{"gene":"RNF146","run_date":"2026-06-10T06:43:37","timeline":{"discoveries":[{"year":2011,"finding":"RNF146 is a RING-domain E3 ubiquitin ligase that directly interacts with poly(ADP-ribose) through its WWE domain, promoting ubiquitylation and proteasomal degradation of PARsylated proteins including Axin, BLZF1, and CASC3. RNF146 acts downstream of tankyrase-dependent PARsylation to couple PARsylation to ubiquitylation, thereby positively regulating Wnt/β-catenin signaling.","method":"RNAi screening, co-immunoprecipitation, proteomics, ubiquitylation assays, cell-based Wnt reporter assays","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (RNAi, Co-IP, mass spectrometry, in-cell ubiquitylation assays) replicated across at least two concurrent independent studies (PMIDs 21478859 and 21799911)","pmids":["21478859"],"is_preprint":false},{"year":2011,"finding":"RNF146, tankyrase, and Axin form a protein complex; RNF146 mediates ubiquitylation of Axin, TNKS1, and TNKS2 targeting all three for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase aggregation at a centrosomal location. Tankyrase auto-PARsylation and RNF146-mediated ubiquitylation are mechanistically coupled in the same complex.","method":"RNAi screening, co-immunoprecipitation, ubiquitylation assays, immunofluorescence/localization studies, proteasome inhibitor experiments","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ubiquitylation assays, and localization experiments in a single study, consistent with independent replication in PMID 21478859","pmids":["21799911"],"is_preprint":false},{"year":2014,"finding":"Iso-ADP-ribose (the smallest internal PAR structural unit) binds between the WWE and RING domains of RNF146, acting as an allosteric signal that converts the RING domain from a catalytically inactive to an active state capable of binding E2 ubiquitin-conjugating enzymes. Crystal structure and biochemical analysis show PAR/iso-ADPr binding induces a major conformational change in RNF146. Additionally, RNF146 directly binds tankyrase via its C-terminal region; disruption of this interaction inhibits Axin turnover in cells.","method":"X-ray crystallography, biochemical binding assays, mutagenesis, E2-binding assays, cell-based Axin stability assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with in vitro reconstitution, mutagenesis, and cell-based validation in a single rigorous study","pmids":["25327252"],"is_preprint":false},{"year":2018,"finding":"RNF146 binds directly to tankyrase (TNKS1/2) via multiple noncanonical tankyrase-binding motifs (TBMs) in its C-terminal region. These extended TBMs (with one or two extra residues between conserved Arg and Gly) mediate weak individual but strong multivalent binding. A crystal structure of the first RNF146 noncanonical TBM with the second ankyrin repeat domain of TNKS was solved.","method":"X-ray crystallography, binding/affinity assays, mutagenesis of TBMs","journal":"Protein science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus biochemical binding validation in a single focused study","pmids":["29604130"],"is_preprint":false},{"year":2016,"finding":"RNF146 and tankyrase2 (TNKS2) maintain the junctional Crumbs complex by promoting degradation of angiomotin (AMOT) family proteins. AMOT proteins are PARylated by TNKS2, which drives their ubiquitylation by RNF146 and subsequent proteasomal degradation. Ablation of RNF146 or tankyrase caused PALS1 (a Crumbs complex component) to relocate from the apical membrane to internal puncta, a phenotype rescued by AMOTL2 knockdown.","method":"RNAi knockdown, co-immunoprecipitation, ubiquitylation assays, immunofluorescence, genetic epistasis (rescue experiments)","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, ubiquitylation assays, localization with functional consequence, and epistatic rescue in a single study","pmids":["27521426"],"is_preprint":false},{"year":2017,"finding":"RANKL represses RNF146 transcription via an NF-κB-related inhibitory element in the RNF146 promoter. RANKL-mediated suppression of RNF146 stabilizes its substrates 3BP2 and AXIN1, triggering SRC activation and attenuation of β-catenin expression, respectively, to coordinate the osteoclast developmental program. RNF146 depletion caused hypersensitivity to LPS-induced TNF-α production in vivo.","method":"Promoter analysis, RNAi, genetic knockout, in vivo LPS challenge, co-immunoprecipitation, substrate stability assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Moderate — promoter analysis combined with KO mice, in vivo experiments, and substrate stabilization assays in a single study","pmids":["28287403"],"is_preprint":false},{"year":2017,"finding":"Loss of RNF146 in osteoblasts stabilizes its substrate AXIN1, impairing WNT3a-induced β-catenin activation and reducing Fgf18 expression. FGF18 was shown to induce TAZ expression required for osteoblast proliferation and differentiation. RNF146 knockout also enhanced adipogenesis in MEFs and caused glucose intolerance with severe osteopenia.","method":"Genetic knockout (Rnf146-/- mice), cell-based Wnt/β-catenin reporter assays, substrate stability assays, osteoblast differentiation assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic KO in mice with multiple cellular and molecular readouts (substrate stabilization, signaling assays, differentiation) in a single study","pmids":["28581440"],"is_preprint":false},{"year":2022,"finding":"TNKS1 and TNKS2 PARylate the mitochondrial adaptor VISA/MAVS at Glu137, priming it for K48-linked polyubiquitination by RNF146 and subsequent proteasomal degradation. This mechanism attenuates innate antiviral signaling. RNF146 deficiency increased RNA virus-triggered interferon induction and impaired viral replication.","method":"Biochemical purification (co-immunoprecipitation), site-directed mutagenesis (Glu137), ubiquitylation assays, RNF146/TNKS knockout cells, in vivo viral infection model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, mutagenesis of modification site, ubiquitylation assays, and in vivo validation in a single study","pmids":["35733260"],"is_preprint":false},{"year":2020,"finding":"Proteome-wide analysis using RNF146 KO and TNKS1/2-double KO cells identified 160 potential RNF146 substrates including 122 potential TNKS-independent substrates. OTUD5 and PARP10 were validated as TNKS1/2-independent substrates of RNF146, and SARDH was validated as a novel substrate of both TNKS1/2 and RNF146.","method":"Proteome profiling (label-free quantification), transcriptome analysis, CRISPR knockout cell lines, Western blot validation","journal":"Molecular & cellular proteomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic proteomics with KO cells and validation of specific substrates by Western blot, single lab","pmids":["32958691"],"is_preprint":false},{"year":2023,"finding":"RNF146 is SUMOylated at K19, K61, K174, and K175 by UBC9/PIAS3/MMS21, with SENP1/2/6 acting as deSUMOylases. SUMOylation (specifically at K19/K175, mediated by UBC9/PIAS3 and removed by SENP1) promotes RNF146 nuclear localization, while deSUMOylation induces cytoplasmic localization. SUMOylation promotes RNF146 association with Axin to accelerate Axin ubiquitylation and degradation, enhancing β-catenin signaling.","method":"Lysine mutagenesis, co-immunoprecipitation, subcellular fractionation/immunofluorescence, ubiquitylation assays, in vitro and in vivo cancer models","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis of SUMO sites, Co-IP, localization