{"gene":"RNF7","run_date":"2026-06-10T06:43:37","timeline":{"discoveries":[{"year":2000,"finding":"SAG/RBX2 binds to Cul1 and the SAG-Cul1 complex has ubiquitin ligase activity promoting poly-ubiquitination of E2/Cdc34 in vitro. This ligase activity is required to rescue lethality caused by ySAG (yeast homolog) deletion, establishing SAG as an essential SCF E3 ubiquitin ligase RING component.","method":"Yeast genetics (targeted gene disruption, tetrad analysis, complementation), in vitro ubiquitin ligase assay, co-immunoprecipitation","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstitution of ligase activity combined with yeast genetic complementation requiring the ligase activity, replicated across multiple experiments","pmids":["10851089"],"is_preprint":false},{"year":2001,"finding":"SAG/RBX2 overexpression promotes S-phase entry and cell growth under serum starvation by inhibiting p27 accumulation through proteasome-dependent degradation, and SAG binds Skp2 (the F-box protein that promotes p27 ubiquitination) in vivo.","method":"Microinjection of SAG mRNA, adenovirus overexpression, DNA transfection, [3H]-thymidine incorporation, co-immunoprecipitation, proteasome inhibitor (MG132) treatment","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple cell-based assays and co-IP showing Skp2 interaction, single lab","pmids":["11255262"],"is_preprint":false},{"year":2002,"finding":"The Ring-H2 finger motif of SAG/CKBBP1/RBX2 is necessary for direct interaction with the CKIIβ subunit and for efficient phosphorylation by CKII; disruption of the Ring-H2 motif suppresses cell proliferation and causes G1/G0 accumulation.","method":"In vitro binding assay with purified CKIIβ, co-immunoprecipitation from cell extracts, cell cycle analysis, stable cell line overexpression","journal":"Journal of biochemistry and molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro binding plus co-IP with mutagenesis, single lab with two orthogonal methods","pmids":["12470599"],"is_preprint":false},{"year":2003,"finding":"CKII phosphorylates SAG/CKBBP1/RBX2 at threonine 10; non-phosphorylatable T10A mutant causes accumulation of IκBα and p27Kip1 via reduced proteasomal degradation, demonstrating that CKII-mediated phosphorylation of SAG is required for efficient degradation of these substrates and G1/S transition.","method":"In vitro kinase assay, site-directed mutagenesis (T10A and T10E substitutions), proteasome inhibitor and CKII inhibitor treatments, Western blotting, cell cycle analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay combined with mutagenesis and functional cell-based readout, single lab with multiple orthogonal methods","pmids":["12748192"],"is_preprint":false},{"year":2004,"finding":"SAG/RBX2 (Rbx2) specifically associates with Cul5 to form Cul5-Rbx2 modules, while RBX1 associates with Cul2; SOCS-box proteins (BC box + Cul5 box) direct interaction with Cul5-Rbx2, whereas VHL-box proteins (BC box + Cul2 box) interact with Cul2-Rbx1. RNAi knockdown of Cul5-Rbx2 (but not Cul2-Rbx1) did not affect VHL-mediated HIF-2α degradation, demonstrating distinct functional specificity.","method":"Co-immunoprecipitation of endogenous complexes, domain-swapping mutagenesis, RNAi knockdown with functional readout (HIF-2α degradation)","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, domain-swapping mutagenesis, and RNAi-based functional validation in a single rigorous study","pmids":["15601820"],"is_preprint":false},{"year":2005,"finding":"ASB family proteins (containing SOCS-box with BC box and Cul5 box) interact specifically with Cul5-Rbx2 but not Cul2 or Rbx1, and ASB-Cul5-Rbx2 complexes have E3 ubiquitin ligase activity.","method":"Co-immunoprecipitation, mutational analysis of BC box and Cul5 box, in vitro ubiquitin ligase assay","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — co-IP with mutagenesis plus in vitro E3 ligase reconstitution, single lab with multiple orthogonal methods","pmids":["16325183"],"is_preprint":false},{"year":2007,"finding":"SAG/RBX2 promotes VHL-mediated HIF-1α ubiquitination and degradation; siRNA silencing of SAG or ROC1 significantly inhibited HIF-1α ubiquitination. SAG forms an in vivo complex with Cul5 and VHL under hypoxia. HIF-1 transcriptionally induces SAG via a consensus HIF-1-binding site in the first intron, establishing an HIF-1–SAG feedback loop.","method":"siRNA silencing, ubiquitination assay, co-immunoprecipitation, luciferase reporter assay, ChIP (chromatin immunoprecipitation)","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, ubiquitination assay, ChIP and reporter assay in a single study with multiple orthogonal methods","pmids":["17828303"],"is_preprint":false},{"year":2007,"finding":"SAG/RBX2 is a transcriptional target of AP-1 (c-Jun); upon induction by AP-1, SAG promotes c-Jun ubiquitination and degradation via SCF-Fbw7, establishing an AP-1/SAG autofeedback loop. SAG siRNA silencing reduced c-Jun polyubiquitination and blocked Fbw7-induced c-Jun degradation.","method":"Luciferase reporter assay, in vitro and in vivo AP-1 binding (EMSA and ChIP), siRNA silencing, polyubiquitination assay, Western blotting","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — ChIP + ubiquitination assay + siRNA rescue in a single study with multiple orthogonal methods","pmids":["17440073"],"is_preprint":false},{"year":2007,"finding":"In transgenic mouse epidermis, SAG/RBX2 targets c-Jun/AP-1 at early stages of skin carcinogenesis (promoting c-Jun degradation and suppressing tumor promotion) and IκBα at later stages (promoting IκBα degradation, activating NF-κB, and reducing apoptosis to enhance tumor growth), demonstrating stage-dependent substrate targeting.","method":"K14 promoter-driven transgenic mouse model, in vitro primary culture, in vivo AP-1 luciferase reporter mouse model, Western blotting, AP-1 activity measurement","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo transgenic model with stage-dependent phenotypic readout, single lab","pmids":["17846172"],"is_preprint":false},{"year":2008,"finding":"SAG/RBX2 promotes degradation of both c-Jun and p27 simultaneously; in a K14-SAG transgenic mouse model, SAG reduced c-Jun and p27 levels and inhibited AP-1 activity after UVB exposure, causing skin hyperplasia without affecting apoptosis or p53/c-Fos/cyclin D1 levels.","method":"Transgenic mouse model, Western blotting, AP-1 activity assay, immunohistochemistry, DNA synthesis rate measurement","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo transgenic model with multiple protein readouts, single lab","pmids":["18258608"],"is_preprint":false},{"year":2008,"finding":"Cullin-box sequences (Cul2-box and Cul5-box) in BC-box proteins directly determine binding specificity for Cul2-Rbx1 vs. Cul5-Rbx2 modules; spacing between BC- and Cullin-boxes is flexible (3–80 aa), and residues conserved in the Cul2-box are a subset of those in the Cul5-box.","method":"Biochemical purification of multisubunit complexes, structure-function mutagenesis, mass spectrometry identification of BC-box proteins","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic structure-function analysis with purification of multiple complexes, single lab","pmids":["18187417"],"is_preprint":false},{"year":2010,"finding":"SAG/RBX2 silencing induces apoptosis with accumulation of NOXA, while SAG overexpression reduces NOXA levels and shortens NOXA protein half-life, identifying NOXA as a substrate of SAG E3 ligase.","method":"siRNA silencing, Western blotting of apoptosis-associated proteins, protein half-life assay, FACS analysis","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — siRNA knockdown and overexpression with protein half-life measurement, single lab","pmids":["20103673"],"is_preprint":false},{"year":2010,"finding":"Sag gene-trap deletion in mouse embryonic stem cells abrogates IκBα degradation and NF-κB activation; IκBα is identified as a direct substrate of SAG-SCF(β-TrCP) E3 ubiquitin ligase. Sag elimination also increases steady-state ROS levels and enhances radiation-induced apoptosis.","method":"Gene-trap strategy (complete Sag elimination), clonogenic survival assay, Western blotting, ROS measurement, NF-κB activation assay, in vitro ubiquitination assay","journal":"Free radical biology & medicine","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic knockout with direct biochemical demonstration of IκBα as substrate via in vitro ubiquitination assay and multiple orthogonal readouts, single lab","pmids":["20638939"],"is_preprint":false},{"year":2011,"finding":"SAG/RBX2 is an essential RING component of SAG-CUL1-FBXW7 E3 ligase that targets NF1 (neurofibromatosis type 1) for ubiquitin-mediated degradation. Sag knockout mice die at E11.5–12.5 with vascular and neural defects associated with NF1 accumulation and RAS inhibition; simultaneous Nf1 deletion partially rescues these defects, establishing NF1 as a physiological SAG substrate.","method":"Conditional knockout mouse, embryonic stem cell differentiation assay, epistasis (double knockout rescue), Western blotting, angiogenesis assays","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis (double KO rescue) combined with in vivo developmental phenotype and biochemical substrate identification, replicated across ES cells and embryos","pmids":["22118770"],"is_preprint":false},{"year":2013,"finding":"Rbx2 (RNF7), as a core subunit of the Cullin5-RING E3 ubiquitin ligase (CRL5) complex, stops neocortical projection neurons at their target layers by facilitating Dab1 ubiquitylation and turnover through SOCS7-CRL5 complexes. Rbx2 mutation causes neocortical and cerebellar ectopias dependent on Dab1 (Reelin pathway). SOCS7-CRL5 is required for neocortical but not cerebellar layering, revealing adaptor-specific functions.","method":"Conditional knockout mouse (Rbx2 mutation), epistasis (Dab1-dependence), ubiquitylation assay (SOCS7-CRL5 stimulates Dab1 ubiquitylation), SOCS7 overexpression rescue experiment","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis combined with ubiquitylation assay and in vivo rescue experiment in a single rigorous study","pmids":["24210661"],"is_preprint":false},{"year":2013,"finding":"Sag endothelial deletion causes embryonic lethality at E15.5 with impaired vasculogenesis; Sag deletion/knockdown in endothelial cells inhibits migration, proliferation, and tube formation, with p27 accumulation responsible for suppression of migration and proliferation.","method":"Conditional endothelial-specific knockout mouse (Tie2-Cre), siRNA knockdown in