{"gene":"RNF43","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2012,"finding":"RNF43 is a transmembrane E3 ubiquitin ligase that selectively ubiquitinates Frizzled receptors at the cell surface, targeting them for lysosomal degradation, thereby reducing Wnt signaling. Deletion of RNF43 (with ZNRF3) in mouse intestinal epithelium induces adenoma formation dependent on paracrine Wnt.","method":"In vitro ubiquitination assays, HEK293T overexpression, RNF43 reconstitution in HCT116 cells, conditional mouse knockout, organoid culture","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 — foundational study with multiple orthogonal methods (cell-based assays, mouse genetics, organoids), 784 citations, independently replicated","pmids":["22895187"],"is_preprint":false},{"year":2013,"finding":"Crystal structures of the RNF43 ectodomain in complex with the Fu1-Fu2 fragment of R-spondin 2 (Rspo2) revealed that a prominent loop in Rspo2 Fu1 clamps into a groove in the RNF43 ectodomain surface, establishing the structural basis of R-spondin-mediated inhibition of RNF43 E3 ligase activity.","method":"X-ray crystallography of ZNRF3 ectodomain, RNF43 ectodomain, and Rspo2(Fu1-Fu2) complexes; biophysical binding assays; cellular Wnt reporter assays","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1 — multiple crystal structures with functional validation in cellular assays","pmids":["24225776"],"is_preprint":false},{"year":2013,"finding":"Crystal structure of RSPO1 bound simultaneously to LGR5 and RNF43 ectodomains showed that RSPO1 is sandwiched between LGR5 and RNF43, with its CRD rod module contacting LGR5 and a hairpin inserted into RNF43. LGR5 does not contact RNF43 but increases RSPO1 affinity for RNF43, establishing LGR5 as an engagement receptor and RNF43 as an effector receptor.","method":"X-ray crystallography of ternary RSPO1-LGR5-RNF43 ectodomain complex; binding affinity measurements","journal":"Genes & Development","confidence":"High","confidence_rationale":"Tier 1 — crystal structure of ternary complex with functional validation","pmids":["23756651"],"is_preprint":false},{"year":2013,"finding":"RNF43 inhibits Wnt/β-catenin signaling by reducing membrane-level Frizzled in pancreatic cancer cells. Inactivating RNF43 mutations increase cell-surface Frizzled and confer dependency on Wnt ligand secretion (Porcupine-dependent) for proliferation.","method":"RNF43 reconstitution in RNF43-mutant pancreatic cancer cell lines, cell-surface Frizzled quantification, Wnt inhibitor (LGK974) sensitivity assays, siRNA knockdown, xenograft models","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 — multiple cell lines, reconstitution, in vivo xenograft, replicated concept from PMID 22895187","pmids":["23847203"],"is_preprint":false},{"year":2015,"finding":"Dishevelled (DVL) acts as a dual-function adaptor required for ZNRF3/RNF43-mediated ubiquitination and degradation of Frizzled (FZD) and LRP6. Physical interaction between ZNRF3/RNF43 and DVL is essential for their Wnt inhibitory activity; the DEP domain of DVL binds FZD, and fusion of DEP to ZNRF3/RNF43 overcomes their DVL dependency.","method":"DVL knockout cells, Co-IP, cell surface FZD/LRP6 quantification, ubiquitination assays, domain-swap experiments (DEP-ZNRF3 fusion)","journal":"Molecular Cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, KO cells, mechanistic domain fusion experiments with multiple orthogonal readouts","pmids":["25891077"],"is_preprint":false},{"year":2015,"finding":"RNF43 suppresses canonical Wnt/β-catenin signaling through its extracellular PA domain interaction with the Frizzled CRD, requiring the intracellular RING finger domain for ubiquitination. RNF43 also suppresses noncanonical Wnt signaling through its C-terminal cytoplasmic region binding the PDZ domain of Dishevelled, independently of the PA and RING domains. Missense mutations in the extracellular portion of RNF43 redirect its localization from endosome to ER, abrogating canonical but not noncanonical Wnt suppression.","method":"Domain deletion/mutation analysis, co-IP, localization studies (immunofluorescence), Wnt reporter assays in HEK293T and cancer cells","journal":"Molecular and Cellular Biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal domain dissection experiments with functional readouts, mechanism for two distinct pathways defined","pmids":["25825523"],"is_preprint":false},{"year":2015,"finding":"RNF43 was detected in the nucleus of human intestinal crypt and colon cancer cells where it physically interacts with TCF4 and tethers it to the nuclear membrane, silencing TCF4 transcriptional activity even in the presence of constitutively active β-catenin. Tumor-associated RING domain mutations disrupt this inhibitory mechanism.","method":"Co-IP of endogenous RNF43 and TCF4, nuclear fractionation, immunofluorescence, Wnt reporter assays, Xenopus embryo overexpression, mutant RNF43 analysis","journal":"Science Signaling","confidence":"Medium","confidence_rationale":"Tier 2-3 — nuclear localization later disputed by antibody artifact concerns (PMID 37023034); mechanistic claim of TCF4 sequestration supported by Co-IP and reporter assays in single study","pmids":["26350900"],"is_preprint":false},{"year":2015,"finding":"Paracrine Wnt secretion from Paneth cells (Wnt3) is the essential driver of Rnf43/Znrf3-null intestinal tumor growth; removal of Paneth cells by Math1 mutation or deletion of Wnt3 inhibits tumorigenesis. Treatment with porcupine inhibitor C59 strongly inhibited RZ-null neoplasia growth while sparing normal crypts.","method":"Genetic epistasis (Math1 KO, Wnt3 KO crossed to Rnf43/Znrf3 KO mice), pharmacological porcupine inhibitor (C59) treatment in vivo","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 — clean genetic epistasis with multiple double-mutant combinations plus pharmacological validation in vivo","pmids":["26023187"],"is_preprint":false},{"year":2008,"finding":"RNF43 localizes to the endoplasmic reticulum and nuclear envelope, exhibits autoubiquitylation activity in a cell-free system with purified recombinant protein, and interacts with HAP95 (a chromatin-associated nuclear envelope protein) as identified by yeast two-hybrid and confirmed by Co-IP.","method":"Indirect immunofluorescence, biochemical fractionation, sucrose density gradient, cell-free autoubiquitylation assay with recombinant MBP-RNF43, yeast two-hybrid, Co-IP","journal":"Experimental Cell Research","confidence":"Medium","confidence_rationale":"Tier 1-3 — in vitro autoubiquitylation is Tier 1; HAP95 interaction by Co-IP is Tier 3; single study","pmids":["18313049"],"is_preprint":false},{"year":2010,"finding":"RNF43 interacts with NEDL1 (a p53-enhancing E3 ligase) and with p53 itself, and suppresses p53-mediated transcriptional activity and UV-induced apoptosis in H1299 cells.","method":"Yeast two-hybrid screening, Co-IP, transcriptional reporter assays, UV apoptosis assay","journal":"Biochemical and Biophysical Research Communications","confidence":"Medium","confidence_rationale":"Tier 3 — yeast two-hybrid and Co-IP with functional reporter readout; single study, single lab","pmids":["21108931"],"is_preprint":false},{"year":2014,"finding":"RNF43 expression is directly regulated as a Wnt target gene through two Wnt-responsive elements (WREs) in intron 2 that bind the TCF4/β-catenin complex, as demonstrated by reporter assays with WRE mutations and ChIP showing TCF4/β-catenin occupancy at both elements.","method":"Luciferase reporter assay with WRE mutants, siRNA knockdown of β-catenin, ChIP assay","journal":"PloS One","confidence":"Medium","confidence_rationale":"Tier 2 — reporter mutagenesis plus ChIP; single lab but two orthogonal methods","pmids":["24466159"],"is_preprint":false},{"year":2020,"finding":"RNF43 E3 ligase activity toward Frizzled requires phosphorylation at a conserved triplet of serine residues. Cancer-associated mutations abrogating this phosphorylation abolish Frizzled degradation and Wnt inhibition while retaining p53 inhibitory function. Phosphomimetic substitutions (Ser→Asp) restored tumor suppressive activity of extracellular oncogenic mutants.","method":"Phosphomutant and phosphomimetic RNF43 constructs, zebrafish development assay, mouse intestinal organoids, co-expression with active Ras for tumorigenesis, Wnt reporter assays","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1-2 — phospho-regulation demonstrated with mutagenesis plus in vivo zebrafish and organoid models; multiple orthogonal readouts","pmids":["32934222"],"is_preprint":false},{"year":2020,"finding":"A subset of RNF43 C-terminal truncating cancer mutations retains Wnt receptor downregulation but traps Casein kinase 1 (CK1) at the plasma membrane via the cytosolic tail, preventing cytoplasmic β-catenin turnover and driving ligand-independent β-catenin-mediated transcription. These mutations confer decreased sensitivity to anti-Wnt therapy.","method":"Gene editing of human colon stem cells, Co-IP of CK1 with truncated RNF43 variants, β-catenin phosphorylation assays, Wnt reporter assays, organoid growth in absence of niche factors","journal":"The EMBO Journal","confidence":"High","confidence_rationale":"Tier 2 — endogenous gene editing, CK1 Co-IP, multiple patient-derived mutation constructs, organoid functional assay; single lab but multiple orthogonal methods","pmids":["32965059"],"is_preprint":false},{"year":2021,"finding":"The deubiquitinase USP42 antagonizes R-spondin-induced clearance of RNF43/ZNRF3 by binding the Dishevelled-interacting region (DIR) of ZNRF3 and deubiquitinating ZNRF3, thereby stalling the R-spondin-LGR4-ZNRF3 ternary complex at the membrane and maintaining Frizzled/LRP6 turnover.","method":"Co-IP of USP42 with ZNRF3, deubiquitination assays, LRP6/FZD cell surface quantification, Wnt reporter assays, siRNA knockdown in colon cancer cells and mouse intestinal organoids","journal":"EMBO Reports","confidence":"High","confidence_rationale":"Tier 2 — Co-IP, enzymatic deubiquitination assay, functional organoid model, multiple orthogonal methods","pmids":["33786993"],"is_preprint":false},{"year":2021,"finding":"RNF43 inhibits noncanonical WNT5A signaling in human cells by interacting with ROR1, ROR2, VANGL1, and VANGL2 receptor complex components. RNF43 triggers ubiquitination and proteasomal degradation of VANGL2 and clathrin-dependent internalization of ROR1, and inhibits ROR2 activation, suppressing WNT5A-driven melanoma invasion and resistance to BRAF/MEK inhibitors.","method":"BioID proximity labeling, Co-IP, ubiquitination assays, VANGL2 degradation assays, ROR1 internalization (clathrin inhibitor), in vitro invasion assays, in vivo xenograft experiments","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — BioID interactome plus Co-IP plus functional ubiquitination and degradation assays plus in vivo validation","pmids":["34702444"],"is_preprint":false},{"year":2021,"finding":"Rnf43 (but not Znrf3) is potently activated by Wnt signaling in oligodendrocyte progenitor cells (OPCs), marks activated OPCs in human MS and HIE, and promotes OPC differentiation/remyelination specifically after injury by negatively regulating Fzd1 receptor presentation at the cell surface.","method":"Conditional Rnf43 KO in OPCs, neonatal hypoxic injury model, adult demyelination model, ex vivo and in vivo remyelination assays, Fzd1 inhibition (UM206)","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with injury-specific phenotype plus pharmacological rescue; multiple in vivo models","pmids":["34390652"],"is_preprint":false},{"year":2024,"finding":"RNF43 