assays, and functional readouts in a single lab study","pmids":["37029301"],"is_preprint":false},{"year":2014,"finding":"In cardiac myocytes, RNF146 acts as a direct interactor of PARP-1. Upon PARP-1 activation by oxidative stress, RNF146 translocates from cytoplasm to nucleus, triggering PARP-1 nuclear exit followed by rapid proteasomal degradation of both PARP-1 and RNF146. RNF146 overexpression protected against oxidant-induced cell death; RNF146 silencing augmented PARP-1-mediated injury.","method":"Co-immunoprecipitation, overexpression/knockdown, immunofluorescence/live imaging, cardiomyocyte injury models","journal":"Molecular medicine (Cambridge, Mass.)","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP, localization experiments, and functional phenotype readout, single lab, two orthogonal methods","pmids":["24842055"],"is_preprint":false},{"year":2020,"finding":"Akt1 suppresses parthanatos in dopaminergic neurons by stimulating CREB-dependent transcriptional activation of the RNF146 gene. RNF146 inhibits PARP1-induced cell death not through its E3 ligase activity but by binding to and sequestering PAR polymer, thereby preventing parthanatos.","method":"Overexpression of constitutively active Akt1, luciferase reporter assays, co-immunoprecipitation/PAR binding assays, RNF146 KO in vivo mouse models, SH-SY5Y cell toxicity assays","journal":"Science signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cell and in vivo models, RNF146 KO, PAR binding assays, single lab","pmids":["33443209"],"is_preprint":false},{"year":2024,"finding":"Upon induction of necroptosis and recruitment by the adaptor TAX1BP1, PARP5A (tankyrase) and RNF146 form liquid-like condensates through multivalent interactions. These condensates perform PARylation and PARylation-dependent ubiquitination (PARdU) of activated RIPK1, predominantly at K376, promoting proteasomal degradation of kinase-activated RIPK1 to restrain necroptosis in mouse embryonic fibroblasts.","method":"Phase separation assays (live imaging), mutagenesis (K376), ubiquitylation assays, co-immunoprecipitation, genetic KO (MEFs), cell death assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods including phase separation imaging, site mutagenesis, ubiquitylation assays, and KO in a single rigorous study","pmids":["38272024"],"is_preprint":false},{"year":2018,"finding":"In Drosophila, Rnf146 functions in the same proteolysis pathway as Tnks for Axin proteolysis in vivo (genetic epistasis), buffering Axin levels to ensure Wingless pathway activation in multiple developmental contexts. However, unlike Tnks, Rnf146 is dispensable for Wingless target gene activation and intestinal stem cell proliferation in the adult midgut during homeostasis, indicating context-dependent requirement for RNF146 in TNKS-mediated Axin degradation.","method":"Drosophila null mutant genetics, biochemical Axin stability assays, epistasis analysis, Wingless reporter assays","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with null mutants and biochemical validation, single lab, Drosophila ortholog","pmids":["30593492"],"is_preprint":false},{"year":2017,"finding":"In Xenopus embryos, zygotic Rnf146 depletion via morpholino causes anteriorized development with increased Otx2 expression (consistent with positive Wnt regulation). Depletion of maternal Rnf146 leads to ventralized development and reduced organizer gene expression, demonstrating Rnf146 is required for Wnt-dependent embryonic pattern formation through negative regulation of Axin1.","method":"Morpholino knockdown, antisense oligonucleotide, in situ hybridization, marker gene expression analysis in Xenopus embryos","journal":"Mechanisms of development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined molecular and morphological readouts in Xenopus ortholog, single lab","pmids":["28807725"],"is_preprint":false},{"year":2021,"finding":"RNF146 promotes ubiquitylation and proteasomal degradation of LKB1 (Liver kinase B1) in cardiomyocytes, thereby suppressing the LKB1-AMPK signaling pathway to promote cardiac hypertrophy. RNF146 KO or knockdown increased AMPK pathway activation in a LKB1-dependent manner.","method":"Genetic KO mice (TAC model), RNAi knockdown, ubiquitylation assays, co-immunoprecipitation, Western blot, in vitro Ang II cardiomyocyte model","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO in vivo plus ubiquitylation assays and epistasis (LKB1 dependence), single lab","pmids":["34856161"],"is_preprint":false},{"year":2022,"finding":"RNF146 promotes ubiquitin-proteasome-mediated degradation of PTEN, thereby activating the AKT/mTOR pathway in hepatocellular carcinoma cells. RNF146 knockdown decreased PTEN ubiquitylation; MG132 (proteasome inhibitor) reversed RNF146-overexpression-induced PTEN reduction. RNF146 is transcriptionally regulated by HIF-1α/HIF-2α.","method":"RNAi knockdown, overexpression, proteasome inhibitor (MG132) rescue, ubiquitylation assays, Western blot, in vivo mouse xenograft","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — ubiquitylation assays and epistasis (PTEN rescue), single lab, no direct protein–protein interaction confirmed","pmids":["35721496"],"is_preprint":false},{"year":2024,"finding":"Inhibition of RNF146 reduces import of proteins into peroxisomes in a manner dependent on stabilization and activity of TNKS/TNKS2, which bind the peroxisomal membrane protein PEX14. Loss of peroxisomes increased TNKS/2- and RNF146-dependent degradation of AXIN1 sufficient to alter β-catenin transcription, linking peroxisome function to Wnt signaling through the RNF146-TNKS axis.","method":"Genome-wide CRISPRi screen, genetic KO/knockdown, peroxisome import assays, substrate stability assays, transcription reporter assays","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPRi screen with mechanistic follow-up (KO, import assays, signaling readouts), single lab","pmids":["38967608"],"is_preprint":false},{"year":2018,"finding":"RNF146 is exported from the nucleus to the cytoplasm via an XPO1 (CRM1)-dependent nuclear export signal. Under angiotensin II (Ang II) stress, RNF146 accumulates in the nucleus; overexpression of XPO1 facilitates nuclear export of RNF146 and protects endothelial cells from Ang II-induced death. Interaction between RNF146 and XPO1 was confirmed by co-immunoprecipitation.","method":"Co-immunoprecipitation, XPO1 inhibitor (KPT-185), overexpression, immunofluorescence/subcellular fractionation, cell viability assays","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and pharmacological inhibition, single lab, limited mechanistic follow-up","pmids":["30029878"],"is_preprint":false},{"year":2025,"finding":"RNF146 promotes ubiquitylation and proteasomal degradation of DAPK1 (death-associated protein kinase 1) in cardiomyocytes, thereby inhibiting ferroptosis and alleviating myocardial ischemia/reperfusion injury. Overexpressing DAPK1 reversed the cardioprotective effects of RNF146 overexpression.","method":"Co-immunoprecipitation, cycloheximide chase, ubiquitylation assays, overexpression/rescue experiments, in vivo MI/RI mouse model","journal":"Cardiovascular toxicology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP, ubiquitylation assay, and epistasis in a single lab study with limited mechanistic depth","pmids":["39953324"],"is_preprint":false},{"year":2025,"finding":"DTX2 and DTX3 (Deltex E3 ligases) monoubiquitylate tankyrase on mono-ADP-ribose (MAR) residues rather than on lysine, creating a hybrid MAR-ubiquitin mark. RNF114 