SwissProt Keywords (SPKW) Unique Terms Project

Bottom line: UniProt keywords were mapped to GO terms (GO_REF:0000043),
and for many genes a keyword was the only source of an annotation. We reviewed
genes whose annotation rested solely on such a keyword, in 12 subprojects that
cover human apoptosis, rhythm and autophagy terms, five model or vector
organisms, phages, viruses and non-Arabidopsis plants (137 genes by the results
table below). Eukaryotic process keywords were the
problem: 79 to 100% of reviewed apoptosis, autophagy, rhythm and S. pombe
meiosis rows were over-annotations, mostly because a gene that regulates or is
merely active during a process was annotated as participating in it. Bacterial
keyword rows were mostly sound (one of four P. putida cases flagged). GOA
retired the SPKW pipeline for all cellular organisms around April 2026, so this
work is now retrospective. The plant subproject shows the cost of that blanket
removal: only about 15% of plant SPKW-unique terms carried real over-annotation
risk, and correct facts such as DELLA gibberellin signaling (RHT1) and patatin
storage activity (PATB1) were lost with the bad ones.

We did this to find which keyword-to-GO mappings are systematically unsafe and
why, so that the same checks can be applied to other mapping sources (the
UniPathway project reuses its closure filter), and to give GOA evidence about
what its retirement discarded.

Overview

This project reviews genes that have GO annotations derived solely from UniProt Keywords (SPKW) via GO_REF:0000043, with no corroborating evidence from experimental, computational, or curator sources. The goal is to identify systematic over-annotation patterns and distinguish legitimate SPKW contributions from problematic mappings.

Key Findings

• Over-annotation rates vary dramatically by organism and term
• Eukaryotic BP terms (apoptosis, meiosis, autophagy, rhythm) show 80-100% over-annotation
• Bacterial annotations are mostly accurate (~5% issues for P. putida)
• Viral annotations are clade-sensitive: phage immune/defense terms are often semantic mismatches, while eukaryotic viral immune-evasion terms may be legitimate but too broad
• Common patterns: process conflation, regulatory vs participatory confusion, caspase substrates
• GOA has retired the SPKW pipeline (≈April 2026): GO_REF:0000043 keyword-to-GO
  annotations have been removed from live GOA for all cellular organisms (verified zero for
  human, mouse, fly, worm, S. pombe, plants; only viruses retain them). The problem this
  project documented is now resolved at the source. Retrospective review of 38 non-Arabidopsis
  plant genes (see PLANTS) shows only ~15% of plant SPKW-unique terms carry
  real over-annotation risk; removal was justified for those, but blanket retirement also
  dropped correct annotations when the keyword was the only carrier of a fact.

Cumulative Results

Subproject	Organism	Total Genes	Reviewed	Issue Rate	Main Pattern
Apoptosis	Human	280	23	87%	Regulatory conflation
Rhythmic Process	Human	146	5	100%	Expression ≠ function
Autophagy	Human	123	14	79%	Signaling over-extension
ANOGA	A. gambiae	5,812	22	Mixed	D7 toxin=100%, immune=17%
SCHPO	S. pombe	1,963	7	100%	ATG-meiosis conflation
DROME	D. melanogaster	2,753	4	50%	Mixed patterns
PSEPK	P. putida	1,098	4	25%	RT defense keyword
ARATH	A. thaliana	8,433	4	75%	Subclade divergence
Virus clades	Viral taxa	54,131	11	55%	Host-context mismatch, specificity
PLANTS	Non-ARATH plants	4,117	38	15% Tier-A	Term-tiering; GOA retired SPKW
BPT4	Phage T4	~300	3	100%	Eukaryote-centric terms
ECO57	E. coli O157	~74,000	2	50%	Toxin vs effector

Methods

See SPKW-METHODOLOGY.md for detailed SQL queries and explanation of closure-based filtering (which reduces false positives by 70%+ in well-curated organisms).

Over-Annotation Patterns Identified

Pattern	Description	Examples	Action
Process conflation	Gene active during process X gets annotated to X	ATG genes → meiosis (S. pombe)	REMOVE
Regulatory conflation	Gene regulates X, annotated to X	AIMP2 → apoptotic process	MODIFY to regulatory term
Caspase substrate	Cleaved by caspases, annotated to apoptosis	AIMP1, BCAP31	REMOVE
Signaling over-extension	4+ steps from direct function	Sin1 → apoptosis	REMOVE
Eukaryote-centric terms	Immune/defense terms for phage-bacteria	T4 DAM → innate immune	REMOVE
Viral host-context mismatch	Same host-pathogen term valid in one viral clade but wrong in another	Phage AcrF8 → innate immune	MODIFY
Toxin vs effector	Effectors incorrectly called toxins	NleB1 (E. coli)	REMOVE
Subclade divergence	Family keyword ignores subfunctionalization	LCR1 (Arabidopsis DEFL)	REMOVE
Kratagonist ≠ toxin	Sequestration ≠ toxin activity	D7 proteins (mosquito)	MODIFY
Enzyme-class keyword → bare process	An activity keyword maps to a generic, substrate-less process term; substrate specificity lives on the MF branch	Methyltransferase → methylation (plant MTases: MET1A, EZ1, CCOAOMT, COQ5)	MARK_OVER / MODIFY

