Focused Hypothesis Report — Horse WEE1 (F6TY09) and "female pronucleus assembly" (GO:0035038)

Hypothesis evaluated: The horse protein F6TY09 participates in female pronucleus assembly (GO:0035038).
Organism: Equus caballus (NCBITaxon:9796). Accession: UniProt F6TY09. Focus type: function_assignment.
Date: 2026-09-08. Analyses use public sequence (UniProt), orthology/annotation (QuickGO/GO, Ensembl Compara references), and primary literature (PubMed).

Executive Judgment

Verdict: REFUTED (over-annotation via paralog confusion).

F6TY09 is unambiguously the somatic WEE1 ortholog in horse (95.4% identity to human WEE1 P30291; gene name WEE1, VGNC:25026), not the oocyte-specific paralog WEE2 / WEE1B. The GO term female pronucleus assembly (GO:0035038) is, in the current Gene Ontology, a WEE2/WEE1B function: across mammals and other vertebrates it is annotated almost exclusively to WEE2 orthologs, its only mammalian experimental support is mouse Wee2 (IMP, PMID:21454751), and WEE1B is oocyte-restricted and molecularly distinct from the widely expressed somatic WEE1. Neither F6TY09 nor human WEE1 (P30291) carries GO:0035038 in current GO; the horse entry that legitimately carries it is the separate horse WEE2 protein A0A9L0SIB9 (605 aa). Assigning female pronucleus assembly to F6TY09 therefore attributes a germline/oocyte paralog function to the somatic mitotic-checkpoint kinase.

Most important caveat: The claim tested is a labeled function assignment; there is no directly observed horse experimental evidence for either WEE1 or WEE2 in female pronucleus assembly — the mammalian evidence is from mouse Wee2 and transferred by orthology. The refutation is of assigning the term to the WEE1 paralog, not a claim that horse WEE2 lacks the function.

Evidence Matrix

GO Curation Implications (lead — requires curator verification)

• Do NOT assign GO:0035038 (female pronucleus assembly) to F6TY09 (horse WEE1). The term is not supported for the somatic WEE1 paralog. If an annotation of GO:0035038 to F6TY09 exists in the review, the recommended action is REMOVE (paralog over-annotation from orthology projection), or at minimum flag as NON-CORE / incorrect paralog.
• Retain the canonical somatic WEE1 terms that F6TY09 legitimately carries:
• MF: protein tyrosine/serine-threonine kinase activity (GO:0004713 IBA; GO:0106310 protein serine kinase per RHEA), ATP binding (GO:0005524).
• BP: negative regulation of G2/M transition of mitotic cell cycle (GO:0010972) — experimentally supported for the ortholog (human WEE1 IDA).
• CC: nucleus (GO:0005634).
• The female-pronucleus/oocyte function should be curated on the WEE2 (WEE1B) record (horse A0A9L0SIB9), not WEE1.
• "protein binding" is not needed here — informative kinase/cell-cycle terms are supported.

GO Decision Table (leads — require curator verification)

Mechanistic Scope

The immediate molecular activity of WEE1 (F6TY09's family) is inhibitory phosphorylation of CDK1 on Tyr15, restraining MPF (CDK1–cyclin B) to enforce the mitotic G2/M checkpoint in somatic cells. Female pronucleus assembly is a germline/fertilization process (assembly of the haploid maternal nucleus of the egg). In vertebrates this germline CDK1-inhibitory role is carried by the oocyte-specific paralog WEE2/WEE1B, which arose from duplication of an ancestral single Wee1 (as retained in Drosophila, where one Dwee1 performs the maternal embryonic function). Thus the hypothesized process is a paralog-partitioned function that does not transfer to the somatic WEE1.

Conflicts and Alternatives

• Paralog confusion (primary alternative, well supported): "female pronucleus assembly" annotations cluster on WEE2, not WEE1. The label "WEE1" on F6TY09 is correct for the somatic gene but the GO term belongs to WEE2.
• Orthology-projection carry-over: GO_REF:0000107 (Ensembl Compara) projects the term across WEE-family orthologs; a pipeline that does not distinguish WEE1 vs WEE2 subtrees can leak the term onto WEE1. This is the most likely origin of the seed claim.
• Drosophila single-Wee1 artifact: because flies have one Wee1 doing both mitotic and maternal roles, naive homology transfer to any mammalian Wee-family protein over-generalizes.
• No isoform rescue: there is no evidence that somatic WEE1 compensates for WEE2 in the oocyte; WEE2 loss causes fertilization failure in humans (WEE2 mutations, e.g. PMID:40830301), indicating WEE1 does not substitute.

