mediated signaling pathway | IDA PMID:19669906 Recruitment of AtWHY1 and AtWHY3 by a distal element upstrea... | MARK AS OVER ANNOTATED | Summary: Annotation based on the finding that SA treatment raises WHY1 and WHY3 transcripts, together with WHY-dependent repression of AtKP1. Reason: The cached abstract supports only induction of WHY3 transcripts by salicylic acid, which is expression evidence (response to SA) rather than participation in the SA signal transduction pathway; no SA-signalling phenotype or step performed by WHY3 is described. The full text was not available, so the row is not removed, but the term appears to overstate the evidence. Supporting Evidence: PMID:19669906 salicylic acid treatment resulted in an increase of AtWHY1 and AtWHY3 transcripts |
| GO:0042644 chloroplast nucleoid | HDA PMID:16326926 pTAC2, -6, and -12 are components of the transcriptionally a... | ACCEPT | Summary: WHY3 identified among proteins of isolated transcriptionally active plastid chromosomes (pTAC / nucleoid fraction). Reason: The chloroplast nucleoid is where WHY3 binds plastid DNA; WHY1/WHY3 are found in DNA-containing complexes in chloroplast extracts. Direct proteomic evidence (paper cached as abstract only; deferring to the curator for the WHY3 identification). Supporting Evidence: PMID:21911368 part of the WHY1/3 population is found in large complexes that contain DNA |
| GO:1990391 DNA repair complex | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic inference that Whirlies are part of a DNA repair complex. Reason: GO:1990391 is defined as a protein complex involved in DNA repair. WHY3 forms Whirly tetramers (and, by analogy with WHY1/WHY2, higher-order 24-mers on long ssDNA) that act in organellar DSB repair, so the claim is defensible, but the complex is a homo-oligomeric ssDNA-binding assembly rather than a defined multi-subunit repair machine, and the term adds little beyond the MF and BP. Kept as non-core. Supporting Evidence: PMID:24192350 reveals that these structurally similar proteins assemble into tetramers PMID:21911368 Whirly proteins are believed to modulate the MMBIR pathway either by binding the resected DNA that forms during processing of DSBs, thus favoring error-free repair through homologous recombination |
| GO:1990391 DNA repair complex | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: ARBA prediction of DNA repair complex. Reason: GO:1990391 is defined as a protein complex involved in DNA repair. WHY3 forms Whirly tetramers (and, by analogy with WHY1/WHY2, higher-order 24-mers on long ssDNA) that act in organellar DSB repair, so the claim is defensible, but the complex is a homo-oligomeric ssDNA-binding assembly rather than a defined multi-subunit repair machine, and the term adds little beyond the MF and BP. Kept as non-core. Supporting Evidence: PMID:24192350 reveals that these structurally similar proteins assemble into tetramers |
| GO:1990391 DNA repair complex | IPI PMID:24192350 A family portrait: structural comparison of the Whirly prote... | KEEP AS NON CORE | Summary: Crystal structure shows WHY3 assembles into a C4 Whirly tetramer like WHY1 and WHY2. Reason: GO:1990391 is defined as a protein complex involved in DNA repair. WHY3 forms Whirly tetramers (and, by analogy with WHY1/WHY2, higher-order 24-mers on long ssDNA) that act in organellar DSB repair, so the claim is defensible, but the complex is a homo-oligomeric ssDNA-binding assembly rather than a defined multi-subunit repair machine, and the term adds little beyond the MF and BP. Kept as non-core. Supporting Evidence: PMID:24192350 reveals that these structurally similar proteins assemble into tetramers PMID:24192350 WHY1, WHY2 and WHY3 all exhibit the canonical Whirly fold. |
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Download this section (compressed HTML)Q: Should WHY1/WHY3 carry negative regulation of DNA recombination (GO:0045910), given that they are proposed to act as antirecombination factors by coating ssDNA, or is the double-strand break repair term sufficient?
Q: Does WHY3 have a physiologically meaningful nuclear pool, and does it act in the nucleus independently of WHY1 (e.g. at the AtKP1 element)?
Q: Does WHY3 compensate for WHY2 in mitochondria in vivo, given its in organello import into mitochondria?
Experiment: Complement why1 why3 with WHY3 alone (wild type and a KGKAAL second-lysine mutant) to test whether WHY3 by itself restores ciprofloxacin resistance and plastid genome stability, independent of WHY1.
Experiment: Use endogenous-promoter WHY3-tagged lines with chloroplast, mitochondrial and nuclear fractionation (with cross-contamination markers) to quantify the WHY3 pool in each compartment.
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