and ZNRF3 preferentially downregulate distinct subsets of Frizzled receptors: RNF43 preferentially targets FZD1/FZD5/FZD7 for endocytosis whereas ZNRF3 preferentially targets FZD6. The transmembrane domain (TMD) of RNF43 is a key molecular determinant for FZD5 endocytosis; swapping TMDs between RNF43 and ZNRF3 redirects their FZD preferences.","method":"FZD endocytosis assays (flow cytometry and imaging), TMD swap domain mutants, cell surface receptor quantification","journal":"Life Science Alliance","confidence":"High","confidence_rationale":"Tier 2 — domain-swap mutagenesis with quantitative endocytosis readouts; mechanistic TMD requirement established","pmids":["38969364"],"is_preprint":false},{"year":2024,"finding":"RNF43 ubiquitinates B-RAF at lysine K499 to promote proteasome-dependent degradation of B-RAF, reducing MEK activity. Phosphorylation of B-RAF at T491 suppresses this ubiquitination by decreasing RNF43–B-RAF interaction. Loss of RNF43 elevates B-RAF/MEK signaling, and MEK plus Wnt inhibitors synergistically suppress RNF43-mutant pancreatic cancer growth.","method":"Co-IP, ubiquitination assay with specific lysine mutants (K499R), phosphorylation site mutagenesis (T491), proteasome inhibitor assays, MEK inhibitor sensitivity in cell lines and in vivo","journal":"Advanced Science","confidence":"High","confidence_rationale":"Tier 1-2 — direct ubiquitination site identified by mutagenesis with Co-IP, phospho-regulation demonstrated, in vivo validation","pmids":["38225722"],"is_preprint":false},{"year":2024,"finding":"ZNRF3/RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and degradation dependent on the RING domain E3 ligase activity. ZNRF3/RNF43 knockout elevates EGFR signaling and promotes tumorigenesis.","method":"Co-IP of ZNRF3/RNF43 with EGFR, ubiquitination assays, ZNRF3/RNF43 overexpression and KO in cancer cells, in vivo tumor growth assays, proteogenomic correlation analysis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — biochemical Co-IP, ubiquitination assay, KO and OE with functional readouts in vitro and in vivo; peer-reviewed publication","pmids":["41960900"],"is_preprint":false},{"year":2022,"finding":"RNF43 G659fs mutant binds the p85 regulatory subunit of PI3K, leading to p85 ubiquitination and degradation, which increases PI3K/mTOR signaling. RNF43 G659fs cells are selectively killed by PI3K/mTOR inhibitors in vitro, in isogenic xenografts, and in patient-derived organoids.","method":"Co-IP of RNF43-G659fs with p85, ubiquitination assay, drug library screen, isogenic cell line and organoid drug sensitivity assays, xenograft experiments","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 2 — direct substrate identification by Co-IP plus ubiquitination assay, functional validation in multiple models including PDO and xenograft","pmids":["35676246"],"is_preprint":false},{"year":2019,"finding":"RNF43 ubiquitinates c-Src-phosphorylated E-cadherin (phospho-Tyr797) at Lys816, promoting its degradation and enabling nuclear β-catenin translocation, thereby facilitating EMT in lung adenocarcinoma. This ubiquitination requires Frizzled 8 (FZD8).","method":"Protein antibody microarray, E3 ligase profiling, Co-IP, ubiquitination site mutagenesis (Y797, K816), immunofluorescence, xenograft models, shRNA knockdown","journal":"BMC Cancer","confidence":"Medium","confidence_rationale":"Tier 2 — specific ubiquitination site identified by mutagenesis; single lab but multiple orthogonal methods; somewhat orthogonal to dominant Wnt-focused RNF43 literature","pmids":["31286874"],"is_preprint":false},{"year":2019,"finding":"RNF43 co-associates with PAR2 (protease-activated receptor 2), promotes its membrane elimination and polyubiquitination, and PAR2 degradation is rescued by R-spondin2 in the presence of LGR5, establishing RNF43 as a negative feedback regulator of PAR2 in colon cancer.","method":"Co-IP, cell surface biotinylation assay for PAR2, polyubiquitination assay, R-spondin/LGR5 rescue experiment, β-catenin reporter assay, AOM/DSS mouse model","journal":"FASEB Journal","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP and surface biotinylation are standard; single lab; PAR2 as substrate not replicated elsewhere","pmids":["36468684"],"is_preprint":false},{"year":2023,"finding":"RNF43 interacts with PD-L1 and augments both K48- and K63-linked ubiquitination of PD-L1 in gastric cancer cell lines, reducing PD-L1 surface expression and enhancing T-cell anti-tumor activity.","method":"Co-IP of RNF43 and PD-L1, ubiquitination linkage-specific assays (K48, K63), T-cell killing assays","journal":"Scandinavian Journal of Immunology","confidence":"Low","confidence_rationale":"Tier 3 — Co-IP and ubiquitination assay; single study, single lab, not replicated","pmids":["39007965"],"is_preprint":false},{"year":2024,"finding":"RNF43 reinforces ubiquitination and proteasomal degradation of NDUFS1 (NADH dehydrogenase Fe-S protein 1) by direct interaction with it, suppressing oxidative phosphorylation and inhibiting endometrial stromal cell viability and migration. RNF43 mRNA stability and expression are regulated by METTL3/IGF2BP2-mediated m6A modification.","method":"Co-IP of RNF43 and NDUFS1, ubiquitination assay, m6A methylation assays (METTL3 overexpression/inhibition, IGF2BP2 RIP), cell viability and migration assays, knockdown/overexpression","journal":"Journal of Cellular Physiology","confidence":"Low","confidence_rationale":"Tier 3 — Co-IP and ubiquitination of NDUFS1 by single lab; m6A regulation of RNF43 mRNA is additional claim; not replicated","pmids":["38988031"],"is_preprint":false},{"year":2008,"finding":"RNF43 interacts with the heterodimer PSF/p54nrb as identified by pull-down and confirmed by Co-IP; co-expression of PSF relocates RNF43 from the nuclear periphery to the nucleoplasm.","method":"Pull-down assay with MS identification, Co-IP, immunofluorescence","journal":"Proteomics","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP confirmation; relocalization noted but functional consequence not established; antibody reliability concerns noted later","pmids":["18655028"],"is_preprint":false},{"year":2020,"finding":"The RNF43 protease-associated (PA) extracellular domain is dispensable for inhibition of canonical Wnt/β-catenin signaling in human cells; RNF43 lacking the PA domain still reduces LRP6/DVL phosphorylation and β-catenin-dependent transcription, and is insensitive to R-spondin1.","method":"TetON-controlled RNF43ΔPA overexpression, CRISPR/Cas9 RNF43/ZNRF3 double KO rescue experiments, Western blot (pLRP6, pDVL, β-catenin), TOPflash luciferase assay","journal":"Cell Communication and Signaling","confidence":"Medium","confidence_rationale":"Tier 2 — controlled expression system plus KO rescue; multiple readouts; single lab","pmids":["32527265"],"is_preprint":false},{"year":2023,"finding":"LGR4, but not LGR5, forms a complex with RNF43/ZNRF3 that provides high-affinity bivalent binding of R-spondin ligands. LGR5 forms homodimers that do not interact with the E3 ligases; co-expression of ZNRF3 with LGR4 dramatically increases monovalent RSPO affinity, whereas co-expression with LGR5 has no effect.","method":"Binding affinity measurements in whole cells, co-expression of LGR4/LGR5 with ZNRF3, bivalent vs monovalent RSPO2 furin domain binding assays","journal":"Scientific Reports","confidence":"Medium","confidence_rationale":"Tier 2 — quantitative binding assays with multiple receptor combinations; single lab","pmids":["37402772"],"is_preprint":false}],"current_model":"RNF43 is a single-pass transmembrane RING-type E3 ubiquitin ligase that acts as a Wnt negative-feedback regulator by ubiquitinating Frizzled receptors (preferentially FZD1/5/7) for lysosomal degradation, a process requiring DVL as a substrate-adaptor and phosphorylation of RNF43 at a conserved serine triplet; R-spondin proteins antagonize RNF43 by recruiting it into an LGR4-RSPO-RNF43 ternary complex (structurally defined by crystal structures) that triggers RNF43 auto-ubiquitination and membrane clearance, counteracted by the deubiquitinase USP42; beyond Frizzled, RNF43 also ubiquitinates B-RAF (K499), EGFR, VANGL2, and PAR2, inhibits noncanonical WNT5A signaling through interactions with ROR1/ROR2/VANGL1/VANGL2, and a class of C-terminal truncating cancer mutations traps CK1 at the plasma membrane to drive ligand-independent β-catenin activation independently of Frizzled regulation."},"narrative":{"teleology":[{"year":2008,"claim":"Initial biochemical characterization established that RNF43 possesses intrinsic E3 ubiquitin ligase (autoubiquitylation) activity and localizes to the ER/nuclear envelope, but its physiological substrates were unknown.","evidence":"Cell-free autoubiquitylation assay with recombinant MBP-RNF43, immunofluorescence, and yeast two-hybrid identification of HAP95 interaction","pmids":["18313049"],"confidence":"Medium","gaps":["HAP95 interaction confirmed by single Co-IP without reciprocal validation","Physiological substrates not identified","ER/nuclear envelope localization later challenged by antibody concerns"]},{"year":2012,"claim":"The breakthrough discovery that RNF43 ubiquitinates Frizzled receptors for lysosomal degradation established its role as a Wnt negative-feedback regulator, and double knockout with ZNRF3 in mouse intestine caused Wnt-dependent adenomas.","evidence":"In vitro ubiquitination assays, HEK293T overexpression, conditional mouse knockout, organoid culture","pmids":["22895187"],"confidence":"High","gaps":["Mechanism of substrate recognition (how RNF43 engages Frizzled) not yet defined","Relative contribution of RNF43 vs ZNRF3 unclear","Adaptor requirements unknown"]},{"year":2013,"claim":"Structural and functional studies defined the R-spondin/LGR/RNF43 ternary complex architecture, revealing how R-spondins bridge LGR receptors and RNF43 to neutralize Frizzled degradation, and showed that cancer-associated RNF43 mutations create Wnt ligand dependency.","evidence":"Crystal structures of RNF43 ectodomain with RSPO2 and ternary RSPO1–LGR5–RNF43 complex; binding assays; RNF43 reconstitution in pancreatic cancer cells with porcupine inhibitor sensitivity","pmids":["24225776","23756651","23847203"],"confidence":"High","gaps":["Whether all R-spondin family members use identical binding mode unresolved","Mechanism of R-spondin-induced RNF43 membrane clearance not determined","Intracellular signaling consequences of ternary complex formation undefined"]},{"year":2014,"claim":"RNF43 was shown to be a direct Wnt/β-catenin target gene regulated by TCF4/β-catenin occupancy at intronic Wnt-responsive elements, completing the negative-feedback loop architecture.","evidence":"Luciferase reporter assay with WRE mutants, ChIP for TCF4/β-catenin at intronic elements","pmids":["24466159"],"confidence":"Medium","gaps":["Regulation in non-intestinal tissues not addressed","Kinetics of feedback induction not measured"]},{"year":2015,"claim":"DVL was identified as an essential adaptor bridging RNF43 to Frizzled substrates, with domain dissection revealing that the PA domain mediates Frizzled CRD interaction for canonical signaling while the C-terminal cytoplasmic region suppresses noncanonical Wnt signaling independently of RING-dependent ubiquitination.","evidence":"DVL knockout cells, Co-IP, DEP-ZNRF3 fusion rescue, domain deletion/mutation analysis, Wnt reporter assays","pmids":["25891077","25825523"],"confidence":"High","gaps":["Identity of the kinase phosphorylating RNF43 to activate it not yet known","Structural basis of DVL–RNF43 