endothelial cells, Matrigel plug angiogenesis assay, in vitro tube formation, Western blotting","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Moderate — tissue-specific genetic knockout with in vivo and in vitro functional readouts plus identification of p27 as mediating substrate, single lab with multiple orthogonal methods","pmids":["24213570"],"is_preprint":false},{"year":2014,"finding":"Biophysical reconstitution of the full-size neddylated and unneddylated SOCS2-EloBC-Cul5-Rbx2 (CRL5SOCS2) complex in vitro; the complex exists as a monomer. Affinities of protein-protein interactions within the complex were measured by isothermal titration calorimetry.","method":"Recombinant protein expression (E. coli and Sf21 insect cells), pulldown from human cell lysates using phospho-GHR peptides, size exclusion chromatography with multi-angle static light scattering, native MS, traveling wave ion mobility MS, ITC","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — full complex reconstitution with multiple orthogonal biophysical methods, single lab","pmids":["25505247"],"is_preprint":false},{"year":2014,"finding":"SAG/RBX2 promotes ubiquitylation and degradation of PHLPP1 and DEPTOR, leading to activation of the PI3K/AKT/mTOR axis. Simultaneous knockdown of PHLPP1 or DEPTOR partially rescues the growth suppression caused by SAG knockdown. Phlpp1 and Deptor accumulate in Sag-null prostate cancer tissues with corresponding inactivation of Akt/mTOR.","method":"Prostate-specific conditional double knockout (Sag/Pten), siRNA knockdown, in vivo and in vitro ubiquitylation assays, Western blotting, epistasis rescue experiment","journal":"Molecular cancer","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vitro ubiquitylation assay combined with in vivo genetic model and epistasis rescue, single lab with multiple orthogonal methods","pmids":["27955654"],"is_preprint":false},{"year":2014,"finding":"SAG/RBX2 knockdown suppresses lung tumorigenesis and causes accumulation of tumor suppressor substrates p21, p27, NOXA, and BIM, inactivates NF-κB and mTOR pathways; growth suppression is partially rescued by simultaneous knockdown of p21 or the mTOR inhibitor DEPTOR, establishing these as functional SAG substrates.","method":"Conditional knockout mouse (KrasG12D-driven lung tumors), siRNA knockdown, epistasis rescue experiments, Western blotting, cell survival assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic model combined with epistasis rescue experiments in vitro, multiple substrates identified, single lab with multiple orthogonal methods","pmids":["24430184"],"is_preprint":false},{"year":2014,"finding":"NEDD4-1 (a HECT-domain E3 ubiquitin ligase) directly binds to the C-terminal RING domain of SAG/RBX2 via its HECT domain and ubiquitylates SAG for proteasome-mediated degradation, thereby identifying SAG as a substrate of NEDD4-1. SAG bridges NEDD4-1 (via its C-terminus) and CUL-5 (via its N-terminus) to form a NEDD4-1/SAG/CUL-5 tri-complex.","method":"Co-immunoprecipitation, domain-mapping (HECT vs. RING domain interactions), protein half-life assay (overexpression and silencing), ubiquitylation assay","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with domain mapping plus ubiquitylation assay, single lab","pmids":["25216516"],"is_preprint":false},{"year":2015,"finding":"Sag deletion induces cellular senescence associated with accumulation of p16 (not p53); mechanistically, Sag deletion causes accumulation of JunB (a SAG-Fbxw7 substrate), a transcription factor that drives p16 transcription. Simultaneous deletion of Cdkn2a (p16 gene) largely rescues senescence, and also rescues Kras(G12D)-induced immortalization abrogated by Sag deletion.","method":"Genetic deletion in mouse embryonic fibroblasts, double knockout epistasis (Sag/Cdkn2a), Western blotting, senescence assays","journal":"Neoplasia","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with double knockout rescue, identification of JunB as substrate, and p16 as the effector, single lab with multiple orthogonal approaches","pmids":["25622904"],"is_preprint":false},{"year":2015,"finding":"RNF7/SAG interacts with PCNA in human cells; this interaction was identified by bimolecular fluorescence complementation screen and validated by co-immunoprecipitation from human cell extracts and by interaction analyses using recombinant proteins.","method":"BiFC screen (bimolecular fluorescence complementation), co-immunoprecipitation from human cell extracts, recombinant protein interaction analysis","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP and recombinant protein interaction as orthogonal validation of BiFC, single lab","pmids":["26030842"],"is_preprint":false},{"year":2015,"finding":"Both Rbx1 and Rbx2 can promote ubiquitination of APOBEC3G (A3G) in vitro as part of the HIV-1 Vif-Cul5 E3 ligase complex; however, in cells, only knockdown of endogenous Rbx2 (not Rbx1) impairs Vif-induced A3G degradation. Rbx2 can dose-dependently inhibit Rbx1 interaction with Cul5.","method":"In vitro ubiquitination assay, co-immunoprecipitation, siRNA knockdown, Western blotting","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro ubiquitination assay combined with co-IP and cellular knockdown, single lab","pmids":["25912140"],"is_preprint":false},{"year":2016,"finding":"SAG/RBX2 selectively binds E2s UBCH10 and UBE2S (which mediate K11 ubiquitin linkage), whereas RBX1 exclusively binds CDC34 and UBCH5C (K48 linkage). SAG-CUL5 promotes K11-linked ubiquitylation and degradation of β-TrCP1; silencing UBCH10 or UBE2S (but not UBCH5C) causes β-TrCP1 accumulation. SAG-CUL5-β-TrCP1 forms a complex under physiological conditions.","method":"Co-immunoprecipitation, E2 binding assays, ubiquitylation assay (K11 linkage-specific), siRNA silencing, protein half-life assay","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal biochemical methods (co-IP, E2 binding, ubiquitylation assay, siRNA) in a single study discriminating SAG from RBX1 biochemically, single lab","pmids":["27910872"],"is_preprint":false},{"year":2016,"finding":"SAG-dependent neddylation in T cells regulates T cell activation, proliferation, and effector cytokine release. SAG T-cell-specific knockout reduces T cell responses and graft-versus-host disease; mechanistically, SAG-mediated effects in T cells are associated with increased SOCS expression (but not NF-κB translocation).","method":"T-cell-specific conditional knockout mouse, in vitro T cell stimulation, in vivo allogeneic bone marrow transplantation model, MLN4924 pharmacological inhibition","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — tissue-specific genetic knockout with in vivo model and mechanistic readout, single lab","pmids":["27543965"],"is_preprint":false},{"year":2017,"finding":"RNF7/SAG functions as an E3 ubiquitin ligase that negatively regulates CARMA2sh (CARD14) NF-κB signaling by regulating the ubiquitination state of MALT1 and NEMO. RNF7 interacts with CARMA2 (identified by yeast two-hybrid), and psoriasis-associated CARMA2sh mutants escape this negative regulation by RNF7.","method":"Yeast two-hybrid screen (identification of interaction), ubiquitination assays for MALT1 and NEMO, functional NF-κB reporter assays","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — yeast two-hybrid plus ubiquitination assays and functional NF-κB readout, single lab","pmids":["29194363"],"is_preprint":false},{"year":2018,"finding":"RBX2 is essential for retinal layering and function: depletion of RBX2 mispositions rod bipolar cells, cone photoreceptors, and Müller glia. SOCS7-CRL5 controls rod bipolar cell and Müller glia positioning via sustained DAB1 (Reelin/RELN) signaling, but cone photoreceptor positioning is SOCS7-independent, revealing distinct CRL5 adaptor utilization. RBX2 depletion also reduces ribbon synapses and disrupts cone photoreceptor function.","method":"Conditional knockout mouse (RBX2 and SOCS7), electrophysiology, immunohistochemistry for synaptic markers, epistasis analysis","journal":"Development","confidence":"High","confidence_rationale":"Tier 2 / Moderate — tissue-specific genetic knockout with functional readout (electrophysiology and synaptic analysis) plus epistasis, single lab","pmids":["29361558"],"is_preprint":false},{"year":2018,"finding":"SAG/RBX2 (Sag) deletion in myeloid cells differentially regulates inflammatory responses: Sag-null macrophages release fewer proinflammatory cytokines, while Sag-null neutrophils release more. SAG depletion alters expression of myeloperoxidase (Mpo) and neutrophil elastase (Elane) in bone marrow cells in response to LPS.","method":"LysM-Cre conditional knockout mouse, LPS challenge in vivo, cytokine measurement (ELISA), gene expression analysis","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-type-specific conditional knockout with in vivo functional readout, single lab","pmids":["30574150"],"is_preprint":false},{"year":2020,"finding":"SAG/RBX2 competes with APC2 for UBE2C/UBE2S binding, acting as a potential endogenous inhibitor of APC/C and regulating G2-to-M progression. SAG knockdown causes premature APC/C activation, mitotic slippage, and resistance to anti-microtubule drugs. Conversely, SAG is itself a substrate of APC/CCDH1 and is degraded at G1 phase; degradation-resistant SAG-R98A/L101A mutant accelerates G1-to-S progression.","method":"Co-immunoprecipitation, competition binding assays, siRNA knockdown, degradation-resistant mutant overexpression, cell cycle and mitotic assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, competition assays, mutagenesis, and functional cell cycle readouts in a single rigorous study with multiple orthogonal methods, single lab","pmids":["32905768"],"is_preprint":false},{"year":2022,"finding":"RNF7/SAG promotes ubiquitination of SOCS1 to activate JAK/STAT3 signaling; STAT3 activation in turn transcriptionally induces RNF7, establishing a STAT3/RNF7 feedback loop. RNF7 overexpression inhibits apoptosis and promotes glycolysis in renal cell carcinoma.","method":"Knockdown and overexpression experiments, ubiquitination assay for SOCS1, Western blotting for JAK/STAT3 pathway, luciferase reporter assay, in vivo xenograft","journal":"Cellular & molecular biology letters","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — ubiquitination assay and functional readouts with reporter assay, single lab","pmids":["35562668"],"is_preprint":false},{"year":2024,"finding":"RBX2/CUL5 (core components of CRL5) localize to mitochondria. RBX2 depletion inhibits mitochondrial ubiquitination and