{"gene":"TRIM25","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2007,"finding":"TRIM25 (EFP) is a RING-finger-dependent E3 ubiquitin ligase that mediates K63-linked ubiquitination of RIG-I CARDs at Lys172, which is required for RIG-I downstream signaling, MAVS binding, and antiviral interferon production. The C-terminal SPRY domain of TRIM25 interacts with the N-terminal CARDs of RIG-I to deliver the ubiquitin moiety. Gene targeting confirmed TRIM25 is essential for RIG-I ubiquitination and IFN-β production in response to RNA virus infection.","method":"Co-immunoprecipitation, in vivo ubiquitination assay, site-directed mutagenesis (K172R), gene knockout (Trim25-/- cells), reporter assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, mutagenesis, gene KO with IFN phenotype), replicated across many subsequent studies","pmids":["17392790"],"is_preprint":false},{"year":2002,"finding":"EFP (TRIM25) is a RING-finger-dependent E3 ubiquitin ligase that targets 14-3-3 sigma for ubiquitin-mediated proteasomal degradation, thereby promoting G2→S cell cycle progression and breast cancer cell proliferation. Loss of EFP in MEFs leads to accumulation of 14-3-3 sigma and reduced cell growth.","method":"In vitro ubiquitination assay, antisense knockdown in MCF7 xenografts, MEF knockout analysis, cell proliferation assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro enzymatic assay plus in vivo genetic loss-of-function, replicated in subsequent studies","pmids":["12075357"],"is_preprint":false},{"year":2005,"finding":"EFP (TRIM25) functions as an ISG15 E3 ligase for 14-3-3 sigma, in addition to its ubiquitin E3 ligase activity. EFP's RING domain is required for ISGylation activity, and EFP expression is interferon-inducible. RNAi knockdown of EFP decreased ISGylation of 14-3-3 sigma in 293T cells and MCF-7 cells upon interferon treatment.","method":"siRNA knockdown, in vivo ISGylation assay, RING domain mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — RING-domain mutagenesis plus RNAi knockdown with functional ISGylation readout in two cell lines","pmids":["16352599"],"is_preprint":false},{"year":2007,"finding":"EFP (TRIM25) undergoes autoISGylation at Lys117, mediated by UbcH6 and UbcH8 E2 enzymes and dependent on the RING domain. AutoISGylation of EFP negatively regulates its ISG15 E3 ligase activity toward 14-3-3 sigma (ISGylation-resistant mutant EFP-K117R shows enhanced ISGylation of 14-3-3 sigma).","method":"Site-directed mutagenesis (K117R), in vivo ISGylation assay, E2 co-expression experiments","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis plus functional assay in single lab, two orthogonal methods","pmids":["17222803"],"is_preprint":false},{"year":2007,"finding":"EFP (TRIM25) mRNA and protein are upregulated by Type I IFN in HeLa and HepG2 cells; the first intron contains a functional ISRE that binds STAT1 (verified by EMSA and ChIP). EFP protein is conjugated with both ubiquitin and ISG15 in an IFN-dependent manner.","method":"Luciferase reporter assay, EMSA, chromatin immunoprecipitation (ChIP), Western blot","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — EMSA and ChIP validate STAT1-ISRE interaction; single lab with multiple methods","pmids":["17069755"],"is_preprint":false},{"year":2011,"finding":"EFP (TRIM25) mediates estrogen-induced degradation of KLF5 protein; EFP interacts with and ubiquitinates KLF5, and EFP knockdown increases KLF5 protein levels while overexpression decreases them even when protein synthesis is blocked.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, overexpression, cycloheximide chase","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and ubiquitination assay plus knockdown/OE with functional protein-level readout, single lab","pmids":["21542805"],"is_preprint":false},{"year":2007,"finding":"EFP (TRIM25) interacts with ERα, ubiquitinates it, and promotes its degradation. In the presence of estrogen, EFP-ERα interaction is enhanced, leading to robust interaction with co-activator Tip60 and activation of ERα transcriptional activity. A dominant negative RING-deleted EFP prolonged ERα half-life and inhibited ERα-mediated transcription.","method":"Co-immunoprecipitation, in vitro and in vivo ubiquitination assay, dominant-negative mutagenesis, transcriptional reporter assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ubiquitination assay plus dominant-negative mutagenesis and functional transcription readout, single lab","pmids":["17418098"],"is_preprint":false},{"year":2012,"finding":"EFP (TRIM25) is the E3 ubiquitin ligase mediating oestrogen-induced degradation of the tumor suppressor ATBF1. EFP interacts with and ubiquitinates ATBF1; knockdown of EFP increases ATBF1 levels and overexpression decreases them.