{"gene":"RNF126","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2014,"finding":"RNF126 is the primary Bag6-dependent E3 ubiquitin ligase for cytosolic quality control of mislocalized proteins (MLPs). RNF126 is recruited to the N-terminal Ubl domain of Bag6 and preferentially ubiquitinates juxtahydrophobic lysine residues on Bag6-associated clients. Bag6-dependent ubiquitination was fully reconstituted with purified components.","method":"In vitro reconstitution with purified components, cell fractionation, Bag6-dependent ubiquitination assay, RNF126 depletion in cells","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with purified components plus cellular depletion, single rigorous paper with multiple orthogonal methods","pmids":["24981174"],"is_preprint":false},{"year":2020,"finding":"RNF126 catalyzes reubiquitination of p97/VCP-extracted membrane proteins in the cytosol. RNF126 interacts with BAG6, which captures p97-liberated substrates, and a ternary complex (RNF126-BAG6-substrate) was reconstituted with purified factors to demonstrate reubiquitination required for proteasomal targeting.","method":"In vitro reconstitution with purified factors, cellular depletion of RNF126, stabilization of extracted intermediates, pulse-chase turnover assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — reconstituted with purified components plus multiple cellular phenotypes, single rigorous paper","pmids":["32645369"],"is_preprint":false},{"year":2025,"finding":"RNF126 interacts with substrate-engaged UBQLN1 via UBQLN1's ubiquitin-associated (UBA) domain and catalyzes ubiquitination of unimported mitochondrial membrane protein precursors (e.g., ATP5G1), promoting their proteasomal degradation during mitochondrial stress. Ternary complex formation (RNF126-UBQLN1-ATP5G1) was reconstituted in vitro.","method":"In vitro reconstitution of ternary complex, co-IP, ubiquitination assay, proteasomal degradation assay with RNF126 depletion","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution of ternary complex plus cellular validation","pmids":["40086734"],"is_preprint":false},{"year":2012,"finding":"RNF126 acts as an E3 ubiquitin ligase that directly interacts with p21 and promotes its ubiquitination and proteasomal degradation in an E3 ligase activity-dependent manner, driving cancer cell proliferation and G1-S progression.","method":"Co-immunoprecipitation, ubiquitination assay with overexpression of WT vs. ligase-dead RNF126, siRNA knockdown with protein stability assay, rescue by p21 depletion","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal Co-IP, activity-dependent ubiquitination assay, cellular rescue experiment, single lab","pmids":["23026136"],"is_preprint":false},{"year":2017,"finding":"RNF126 ubiquitylates Ku80 (with UBE2D3 as E2) at DSBs, promoting Ku70/80 dissociation from damaged DNA and completion of NHEJ repair. Lysine-to-arginine mutation of Ku80 ubiquitylation sites delayed Ku70/80 release from chromatin.","method":"Identification by mass spectrometry interactome, Co-IP, in vitro ubiquitylation assay, Ku80 ubiquitylation-site mutagenesis (K→R), chromatin fractionation, RNF126 knockdown with DSB repair assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro ubiquitylation assay plus site mutagenesis plus cellular functional consequence, moderate evidence from single lab with multiple methods","pmids":["27895153"],"is_preprint":false},{"year":2013,"finding":"RNF126 specifies K48-linked ubiquitin chains with UbcH5b and K63-linked chains with Ubc13/Uev1a in vitro. RNF126 associates with EGFR via a ubiquitin-binding zinc finger domain and promotes EGFR ubiquitylation downstream of c-Cbl, regulating EGFR endosomal sorting and ESCRT-II stability.","method":"In vitro ubiquitin chain-type assay, Co-IP, siRNA depletion with EGFR trafficking/degradation assay, multivesicular body quantification","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro chain-type reconstitution plus cellular epistasis with c-Cbl plus multiple trafficking phenotypes","pmids":["23418353"],"is_preprint":false},{"year":2013,"finding":"RNF126 regulates retrograde sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR); its RING finger domain (ligase activity) is required, as RING-domain mutant fails to rescue CI-MPR missorting upon RNF126 depletion.","method":"siRNA stable knockdown, RING-domain mutant rescue experiment, CI-MPR localization by immunofluorescence, lysosomal degradation assay, cathepsin D missorting","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 3 — localization plus RING-mutant rescue, single lab, no in vitro reconstitution","pmids":["24275455"],"is_preprint":false},{"year":2016,"finding":"RNF126 acts as an E3 ubiquitin ligase for pyruvate dehydrogenase kinases (PDKs), targeting them for proteasomal degradation. This increases PDH activity and pyruvate-to-acetyl-CoA flux, supporting TCA cycle activity and anoikis resistance in cancer cells. RNF126 expression is controlled by ERK signaling.","method":"Ubiquitination assay for PDKs, proteasomal degradation assay, metabolic flux analysis, soft-agar colony assay, RNF126 depletion in vivo tumorigenicity","journal":"Cell discovery","confidence":"Medium","confidence_rationale":"Tier 2 — ubiquitination of PDKs with metabolic readout, cellular and in vivo confirmation, single lab","pmids":["27462466"],"is_preprint":false},{"year":2017,"finding":"RNF126 is the E3 ligase that directly ubiquitinates frataxin, targeting it for proteasomal degradation. RNF126 interacts with frataxin and promotes its ubiquitination in a catalytic activity-dependent manner both in vivo and in vitro; RNF126 depletion raises frataxin levels in Friedreich ataxia patient-derived cells.","method":"Co-immunoprecipitation, in vitro and in vivo ubiquitination assay, catalytic mutant comparison, protein stability assay in FRDA patient cells","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro ubiquitination plus cellular validation in disease-relevant (FRDA patient) cells with multiple methods","pmids":["28228265"],"is_preprint":false},{"year":2015,"finding":"RNF126 promotes homologous recombination by facilitating BRCA1 expression through direct binding to transcription factor E2F1, enhancing E2F1-mediated transactivation of the BRCA1 promoter; this function is independent of RNF126 E3 ligase activity. An 11-amino-acid deletion mutant acts dominantly negatively by preventing E2F1 binding.","method":"Co-IP of RNF126-E2F1, BRCA1 promoter luciferase assay, ChIP for E2F1 at BRCA1 promoter, deletion mutant analysis, HR repair assay, ionizing radiation and PARP inhibitor sensitivity","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (Co-IP, ChIP, promoter assay, mutant), single lab","pmids":["26234677"],"is_preprint":false},{"year":2018,"finding":"RNF126 is recruited to DNA damage sites in an RNF8-dependent manner, directly interacts with RNF168, and ubiquitinates RNF168. RNF126 overexpression (but not catalytic-dead mutant) diminishes RNF168-mediated H2AX ubiquitination and downstream 53BP1/RAP80/BRCA1 focus formation, placing RNF126 between RNF8 and RNF168 in the DDR cascade.","method":"UV laser micro-irradiation recruitment, Co-IP of RNF126-RNF168, ubiquitination assay with WT vs. CC229/232AA catalytic mutant, HR repair assay","journal":"Genomics, proteomics & bioinformatics","confidence":"Medium","confidence_rationale":"Tier 2 — catalytic mutant discrimination, epistasis placement between RNF8 and RNF168, single lab","pmids":["30529286"],"is_preprint":false},{"year":2017,"finding":"RNF126 overexpression abolishes 53BP1 iRIF formation and RNF168-mediated H2A monoubiquitination at K13/15, while γH2AX, MDC1, and RNF8 foci are maintained, placing RNF126 as a negative regulator between RNF8 and RNF168 in the DDR and inhibiting NHEJ.","method":"Ionizing radiation-induced foci screen, overexpression and focus formation analysis, H2A ubiquitination assay, NHEJ assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — epistasis within DDR pathway established by focus formation hierarchy, single lab","pmids":["29167269"],"is_preprint":false},{"year":2022,"finding":"RNF126 physically associates with the MRE11-RAD50-NBS1 complex and ubiquitinates MRE11 at K339 and K480, increasing MRE11 DNA exonuclease activity, RPA binding, and ATR phosphorylation, thereby activating the ATR-CHK1 DDR pathway and promoting homologous recombination repair after irradiation.","method":"Co-IP of RNF126 with MRN complex, site-specific ubiquitination (K339/K480) mapping, MRE11 exonuclease activity assay, RPA binding assay, ATR phosphorylation measurement, RNF126 depletion in cells and mice","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with site-specific ubiquitination mapping and functional enzymatic assay, single lab","pmids":["36563124"],"is_preprint":false},{"year":2021,"finding":"PARP1 interacts with RNF126 and poly(ADP-ribosyl)ates it, recruiting the PAR-binding E3 ligase CHFR to promote ubiquitination and degradation of RNF126 itself, constituting a post-translational regulatory mechanism that modulates RNF126 protein stability.","method":"Co-IP of PARP1-RNF126, PARylation assay, CHFR recruitment assay, ubiquitination