WHIRLY2 (WHY2, At1g71260) is a mitochondrially targeted member of the plant-specific Whirly family of single-stranded DNA-binding proteins. The mature protein assembles into a tetramer of Whirly domains that binds ssDNA with nanomolar affinity and little sequence specificity, holding the strand between adjacent subunits so it cannot anneal. In mitochondria WHY2 binds mtDNA along the entire genome and is associated with nucleoids. Loss of WHY2 causes microhomology-mediated rearrangements of the mitochondrial genome after double-strand breaks, and the protein blocks microhomology-mediated end-joining by organellar DNA polymerases in vitro, consistent with a role in protecting resected DNA ends and favouring accurate repair. why2 mutants also show disorganised nucleoids, altered mitochondrial ultrastructure and reduced seed germination, while overexpression lowers mtDNA content and mitochondrial function.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003677 DNA binding | IEA GO_REF:0000120 | MODIFY | Summary: Generic DNA binding from combined automated pipelines (ARBA/InterPro IPR009044). WHY2 is shown to bind single-stranded DNA specifically. Reason: True but less specific than the evidence: recombinant AtWHY2 binds ssDNA with nanomolar affinity, and the Whirly binding surface is built to exclude a second strand. Refine to single-stranded DNA binding, which the gene already carries by IDA and IBA. Proposed replacements: single-stranded DNA binding Supporting Evidence: PMID:30698803 AtOSB2 and AtWhirly2 bind to a pssDNA-10 substrate with dissociation constants (Kd) of 8 and 22 nM respectively |
| GO:0003677 DNA binding | ISS PMID:15967440 DNA-binding proteins of the Whirly family in Arabidopsis tha... | MODIFY | Summary: ISS DNA binding from similarity to potato p24/StWHY1 (GenBank AF233342), made in the paper describing the Arabidopsis Whirly family. Reason: The similarity transfer is sound, but the Whirly family, and AtWHY2 itself, bind single-stranded rather than generic DNA. Refine to single-stranded DNA binding (already supported here by IDA). Proposed replacements: single-stranded DNA binding Supporting Evidence: PMID:30698803 AtOSB2 and AtWhirly2 bind to a pssDNA-10 substrate with dissociation constants (Kd) of 8 and 22 nM respectively PMID:24192350 We also observed that the residues involved in ssDNA binding were either conserved or replaced by residues with similar biochemical propensities at equivalent positions in WHY1, WHY2 or WHY3 |
| GO:0003697 single-stranded DNA binding | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA for single-stranded DNA binding from the Whirly family node; AT1G71260 (WHY2) and AT1G14410 (WHY1) are among the descendants with experimental evidence. Reason: Core function of the Whirly family, and directly measured for AtWHY2 (Kd 22 nM on a partial-duplex ssDNA substrate). WHY2 appearing in its own With/From is expected: its own experimental annotation helped place the node. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN008423852 SUPPORTS TRANSFER Node seeded by Arabidopsis Whirly experimental ssDNA-binding data, including WHY2 itself. Supporting Evidence: PMID:30698803 AtOSB2 and AtWhirly2 bind to a pssDNA-10 substrate with dissociation constants (Kd) of 8 and 22 nM respectively PMID:24192350 We also observed that the residues involved in ssDNA binding were either conserved or replaced by residues with similar biochemical propensities at equivalent positions in WHY1, WHY2 or WHY3 |
| GO:0003697 single-stranded DNA binding | IDA PMID:30698803 Plant organellar DNA polymerases repair double-stranded brea... | ACCEPT | Summary: Direct binding assays (EMSA, fluorescence anisotropy) with recombinant AtWhirly2 on ssDNA and partial-duplex substrates. Reason: Full text read: AtWhirly2 binds ssDNA with nanomolar affinity as a tetramer and competes with polymerases for ssDNA overhangs. This is the core molecular function. Supporting Evidence: PMID:30698803 AtOSB2 and AtWhirly2 bind to a pssDNA-10 substrate with dissociation constants (Kd) of 8 and 22 nM respectively PMID:30698803 An EMSA on a labeled oligonucleotide of 70 nts illustrates the high binding affinity of AtWhirly2 for single-stranded DNA |
| GO:0003697 single-stranded DNA binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the Whirly domain (IPR013742) to single-stranded DNA binding. Reason: Mapping is appropriate for the Whirly fold and is confirmed experimentally for WHY2. Supporting Evidence: PMID:30698803 AtOSB2 and AtWhirly2 bind to a pssDNA-10 substrate with dissociation constants (Kd) of 8 and 22 nM respectively PMID:24192350 In the WHY2 structure, the four proteins present in the asymmetric unit form a Whirly tetramer with fourfold symmetry |