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, overexpression experiments","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus ubiquitination and knockdown/OE functional readout, single lab","pmids":["22452784"],"is_preprint":false},{"year":2014,"finding":"USP15 deubiquitylates TRIM25, preventing LUBAC-mediated K48-linked ubiquitination and proteasomal degradation of TRIM25. USP15 was identified as a TRIM25 interaction partner by protein purification and mass spectrometry. Catalytically inactive USP15 failed to stabilize TRIM25. USP15 knockdown enhanced TRIM25 ubiquitination, reduced IFN production, and enhanced viral replication.","method":"Protein purification, mass spectrometry, Co-IP, siRNA knockdown, catalytic mutant (USP15-C269A), IFN reporter assay","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mass spectrometry-based interactome, catalytic mutant validation, functional phenotype (IFN and viral replication), single lab with multiple orthogonal methods","pmids":["24399297"],"is_preprint":false},{"year":2015,"finding":"Dengue virus subgenomic flavivirus RNA (sfRNA) binds TRIM25 in a sequence-dependent manner and prevents its deubiquitylation, which is critical for sustained and amplified RIG-I-induced type I IFN expression. PR-2B DENV-2 produced more sfRNA relative to genomic RNA and showed greater epidemiological fitness via this mechanism.","method":"RNA-protein binding assay, ubiquitination/deubiquitylation assay, viral fitness comparison between DENV clades","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct RNA-protein binding demonstrated, functional consequence on TRIM25 deubiquitylation and IFN expression established, single lab with multiple methods","pmids":["26138103"],"is_preprint":false},{"year":2015,"finding":"TRIM25 has a dual role in the p53/Mdm2 circuit: TRIM25 inhibits ubiquitination and proteasomal degradation of both p53 and Mdm2, increasing their abundance. Despite increased p53 levels, TRIM25 inhibits p53 transcriptional activity by interfering with p300-Mdm2 interaction (a critical step for p53 polyubiquitination). TRIM25 knockdown increases p53 acetylation and p53-dependent cell death. In medaka, TRIM25 knockdown-induced apoptosis was rescued by co-knockdown of p53.","method":"Co-immunoprecipitation, ubiquitination assay, genetic epistasis (medaka knockdown rescue), p53 transcriptional activity assay, apoptosis assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, and in vivo epistasis in medaka; single lab with multiple methods","pmids":["25728675"],"is_preprint":false},{"year":2014,"finding":"TRIM25 acts as an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation: it binds to the conserved terminal loop (CTL) of pre-let-7 and activates TuT4, allowing more efficient Lin28a-mediated uridylation. This function was identified via RNA pull-downs coupled with quantitative mass spectrometry.","method":"RNA pull-down, quantitative mass spectrometry, uridylation assay, RNA binding experiments","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro uridylation assay plus RNA pull-down and MS identification, single lab","pmids":["25457611"],"is_preprint":false},{"year":2017,"finding":"TRIM25 enhances ZAP's antiviral activity by interacting with ZAP through the SPRY domain. Both TRIM25's RING domain (ligase activity) and coiled-coil domain (oligomerization) are required. TRIM25 increases K48- and K63-linked polyubiquitination of ZAP and is critical for ZAP's ability to inhibit translation of incoming Sindbis virus genome.","method":"Co-immunoprecipitation, genome-wide RNAi screen, RING and coiled-coil domain deletion mutants, viral replication