and RNF166 recognize this hybrid mark and further diubiquitylate it near the ADP-ribose addition site. This ubiquitylation of MAR prevents PAR chain extension on tankyrase, thereby antagonizing RNF146-mediated PARdU and stabilizing tankyrase. Thus, RNF146-dependent tankyrase degradation is counteracted by this MAR-ubiquitin hybrid mechanism.","method":"Biochemical ubiquitylation assays, mass spectrometry (identification of ubiquitin-MAR hybrid), cell-based tankyrase stability assays, domain binding studies","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab, biochemical assays without full independent replication; novel mechanism warrants inclusion but confidence limited","pmids":["bio_10.1101_2025.04.09.648013"],"is_preprint":true},{"year":2025,"finding":"The WWE domain of RNF146, when expressed as an EGFP fusion, functions as a genetically encoded probe for detecting PAR chains (specifically via iso-ADP-ribose recognition) at DNA damage sites in live cells, demonstrating unique PAR dynamics compared to other PAR-binding domains.","method":"Live-cell imaging (EGFP-WWE domain fusion), structural prediction, DNA damage induction, comparison across WWE domain variants","journal":"DNA repair","confidence":"Low","confidence_rationale":"Tier 3 / Weak — live imaging tool development study; establishes WWE domain PAR-binding specificity in cells but primarily a methods/probe paper, single lab","pmids":["40403420"],"is_preprint":false}],"current_model":"RNF146 is a RING-domain E3 ubiquitin ligase that is allosterically activated by binding poly(ADP-ribose) (PAR) through its WWE domain, which induces a conformational change enabling productive E2 engagement; it is recruited to substrates (including Axin, 3BP2, AMOT, VISA/MAVS, RIPK1, LKB1, DAPK1, PTEN, and PARP1) after they are PARylated by tankyrase (TNKS1/2) — to which RNF146 directly binds via noncanonical tankyrase-binding motifs — thereby coupling PARylation to K48-linked polyubiquitination and proteasomal degradation of these targets to regulate Wnt/β-catenin, Hippo, innate immune, and cell death signaling pathways."},"narrative":{"mechanistic_narrative":"RNF146 is a RING-domain E3 ubiquitin ligase that couples tankyrase-dependent poly(ADP-ribosyl)ation (PARylation) to K48-linked polyubiquitination and proteasomal degradation, thereby acting as the destruction arm of the PARylation-dependent ubiquitination (PARdU) axis across Wnt/β-catenin, Hippo, innate immune, and cell-death signaling [PMID:21478859, PMID:21799911]. Its WWE domain directly binds poly(ADP-ribose) and the smallest internal PAR unit, iso-ADP-ribose, which lodges between the WWE and RING domains and allosterically converts the catalytically inactive RING into an active E2-engaging state; in parallel, multiple noncanonical extended tankyrase-binding motifs in its C-terminus mediate weak-but-multivalent binding to the ankyrin repeats of TNKS1/2, recruiting RNF146 to PARylated substrates [PMID:25327252, PMID:29604130]. Through this mechanism RNF146 forms a complex with tankyrase and ubiquitylates targets after they are PARylated by TNKS: it degrades Axin to promote Wnt/β-catenin signaling [PMID:21478859, PMID:21799911], angiomotin-family proteins to maintain the junctional Crumbs complex [PMID:27521426], PARylated VISA/MAVS to attenuate antiviral interferon induction [PMID:35733260], and activated RIPK1 within TAX1BP1-nucleated tankyrase/RNF146 condensates to restrain necroptosis [PMID:38272024]. Substrate profiling in TNKS-double-knockout cells shows RNF146 also has tankyrase-independent substrates such as OTUD5 and PARP10 [PMID:32958691]. RNF146 activity is further tuned by SUMOylation, which drives nuclear localization and accelerates Axin turnover [PMID:37029301], and its physiology is documented in vivo, where loss stabilizes Axin1 and 3BP2 to reprogram osteoclast/osteoblast development, glucose handling, and inflammatory responses [PMID:28287403, PMID:28581440]. RNF146 additionally protects against PARP1-driven parthanatos by sequestering PAR polymer independently of its ligase activity [PMID:33443209].","teleology":[{"year":2011,"claim":"Established RNF146 as the molecular bridge that converts tankyrase-dependent PARylation into ubiquitin-mediated degradation, answering how a PAR signal is read and translated into protein turnover.","evidence":"RNAi screening, Co-IP, proteomics, ubiquitylation and Wnt reporter assays identifying WWE-domain PAR binding and degradation of Axin, BLZF1, CASC3","pmids":["21478859","21799911"],"confidence":"High","gaps":["Structural basis of PAR-induced activation not yet defined","Full substrate repertoire unknown"]},{"year":2014,"claim":"Resolved how PAR binding activates RNF146 — iso-ADP-ribose binding between WWE and RING domains allosterically licenses E2 engagement, and a C-terminal region directly binds tankyrase.","evidence":"X-ray crystallography with biochemical, mutagenesis, E2-binding, and cell-based Axin stability assays","pmids":["25327252"],"confidence":"High","gaps":["Did not enumerate which substrates require allosteric activation versus tankyrase tethering"]},{"year":2018,"claim":"Defined the architecture of the RNF146–tankyrase interaction as multivalent binding through noncanonical extended tankyrase-binding motifs to TNKS ankyrin repeats.","evidence":"Crystal structure of a noncanonical TBM with TNKS ARD plus affinity assays and TBM mutagenesis","pmids":["29604130"],"confidence":"High","gaps":["Stoichiometry and cooperativity within the full complex not fully resolved"]},{"year":2016,"claim":"Extended RNF146 function beyond Wnt by showing it degrades PARylated angiomotin proteins to maintain apical Crumbs/PALS1 polarity, linking the PARdU axis to junctional/Hippo biology.","evidence":"RNAi, reciprocal Co-IP, ubiquitylation assays, immunofluorescence, and epistatic rescue by AMOTL2 knockdown","pmids":["27521426"],"confidence":"High","gaps":["Direct connection to downstream Hippo transcriptional output not measured"]},{"year":2017,"claim":"Demonstrated in vivo physiological roles by showing transcriptional repression of RNF146 and substrate stabilization (3BP2, Axin1) coordinate osteoclast/osteoblast programs, metabolism, and inflammatory responses.","evidence":"Promoter analysis, knockout mice, in vivo LPS challenge, Wnt reporter and substrate stability assays","pmids":["28287403","28581440"],"confidence":"High","gaps":["Tissue-specific substrate priorities incompletely defined"]},{"year":2017,"claim":"Showed using vertebrate (Xenopus) and invertebrate (Drosophila) genetics that RNF146 is required for Wnt-dependent patterning and Axin proteolysis but is dispensable in some homeostatic contexts, establishing context-dependence of the TNKS–RNF146 axis.","evidence":"Morpholino knockdown with marker analysis in Xenopus; null-mutant genetics and epistasis in Drosophila","pmids":["28807725","30593492"],"confidence":"Medium","gaps":["Molecular basis for context-dependent dispensability unknown"]},{"year":2020,"claim":"Distinguished tankyrase-dependent from tankyrase-independent RNF146 substrates proteome-wide, broadening its substrate landscape beyond the canonical PARdU model.","evidence":"Label-free proteomics with RNF146 KO and TNKS1/2-double KO cells, validating OTUD5, PARP10, and SARDH","pmids":["32958691"],"confidence":"Medium","gaps":["Mechanism of PAR-independent substrate recognition unresolved","Many of the 160 candidates unvalidated"]},{"year":2020,"claim":"Revealed a ligase-independent protective function: RNF146 sequesters