interaction unresolved","Mechanism of RING-independent noncanonical suppression unclear"]},{"year":2015,"claim":"Genetic epistasis demonstrated that paracrine Wnt3 from Paneth cells is the essential proliferative signal in RNF43/ZNRF3-null intestinal tumors, validating porcupine inhibition as a therapeutic strategy.","evidence":"Genetic crosses of Rnf43/Znrf3 KO with Math1 KO and Wnt3 KO mice; porcupine inhibitor C59 treatment in vivo","pmids":["26023187"],"confidence":"High","gaps":["Applicability to human RNF43-mutant cancers not directly demonstrated","Whether all RNF43 mutation classes are equally porcupine-sensitive unknown"]},{"year":2019,"claim":"RNF43 substrate scope was expanded beyond Frizzled to include PAR2 and phospho-E-cadherin, suggesting broader membrane protein quality control functions.","evidence":"Co-IP and ubiquitination assays for PAR2 (with R-spondin/LGR5 rescue) and E-cadherin (K816 site mutagenesis), xenograft models","pmids":["36468684","31286874"],"confidence":"Medium","gaps":["PAR2 as substrate not independently replicated","E-cadherin ubiquitination requirement for FZD8 is unusual and unexplained mechanistically","Relative importance of non-Frizzled substrates in physiology unknown"]},{"year":2020,"claim":"The critical regulatory mechanism of RNF43 phosphorylation at a conserved serine triplet was defined: phosphorylation is required for Frizzled ubiquitination, and cancer mutations that abolish it selectively lose tumor-suppressive but not p53-inhibitory function. Separately, C-terminal truncating mutations were shown to trap CK1 at the plasma membrane, driving ligand-independent β-catenin activation.","evidence":"Phosphomutant/phosphomimetic constructs in zebrafish and organoids; gene-edited human colon stem cells with CK1 Co-IP and β-catenin phosphorylation assays","pmids":["32934222","32965059"],"confidence":"High","gaps":["Identity of the kinase(s) phosphorylating the serine triplet unknown","CK1 trapping mechanism (direct binding vs indirect) not structurally resolved","Whether phosphorylation and CK1-trapping mutations co-occur in tumors not examined"]},{"year":2021,"claim":"USP42 was identified as the deubiquitinase counteracting R-spondin-induced RNF43/ZNRF3 membrane clearance, and RNF43 was shown to suppress noncanonical WNT5A signaling by ubiquitinating VANGL2 and internalizing ROR1, establishing dual canonical/noncanonical tumor-suppressive functions.","evidence":"Co-IP and deubiquitination assays for USP42–ZNRF3; BioID proximity labeling plus Co-IP and degradation assays for ROR1/ROR2/VANGL interactions; invasion and xenograft models","pmids":["33786993","34702444"],"confidence":"High","gaps":["Whether USP42 acts on RNF43 directly (in addition to ZNRF3) not established","Structural basis of RNF43–ROR/VANGL interaction unknown","In vivo relevance of noncanonical suppression in normal tissue homeostasis not tested"]},{"year":2021,"claim":"A non-redundant role for RNF43 (distinct from ZNRF3) in oligodendrocyte progenitor differentiation was demonstrated: RNF43 is Wnt-induced specifically after injury and promotes remyelination by clearing Fzd1.","evidence":"Conditional Rnf43 KO in OPCs, neonatal hypoxic injury model, adult demyelination model, Fzd1 pharmacological inhibition rescue","pmids":["34390652"],"confidence":"High","gaps":["Whether other Frizzled subtypes contribute to OPC phenotype not resolved","Human translational relevance limited to expression data"]},{"year":2024,"claim":"RNF43 and ZNRF3 were shown to have distinct Frizzled substrate preferences dictated by the transmembrane domain, and RNF43 was found to ubiquitinate B-RAF at K499 and EGFR, broadening its tumor-suppressive reach beyond Wnt receptors.","evidence":"TMD swap domain mutants with quantitative FZD endocytosis assays; K499R B-RAF mutagenesis with Co-IP; EGFR Co-IP and ubiquitination assays in KO/OE systems","pmids":["38969364","38225722","41960900"],"confidence":"High","gaps":["Structural basis of TMD-mediated Frizzled selectivity unknown","Physiological contexts where B-RAF and EGFR ubiquitination by RNF43 are rate-limiting undefined","Whether all non-Frizzled substrates require DVL adaptor function untested"]},{"year":null,"claim":"Key open questions include the identity of the kinase(s) phosphorylating the RNF43 serine triplet, the structural basis of TMD-determined Frizzled selectivity, whether non-Frizzled substrates (B-RAF, EGFR, VANGL2) use the same DVL-dependent adaptor mechanism, and the in vivo tissue-specific hierarchy among RNF43's multiple substrates.","evidence":"","pmids":[],"confidence":"Low","gaps":["Kinase for serine triplet phosphorylation unidentified","No structural model of full-length RNF43 in complex with any Frizzled","Relative in vivo importance of Wnt-dependent vs Wnt-independent substrates unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,4,5,11,14,17,18,19]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,8,11,17]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,3,12,16]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,5]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[5,8]},{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3,4,5,7,11,12,14,15,16]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,4,8,11,14,17,18]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,7,12,17,19]}],"complexes":["RSPO-LGR4-RNF43 ternary complex"],"partners":["FZD5","DVL2","LGR4","BRAF","EGFR","VANGL2","ROR1","CSNK1A1"],"other_free_text":[]},"mechanistic_narrative":"RNF43 is a transmembrane RING-type E3 ubiquitin ligase that functions as a central negative-feedback regulator of Wnt signaling by ubiquitinating Frizzled receptors—preferentially FZD1, FZD5, and FZD7—for lysosomal degradation, a process requiring Dishevelled (DVL) as a substrate adaptor and phosphorylation at a conserved serine triplet [PMID:22895187, PMID:25891077, PMID:32934222, PMID:38969364]. R-spondin ligands antagonize RNF43 by forming a ternary complex with LGR4 and the RNF43 ectodomain, neutralizing its Frizzled-clearing activity and potentiating Wnt signaling; this interaction is counteracted by the deubiquitinase USP42 [PMID:23756651, PMID:24225776, PMID:33786993, PMID:37402772]. Beyond Frizzled, RNF43 ubiquitinates additional substrates including B-RAF at K499, EGFR, and VANGL2, and suppresses noncanonical WNT5A signaling through interactions with ROR1/ROR2/VANGL1/VANGL2 [PMID:34702444, PMID:38225722, PMID:41960900]. Cancer-associated C-terminal truncating mutations can retain Frizzled regulation yet trap CK1 at the plasma membrane, driving ligand-independent β-catenin activation and resistance to anti-Wnt therapies [PMID:32965059]."},"prefetch_data":{"uniprot":{"accession":"Q68DV7","full_name":"E3 ubiquitin-protein ligase RNF43","aliases":["RING finger protein 43","RING-type E3 ubiquitin transferase RNF43"],"length_aa":783,"mass_kda":85.7,"function":"E3 ubiquitin-protein ligase that acts as a negative regulator of the Wnt signaling pathway by mediating the ubiquitination, endocytosis and subsequent degradation of Wnt receptor complex components Frizzled. Acts on both canonical and non-canonical Wnt signaling pathway (PubMed:18313049, PubMed:22575959, PubMed:22895187). Along with RSPO2 and ZNRF3, constitutes a master switch that governs limb specification (By similarity)","subcellular_location":"Cell membrane; Endoplasmic reticulum membrane; Nucleus envelope","url":"https://www.uniprot.org/uniprotkb/Q68DV7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RNF43","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RNF43","total_profiled":1310},"omim":[{"mim_id":"620946","title":"UBIQUITIN-SPECIFIC PEPTIDASE 42; USP42","url":"https://www.omim.org/entry/620946"},{"mim_id":"618022","title":"HUMEROFEMORAL HYPOPLASIA WITH RADIOTIBIAL RAY DEFICIENCY; HHRRD","url":"https://www.omim.org/entry/618022"},{"mim_id":"618021","title":"TETRAAMELIA SYNDROME 2; TETAMS2","url":"https://www.omim.org/entry/618021"},{"mim_id":"617108","title":"SESSILE SERRATED POLYPOSIS CANCER SYNDROME; SSPCS","url":"https://www.omim.org/entry/617108"},{"mim_id":"612482","title":"RING FINGER PROTEIN 43; RNF43","url":"https://www.omim.org/entry/612482"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"intestine","ntpm":21.4}],"url":"https://www.proteinatlas.org/search/RNF43"},"hgnc":{"alias_symbol":["FLJ20315","RNF124","DKFZp781H0392","URCC"],"prev_symbol":[]},"alphafold":{"accession":"Q68DV7","domains":[{"cath_id":"3.50.30.30","chopping":"44-192","consensus_level":"high","plddt":87.356,"start":44,"end":192},{"cath_id":"3.30.40.10","chopping":"233-249_271-319","consensus_level":"medium","plddt":84.2442,"start":233,"end":319},{"cath_id":"1.20.5","chopping":"194-223","consensus_level":"medium","plddt":81.6147,"start":194,"end":223}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q68DV7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q68DV7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q68DV7-F1-predicted_aligned_error_v6.png","plddt_mean":55.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RNF43","jax_strain_url":"https://www.jax.org/strain/search?query=RNF43"},"sequence":{"accession":"Q68DV7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q68DV7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q68DV7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q68DV7"}},"corpus_meta":[{"pmid":"22895187","id":"PMC_22895187","title":"Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors.","date":"2012","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/22895187","citation_count":784,"is_preprint":false},{"pmid":"24532711","id":"PMC_24532711","title":"The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength.","date":"2014","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/24532711","citation_count":523,"is_preprint":false},{"pmid":"23847203","id":"PMC_23847203","title":"Inactivating mutations of RNF43 confer Wnt dependency in pancreatic ductal adenocarcinoma.","date":"2013","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/23847203","citation_count":355,"is_preprint":false},{"pmid":"27088858","id":"PMC_27088858","title":"The RSPO-LGR4/5-ZNRF3/RNF43 module controls liver zonation and size.","date":"2016","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/27088858","citation_count":265,"is_preprint":false},{"pmid":"27869803","id":"PMC_27869803","title":"Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors.","date":"2016","source":"Nature medicine","url":"https://pubmed.ncbi.nlm.nih.gov/27869803","citation_count":230,"is_preprint":false},{"pmid":"24225776","id":"PMC_24225776","title":"Structural and molecular basis of ZNRF3/RNF43 transmembrane ubiquitin ligase inhibition by the Wnt agonist R-spondin.","date":"2013","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/24225776","citation_count":162,"is_preprint":false},{"pmid":"25891077","id":"PMC_25891077","title":"Dishevelled promotes Wnt receptor degradation through recruitment of ZNRF3/RNF43 E3 ubiquitin ligases.","date":"2015","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/25891077","citation_count":161,"is_preprint":false},{"pmid":"27329244","id":"PMC_27329244","title":"RNF43 germline and somatic mutation in serrated neoplasia pathway and its association with BRAF mutation.","date":"2016","source":"Gut","url":"https://pubmed.ncbi.nlm.nih.gov/27329244","citation_count":159,"is_preprint":false},{"pmid":"27338477","id":"PMC_27338477","title":"Control of Wnt Receptor Turnover by R-spondin-ZNRF3/RNF43 Signaling Module and Its Dysregulation in