turnover, impairs mitochondrial membrane potential and respiration, and causes cardiomyocyte death. In vivo cardiac-specific Rbx2 deletion suppresses mitophagic activity, causes accumulation of damaged mitochondria, and leads to dilated cardiomyopathy and heart failure. RBX2 controls PINK1 stability in mitochondria. This Parkin-independent mitophagic function does not require Parkin (Parkin deletion has no impact on cardiomyopathy in RBX2-deficient hearts).","method":"Subcellular fractionation, immunostaining, immunogold electron microscopy (RBX2/CUL5 mitochondrial localization), cardiomyocyte-specific and adult heart conditional knockout mice, in vitro mitochondrial function assays (membrane potential, respiration), mitophagy assays, proteomics, RNA-sequencing, Western blotting","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct localization by immunogold EM, multiple genetic knockouts (tissue-specific, inducible, and double KO), proteomics, and functional mitochondrial assays, all in a single rigorous study","pmids":["38873758"],"is_preprint":false},{"year":2025,"finding":"YTHDF1 (m6A reader) promotes RNF7 translation in an m6A-dependent manner; elevated RNF7 then promotes degradation of the CDK inhibitor p27, driving prostate cancer cell proliferation. MLN4924 (neddylation inhibitor) inhibits prostate cancer progression in vitro and in vivo through this axis.","method":"YTHDF1 knockdown/overexpression, p27 protein stability assay, E3 ligase screening (candidate p27-targeting ligases), xenograft and organoid models, in vitro and in vivo drug treatment","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — candidate E3 screening with functional rescue experiments and organoid validation, single lab","pmids":["40251202"],"is_preprint":false},{"year":2025,"finding":"Let-7 microRNA directly binds a conserved motif in the 3' UTR of RBX2, reducing its translation and thereby diminishing CRL5 activity. Restoring RBX2 levels rescues pyramidal neuron positioning (without altering let-7-induced fate effects), demonstrating that let-7 regulates RBX2-CRL5 to control neocortical neuron migration independently of fate specification.","method":"In utero electroporation, let-7 overexpression and RBX2 rescue experiments in mouse neocortex, 3' UTR reporter assays, live imaging of neuronal migration","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 / Moderate — 3'UTR reporter + in vivo epistasis rescue + live imaging of neuronal migration, single lab with multiple orthogonal methods","pmids":["42012943"],"is_preprint":false}],"current_model":"RNF7 (SAG/RBX2/ROC2) is a RING-domain protein that functions as the catalytic RING subunit of Cullin-RING E3 ubiquitin ligases (CRL1 and preferentially CRL5): it selectively associates with CUL5 (via SOCS-box proteins whose Cul5-box determines specificity) or CUL1, recruits K11-linked E2 enzymes UBCH10/UBE2S, and ubiquitylates multiple substrates including p27, IκBα, c-Jun, NF1, NOXA, HIF-1α, DEPTOR, PHLPP1, β-TrCP1, SOCS1, JunB, and PINK1; it is itself regulated by CKII-mediated phosphorylation at T10 (which is required for efficient substrate degradation), by NEDD4-1-mediated ubiquitination and degradation, by APC/CCDH1 at G1, and at the transcriptional level by HIF-1 and AP-1; RNF7 also localizes to mitochondria as part of CRL5 to drive Parkin-independent, PINK1-dependent mitophagy essential for cardiac homeostasis, and in the developing brain its activity is post-transcriptionally restrained by let-7 miRNA to regulate CRL5-SOCS7-dependent Dab1 ubiquitination and Reelin signaling to control neuronal migration and laminar positioning."},"narrative":{"mechanistic_narrative":"RNF7 (SAG/RBX2/ROC2) is the catalytic RING subunit of Cullin-RING E3 ubiquitin ligases, providing the essential ligase activity that drives substrate polyubiquitination and proteasomal turnover across cell-cycle, signaling, and developmental programs [PMID:10851089, PMID:22118770]. It partitions cullin specificity by selectively associating with CUL5 (recruited through the Cul5-box of SOCS-box adaptor proteins such as ASB and SOCS2/SOCS7) while RBX1 pairs with CUL2, and it also serves as the RING component of SCF-type CUL1 complexes (e.g., with FBXW7/Fbw7) [PMID:15601820, PMID:16325183, PMID:22118770]. Distinct from RBX1, RNF7 selectively engages the K11-linkage E2 enzymes UBCH10 and UBE2S to assemble K11-linked chains [PMID:27910872]. Through these complexes RNF7 targets a broad substrate set governing proliferation, survival, and signaling — p27 and IκBα [PMID:12748192], c-Jun via SCF-Fbw7 [PMID:17440073], NF1 [PMID:22118770], NOXA [PMID:20103673], PHLPP1 and DEPTOR to activate the PI3K/AKT/mTOR axis [PMID:27955654], JunB controlling p16-dependent senescence [PMID:25622904], and β-TrCP1 [PMID:27910872] — and it operates within feedback loops in which it is transcriptionally induced by HIF-1, AP-1/c-Jun, and STAT3 while degrading components of those same pathways [PMID:17828303, PMID:17440073, PMID:35562668]. Its activity is post-translationally tuned by CKII-mediated phosphorylation at Thr10, required for efficient substrate degradation [PMID:12748192], and by APC/C-CDH1, which both is antagonized by RNF7 in mitosis and degrades RNF7 at G1 [PMID:32905768]. Beyond cytoplasmic ligase functions, RNF7 localizes as part of CRL5 to mitochondria to drive Parkin-independent, PINK1-dependent mitophagy essential for cardiac homeostasis [PMID:38873758], and in the developing nervous system its CRL5-SOCS7 activity ubiquitylates Dab1 to terminate Reelin signaling and control neuronal lamination, an activity restrained by let-7 miRNA [PMID:24210661, PMID:29361558, PMID:42012943].","teleology":[{"year":2000,"claim":"Established that RNF7/SAG is not a passive scaffold but the functional RING ligase subunit of an SCF complex, answering whether it provides catalytic activity essential for viability.","evidence":"Yeast genetics with complementation requiring ligase activity, in vitro ubiquitin ligase assay, and co-IP with Cul1","pmids":["10851089"],"confidence":"High","gaps":["Did not define mammalian substrate range","Did not distinguish CUL1 vs other cullin partners"]},{"year":2001,"claim":"Linked RNF7 to cell-cycle control by showing it promotes S-phase entry through proteasomal degradation of p27, identifying Skp2 as the relevant F-box adaptor.","evidence":"mRNA microinjection, adenoviral overexpression, thymidine incorporation, co-IP, and MG132 treatment","pmids":["11255262"],"confidence":"Medium","gaps":["Did not reconstitute direct p27 ubiquitination by SAG complex","Single-lab cell-based correlation"]},{"year":2002,"claim":"Connected the RING-H2 motif to CKII regulation, showing the motif mediates CKIIβ binding and is required for proliferation.","evidence":"In vitro binding with purified CKIIβ, co-IP, mutagenesis, and cell-cycle analysis","pmids":["12470599"],"confidence":"Medium","gaps":["Did not identify the phosphorylated residue","Mechanistic link between phosphorylation and ligase output unresolved"]},{"year":2003,"claim":"Resolved how CKII modulates RNF7 by mapping phosphorylation to Thr10 and showing it is required for efficient degradation of IκBα and p27.","evidence":"In vitro kinase assay, T10A/T10E mutagenesis, proteasome/CKII inhibition, and cell-cycle readout","pmids":["12748192"],"confidence":"High","gaps":["Did not show how phosphorylation alters complex assembly or E2 engagement"]},{"year":2005,"claim":"Defined the structural basis for cullin specificity, demonstrating SAG/RBX2 pairs with CUL5 (not CUL2) and that the SOCS-box Cul5-box determines this partitioning.","evidence":"Reciprocal co-IP, domain-swapping mutagenesis, RNAi functional readout, and analysis of ASB-family adaptors with in vitro ligase reconstitution","pmids":["15601820","16325183"],"confidence":"High","gaps":["Did not enumerate the full SOCS-box adaptor repertoire","Did not address in vivo substrate consequences"]},{"year":2007,"claim":"Embedded RNF7 in transcription-coupled feedback loops, showing HIF-1 and AP-1/c-Jun induce RNF7, which in turn degrades HIF-1α and c-Jun.","evidence":"siRNA silencing, ubiquitination assays, co-IP, ChIP, EMSA, and luciferase reporters across two studies","pmids":["17828303","17440073"],"confidence":"High","gaps":["Did not quantify physiological balance of these competing loops","Adaptor identity for HIF-1α not fully resolved beyond VHL context"]},{"year":2008,"claim":"Demonstrated context- and stage-dependent substrate targeting in vivo, with RNF7 degrading c-Jun early and IκBα later during skin carcinogenesis.","evidence":"K14-driven transgenic mouse models, AP-1 reporter mice, UVB exposure, and protein-level readouts","pmids":["17846172","18258608"],"confidence":"Medium","gaps":["Mechanism of temporal substrate switching unexplained","Single-lab transgenic system"]},{"year":2010,"claim":"Established genetic, knockout-level proof that IκBα is a direct SAG-SCF(β-TrCP) substrate and linked RNF7 loss to ROS accumulation and apoptosis, plus identified NOXA as a survival-controlling substrate.","evidence":"Gene-trap Sag elimination in ES cells, in vitro ubiquitination, ROS and NF-κB assays; separate siRNA/half-life study for NOXA","pmids":["20638939","20103673"],"confidence":"High","gaps":["NOXA finding was Medium-confidence and lacked in vitro ubiquitination","Adaptor for NOXA not identified"]},{"year":2011,"claim":"Defined an essential developmental role by showing SAG-CUL1-FBXW7 targets NF1, with Sag-null embryonic lethality partially rescued by Nf1 deletion.","evidence":"Conditional knockout mice, ES-cell differentiation, double-knockout epistasis, and biochemical substrate identification","pmids":["22118770"],"confidence":"High","gaps":["Did not separate CUL1- vs CUL5-dependent embryonic functions"]},{"year":2013,"claim":"Revealed adaptor-specific neurodevelopmental functions, showing CRL5-SOCS7 ubiquitylates Dab1 to terminate Reelin signaling and control neocortical layering, while parallel work tied SAG/p27 to vasculogenesis.","evidence":"Rbx2 conditional knockouts, Dab1-dependence epistasis, SOCS7 rescue and ubiquitylation assays; endothelial Tie2-Cre knockout with angiogenesis assays","pmids":["24210661","24213570"],"confidence":"High","gaps":["Did not explain region-specific adaptor selection (neocortex vs cerebellum)"]},{"year":2014,"claim":"Expanded the oncogenic substrate network and provided biophysical complex reconstitution, showing RNF7 degrades PHLPP1/DEPTOR to activate AKT/mTOR and degrades p21/p27/NOXA/BIM/DEPTOR in lung tumors; also defined NEDD4-1 as a