PAR polymer to block PARP1-driven parthanatos, downstream of Akt1/CREB transcriptional control.","evidence":"Constitutively active Akt1, luciferase reporters, PAR-binding assays, RNF146 KO mice, and SH-SY5Y toxicity assays","pmids":["33443209"],"confidence":"Medium","gaps":["Relationship between sequestration and catalytic functions in the same cell not delineated"]},{"year":2022,"claim":"Placed RNF146 in innate antiviral control by showing it degrades TNKS-PARylated VISA/MAVS (at Glu137) to dampen interferon induction.","evidence":"Co-IP, Glu137 mutagenesis, ubiquitylation assays, RNF146/TNKS KO cells, in vivo viral infection","pmids":["35733260"],"confidence":"High","gaps":["Temporal regulation during infection not mapped"]},{"year":2024,"claim":"Showed that adaptor-nucleated tankyrase/RNF146 liquid-like condensates execute PARdU of activated RIPK1 to restrain necroptosis, adding biomolecular condensation as an organizing principle of the axis.","evidence":"Phase-separation live imaging, K376 mutagenesis, ubiquitylation assays, Co-IP, and KO in MEFs","pmids":["38272024"],"confidence":"High","gaps":["Whether condensate formation is required for other substrates is untested"]},{"year":2024,"claim":"Connected organelle homeostasis to the axis by showing peroxisome loss enhances TNKS/RNF146-dependent Axin1 degradation and Wnt output, with TNKS binding PEX14.","evidence":"Genome-wide CRISPRi screen with KO/knockdown, peroxisome import and substrate stability assays, and transcription reporters","pmids":["38967608"],"confidence":"Medium","gaps":["Direct mechanism linking peroxisome status to TNKS activity unclear"]},{"year":2023,"claim":"Identified SUMOylation as a regulatory layer controlling RNF146 nucleocytoplasmic distribution and its efficiency in degrading Axin.","evidence":"Lysine mutagenesis, Co-IP, fractionation/immunofluorescence, ubiquitylation assays, and cancer models","pmids":["37029301"],"confidence":"Medium","gaps":["Single-lab study; physiological triggers of RNF146 SUMOylation not defined"]},{"year":2025,"claim":"Cardiac studies expanded the substrate set (LKB1, PTEN, DAPK1) linking RNF146 degradation to AMPK suppression, AKT/mTOR activation, and ferroptosis/hypertrophy outcomes.","evidence":"Co-IP, cycloheximide chase, ubiquitylation assays, KO/overexpression and in vivo cardiac injury models","pmids":["34856161","35721496","39953324"],"confidence":"Medium","gaps":["Several lack confirmed direct interaction or PAR-dependence","Low-confidence single-lab reports for DAPK1"]},{"year":2025,"claim":"Characterized antagonism of RNF146 by a MAR-ubiquitin hybrid mark on tankyrase, showing how PAR chain extension — and thus PARdU — can be blocked to stabilize tankyrase.","evidence":"Biochemical ubiquitylation assays, mass spectrometry, and cell-based stability assays (preprint)","pmids":["bio_10.1101_2025.04.09.648013"],"confidence":"Low","gaps":["Preprint not independently replicated","Physiological relevance of the antagonism untested"]},{"year":null,"claim":"How RNF146 selects among its many substrates in specific tissues, balances catalytic versus PAR-sequestering functions, and is regulated spatially (condensates, SUMOylation, export) remains incompletely integrated.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unified model of substrate prioritization across tissues","Switch between ligase and sequestration modes undefined","In vivo relevance of condensate-based PARdU beyond necroptosis unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,7,12]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,7,12]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,2]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[11]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[2,21]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,9,10]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[9,10,18]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,4]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,7,12]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[7]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[11,12,19]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[5,6,13,14]}],"complexes":["RNF146–tankyrase(TNKS1/2)–Axin complex","TAX1BP1-nucleated tankyrase/RNF146 condensate"],"partners":["TNKS1","TNKS2","AXIN1","PARP1","AMOTL2","XPO1","MAVS","RIPK1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NTX7","full_name":"E3 ubiquitin-protein ligase RNF146","aliases":["Dactylidin","Iduna","RING finger protein 146","RING-type E3 ubiquitin transferase RNF146"],"length_aa":359,"mass_kda":39.0,"function":"E3 ubiquitin-protein ligase that specifically binds poly-ADP-ribosylated (PARsylated) proteins and mediates their ubiquitination and subsequent degradation (PubMed:21478859, PubMed:21799911, PubMed:22267412). May regulate many important biological processes, such as cell survival and DNA damage response (PubMed:21825151, PubMed:22267412). Acts as an activator of the Wnt signaling pathway by mediating the ubiquitination of PARsylated AXIN1 and AXIN2, 2 key components of the beta-catenin destruction complex (PubMed:21478859, PubMed:21799911). Acts in cooperation with tankyrase proteins (TNKS and TNKS2), which mediate PARsylation of target proteins AXIN1, AXIN2, BLZF1, CASC3, TNKS and TNKS2 (PubMed:21799911). Recognizes and binds tankyrase-dependent PARsylated proteins via its WWE domain and mediates their ubiquitination, leading to their degradation (PubMed:21799911). Different ubiquitin linkage types have been observed: TNKS2 undergoes ubiquitination at 'Lys-48' and 'Lys-63', while AXIN1 is only ubiquitinated at 'Lys-48' (PubMed:21799911). May regulate TNKS and TNKS2 subcellular location, preventing aggregation at a centrosomal location (PubMed:21799911). Neuroprotective protein (PubMed:21602803). Protects the brain against N-methyl-D-aspartate (NMDA) receptor-mediated glutamate excitotoxicity and ischemia, by interfering with PAR-induced cell death, called parthanatos (By similarity). Prevents nuclear translocation of AIFM1 in a PAR-binding dependent manner (By similarity). Does not affect PARP1 activation (By similarity). Protects against cell death induced by DNA damaging agents, such as N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and rescues cells from G1 arrest (By similarity). Promotes cell survival after gamma-irradiation (PubMed:21825151). Facilitates DNA repair (PubMed:21825151)","subcellular_location":"Cytoplasm, cytosol; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9NTX7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RNF146","classification":"Not Classified","n_dependent_lines":23,"n_total_lines":1208,"dependency_fraction":0.01903973509933775},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PARP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/RNF146","total_profiled":1310},"omim":[{"mim_id":"620871","title":"DNA DAMAGE-INDUCIBLE 1 HOMOLOG 2; DDI2","url":"https://www.omim.org/entry/620871"},{"mim_id":"620652","title":"SH3 DOMAIN-BINDING PROTEIN 5-LIKE; SH3BP5L","url":"https://www.omim.org/entry/620652"},{"mim_id":"612137","title":"RING FINGER PROTEIN 146; RNF146","url":"https://www.omim.org/entry/612137"},{"mim_id":"605612","title":"SH3 DOMAIN-BINDING PROTEIN 5; SH3BP5","url":"https://www.omim.org/entry/605612"},{"mim_id":"603303","title":"TANKYRASE; TNKS","url":"https://www.omim.org/entry/603303"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RNF146"},"hgnc":{"alias_symbol":["DKFZp434O1427","dactylidin","dJ351K20.1"],"prev_symbol":[]},"alphafold":{"accession":"Q9NTX7","domains":[{"cath_id":"3.30.40.10","chopping":"35-89","consensus_level":"high","plddt":95.0798,"start":35,"end":89},{"cath_id":"3.30.720.50","chopping":"101-180","consensus_level":"high","plddt":95.5171,"start":101,"end":180}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NTX7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NTX7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NTX7-F1-predicted_aligned_error_v6.png","plddt_mean":66.