| GO:0003729 mRNA binding | HDA PMID:27729395 In Planta Determination of the mRNA-Binding Proteome of Arab... | KEEP AS NON CORE | Summary: High-throughput mRNA interactome capture from etiolated seedlings recovered Whirly-domain proteins. Reason: Interactome capture (oligo-dT pull-down after UV crosslinking) supports RNA association, and Whirly proteins in maize/barley plastids bind RNA, but no AtWHY2-specific RNA target or function in mitochondria is known. Kept as a non-core activity; cached text is abstract-only. Supporting Evidence: PMID:27729395 emerging and potentially novel RBDs, including WHIRLY, LIM, ALBA, DUF1296, and |
| GO:0003729 mRNA binding | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT IBA for mRNA binding from the Whirly family node. Reason: Consistent with the WHY1/WHY2/WHY3 interactome-capture data from which the node draws, but peripheral to the ssDNA-binding role of WHY2 in mitochondrial genome maintenance. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN008423852 SUPPORTS TRANSFER Arabidopsis Whirly descendants were captured in RNA interactome studies. 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:0003729 mRNA binding | IDA PMID:32344669 Discovering the RNA-Binding Proteome of Plant Leaves with an... | KEEP AS NON CORE | Summary: Leaf RNA interactome capture (ptRIC) assigned RNA-binding activity to all Whirly-domain proteins. Reason: Direct but high-throughput evidence of RNA association; the biological role of RNA binding by WHY2 is unknown, so 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:0005515 protein binding | IPI PMID:32612234 Extensive signal integration by the phytohormone protein net... | REMOVE | Summary: Binary interaction with GWD1 (Q9SAC6, alpha-glucan water dikinase) from the phytohormone interactome Y2H screen. Reason: Bare protein binding is uninformative under the project protein-binding policy. GWD1 is a plastid starch enzyme and WHY2 is mitochondrial, so the interaction is unlikely to be physiological in any case. |
| GO:0005634 nucleus | IDA NOT PMID:15967440 DNA-binding proteins of the Whirly family in Arabidopsis tha... | ACCEPT | Summary: NOT nucleus: GFP fusions placed WHY2 in mitochondria, not in nuclei. Reason: Consistent with all localisation data (GFP fusions, import assays, stable GFP lines in PMID:32490348). Cached text is abstract-only but the negative claim agrees with the positive mitochondrial result. Supporting Evidence: PMID:15967440 functionality of these sequences was demonstrated by in vitro import assays into PMID:32490348 In Figure 2c, it is shown that WHIRLY2:GFP clearly colocalize with the mitochondria‐specific dye TMRM |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA for mitochondrion (is_active_in) seeded by WHY2 itself. Reason: Mitochondrion is a root organelle term and informative. The IBA rests on WHY2's own experimental localisation, and the claim holds (transit peptide, import, GFP). The sub-compartment (nucleoid) is added as a separate NEW row rather than substituted. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN008423852 SUPPORTS TRANSFER Seeded by WHY2's own direct mitochondrial localisation. Supporting Evidence: PMID:32490348 In Figure 2c, it is shown that WHIRLY2:GFP clearly colocalize with the mitochondria‐specific dye TMRM file:ARATH/WHY2/WHY2-uniprot.txt /note="Mitochondrion" |
| GO:0005739 mitochondrion | IDA PMID:15967440 DNA-binding proteins of the Whirly family in Arabidopsis tha... | ACCEPT | Summary: In vitro import into isolated mitochondria and transient GFP fusions in protoplasts/onion cells. Reason: Direct evidence from an assay that resolves the organelle; matches the predicted transit peptide and later stable GFP lines. Supporting Evidence: PMID:15967440 functionality of these sequences was demonstrated by in vitro import assays into file:ARATH/WHY2/WHY2-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000269|PubMed:15967440, |
| GO:0005739 mitochondrion | IDA PMID:32490348 WHIRLY2 plays a key role in mitochondria morphology, dynamic... | ACCEPT | Summary: Stable WHIRLY2:GFP lines co-localise with the mitochondrial dye TMRM in roots, leaves and hypocotyls. Reason: Full text read; direct in planta localisation. Supporting Evidence: PMID:32490348 In Figure 2c, it is shown that WHIRLY2:GFP clearly colocalize with the mitochondria‐specific dye TMRM PMID:32490348 Within the mitochondria, WHIRLY2:GFP fluorescence was not equally distributed but appeared in punctiform spots. |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (SL-0173 Mitochondrion). Reason: The UniProt location is based on three experimental papers and is correct. Supporting Evidence: file:ARATH/WHY2/WHY2-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000269|PubMed:15967440, |