Cancer.","date":"2016","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/27338477","citation_count":144,"is_preprint":false},{"pmid":"26023187","id":"PMC_26023187","title":"Porcupine inhibitor suppresses paracrine Wnt-driven growth of Rnf43;Znrf3-mutant neoplasia.","date":"2015","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/26023187","citation_count":140,"is_preprint":false},{"pmid":"21818642","id":"PMC_21818642","title":"Ginger (Zingiber officinale) reduces acute chemotherapy-induced nausea: a URCC CCOP study of 576 patients.","date":"2011","source":"Supportive care in cancer : official journal of the Multinational Association of Supportive Care in Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/21818642","citation_count":135,"is_preprint":false},{"pmid":"23756651","id":"PMC_23756651","title":"The structural basis of R-spondin recognition by LGR5 and RNF43.","date":"2013","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/23756651","citation_count":124,"is_preprint":false},{"pmid":"26924569","id":"PMC_26924569","title":"Frequent PTPRK-RSPO3 fusions and RNF43 mutations in colorectal traditional serrated adenoma.","date":"2016","source":"The Journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/26924569","citation_count":107,"is_preprint":false},{"pmid":"23096461","id":"PMC_23096461","title":"RNF43 is a tumour suppressor gene mutated in mucinous tumours of the ovary.","date":"2013","source":"The Journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/23096461","citation_count":100,"is_preprint":false},{"pmid":"27661107","id":"PMC_27661107","title":"RNF43 and ZNRF3 are commonly altered in serrated pathway colorectal tumorigenesis.","date":"2016","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/27661107","citation_count":91,"is_preprint":false},{"pmid":"27512631","id":"PMC_27512631","title":"KRAS, GNAS, and RNF43 mutations in intraductal papillary mucinous neoplasm of the pancreas: a meta-analysis.","date":"2016","source":"SpringerPlus","url":"https://pubmed.ncbi.nlm.nih.gov/27512631","citation_count":82,"is_preprint":false},{"pmid":"16198982","id":"PMC_16198982","title":"5-Hydroxytryptamine-receptor antagonists versus prochlorperazine for control of delayed nausea caused by doxorubicin: a URCC CCOP randomised controlled trial.","date":"2005","source":"The Lancet. Oncology","url":"https://pubmed.ncbi.nlm.nih.gov/16198982","citation_count":79,"is_preprint":false},{"pmid":"25825523","id":"PMC_25825523","title":"Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling.","date":"2015","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/25825523","citation_count":77,"is_preprint":false},{"pmid":"26350900","id":"PMC_26350900","title":"The E3 ligase RNF43 inhibits Wnt signaling downstream of mutated β-catenin by sequestering TCF4 to the nuclear membrane.","date":"2015","source":"Science signaling","url":"https://pubmed.ncbi.nlm.nih.gov/26350900","citation_count":66,"is_preprint":false},{"pmid":"34129813","id":"PMC_34129813","title":"ZNRF3 and RNF43 cooperate to safeguard metabolic liver zonation and hepatocyte proliferation.","date":"2021","source":"Cell stem cell","url":"https://pubmed.ncbi.nlm.nih.gov/34129813","citation_count":64,"is_preprint":false},{"pmid":"25081753","id":"PMC_25081753","title":"Clinicopathological significance of somatic RNF43 mutation and aberrant expression of ring finger protein 43 in intraductal papillary mucinous neoplasms of the pancreas.","date":"2014","source":"Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc","url":"https://pubmed.ncbi.nlm.nih.gov/25081753","citation_count":58,"is_preprint":false},{"pmid":"31811196","id":"PMC_31811196","title":"The most common RNF43 mutant G659Vfs*41 is fully functional in inhibiting Wnt signaling and unlikely to play a role in tumorigenesis.","date":"2019","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/31811196","citation_count":56,"is_preprint":false},{"pmid":"25996295","id":"PMC_25996295","title":"The nucleoprotein of influenza A virus induces p53 signaling and apoptosis via attenuation of host ubiquitin ligase RNF43.","date":"2015","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/25996295","citation_count":55,"is_preprint":false},{"pmid":"25961913","id":"PMC_25961913","title":"Brg-1 targeting of novel miR550a-5p/RNF43/Wnt signaling axis regulates colorectal cancer metastasis.","date":"2015","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/25961913","citation_count":54,"is_preprint":false},{"pmid":"34973294","id":"PMC_34973294","title":"Loss of Rnf43 Accelerates Kras-Mediated Neoplasia and Remodels the Tumor Immune Microenvironment in Pancreatic Adenocarcinoma.","date":"2021","source":"Gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/34973294","citation_count":52,"is_preprint":false},{"pmid":"21108931","id":"PMC_21108931","title":"RNF43 interacts with NEDL1 and regulates p53-mediated transcription.","date":"2010","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/21108931","citation_count":52,"is_preprint":false},{"pmid":"15492824","id":"PMC_15492824","title":"A novel oncoprotein RNF43 functions in an autocrine manner in colorectal cancer.","date":"2004","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/15492824","citation_count":52,"is_preprint":false},{"pmid":"32934222","id":"PMC_32934222","title":"A phospho-switch controls RNF43-mediated degradation of Wnt receptors to suppress tumorigenesis.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/32934222","citation_count":51,"is_preprint":false},{"pmid":"35039505","id":"PMC_35039505","title":"RNF43/ZNRF3 loss predisposes to hepatocellular-carcinoma by impairing liver regeneration and altering the liver lipid metabolic ground-state.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/35039505","citation_count":49,"is_preprint":false},{"pmid":"27514024","id":"PMC_27514024","title":"Dysregulated Wnt signalling and recurrent mutations of the tumour suppressor RNF43 in early gastric carcinogenesis.","date":"2016","source":"The Journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/27514024","citation_count":48,"is_preprint":false},{"pmid":"18313049","id":"PMC_18313049","title":"A cancer-associated RING finger protein, RNF43, is a ubiquitin ligase that interacts with a nuclear protein, HAP95.","date":"2008","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/18313049","citation_count":46,"is_preprint":false},{"pmid":"27081527","id":"PMC_27081527","title":"A deleterious RNF43 germline mutation in a severely affected serrated polyposis kindred.","date":"2015","source":"Human genome variation","url":"https://pubmed.ncbi.nlm.nih.gov/27081527","citation_count":46,"is_preprint":false},{"pmid":"25937466","id":"PMC_25937466","title":"ZNRF3/RNF43--A direct linkage of extracellular recognition and E3 ligase activity to modulate cell surface signalling.","date":"2015","source":"Progress in biophysics and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/25937466","citation_count":44,"is_preprint":false},{"pmid":"33067269","id":"PMC_33067269","title":"The Functional Landscape of Patient-Derived RNF43 Mutations Predicts Sensitivity to Wnt Inhibition.","date":"2020","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/33067269","citation_count":44,"is_preprint":false},{"pmid":"33786993","id":"PMC_33786993","title":"USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling.","date":"2021","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/33786993","citation_count":43,"is_preprint":false},{"pmid":"32965059","id":"PMC_32965059","title":"RNF43 truncations trap CK1 to drive niche-independent self-renewal in cancer.","date":"2020","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/32965059","citation_count":43,"is_preprint":false},{"pmid":"35676246","id":"PMC_35676246","title":"RNF43 G659fs is an oncogenic colorectal cancer mutation and sensitizes tumor cells to PI3K/mTOR inhibition.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/35676246","citation_count":41,"is_preprint":false},{"pmid":"29756208","id":"PMC_29756208","title":"Impact of loss-of-function mutations at the RNF43 locus on colorectal cancer development and progression.","date":"2018","source":"The Journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/29756208","citation_count":39,"is_preprint":false},{"pmid":"32103169","id":"PMC_32103169","title":"Commonly observed RNF43 mutations retain functionality in attenuating Wnt/β-catenin signaling and unlikely confer Wnt-dependency onto colorectal cancers.","date":"2020","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/32103169","citation_count":39,"is_preprint":false},{"pmid":"35006616","id":"PMC_35006616","title":"The RSPO-LGR4/5-ZNRF3/RNF43 module in liver homeostasis, regeneration, and disease.","date":"2022","source":"Hepatology (Baltimore, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/35006616","citation_count":38,"is_preprint":false},{"pmid":"27305845","id":"PMC_27305845","title":"RNF43 Is an Early and Specific Mutated Gene in the Serrated Pathway, With Increased Frequency in Traditional Serrated Adenoma and Its Associated Malignancy.","date":"2016","source":"The American journal of surgical pathology","url":"https://pubmed.ncbi.nlm.nih.gov/27305845","citation_count":38,"is_preprint":false},{"pmid":"34702444","id":"PMC_34702444","title":"RNF43 inhibits WNT5A-driven signaling and suppresses melanoma invasion and resistance to the targeted therapy.","date":"2021","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/34702444","citation_count":36,"is_preprint":false},{"pmid":"34390652","id":"PMC_34390652","title":"Oligodendroglial ring finger protein Rnf43 is an essential injury-specific regulator of oligodendrocyte maturation.","date":"2021","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/34390652","citation_count":34,"is_preprint":false},{"pmid":"35040131","id":"PMC_35040131","title":"Characterization of RNF43 frameshift mutations that drive Wnt ligand- and R-spondin-dependent colon cancer.","date":"2022","source":"The Journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/35040131","citation_count":34,"is_preprint":false},{"pmid":"33188943","id":"PMC_33188943","title":"Loss of RNF43 Function Contributes to Gastric Carcinogenesis by Impairing DNA Damage Response.","date":"2020","source":"Cellular and molecular gastroenterology and hepatology","url":"https://pubmed.ncbi.nlm.nih.gov/33188943","citation_count":31,"is_preprint":false},{"pmid":"32236609","id":"PMC_32236609","title":"RNF43 mutation is associated with aggressive tumor biology along with BRAF V600E mutation in right-sided colorectal cancer.","date":"2020","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/32236609","citation_count":29,"is_preprint":false},{"pmid":"30380024","id":"PMC_30380024","title":"Loss of endogenous RNF43 function enhances proliferation and tumour growth of intestinal and gastric cells.","date":"2019","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/30380024","citation_count":29,"is_preprint":false},{"pmid":"35254413","id":"PMC_35254413","title":"Comprehensive Analysis of R-Spondin Fusions and RNF43 Mutations Implicate Novel Therapeutic Options in Colorectal Cancer.","date":"2022","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/35254413","citation_count":28,"is_preprint":false},{"pmid":"37643759","id":"PMC_37643759","title":"RNF43 and ZNRF3 in Wnt Signaling - A Master Regulator at the Membrane.","date":"2023","source":"International journal of stem cells","url":"https://pubmed.ncbi.nlm.nih.gov/37643759","citation_count":28,"is_preprint":false},{"pmid":"27864998","id":"PMC_27864998","title":"RNF43 mutation frequently occurs with GNAS mutation and mucin hypersecretion in intraductal papillary neoplasms of the bile duct.","date":"2017","source":"Histopathology","url":"https://pubmed.ncbi.nlm.nih.gov/27864998","citation_count":28,"is_preprint":false},{"pmid":"24466159","id":"PMC_24466159","title":"Identification of two Wnt-responsive elements in the intron of RING finger protein 43 (RNF43) gene.