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RNF146","jax_strain_url":"https://www.jax.org/strain/search?query=RNF146"},"sequence":{"accession":"Q9NTX7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NTX7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NTX7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NTX7"}},"corpus_meta":[{"pmid":"21478859","id":"PMC_21478859","title":"RNF146 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genome-wide screen links peroxisome regulation with Wnt signaling through RNF146 and TNKS/2.","date":"2024","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/38967608","citation_count":5,"is_preprint":false},{"pmid":"38237916","id":"PMC_38237916","title":"Iso-ADP-Ribose Fluorescence Polarization Probe for the Screening of RNF146 WWE Domain Inhibitors.","date":"2024","source":"ACS chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/38237916","citation_count":2,"is_preprint":false},{"pmid":"39953324","id":"PMC_39953324","title":"RNF146 Alleviates Myocardial Ischemia/Reperfusion Injury by Regulating the Ubiquitination-Mediated Degradation of DAPK1 to Inhibit Ferroptosis.","date":"2025","source":"Cardiovascular toxicology","url":"https://pubmed.ncbi.nlm.nih.gov/39953324","citation_count":2,"is_preprint":false},{"pmid":"38234836","id":"PMC_38234836","title":"Overexpression of the WWE domain of RNF146 modulates poly-(ADP)-ribose dynamics at sites of DNA 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directly interacts with poly(ADP-ribose) through its WWE domain, promoting ubiquitylation and proteasomal degradation of PARsylated proteins including Axin, BLZF1, and CASC3. RNF146 acts downstream of tankyrase-dependent PARsylation to couple PARsylation to ubiquitylation, thereby positively regulating Wnt/β-catenin signaling.\",\n      \"method\": \"RNAi screening, co-immunoprecipitation, proteomics, ubiquitylation assays, cell-based Wnt reporter assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (RNAi, Co-IP, mass spectrometry, in-cell ubiquitylation assays) replicated across at least two concurrent independent studies (PMIDs 21478859 and 21799911)\",\n      \"pmids\": [\"21478859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RNF146, tankyrase, and Axin form a protein complex; RNF146 mediates ubiquitylation of Axin, TNKS1, and TNKS2 targeting all three for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase aggregation at a centrosomal location. Tankyrase auto-PARsylation and RNF146-mediated ubiquitylation are mechanistically coupled in the same complex.\",\n      \"method\": \"RNAi screening, co-immunoprecipitation, ubiquitylation assays, immunofluorescence/localization studies, proteasome inhibitor experiments\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ubiquitylation assays, and localization experiments in a single study, consistent with independent replication in PMID 21478859\",\n      \"pmids\": [\"21799911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Iso-ADP-ribose (the smallest internal PAR structural unit) binds between the WWE and RING domains of RNF146, acting as an allosteric signal that converts the RING domain from a catalytically inactive to an active state capable of binding E2 ubiquitin-conjugating enzymes. Crystal structure and biochemical analysis show PAR/iso-ADPr binding induces a major conformational change in RNF146. Additionally, RNF146 directly binds tankyrase via its C-terminal region; disruption of this interaction inhibits Axin turnover in cells.\",\n      \"method\": \"X-ray crystallography, biochemical binding assays, mutagenesis, E2-binding assays, cell-based Axin stability assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with in vitro reconstitution, mutagenesis, and cell-based validation in a single rigorous study\",\n      \"pmids\": [\"25327252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RNF146 binds directly to tankyrase (TNKS1/2) via multiple noncanonical tankyrase-binding motifs (TBMs) in its C-terminal region. These extended TBMs (with one or two extra residues between conserved Arg and Gly) mediate weak individual but strong multivalent binding. A crystal structure of the first RNF146 noncanonical TBM with the second ankyrin repeat domain of TNKS was solved.\",\n      \"method\": \"X-ray crystallography, binding/affinity assays, mutagenesis of TBMs\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus biochemical binding validation in a single focused study\",\n      \"pmids\": [\"29604130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RNF146 and tankyrase2 (TNKS2) maintain the junctional Crumbs complex by promoting degradation of angiomotin (AMOT) family proteins. AMOT proteins are PARylated by TNKS2, which drives their ubiquitylation by RNF146 and subsequent proteasomal degradation. Ablation of RNF146 or tankyrase caused PALS1 (a Crumbs complex component) to relocate from the apical membrane to internal puncta, a phenotype rescued by AMOTL2 knockdown.\",\n      \"method\": \"RNAi knockdown, co-immunoprecipitation, ubiquitylation assays, immunofluorescence, genetic epistasis (rescue experiments)\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, ubiquitylation assays, localization with functional consequence, and epistatic rescue in a single study\",\n      \"pmids\": [\"27521426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RANKL represses RNF146 transcription via an NF-κB-related inhibitory element in the RNF146 promoter. RANKL-mediated suppression of RNF146 stabilizes its substrates 3BP2 and AXIN1, triggering SRC activation and attenuation of β-catenin expression, respectively, to coordinate the osteoclast developmental program. RNF146 depletion caused hypersensitivity to LPS-induced TNF-α production in vivo.\",\n      \"method\": \"Promoter analysis, RNAi, genetic knockout, in vivo LPS challenge, co-immunoprecipitation, substrate stability assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter analysis combined with KO mice, in vivo experiments, and substrate stabilization assays in a single study\",\n      \"pmids\": [\"28287403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Loss of RNF146 in osteoblasts stabilizes its substrate AXIN1, impairing WNT3a-induced β-catenin activation and reducing Fgf18 expression. FGF18 was shown to induce TAZ expression required for osteoblast proliferation and differentiation. RNF146 knockout also enhanced adipogenesis in MEFs and caused glucose intolerance with severe osteopenia.\",\n      \"method\": \"Genetic knockout (Rnf146-/- mice), cell-based Wnt/β-catenin reporter assays, substrate stability assays, osteoblast differentiation assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO in mice with multiple cellular and molecular readouts (substrate stabilization, signaling assays, differentiation) in a single study\",\n      \"pmids\": [\"28581440\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TNKS1 and TNKS2 PARylate the mitochondrial adaptor VISA/MAVS at Glu137, priming it for K48-linked polyubiquitination by RNF146 and subsequent proteasomal degradation. This mechanism attenuates innate antiviral signaling. RNF146 deficiency increased RNA virus-triggered interferon induction and impaired viral replication.