| GO:0006281 DNA repair | IBA GO_REF:0000033 | MODIFY | Summary: PAINT IBA for DNA repair from the Whirly family node (WHY1, WHY2, WHY3 descendants). Reason: The Whirly-family role in organelle DNA repair is sound, but the evidence is specifically about double-strand breaks, and for WHY2 the effect is restricted to the mitochondrial genome. GO asks that mitochondrial DSB repair be annotated to GO:0097551 (is_a GO:0006302 is_a GO:0006281, checked in OLS). Propagation Review Root cause: TERM SCOPING PROBLEM Sources checked: PANTHER:PTN008423852 SUPPORTS TRANSFER Family node is right; the term is broader than the descendant evidence, which concerns DSB repair in each Whirly's own organelle. Proposed replacements: mitochondrial double-strand break repair Supporting Evidence: PMID:20551348 These results indicate that, upon induction of DSBs, why2-1 plants accumulate DNA rearrangements in the mitochondrial genome more readily than do wild-type plants. PMID:30698803 Only AtWhirly2 and AtOSBs, however, completely block MMEJ by AtPolIs. |
| GO:0006281 DNA repair | IEA GO_REF:0000117 | MODIFY | Summary: ARBA machine-learning rule assigning DNA repair. Reason: Same claim as the experimental rows; refine to mitochondrial double-strand break repair, which the experimental data specifically support. Proposed replacements: mitochondrial double-strand break repair Supporting Evidence: PMID:20551348 These results indicate that, upon induction of DSBs, why2-1 plants accumulate DNA rearrangements in the mitochondrial genome more readily than do wild-type plants. |
| GO:0006281 DNA repair | IMP PMID:20551348 Crystal structures of DNA-Whirly complexes and their role in... | MODIFY | Summary: IMP (acts upstream of or within): why2-1 and why2-2 mutants accumulate microhomology-mediated mtDNA rearrangements after ciprofloxacin-induced DSBs. Reason: Full text read. The mutant phenotype is specific to DSBs in the mitochondrial genome (plastid genome unaffected); refine to mitochondrial double-strand break repair. Together with the biochemistry (WHY2 binds resected ssDNA and blocks MMEJ), WHY2 performs a step of the process rather than being merely required for it. Proposed replacements: mitochondrial double-strand break repair Supporting Evidence: PMID:20551348 These results indicate that, upon induction of DSBs, why2-1 plants accumulate DNA rearrangements in the mitochondrial genome more readily than do wild-type plants. 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 |
| GO:0006281 DNA repair | IMP PMID:20551348 Crystal structures of DNA-Whirly complexes and their role in... | MODIFY | Summary: IMP (involved in) from the same why2 mutant ciprofloxacin experiments. Reason: As for the companion row: the evidence concerns repair of DSBs in mitochondrial DNA, and WHY2 acts directly by binding the ssDNA of resected breaks, favouring accurate repair over error-prone MMBIR/MMEJ. Proposed replacements: mitochondrial double-strand break repair Supporting Evidence: PMID:20551348 These results indicate that, upon induction of DSBs, why2-1 plants accumulate DNA rearrangements in the mitochondrial genome more readily than do wild-type plants. PMID:30698803 Only AtWhirly2 and AtOSBs, however, completely block MMEJ by AtPolIs. |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro2GO mapping from Whirly / PC4-like ssDNA-binding domains to regulation of DNA-templated transcription. Reason: Transcription-factor activity was shown for nuclear StWHY1/AtWHY1, not for WHY2. WHY2 is mitochondrial; reduced mitochondrial transcripts in overexpressors were attributed to lower mtDNA content, and a transcriptional role is only speculated. Family-level mapping over-reaches for this member. Supporting Evidence: PMID:18423020 hence a role in transcription of mtDNA-encoded genes for AtWhy2 remains possible file:ARATH/WHY2/WHY2-deep-research-manual.md Defense response / nuclear transcription: these roles were described for potato StWHY1 and Arabidopsis AtWHY1 |
| GO:0006952 defense response | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro2GO mapping from the Whirly domain to defense response. Reason: Defense-gene regulation is a property of nuclear WHY1 (potato and Arabidopsis); no study links WHY2 to defense. The mapping applies a member-specific role to the whole family. Supporting Evidence: PMID:15708347 Arabidopsis and potato Whirly orthologs act as transcription factors that file:ARATH/WHY2/WHY2-deep-research-manual.md Defense response / nuclear transcription: these roles were described for potato StWHY1 and Arabidopsis AtWHY1 |
