# ASSAY_TO_FUNCTION rubric — what GO annotation each readout class licenses. # # Machine-readable companion to RUBRIC.md. Grounded in the publications-corpus # mining (see ASSAY_TO_FUNCTION.md): for each convergent phenotypic readout we # record the GO aspect it may license, whether it supports a direct # (`involved_in`) vs only a regulatory/response framing, the default core # status, and the discriminator a curator uses to decide whether a positive # readout reflects a *core* function or merely a peripheral/indirect effect. # # General principle (applies to every `phenotypic` + `high`-convergence class): # A readout that reports the *state* of process P measures a downstream # consequence, not the gene product's molecular activity. It therefore # licenses at most a BP term ("response to P" / "regulation of P") and that # annotation defaults to NON-CORE. It does NOT license an MF term, and does # NOT license a process more distal than what the readout directly reports. # Promote to core only when independent evidence places the gene product in # the recognized core machinery / sensor set for P (see `promote_to_core_if`). general_principle: licenses_aspect: [BP] # CC where the readout is an organelle-health probe never_licenses_aspect: [MF] # a state readout does not measure molecular activity default_core_status: non_core default_framing: regulation_or_response # not direct `involved_in` effector role promote_to_core_if: >- independent evidence (genetics, biochemistry, structure) shows the gene product is a direct effector or sensor within the recognized machinery for the process, not merely a perturbation that shifts the readout. mf_prohibition_scope: >- CALIBRATED (Tier-1 re-review, REREVIEW_TIER1.md): the "never MF" rule targets *regulatory/process-like* MF inferred from a pathway reporter (e.g. transcription coactivator/corepressor activity) for a gene outside the relevant machinery. It does NOT apply to direct binding or catalytic MF (e.g. "calcium ion binding", enzyme activities), which are established by their own assays regardless of any co-reported state readout. readout_rubric: APOPTOSIS_CASPASE: example_probes: [CellEvent, caspase-3/7 activity, Annexin V, TUNEL, PARP cleavage] licenses_aspect: [BP] never_licenses_aspect: [MF] default_core_status: non_core licensed_terms_examples: [GO:0006915, GO:0043065, GO:0043066, GO:0097190] promote_to_core_if: >- gene is a recognized apoptosis effector/regulator (BCL2 family, caspases, IAPs, APAF1, BH3-only proteins). do_not: - Annotate apoptosis as core for signaling/metabolic/structural genes that merely shift caspase readout downstream of their real function. - Use a tissue/context-specific apoptosis child term as evidence of a core role. corpus_examples: core_accept: - {gene: BCL2, term: negative regulation of apoptotic process, action: ACCEPT} - {gene: AREL1, term: negative regulation of apoptotic process, action: ACCEPT} non_core_or_over: - {gene: Akt1, term: negative regulation of intrinsic apoptotic signaling pathway, action: KEEP_AS_NON_CORE} - {gene: BAG3, term: negative regulation of striated muscle cell apoptotic process, action: KEEP_AS_NON_CORE} VIABILITY_PROLIFERATION: example_probes: [MTT, MTS, CCK-8, resazurin, BrdU, EdU, Ki-67, colony formation] licenses_aspect: [BP] never_licenses_aspect: [MF] default_core_status: non_core licensed_terms_examples: [GO:0008283, GO:0042127] promote_to_core_if: >- gene is core cell-cycle/division machinery (cyclins, CDKs, replication/ mitotic apparatus) acting directly on