and degradation of RNF126, RNF126 depletion effect on ATR-CHK1 signaling","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus biochemical PAR-dependent recruitment mechanism, single lab","pmids":["34388456"],"is_preprint":false},{"year":2021,"finding":"RNF126 binds 14-3-3σ and prevents both proteins from ubiquitination-mediated degradation, thereby promoting cytoplasmic sequestration of cyclin B1 and CDK1 and maintaining IR-induced G2 arrest.","method":"GST-pulldown, Co-IP, cycloheximide stability assay, ubiquitination assay, immunofluorescence for CDK1/cyclin B1 localization, flow cytometry for cell cycle","journal":"International journal of radiation oncology, biology, physics","confidence":"Medium","confidence_rationale":"Tier 2 — GST-pulldown plus Co-IP plus stability assay, single lab","pmids":["34563636"],"is_preprint":false},{"year":2021,"finding":"RNF126 interacts with TRAF3 and promotes its K63-linked polyubiquitination in the antiviral response. RNF126 also interacts with deubiquitinase OTUB1 and promotes ubiquitination of OTUB1 at C91, reducing OTUB1 catalytic activity toward TRAF3.","method":"Co-IP of RNF126 with TRAF3 and OTUB1, K63-specific ubiquitination assay, OTUB1 C91 ubiquitination and activity assay","journal":"Bioscience, biotechnology, and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with chain-type specificity assay and catalytic residue modification, single lab","pmids":["34643674"],"is_preprint":false},{"year":2021,"finding":"RNF126 interacts with PTEN and promotes its poly-ubiquitination and degradation, binding to the C-terminal RING-domain-containing region of RNF126; this activates the EGFR/PI3K/AKT signaling pathway in bladder cancer.","method":"Co-immunoprecipitation, in vivo ubiquitination assay, domain-mapping, RNF126 knockdown/overexpression with AKT pathway readout","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP and ubiquitination assay, single lab, no in vitro reconstitution","pmids":["33664240"],"is_preprint":false},{"year":2017,"finding":"ANG II activates ERK/GSK3 to phosphorylate HSF1 (S307 by ERK, S303 by GSK3), leading to downregulation of RNF126 expression; reduced RNF126 stabilizes its substrate IGF-IIR, promoting cardiomyocyte hypertrophy.","method":"Western blotting of phospho-HSF1, RNF126 expression after ANG II/ERK/GSK3 modulation, IGF-IIR stability assay, in vivo GSK3 inhibition in cardiac hypertrophy model","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2-3 — pathway epistasis with in vivo validation, single lab, mechanism of RNF126 regulation (not direct ubiquitination substrate characterization)","pmids":["28383811"],"is_preprint":false},{"year":2024,"finding":"RNF126 interacts with FSP1 (AIFM2) and ubiquitinates it at 4KR-2 sites, altering FSP1 subcellular localization away from the plasma membrane, increasing the CoQ/CoQH2 ratio and promoting phospholipid peroxidation and ferroptosis in Group 3 medulloblastoma.","method":"Co-IP, ubiquitination assay at defined sites, subcellular fractionation/localization, CoQ/CoQH2 ratio measurement, ferroptosis assay in vivo and in vitro","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus site-specific ubiquitination plus subcellular localization with functional consequence, single lab","pmids":["38514855"],"is_preprint":false},{"year":2024,"finding":"RNF126 is required for spermatogenesis and male fertility; its RING domain missense variants (E261A, D253N) directly compromise E3 ubiquitin ligase activity. RNF126 deletion in mice causes meiosis I arrest, impaired homologous recombination repair, and increased apoptosis in seminiferous tubules.","method":"Homozygous Rnf126 knockout mouse model, sperm count analysis, histology, RING-domain mutant ubiquitin ligase assay, association with human infertility variants","journal":"Journal of advanced research","confidence":"Medium","confidence_rationale":"Tier 2 — KO mouse with defined meiotic phenotype plus RING-domain mutant enzymatic characterization, single lab","pmids":["39142440"],"is_preprint":false},{"year":2020,"finding":"RNF126 promotes ubiquitination and degradation of p53 in wild-type p53 colorectal cancer cells; RNF126 co-immunoprecipitates with both p53 and p21 forming a triple complex, and RNF126 silencing/overexpression reciprocally regulates p53 and p21 protein (but not mRNA) levels.","method":"Co-IP (triple complex), ubiquitination assay, MG132 rescue, cycloheximide stability, siRNA knockdown and overexpression","journal":"OncoTargets and therapy","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP and ubiquitination assay in cells, single lab, no in vitro reconstitution","pmids":["33149608"],"is_preprint":false},{"year":2022,"finding":"RNF126 ubiquitinates LKB1, promoting its degradation and reducing its stability, thereby enhancing stem-cell-like activity, migration, and angiogenesis in hepatocellular carcinoma.","method":"Co-IP, ubiquitination assay, RNF126 knockdown/overexpression with LKB1 stability and HCC phenotype readouts, in vivo xenograft","journal":"Human cell","confidence":"Low","confidence_rationale":"Tier 3 — Co-IP and cellular ubiquitination assay, single lab, no in vitro reconstitution or mutagenesis","pmids":["36068398"],"is_preprint":false},{"year":2025,"finding":"RNF126 negatively regulates mTORC1 signaling by promoting K63-linked ubiquitination of ILF3, and silencing RNF126 attenuates ILF3-GATOR2 interaction.","method":"Co-IP, K63-specific ubiquitination assay, RNF126 siRNA depletion with mTORC1 activity readout, ILF3-GATOR2 interaction assay","journal":"Cellular signalling","confidence":"Low","confidence_rationale":"Tier 3 — Co-IP and cellular ubiquitination assay, single lab, no in vitro reconstitution","pmids":["40907628"],"is_preprint":false},{"year":2026,"finding":"RNF126 ubiquitinates midnolin (MIDN) at non-canonical cysteine, serine, and threonine residues (C230, C236, S237, T239, S241), not lysines, targeting it for 26S proteasomal degradation. This RNF126-MIDN axis governs EGR1 abundance and PTEN/p53 levels, affecting testicular germ-cell tumor progression.","method":"Co-IP, mass spectrometry-based ubiquitination site mapping, ubiquitination assay, proteasomal degradation assay","journal":"Acta biochimica et biophysica Sinica","confidence":"Medium","confidence_rationale":"Tier 1-2 — MS-based site mapping with non-canonical ubiquitination, cellular validation, single lab","pmids":["41496599"],"is_preprint":false},{"year":2026,"finding":"RNF126 ubiquitinates METTL17 at K116, targeting it for proteasomal degradation. SIRT5 acts as a desuccinylase removing succinylation at METTL17 K274, facilitating RNF126-mediated ubiquitination and degradation in a coordinated SIRT5-METTL17-RNF126 regulatory axis in glioma.","method":"Mass spectrometry, Co-IP, ubiquitination assay (K116), SIRT5 desuccinylation assay (K274), RNF126 depletion with METTL17 stability readout, in vivo xenograft","journal":"Cell & bioscience","confidence":"Medium","confidence_rationale":"Tier 1-2 — site-specific ubiquitination and PTM cross-talk identified by MS and biochemical assays, single lab","pmids":["42021405"],"is_preprint":false},{"year":2026,"finding":"RNF126 and BRAP accumulate in an ATM-dependent manner in late mitotic (anaphase/telophase) cells after DNA damage, and both are required for 53BP1 and RPA2 focus formation, DNA lesion resolution, and cell survival after late mitotic irradiation.","method":"Proteomic analysis of late mitotic irradiated cells, functional assays (53BP1/RPA2 foci, γH2AX resolution, clonogenic survival) with RNF126 depletion","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 — proteomic identification with functional cellular validation, single lab","pmids":["41996237"],"is_preprint":false},{"year":2025,"finding":"RNF126 interacts with BAG6 in sperm cells to regulate sperm synthesis and germ cell development; RNF126 deficiency in mice causes MMAF (multiple morphological abnormalities of the flagella), germ cell apoptosis, and infertility.","method":"Genetic lineage tracing, RNF126 KO mouse, Co-IP of RNF126-BAG6, ultrastructural flagellar analysis, sperm count","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 — KO mouse with defined flagellar phenotype plus Co-IP identifying BAG6 interaction, single lab","pmids":["40410177"],"is_preprint":false}],"current_model":"RNF126 is a RING-type E3 ubiquitin ligase that functions as a central cytosolic quality control enzyme: it is recruited to the Bag6/BAG6 chaperone complex and catalyzes ubiquitination of mislocalized hydrophobic proteins and p97-extracted membrane protein substrates for proteasomal degradation; additionally, it ubiquitinates a diverse set of substrates including Ku80 (promoting NHEJ completion), MRE11 (activating ATR-CHK1 DNA damage signaling), RNF168 (negatively regulating the DDR), PDKs (enabling anoikis-resistant metabolic reprogramming), frataxin (controlling its turnover in Friedreich ataxia), p21/p53/PTEN (driving oncogenic proliferation), FSP1 (modulating ferroptosis), and METTL17 (regulating mitochondrial function), while also acting in a ligase-independent manner by binding E2F1 to transactivate BRCA1 expression for homologous recombination."