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24466159","citation_count":26,"is_preprint":false},{"pmid":"31286874","id":"PMC_31286874","title":"RNF43 ubiquitinates and degrades phosphorylated E-cadherin by c-Src to facilitate epithelial-mesenchymal transition in lung adenocarcinoma.","date":"2019","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/31286874","citation_count":26,"is_preprint":false},{"pmid":"26297255","id":"PMC_26297255","title":"Frequent frameshift mutations in 2 mononucleotide repeats of RNF43 gene and its regional heterogeneity in gastric and colorectal cancers.","date":"2015","source":"Human pathology","url":"https://pubmed.ncbi.nlm.nih.gov/26297255","citation_count":22,"is_preprint":false},{"pmid":"24001777","id":"PMC_24001777","title":"RNF43 mutations are recurrent in Chinese patients with mucinous ovarian carcinoma but absent in other subtypes of ovarian cancer.","date":"2013","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/24001777","citation_count":22,"is_preprint":false},{"pmid":"29018044","id":"PMC_29018044","title":"Rnf43.","date":"2017","source":"Journal of clinical pathology","url":"https://pubmed.ncbi.nlm.nih.gov/29018044","citation_count":20,"is_preprint":false},{"pmid":"18632524","id":"PMC_18632524","title":"A Phase II/III Randomized, Placebo-Controlled, Double-Blind Clinical Trial of Ginger (Zingiber officinale) for Nausea Caused by Chemotherapy for Cancer: A Currently Accruing URCC CCOP Cancer Control Study.","date":"2007","source":"Supportive cancer therapy","url":"https://pubmed.ncbi.nlm.nih.gov/18632524","citation_count":20,"is_preprint":false},{"pmid":"32527265","id":"PMC_32527265","title":"Protease associated domain of RNF43 is not necessary for the suppression of Wnt/β-catenin signaling in human cells.","date":"2020","source":"Cell communication and signaling : CCS","url":"https://pubmed.ncbi.nlm.nih.gov/32527265","citation_count":20,"is_preprint":false},{"pmid":"33938624","id":"PMC_33938624","title":"Ub and Dub of RNF43/ZNRF3 in the WNT signalling pathway.","date":"2021","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/33938624","citation_count":17,"is_preprint":false},{"pmid":"33569855","id":"PMC_33569855","title":"Post-translational Wnt receptor regulation: Is the fog slowly clearing?: The molecular mechanism of RNF43/ZNRF3 ubiquitin ligases is not yet fully elucidated and still controversial.","date":"2021","source":"BioEssays : news and reviews in molecular, cellular and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/33569855","citation_count":16,"is_preprint":false},{"pmid":"33098683","id":"PMC_33098683","title":"RNF43 mutation analysis in serrated polyposis, sporadic serrated polyps and Lynch syndrome polyps.","date":"2020","source":"Histopathology","url":"https://pubmed.ncbi.nlm.nih.gov/33098683","citation_count":16,"is_preprint":false},{"pmid":"36823311","id":"PMC_36823311","title":"The tumor biological significance of RNF43 and LRP1B in gastric cancer is complex and context-dependent.","date":"2023","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/36823311","citation_count":15,"is_preprint":false},{"pmid":"37402772","id":"PMC_37402772","title":"LGR4 and LGR5 form distinct homodimers that only LGR4 complexes with RNF43/ZNRF3 to provide high affinity binding of R-spondin ligands.","date":"2023","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/37402772","citation_count":15,"is_preprint":false},{"pmid":"28573495","id":"PMC_28573495","title":"RNF43 is mutated less frequently in Lynch Syndrome compared with sporadic microsatellite unstable colorectal cancers.","date":"2018","source":"Familial cancer","url":"https://pubmed.ncbi.nlm.nih.gov/28573495","citation_count":15,"is_preprint":false},{"pmid":"34637826","id":"PMC_34637826","title":"CircRNA-IGLL1/miR-15a/RNF43 axis mediates ammonia-induced autophagy in broilers jejunum via Wnt/β-catenin pathway.","date":"2021","source":"Environmental pollution (Barking, Essex : 1987)","url":"https://pubmed.ncbi.nlm.nih.gov/34637826","citation_count":14,"is_preprint":false},{"pmid":"29293510","id":"PMC_29293510","title":"A phase I clinical trial of RNF43 peptide-related immune cell therapy combined with low-dose cyclophosphamide in patients with advanced solid tumors.","date":"2018","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/29293510","citation_count":14,"is_preprint":false},{"pmid":"38969364","id":"PMC_38969364","title":"E3 ligases RNF43 and ZNRF3 display differential specificity for endocytosis of Frizzled receptors.","date":"2024","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/38969364","citation_count":13,"is_preprint":false},{"pmid":"34995498","id":"PMC_34995498","title":"RNF43/ZNRF3 negatively regulates taste tissue homeostasis and positively regulates dorsal lingual epithelial tissue homeostasis.","date":"2022","source":"Stem cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/34995498","citation_count":13,"is_preprint":false},{"pmid":"18655028","id":"PMC_18655028","title":"Proteomic identification of a PSF/p54nrb heterodimer as RNF43 oncoprotein-interacting proteins.","date":"2008","source":"Proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/18655028","citation_count":13,"is_preprint":false},{"pmid":"30884828","id":"PMC_30884828","title":"Mutated Rnf43 Aggravates Helicobacter Pylori-Induced Gastric Pathology.","date":"2019","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/30884828","citation_count":13,"is_preprint":false},{"pmid":"31122752","id":"PMC_31122752","title":"RNF43 frameshift mutations contribute to tumourigenesis in right-sided colon cancer.","date":"2019","source":"Pathology, research and practice","url":"https://pubmed.ncbi.nlm.nih.gov/31122752","citation_count":13,"is_preprint":false},{"pmid":"36468684","id":"PMC_36468684","title":"RNF43 induces the turnover of protease-activated receptor 2 in colon cancer.","date":"2023","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/36468684","citation_count":12,"is_preprint":false},{"pmid":"35229994","id":"PMC_35229994","title":"Deficient Rnf43 potentiates hyperactive Kras-mediated pancreatic preneoplasia initiation and malignant transformation.","date":"2022","source":"Animal models and experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35229994","citation_count":10,"is_preprint":false},{"pmid":"34733364","id":"PMC_34733364","title":"RNF43 overexpression attenuates the Wnt/β-catenin signalling pathway to suppress tumour progression in cholangiocarcinoma.","date":"2021","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/34733364","citation_count":10,"is_preprint":false},{"pmid":"28789449","id":"PMC_28789449","title":"RT-qPCR analysis of the tumor antigens TOMM34 and RNF43 in samples extracted from paraffin-embedded specimens of colorectal cancer.","date":"2017","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/28789449","citation_count":9,"is_preprint":false},{"pmid":"34541672","id":"PMC_34541672","title":"RNF43 pathogenic Germline variant in a family with colorectal cancer.","date":"2021","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34541672","citation_count":9,"is_preprint":false},{"pmid":"38988031","id":"PMC_38988031","title":"m6A methylation of RNF43 inhibits the progression of endometriosis through regulating oxidative phosphorylation via NDUFS1.","date":"2024","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/38988031","citation_count":8,"is_preprint":false},{"pmid":"26823834","id":"PMC_26823834","title":"Association of RNF43 with cell cycle proteins involved in p53 pathway.","date":"2015","source":"International journal of clinical and experimental pathology","url":"https://pubmed.ncbi.nlm.nih.gov/26823834","citation_count":8,"is_preprint":false},{"pmid":"35487932","id":"PMC_35487932","title":"RNF43 R117fs mutant positively regulates Wnt/β-catenin signaling by failing to internalize FZD expressed on the cell surface.","date":"2022","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/35487932","citation_count":8,"is_preprint":false},{"pmid":"38058823","id":"PMC_38058823","title":"RNF43 mutation as a predictor of immunotherapeutic efficacy in colorectal cancer.","date":"2023","source":"American journal of cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/38058823","citation_count":8,"is_preprint":false},{"pmid":"35066614","id":"PMC_35066614","title":"RNF43 as a predictor of malignant transformation of pancreatic mucinous cystic neoplasm.","date":"2022","source":"Virchows Archiv : an international journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/35066614","citation_count":8,"is_preprint":false},{"pmid":"38063999","id":"PMC_38063999","title":"Inherited BRCA1 and RNF43 pathogenic variants in a familial colorectal cancer type X family.","date":"2023","source":"Familial cancer","url":"https://pubmed.ncbi.nlm.nih.gov/38063999","citation_count":7,"is_preprint":false},{"pmid":"37797285","id":"PMC_37797285","title":"Predictive Impact of RNF43 Mutations in Patients With Proficient Mismatch Repair/Microsatellite Stable BRAFV600E-Mutated Metastatic Colorectal Cancer Treated With Target Therapy or Chemotherapy.","date":"2023","source":"JCO precision oncology","url":"https://pubmed.ncbi.nlm.nih.gov/37797285","citation_count":6,"is_preprint":false},{"pmid":"39551397","id":"PMC_39551397","title":"RNF43 and ZNRF3: Versatile regulators at the membrane and their role in cancer.","date":"2024","source":"Biochimica et biophysica acta. Reviews on cancer","url":"https://pubmed.ncbi.nlm.nih.gov/39551397","citation_count":5,"is_preprint":false},{"pmid":"38225722","id":"PMC_38225722","title":"RNF43 Inactivation Enhances the B-RAF/MEK Signaling and Creates a Combinatory Therapeutic Target in Cancer Cells.","date":"2024","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/38225722","citation_count":5,"is_preprint":false},{"pmid":"39125653","id":"PMC_39125653","title":"New Target(s) for RNF43 Regulation: Implications for Therapeutic Strategies.","date":"2024","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/39125653","citation_count":5,"is_preprint":false},{"pmid":"37023034","id":"PMC_37023034","title":"Issues with RNF43 antibodies to reliably detect intracellular location.","date":"2023","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/37023034","citation_count":5,"is_preprint":false},{"pmid":"38394466","id":"PMC_38394466","title":"Association of RNF43 Genetic Alterations With BRAFV600E and MSIhigh in Colorectal Cancer.","date":"2024","source":"JCO precision oncology","url":"https://pubmed.ncbi.nlm.nih.gov/38394466","citation_count":5,"is_preprint":false},{"pmid":"40000409","id":"PMC_40000409","title":"Structure-Guided Development of Chemically Tailored Peptide Binders of RNF43/ZNRF3 to Enable Versatile Design of Membrane Protein-Targeting PROTACs.","date":"2025","source":"Angewandte Chemie (International ed. in English)","url":"https://pubmed.ncbi.nlm.nih.gov/40000409","citation_count":5,"is_preprint":false},{"pmid":"39546056","id":"PMC_39546056","title":"Germline pathogenic variants in RNF43 in patients with and without serrated polyposis syndrome.","date":"2024","source":"Familial cancer","url":"https://pubmed.ncbi.nlm.nih.gov/39546056","citation_count":4,"is_preprint":false},{"pmid":"36889779","id":"PMC_36889779","title":"RNF43 Suppressed Triple-Negative Breast Cancer Progression by Inhibiting Wnt/beta-Catenin Pathway.","date":"2023","source":"Annals of clinical and laboratory science","url":"https://pubmed.ncbi.nlm.nih.gov/36889779","citation_count":4,"is_preprint":false},{"pmid":"38260423","id":"PMC_38260423","title":"Loss of ZNRF3/RNF43 Unleashes EGFR in Cancer.