regulator that degrades RNF7.","evidence":"Conditional knockout tumor models, in vivo/in vitro ubiquitylation, epistasis rescue, full CRL5SOCS2 reconstitution with ITC/native MS, and NEDD4-1 domain-mapping/ubiquitylation","pmids":["27955654","24430184","25505247","25216516"],"confidence":"High","gaps":["NEDD4-1 regulation was Medium-confidence single lab","Physiological balance of pro- vs anti-tumor substrates context-dependent"]},{"year":2015,"claim":"Linked RNF7 to senescence and identified its physical interaction with PCNA and its E2/cullin partner discrimination from RBX1.","evidence":"Sag/Cdkn2a double-knockout epistasis with JunB substrate identification; BiFC plus co-IP for PCNA; in vitro and cellular APOBEC3G/Vif-Cul5 ubiquitination assays","pmids":["25622904","26030842","25912140"],"confidence":"High","gaps":["Functional consequence of PCNA interaction not defined (Medium-confidence)","A3G work used a viral hijacked complex"]},{"year":2016,"claim":"Defined the biochemical individuality of RNF7 versus RBX1 by showing it selectively binds K11-linkage E2s UBCH10/UBE2S to degrade β-TrCP1, and characterized SAG-dependent neddylation in T-cell function.","evidence":"Co-IP, E2 binding and K11-specific ubiquitylation assays, siRNA; T-cell conditional knockout with GVHD model and MLN4924","pmids":["27910872","27543965"],"confidence":"High","gaps":["T-cell mechanism (SOCS expression link) was Medium-confidence correlation","Did not define which adaptor selects β-TrCP1"]},{"year":2017,"claim":"Identified a negative-regulatory role of RNF7 in CARMA2/CARD14 NF-κB signaling via MALT1 and NEMO ubiquitination, with psoriasis-associated mutants escaping regulation.","evidence":"Yeast two-hybrid, MALT1/NEMO ubiquitination assays, and NF-κB reporters","pmids":["29194363"],"confidence":"Medium","gaps":["Direct vs indirect ubiquitination of MALT1/NEMO not fully separated","Single-lab system"]},{"year":2018,"claim":"Extended adaptor-specific CRL5 function to retinal layering, showing SOCS7-dependent Dab1 control of bipolar/Müller positioning and a SOCS7-independent cone photoreceptor function.","evidence":"RBX2 and SOCS7 conditional knockouts, electrophysiology, synaptic marker IHC, and epistasis","pmids":["29361558"],"confidence":"High","gaps":["Adaptor responsible for SOCS7-independent cone function unidentified"]},{"year":2020,"claim":"Uncovered reciprocal regulation with APC/C, showing RNF7 competes for UBE2C/UBE2S to inhibit APC/C in mitosis while being degraded by APC/C-CDH1 at G1.","evidence":"Co-IP, competition binding, siRNA, degradation-resistant mutants, and cell-cycle/mitotic assays","pmids":["32905768"],"confidence":"High","gaps":["Did not establish how the E2 competition is balanced under normal cycling"]},{"year":2024,"claim":"Established a non-canonical mitochondrial function, showing CRL5/RBX2 localizes to mitochondria and drives Parkin-independent, PINK1-dependent mitophagy essential to prevent dilated cardiomyopathy.","evidence":"Subcellular fractionation, immunogold EM, multiple cardiac conditional/inducible and Parkin double knockouts, proteomics, and mitochondrial function assays","pmids":["38873758"],"confidence":"High","gaps":["Direct demonstration of RBX2-mediated PINK1 ubiquitination chemistry not fully detailed","Adaptor recruiting mitochondrial substrates unspecified"]},{"year":2025,"claim":"Defined post-transcriptional control of RNF7, showing m6A/YTHDF1 promotes its translation in prostate cancer and let-7 miRNA restrains RBX2 translation to control neocortical migration.","evidence":"YTHDF1 knockdown/overexpression with p27 stability and organoid/xenograft models; let-7 3'UTR reporters, in utero electroporation rescue, and live migration imaging","pmids":["40251202","42012943"],"confidence":"High","gaps":["YTHDF1 axis was Medium-confidence candidate-screen based","Interplay between m6A and let-7 control not jointly examined"]},{"year":null,"claim":"How adaptor selection, cullin choice (CUL1 vs CUL5), and E2/linkage usage are dynamically coordinated to specify substrate sets across tissues and subcellular compartments remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No unified structural model integrating mitochondrial vs cytoplasmic complexes","Rules governing context-dependent substrate prioritization not established","Mitochondrial substrate adaptors uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,5,13,23]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,12,17,23]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,28]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[30]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4,6]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,12,23]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1,3,28]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[17,25,29]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[13,14,26,32]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[30]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[24,27]}],"complexes":["CRL5 (CUL5-RBX2)","SCF (CUL1-RBX2)","CRL5-SOCS7","SCF-FBXW7"],"partners":["CUL5","CUL1","SOCS7","FBXW7","UBE2C","UBE2S","NEDD4-1","PCNA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UBF6","full_name":"RING-box protein 2","aliases":["CKII beta-binding protein 1","CKBBP1","RING finger protein 7","Regulator of cullins 2","Sensitive to apoptosis gene protein"],"length_aa":113,"mass_kda":12.7,"function":"Catalytic component of multiple cullin-5-RING E3 ubiquitin-protein ligase complexes (ECS complexes), which mediate the ubiquitination and subsequent proteasomal degradation of target proteins (PubMed:21980433, PubMed:33268465, PubMed:38418882, PubMed:38574733, PubMed:35512830, PubMed:40440427). It is thereby involved in various biological processes, such as cell cycle progression, signal transduction and transcription (PubMed:21980433, PubMed:33268465, PubMed:38418882, PubMed:38574733). The functional specificity of the E3 ubiquitin-protein ligase ECS complexes depend on the variable SOCS box-containing substrate recognition component (PubMed:21980433, PubMed:33268465). Within ECS complexes, RNF7/RBX2 recruits the E2 ubiquitination enzyme to the complex via its RING-type and brings it into close proximity to the substrate (PubMed:34518685). Catalytic subunit of various SOCS-containing ECS complexes, such as the ECS(SOCS7) complex, that regulate reelin signaling by mediating ubiquitination and degradation of DAB1 (By similarity). The ECS(SOCS2) complex mediates the ubiquitination and subsequent proteasomal degradation of phosphorylated EPOR and GHR (PubMed:21980433, PubMed:25505247). Promotes ubiquitination and degradation of NF1, thereby regulating Ras protein signal transduction (By similarity). As part of the ECS(ASB9) complex, catalyzes ubiquitination and degradation of CKB (PubMed:33268465). The ECS(SPSB3) complex catalyzes ubiquitination of nuclear CGAS (PubMed:38418882). As part of the ECS(RAB40C) complex, mediates ANKRD28 ubiquitination and degradation, thereby inhibiting protein phosphatase 6 (PP6) complex activity and focal adhesion assembly during cell migration (PubMed:35512830). The ECS(ASB7) complex acts a negative regulator of H3K9me3 histone mark by mediating ubiquitination and degradation of SUV39H1 (PubMed:40440427). As part of some ECS complex, catalyzes 'Lys-11'-linked ubiquitination and degradation of BTRC (PubMed:27910872). ECS complexes and ARIH2 collaborate in tandem to mediate ubiquitination of target proteins; ARIH2 mediating addition of the first ubiquitin on CRLs targets (PubMed:34518685, PubMed:38418882). Specifically catalyzes the neddylation of CUL5 via its interaction with UBE2F (PubMed:19250909). Does not catalyze neddylation of other cullins (CUL1, CUL2, CUL3, CUL4A or CUL4B) (PubMed:19250909). May play a role in protecting cells from apoptosis induced by redox agents (PubMed:10082581) Inactive (Microbial infection) Following infection by HIV-1 virus, catalytic component of a cullin-5-RING E3 ubiquitin-protein ligase complex (ECS complex) hijacked by the HIV-1 Vif protein, which catalyzes ubiquitination and degradation of APOBEC3F and APOBEC3G","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9UBF6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RNF7","classification":"Not Classified","n_dependent_lines":20,"n_total_lines":1208,"dependency_fraction":0.016556291390728478},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RNF7","total_profiled":1310},"omim":[{"mim_id":"620077","title":"PROTEIN-L-ISOASPARTATE (D-ASPARTATE) O-METHYLTRANSFERASE DOMAIN-CONTAINING PROTEIN 2; PCMTD2","url":"https://www.omim.org/entry/620077"},{"mim_id":"617700","title":"UBIQUITIN-CONJUGATING ENZYME E2 F; UBE2F","url":"https://www.omim.org/entry/617700"},{"mim_id":"615052","title":"ANKYRIN REPEAT- AND SOCS BOX-CONTAINING PROTEIN 7; ASB7","url":"https://www.omim.org/entry/615052"},{"mim_id":"615051","title":"ANKYRIN REPEAT- AND SOCS BOX-CONTAINING PROTEIN 6; ASB6","url":"https://www.omim.org/entry/615051"},{"mim_id":"605759","title":"ANKYRIN REPEAT- AND SOCS BOX-CONTAINING PROTEIN 2; ASB2","url":"https://www.omim.org/entry/605759"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RNF7"},"hgnc":{"alias_symbol":["SAG","ROC2","CKBBP1","RBX2"],"prev_symbol":[]},"alphafold":{"accession":"Q9UBF6","domains":[{"cath_id":"3.30.40.10","chopping":"61-111","consensus_level":"high","plddt":94.0553,"start":61,"end":111}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UBF6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UBF6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UBF6-F1-predicted_aligned_error_v6.png","plddt_mean":81.