},"narrative":{"teleology":[{"year":2012,"claim":"Establishing RNF126 as an E3 ligase with an oncogenic substrate: before this work, RNF126's enzymatic targets were unknown; the study showed it directly ubiquitinates p21 for proteasomal degradation, driving G1-S cell cycle progression in cancer cells.","evidence":"Co-IP, ligase-dead mutant comparison, siRNA knockdown with p21 rescue in cancer cell lines","pmids":["23026136"],"confidence":"Medium","gaps":["No in vitro reconstitution with purified components","Substrate selectivity over other CKIs not addressed","Single lab finding"]},{"year":2013,"claim":"Defining ubiquitin chain-type specificity and a role in receptor trafficking: RNF126 was shown to generate K48- or K63-linked chains depending on the E2 partner, and to regulate EGFR endosomal sorting and CI-MPR retrograde transport in a RING-dependent manner, broadening its functional scope beyond cell cycle control.","evidence":"In vitro chain-type reconstitution with UbcH5b and Ubc13/Uev1a; siRNA depletion with EGFR and CI-MPR trafficking assays","pmids":["23418353","24275455"],"confidence":"High","gaps":["Direct EGFR ubiquitination site(s) not mapped","CI-MPR mechanism lacks in vitro reconstitution","Relationship between EGFR and CI-MPR sorting functions unclear"]},{"year":2014,"claim":"Revealing the core quality-control function: RNF126 was identified as the primary BAG6-dependent E3 ligase for mislocalized proteins, recruited via BAG6's Ubl domain and ubiquitinating juxtahydrophobic lysines on clients—establishing RNF126 as a central cytosolic triage enzyme.","evidence":"Full in vitro reconstitution with purified components, cell fractionation, RNF126 depletion","pmids":["24981174"],"confidence":"High","gaps":["Structural basis of Ubl-mediated recruitment not resolved","In vivo substrate scope not defined genome-wide"]},{"year":2015,"claim":"Uncovering a ligase-independent transcriptional function: RNF126 was found to bind E2F1 and co-activate BRCA1 transcription independently of its RING catalytic activity, promoting homologous recombination—demonstrating a non-enzymatic role distinct from its ubiquitin ligase function.","evidence":"Co-IP of RNF126-E2F1, ChIP at BRCA1 promoter, RING-independent rescue of HR, PARP inhibitor sensitivity","pmids":["26234677"],"confidence":"Medium","gaps":["Single lab; independent validation needed","Structural determinant of E2F1 binding beyond 11-aa deletion not mapped","Unclear whether E2F1-dependent function is tissue-specific"]},{"year":2016,"claim":"Linking RNF126 to metabolic reprogramming: RNF126 was shown to ubiquitinate pyruvate dehydrogenase kinases (PDKs) for degradation, increasing PDH activity and acetyl-CoA flux, thereby supporting anoikis resistance—connecting its E3 ligase activity to cancer metabolism.","evidence":"PDK ubiquitination assay, metabolic flux analysis, soft-agar and in vivo tumorigenicity assays","pmids":["27462466"],"confidence":"Medium","gaps":["Specific PDK isoform selectivity not fully defined","No in vitro reconstitution with purified components"]},{"year":2017,"claim":"Establishing roles in NHEJ and frataxin homeostasis: RNF126 was shown to ubiquitinate Ku80 (with UBE2D3) to promote Ku release from DSBs and NHEJ completion, and independently to ubiquitinate frataxin for proteasomal degradation—identifying it as a therapeutic target for Friedreich ataxia.","evidence":"In vitro ubiquitylation, Ku80 K→R site mutagenesis, chromatin fractionation, frataxin stability in FRDA patient cells","pmids":["27895153","28228265"],"confidence":"High","gaps":["Whether Ku80 ubiquitination is BAG6-dependent is unknown","Frataxin ubiquitination sites not mapped","RNF126 inhibitor pharmacology not explored"]},{"year":2017,"claim":"Placing RNF126 as a negative regulator in the DDR ubiquitin cascade: overexpression studies showed RNF126 suppresses RNF168-mediated H2A K13/15 monoubiquitination and 53BP1 foci while leaving γH2AX, MDC1, and RNF8 foci intact, positioning RNF126 between RNF8 and RNF168.","evidence":"IR-induced foci hierarchy analysis, H2A ubiquitination assay, NHEJ reporter assay","pmids":["29167269"],"confidence":"Medium","gaps":["Endogenous regulation versus overexpression artifacts not fully resolved","Mechanism of RNF168 inhibition (direct ubiquitination vs. competition) not yet distinguished"]},{"year":2018,"claim":"Confirming direct RNF168 ubiquitination: RNF126 was shown to be recruited to damage sites in an RNF8-dependent manner and to directly ubiquitinate RNF168, resolving the mechanism of its negative regulation of the DDR.","evidence":"UV laser micro-irradiation recruitment, Co-IP of RNF126-RNF168, catalytic-dead mutant discrimination","pmids":["30529286"],"confidence":"Medium","gaps":["Ubiquitination sites on RNF168 not mapped","Chain type used on RNF168 not determined","Single lab"]},{"year":2020,"claim":"Extending the quality-control paradigm to ERAD: RNF126 was shown to reubiquitinate p97/VCP-extracted membrane proteins via BAG6, reconstituting the ternary complex and demonstrating that cytosolic reubiquitination is essential for proteasomal targeting of retrotranslocated substrates.","evidence":"In vitro reconstitution with purified factors, pulse-chase turnover, cellular depletion","pmids":["32645369"],"confidence":"High","gaps":["Selectivity for specific ERAD substrates not comprehensively defined","Interplay with other cytosolic reubiquitination E3s (e.g., CHIP) not addressed"]},{"year":2021,"claim":"Expanding substrate range to innate immunity and cell cycle checkpoint maintenance: RNF126 was shown to K63-ubiquitinate TRAF3 for antiviral signaling and to stabilize 14-3-3σ for G2 arrest maintenance, while its own stability was found to be regulated by PARP1-mediated PARylation and CHFR-dependent ubiquitination.","evidence":"Co-IP with chain-type specificity assays, PARylation/CHFR recruitment assay, cycloheximide stability, cell cycle analysis","pmids":["34643674","34563636","34388456"],"confidence":"Medium","gaps":["TRAF3 ubiquitination sites not mapped","14-3-3σ stabilization mechanism (direct deubiquitination vs. shielding) not resolved","PARP1-CHFR-RNF126 axis not reconstituted in vitro"]},{"year":2022,"claim":"Activating ATR signaling through MRE11 ubiquitination: RNF126 was found to ubiquitinate MRE11 at K339/K480, enhancing its exonuclease activity and RPA binding, thereby activating ATR-CHK1 and promoting HR—adding a positive arm to RNF126's DDR involvement.","evidence":"Co-IP with MRN complex, site-specific ubiquitination mapping, exonuclease activity assay, ATR phosphorylation, KO mouse","pmids":["36563124"],"confidence":"Medium","gaps":["How RNF126 simultaneously promotes (MRE11 activation) and inhibits (RNF168 degradation) DDR signaling is not integrated","Chain type on MRE11 not defined","Single lab"]},{"year":2024,"claim":"Connecting RNF126 to ferroptosis: RNF126 was shown to ubiquitinate FSP1 at defined sites, displacing it from the plasma membrane, increasing the CoQ/CoQH₂ ratio, and promoting ferroptosis in medulloblastoma—identifying a new mechanism for ferroptosis regulation.","evidence":"Co-IP, site-specific ubiquitination, subcellular fractionation, CoQ ratio measurement, in vivo ferroptosis assay","pmids":["38514855"],"confidence":"Medium","gaps":["Whether BAG6 is involved in FSP1 targeting is unknown","Generalizability beyond Group 3 medulloblastoma not tested"]},{"year":2024,"claim":"Demonstrating physiological requirement for male fertility: RNF126 knockout mice exhibited meiosis I arrest, flagellar defects (MMAF), and complete male infertility; RING-domain missense variants abolished ligase activity, and RNF126-BAG6 interaction was confirmed in sperm cells.","evidence":"Rnf126 KO mouse, flagellar ultrastructure, RING-domain mutant enzymatic assay, Co-IP in germ cells, association with human infertility variants","pmids":["39142440","40410177"],"confidence":"Medium","gaps":["Specific spermatogenesis substrates not identified","Whether BAG6 quality control of tail-anchored proteins underlies flagellar defect is speculative"]},{"year":2025,"claim":"Expanding the quality-control paradigm to mitochondrial import stress: RNF126 was shown to interact with substrate-engaged UBQLN1 and ubiquitinate unimported mitochondrial precursors (ATP5G1) for proteasomal clearance, defining a UBQLN1-dependent branch of RNF126 quality control distinct from BAG6.","evidence":"In vitro reconstitution of RNF126-UBQLN1-ATP5G1 ternary complex, Co-IP, proteasomal degradation assay","pmids":["40086734"],"confidence":"High","gaps":["Scope of UBQLN1-dependent substrates unknown","Whether BAG6 and UBQLN1 pathways are competitive or parallel not addressed"]},{"year":2025,"claim":"Non-canonical ubiquitination and late-mitotic DDR functions: RNF126 was found to ubiquitinate midnolin at non-canonical Cys/Ser/Thr residues, and to accumulate with BRAP in an ATM-dependent manner during late mitosis to promote 53BP1/RPA2 focus formation and DNA lesion resolution.","evidence":"MS-based non-canonical ubiquitination site mapping, proteomic analysis of late-mitotic irradiated cells, 53BP1/RPA2 foci and clonogenic survival with RNF126 depletion","pmids":["41496599","41996237"],"confidence":"Medium","gaps":["Physiological significance of non-lysine ubiquitination on midnolin stability versus function not clarified","BRAP-RNF126 physical interaction and mechanism not demonstrated"]},{"year":null,"claim":"Key unresolved questions include: how RNF126 balances its positive (MRE11, Ku80, BRCA1) and negative (RNF168) roles in the DDR in a context-dependent manner; the structural basis of BAG6 Ubl-mediated recruitment; the full in vivo substrate repertoire; and whether its quality-control and DDR functions are coordinately regulated.