the cycle. do_not: - Annotate "regulation of cell population proliferation" as core for genes whose proliferation effect is indirect (housekeeping, cytoskeletal, metabolic). This is the single most frequent over-annotation in the corpus. corpus_examples: core_accept: - {gene: CDK1, term: G2/M transition of mitotic cell cycle, action: ACCEPT} non_core_or_over: - {gene: ACTB, term: positive regulation of cell population proliferation, action: MARK_AS_OVER_ANNOTATED} - {gene: AKT1, term: positive regulation of G1/S transition of mitotic cell cycle, action: KEEP_AS_NON_CORE} OXIDATIVE_STRESS_ROS: example_probes: [CellROX, H2DCFDA/DCF, MitoSOX, dihydroethidium, roGFP, HyPer, Amplex Red] licenses_aspect: [BP] never_licenses_aspect: [MF] default_core_status: non_core licensed_terms_examples: [GO:0006979, GO:0034599] promote_to_core_if: >- gene is a direct antioxidant enzyme / redox sensor (catalase, SOD, peroxiredoxin, glutathione/thioredoxin system, KEAP1/NRF2). do_not: - Annotate "response to oxidative stress" for genes whose perturbation merely raises ROS as a secondary consequence (e.g. organelle-biogenesis factors). ROS is the most convergent readout of all. corpus_examples: core_accept: - {gene: TP53, term: positive regulation of reactive oxygen species metabolic process, action: ACCEPT} - {gene: RIM15, term: cellular response to oxidative stress, action: ACCEPT} non_core_or_over: - {gene: PEX10, term: cellular response to reactive oxygen species, action: MARK_AS_OVER_ANNOTATED} - {gene: PEX13, term: cellular response to reactive oxygen species, action: MARK_AS_OVER_ANNOTATED} AUTOPHAGY_FLUX: example_probes: [GFP-LC3, mCherry-GFP-LC3, LC3-II, p62/SQSTM1, autophagic flux] licenses_aspect: [BP, CC] never_licenses_aspect: [MF] default_core_status: context_dependent # core for ATG machinery, else non-core licensed_terms_examples: [GO:0006914, GO:0010506, GO:0000045] promote_to_core_if: >- gene is core autophagy machinery (ATG proteins, ULK/BECN1 complexes, LC3/ATG8 conjugation system). do_not: - Annotate mitophagy/selective-autophagy child terms from a generic LC3 flux readout without evidence of the specific selective pathway. corpus_examples: core_accept: - {gene: AMBRA1, term: autophagosome assembly, action: ACCEPT} - {gene: AMBRA1, term: positive regulation of autophagy, action: ACCEPT} non_core_or_over: - {gene: ABI2, term: mitophagy, action: MARK_AS_OVER_ANNOTATED} - {gene: ATG2A, term: positive regulation of autophagosome assembly, action: MODIFY} MITO_MEMBRANE_POTENTIAL: example_probes: [TMRM, TMRE, JC-1, MitoTracker, mitochondrial membrane potential] licenses_aspect: [CC, BP] # mostly CC (localization); elevated MODIFY rate never_licenses_aspect: [MF] default_core_status: non_core licensed_terms_examples: [GO:0005739, GO:0007005] promote_to_core_if: >- gene is a direct component of the respiratory chain / mitochondrial bioenergetic or import machinery. do_not: - Rest on generic `mitochondrion` (GO:0005739) when a sub-mitochondrial compartment is known; this readout class shows the highest MODIFY rate. - Infer a bioenergetic *function* from a membrane-potential shift alone. corpus_examples: core_accept: - {gene: ACAD9, term: mitochondrion, action: ACCEPT} - {gene: ABCB7, term: mitochondrion, action: ACCEPT} non_core_or_over: - {gene: AFG3L2, term: mitochondrion, action: MODIFY} - {gene: ATP5MC1, term: mitochondrion, action: KEEP_AS_NON_CORE} UPR_ER_STRESS: example_probes: [5xUPRE, XBP1 splicing, ERSE/ERAI, BiP/GRP78/CHOP/ATF4 reporters] licenses_aspect: [BP] never_licenses_aspect: [MF] default_core_status: non_core licensed_terms_examples: [GO:0006986, GO:0030968, GO:0034976] promote_to_core_if: >- gene is a UPR transducer/sensor (IRE1, PERK, ATF6, XBP1) or master ER chaperone (BiP/HSPA5) acting directly in the response. do_not: - Assign `unfolded protein binding` (MF) from a UPR reporter — the reporter reports pathway state, not chaperone activity (see IRE1 example). - Annotate UPR as core for client/cargo proteins that merely burden the ER. corpus_examples: core_accept: - {gene: ATF4, term: PERK-mediated unfolded protein response, action: ACCEPT} - {gene: ATF4, term: response to endoplasmic reticulum stress, action: ACCEPT} non_core_or_over: - {gene: HSPA1A, term: endoplasmic reticulum unfolded protein response, action: KEEP_AS_NON_CORE} - {gene: IRE1, term: unfolded protein binding, action: MARK_AS_OVER_ANNOTATED} # MF over-annotation CALCIUM_FLUX: example_probes: [Fluo-4, Fura-2, GCaMP, calcium imaging] licenses_aspect: [BP] never_licenses_aspect: [MF] # MF only via independent Ca2+-binding evidence default_core_status: non_core licensed_terms_examples: [GO:0019722, GO:0070588] promote_to_core_if: >- gene is a Ca2+ channel/pump/sensor with direct ion-handling or Ca2+-binding (EF-hand) evidence. do_not: - Annotate "regulation of cytosolic calcium ion concentration" as core for genes that shift Ca2+ indirectly. corpus_examples: core_accept: - {gene: Calm2, term: calcium-dependent protein binding, action: ACCEPT} # MF justified by EF-hand, not the imaging - {gene: HMGB1, term: positive regulation of cytosolic calcium ion concentration, action: ACCEPT} non_core_or_over: - {gene: CDH23, term: regulation of cytosolic calcium ion concentration, action: MARK_AS_OVER_ANNOTATED} - {gene: BCL2, term: regulation of calcium ion transport, action: KEEP_AS_NON_CORE} IRON_PROBE: example_probes: [FeRhoNox, calcein quenching, labile iron pool] licenses_aspect: [BP] never_licenses_aspect: [MF] default_core_status: non_core licensed_terms_examples: [GO:0006879, GO:0055072] promote_to_core_if: >- gene is a direct iron transporter, Fe-S cluster biogenesis factor, or iron-storage/regulatory protein. do_not: - Annotate iron homeostasis as core for genes with only a downstream labile- iron-pool effect. corpus_examples: core_accept: - {gene: GLRX3, term: intracellular iron ion homeostasis, action: ACCEPT} - {gene: ABCB7, term: intracellular iron ion homeostasis, action: ACCEPT} non_core_or_over: - {gene: GLRX5, term: intracellular iron ion homeostasis, action: KEEP_AS_NON_CORE} pH_PROBE: example_probes: [pHrodo, pHluorin, SNARF, BCECF] licenses_aspect: [BP] never_licenses_aspect: [MF] default_core_status: non_core promote_to_core_if: >- gene is a direct proton pump/transporter or acidification machinery component. do_not: - Infer transport function from a compartment-pH shift alone. corpus_examples: {} # too few aligned examples in current corpus to cite TRANSCRIPTIONAL_REPORTER: example_probes: [luciferase reporter, promoter-reporter, NF-kB/ARE/HSE/SRE/CRE reporters] licenses_aspect: [BP, MF] # MF (TF activity) only for bona fide TFs — see note never_licenses_aspect: [] default_core_status: context_dependent note: >- Unlike the other hubs, a reporter assay legitimately supports an MF `DNA-binding transcription factor activity` term FOR A GENUINE TF (corpus: ATF2, ASCL1, ARNT — mostly ACCEPT). For non-TF genes a reporter shift reports pathway output only and licenses at most a BP regulation term, default non-core. promote_to_core_if: >- gene is a sequence-specific