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38260423","citation_count":4,"is_preprint":false},{"pmid":"37409251","id":"PMC_37409251","title":"RNF43 is associated with genomic features and clinical outcome in BRAF mutant colorectal cancer.","date":"2023","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/37409251","citation_count":4,"is_preprint":false},{"pmid":"26240024","id":"PMC_26240024","title":"The ubiquitin ligase RNF43 downregulation increases membrane expression of frizzled receptor in pancreatic ductal adenocarcinoma.","date":"2015","source":"Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/26240024","citation_count":4,"is_preprint":false},{"pmid":"39146560","id":"PMC_39146560","title":"RNF43 in cancer: Molecular understanding and clinical significance in immunotherapy.","date":"2024","source":"The journal of gene medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39146560","citation_count":3,"is_preprint":false},{"pmid":"35907983","id":"PMC_35907983","title":"Exome sequencing revealed comparable frequencies of RNF43 and BRAF mutations in Middle Eastern colorectal cancer.","date":"2022","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/35907983","citation_count":3,"is_preprint":false},{"pmid":"39007965","id":"PMC_39007965","title":"PD-L1 expression downregulation by RNF43 in gastric carcinoma enhances antitumour activity of T cells.","date":"2023","source":"Scandinavian journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/39007965","citation_count":2,"is_preprint":false},{"pmid":"41130209","id":"PMC_41130209","title":"STT3A is essential for Wnt signaling and represents a target for cancers driven by RNF43 deficiency.","date":"2025","source":"Cell chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/41130209","citation_count":2,"is_preprint":false},{"pmid":"38622664","id":"PMC_38622664","title":"Selective epigenetic alterations in RNF43 in pancreatic exocrine cells from high-fat-diet-induced obese mice; implications for pancreatic cancer.","date":"2024","source":"BMC research notes","url":"https://pubmed.ncbi.nlm.nih.gov/38622664","citation_count":2,"is_preprint":false},{"pmid":"38026183","id":"PMC_38026183","title":"The fusion gene hsf5-rnf43 in Nile tilapia: A potential regulator in the maintenance of testis function and sexual differentiation.","date":"2023","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/38026183","citation_count":1,"is_preprint":false},{"pmid":"41960900","id":"PMC_41960900","title":"Loss of ZNRF3/RNF43 unleashes EGFR in cancer.","date":"2026","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/41960900","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":50914,"output_tokens":7053,"usd":0.129269},"stage2":{"model":"claude-opus-4-6","input_tokens":10733,"output_tokens":3521,"usd":0.212535},"total_usd":0.341804,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"RNF43 is a transmembrane E3 ubiquitin ligase that selectively ubiquitinates Frizzled receptors at the cell surface, targeting them for lysosomal degradation, thereby reducing Wnt signaling. Deletion of RNF43 (with ZNRF3) in mouse intestinal epithelium induces adenoma formation dependent on paracrine Wnt.\",\n      \"method\": \"In vitro ubiquitination assays, HEK293T overexpression, RNF43 reconstitution in HCT116 cells, conditional mouse knockout, organoid culture\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — foundational study with multiple orthogonal methods (cell-based assays, mouse genetics, organoids), 784 citations, independently replicated\",\n      \"pmids\": [\"22895187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structures of the RNF43 ectodomain in complex with the Fu1-Fu2 fragment of R-spondin 2 (Rspo2) revealed that a prominent loop in Rspo2 Fu1 clamps into a groove in the RNF43 ectodomain surface, establishing the structural basis of R-spondin-mediated inhibition of RNF43 E3 ligase activity.\",\n      \"method\": \"X-ray crystallography of ZNRF3 ectodomain, RNF43 ectodomain, and Rspo2(Fu1-Fu2) complexes; biophysical binding assays; cellular Wnt reporter assays\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple crystal structures with functional validation in cellular assays\",\n      \"pmids\": [\"24225776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structure of RSPO1 bound simultaneously to LGR5 and RNF43 ectodomains showed that RSPO1 is sandwiched between LGR5 and RNF43, with its CRD rod module contacting LGR5 and a hairpin inserted into RNF43. LGR5 does not contact RNF43 but increases RSPO1 affinity for RNF43, establishing LGR5 as an engagement receptor and RNF43 as an effector receptor.\",\n      \"method\": \"X-ray crystallography of ternary RSPO1-LGR5-RNF43 ectodomain complex; binding affinity measurements\",\n      \"journal\": \"Genes & Development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure of ternary complex with functional validation\",\n      \"pmids\": [\"23756651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RNF43 inhibits Wnt/β-catenin signaling by reducing membrane-level Frizzled in pancreatic cancer cells. Inactivating RNF43 mutations increase cell-surface Frizzled and confer dependency on Wnt ligand secretion (Porcupine-dependent) for proliferation.\",\n      \"method\": \"RNF43 reconstitution in RNF43-mutant pancreatic cancer cell lines, cell-surface Frizzled quantification, Wnt inhibitor (LGK974) sensitivity assays, siRNA knockdown, xenograft models\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple cell lines, reconstitution, in vivo xenograft, replicated concept from PMID 22895187\",\n      \"pmids\": [\"23847203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Dishevelled (DVL) acts as a dual-function adaptor required for ZNRF3/RNF43-mediated ubiquitination and degradation of Frizzled (FZD) and LRP6. Physical interaction between ZNRF3/RNF43 and DVL is essential for their Wnt inhibitory activity; the DEP domain of DVL binds FZD, and fusion of DEP to ZNRF3/RNF43 overcomes their DVL dependency.\",\n      \"method\": \"DVL knockout cells, Co-IP, cell surface FZD/LRP6 quantification, ubiquitination assays, domain-swap experiments (DEP-ZNRF3 fusion)\",\n      \"journal\": \"Molecular Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, KO cells, mechanistic domain fusion experiments with multiple orthogonal readouts\",\n      \"pmids\": [\"25891077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RNF43 suppresses canonical Wnt/β-catenin signaling through its extracellular PA domain interaction with the Frizzled CRD, requiring the intracellular RING finger domain for ubiquitination. RNF43 also suppresses noncanonical Wnt signaling through its C-terminal cytoplasmic region binding the PDZ domain of Dishevelled, independently of the PA and RING domains. Missense mutations in the extracellular portion of RNF43 redirect its localization from endosome to ER, abrogating canonical but not noncanonical Wnt suppression.\",\n      \"method\": \"Domain deletion/mutation analysis, co-IP, localization studies (immunofluorescence), Wnt reporter assays in HEK293T and cancer cells\",\n      \"journal\": \"Molecular and Cellular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal domain dissection experiments with functional readouts, mechanism for two distinct pathways defined\",\n      \"pmids\": [\"25825523\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RNF43 was detected in the nucleus of human intestinal crypt and colon cancer cells where it physically interacts with TCF4 and tethers it to the nuclear membrane, silencing TCF4 transcriptional activity even in the presence of constitutively active β-catenin. Tumor-associated RING domain mutations disrupt this inhibitory mechanism.\",\n      \"method\": \"Co-IP of endogenous RNF43 and TCF4, nuclear fractionation, immunofluorescence, Wnt reporter assays, Xenopus embryo overexpression, mutant RNF43 analysis\",\n      \"journal\": \"Science Signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — nuclear localization later disputed by antibody artifact concerns (PMID 37023034); mechanistic claim of TCF4 sequestration supported by Co-IP and reporter assays in single study\",\n      \"pmids\": [\"26350900\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Paracrine Wnt secretion from Paneth cells (Wnt3) is the essential driver of Rnf43/Znrf3-null intestinal tumor growth; removal of Paneth cells by Math1 mutation or deletion of Wnt3 inhibits tumorigenesis. Treatment with porcupine inhibitor C59 strongly inhibited RZ-null neoplasia growth while sparing normal crypts.\",\n      \"method\": \"Genetic epistasis (Math1 KO, Wnt3 KO crossed to Rnf43/Znrf3 KO mice), pharmacological porcupine inhibitor (C59) treatment in vivo\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic epistasis with multiple double-mutant combinations plus pharmacological validation in vivo\",\n      \"pmids\": [\"26023187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"RNF43 localizes to the endoplasmic reticulum and nuclear envelope, exhibits autoubiquitylation activity in a cell-free system with purified recombinant protein, and interacts with HAP95 (a chromatin-associated nuclear envelope protein) as identified by yeast two-hybrid and confirmed by Co-IP.\",\n      \"method\": \"Indirect immunofluorescence, biochemical fractionation, sucrose density gradient, cell-free autoubiquitylation assay with recombinant MBP-RNF43, yeast two-hybrid, Co-IP\",\n      \"journal\": \"Experimental Cell Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-3 — in vitro autoubiquitylation is Tier 1; HAP95 interaction by Co-IP is Tier 3; single study\",\n      \"pmids\": [\"18313049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RNF43 interacts with NEDL1 (a p53-enhancing E3 ligase) and with p53 itself, and suppresses p53-mediated transcriptional activity and UV-induced apoptosis in H1299 cells.\",\n      \"method\": \"Yeast two-hybrid screening, Co-IP, transcriptional reporter assays, UV apoptosis assay\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — yeast two-hybrid and Co-IP with functional reporter readout; single study, single lab\",\n      \"pmids\": [\"21108931\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RNF43 expression is directly regulated as a Wnt target gene through two Wnt-responsive elements (WREs) in intron 2 that bind the TCF4/β-catenin complex, as demonstrated by reporter assays with WRE mutations and ChIP showing TCF4/β-catenin occupancy at both elements.\",\n      \"method\": \"Luciferase reporter assay with WRE mutants, siRNA knockdown of β-catenin, ChIP assay\",\n      \"journal\": \"PloS One\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reporter mutagenesis plus ChIP; single lab but two orthogonal methods\",\n      \"pmids\": [\"24466159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RNF43 E3 ligase activity toward Frizzled requires phosphorylation at a conserved triplet of serine residues. Cancer-associated mutations abrogating this phosphorylation abolish Frizzled degradation and Wnt inhibition while retaining p53 inhibitory function. Phosphomimetic substitutions (Ser→Asp) restored tumor suppressive activity of extracellular oncogenic mutants.