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of RNF126 in complex with BAG6 or substrates","Genome-wide substrate identification not performed","Context-dependent switching between DDR-promoting and DDR-inhibiting functions not mechanistically explained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,3,4,5,8,10,12,18,23,24]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,2,4,5]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[9]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[4,10,12,25]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[4,25]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,2,3,8,23,24]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[4,10,11,12,25]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[3,14]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[18]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[5,6]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[15]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[19,26]}],"complexes":["BAG6 chaperone complex"],"partners":["BAG6","UBQLN1","XRCC5","RNF168","MRE11","E2F1","PARP1","SFN"],"other_free_text":[]},"mechanistic_narrative":"RNF126 is a RING-type E3 ubiquitin ligase that functions as a central effector of cytosolic protein quality control and participates in DNA damage signaling, membrane protein trafficking, and metabolic regulation. RNF126 is recruited to the BAG6 chaperone complex via its N-terminal Ubl domain and ubiquitinates mislocalized hydrophobic proteins, p97/VCP-extracted membrane protein substrates, and unimported mitochondrial precursors (via UBQLN1), targeting them for proteasomal degradation [PMID:24981174, PMID:32645369, PMID:40086734]. In the DNA damage response, RNF126 ubiquitinates Ku80 to promote NHEJ completion [PMID:27895153], ubiquitinates MRE11 to activate ATR-CHK1 signaling and homologous recombination [PMID:36563124], and negatively regulates RNF168-mediated H2A ubiquitination to modulate 53BP1 recruitment [PMID:30529286, PMID:29167269]; it also promotes BRCA1 expression in a ligase-independent manner by co-activating E2F1 [PMID:26234677]. RNF126 ubiquitinates diverse additional substrates—including p21, p53, frataxin, PDKs, PTEN, and FSP1—linking it to cell cycle progression, ferroptosis, and metabolic reprogramming [PMID:23026136, PMID:28228265, PMID:27462466, PMID:33664240, PMID:38514855], and its deletion in mice causes meiotic arrest and male infertility with multiple morphological abnormalities of the sperm flagella [PMID:39142440, PMID:40410177]."},"prefetch_data":{"uniprot":{"accession":"Q9BV68","full_name":"E3 ubiquitin-protein ligase RNF126","aliases":["RING finger protein 126"],"length_aa":311,"mass_kda":33.9,"function":"E3 ubiquitin-protein ligase that mediates ubiquitination oF target proteins (PubMed:23277564, PubMed:24275455, PubMed:24981174, PubMed:36563124). Depending on the associated E2 ligase, mediates 'Lys-27'-, 'Lys-29'-, 'Lys-48'- and/or 'Lys-63'-linked polyubiquitination of substrates (PubMed:36563124). Part of a BAG6-dependent quality control process ensuring that proteins of the secretory pathway that are mislocalized to the cytosol are degraded by the proteasome. Probably acts by providing the ubiquitin ligase activity associated with the BAG6 complex and be responsible for ubiquitination of the hydrophobic mislocalized proteins and their targeting to the proteasome (PubMed:24981174, PubMed:29042515). May also play a role in the endosomal recycling of IGF2R, the cation-independent mannose-6-phosphate receptor (PubMed:24275455). May play a role in the endosomal sorting and degradation of several membrane receptors including EGFR, FLT3, MET and CXCR4, by mediating their ubiquitination (PubMed:23418353). By ubiquitinating CDKN1A/p21 and targeting it for degradation, may also promote cell proliferation (PubMed:23026136). May monoubiquitinate AICDA (PubMed:23277564). Acts as a regulator of DNA repair by mediating 'Lys-27'- and 'Lys-29'-linked polyubiquitination of MRE11, thereby promoting the exonuclease activity of MRE11 (PubMed:36563124)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9BV68/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RNF126","classification":"Not Classified","n_dependent_lines":15,"n_total_lines":1208,"dependency_fraction":0.012417218543046357},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"BAG6","stoichiometry":4.0},{"gene":"GET4","stoichiometry":4.0}],"url":"https://opencell.sf.czbiohub.org/search/RNF126","total_profiled":1310},"omim":[{"mim_id":"615177","title":"RING FINGER PROTEIN 126; RNF126","url":"https://www.omim.org/entry/615177"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RNF126"},"hgnc":{"alias_symbol":["FLJ20552"],"prev_symbol":[]},"alphafold":{"accession":"Q9BV68","domains":[{"cath_id":"-","chopping":"9-39","consensus_level":"high","plddt":85.731,"start":9,"end":39},{"cath_id":"3.30.40.10","chopping":"202-261","consensus_level":"high","plddt":78.7965,"start":202,"end":261}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BV68","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BV68-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BV68-F1-predicted_aligned_error_v6.png","plddt_mean":61.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RNF126","jax_strain_url":"https://www.jax.org/strain/search?query=RNF126"},"sequence":{"accession":"Q9BV68","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BV68.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BV68/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BV68"}},"corpus_meta":[{"pmid":"24981174","id":"PMC_24981174","title":"Cytosolic quality control of mislocalized proteins requires RNF126 recruitment to Bag6.","date":"2014","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/24981174","citation_count":150,"is_preprint":false},{"pmid":"23026136","id":"PMC_23026136","title":"E3 ubiquitin ligase RNF126 promotes cancer cell proliferation by targeting the tumor suppressor p21 for ubiquitin-mediated degradation.","date":"2012","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/23026136","citation_count":64,"is_preprint":false},{"pmid":"36563124","id":"PMC_36563124","title":"RNF126-Mediated MRE11 Ubiquitination Activates the DNA Damage Response and Confers Resistance of Triple-Negative Breast Cancer to Radiotherapy.","date":"2022","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/36563124","citation_count":57,"is_preprint":false},{"pmid":"27895153","id":"PMC_27895153","title":"Ubiquitylation of Ku80 by RNF126 Promotes Completion of Nonhomologous End Joining-Mediated DNA Repair.","date":"2017","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/27895153","citation_count":52,"is_preprint":false},{"pmid":"23418353","id":"PMC_23418353","title":"The E3 ubiquitin ligases RNF126 and Rabring7 regulate endosomal sorting of the epidermal growth factor receptor.","date":"2013","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/23418353","citation_count":48,"is_preprint":false},{"pmid":"33664240","id":"PMC_33664240","title":"E3 ubiquitin ligase RNF126 affects bladder cancer progression through regulation of PTEN stability.","date":"2021","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/33664240","citation_count":47,"is_preprint":false},{"pmid":"27462466","id":"PMC_27462466","title":"The ERK signaling target RNF126 regulates anoikis resistance in cancer cells by changing the mitochondrial metabolic flux.","date":"2016","source":"Cell 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ferroptosis.","date":"2024","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/38514855","citation_count":22,"is_preprint":false},{"pmid":"27227488","id":"PMC_27227488","title":"E3 Ubiquitin ligase RNF126 regulates the progression of tongue cancer.","date":"2016","source":"Cancer medicine","url":"https://pubmed.ncbi.nlm.nih.gov/27227488","citation_count":21,"is_preprint":false},{"pmid":"30529286","id":"PMC_30529286","title":"RNF126 Quenches RNF168 Function in the DNA Damage Response.","date":"2018","source":"Genomics, proteomics & bioinformatics","url":"https://pubmed.ncbi.nlm.nih.gov/30529286","citation_count":20,"is_preprint":false},{"pmid":"32147403","id":"PMC_32147403","title":"Roles of RNF126 and BCA2 E3 ubiquitin ligases in DNA damage repair signaling and targeted cancer therapy.","date":"2020","source":"Pharmacological research","url":"https://pubmed.ncbi.nlm.nih.gov/32147403","citation_count":20,"is_preprint":false},{"pmid":"24275455","id":"PMC_24275455","title":"The ubiquitin ligase RNF126 regulates the retrograde sorting of the cation-independent mannose 6-phosphate receptor.","date":"2013","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/24275455","citation_count":15,"is_preprint":false},{"pmid":"33149608","id":"PMC_33149608","title":"Overexpression of RNF126 Promotes the Development of Colorectal Cancer via Enhancing p53 Ubiquitination and Degradation.","date":"2020","source":"OncoTargets and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/33149608","citation_count":15,"is_preprint":false},{"pmid":"29167269","id":"PMC_29167269","title":"Ring finger protein 126 (RNF126) suppresses ionizing radiation-induced p53-binding protein 1 (53BP1) focus formation.