Knowledge Gaps

1. Direct horse evidence: None found for either horse WEE1 or WEE2 in female pronucleus assembly. Resolving would require horse oocyte/zygote expression or perturbation data. Matters because all mammalian support is mouse Wee2 + orthology.
2. Exact provenance of the seed claim: Whether the review's GO:0035038 assignment came from an Ensembl projection or a mislabeled source was inferred, not directly traced in the (blinded) review file.
3. WEE2 necessity vs sufficiency for the term itself: The GO term rests on one mouse Wee2 IMP (PMID:21454751); the assay specifics (necessity/sufficiency) were not re-examined here.

Discriminating Tests

• Phylogenetic tree of vertebrate WEE1 vs WEE2 placing F6TY09 — will place it firmly in the WEE1 clade (already implied by 95.4% vs ~50% identity).
• Tissue expression (horse/mammalian): WEE1 ubiquitous vs WEE2 oocyte-restricted (RNA-seq/GTEx-type data) — separates the paralogs functionally.
• Reciprocal-best-hit / synteny of F6TY09 vs human WEE1 and WEE2 loci to confirm 1:1 WEE1 orthology.
• Oocyte perturbation specifically knocking down WEE1 vs WEE2 (as in Oh et al. 2010) — only WEE2 knockdown affects meiotic/pronuclear phenotypes.

Curation Leads (require curator verification)

• Action: Remove / reject GO:0035038 on F6TY09 (WEE1); reassign the concept to horse WEE2 (A0A9L0SIB9) if desired.
• Candidate references to verify: PMID:16169490 (WEE1B oocyte-specific), PMID:23616086 (WEE2=WEE1B meiotic-arrest kinase), PMID:20083600 (Wee1B oocyte meiosis), PMID:21454751 (mouse Wee2 IMP underlying GO:0035038), PMID:10790391 (Drosophila single Wee1 maternal role).
• Candidate correct terms for F6TY09: GO:0010972 (negative regulation of G2/M transition of mitotic cell cycle), GO:0004713/GO:0106310 (kinase activity), GO:0005634 (nucleus).
• Suggested question for curators: Did the GO:0035038 assignment originate from an Ensembl Compara projection that did not separate WEE1 vs WEE2 subtrees?
• Suggested experiment: paralog-specific expression/knockdown in equine oocytes to obtain direct horse evidence.

Analysis Provenance (artifacts)

• wee_identity_matrix.csv — computed pairwise % identity matrix (7 sequences).
• WEE1/WEE2 identity heatmap (auto-saved plot) with clade boxes.

The discriminating clade test proposed for this hypothesis has now been executed: F6TY09 partitions cleanly into the WEE1 clade (~90–95% intra-clade) and is ~50% identical to every WEE2 ortholog, which form their own clade. GO:0035038 tracks the WEE2 clade.

{{figure:plot_1.png|caption=Pairwise global-identity heatmap of WEE1 and WEE2 orthologs. F6TY09 (horse WEE1) sits inside the WEE1 clade (~90–95% identity to human/mouse WEE1) and is only ~50% identical to every WEE2/WEE1B ortholog, which form a separate clade. Female pronucleus assembly (GO:0035038) tracks the WEE2 clade, not F6TY09.}}

Reproducibility / Methods

• Sequence verified: 646 aa; SHA-256 f46f1c6be9cdfa7025987fbd2b3d5f5a49a4e7cbb69d75d9101e8b34234429d4 (matches frozen input; computed with a pure-Python SHA-256).
• Alignments: Needleman-Wunsch (match +1, mismatch −1, gap −2), sequences fetched from UniProt REST (P30291 646 aa, P0C1S8 567 aa; A0A9L0SIB9 605 aa).
• Annotations: QuickGO REST (/ontology/go/terms/GO:0035038, /annotation/search), retrieved 2026-09-08.
• Literature: PubMed.
• No local ai-gene-review analyses or existing judgments were consulted.

Artifacts

• OpenScientist final report
• OpenScientist final report
• OpenScientist plot 1
  
• OpenScientist wee identity matrix