Legitimate SPKW Contributions

Not all SPKW-unique annotations are over-annotations:

• Antimicrobial peptides/lysozymes (D. melanogaster) - "killing of cells" is correct
• Arsenic/antibiotic resistance genes (P. putida) - direct functional annotations
• Conserved functions (Ced-12/ELMO in D. mel) - SPKW captures known biology missing from experimental annotations
• Core circadian genes (ELF4 in Arabidopsis) - accurate but redundant with specific terms
• DELLA growth repressors (RHT1/"Reduced height" in wheat) - "GA signaling pathway" is the core function of a DELLA; retirement was collateral damage (left only "nucleus")
• Storage proteins (patatin PATB1 in potato) - "nutrient reservoir activity" is patatin's defining role; current GOA has no storage term after retirement
• Viral life-cycle terms (phage DGR reverse transcriptase, phage quorum-sensing peptide, influenza M2) - often accurate but may need more specific viral or molecular-function terms

Project Status

• Started: 2025-12-23
• Last updated: 2026-05-30
• Total genes reviewed: 137 across 12 subprojects (sum of the results table above)
• Compiled data: spkw_reviewed_genes.csv was not archived in this repository; the reviewed-gene summary is retained below.

Phase 1 (Original)

• [x] MAP3K5 - COMPLETE
• [x] PHF23 - COMPLETE
• [x] SIRT2 - COMPLETE

Subprojects

• [x] Apoptosis - 23/280 reviewed
• [x] Rhythmic Process - 5/146 reviewed
• [x] Autophagy - 14/123 reviewed
• [x] ANOGA - D7 + immune genes
• [x] SCHPO - ATG-meiosis pattern
• [x] DROME - Case studies
• [x] PSEPK - Bacterial control
• [x] ARATH - Plant patterns
• [x] Virus clades - Virus-wide and clade-specific patterns
• [x] PLANTS - Non-Arabidopsis crops (38 genes, 14 species); term-tier classification + retrospective validation + full Tier-A keyword-watch-list sweep (methylation, developmental, defense, nodulation, hormone-signaling x6, long-tail) + verbatim-quote integrity audit
• [x] BPT4 - Phage semantics
• [x] ECO57 - Toxin/effector

Curation Recommendations

1. Check regulatory vs participatory - many genes regulate processes but don't participate IN them
2. Consider organism biology - same GO term can have different validity across taxa
3. Distinguish toxins from effectors - direct cytotoxicity vs signaling modulation
4. Validate family-level keywords - subfunctionalization can invalidate family annotations
5. Expression ≠ function - upregulation during a process doesn't mean functional involvement
6. Check viral host context - phage-bacterium interactions need different terms from eukaryotic viral immune evasion

Swiss-Prot vs TrEMBL Analysis

Key finding: Keywords on Swiss-Prot entries are manually assigned by curators, not by ARBA/UniRule automatic systems. This means:

Organism	Swiss-Prot %	Implication
Human	99.6%	Over-annotations reflect manual curator keyword choices
T4 Phage	99.6%	Same - curators chose these keywords
E. coli O157	88.6%	Mostly manual
P. putida	32.2%	Mixed manual/automatic
Virus (all)	13.1%	Mostly TrEMBL; errors may reflect mapping or automatic keyword assignment
A. gambiae	3.8%	Mostly automatic keyword assignment

Of 71 genes with over-annotation issues: 70 are Swiss-Prot (99%)

For reviewed high-confidence organism batches, this confirms the problem is usually in the KW→GO mapping layer, not keyword assignment. Virus-wide analysis is different because most candidates are TrEMBL. See SPKW-METHODOLOGY.md and SPKW-VIRUS.md for stratification queries.