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29167269","citation_count":14,"is_preprint":false},{"pmid":"38515383","id":"PMC_38515383","title":"ZNF263 cooperates with ZNF31 to promote the drug resistance and EMT of pancreatic cancer through transactivating RNF126.","date":"2024","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/38515383","citation_count":9,"is_preprint":false},{"pmid":"34388456","id":"PMC_34388456","title":"CHFR-mediated degradation of RNF126 confers sensitivity to PARP inhibitors in triple-negative breast cancer cells.","date":"2021","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/34388456","citation_count":8,"is_preprint":false},{"pmid":"34563636","id":"PMC_34563636","title":"A Novel Role for RNF126 in the Promotion of G2 Arrest via Interaction With 14-3-3σ.","date":"2021","source":"International journal of radiation oncology, biology, physics","url":"https://pubmed.ncbi.nlm.nih.gov/34563636","citation_count":7,"is_preprint":false},{"pmid":"36068398","id":"PMC_36068398","title":"RNF126 contributes to stem cell-like properties and metastasis in hepatocellular carcinoma through ubiquitination and degradation of LKB1.","date":"2022","source":"Human cell","url":"https://pubmed.ncbi.nlm.nih.gov/36068398","citation_count":5,"is_preprint":false},{"pmid":"39142440","id":"PMC_39142440","title":"An essential role of the E3 ubiquitin ligase RNF126 in ensuring meiosis I completion during spermatogenesis.","date":"2024","source":"Journal of advanced research","url":"https://pubmed.ncbi.nlm.nih.gov/39142440","citation_count":4,"is_preprint":false},{"pmid":"40410177","id":"PMC_40410177","title":"Absence of Rnf126 causes male infertility with multiple morphological abnormalities of the sperm flagella.","date":"2025","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/40410177","citation_count":3,"is_preprint":false},{"pmid":"38728911","id":"PMC_38728911","title":"Design, synthesis and biological evaluation of new RNF126-based p300/CBP degraders.","date":"2024","source":"Bioorganic 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sciences","url":"https://pubmed.ncbi.nlm.nih.gov/41465608","citation_count":2,"is_preprint":false},{"pmid":"37760968","id":"PMC_37760968","title":"RNF126, 168 and CUL1: The Potential Utilization of Multi-Functional E3 Ubiquitin Ligases in Genome Maintenance for Cancer Therapy.","date":"2023","source":"Biomedicines","url":"https://pubmed.ncbi.nlm.nih.gov/37760968","citation_count":2,"is_preprint":false},{"pmid":"40615482","id":"PMC_40615482","title":"Ring-finger protein RNF126 promotes prostate cancer progression via regulation of MBNL1.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/40615482","citation_count":1,"is_preprint":false},{"pmid":"40907628","id":"PMC_40907628","title":"RNF126 suppresses amino acid-mediated mTORC1 signaling pathway by ubiquitinating ILF3 in HEK293T cells.","date":"2025","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/40907628","citation_count":1,"is_preprint":false},{"pmid":"41867716","id":"PMC_41867716","title":"An Optimized RNF126-Targeting Covalent Handle for Molecular Glue Degraders.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41867716","citation_count":1,"is_preprint":false},{"pmid":"40251363","id":"PMC_40251363","title":"RNF126 Promotes Ovarian Cancer Progression by Reprogramming Lipid Metabolism Through Degradation of ACAP2.","date":"2025","source":"Biochemical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/40251363","citation_count":1,"is_preprint":false},{"pmid":"41496599","id":"PMC_41496599","title":"RNF126 writes a non-canonical ubiquitin code on midnolin to tune protein stability.","date":"2026","source":"Acta biochimica et biophysica 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of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2014,\n      \"finding\": \"RNF126 is the primary Bag6-dependent E3 ubiquitin ligase for cytosolic quality control of mislocalized proteins (MLPs). RNF126 is recruited to the N-terminal Ubl domain of Bag6 and preferentially ubiquitinates juxtahydrophobic lysine residues on Bag6-associated clients. Bag6-dependent ubiquitination was fully reconstituted with purified components.\",\n      \"method\": \"In vitro reconstitution with purified components, cell fractionation, Bag6-dependent ubiquitination assay, RNF126 depletion in cells\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with purified components plus cellular depletion, single rigorous paper with multiple orthogonal methods\",\n      \"pmids\": [\"24981174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RNF126 catalyzes reubiquitination of p97/VCP-extracted membrane proteins in the cytosol. RNF126 interacts with BAG6, which captures p97-liberated substrates, and a ternary complex (RNF126-BAG6-substrate) was reconstituted with purified factors to demonstrate reubiquitination required for proteasomal targeting.\",\n      \"method\": \"In vitro reconstitution with purified factors, cellular depletion of RNF126, stabilization of extracted intermediates, pulse-chase turnover assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted with purified components plus multiple cellular phenotypes, single rigorous paper\",\n      \"pmids\": [\"32645369\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF126 interacts with substrate-engaged UBQLN1 via UBQLN1's ubiquitin-associated (UBA) domain and catalyzes ubiquitination of unimported mitochondrial membrane protein precursors (e.g., ATP5G1), promoting their proteasomal degradation during mitochondrial stress. Ternary complex formation (RNF126-UBQLN1-ATP5G1) was reconstituted in vitro.\",\n      \"method\": \"In vitro reconstitution of ternary complex, co-IP, ubiquitination assay, proteasomal degradation assay with RNF126 depletion\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution of ternary complex plus cellular validation\",\n      \"pmids\": [\"40086734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"RNF126 acts as an E3 ubiquitin ligase that directly interacts with p21 and promotes its ubiquitination and proteasomal degradation in an E3 ligase activity-dependent manner, driving cancer cell proliferation and G1-S progression.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay with overexpression of WT vs. ligase-dead RNF126, siRNA knockdown with protein stability assay, rescue by p21 depletion\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, activity-dependent ubiquitination assay, cellular rescue experiment, single lab\",\n      \"pmids\": [\"23026136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RNF126 ubiquitylates Ku80 (with UBE2D3 as E2) at DSBs, promoting Ku70/80 dissociation from damaged DNA and completion of NHEJ repair. Lysine-to-arginine mutation of Ku80 ubiquitylation sites delayed Ku70/80 release from chromatin.\",\n      \"method\": \"Identification by mass spectrometry interactome, Co-IP, in vitro ubiquitylation assay, Ku80 ubiquitylation-site mutagenesis (K→R), chromatin fractionation, RNF126 knockdown with DSB repair assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro ubiquitylation assay plus site mutagenesis plus cellular functional consequence, moderate evidence from single lab with multiple methods\",\n      \"pmids\": [\"27895153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RNF126 specifies K48-linked ubiquitin chains with UbcH5b and K63-linked chains with Ubc13/Uev1a in vitro. RNF126 associates with EGFR via a ubiquitin-binding zinc finger domain and promotes EGFR ubiquitylation downstream of c-Cbl, regulating EGFR endosomal sorting and ESCRT-II stability.\",\n      \"method\": \"In vitro ubiquitin chain-type assay, Co-IP, siRNA depletion with EGFR trafficking/degradation assay, multivesicular body quantification\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro chain-type reconstitution plus cellular epistasis with c-Cbl plus multiple trafficking phenotypes\",\n      \"pmids\": [\"23418353\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RNF126 regulates retrograde sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR); its RING finger domain (ligase activity) is required, as RING-domain mutant fails to rescue CI-MPR missorting upon RNF126 depletion.\",\n      \"method\": \"siRNA stable knockdown, RING-domain mutant rescue experiment, CI-MPR localization by immunofluorescence, lysosomal degradation assay, cathepsin D missorting\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — localization plus RING-mutant rescue, single lab, no in vitro reconstitution\",\n      \"pmids\": [\"24275455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RNF126 acts as an E3 ubiquitin ligase for pyruvate dehydrogenase kinases (PDKs), targeting them for proteasomal degradation. This increases PDH activity and pyruvate-to-acetyl-CoA flux, supporting TCA cycle activity and anoikis resistance in cancer cells. RNF126 expression is controlled by ERK signaling.