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RNF7","jax_strain_url":"https://www.jax.org/strain/search?query=RNF7"},"sequence":{"accession":"Q9UBF6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UBF6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UBF6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UBF6"}},"corpus_meta":[{"pmid":"15601820","id":"PMC_15601820","title":"VHL-box and 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a molecular hallmark in cancer.","date":"2023","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/37820940","citation_count":6,"is_preprint":false},{"pmid":"36920202","id":"PMC_36920202","title":"RNF7 Induces Skeletal Muscle Cell Apoptosis and Arrests Cell Autophagy via Upregulation of THBS1 and Inactivation of the PI3K/Akt Signaling Pathway in a Rat Sepsis Model.","date":"2023","source":"Infection and immunity","url":"https://pubmed.ncbi.nlm.nih.gov/36920202","citation_count":6,"is_preprint":false},{"pmid":"30269386","id":"PMC_30269386","title":"Comparative Analysis of cul5 and rbx2 Expression in the Developing and Adult Murine Brain and Their Essentiality During Mouse Embryogenesis.","date":"2018","source":"Developmental dynamics : an official publication of the American Association of Anatomists","url":"https://pubmed.ncbi.nlm.nih.gov/30269386","citation_count":5,"is_preprint":false},{"pmid":"12470599","id":"PMC_12470599","title":"The Ring-H2 finger motif of CKBBP1/SAG is necessary for interaction with protein kinase CKII and optimal cell proliferation.","date":"2002","source":"Journal of biochemistry and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/12470599","citation_count":4,"is_preprint":false},{"pmid":"36644576","id":"PMC_36644576","title":"RNF7 Facilitated the Tumorigenesis of Pancreatic Cancer by Activating PI3K/Akt Signaling Pathway.","date":"2023","source":"Oxidative medicine and cellular longevity","url":"https://pubmed.ncbi.nlm.nih.gov/36644576","citation_count":3,"is_preprint":false},{"pmid":"38464205","id":"PMC_38464205","title":"The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38464205","citation_count":2,"is_preprint":false},{"pmid":"41478481","id":"PMC_41478481","title":"The ubiquitin conjugating enzyme E2 F (UBE2F)-RING-box protein 2 (RBX2)-mediated neddylation of Cullin5 facilitates pseudorabies virus replication.","date":"2025","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/41478481","citation_count":2,"is_preprint":false},{"pmid":"39783768","id":"PMC_39783768","title":"RNF7-Mediated ROS Targets Malignant Phenotype and Radiotherapy Sensitivity in Glioma With Different IDH1 Genotypes.","date":"2025","source":"Molecular carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/39783768","citation_count":1,"is_preprint":false},{"pmid":"36745565","id":"PMC_36745565","title":"Sag/Rbx2 Partial Inactivation Sensitizes Mice to Radiation and Radiation-Induced Tumorigenesis1.","date":"2023","source":"Radiation research","url":"https://pubmed.ncbi.nlm.nih.gov/36745565","citation_count":1,"is_preprint":false},{"pmid":"41000670","id":"PMC_41000670","title":"Uncoupling Neocortical Neuron Fate and Migration via a Let-7-RBX2 Axis.","date":"2025","source":"bioRxiv : the preprint server for 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Reviews on cancer","url":"https://pubmed.ncbi.nlm.nih.gov/41610919","citation_count":0,"is_preprint":false},{"pmid":"39505338","id":"PMC_39505338","title":"[High RNF7 expression enhances PD-1 resistance of non-small cell lung cancer cells by promoting CXCL1 expression and myeloid-derived suppressor cell recruitment via activating NF-κB signaling].","date":"2024","source":"Nan fang yi ke da xue xue bao = Journal of Southern Medical University","url":"https://pubmed.ncbi.nlm.nih.gov/39505338","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.09.11.675676","title":"Uncoupling Neocortical Neuron Fate and Migration via a Let-7–RBX2 Axis","date":"2025-09-16","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.11.675676","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.11.03.621734","title":"A Perturbation Cell Atlas of Human Induced Pluripotent Stem Cells","date":"2024-11-04","source":"bioRxiv","url":"https://doi.org/10.1101/2024.11.03.621734","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":30650,"output_tokens":8919,"usd":0.112867,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":18727,"output_tokens":5803,"usd":0.119355,"stage2_stop_reason":"end_turn"},"total_usd":0.232222,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"SAG/RBX2 binds to Cul1 and the SAG-Cul1 complex has ubiquitin ligase activity promoting poly-ubiquitination of E2/Cdc34 in vitro. This ligase activity is required to rescue lethality caused by ySAG (yeast homolog) deletion, establishing SAG as an essential SCF E3 ubiquitin ligase RING component.\",\n      \"method\": \"Yeast genetics (targeted gene disruption, tetrad analysis, complementation), in vitro ubiquitin ligase assay, co-immunoprecipitation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstitution of ligase activity combined with yeast genetic complementation requiring the ligase activity, replicated across multiple experiments\",\n      \"pmids\": [\"10851089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SAG/RBX2 overexpression promotes S-phase entry and cell growth under serum starvation by inhibiting p27 accumulation through proteasome-dependent degradation, and SAG binds Skp2 (the F-box protein that promotes p27 ubiquitination) in vivo.\",\n      \"method\": \"Microinjection of SAG mRNA, adenovirus overexpression, DNA transfection, [3H]-thymidine incorporation, co-immunoprecipitation, proteasome inhibitor (MG132) treatment\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple cell-based assays and co-IP showing Skp2 interaction, single lab\",\n      \"pmids\": [\"11255262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The Ring-H2 finger motif of SAG/CKBBP1/RBX2 is necessary for direct interaction with the CKIIβ subunit and for efficient phosphorylation by CKII; disruption of the Ring-H2 motif suppresses cell proliferation and causes G1/G0 accumulation.\",\n      \"method\": \"In vitro binding assay with purified CKIIβ, co-immunoprecipitation from cell extracts, cell cycle analysis, stable cell line overexpression\",\n      \"journal\": \"Journal of biochemistry and molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro binding plus co-IP with mutagenesis, single lab with two orthogonal methods\",\n      \"pmids\": [\"12470599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CKII phosphorylates SAG/CKBBP1/RBX2 at threonine 10; non-phosphorylatable T10A mutant causes accumulation of IκBα and p27Kip1 via reduced proteasomal degradation, demonstrating that CKII-mediated phosphorylation of SAG is required for efficient degradation of these substrates and G1/S transition.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis (T10A and T10E substitutions), proteasome inhibitor and CKII inhibitor treatments, Western blotting, cell cycle analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay combined with mutagenesis and functional cell-based readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"12748192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"SAG/RBX2 (Rbx2) specifically associates with Cul5 to form Cul5-Rbx2 modules, while RBX1 associates with Cul2; SOCS-box proteins (BC box + Cul5 box) direct interaction with Cul5-Rbx2, whereas VHL-box proteins (BC box + Cul2 box) interact with Cul2-Rbx1. RNAi knockdown of Cul5-Rbx2 (but not Cul2-Rbx1) did not affect VHL-mediated HIF-2α degradation, demonstrating distinct functional specificity.\",\n      \"method\": \"Co-immunoprecipitation of endogenous complexes, domain-swapping mutagenesis, RNAi knockdown with functional readout (HIF-2α degradation)\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, domain-swapping mutagenesis, and RNAi-based functional validation in a single rigorous study\",\n      \"pmids\": [\"15601820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ASB family proteins (containing SOCS-box with BC box and Cul5 box) interact specifically with Cul5-Rbx2 but not Cul2 or Rbx1, and ASB-Cul5-Rbx2 complexes have E3 ubiquitin ligase activity.\",\n      \"method\": \"Co-immunoprecipitation, mutational analysis of BC box and Cul5 box, in vitro ubiquitin ligase assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — co-IP with mutagenesis plus in vitro E3 ligase reconstitution, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"16325183\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SAG/RBX2 promotes VHL-mediated HIF-1α ubiquitination and degradation; siRNA silencing of SAG or ROC1 significantly inhibited HIF-1α ubiquitination. SAG forms an in vivo complex with Cul5 and VHL under hypoxia. HIF-1 transcriptionally induces SAG via a consensus HIF-1-binding site in the first intron, establishing an HIF-1–SAG feedback loop.\",\n      \"method\": \"siRNA silencing, ubiquitination assay, co-immunoprecipitation, luciferase reporter assay, ChIP (chromatin immunoprecipitation)\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, ubiquitination assay, ChIP and reporter assay in a single study with multiple orthogonal methods\",\n      \"pmids\": [\"17828303\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SAG/RBX2 is a transcriptional target of AP-1 (c-Jun); upon induction by AP-1, SAG promotes c-Jun ubiquitination and degradation via SCF-Fbw7, establishing an AP-1/SAG autofeedback loop. SAG siRNA silencing reduced c-Jun polyubiquitination and blocked Fbw7-induced c-Jun degradation.\",\n      \"method\": \"Luciferase reporter assay, in vitro and in vivo AP-1 binding (EMSA and ChIP), siRNA silencing, polyubiquitination assay, Western blotting\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP + ubiquitination assay + siRNA rescue in a single study with multiple orthogonal methods\",\n      \"pmids\": [\"17440073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In transgenic mouse epidermis, SAG/RBX2 targets c-Jun/AP-1 at early stages of skin carcinogenesis (promoting c-Jun degradation and suppressing tumor promotion) and IκBα at later stages (promoting IκBα degradation, activating NF-κB, and reducing apoptosis to enhance tumor growth), demonstrating stage-dependent substrate targeting.\",\n      \"method\": \"K14 promoter-driven transgenic mouse model, in vitro primary culture, in vivo AP-1 luciferase reporter mouse model, Western blotting, AP-1 activity measurement\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo transgenic model with stage-dependent phenotypic readout, single lab\",\n      \"pmids\": [\"17846172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"SAG/RBX2 promotes degradation of both c-Jun and p27 simultaneously; in a K14-SAG transgenic mouse model, SAG reduced c-Jun and p27 levels and inhibited AP-1 activity after UVB exposure, causing skin hyperplasia without affecting apoptosis or p53/c-Fos/cyclin D1 levels.\",\n      \"method\": \"Transgenic mouse model, Western blotting, AP-1 activity assay, immunohistochemistry, DNA synthesis rate measurement\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo transgenic model with multiple protein readouts, single lab\",\n      \"pmids\": [\"18258608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Cullin-box sequences (Cul2-box and Cul5-box) in BC-box proteins directly determine binding specificity for Cul2-Rbx1 vs. Cul5-Rbx2 modules; spacing between BC- and Cullin-boxes is flexible (3–80 aa), and residues conserved in the Cul2-box are a subset of those in the Cul5-box.