---

Session Notes

2026-05-30

• Added a keyword-level view to SPKW-METHODOLOGY.md: reverse-map
  SPKW-unique GO terms to their source UniProt keywords via the public keyword2go
  (external2go) mapping (the GAF stores only the GO term). Produced a tier-annotated
  watch-list of the ~30 process/role keywords that drive plant over-annotation; it is
  organism-independent (same keywords drove the human/pombe/Arabidopsis subprojects).
• Reviewed the largest unreviewed Tier A keyword, Methyltransferase → methylation
  (GO:0032259) (92 plant genes), across 4 substrate classes: MET1A (rice, DNA),
  EZ1 (maize, histone H3K27), CCOAOMT (potato, lignin caffeoyl-CoA), COQ5 (rice, ubiquinone).
• Finding: Methyltransferase → methylation is a reliable, mechanistically-explained
  over-annotation (4/4 flagged; 3 MARK_OVER, 1 MODIFY). GO keeps methylation
  substrate-specificity on the molecular-function branch and has obsoleted the specific
  process terms (DNA methylation GO:0006306, histone methylation GO:0016571, H3K27
  methylation GO:0070734), so the bare methylation (GO:0032259) is structurally doomed to be
  redundant with the specific …-methyltransferase activity MF the gene already carries.
• Bonus: the review pass also caught two unrelated over-predictions — CCOAOMT circadian
  rhythm (ARBA) and EZ1 single-stranded RNA binding (ortholog transfer), both REMOVEd.
• Keyword-watch-list sweep (12 more genes across the next three Tier A keyword classes):
• Developmental (Differentiation/Flowering): GI, HD3A (REMOVE cell differentiation;
  MODIFY flower development on flowering-TIME genes — the ELF4 pattern), MADS3
  (MODIFY→specification of stamen identity), FEA2 (MODIFY→meristem maintenance). "cell
  differentiation" is a catch-all that's wrong or too coarse.
• Defense/killing (Plant defense/toxin): XA21 R-gene (MODIFY→defense response to
  bacterium), CPS4 phytoalexin synthase (MARK_OVER, STS3 enzyme-vs-product), O6/b-32 RIP
  (toxin activity legitimate, kept→rRNA N-glycosylase), CHIB chitinase (mixed).
• Nodulation: NSP1 (ACCEPT — legitimate core Nod-signaling TF, removal=collateral damage),
  CCAMK (MODIFY→arbuscular mycorrhizal association — rice doesn't nodulate; PPC16-style
  organism-context error), LBA leghemoglobin (O2-carrier ACCEPT, nodulation MARK_OVER),
  ENOD2A (MARK_OVER, expression marker).
• Meta-rule confirmed: a process/role keyword is only as good as the gene's position
  relative to that process → (1) core component → keep; (2) wrong organism/pathway → MODIFY;
  (3) right area wrong altitude → MODIFY-to-specific; (4) expression/component ≠ function →
  MARK_OVER/REMOVE. PLANTS now 30 genes / 14 species (added common bean).
• Hormone-signaling-subtype batch (4 genes; completes all six major plant hormones):
  VP1 (maize, ABA B3 TF) MODIFY→regulation of/response to ABA (responsive effector); CKX2
  (rice cytokinin dehydrogenase) MARK_OVER — a catabolic enzyme that degrades the hormone,
  not a signaling component (cleanest case); TUD1 (rice BR U-box E3 ligase) ACCEPT (legitimate
  component); EIL2 (rice ethylene EIN3-like TF) ACCEPT (legitimate; collateral damage). With
  auxin (ABP1) and GA (RHT1/DELLA), one keyword ("X signaling pathway") yields keep / MODIFY /
  remove purely by the gene's pathway position (transduction component vs responsive effector vs
  hormone-metabolism enzyme). PLANTS now 34 genes / 14 species; the full high/medium-value
  Tier A watch-list is covered.
• Long-tail keyword batch (4 genes): PG2 (tomato polygalacturonase) fruit ripening MARK_OVER
  (enzyme-vs-process); JOKA2 (potato NBR1-like) autophagy MODIFY->macroautophagy (legitimate
  autophagy gene — keyword reputation ≠ gene verdict); LSD1 (rice) HR MODIFY->negative
  regulation of HR; DET1 (tomato) light signaling MODIFY->negative regulation of
  photomorphogenesis. New variant: regulatory-direction inversion — LSD1/DET1 are negative
  regulators of the very process their keyword names. PLANTS now 38 genes / 14 species.
• Data-integrity audit (important): the reference-validator
  (conf/reference_validator_config.yaml) lists file in skip_prefixes, so file:
  supporting-text is NEVER verbatim-checked by just validate — only PMID: quotes are. An
  audit found 265/550 (~48%) of file: quotes across all SPKW-PLANTS reviews (incl. the
  original 9 and the EME1 exemplar) were non-verbatim — fabricated/paraphrased/markdown-mangled —
  yet had passed validation. Fixed every one (replaced with exact substrings or verbatim
  primary-PMID quotes; removed a few genuinely fabricated claims) with zero annotation-action
  changes (curation was sound; provenance was not). Re-verified with the validator's own
  matching logic: 0/536 file: quotes non-verbatim across all 38 genes. Lesson recorded:
  prefer PMID quotes; grep-verify any file: quote.