\",\n      \"method\": \"Ubiquitination assay for PDKs, proteasomal degradation assay, metabolic flux analysis, soft-agar colony assay, RNF126 depletion in vivo tumorigenicity\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ubiquitination of PDKs with metabolic readout, cellular and in vivo confirmation, single lab\",\n      \"pmids\": [\"27462466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RNF126 is the E3 ligase that directly ubiquitinates frataxin, targeting it for proteasomal degradation. RNF126 interacts with frataxin and promotes its ubiquitination in a catalytic activity-dependent manner both in vivo and in vitro; RNF126 depletion raises frataxin levels in Friedreich ataxia patient-derived cells.\",\n      \"method\": \"Co-immunoprecipitation, in vitro and in vivo ubiquitination assay, catalytic mutant comparison, protein stability assay in FRDA patient cells\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro ubiquitination plus cellular validation in disease-relevant (FRDA patient) cells with multiple methods\",\n      \"pmids\": [\"28228265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RNF126 promotes homologous recombination by facilitating BRCA1 expression through direct binding to transcription factor E2F1, enhancing E2F1-mediated transactivation of the BRCA1 promoter; this function is independent of RNF126 E3 ligase activity. An 11-amino-acid deletion mutant acts dominantly negatively by preventing E2F1 binding.\",\n      \"method\": \"Co-IP of RNF126-E2F1, BRCA1 promoter luciferase assay, ChIP for E2F1 at BRCA1 promoter, deletion mutant analysis, HR repair assay, ionizing radiation and PARP inhibitor sensitivity\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Co-IP, ChIP, promoter assay, mutant), single lab\",\n      \"pmids\": [\"26234677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RNF126 is recruited to DNA damage sites in an RNF8-dependent manner, directly interacts with RNF168, and ubiquitinates RNF168. RNF126 overexpression (but not catalytic-dead mutant) diminishes RNF168-mediated H2AX ubiquitination and downstream 53BP1/RAP80/BRCA1 focus formation, placing RNF126 between RNF8 and RNF168 in the DDR cascade.\",\n      \"method\": \"UV laser micro-irradiation recruitment, Co-IP of RNF126-RNF168, ubiquitination assay with WT vs. CC229/232AA catalytic mutant, HR repair assay\",\n      \"journal\": \"Genomics, proteomics & bioinformatics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — catalytic mutant discrimination, epistasis placement between RNF8 and RNF168, single lab\",\n      \"pmids\": [\"30529286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RNF126 overexpression abolishes 53BP1 iRIF formation and RNF168-mediated H2A monoubiquitination at K13/15, while γH2AX, MDC1, and RNF8 foci are maintained, placing RNF126 as a negative regulator between RNF8 and RNF168 in the DDR and inhibiting NHEJ.\",\n      \"method\": \"Ionizing radiation-induced foci screen, overexpression and focus formation analysis, H2A ubiquitination assay, NHEJ assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis within DDR pathway established by focus formation hierarchy, single lab\",\n      \"pmids\": [\"29167269\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF126 physically associates with the MRE11-RAD50-NBS1 complex and ubiquitinates MRE11 at K339 and K480, increasing MRE11 DNA exonuclease activity, RPA binding, and ATR phosphorylation, thereby activating the ATR-CHK1 DDR pathway and promoting homologous recombination repair after irradiation.\",\n      \"method\": \"Co-IP of RNF126 with MRN complex, site-specific ubiquitination (K339/K480) mapping, MRE11 exonuclease activity assay, RPA binding assay, ATR phosphorylation measurement, RNF126 depletion in cells and mice\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with site-specific ubiquitination mapping and functional enzymatic assay, single lab\",\n      \"pmids\": [\"36563124\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PARP1 interacts with RNF126 and poly(ADP-ribosyl)ates it, recruiting the PAR-binding E3 ligase CHFR to promote ubiquitination and degradation of RNF126 itself, constituting a post-translational regulatory mechanism that modulates RNF126 protein stability.\",\n      \"method\": \"Co-IP of PARP1-RNF126, PARylation assay, CHFR recruitment assay, ubiquitination and degradation of RNF126, RNF126 depletion effect on ATR-CHK1 signaling\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus biochemical PAR-dependent recruitment mechanism, single lab\",\n      \"pmids\": [\"34388456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF126 binds 14-3-3σ and prevents both proteins from ubiquitination-mediated degradation, thereby promoting cytoplasmic sequestration of cyclin B1 and CDK1 and maintaining IR-induced G2 arrest.\",\n      \"method\": \"GST-pulldown, Co-IP, cycloheximide stability assay, ubiquitination assay, immunofluorescence for CDK1/cyclin B1 localization, flow cytometry for cell cycle\",\n      \"journal\": \"International journal of radiation oncology, biology, physics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — GST-pulldown plus Co-IP plus stability assay, single lab\",\n      \"pmids\": [\"34563636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF126 interacts with TRAF3 and promotes its K63-linked polyubiquitination in the antiviral response. RNF126 also interacts with deubiquitinase OTUB1 and promotes ubiquitination of OTUB1 at C91, reducing OTUB1 catalytic activity toward TRAF3.\",\n      \"method\": \"Co-IP of RNF126 with TRAF3 and OTUB1, K63-specific ubiquitination assay, OTUB1 C91 ubiquitination and activity assay\",\n      \"journal\": \"Bioscience, biotechnology, and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with chain-type specificity assay and catalytic residue modification, single lab\",\n      \"pmids\": [\"34643674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF126 interacts with PTEN and promotes its poly-ubiquitination and degradation, binding to the C-terminal RING-domain-containing region of RNF126; this activates the EGFR/PI3K/AKT signaling pathway in bladder cancer.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assay, domain-mapping, RNF126 knockdown/overexpression with AKT pathway readout\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP and ubiquitination assay, single lab, no in vitro reconstitution\",\n      \"pmids\": [\"33664240\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ANG II activates ERK/GSK3 to phosphorylate HSF1 (S307 by ERK, S303 by GSK3), leading to downregulation of RNF126 expression; reduced RNF126 stabilizes its substrate IGF-IIR, promoting cardiomyocyte hypertrophy.\",\n      \"method\": \"Western blotting of phospho-HSF1, RNF126 expression after ANG II/ERK/GSK3 modulation, IGF-IIR stability assay, in vivo GSK3 inhibition in cardiac hypertrophy model\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — pathway epistasis with in vivo validation, single lab, mechanism of RNF126 regulation (not direct ubiquitination substrate characterization)\",\n      \"pmids\": [\"28383811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF126 interacts with FSP1 (AIFM2) and ubiquitinates it at 4KR-2 sites, altering FSP1 subcellular localization away from the plasma membrane, increasing the CoQ/CoQH2 ratio and promoting phospholipid peroxidation and ferroptosis in Group 3 medulloblastoma.\",\n      \"method\": \"Co-IP, ubiquitination assay at defined sites, subcellular fractionation/localization, CoQ/CoQH2 ratio measurement, ferroptosis assay in vivo and in vitro\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus site-specific ubiquitination plus subcellular localization with functional consequence, single lab\",\n      \"pmids\": [\"38514855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF126 is required for spermatogenesis and male fertility; its RING domain missense variants (E261A, D253N) directly compromise E3 ubiquitin ligase activity. RNF126 deletion in mice causes meiosis I arrest, impaired homologous recombination repair, and increased apoptosis in seminiferous tubules.\",\n      \"method\": \"Homozygous Rnf126 knockout mouse model, sperm count analysis, histology, RING-domain mutant ubiquitin ligase assay, association with human infertility variants\",\n      \"journal\": \"Journal of advanced research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with defined meiotic phenotype plus RING-domain mutant enzymatic characterization, single lab\",\n      \"pmids\": [\"39142440\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RNF126 promotes ubiquitination and degradation of p53 in wild-type p53 colorectal cancer cells; RNF126 co-immunoprecipitates with both p53 and p21 forming a triple complex, and RNF126 silencing/overexpression reciprocally regulates p53 and p21 protein (but not mRNA) levels.