\",\n      \"method\": \"Biochemical purification of multisubunit complexes, structure-function mutagenesis, mass spectrometry identification of BC-box proteins\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic structure-function analysis with purification of multiple complexes, single lab\",\n      \"pmids\": [\"18187417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"SAG/RBX2 silencing induces apoptosis with accumulation of NOXA, while SAG overexpression reduces NOXA levels and shortens NOXA protein half-life, identifying NOXA as a substrate of SAG E3 ligase.\",\n      \"method\": \"siRNA silencing, Western blotting of apoptosis-associated proteins, protein half-life assay, FACS analysis\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — siRNA knockdown and overexpression with protein half-life measurement, single lab\",\n      \"pmids\": [\"20103673\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Sag gene-trap deletion in mouse embryonic stem cells abrogates IκBα degradation and NF-κB activation; IκBα is identified as a direct substrate of SAG-SCF(β-TrCP) E3 ubiquitin ligase. Sag elimination also increases steady-state ROS levels and enhances radiation-induced apoptosis.\",\n      \"method\": \"Gene-trap strategy (complete Sag elimination), clonogenic survival assay, Western blotting, ROS measurement, NF-κB activation assay, in vitro ubiquitination assay\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout with direct biochemical demonstration of IκBα as substrate via in vitro ubiquitination assay and multiple orthogonal readouts, single lab\",\n      \"pmids\": [\"20638939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SAG/RBX2 is an essential RING component of SAG-CUL1-FBXW7 E3 ligase that targets NF1 (neurofibromatosis type 1) for ubiquitin-mediated degradation. Sag knockout mice die at E11.5–12.5 with vascular and neural defects associated with NF1 accumulation and RAS inhibition; simultaneous Nf1 deletion partially rescues these defects, establishing NF1 as a physiological SAG substrate.\",\n      \"method\": \"Conditional knockout mouse, embryonic stem cell differentiation assay, epistasis (double knockout rescue), Western blotting, angiogenesis assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis (double KO rescue) combined with in vivo developmental phenotype and biochemical substrate identification, replicated across ES cells and embryos\",\n      \"pmids\": [\"22118770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Rbx2 (RNF7), as a core subunit of the Cullin5-RING E3 ubiquitin ligase (CRL5) complex, stops neocortical projection neurons at their target layers by facilitating Dab1 ubiquitylation and turnover through SOCS7-CRL5 complexes. Rbx2 mutation causes neocortical and cerebellar ectopias dependent on Dab1 (Reelin pathway). SOCS7-CRL5 is required for neocortical but not cerebellar layering, revealing adaptor-specific functions.\",\n      \"method\": \"Conditional knockout mouse (Rbx2 mutation), epistasis (Dab1-dependence), ubiquitylation assay (SOCS7-CRL5 stimulates Dab1 ubiquitylation), SOCS7 overexpression rescue experiment\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis combined with ubiquitylation assay and in vivo rescue experiment in a single rigorous study\",\n      \"pmids\": [\"24210661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Sag endothelial deletion causes embryonic lethality at E15.5 with impaired vasculogenesis; Sag deletion/knockdown in endothelial cells inhibits migration, proliferation, and tube formation, with p27 accumulation responsible for suppression of migration and proliferation.\",\n      \"method\": \"Conditional endothelial-specific knockout mouse (Tie2-Cre), siRNA knockdown in endothelial cells, Matrigel plug angiogenesis assay, in vitro tube formation, Western blotting\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — tissue-specific genetic knockout with in vivo and in vitro functional readouts plus identification of p27 as mediating substrate, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"24213570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Biophysical reconstitution of the full-size neddylated and unneddylated SOCS2-EloBC-Cul5-Rbx2 (CRL5SOCS2) complex in vitro; the complex exists as a monomer. Affinities of protein-protein interactions within the complex were measured by isothermal titration calorimetry.\",\n      \"method\": \"Recombinant protein expression (E. coli and Sf21 insect cells), pulldown from human cell lysates using phospho-GHR peptides, size exclusion chromatography with multi-angle static light scattering, native MS, traveling wave ion mobility MS, ITC\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — full complex reconstitution with multiple orthogonal biophysical methods, single lab\",\n      \"pmids\": [\"25505247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SAG/RBX2 promotes ubiquitylation and degradation of PHLPP1 and DEPTOR, leading to activation of the PI3K/AKT/mTOR axis. Simultaneous knockdown of PHLPP1 or DEPTOR partially rescues the growth suppression caused by SAG knockdown. Phlpp1 and Deptor accumulate in Sag-null prostate cancer tissues with corresponding inactivation of Akt/mTOR.\",\n      \"method\": \"Prostate-specific conditional double knockout (Sag/Pten), siRNA knockdown, in vivo and in vitro ubiquitylation assays, Western blotting, epistasis rescue experiment\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro ubiquitylation assay combined with in vivo genetic model and epistasis rescue, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"27955654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SAG/RBX2 knockdown suppresses lung tumorigenesis and causes accumulation of tumor suppressor substrates p21, p27, NOXA, and BIM, inactivates NF-κB and mTOR pathways; growth suppression is partially rescued by simultaneous knockdown of p21 or the mTOR inhibitor DEPTOR, establishing these as functional SAG substrates.\",\n      \"method\": \"Conditional knockout mouse (KrasG12D-driven lung tumors), siRNA knockdown, epistasis rescue experiments, Western blotting, cell survival assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic model combined with epistasis rescue experiments in vitro, multiple substrates identified, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"24430184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NEDD4-1 (a HECT-domain E3 ubiquitin ligase) directly binds to the C-terminal RING domain of SAG/RBX2 via its HECT domain and ubiquitylates SAG for proteasome-mediated degradation, thereby identifying SAG as a substrate of NEDD4-1. SAG bridges NEDD4-1 (via its C-terminus) and CUL-5 (via its N-terminus) to form a NEDD4-1/SAG/CUL-5 tri-complex.\",\n      \"method\": \"Co-immunoprecipitation, domain-mapping (HECT vs. RING domain interactions), protein half-life assay (overexpression and silencing), ubiquitylation assay\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with domain mapping plus ubiquitylation assay, single lab\",\n      \"pmids\": [\"25216516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Sag deletion induces cellular senescence associated with accumulation of p16 (not p53); mechanistically, Sag deletion causes accumulation of JunB (a SAG-Fbxw7 substrate), a transcription factor that drives p16 transcription. Simultaneous deletion of Cdkn2a (p16 gene) largely rescues senescence, and also rescues Kras(G12D)-induced immortalization abrogated by Sag deletion.\",\n      \"method\": \"Genetic deletion in mouse embryonic fibroblasts, double knockout epistasis (Sag/Cdkn2a), Western blotting, senescence assays\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with double knockout rescue, identification of JunB as substrate, and p16 as the effector, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"25622904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RNF7/SAG interacts with PCNA in human cells; this interaction was identified by bimolecular fluorescence complementation screen and validated by co-immunoprecipitation from human cell extracts and by interaction analyses using recombinant proteins.\",\n      \"method\": \"BiFC screen (bimolecular fluorescence complementation), co-immunoprecipitation from human cell extracts, recombinant protein interaction analysis\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP and recombinant protein interaction as orthogonal validation of BiFC, single lab\",\n      \"pmids\": [\"26030842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Both Rbx1 and Rbx2 can promote ubiquitination of APOBEC3G (A3G) in vitro as part of the HIV-1 Vif-Cul5 E3 ligase complex; however, in cells, only knockdown of endogenous Rbx2 (not Rbx1) impairs Vif-induced A3G degradation. Rbx2 can dose-dependently inhibit Rbx1 interaction with Cul5.\",\n      \"method\": \"In vitro ubiquitination assay, co-immunoprecipitation, siRNA knockdown, Western blotting\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro ubiquitination assay combined with co-IP and cellular knockdown, single lab\",\n      \"pmids\": [\"25912140\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SAG/RBX2 selectively binds E2s UBCH10 and UBE2S (which mediate K11 ubiquitin linkage), whereas RBX1 exclusively binds CDC34 and UBCH5C (K48 linkage). SAG-CUL5 promotes K11-linked ubiquitylation and degradation of β-TrCP1; silencing UBCH10 or UBE2S (but not UBCH5C) causes β-TrCP1 accumulation. SAG-CUL5-β-TrCP1 forms a complex under physiological conditions.\",\n      \"method\": \"Co-immunoprecipitation, E2 binding assays, ubiquitylation assay (K11 linkage-specific), siRNA silencing, protein half-life assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal biochemical methods (co-IP, E2 binding, ubiquitylation assay, siRNA) in a single study discriminating SAG from RBX1 biochemically, single lab\",\n      \"pmids\": [\"27910872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SAG-dependent neddylation in T cells regulates T cell activation, proliferation, and effector cytokine release. SAG T-cell-specific knockout reduces T cell responses and graft-versus-host disease; mechanistically, SAG-mediated effects in T cells are associated with increased SOCS expression (but not NF-κB translocation).