assay, translation inhibition assay","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — genome-wide screen with stringent confirmation, domain mutagenesis, and functional translation inhibition readout, single lab with multiple orthogonal methods","pmids":["28060952"],"is_preprint":false},{"year":2017,"finding":"TRIM25 is required for the antiviral activity of ZAP; TRIM25 E3 ligase activity is required for this function. TRIM25 mediates ZAP ubiquitination and modulates ZAP's target RNA-binding activity. Downregulation of endogenous ubiquitin or overexpression of OTUB1 deubiquitinase impaired ZAP's antiviral activity.","method":"siRNA knockdown, E3 ligase mutant, ubiquitination assay, RNA-binding assay, viral replication assay","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown plus mutant E3 ligase and RNA-binding assay, single lab","pmids":["28202764"],"is_preprint":false},{"year":2017,"finding":"Nuclear TRIM25 specifically binds to influenza A virus ribonucleoproteins (vRNPs) and inhibits viral RNA synthesis independently of its ubiquitin ligase activity and the interferon pathway. TRIM25 does not block initiation of viral mRNA synthesis but prevents movement of RNA into the polymerase complex (blocks RNA chain elongation). NS1 can inhibit this nuclear TRIM25 function.","method":"Nuclear fractionation, Co-IP with vRNPs, RNA synthesis assay, ubiquitin ligase-dead mutant, viral replication assay","journal":"Cell host & microbe","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct vRNP binding shown, ligase-independent mechanism established by mutant analysis, single lab","pmids":["29107643"],"is_preprint":false},{"year":2018,"finding":"Crystal structure of the human TRIM25 coiled-coil–PRYSPRY module and of complexes with influenza NS1 revealed that NS1 binding to the coiled-coil domain interferes with correct positioning of the PRYSPRY domain required for substrate ubiquitination, explaining how NS1 suppresses RIG-I ubiquitination. NS1 binding does not affect RING dimerization or unanchored K63-linked poly-Ub chain synthesis.","method":"X-ray crystallography, in vitro ubiquitination assay, structural mutagenesis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional validation by in vitro ubiquitination assay and mutagenesis, single lab","pmids":["29739942"],"is_preprint":false},{"year":2018,"finding":"TRIM25 binds both single-stranded and double-stranded RNA. Multiple regions including the C-terminal SPRY domain and a lysine-rich linker motif contribute to RNA binding. RNA binding modulates TRIM25's ubiquitination activity in vitro, its subcellular localization in cells, and its antiviral activity.","method":"In vitro RNA binding assay (gel shift, filter binding), in vitro ubiquitination assay, subcellular fractionation/localization, antiviral reporter assay, domain mutagenesis","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro assays with mutagenesis plus localization and antiviral function, single lab","pmids":["30342007"],"is_preprint":false},{"year":2018,"finding":"TRIM25 interacts with G3BP2 and modulates p53 via the G3BP2/RanBP2-mediated p53 nuclear export mechanism in prostate cancer. TRIM25 knockdown activates p53 downstream cell cycle inhibition and apoptosis, while overexpression promotes proliferation. TRIM25 is required for G3BP2/RanBP2-mediated sumoylation of p53 leading to its cytoplasmic localization.","method":"Co-immunoprecipitation, siRNA knockdown, overexpression, apoptosis/cell cycle assays, xenograft model","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus KD/OE with functional phenotype and in vivo model, single lab","pmids":["29379164"],"is_preprint":false},{"year":2017,"finding":"Upon viral infection, TRIM25 is redistributed into cytoplasmic dots associated with stress granules, while RIG-I associates with TRIM25/stress granules and then with mitochondrial MAVS. MAVS competes with TRIM25 for RIG-I binding, suggesting that upon TRIM25-mediated K63-ubiquitination, RIG-I moves from TRIM25 to MAVS at mitochondria. Influenza NS1 inhibits TRIM25 homocomplex formation but not RIG-I/TRIM25 heterocomplex formation.","method":"Bimolecular fluorescence complementation (BiFC), super-resolution