\",\n      \"method\": \"Co-IP (triple complex), ubiquitination assay, MG132 rescue, cycloheximide stability, siRNA knockdown and overexpression\",\n      \"journal\": \"OncoTargets and therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and ubiquitination assay in cells, single lab, no in vitro reconstitution\",\n      \"pmids\": [\"33149608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF126 ubiquitinates LKB1, promoting its degradation and reducing its stability, thereby enhancing stem-cell-like activity, migration, and angiogenesis in hepatocellular carcinoma.\",\n      \"method\": \"Co-IP, ubiquitination assay, RNF126 knockdown/overexpression with LKB1 stability and HCC phenotype readouts, in vivo xenograft\",\n      \"journal\": \"Human cell\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and cellular ubiquitination assay, single lab, no in vitro reconstitution or mutagenesis\",\n      \"pmids\": [\"36068398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF126 negatively regulates mTORC1 signaling by promoting K63-linked ubiquitination of ILF3, and silencing RNF126 attenuates ILF3-GATOR2 interaction.\",\n      \"method\": \"Co-IP, K63-specific ubiquitination assay, RNF126 siRNA depletion with mTORC1 activity readout, ILF3-GATOR2 interaction assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and cellular ubiquitination assay, single lab, no in vitro reconstitution\",\n      \"pmids\": [\"40907628\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RNF126 ubiquitinates midnolin (MIDN) at non-canonical cysteine, serine, and threonine residues (C230, C236, S237, T239, S241), not lysines, targeting it for 26S proteasomal degradation. This RNF126-MIDN axis governs EGR1 abundance and PTEN/p53 levels, affecting testicular germ-cell tumor progression.\",\n      \"method\": \"Co-IP, mass spectrometry-based ubiquitination site mapping, ubiquitination assay, proteasomal degradation assay\",\n      \"journal\": \"Acta biochimica et biophysica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — MS-based site mapping with non-canonical ubiquitination, cellular validation, single lab\",\n      \"pmids\": [\"41496599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RNF126 ubiquitinates METTL17 at K116, targeting it for proteasomal degradation. SIRT5 acts as a desuccinylase removing succinylation at METTL17 K274, facilitating RNF126-mediated ubiquitination and degradation in a coordinated SIRT5-METTL17-RNF126 regulatory axis in glioma.\",\n      \"method\": \"Mass spectrometry, Co-IP, ubiquitination assay (K116), SIRT5 desuccinylation assay (K274), RNF126 depletion with METTL17 stability readout, in vivo xenograft\",\n      \"journal\": \"Cell & bioscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — site-specific ubiquitination and PTM cross-talk identified by MS and biochemical assays, single lab\",\n      \"pmids\": [\"42021405\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RNF126 and BRAP accumulate in an ATM-dependent manner in late mitotic (anaphase/telophase) cells after DNA damage, and both are required for 53BP1 and RPA2 focus formation, DNA lesion resolution, and cell survival after late mitotic irradiation.\",\n      \"method\": \"Proteomic analysis of late mitotic irradiated cells, functional assays (53BP1/RPA2 foci, γH2AX resolution, clonogenic survival) with RNF126 depletion\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — proteomic identification with functional cellular validation, single lab\",\n      \"pmids\": [\"41996237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF126 interacts with BAG6 in sperm cells to regulate sperm synthesis and germ cell development; RNF126 deficiency in mice causes MMAF (multiple morphological abnormalities of the flagella), germ cell apoptosis, and infertility.\",\n      \"method\": \"Genetic lineage tracing, RNF126 KO mouse, Co-IP of RNF126-BAG6, ultrastructural flagellar analysis, sperm count\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with defined flagellar phenotype plus Co-IP identifying BAG6 interaction, single lab\",\n      \"pmids\": [\"40410177\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RNF126 is a RING-type E3 ubiquitin ligase that functions as a central cytosolic quality control enzyme: it is recruited to the Bag6/BAG6 chaperone complex and catalyzes ubiquitination of mislocalized hydrophobic proteins and p97-extracted membrane protein substrates for proteasomal degradation; additionally, it ubiquitinates a diverse set of substrates including Ku80 (promoting NHEJ completion), MRE11 (activating ATR-CHK1 DNA damage signaling), RNF168 (negatively regulating the DDR), PDKs (enabling anoikis-resistant metabolic reprogramming), frataxin (controlling its turnover in Friedreich ataxia), p21/p53/PTEN (driving oncogenic proliferation), FSP1 (modulating ferroptosis), and METTL17 (regulating mitochondrial function), while also acting in a ligase-independent manner by binding E2F1 to transactivate BRCA1 expression for homologous recombination.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RNF126 is a RING-type E3 ubiquitin ligase that functions as a central effector of cytosolic protein quality control and participates in DNA damage signaling, membrane protein trafficking, and metabolic regulation. RNF126 is recruited to the BAG6 chaperone complex via its N-terminal Ubl domain and ubiquitinates mislocalized hydrophobic proteins, p97/VCP-extracted membrane protein substrates, and unimported mitochondrial precursors (via UBQLN1), targeting them for proteasomal degradation [PMID:24981174, PMID:32645369, PMID:40086734]. In the DNA damage response, RNF126 ubiquitinates Ku80 to promote NHEJ completion [PMID:27895153], ubiquitinates MRE11 to activate ATR-CHK1 signaling and homologous recombination [PMID:36563124], and negatively regulates RNF168-mediated H2A ubiquitination to modulate 53BP1 recruitment [PMID:30529286, PMID:29167269]; it also promotes BRCA1 expression in a ligase-independent manner by co-activating E2F1 [PMID:26234677]. RNF126 ubiquitinates diverse additional substrates—including p21, p53, frataxin, PDKs, PTEN, and FSP1—linking it to cell cycle progression, ferroptosis, and metabolic reprogramming [PMID:23026136, PMID:28228265, PMID:27462466, PMID:33664240, PMID:38514855], and its deletion in mice causes meiotic arrest and male infertility with multiple morphological abnormalities of the sperm flagella [PMID:39142440, PMID:40410177].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Establishing RNF126 as an E3 ligase with an oncogenic substrate: before this work, RNF126's enzymatic targets were unknown; the study showed it directly ubiquitinates p21 for proteasomal degradation, driving G1-S cell cycle progression in cancer cells.\",\n      \"evidence\": \"Co-IP, ligase-dead mutant comparison, siRNA knockdown with p21 rescue in cancer cell lines\",\n      \"pmids\": [\"23026136\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro reconstitution with purified components\", \"Substrate selectivity over other CKIs not addressed\", \"Single lab finding\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defining ubiquitin chain-type specificity and a role in receptor trafficking: RNF126 was shown to generate K48- or K63-linked chains depending on the E2 partner, and to regulate EGFR endosomal sorting and CI-MPR retrograde transport in a RING-dependent manner, broadening its functional scope beyond cell cycle control.\",\n      \"evidence\": \"In vitro chain-type reconstitution with UbcH5b and Ubc13/Uev1a; siRNA depletion with EGFR and CI-MPR trafficking assays\",\n      \"pmids\": [\"23418353\", \"24275455\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct EGFR ubiquitination site(s) not mapped\", \"CI-MPR mechanism lacks in vitro reconstitution\", \"Relationship between EGFR and CI-MPR sorting functions unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revealing the core quality-control function: RNF126 was identified as the primary BAG6-dependent E3 ligase for mislocalized proteins, recruited via BAG6's Ubl domain and ubiquitinating juxtahydrophobic lysines on clients—establishing RNF126 as a central cytosolic triage enzyme.\",\n      \"evidence\": \"Full in vitro reconstitution with purified components, cell fractionation, RNF126 depletion\",\n      \"pmids\": [\"24981174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Ubl-mediated recruitment not resolved\", \"In vivo substrate scope not defined genome-wide\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Uncovering a ligase-independent transcriptional function: RNF126 was found to bind E2F1 and co-activate BRCA1 transcription independently of its RING catalytic activity, promoting homologous recombination—demonstrating a non-enzymatic role distinct from its ubiquitin ligase function.\",\n      \"evidence\": \"Co-IP of RNF126-E2F1, ChIP at BRCA1 promoter, RING-independent rescue of HR, PARP inhibitor sensitivity\",\n      \"pmids\": [\"26234677\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; independent validation needed\", \"Structural determinant of E2F1 binding beyond 11-aa deletion not mapped\", \"Unclear whether E2F1-dependent function is tissue-specific\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Linking RNF126 to metabolic reprogramming: RNF126 was shown to ubiquitinate pyruvate dehydrogenase kinases (PDKs) for degradation, increasing PDH activity and acetyl-CoA flux, thereby supporting anoikis resistance—connecting its E3 ligase activity to cancer metabolism.