\",\n      \"method\": \"Biochemical purification (co-immunoprecipitation), site-directed mutagenesis (Glu137), ubiquitylation assays, RNF146/TNKS knockout cells, in vivo viral infection model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, mutagenesis of modification site, ubiquitylation assays, and in vivo validation in a single study\",\n      \"pmids\": [\"35733260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Proteome-wide analysis using RNF146 KO and TNKS1/2-double KO cells identified 160 potential RNF146 substrates including 122 potential TNKS-independent substrates. OTUD5 and PARP10 were validated as TNKS1/2-independent substrates of RNF146, and SARDH was validated as a novel substrate of both TNKS1/2 and RNF146.\",\n      \"method\": \"Proteome profiling (label-free quantification), transcriptome analysis, CRISPR knockout cell lines, Western blot validation\",\n      \"journal\": \"Molecular & cellular proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic proteomics with KO cells and validation of specific substrates by Western blot, single lab\",\n      \"pmids\": [\"32958691\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RNF146 is SUMOylated at K19, K61, K174, and K175 by UBC9/PIAS3/MMS21, with SENP1/2/6 acting as deSUMOylases. SUMOylation (specifically at K19/K175, mediated by UBC9/PIAS3 and removed by SENP1) promotes RNF146 nuclear localization, while deSUMOylation induces cytoplasmic localization. SUMOylation promotes RNF146 association with Axin to accelerate Axin ubiquitylation and degradation, enhancing β-catenin signaling.\",\n      \"method\": \"Lysine mutagenesis, co-immunoprecipitation, subcellular fractionation/immunofluorescence, ubiquitylation assays, in vitro and in vivo cancer models\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis of SUMO sites, Co-IP, localization assays, and functional readouts in a single lab study\",\n      \"pmids\": [\"37029301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In cardiac myocytes, RNF146 acts as a direct interactor of PARP-1. Upon PARP-1 activation by oxidative stress, RNF146 translocates from cytoplasm to nucleus, triggering PARP-1 nuclear exit followed by rapid proteasomal degradation of both PARP-1 and RNF146. RNF146 overexpression protected against oxidant-induced cell death; RNF146 silencing augmented PARP-1-mediated injury.\",\n      \"method\": \"Co-immunoprecipitation, overexpression/knockdown, immunofluorescence/live imaging, cardiomyocyte injury models\",\n      \"journal\": \"Molecular medicine (Cambridge, Mass.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP, localization experiments, and functional phenotype readout, single lab, two orthogonal methods\",\n      \"pmids\": [\"24842055\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Akt1 suppresses parthanatos in dopaminergic neurons by stimulating CREB-dependent transcriptional activation of the RNF146 gene. RNF146 inhibits PARP1-induced cell death not through its E3 ligase activity but by binding to and sequestering PAR polymer, thereby preventing parthanatos.\",\n      \"method\": \"Overexpression of constitutively active Akt1, luciferase reporter assays, co-immunoprecipitation/PAR binding assays, RNF146 KO in vivo mouse models, SH-SY5Y cell toxicity assays\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cell and in vivo models, RNF146 KO, PAR binding assays, single lab\",\n      \"pmids\": [\"33443209\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Upon induction of necroptosis and recruitment by the adaptor TAX1BP1, PARP5A (tankyrase) and RNF146 form liquid-like condensates through multivalent interactions. These condensates perform PARylation and PARylation-dependent ubiquitination (PARdU) of activated RIPK1, predominantly at K376, promoting proteasomal degradation of kinase-activated RIPK1 to restrain necroptosis in mouse embryonic fibroblasts.\",\n      \"method\": \"Phase separation assays (live imaging), mutagenesis (K376), ubiquitylation assays, co-immunoprecipitation, genetic KO (MEFs), cell death assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods including phase separation imaging, site mutagenesis, ubiquitylation assays, and KO in a single rigorous study\",\n      \"pmids\": [\"38272024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In Drosophila, Rnf146 functions in the same proteolysis pathway as Tnks for Axin proteolysis in vivo (genetic epistasis), buffering Axin levels to ensure Wingless pathway activation in multiple developmental contexts. However, unlike Tnks, Rnf146 is dispensable for Wingless target gene activation and intestinal stem cell proliferation in the adult midgut during homeostasis, indicating context-dependent requirement for RNF146 in TNKS-mediated Axin degradation.\",\n      \"method\": \"Drosophila null mutant genetics, biochemical Axin stability assays, epistasis analysis, Wingless reporter assays\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with null mutants and biochemical validation, single lab, Drosophila ortholog\",\n      \"pmids\": [\"30593492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In Xenopus embryos, zygotic Rnf146 depletion via morpholino causes anteriorized development with increased Otx2 expression (consistent with positive Wnt regulation). Depletion of maternal Rnf146 leads to ventralized development and reduced organizer gene expression, demonstrating Rnf146 is required for Wnt-dependent embryonic pattern formation through negative regulation of Axin1.\",\n      \"method\": \"Morpholino knockdown, antisense oligonucleotide, in situ hybridization, marker gene expression analysis in Xenopus embryos\",\n      \"journal\": \"Mechanisms of development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined molecular and morphological readouts in Xenopus ortholog, single lab\",\n      \"pmids\": [\"28807725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF146 promotes ubiquitylation and proteasomal degradation of LKB1 (Liver kinase B1) in cardiomyocytes, thereby suppressing the LKB1-AMPK signaling pathway to promote cardiac hypertrophy. RNF146 KO or knockdown increased AMPK pathway activation in a LKB1-dependent manner.\",\n      \"method\": \"Genetic KO mice (TAC model), RNAi knockdown, ubiquitylation assays, co-immunoprecipitation, Western blot, in vitro Ang II cardiomyocyte model\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO in vivo plus ubiquitylation assays and epistasis (LKB1 dependence), single lab\",\n      \"pmids\": [\"34856161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF146 promotes ubiquitin-proteasome-mediated degradation of PTEN, thereby activating the AKT/mTOR pathway in hepatocellular carcinoma cells. RNF146 knockdown decreased PTEN ubiquitylation; MG132 (proteasome inhibitor) reversed RNF146-overexpression-induced PTEN reduction. RNF146 is transcriptionally regulated by HIF-1α/HIF-2α.\",\n      \"method\": \"RNAi knockdown, overexpression, proteasome inhibitor (MG132) rescue, ubiquitylation assays, Western blot, in vivo mouse xenograft\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — ubiquitylation assays and epistasis (PTEN rescue), single lab, no direct protein–protein interaction confirmed\",\n      \"pmids\": [\"35721496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Inhibition of RNF146 reduces import of proteins into peroxisomes in a manner dependent on stabilization and activity of TNKS/TNKS2, which bind the peroxisomal membrane protein PEX14. Loss of peroxisomes increased TNKS/2- and RNF146-dependent degradation of AXIN1 sufficient to alter β-catenin transcription, linking peroxisome function to Wnt signaling through the RNF146-TNKS axis.