microscopy, live-cell imaging, Co-IP","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BiFC and super-resolution imaging with endogenous and overexpressed proteins, functional context established, single lab","pmids":["27807226"],"is_preprint":false},{"year":2019,"finding":"NLRP12 binds TRIM25 through its nucleotide-binding domain, preventing TRIM25-mediated K63-linked ubiquitination and activation of RIG-I. NLRP12 also enhances RNF125-mediated K48-linked degradative ubiquitination of RIG-I. VSV infection downregulates NLRP12 to allow RIG-I activation. Myeloid-specific Nlrp12-deficient mice show heightened IFN and TNF responses and are more resistant to VSV.","method":"Co-immunoprecipitation, ubiquitination assay, conditional knockout mice, viral infection assay","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay with in vivo mouse KO validation, single lab with multiple orthogonal methods","pmids":["30902577"],"is_preprint":false},{"year":2019,"finding":"TRIM25 deletion does not affect the IFN response to influenza A/B, Sendai virus, or RIG-I agonists in mouse or human cell lines, and does not affect RIG-I ubiquitination endogenously, in contrast to RIPLET deletion which completely abrogates RIG-I-dependent IFN responses. Despite this, TRIM25 loss increases susceptibility to IAV infection in vivo, suggesting an alternative antiviral role.","method":"CRISPR knockout (Trim25, Riplet, Rig-i), IFN reporter assay, viral replication assay, in vivo mouse infection","journal":"Immunology and cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean CRISPR KO in multiple cell lines (mouse and human) plus in vivo, replicating negative result for TRIM25 in endogenous RIG-I ubiquitination; challenges prior paradigm","pmids":["31335993"],"is_preprint":false},{"year":2019,"finding":"MAP3K13 phosphorylates TRIM25 at Ser12, decreasing its polyubiquitination and proteasomal degradation. Stabilized TRIM25 then ubiquitinates FBXW7α at Lys412, preventing Myc ubiquitination and promoting Myc protein stability and tumor development.","method":"Co-immunoprecipitation, in vitro kinase assay, ubiquitination assay, phosphorylation site mutagenesis, xenograft tumor model","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase assay and ubiquitination assay with site mutagenesis and in vivo tumor model, single lab","pmids":["31186535"],"is_preprint":false},{"year":2019,"finding":"The lncRNA Lnczc3h7a binds to both TRIM25 and activated RIG-I, serving as a molecular scaffold that stabilizes the RIG-I–TRIM25 complex at early stages of viral infection. This facilitates TRIM25-mediated K63-linked ubiquitination of RIG-I and promotes downstream antiviral signaling. Depletion of Lnczc3h7a impairs RIG-I signaling in vitro and in vivo.","method":"RNA immunoprecipitation, Co-IP, in vitro ubiquitination assay, lncRNA knockdown in vivo","journal":"Nature immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNA-protein Co-IP plus ubiquitination and in vivo knockdown, single lab; the lncRNA as scaffold is a new interaction mode for TRIM25","pmids":["31036902"],"is_preprint":false},{"year":2020,"finding":"TRIM25 directly ubiquitinates Keap1, leading to its proteasomal degradation and consequent Nrf2 activation, which bolsters antioxidant defense and promotes ER-associated degradation and cell survival in hepatocellular carcinoma. Depletion of TRIM25 causes ER stress and attenuates tumor growth in vitro and in vivo.","method":"Co-immunoprecipitation, ubiquitination assay, TRIM25 knockout/knockdown, xenograft model, ER stress assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct ubiquitination assay plus KO with ER stress and in vivo tumor phenotype, single lab with multiple orthogonal methods","pmids":["31953436"],"is_preprint":false},{"year":2020,"finding":"OTUD5 deubiquitinase deubiquitinates TRIM25, altering its ubiquitination level. OTUD5 depletion leads to enhanced TRIM25 transcriptional activity and inhibited PML expression, promoting tumor growth.","method":"RNAi screen, Co-immunoprecipitation, ubiquitination assay, xenograft model","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — RNAi screen plus Co-IP and ubiquitination assay with in vivo tumor model, single lab","pmids":["32826889"],"is_preprint":false},{"year":2020,"finding":"TRIM25 interacts with TRAF2 and promotes K63-linked polyubiquitination of TRAF2, enhancing TNF-α-induced NF-κB activation. TRIM25 bridges the interaction between TRAF2 and TAK1 or IKKβ. Knockdown of TRIM25 reduces NF-κB signaling.