2026-05-29

• Extended PLANTS with five more genes across four new species
  (maize, wheat, the moss Physcomitrium patens, potato), bringing PLANTS to 14 genes /
  13 species. Selected from the plant.ddb closure-filtered TRUE SPKW-unique set
  (spkw_plants_unique table) to stress-test the highest-risk Tier A keyword classes.
• Reviewed (falcon deep research + full ai-review.yaml, all just validate-clean):
• RHT1 (wheat DELLA, GA signaling) → MODIFY→neg. regulation of GA signaling; removal
  NOT justified (core repressor function lost — strongest collateral-damage case yet).
• ABP1 (maize, auxin signaling) → MARK_AS_OVER_ANNOTATED→response to auxin; removal
  justified (contested receptor), but auxin binding kept as core.
• MPK4a (moss, innate immunity/defense) → MARK_AS_OVER_ANNOTATED ×2; removal justified
  (correct but redundant with experimental PRR-signaling-pathway).
• AM1 (maize AMEIOTIC1, chromosome segregation/cell division) → REMOVE / MARK_OVER;
  removal justified (over-broad/mis-placed; specific meiotic terms carry the biology).
• PATB1 (potato patatin, nutrient reservoir/lipid catabolic/defense) → ACCEPT/ACCEPT/
  MARK_OVER; mixed — storage role is collateral damage, defense over-annotated.
• New finding — hormone-signaling keywords cut both ways. Tier alone does not decide a
  hormone-signaling term; the gene's pathway position does. RHT1 (DELLA, a transduction
  component) refines the earlier ARF19/PARA rule (those were merely hormone-responsive).
  Running collateral-damage tally now five: CASP1, EME1, PR1B1, RHT1, PATB1.

2026-05-21

• Added PLANTS subproject: SPKW over-annotation in non-Arabidopsis
  Viridiplantae.
• Built plant.ddb (go-db, make plant) from the Sept 2025 goa_uniprot_gcrp snapshot;
  40.4M annotations, 26,493 SwissProt accessions.
• Closure-filtered TRUE SPKW-unique query (non-ARATH, SwissProt-only): 6,678 annotations,
  4,117 genes, 214 terms — 74% closure reduction; aspect F 54% / P 37% / C 8%. Rice
  dominates (1,927 genes); 200+ species represented.
• Discovered GOA retired GO_REF:0000043 for all cellular organisms since the snapshot.
  Reframed PLANTS as a retrospective validation study.
• Reviewed 4 genes (rice EME1, soybean PPC16, tobacco PARA, tomato PR1B1) — all 4 SPKW-unique
  annotations were over-annotations; GOA's removal justified for every headline term, but
  EME1 (endonuclease activity) and PR1B1 (defense response to fungus) lost correct biology.
• Classified all 214 SPKW-unique terms into 4 tiers: A over-annotation-risk (15%, 29 terms),
  B broad cofactor/enzyme-class MF (48%), C specific informative (31%), D context-dependent
  (6%). Only ~15% carry real over-annotation risk; ~79% (B+C) are correct — "SPKW-unique"
  is not a synonym for "over-annotation".
• Reviewed 5 more genes across broader taxa (grape STS3, Medicago NFP, poplar METK1,
  Chlamydomonas psaC, sorghum CASP1) — 9 genes / 9 species total, sampling all 4 tiers.
  The tier predicts the verdict: every Tier A removal was justified; the Tier C removal
  (CASP1, cell wall organization) discarded correct plant-specific biology.

2026-02-04

• Researched UniProt keyword assignment process (confirmed: Swiss-Prot = manual)
• Created spkw_reviewed_genes.csv compiling 95 reviewed genes
• Added Swiss-Prot vs TrEMBL stratification to methodology
• Cross-species analysis: issue rates 10-40%, all Swiss-Prot dominated

2026-05-21

• Added SPKW-VIRUS.md as the virus-wide and clade-specific counterpart to the organism SPKW subprojects
• Quantified virus.ddb: 180,680 SPKW annotations, 135,117 naive SPKW-unique annotations, and 80,218 closure-filtered SPKW-unique annotations
• Summarized 11 existing viral gene reviews across phage, anti-CRISPR, influenza, phage quorum-sensing, and DGR cases

Slides

• Slides (Marp source: SPKW-slides.md) — AI generated