DNA-binding transcription factor or transcriptional coregulator with direct evidence. do_not: - Assign TF-activity MF terms to signaling/upstream genes that move a reporter without binding DNA. corpus_examples: core_accept: - {gene: ATF2, term: "DNA-binding transcription factor activity, RNA polymerase II-specific", action: ACCEPT} - {gene: ASCL1, term: "DNA-binding transcription repressor activity, RNA polymerase II-specific", action: ACCEPT} non_core_or_over: - {gene: AIP, term: transcription coactivator activity, action: MARK_AS_OVER_ANNOTATED} # ---- Second-pass readout classes (added in the catalog extension) ---- # All confirm the general principle: BP/CC aspect, never MF, default non-core, # promote to core only for the recognized machinery. Worked corpus contrasts # are in RUBRIC.md. CELL_MIGRATION_INVASION: example_probes: [scratch/wound-healing assay, Transwell, Boyden chamber, Matrigel invasion] licenses_aspect: [BP] never_licenses_aspect: [MF] default_core_status: non_core licensed_terms_examples: [GO:0016477, GO:0030335, GO:0048870, GO:0006935] promote_to_core_if: >- gene is in the cytoskeletal/adhesion-turnover motility machinery, OR is a guidance ligand/chemokine whose *signature* function is directed motility (signature-vs-incidental axis). do_not: - Demote a dedicated chemokine/growth-factor's chemotaxis/migration term to non-core -- that is its signature output (CCL11, PDGFA/B; already core). - Annotate migration as core for a transcription factor or metabolic gene whose migration effect is a downstream transcriptional/indirect consequence. corpus_examples: core_accept: - {gene: CCL11, term: cell chemotaxis, action: ACCEPT} # chemokine signature - {gene: PDGFB, term: cell chemotaxis, action: ACCEPT} # in core_functions non_core_or_over: - {gene: STAT3, term: positive regulation of cell migration, action: UNDECIDED} # TF; downstream; deferred to expert review CELL_ADHESION_SPREADING: example_probes: [cell adhesion assay, spreading assay, attachment assay] licenses_aspect: [BP] never_licenses_aspect: [MF] default_core_status: non_core licensed_terms_examples: [GO:0007155, GO:0007160, GO:0034446, GO:0098609] promote_to_core_if: >- gene is an integrin/adhesion receptor or ECM/matrix component that directly mediates adhesion. corpus_examples: core_accept: - {gene: FN1, term: substrate adhesion-dependent cell spreading, action: ACCEPT} MEMBRANE_TRAFFICKING_ENDOCYTOSIS: example_probes: [transferrin uptake, FM4-64, dextran uptake, DiI-LDL, endocytosis assay] licenses_aspect: [BP, CC] never_licenses_aspect: [MF] default_core_status: context_dependent licensed_terms_examples: [GO:0006897, GO:0006898, GO:0016192, GO:0006887] promote_to_core_if: >- gene is clathrin/adaptor/Rab/ESCRT trafficking machinery, or the receptor that is itself internalized. corpus_examples: core_accept: - {gene: CLTC, term: clathrin-dependent endocytosis, action: ACCEPT} - {gene: TFRC, term: receptor-mediated endocytosis, action: ACCEPT} SECRETION_DEGRANULATION: example_probes: [LDH release, CD107a, beta-hexosaminidase, degranulation assay, insulin secretion assay] licenses_aspect: [BP] never_licenses_aspect: [MF] default_core_status: non_core licensed_terms_examples: [GO:0046903, GO:0032940, GO:0002576, GO:0043299] promote_to_core_if: >- gene is SNARE/exocytic machinery or a dedicated secretagogue. corpus_examples: {} # under-powered in current corpus (1 aligned) METABOLIC_FLUX: example_probes: [Seahorse, oxygen consumption rate, ECAR, 2-NBDG glucose uptake, mito stress test] licenses_aspect: [BP] never_licenses_aspect: [MF] default_core_status: non_core licensed_terms_examples: [GO:0006006, GO:0006096, GO:0045333, GO:0006119] promote_to_core_if: >- gene is a glycolytic/OXPHOS enzyme or a glucose transporter acting directly. corpus_examples: {} # under-powered in current corpus (3 aligned) DNA_DAMAGE_FOCI: example_probes: [gamma-H2AX foci, comet assay, 53BP1 foci, RAD51 foci] licenses_aspect: [BP, CC] never_licenses_aspect: [MF] default_core_status: context_dependent licensed_terms_examples: [GO:0006974, GO:0006281, GO:0006302, GO:0042770] promote_to_core_if: >- gene is DDR/repair machinery (ATM/ATR, BRCA1/2, RAD51, 53BP1, MRN, etc.). do_not: - Annotate "DNA damage response" as core for a gene that merely shows increased gammaH2AX foci after perturbation. corpus_examples: core_accept: - {gene: BRCA1, term: double-strand break repair, action: ACCEPT} - {gene: CHD1, term: double-strand break repair via homologous recombination, action: ACCEPT} SENESCENCE: example_probes: [SA-beta-gal, SASP] licenses_aspect: [BP] never_licenses_aspect: [MF] default_core_status: non_core licensed_terms_examples: [GO:0090398, GO:0007569, GO:0001302] promote_to_core_if: >- gene is a core senescence effector (p53/p21, p16-RB axis). corpus_examples: core_accept: - {gene: TP53, term: positive regulation of cellular senescence, action: ACCEPT} WNT_REPORTER: example_probes: [TOPFlash, FOPFlash, TCF/LEF reporter] licenses_aspect: [BP] never_licenses_aspect: [MF] # MF only for the TCF/LEF TF itself default_core_status: context_dependent licensed_terms_examples: [GO:0016055, GO:0060070, GO:0090263] promote_to_core_if: >- gene is a Wnt pathway component (ligand, Frizzled/LRP receptor, destruction complex, or TCF/LEF). corpus_examples: core_accept: - {gene: CTNNB1, term: canonical Wnt signaling pathway, action: ACCEPT} - {gene: FZD7, term: canonical Wnt signaling pathway, action: ACCEPT} NFKB_REPORTER: example_probes: [NF-kB luciferase reporter] licenses_aspect: [BP] never_licenses_aspect: [MF] default_core_status: context_dependent licensed_terms_examples: [GO:0007249, GO:0038061, GO:0051092] promote_to_core_if: >- gene is an NF-kB pathway component (RelA/p50, IkB, IKK, upstream adaptor such as TRAF6). corpus_examples: core_accept: - {gene: TRAF6, term: positive regulation of canonical NF-kappaB signal transduction, action: ACCEPT} HYPOXIA_HIF: example_probes: [HRE-luciferase reporter, HIF-1alpha stabilization, pimonidazole, EF5] licenses_aspect: [BP] never_licenses_aspect: [MF] default_core_status: non_core licensed_terms_examples: [GO:0001666, GO:0071456, GO:0036294] promote_to_core_if: >- gene is HIF subunit / PHD / VHL oxygen-sensing machinery. corpus_examples: core_accept: - {gene: ARNT, term: cellular response to hypoxia, action: ACCEPT} # HIF-1beta NOTCH_REPORTER: example_probes: [RBP-J/CSL reporter, CBF-1 reporter, HES1 reporter, TP-1 luciferase] licenses_aspect: [BP] never_licenses_aspect: [MF] default_core_status: context_dependent licensed_terms_examples: [GO:0007219, GO:0045747] promote_to_core_if: >- gene is a Notch receptor/ligand or part of the CSL transcription complex. corpus_examples: {} # under-powered (0 aligned) HIPPO_TEAD_REPORTER: example_probes: [8xGTIIC reporter, TEAD luciferase, YAP/TAZ reporter] licenses_aspect: [BP] never_licenses_aspect: [MF] default_core_status: context_dependent licensed_terms_examples: [GO:0035329, GO:0035330] promote_to_core_if: >- gene is a Hippo kinase-cassette component or YAP/TAZ/TEAD. corpus_examples: {} # under-powered (0 aligned)