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, NF-κB reporter assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus ubiquitination assay and functional NF-κB reporter, single lab","pmids":["32024699"],"is_preprint":false},{"year":2021,"finding":"TRIM25 directly interacts with RIP3 through its SPRY domain and mediates K48-linked polyubiquitination of RIP3 at Lys501 via its RING domain, promoting proteasomal degradation of RIP3 and inhibiting TNF-induced cell necrosis. TRIM25 deficiency inhibits RIP3 ubiquitination and promotes necrosis.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, site-directed mutagenesis (K501R), TRIM25 knockout, cell necrosis assay","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and in vitro ubiquitination with mutagenesis and KO functional phenotype, single lab","pmids":["33953350"],"is_preprint":false},{"year":2021,"finding":"TRIM25 activates AKT/mTOR signaling in NSCLC by binding PTEN and mediating K63-linked ubiquitination at K266, which prevents PTEN plasma membrane translocation and reduces its phosphatase activity, thereby accumulating PI(3,4,5)P3.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K266), PTEN localization assay, phosphatase activity assay","journal":"Acta pharmacologica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination with mutagenesis, and functional localization/phosphatase assay, single lab","pmids":["33931764"],"is_preprint":false},{"year":2018,"finding":"TRIM25 is a novel E3 ubiquitin ligase of PPARγ that directly ubiquitinates PPARγ, leading to its proteasome-dependent degradation. Stable TRIM25 expression suppresses adipocyte differentiation in 3T3-L1 cells; TRIM25 knockout MEFs show increased adipocyte differentiation capability.","method":"Co-immunoprecipitation, ubiquitination assay, TRIM25 knockout MEFs, adipocyte differentiation assay","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct ubiquitination assay with KO MEF phenotype, single lab","pmids":["30323259"],"is_preprint":false},{"year":2022,"finding":"TRIM25 interacts with the EBOV vRNP, undergoes autoubiquitination, and ubiquitinates viral nucleoprotein (NP). TRIM25 is recruited to incoming vRNPs shortly after cell entry and causes dissociation of NP from vRNA. TRIM25 antiviral activity against EBOV requires ZAP and is modulated by CpG content of viral genome.","method":"Co-immunoprecipitation, ubiquitination assay, confocal microscopy (TRIM25 recruitment to vRNPs), TRIM25 knockout, ISG screen","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay with localization and KO functional readout, single lab","pmids":["35533151"],"is_preprint":false},{"year":2022,"finding":"TRIM25 binds and destabilizes influenza A virus mRNAs independently of its E3 ubiquitin ligase activity and independently of the RIG-I/IFN pathway. Direct tethering of TRIM25 to RNA is sufficient to downregulate the targeted RNA. TRIM25 RNA-binding-deficient mutant (ΔRBD) and RING-dead mutant still inhibited IAV replication. TRIM25 is not required for RIG-I pathway activation by IAV-derived 5'-triphosphate RNA.","method":"TRIM25 mutant analysis (ΔRBD, ΔRING), tethering assay, viral replication assay, IFN pathway reporter assay, RNA stability assay","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple domain mutants with viral replication and RNA stability readout, single lab","pmids":["35736141"],"is_preprint":false},{"year":2022,"finding":"TRIM25 ubiquitinates multiple substrates identified by a substrate-trapping R54P catalytic mutant, including G3BP1/2 (stress granule formation), UPF1 (NMD), NME1 (nucleoside synthesis), and PABPC4 (mRNA translation/stability). The R54P mutation abolishes TRIM25 inhibition of alphaviruses independently of the host IFN response, indicating direct ubiquitination-dependent antiviral activity.","method":"Substrate-trapping mutagenesis (R54P), mass spectrometry, knockdown of interactors, viral