\",\n      \"method\": \"Genome-wide CRISPRi screen, genetic KO/knockdown, peroxisome import assays, substrate stability assays, transcription reporter assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPRi screen with mechanistic follow-up (KO, import assays, signaling readouts), single lab\",\n      \"pmids\": [\"38967608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RNF146 is exported from the nucleus to the cytoplasm via an XPO1 (CRM1)-dependent nuclear export signal. Under angiotensin II (Ang II) stress, RNF146 accumulates in the nucleus; overexpression of XPO1 facilitates nuclear export of RNF146 and protects endothelial cells from Ang II-induced death. Interaction between RNF146 and XPO1 was confirmed by co-immunoprecipitation.\",\n      \"method\": \"Co-immunoprecipitation, XPO1 inhibitor (KPT-185), overexpression, immunofluorescence/subcellular fractionation, cell viability assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and pharmacological inhibition, single lab, limited mechanistic follow-up\",\n      \"pmids\": [\"30029878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF146 promotes ubiquitylation and proteasomal degradation of DAPK1 (death-associated protein kinase 1) in cardiomyocytes, thereby inhibiting ferroptosis and alleviating myocardial ischemia/reperfusion injury. Overexpressing DAPK1 reversed the cardioprotective effects of RNF146 overexpression.\",\n      \"method\": \"Co-immunoprecipitation, cycloheximide chase, ubiquitylation assays, overexpression/rescue experiments, in vivo MI/RI mouse model\",\n      \"journal\": \"Cardiovascular toxicology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP, ubiquitylation assay, and epistasis in a single lab study with limited mechanistic depth\",\n      \"pmids\": [\"39953324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DTX2 and DTX3 (Deltex E3 ligases) monoubiquitylate tankyrase on mono-ADP-ribose (MAR) residues rather than on lysine, creating a hybrid MAR-ubiquitin mark. RNF114 and RNF166 recognize this hybrid mark and further diubiquitylate it near the ADP-ribose addition site. This ubiquitylation of MAR prevents PAR chain extension on tankyrase, thereby antagonizing RNF146-mediated PARdU and stabilizing tankyrase. Thus, RNF146-dependent tankyrase degradation is counteracted by this MAR-ubiquitin hybrid mechanism.\",\n      \"method\": \"Biochemical ubiquitylation assays, mass spectrometry (identification of ubiquitin-MAR hybrid), cell-based tankyrase stability assays, domain binding studies\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab, biochemical assays without full independent replication; novel mechanism warrants inclusion but confidence limited\",\n      \"pmids\": [\"bio_10.1101_2025.04.09.648013\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The WWE domain of RNF146, when expressed as an EGFP fusion, functions as a genetically encoded probe for detecting PAR chains (specifically via iso-ADP-ribose recognition) at DNA damage sites in live cells, demonstrating unique PAR dynamics compared to other PAR-binding domains.\",\n      \"method\": \"Live-cell imaging (EGFP-WWE domain fusion), structural prediction, DNA damage induction, comparison across WWE domain variants\",\n      \"journal\": \"DNA repair\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — live imaging tool development study; establishes WWE domain PAR-binding specificity in cells but primarily a methods/probe paper, single lab\",\n      \"pmids\": [\"40403420\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RNF146 is a RING-domain E3 ubiquitin ligase that is allosterically activated by binding poly(ADP-ribose) (PAR) through its WWE domain, which induces a conformational change enabling productive E2 engagement; it is recruited to substrates (including Axin, 3BP2, AMOT, VISA/MAVS, RIPK1, LKB1, DAPK1, PTEN, and PARP1) after they are PARylated by tankyrase (TNKS1/2) — to which RNF146 directly binds via noncanonical tankyrase-binding motifs — thereby coupling PARylation to K48-linked polyubiquitination and proteasomal degradation of these targets to regulate Wnt/β-catenin, Hippo, innate immune, and cell death signaling pathways.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RNF146 is a RING-domain E3 ubiquitin ligase that couples tankyrase-dependent poly(ADP-ribosyl)ation (PARylation) to K48-linked polyubiquitination and proteasomal degradation, thereby acting as the destruction arm of the PARylation-dependent ubiquitination (PARdU) axis across Wnt/\\u03b2-catenin, Hippo, innate immune, and cell-death signaling [#0, #1]. Its WWE domain directly binds poly(ADP-ribose) and the smallest internal PAR unit, iso-ADP-ribose, which lodges between the WWE and RING domains and allosterically converts the catalytically inactive RING into an active E2-engaging state; in parallel, multiple noncanonical extended tankyrase-binding motifs in its C-terminus mediate weak-but-multivalent binding to the ankyrin repeats of TNKS1/2, recruiting RNF146 to PARylated substrates [#2, #3]. Through this mechanism RNF146 forms a complex with tankyrase and ubiquitylates targets after they are PARylated by TNKS: it degrades Axin to promote Wnt/\\u03b2-catenin signaling [#0, #1], angiomotin-family proteins to maintain the junctional Crumbs complex [#4], PARylated VISA/MAVS to attenuate antiviral interferon induction [#7], and activated RIPK1 within TAX1BP1-nucleated tankyrase/RNF146 condensates to restrain necroptosis [#12]. Substrate profiling in TNKS-double-knockout cells shows RNF146 also has tankyrase-independent substrates such as OTUD5 and PARP10 [#8]. RNF146 activity is further tuned by SUMOylation, which drives nuclear localization and accelerates Axin turnover [#9], and its physiology is documented in vivo, where loss stabilizes Axin1 and 3BP2 to reprogram osteoclast/osteoblast development, glucose handling, and inflammatory responses [#5, #6]. RNF146 additionally protects against PARP1-driven parthanatos by sequestering PAR polymer independently of its ligase activity [#11].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Established RNF146 as the molecular bridge that converts tankyrase-dependent PARylation into ubiquitin-mediated degradation, answering how a PAR signal is read and translated into protein turnover.\",\n      \"evidence\": \"RNAi screening, Co-IP, proteomics, ubiquitylation and Wnt reporter assays identifying WWE-domain PAR binding and degradation of Axin, BLZF1, CASC3\",\n      \"pmids\": [\"21478859\", \"21799911\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of PAR-induced activation not yet defined\", \"Full substrate repertoire unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Resolved how PAR binding activates RNF146 \\u2014 iso-ADP-ribose binding between WWE and RING domains allosterically licenses E2 engagement, and a C-terminal region directly binds tankyrase.