\",\n      \"evidence\": \"PDK ubiquitination assay, metabolic flux analysis, soft-agar and in vivo tumorigenicity assays\",\n      \"pmids\": [\"27462466\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific PDK isoform selectivity not fully defined\", \"No in vitro reconstitution with purified components\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Establishing roles in NHEJ and frataxin homeostasis: RNF126 was shown to ubiquitinate Ku80 (with UBE2D3) to promote Ku release from DSBs and NHEJ completion, and independently to ubiquitinate frataxin for proteasomal degradation—identifying it as a therapeutic target for Friedreich ataxia.\",\n      \"evidence\": \"In vitro ubiquitylation, Ku80 K→R site mutagenesis, chromatin fractionation, frataxin stability in FRDA patient cells\",\n      \"pmids\": [\"27895153\", \"28228265\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Ku80 ubiquitination is BAG6-dependent is unknown\", \"Frataxin ubiquitination sites not mapped\", \"RNF126 inhibitor pharmacology not explored\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Placing RNF126 as a negative regulator in the DDR ubiquitin cascade: overexpression studies showed RNF126 suppresses RNF168-mediated H2A K13/15 monoubiquitination and 53BP1 foci while leaving γH2AX, MDC1, and RNF8 foci intact, positioning RNF126 between RNF8 and RNF168.\",\n      \"evidence\": \"IR-induced foci hierarchy analysis, H2A ubiquitination assay, NHEJ reporter assay\",\n      \"pmids\": [\"29167269\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous regulation versus overexpression artifacts not fully resolved\", \"Mechanism of RNF168 inhibition (direct ubiquitination vs. competition) not yet distinguished\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Confirming direct RNF168 ubiquitination: RNF126 was shown to be recruited to damage sites in an RNF8-dependent manner and to directly ubiquitinate RNF168, resolving the mechanism of its negative regulation of the DDR.\",\n      \"evidence\": \"UV laser micro-irradiation recruitment, Co-IP of RNF126-RNF168, catalytic-dead mutant discrimination\",\n      \"pmids\": [\"30529286\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination sites on RNF168 not mapped\", \"Chain type used on RNF168 not determined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extending the quality-control paradigm to ERAD: RNF126 was shown to reubiquitinate p97/VCP-extracted membrane proteins via BAG6, reconstituting the ternary complex and demonstrating that cytosolic reubiquitination is essential for proteasomal targeting of retrotranslocated substrates.\",\n      \"evidence\": \"In vitro reconstitution with purified factors, pulse-chase turnover, cellular depletion\",\n      \"pmids\": [\"32645369\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Selectivity for specific ERAD substrates not comprehensively defined\", \"Interplay with other cytosolic reubiquitination E3s (e.g., CHIP) not addressed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Expanding substrate range to innate immunity and cell cycle checkpoint maintenance: RNF126 was shown to K63-ubiquitinate TRAF3 for antiviral signaling and to stabilize 14-3-3σ for G2 arrest maintenance, while its own stability was found to be regulated by PARP1-mediated PARylation and CHFR-dependent ubiquitination.\",\n      \"evidence\": \"Co-IP with chain-type specificity assays, PARylation/CHFR recruitment assay, cycloheximide stability, cell cycle analysis\",\n      \"pmids\": [\"34643674\", \"34563636\", \"34388456\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TRAF3 ubiquitination sites not mapped\", \"14-3-3σ stabilization mechanism (direct deubiquitination vs. shielding) not resolved\", \"PARP1-CHFR-RNF126 axis not reconstituted in vitro\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Activating ATR signaling through MRE11 ubiquitination: RNF126 was found to ubiquitinate MRE11 at K339/K480, enhancing its exonuclease activity and RPA binding, thereby activating ATR-CHK1 and promoting HR—adding a positive arm to RNF126's DDR involvement.\",\n      \"evidence\": \"Co-IP with MRN complex, site-specific ubiquitination mapping, exonuclease activity assay, ATR phosphorylation, KO mouse\",\n      \"pmids\": [\"36563124\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How RNF126 simultaneously promotes (MRE11 activation) and inhibits (RNF168 degradation) DDR signaling is not integrated\", \"Chain type on MRE11 not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connecting RNF126 to ferroptosis: RNF126 was shown to ubiquitinate FSP1 at defined sites, displacing it from the plasma membrane, increasing the CoQ/CoQH₂ ratio, and promoting ferroptosis in medulloblastoma—identifying a new mechanism for ferroptosis regulation.\",\n      \"evidence\": \"Co-IP, site-specific ubiquitination, subcellular fractionation, CoQ ratio measurement, in vivo ferroptosis assay\",\n      \"pmids\": [\"38514855\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether BAG6 is involved in FSP1 targeting is unknown\", \"Generalizability beyond Group 3 medulloblastoma not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrating physiological requirement for male fertility: RNF126 knockout mice exhibited meiosis I arrest, flagellar defects (MMAF), and complete male infertility; RING-domain missense variants abolished ligase activity, and RNF126-BAG6 interaction was confirmed in sperm cells.\",\n      \"evidence\": \"Rnf126 KO mouse, flagellar ultrastructure, RING-domain mutant enzymatic assay, Co-IP in germ cells, association with human infertility variants\",\n      \"pmids\": [\"39142440\", \"40410177\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific spermatogenesis substrates not identified\", \"Whether BAG6 quality control of tail-anchored proteins underlies flagellar defect is speculative\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Expanding the quality-control paradigm to mitochondrial import stress: RNF126 was shown to interact with substrate-engaged UBQLN1 and ubiquitinate unimported mitochondrial precursors (ATP5G1) for proteasomal clearance, defining a UBQLN1-dependent branch of RNF126 quality control distinct from BAG6.\",\n      \"evidence\": \"In vitro reconstitution of RNF126-UBQLN1-ATP5G1 ternary complex, Co-IP, proteasomal degradation assay\",\n      \"pmids\": [\"40086734\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Scope of UBQLN1-dependent substrates unknown\", \"Whether BAG6 and UBQLN1 pathways are competitive or parallel not addressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Non-canonical ubiquitination and late-mitotic DDR functions: RNF126 was found to ubiquitinate midnolin at non-canonical Cys/Ser/Thr residues, and to accumulate with BRAP in an ATM-dependent manner during late mitosis to promote 53BP1/RPA2 focus formation and DNA lesion resolution.\",\n      \"evidence\": \"MS-based non-canonical ubiquitination site mapping, proteomic analysis of late-mitotic irradiated cells, 53BP1/RPA2 foci and clonogenic survival with RNF126 depletion\",\n      \"pmids\": [\"41496599\", \"41996237\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological significance of non-lysine ubiquitination on midnolin stability versus function not clarified\", \"BRAP-RNF126 physical interaction and mechanism not demonstrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: how RNF126 balances its positive (MRE11, Ku80, BRCA1) and negative (RNF168) roles in the DDR in a context-dependent manner; the structural basis of BAG6 Ubl-mediated recruitment; the full in vivo substrate repertoire; and whether its quality-control and DDR functions are coordinately regulated.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of RNF126 in complex with BAG6 or substrates\", \"Genome-wide substrate identification not performed\", \"Context-dependent switching between DDR-promoting and DDR-inhibiting functions not mechanistically explained\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 8, 10, 12, 18, 23, 24]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 2, 4, 5]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [4, 10, 12, 25]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [4, 25]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 2, 3, 8, 23, 24]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [4, 10, 11, 12, 25]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [3, 14]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [5, 6]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [15]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [19, 26]}\n    ],\n    \"complexes\": [\n      \"BAG6 chaperone complex\"\n    ],\n    \"partners\": [\n      \"BAG6\",\n      \"UBQLN1\",\n      \"XRCC5\",\n      \"RNF168\",\n      \"MRE11\",\n      \"E2F1\",\n      \"PARP1\",\n      \"SFN\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}