replication assay","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — substrate-trap MS approach with functional confirmation by knockdown and viral assay, single lab","pmids":["36067236"],"is_preprint":false},{"year":2024,"finding":"TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the stress granule core protein G3BP1 in a dsRNA-dependent manner. This co-condensation into antiviral stress granules significantly enhances TRIM25's ubiquitination activity toward multiple antiviral proteins localized in SGs, and is critical for activating the RIG-I signaling pathway to restrain RNA virus infection.","method":"LLPS assay, co-immunoprecipitation, live-cell imaging, ubiquitination assay, TRIM25 knockout, viral replication assay","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — LLPS demonstrated in vitro and in cells, ubiquitination enhanced in condensates shown functionally, single lab","pmids":["38750080"],"is_preprint":false},{"year":2024,"finding":"TRIM25's RNA-binding residues were comprehensively mapped using biophysical techniques, identifying key residues required for RNA interaction. A RNA-binding-deficient mutant (TRIM25-m9) was developed. iCLIP2 in virus-infected and uninfected cells showed TRIM25 binds specifically to viral RNA, and RNA binding is critical for TRIM25 antiviral activity.","method":"iCLIP2, biophysical RNA-binding assays, mutagenesis (TRIM25-m9), antiviral activity assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple biophysical methods plus iCLIP2 transcriptome-wide mapping and functional mutagenesis, single lab","pmids":["39353916"],"is_preprint":false},{"year":2025,"finding":"TRIM25 is a key suppressor of exogenous (LNP-delivered) mRNA turnover; it is activated by acidic pH (protons released from ruptured endosomes), increasing its RNA affinity and inducing turnover of both linear and circular exogenous mRNAs. The endoribonucleases N4BP1 and KHNYN and antiviral protein ZAP act redundantly downstream of TRIM25 in this surveillance pathway. N1-methylpseudouridine modification reduces TRIM25's RNA binding, enabling RNAs to evade suppression.","method":"Genome-wide CRISPR screen, RNA binding assay at varying pH, mRNA stability assay, KO of pathway components","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — genome-wide CRISPR screen with functional validation, pH-dependent binding mechanism biochemically demonstrated, multiple orthogonal approaches, single lab","pmids":["40179174"],"is_preprint":false},{"year":2021,"finding":"TRIM25 inhibits IBDV replication by specifically interacting with and mediating K27-linked polyubiquitination of viral structural protein VP3 at Lys854, promoting its proteasomal degradation.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K854R), viral replication assay in vitro and in vivo","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay with mutagenesis and in vivo confirmation, single lab","pmids":["34516573"],"is_preprint":false},{"year":2019,"finding":"NDR2 kinase directly associates with both RIG-I and TRIM25, facilitating the RIG-I–TRIM25 complex and enhancing TRIM25-mediated K63-linked polyubiquitination of RIG-I. Both wild-type and kinase-inactive NDR2 potentiate antiviral IFN production, indicating the scaffolding rather than kinase activity mediates this effect.","method":"Co-immunoprecipitation, ubiquitination assay, conditional knockout mice (Lysm+NDR2f/f), viral replication assay","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay with conditional KO mouse model, single lab","pmids":["30775439"],"is_preprint":false},{"year":2016,"finding":"TRIM25 (EFP) ubiquitinates and promotes degradation of ERG transcription factor in prostate cancer. TRIM25 binds full-length ERG and N-terminally truncated TMPRSS2-ERG fusion variants. TRIM25 polyubiquitinates ERG in vitro; inactivation of TRIM25 stabilizes ERG. ERG upregulates TRIM25 expression, creating a regulatory feedback.