\",\n      \"method\": \"T-cell-specific conditional knockout mouse, in vitro T cell stimulation, in vivo allogeneic bone marrow transplantation model, MLN4924 pharmacological inhibition\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — tissue-specific genetic knockout with in vivo model and mechanistic readout, single lab\",\n      \"pmids\": [\"27543965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RNF7/SAG functions as an E3 ubiquitin ligase that negatively regulates CARMA2sh (CARD14) NF-κB signaling by regulating the ubiquitination state of MALT1 and NEMO. RNF7 interacts with CARMA2 (identified by yeast two-hybrid), and psoriasis-associated CARMA2sh mutants escape this negative regulation by RNF7.\",\n      \"method\": \"Yeast two-hybrid screen (identification of interaction), ubiquitination assays for MALT1 and NEMO, functional NF-κB reporter assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — yeast two-hybrid plus ubiquitination assays and functional NF-κB readout, single lab\",\n      \"pmids\": [\"29194363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RBX2 is essential for retinal layering and function: depletion of RBX2 mispositions rod bipolar cells, cone photoreceptors, and Müller glia. SOCS7-CRL5 controls rod bipolar cell and Müller glia positioning via sustained DAB1 (Reelin/RELN) signaling, but cone photoreceptor positioning is SOCS7-independent, revealing distinct CRL5 adaptor utilization. RBX2 depletion also reduces ribbon synapses and disrupts cone photoreceptor function.\",\n      \"method\": \"Conditional knockout mouse (RBX2 and SOCS7), electrophysiology, immunohistochemistry for synaptic markers, epistasis analysis\",\n      \"journal\": \"Development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — tissue-specific genetic knockout with functional readout (electrophysiology and synaptic analysis) plus epistasis, single lab\",\n      \"pmids\": [\"29361558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SAG/RBX2 (Sag) deletion in myeloid cells differentially regulates inflammatory responses: Sag-null macrophages release fewer proinflammatory cytokines, while Sag-null neutrophils release more. SAG depletion alters expression of myeloperoxidase (Mpo) and neutrophil elastase (Elane) in bone marrow cells in response to LPS.\",\n      \"method\": \"LysM-Cre conditional knockout mouse, LPS challenge in vivo, cytokine measurement (ELISA), gene expression analysis\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-type-specific conditional knockout with in vivo functional readout, single lab\",\n      \"pmids\": [\"30574150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SAG/RBX2 competes with APC2 for UBE2C/UBE2S binding, acting as a potential endogenous inhibitor of APC/C and regulating G2-to-M progression. SAG knockdown causes premature APC/C activation, mitotic slippage, and resistance to anti-microtubule drugs. Conversely, SAG is itself a substrate of APC/CCDH1 and is degraded at G1 phase; degradation-resistant SAG-R98A/L101A mutant accelerates G1-to-S progression.\",\n      \"method\": \"Co-immunoprecipitation, competition binding assays, siRNA knockdown, degradation-resistant mutant overexpression, cell cycle and mitotic assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, competition assays, mutagenesis, and functional cell cycle readouts in a single rigorous study with multiple orthogonal methods, single lab\",\n      \"pmids\": [\"32905768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF7/SAG promotes ubiquitination of SOCS1 to activate JAK/STAT3 signaling; STAT3 activation in turn transcriptionally induces RNF7, establishing a STAT3/RNF7 feedback loop. RNF7 overexpression inhibits apoptosis and promotes glycolysis in renal cell carcinoma.\",\n      \"method\": \"Knockdown and overexpression experiments, ubiquitination assay for SOCS1, Western blotting for JAK/STAT3 pathway, luciferase reporter assay, in vivo xenograft\",\n      \"journal\": \"Cellular & molecular biology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — ubiquitination assay and functional readouts with reporter assay, single lab\",\n      \"pmids\": [\"35562668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RBX2/CUL5 (core components of CRL5) localize to mitochondria. RBX2 depletion inhibits mitochondrial ubiquitination and turnover, impairs mitochondrial membrane potential and respiration, and causes cardiomyocyte death. In vivo cardiac-specific Rbx2 deletion suppresses mitophagic activity, causes accumulation of damaged mitochondria, and leads to dilated cardiomyopathy and heart failure. RBX2 controls PINK1 stability in mitochondria. This Parkin-independent mitophagic function does not require Parkin (Parkin deletion has no impact on cardiomyopathy in RBX2-deficient hearts).\",\n      \"method\": \"Subcellular fractionation, immunostaining, immunogold electron microscopy (RBX2/CUL5 mitochondrial localization), cardiomyocyte-specific and adult heart conditional knockout mice, in vitro mitochondrial function assays (membrane potential, respiration), mitophagy assays, proteomics, RNA-sequencing, Western blotting\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct localization by immunogold EM, multiple genetic knockouts (tissue-specific, inducible, and double KO), proteomics, and functional mitochondrial assays, all in a single rigorous study\",\n      \"pmids\": [\"38873758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"YTHDF1 (m6A reader) promotes RNF7 translation in an m6A-dependent manner; elevated RNF7 then promotes degradation of the CDK inhibitor p27, driving prostate cancer cell proliferation. MLN4924 (neddylation inhibitor) inhibits prostate cancer progression in vitro and in vivo through this axis.\",\n      \"method\": \"YTHDF1 knockdown/overexpression, p27 protein stability assay, E3 ligase screening (candidate p27-targeting ligases), xenograft and organoid models, in vitro and in vivo drug treatment\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — candidate E3 screening with functional rescue experiments and organoid validation, single lab\",\n      \"pmids\": [\"40251202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Let-7 microRNA directly binds a conserved motif in the 3' UTR of RBX2, reducing its translation and thereby diminishing CRL5 activity. Restoring RBX2 levels rescues pyramidal neuron positioning (without altering let-7-induced fate effects), demonstrating that let-7 regulates RBX2-CRL5 to control neocortical neuron migration independently of fate specification.\",\n      \"method\": \"In utero electroporation, let-7 overexpression and RBX2 rescue experiments in mouse neocortex, 3' UTR reporter assays, live imaging of neuronal migration\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — 3'UTR reporter + in vivo epistasis rescue + live imaging of neuronal migration, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"42012943\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RNF7 (SAG/RBX2/ROC2) is a RING-domain protein that functions as the catalytic RING subunit of Cullin-RING E3 ubiquitin ligases (CRL1 and preferentially CRL5): it selectively associates with CUL5 (via SOCS-box proteins whose Cul5-box determines specificity) or CUL1, recruits K11-linked E2 enzymes UBCH10/UBE2S, and ubiquitylates multiple substrates including p27, IκBα, c-Jun, NF1, NOXA, HIF-1α, DEPTOR, PHLPP1, β-TrCP1, SOCS1, JunB, and PINK1; it is itself regulated by CKII-mediated phosphorylation at T10 (which is required for efficient substrate degradation), by NEDD4-1-mediated ubiquitination and degradation, by APC/CCDH1 at G1, and at the transcriptional level by HIF-1 and AP-1; RNF7 also localizes to mitochondria as part of CRL5 to drive Parkin-independent, PINK1-dependent mitophagy essential for cardiac homeostasis, and in the developing brain its activity is post-transcriptionally restrained by let-7 miRNA to regulate CRL5-SOCS7-dependent Dab1 ubiquitination and Reelin signaling to control neuronal migration and laminar positioning.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RNF7 (SAG/RBX2/ROC2) is the catalytic RING subunit of Cullin-RING E3 ubiquitin ligases, providing the essential ligase activity that drives substrate polyubiquitination and proteasomal turnover across cell-cycle, signaling, and developmental programs [#0, #13]. It partitions cullin specificity by selectively associating with CUL5 (recruited through the Cul5-box of SOCS-box adaptor proteins such as ASB and SOCS2/SOCS7) while RBX1 pairs with CUL2, and it also serves as the RING component of SCF-type CUL1 complexes (e.g., with FBXW7/Fbw7) [#4, #5, #13]. Distinct from RBX1, RNF7 selectively engages the K11-linkage E2 enzymes UBCH10 and UBE2S to assemble K11-linked chains [#23]. Through these complexes RNF7 targets a broad substrate set governing proliferation, survival, and signaling — p27 and IκBα [#3], c-Jun via SCF-Fbw7 [#7], NF1 [#13], NOXA [#11], PHLPP1 and DEPTOR to activate the PI3K/AKT/mTOR axis [#17], JunB controlling p16-dependent senescence [#20], and β-TrCP1 [#23] — and it operates within feedback loops in which it is transcriptionally induced by HIF-1, AP-1/c-Jun, and STAT3 while degrading components of those same pathways [#6, #7, #29]. Its activity is post-translationally tuned by CKII-mediated phosphorylation at Thr10, required for efficient substrate degradation [#3], and by APC/C-CDH1, which both is antagonized by RNF7 in mitosis and degrades RNF7 at G1 [#28]. Beyond cytoplasmic ligase functions, RNF7 localizes as part of CRL5 to mitochondria to drive Parkin-independent, PINK1-dependent mitophagy essential for cardiac homeostasis [#30], and in the developing nervous system its CRL5-SOCS7 activity ubiquitylates Dab1 to terminate Reelin signaling and control neuronal lamination, an activity restrained by let-7 miRNA [#14, #26, #32].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established that RNF7/SAG is not a passive scaffold but the functional RING ligase subunit of an SCF complex, answering whether it provides catalytic activity essential for viability.\",\n      \"evidence\": \"Yeast genetics with complementation requiring ligase activity, in vitro ubiquitin ligase assay, and co-IP with Cul1\",\n      \"pmids\": [\"10851089\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define mammalian substrate range\", \"Did not distinguish CUL1 vs other cullin partners\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Linked RNF7 to cell-cycle control by showing it promotes S-phase entry through proteasomal degradation of p27, identifying Skp2 as the relevant F-box adaptor.