\",\n      \"method\": \"Phosphomutant and phosphomimetic RNF43 constructs, zebrafish development assay, mouse intestinal organoids, co-expression with active Ras for tumorigenesis, Wnt reporter assays\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — phospho-regulation demonstrated with mutagenesis plus in vivo zebrafish and organoid models; multiple orthogonal readouts\",\n      \"pmids\": [\"32934222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A subset of RNF43 C-terminal truncating cancer mutations retains Wnt receptor downregulation but traps Casein kinase 1 (CK1) at the plasma membrane via the cytosolic tail, preventing cytoplasmic β-catenin turnover and driving ligand-independent β-catenin-mediated transcription. These mutations confer decreased sensitivity to anti-Wnt therapy.\",\n      \"method\": \"Gene editing of human colon stem cells, Co-IP of CK1 with truncated RNF43 variants, β-catenin phosphorylation assays, Wnt reporter assays, organoid growth in absence of niche factors\",\n      \"journal\": \"The EMBO Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — endogenous gene editing, CK1 Co-IP, multiple patient-derived mutation constructs, organoid functional assay; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"32965059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The deubiquitinase USP42 antagonizes R-spondin-induced clearance of RNF43/ZNRF3 by binding the Dishevelled-interacting region (DIR) of ZNRF3 and deubiquitinating ZNRF3, thereby stalling the R-spondin-LGR4-ZNRF3 ternary complex at the membrane and maintaining Frizzled/LRP6 turnover.\",\n      \"method\": \"Co-IP of USP42 with ZNRF3, deubiquitination assays, LRP6/FZD cell surface quantification, Wnt reporter assays, siRNA knockdown in colon cancer cells and mouse intestinal organoids\",\n      \"journal\": \"EMBO Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, enzymatic deubiquitination assay, functional organoid model, multiple orthogonal methods\",\n      \"pmids\": [\"33786993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF43 inhibits noncanonical WNT5A signaling in human cells by interacting with ROR1, ROR2, VANGL1, and VANGL2 receptor complex components. RNF43 triggers ubiquitination and proteasomal degradation of VANGL2 and clathrin-dependent internalization of ROR1, and inhibits ROR2 activation, suppressing WNT5A-driven melanoma invasion and resistance to BRAF/MEK inhibitors.\",\n      \"method\": \"BioID proximity labeling, Co-IP, ubiquitination assays, VANGL2 degradation assays, ROR1 internalization (clathrin inhibitor), in vitro invasion assays, in vivo xenograft experiments\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — BioID interactome plus Co-IP plus functional ubiquitination and degradation assays plus in vivo validation\",\n      \"pmids\": [\"34702444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Rnf43 (but not Znrf3) is potently activated by Wnt signaling in oligodendrocyte progenitor cells (OPCs), marks activated OPCs in human MS and HIE, and promotes OPC differentiation/remyelination specifically after injury by negatively regulating Fzd1 receptor presentation at the cell surface.\",\n      \"method\": \"Conditional Rnf43 KO in OPCs, neonatal hypoxic injury model, adult demyelination model, ex vivo and in vivo remyelination assays, Fzd1 inhibition (UM206)\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with injury-specific phenotype plus pharmacological rescue; multiple in vivo models\",\n      \"pmids\": [\"34390652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF43 and ZNRF3 preferentially downregulate distinct subsets of Frizzled receptors: RNF43 preferentially targets FZD1/FZD5/FZD7 for endocytosis whereas ZNRF3 preferentially targets FZD6. The transmembrane domain (TMD) of RNF43 is a key molecular determinant for FZD5 endocytosis; swapping TMDs between RNF43 and ZNRF3 redirects their FZD preferences.\",\n      \"method\": \"FZD endocytosis assays (flow cytometry and imaging), TMD swap domain mutants, cell surface receptor quantification\",\n      \"journal\": \"Life Science Alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain-swap mutagenesis with quantitative endocytosis readouts; mechanistic TMD requirement established\",\n      \"pmids\": [\"38969364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF43 ubiquitinates B-RAF at lysine K499 to promote proteasome-dependent degradation of B-RAF, reducing MEK activity. Phosphorylation of B-RAF at T491 suppresses this ubiquitination by decreasing RNF43–B-RAF interaction. Loss of RNF43 elevates B-RAF/MEK signaling, and MEK plus Wnt inhibitors synergistically suppress RNF43-mutant pancreatic cancer growth.\",\n      \"method\": \"Co-IP, ubiquitination assay with specific lysine mutants (K499R), phosphorylation site mutagenesis (T491), proteasome inhibitor assays, MEK inhibitor sensitivity in cell lines and in vivo\",\n      \"journal\": \"Advanced Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct ubiquitination site identified by mutagenesis with Co-IP, phospho-regulation demonstrated, in vivo validation\",\n      \"pmids\": [\"38225722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZNRF3/RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and degradation dependent on the RING domain E3 ligase activity. ZNRF3/RNF43 knockout elevates EGFR signaling and promotes tumorigenesis.\",\n      \"method\": \"Co-IP of ZNRF3/RNF43 with EGFR, ubiquitination assays, ZNRF3/RNF43 overexpression and KO in cancer cells, in vivo tumor growth assays, proteogenomic correlation analysis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — biochemical Co-IP, ubiquitination assay, KO and OE with functional readouts in vitro and in vivo; peer-reviewed publication\",\n      \"pmids\": [\"41960900\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF43 G659fs mutant binds the p85 regulatory subunit of PI3K, leading to p85 ubiquitination and degradation, which increases PI3K/mTOR signaling. RNF43 G659fs cells are selectively killed by PI3K/mTOR inhibitors in vitro, in isogenic xenografts, and in patient-derived organoids.\",\n      \"method\": \"Co-IP of RNF43-G659fs with p85, ubiquitination assay, drug library screen, isogenic cell line and organoid drug sensitivity assays, xenograft experiments\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct substrate identification by Co-IP plus ubiquitination assay, functional validation in multiple models including PDO and xenograft\",\n      \"pmids\": [\"35676246\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RNF43 ubiquitinates c-Src-phosphorylated E-cadherin (phospho-Tyr797) at Lys816, promoting its degradation and enabling nuclear β-catenin translocation, thereby facilitating EMT in lung adenocarcinoma. This ubiquitination requires Frizzled 8 (FZD8).\",\n      \"method\": \"Protein antibody microarray, E3 ligase profiling, Co-IP, ubiquitination site mutagenesis (Y797, K816), immunofluorescence, xenograft models, shRNA knockdown\",\n      \"journal\": \"BMC Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — specific ubiquitination site identified by mutagenesis; single lab but multiple orthogonal methods; somewhat orthogonal to dominant Wnt-focused RNF43 literature\",\n      \"pmids\": [\"31286874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RNF43 co-associates with PAR2 (protease-activated receptor 2), promotes its membrane elimination and polyubiquitination, and PAR2 degradation is rescued by R-spondin2 in the presence of LGR5, establishing RNF43 as a negative feedback regulator of PAR2 in colon cancer.\",\n      \"method\": \"Co-IP, cell surface biotinylation assay for PAR2, polyubiquitination assay, R-spondin/LGR5 rescue experiment, β-catenin reporter assay, AOM/DSS mouse model\",\n      \"journal\": \"FASEB Journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and surface biotinylation are standard; single lab; PAR2 as substrate not replicated elsewhere\",\n      \"pmids\": [\"36468684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RNF43 interacts with PD-L1 and augments both K48- and K63-linked ubiquitination of PD-L1 in gastric cancer cell lines, reducing PD-L1 surface expression and enhancing T-cell anti-tumor activity.\",\n      \"method\": \"Co-IP of RNF43 and PD-L1, ubiquitination linkage-specific assays (K48, K63), T-cell killing assays\",\n      \"journal\": \"Scandinavian Journal of Immunology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and ubiquitination assay; single study, single lab, not replicated\",\n      \"pmids\": [\"39007965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF43 reinforces ubiquitination and proteasomal degradation of NDUFS1 (NADH dehydrogenase Fe-S protein 1) by direct interaction with it, suppressing oxidative phosphorylation and inhibiting endometrial stromal cell viability and migration. RNF43 mRNA stability and expression are regulated by METTL3/IGF2BP2-mediated m6A modification.\",\n      \"method\": \"Co-IP of RNF43 and NDUFS1, ubiquitination assay, m6A methylation assays (METTL3 overexpression/inhibition, IGF2BP2 RIP), cell viability and migration assays, knockdown/overexpression\",\n      \"journal\": \"Journal of Cellular Physiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and ubiquitination of NDUFS1 by single lab; m6A regulation of RNF43 mRNA is additional claim; not replicated\",\n      \"pmids\": [\"38988031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"RNF43 interacts with the heterodimer PSF/p54nrb as identified by pull-down and confirmed by Co-IP; co-expression of PSF relocates RNF43 from the nuclear periphery to the nucleoplasm.\",\n      \"method\": \"Pull-down assay with MS identification, Co-IP, immunofluorescence\",\n      \"journal\": \"Proteomics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP confirmation; relocalization noted but functional consequence not established; antibody reliability concerns noted later\",\n      \"pmids\": [\"18655028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The RNF43 protease-associated (PA) extracellular domain is dispensable for inhibition of canonical Wnt/β-catenin signaling in human cells; RNF43 lacking the PA domain still reduces LRP6/DVL phosphorylation and β-catenin-dependent transcription, and is insensitive to R-spondin1.\",\n      \"method\": \"TetON-controlled RNF43ΔPA overexpression, CRISPR/Cas9 RNF43/ZNRF3 double KO rescue experiments, Western blot (pLRP6, pDVL, β-catenin), TOPflash luciferase assay\",\n      \"journal\": \"Cell Communication and Signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — controlled expression system plus KO rescue; multiple readouts; single lab\",\n      \"pmids\": [\"32527265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LGR4, but not LGR5, forms a complex with RNF43/ZNRF3 that provides high-affinity bivalent binding of R-spondin ligands. LGR5 forms homodimers that do not interact with the E3 ligases; co-expression of ZNRF3 with LGR4 dramatically increases monovalent RSPO affinity, whereas co-expression with LGR5 has no effect.