| GO:0006952 defense response | TAS PMID:15708347 Whirly transcription factors: defense gene regulation and be... | MARK AS OVER ANNOTATED | Summary: TAS from a 2005 review of Whirly transcription factors in defense gene regulation. Reason: The review (abstract-only in cache) attributes defense roles to AtWhy1 and StWhy1. WHY2 was not tested in defense assays, and its mitochondrial location argues against a direct role; treated as a family-level over-annotation consistent with the IEA row. Supporting Evidence: PMID:15708347 Arabidopsis and potato Whirly orthologs act as transcription factors that file:ARATH/WHY2/WHY2-deep-research-manual.md Defense response / nuclear transcription: these roles were described for potato StWHY1 and Arabidopsis AtWHY1 |
| GO:0009507 chloroplast | ISM GO_REF:0000122 | REMOVE | Summary: ISM chloroplast from AtSubP prediction. Reason: Electronic targeting prediction contradicted by experiment: import assays, transient and stable GFP fusions place WHY2 in mitochondria, and why2 mutants affect only the mitochondrial genome. Dual targeting was found for WHY3, not WHY2. Supporting Evidence: PMID:32490348 In Figure 2c, it is shown that WHIRLY2:GFP clearly colocalize with the mitochondria‐specific dye TMRM 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 |
| GO:1990391 DNA repair complex | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT IBA for DNA repair complex (part_of) from the Whirly node; reflects the ComplexPortal WHY homotetramer entries. Reason: WHY2 forms a homotetramer (crystal structure, gel filtration) and ComplexPortal records it as a DNA repair complex; the claim holds but is peripheral to the location and activity that define WHY2. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN008423852 SUPPORTS TRANSFER All three Arabidopsis Whirlies crystallise as tetramers. Supporting Evidence: PMID:24192350 In the WHY2 structure, the four proteins present in the asymmetric unit form a Whirly tetramer with fourfold symmetry |
| GO:1990391 DNA repair complex | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: ARBA rule assigning DNA repair complex. Reason: Consistent with the structural and ComplexPortal evidence for the WHY2 homotetramer; non-core. Supporting Evidence: PMID:24192350 In the WHY2 structure, the four proteins present in the asymmetric unit form a Whirly tetramer with fourfold symmetry |
| GO:1990391 DNA repair complex | IPI PMID:24192350 A family portrait: structural comparison of the Whirly prote... | KEEP AS NON CORE | Summary: IPI from the AtWHY2 crystal structure (PDB 4KOP) showing tetramer assembly, used by ComplexPortal for CPX-3584. Reason: Full text read; AtWHY2 is a tetramer, the assembly that binds ssDNA. The complex is real, but the "DNA repair complex" label is a ComplexPortal classification of the homotetramer, not a distinct repair machine. Supporting Evidence: PMID:24192350 In the WHY2 structure, the four proteins present in the asymmetric unit form a Whirly tetramer with fourfold symmetry |
| GO:0042645 mitochondrial nucleoid | IC PMID:32490348 WHIRLY2 plays a key role in mitochondria morphology, dynamic... | NEW | Summary: Proposed sub-compartment location: WHY2 associates with mtDNA throughout the genome and appears as punctate intramitochondrial foci. Reason: Root organelle policy: three independent lines agree - mtDNA immunoprecipitation along the whole genome (PMID:18423020), punctate WHIRLY2:GFP foci with disorganised nucleoids in why2-1 (PMID:32490348), and a ssDNA-binding activity that only makes sense at mtDNA. Added beside, not instead of, the mitochondrion rows. Supporting Evidence: PMID:18423020 Binding sites seem to occur along the length of the mitochondrial genome since we were unable to detect a region where Whirlies are not bound to mtDNA. PMID:32490348 Within the mitochondria, WHIRLY2:GFP fluorescence was not equally distributed but appeared in punctiform spots. PMID:32490348 The data presented in this study confirm the nucleoid localization of WHIRLY2 but also support a structural role in mitochondria nucleoid organization. |
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Download this section (compressed HTML)Q: Does WHY2 promote homologous recombination with ODB1/RAD52-1 at mitochondrial DSBs, or act only by sequestering ssDNA from MMEJ-competent polymerases?
Q: Is the RNA binding seen in interactome capture relevant to mitochondrial RNA metabolism, and which transcripts does WHY2 bind?
Q: Does WHY3 dual targeting to mitochondria fully explain the weak why2 phenotype, and does a why2 why3 double mutant show spontaneous mtDNA rearrangements?
Experiment: Ciprofloxacin treatment of why2 why3 and why2 odb1 double mutants with deep sequencing of mtDNA junctions to separate redundancy from pathway position.
Experiment: Super-resolution co-imaging of WHIRLY2:GFP with a mtDNA stain (SYBR Green/PicoGreen) to confirm nucleoid localisation directly.
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