\",\n      \"evidence\": \"mRNA microinjection, adenoviral overexpression, thymidine incorporation, co-IP, and MG132 treatment\",\n      \"pmids\": [\"11255262\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not reconstitute direct p27 ubiquitination by SAG complex\", \"Single-lab cell-based correlation\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Connected the RING-H2 motif to CKII regulation, showing the motif mediates CKIIβ binding and is required for proliferation.\",\n      \"evidence\": \"In vitro binding with purified CKIIβ, co-IP, mutagenesis, and cell-cycle analysis\",\n      \"pmids\": [\"12470599\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not identify the phosphorylated residue\", \"Mechanistic link between phosphorylation and ligase output unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Resolved how CKII modulates RNF7 by mapping phosphorylation to Thr10 and showing it is required for efficient degradation of IκBα and p27.\",\n      \"evidence\": \"In vitro kinase assay, T10A/T10E mutagenesis, proteasome/CKII inhibition, and cell-cycle readout\",\n      \"pmids\": [\"12748192\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not show how phosphorylation alters complex assembly or E2 engagement\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined the structural basis for cullin specificity, demonstrating SAG/RBX2 pairs with CUL5 (not CUL2) and that the SOCS-box Cul5-box determines this partitioning.\",\n      \"evidence\": \"Reciprocal co-IP, domain-swapping mutagenesis, RNAi functional readout, and analysis of ASB-family adaptors with in vitro ligase reconstitution\",\n      \"pmids\": [\"15601820\", \"16325183\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not enumerate the full SOCS-box adaptor repertoire\", \"Did not address in vivo substrate consequences\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Embedded RNF7 in transcription-coupled feedback loops, showing HIF-1 and AP-1/c-Jun induce RNF7, which in turn degrades HIF-1α and c-Jun.\",\n      \"evidence\": \"siRNA silencing, ubiquitination assays, co-IP, ChIP, EMSA, and luciferase reporters across two studies\",\n      \"pmids\": [\"17828303\", \"17440073\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not quantify physiological balance of these competing loops\", \"Adaptor identity for HIF-1α not fully resolved beyond VHL context\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated context- and stage-dependent substrate targeting in vivo, with RNF7 degrading c-Jun early and IκBα later during skin carcinogenesis.\",\n      \"evidence\": \"K14-driven transgenic mouse models, AP-1 reporter mice, UVB exposure, and protein-level readouts\",\n      \"pmids\": [\"17846172\", \"18258608\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of temporal substrate switching unexplained\", \"Single-lab transgenic system\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Established genetic, knockout-level proof that IκBα is a direct SAG-SCF(β-TrCP) substrate and linked RNF7 loss to ROS accumulation and apoptosis, plus identified NOXA as a survival-controlling substrate.\",\n      \"evidence\": \"Gene-trap Sag elimination in ES cells, in vitro ubiquitination, ROS and NF-κB assays; separate siRNA/half-life study for NOXA\",\n      \"pmids\": [\"20638939\", \"20103673\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"NOXA finding was Medium-confidence and lacked in vitro ubiquitination\", \"Adaptor for NOXA not identified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined an essential developmental role by showing SAG-CUL1-FBXW7 targets NF1, with Sag-null embryonic lethality partially rescued by Nf1 deletion.\",\n      \"evidence\": \"Conditional knockout mice, ES-cell differentiation, double-knockout epistasis, and biochemical substrate identification\",\n      \"pmids\": [\"22118770\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not separate CUL1- vs CUL5-dependent embryonic functions\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Revealed adaptor-specific neurodevelopmental functions, showing CRL5-SOCS7 ubiquitylates Dab1 to terminate Reelin signaling and control neocortical layering, while parallel work tied SAG/p27 to vasculogenesis.\",\n      \"evidence\": \"Rbx2 conditional knockouts, Dab1-dependence epistasis, SOCS7 rescue and ubiquitylation assays; endothelial Tie2-Cre knockout with angiogenesis assays\",\n      \"pmids\": [\"24210661\", \"24213570\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not explain region-specific adaptor selection (neocortex vs cerebellum)\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Expanded the oncogenic substrate network and provided biophysical complex reconstitution, showing RNF7 degrades PHLPP1/DEPTOR to activate AKT/mTOR and degrades p21/p27/NOXA/BIM/DEPTOR in lung tumors; also defined NEDD4-1 as a regulator that degrades RNF7.\",\n      \"evidence\": \"Conditional knockout tumor models, in vivo/in vitro ubiquitylation, epistasis rescue, full CRL5SOCS2 reconstitution with ITC/native MS, and NEDD4-1 domain-mapping/ubiquitylation\",\n      \"pmids\": [\"27955654\", \"24430184\", \"25505247\", \"25216516\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"NEDD4-1 regulation was Medium-confidence single lab\", \"Physiological balance of pro- vs anti-tumor substrates context-dependent\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Linked RNF7 to senescence and identified its physical interaction with PCNA and its E2/cullin partner discrimination from RBX1.\",\n      \"evidence\": \"Sag/Cdkn2a double-knockout epistasis with JunB substrate identification; BiFC plus co-IP for PCNA; in vitro and cellular APOBEC3G/Vif-Cul5 ubiquitination assays\",\n      \"pmids\": [\"25622904\", \"26030842\", \"25912140\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of PCNA interaction not defined (Medium-confidence)\", \"A3G work used a viral hijacked complex\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined the biochemical individuality of RNF7 versus RBX1 by showing it selectively binds K11-linkage E2s UBCH10/UBE2S to degrade β-TrCP1, and characterized SAG-dependent neddylation in T-cell function.\",\n      \"evidence\": \"Co-IP, E2 binding and K11-specific ubiquitylation assays, siRNA; T-cell conditional knockout with GVHD model and MLN4924\",\n      \"pmids\": [\"27910872\", \"27543965\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"T-cell mechanism (SOCS expression link) was Medium-confidence correlation\", \"Did not define which adaptor selects β-TrCP1\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified a negative-regulatory role of RNF7 in CARMA2/CARD14 NF-κB signaling via MALT1 and NEMO ubiquitination, with psoriasis-associated mutants escaping regulation.\",\n      \"evidence\": \"Yeast two-hybrid, MALT1/NEMO ubiquitination assays, and NF-κB reporters\",\n      \"pmids\": [\"29194363\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect ubiquitination of MALT1/NEMO not fully separated\", \"Single-lab system\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended adaptor-specific CRL5 function to retinal layering, showing SOCS7-dependent Dab1 control of bipolar/Müller positioning and a SOCS7-independent cone photoreceptor function.\",\n      \"evidence\": \"RBX2 and SOCS7 conditional knockouts, electrophysiology, synaptic marker IHC, and epistasis\",\n      \"pmids\": [\"29361558\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Adaptor responsible for SOCS7-independent cone function unidentified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Uncovered reciprocal regulation with APC/C, showing RNF7 competes for UBE2C/UBE2S to inhibit APC/C in mitosis while being degraded by APC/C-CDH1 at G1.\",\n      \"evidence\": \"Co-IP, competition binding, siRNA, degradation-resistant mutants, and cell-cycle/mitotic assays\",\n      \"pmids\": [\"32905768\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish how the E2 competition is balanced under normal cycling\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established a non-canonical mitochondrial function, showing CRL5/RBX2 localizes to mitochondria and drives Parkin-independent, PINK1-dependent mitophagy essential to prevent dilated cardiomyopathy.\",\n      \"evidence\": \"Subcellular fractionation, immunogold EM, multiple cardiac conditional/inducible and Parkin double knockouts, proteomics, and mitochondrial function assays\",\n      \"pmids\": [\"38873758\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct demonstration of RBX2-mediated PINK1 ubiquitination chemistry not fully detailed\", \"Adaptor recruiting mitochondrial substrates unspecified\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined post-transcriptional control of RNF7, showing m6A/YTHDF1 promotes its translation in prostate cancer and let-7 miRNA restrains RBX2 translation to control neocortical migration.\",\n      \"evidence\": \"YTHDF1 knockdown/overexpression with p27 stability and organoid/xenograft models; let-7 3'UTR reporters, in utero electroporation rescue, and live migration imaging\",\n      \"pmids\": [\"40251202\", \"42012943\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"YTHDF1 axis was Medium-confidence candidate-screen based\", \"Interplay between m6A and let-7 control not jointly examined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How adaptor selection, cullin choice (CUL1 vs CUL5), and E2/linkage usage are dynamically coordinated to specify substrate sets across tissues and subcellular compartments remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No unified structural model integrating mitochondrial vs cytoplasmic complexes\", \"Rules governing context-dependent substrate prioritization not established\", \"Mitochondrial substrate adaptors uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 5, 13, 23]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 12, 17, 23]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 28]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [30]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 12, 23]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1, 3, 28]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [17, 25, 29]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [13, 14, 26, 32]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [30]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [24, 27]}\n    ],\n    \"complexes\": [\"CRL5 (CUL5-RBX2)\", \"SCF (CUL1-RBX2)\", \"CRL5-SOCS7\", \"SCF-FBXW7\"],\n    \"partners\": [\"CUL5\", \"CUL1\", \"SOCS7\", \"FBXW7\", \"UBE2C\", \"UBE2S\", \"NEDD4-1\", \"PCNA\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}