\",\n      \"method\": \"Binding affinity measurements in whole cells, co-expression of LGR4/LGR5 with ZNRF3, bivalent vs monovalent RSPO2 furin domain binding assays\",\n      \"journal\": \"Scientific Reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — quantitative binding assays with multiple receptor combinations; single lab\",\n      \"pmids\": [\"37402772\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RNF43 is a single-pass transmembrane RING-type E3 ubiquitin ligase that acts as a Wnt negative-feedback regulator by ubiquitinating Frizzled receptors (preferentially FZD1/5/7) for lysosomal degradation, a process requiring DVL as a substrate-adaptor and phosphorylation of RNF43 at a conserved serine triplet; R-spondin proteins antagonize RNF43 by recruiting it into an LGR4-RSPO-RNF43 ternary complex (structurally defined by crystal structures) that triggers RNF43 auto-ubiquitination and membrane clearance, counteracted by the deubiquitinase USP42; beyond Frizzled, RNF43 also ubiquitinates B-RAF (K499), EGFR, VANGL2, and PAR2, inhibits noncanonical WNT5A signaling through interactions with ROR1/ROR2/VANGL1/VANGL2, and a class of C-terminal truncating cancer mutations traps CK1 at the plasma membrane to drive ligand-independent β-catenin activation independently of Frizzled regulation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RNF43 is a transmembrane RING-type E3 ubiquitin ligase that functions as a central negative-feedback regulator of Wnt signaling by ubiquitinating Frizzled receptors—preferentially FZD1, FZD5, and FZD7—for lysosomal degradation, a process requiring Dishevelled (DVL) as a substrate adaptor and phosphorylation at a conserved serine triplet [PMID:22895187, PMID:25891077, PMID:32934222, PMID:38969364]. R-spondin ligands antagonize RNF43 by forming a ternary complex with LGR4 and the RNF43 ectodomain, neutralizing its Frizzled-clearing activity and potentiating Wnt signaling; this interaction is counteracted by the deubiquitinase USP42 [PMID:23756651, PMID:24225776, PMID:33786993, PMID:37402772]. Beyond Frizzled, RNF43 ubiquitinates additional substrates including B-RAF at K499, EGFR, and VANGL2, and suppresses noncanonical WNT5A signaling through interactions with ROR1/ROR2/VANGL1/VANGL2 [PMID:34702444, PMID:38225722, PMID:41960900]. Cancer-associated C-terminal truncating mutations can retain Frizzled regulation yet trap CK1 at the plasma membrane, driving ligand-independent β-catenin activation and resistance to anti-Wnt therapies [PMID:32965059].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Initial biochemical characterization established that RNF43 possesses intrinsic E3 ubiquitin ligase (autoubiquitylation) activity and localizes to the ER/nuclear envelope, but its physiological substrates were unknown.\",\n      \"evidence\": \"Cell-free autoubiquitylation assay with recombinant MBP-RNF43, immunofluorescence, and yeast two-hybrid identification of HAP95 interaction\",\n      \"pmids\": [\"18313049\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"HAP95 interaction confirmed by single Co-IP without reciprocal validation\", \"Physiological substrates not identified\", \"ER/nuclear envelope localization later challenged by antibody concerns\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"The breakthrough discovery that RNF43 ubiquitinates Frizzled receptors for lysosomal degradation established its role as a Wnt negative-feedback regulator, and double knockout with ZNRF3 in mouse intestine caused Wnt-dependent adenomas.\",\n      \"evidence\": \"In vitro ubiquitination assays, HEK293T overexpression, conditional mouse knockout, organoid culture\",\n      \"pmids\": [\"22895187\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of substrate recognition (how RNF43 engages Frizzled) not yet defined\", \"Relative contribution of RNF43 vs ZNRF3 unclear\", \"Adaptor requirements unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Structural and functional studies defined the R-spondin/LGR/RNF43 ternary complex architecture, revealing how R-spondins bridge LGR receptors and RNF43 to neutralize Frizzled degradation, and showed that cancer-associated RNF43 mutations create Wnt ligand dependency.\",\n      \"evidence\": \"Crystal structures of RNF43 ectodomain with RSPO2 and ternary RSPO1–LGR5–RNF43 complex; binding assays; RNF43 reconstitution in pancreatic cancer cells with porcupine inhibitor sensitivity\",\n      \"pmids\": [\"24225776\", \"23756651\", \"23847203\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether all R-spondin family members use identical binding mode unresolved\", \"Mechanism of R-spondin-induced RNF43 membrane clearance not determined\", \"Intracellular signaling consequences of ternary complex formation undefined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"RNF43 was shown to be a direct Wnt/β-catenin target gene regulated by TCF4/β-catenin occupancy at intronic Wnt-responsive elements, completing the negative-feedback loop architecture.\",\n      \"evidence\": \"Luciferase reporter assay with WRE mutants, ChIP for TCF4/β-catenin at intronic elements\",\n      \"pmids\": [\"24466159\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Regulation in non-intestinal tissues not addressed\", \"Kinetics of feedback induction not measured\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"DVL was identified as an essential adaptor bridging RNF43 to Frizzled substrates, with domain dissection revealing that the PA domain mediates Frizzled CRD interaction for canonical signaling while the C-terminal cytoplasmic region suppresses noncanonical Wnt signaling independently of RING-dependent ubiquitination.\",\n      \"evidence\": \"DVL knockout cells, Co-IP, DEP-ZNRF3 fusion rescue, domain deletion/mutation analysis, Wnt reporter assays\",\n      \"pmids\": [\"25891077\", \"25825523\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the kinase phosphorylating RNF43 to activate it not yet known\", \"Structural basis of DVL–RNF43 interaction unresolved\", \"Mechanism of RING-independent noncanonical suppression unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Genetic epistasis demonstrated that paracrine Wnt3 from Paneth cells is the essential proliferative signal in RNF43/ZNRF3-null intestinal tumors, validating porcupine inhibition as a therapeutic strategy.\",\n      \"evidence\": \"Genetic crosses of Rnf43/Znrf3 KO with Math1 KO and Wnt3 KO mice; porcupine inhibitor C59 treatment in vivo\",\n      \"pmids\": [\"26023187\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Applicability to human RNF43-mutant cancers not directly demonstrated\", \"Whether all RNF43 mutation classes are equally porcupine-sensitive unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"RNF43 substrate scope was expanded beyond Frizzled to include PAR2 and phospho-E-cadherin, suggesting broader membrane protein quality control functions.\",\n      \"evidence\": \"Co-IP and ubiquitination assays for PAR2 (with R-spondin/LGR5 rescue) and E-cadherin (K816 site mutagenesis), xenograft models\",\n      \"pmids\": [\"36468684\", \"31286874\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"PAR2 as substrate not independently replicated\", \"E-cadherin ubiquitination requirement for FZD8 is unusual and unexplained mechanistically\", \"Relative importance of non-Frizzled substrates in physiology unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"The critical regulatory mechanism of RNF43 phosphorylation at a conserved serine triplet was defined: phosphorylation is required for Frizzled ubiquitination, and cancer mutations that abolish it selectively lose tumor-suppressive but not p53-inhibitory function. Separately, C-terminal truncating mutations were shown to trap CK1 at the plasma membrane, driving ligand-independent β-catenin activation.\",\n      \"evidence\": \"Phosphomutant/phosphomimetic constructs in zebrafish and organoids; gene-edited human colon stem cells with CK1 Co-IP and β-catenin phosphorylation assays\",\n      \"pmids\": [\"32934222\", \"32965059\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the kinase(s) phosphorylating the serine triplet unknown\", \"CK1 trapping mechanism (direct binding vs indirect) not structurally resolved\", \"Whether phosphorylation and CK1-trapping mutations co-occur in tumors not examined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"USP42 was identified as the deubiquitinase counteracting R-spondin-induced RNF43/ZNRF3 membrane clearance, and RNF43 was shown to suppress noncanonical WNT5A signaling by ubiquitinating VANGL2 and internalizing ROR1, establishing dual canonical/noncanonical tumor-suppressive functions.\",\n      \"evidence\": \"Co-IP and deubiquitination assays for USP42–ZNRF3; BioID proximity labeling plus Co-IP and degradation assays for ROR1/ROR2/VANGL interactions; invasion and xenograft models\",\n      \"pmids\": [\"33786993\", \"34702444\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether USP42 acts on RNF43 directly (in addition to ZNRF3) not established\", \"Structural basis of RNF43–ROR/VANGL interaction unknown\", \"In vivo relevance of noncanonical suppression in normal tissue homeostasis not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A non-redundant role for RNF43 (distinct from ZNRF3) in oligodendrocyte progenitor differentiation was demonstrated: RNF43 is Wnt-induced specifically after injury and promotes remyelination by clearing Fzd1.\",\n      \"evidence\": \"Conditional Rnf43 KO in OPCs, neonatal hypoxic injury model, adult demyelination model, Fzd1 pharmacological inhibition rescue\",\n      \"pmids\": [\"34390652\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other Frizzled subtypes contribute to OPC phenotype not resolved\", \"Human translational relevance limited to expression data\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"RNF43 and ZNRF3 were shown to have distinct Frizzled substrate preferences dictated by the transmembrane domain, and RNF43 was found to ubiquitinate B-RAF at K499 and EGFR, broadening its tumor-suppressive reach beyond Wnt receptors.\",\n      \"evidence\": \"TMD swap domain mutants with quantitative FZD endocytosis assays; K499R B-RAF mutagenesis with Co-IP; EGFR Co-IP and ubiquitination assays in KO/OE systems\",\n      \"pmids\": [\"38969364\", \"38225722\", \"41960900\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of TMD-mediated Frizzled selectivity unknown\", \"Physiological contexts where B-RAF and EGFR ubiquitination by RNF43 are rate-limiting undefined\", \"Whether all non-Frizzled substrates require DVL adaptor function untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include the identity of the kinase(s) phosphorylating the RNF43 serine triplet, the structural basis of TMD-determined Frizzled selectivity, whether non-Frizzled substrates (B-RAF, EGFR, VANGL2) use the same DVL-dependent adaptor mechanism, and the in vivo tissue-specific hierarchy among RNF43's multiple substrates.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Kinase for serine triplet phosphorylation unidentified\", \"No structural model of full-length RNF43 in complex with any Frizzled\", \"Relative in vivo importance of Wnt-dependent vs Wnt-independent substrates unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 4, 5, 11, 14, 17, 18, 19]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 8, 11, 17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 3, 12, 16]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [5, 8]},\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3, 4, 5, 7, 11, 12, 14, 15, 16]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 4, 8, 11, 14, 17, 18]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 7, 12, 17, 19]}\n    ],\n    \"complexes\": [\n      \"RSPO-LGR4-RNF43 ternary complex\"\n    ],\n    \"partners\": [\n      \"FZD5\",\n      \"DVL2\",\n      \"LGR4\",\n      \"BRAF\",\n      \"EGFR\",\n      \"VANGL2\",\n      \"ROR1\",\n      \"CSNK1A1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}