\",\n      \"evidence\": \"X-ray crystallography with biochemical, mutagenesis, E2-binding, and cell-based Axin stability assays\",\n      \"pmids\": [\"25327252\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not enumerate which substrates require allosteric activation versus tankyrase tethering\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined the architecture of the RNF146\\u2013tankyrase interaction as multivalent binding through noncanonical extended tankyrase-binding motifs to TNKS ankyrin repeats.\",\n      \"evidence\": \"Crystal structure of a noncanonical TBM with TNKS ARD plus affinity assays and TBM mutagenesis\",\n      \"pmids\": [\"29604130\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and cooperativity within the full complex not fully resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended RNF146 function beyond Wnt by showing it degrades PARylated angiomotin proteins to maintain apical Crumbs/PALS1 polarity, linking the PARdU axis to junctional/Hippo biology.\",\n      \"evidence\": \"RNAi, reciprocal Co-IP, ubiquitylation assays, immunofluorescence, and epistatic rescue by AMOTL2 knockdown\",\n      \"pmids\": [\"27521426\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct connection to downstream Hippo transcriptional output not measured\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated in vivo physiological roles by showing transcriptional repression of RNF146 and substrate stabilization (3BP2, Axin1) coordinate osteoclast/osteoblast programs, metabolism, and inflammatory responses.\",\n      \"evidence\": \"Promoter analysis, knockout mice, in vivo LPS challenge, Wnt reporter and substrate stability assays\",\n      \"pmids\": [\"28287403\", \"28581440\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific substrate priorities incompletely defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed using vertebrate (Xenopus) and invertebrate (Drosophila) genetics that RNF146 is required for Wnt-dependent patterning and Axin proteolysis but is dispensable in some homeostatic contexts, establishing context-dependence of the TNKS\\u2013RNF146 axis.\",\n      \"evidence\": \"Morpholino knockdown with marker analysis in Xenopus; null-mutant genetics and epistasis in Drosophila\",\n      \"pmids\": [\"28807725\", \"30593492\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis for context-dependent dispensability unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Distinguished tankyrase-dependent from tankyrase-independent RNF146 substrates proteome-wide, broadening its substrate landscape beyond the canonical PARdU model.\",\n      \"evidence\": \"Label-free proteomics with RNF146 KO and TNKS1/2-double KO cells, validating OTUD5, PARP10, and SARDH\",\n      \"pmids\": [\"32958691\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of PAR-independent substrate recognition unresolved\", \"Many of the 160 candidates unvalidated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed a ligase-independent protective function: RNF146 sequesters PAR polymer to block PARP1-driven parthanatos, downstream of Akt1/CREB transcriptional control.\",\n      \"evidence\": \"Constitutively active Akt1, luciferase reporters, PAR-binding assays, RNF146 KO mice, and SH-SY5Y toxicity assays\",\n      \"pmids\": [\"33443209\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relationship between sequestration and catalytic functions in the same cell not delineated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed RNF146 in innate antiviral control by showing it degrades TNKS-PARylated VISA/MAVS (at Glu137) to dampen interferon induction.\",\n      \"evidence\": \"Co-IP, Glu137 mutagenesis, ubiquitylation assays, RNF146/TNKS KO cells, in vivo viral infection\",\n      \"pmids\": [\"35733260\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Temporal regulation during infection not mapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed that adaptor-nucleated tankyrase/RNF146 liquid-like condensates execute PARdU of activated RIPK1 to restrain necroptosis, adding biomolecular condensation as an organizing principle of the axis.\",\n      \"evidence\": \"Phase-separation live imaging, K376 mutagenesis, ubiquitylation assays, Co-IP, and KO in MEFs\",\n      \"pmids\": [\"38272024\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether condensate formation is required for other substrates is untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected organelle homeostasis to the axis by showing peroxisome loss enhances TNKS/RNF146-dependent Axin1 degradation and Wnt output, with TNKS binding PEX14.\",\n      \"evidence\": \"Genome-wide CRISPRi screen with KO/knockdown, peroxisome import and substrate stability assays, and transcription reporters\",\n      \"pmids\": [\"38967608\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mechanism linking peroxisome status to TNKS activity unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified SUMOylation as a regulatory layer controlling RNF146 nucleocytoplasmic distribution and its efficiency in degrading Axin.\",\n      \"evidence\": \"Lysine mutagenesis, Co-IP, fractionation/immunofluorescence, ubiquitylation assays, and cancer models\",\n      \"pmids\": [\"37029301\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study; physiological triggers of RNF146 SUMOylation not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cardiac studies expanded the substrate set (LKB1, PTEN, DAPK1) linking RNF146 degradation to AMPK suppression, AKT/mTOR activation, and ferroptosis/hypertrophy outcomes.\",\n      \"evidence\": \"Co-IP, cycloheximide chase, ubiquitylation assays, KO/overexpression and in vivo cardiac injury models\",\n      \"pmids\": [\"34856161\", \"35721496\", \"39953324\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Several lack confirmed direct interaction or PAR-dependence\", \"Low-confidence single-lab reports for DAPK1\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Characterized antagonism of RNF146 by a MAR-ubiquitin hybrid mark on tankyrase, showing how PAR chain extension \\u2014 and thus PARdU \\u2014 can be blocked to stabilize tankyrase.\",\n      \"evidence\": \"Biochemical ubiquitylation assays, mass spectrometry, and cell-based stability assays (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.04.09.648013\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprint not independently replicated\", \"Physiological relevance of the antagonism untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RNF146 selects among its many substrates in specific tissues, balances catalytic versus PAR-sequestering functions, and is regulated spatially (condensates, SUMOylation, export) remains incompletely integrated.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified model of substrate prioritization across tissues\", \"Switch between ligase and sequestration modes undefined\", \"In vivo relevance of condensate-based PARdU beyond necroptosis unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 7, 12]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 7, 12]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [2, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 9, 10]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [9, 10, 18]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 7, 12]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [11, 12, 19]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [5, 6, 13, 14]}\n    ],\n    \"complexes\": [\"RNF146\\u2013tankyrase(TNKS1/2)\\u2013Axin complex\", \"TAX1BP1-nucleated tankyrase/RNF146 condensate\"],\n    \"partners\": [\"TNKS1\", \"TNKS2\", \"AXIN1\", \"PARP1\", \"AMOTL2\", \"XPO1\", \"MAVS\", \"RIPK1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}