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, TRIM25 inactivation, ERG protein stability assay","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro ubiquitination plus Co-IP and genetic inactivation, single lab","pmids":["27626314"],"is_preprint":false},{"year":2024,"finding":"UBC12 transfers NEDD8 to TRIM25 at K117, and this neddylation modification reduces steric hindrance in the TRIM25 RING domain, facilitating TRIM25 binding to ubiquitylated substrates. Neddylated TRIM25 then promotes K63-polyubiquitination of TFEB, increasing TFEB nuclear translocation and autophagy gene transcription, thereby reducing TNBC sensitivity to paclitaxel.","method":"Co-IP, Western blot, molecular docking and dynamics simulation, ubiquitination assay, site mutagenesis (K117), xenograft model","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and ubiquitination assay with site mutagenesis and in vivo model; molecular dynamics as supporting but not definitive evidence","pmids":["38926803"],"is_preprint":false}],"current_model":"TRIM25 is a multi-functional RING-type E3 ubiquitin ligase (and ISG15 E3 ligase) that primarily acts by mediating K63-linked ubiquitination of RIG-I CARDs (via SPRY domain–CARD interaction) to activate antiviral innate immunity, but also ubiquitinates a broad range of substrates including 14-3-3 sigma (K48, proteasomal), Keap1/Nrf2 axis, PTEN, RIP3, PPARγ, ERG, TRAF2, and viral proteins; its activity is regulated by RNA binding (which enhances ubiquitination activity and is required for antiviral function), by pH-dependent RNA affinity, by liquid-liquid phase separation with G3BP1 at antiviral stress granules, by USP15-mediated deubiquitination that prevents LUBAC/K48-linked degradation, by OTUD5-mediated deubiquitination, by neddylation at K117, and by MAP3K13-mediated phosphorylation at Ser12; TRIM25 is also inhibited by viral proteins (influenza NS1, SARS-CoV-2 N, dengue sfRNA, paramyxovirus V proteins) that disrupt SPRY–CARD interactions or TRIM25 deubiquitylation, and—contrary to the original paradigm—endogenous TRIM25 is dispensable for RIG-I-dependent IFN responses, where RIPLET is the essential E3 ligase."},"narrative":{"mechanistic_narrative":"TRIM25 (EFP) is a RING-type E3 ubiquitin ligase that operates at the interface of antiviral innate immunity, RNA surveillance, and cell-growth control, exerting its effects both through substrate ubiquitination and through ligase-independent RNA binding [PMID:17392790, PMID:12075357, PMID:35736141]. In the canonical antiviral arm, TRIM25 mediates K63-linked ubiquitination of the RIG-I CARDs at Lys172 via a SPRY domain–CARD interaction, a modification originally defined as required for MAVS engagement and IFN-β production [PMID:17392790]; this reaction is scaffolded and tuned by accessory factors including the lncRNA Lnczc3h7a, which stabilizes the RIG-I–TRIM25 complex, and the NDR2 kinase acting as a non-catalytic scaffold [PMID:31036902, PMID:30775439], and is restrained by NLRP12 binding [PMID:30902577]. A substantial body of work establishes that TRIM25 is itself a sequence-specific RNA-binding protein: it binds single- and double-stranded RNA through its SPRY domain and a lysine-rich linker, binds viral RNA in cells, and this RNA engagement modulates its ubiquitination activity, localization, and antiviral function [PMID:30342007, PMID:39353916]. RNA-dependent regulation extends to liquid–liquid phase separation with G3BP1 in antiviral stress granules, which concentrates and enhances TRIM25 ligase activity [PMID:38750080], and to a pH-sensitive RNA-surveillance pathway in which acidic conditions raise TRIM25 RNA affinity to trigger turnover of exogenous mRNAs through the redundant nucleases N4BP1, KHNYN, and ZAP [PMID:40179174]. TRIM25 also restricts viruses by ligase-independent mechanisms—binding influenza vRNPs to block viral RNA synthesis and destabilizing viral mRNAs—and partners with ZAP to enforce antiviral activity [PMID:29107643, PMID:35736141, PMID:28060952]. Beyond immunity, TRIM25 was first characterized as an estrogen-responsive ligase driving proteasomal degradation of 14-3-3 sigma to promote cell-cycle progression and also functions as an ISG15 E3 ligase [PMID:12075357, PMID:16352599]. It ubiquitinates a broad substrate set spanning oncogenic and tumor-suppressor pathways, including Keap1 (activating Nrf2 antioxidant defense) [PMID:31953436], PTEN (K63 ubiquitination restraining its membrane localization to activate AKT/mTOR) [PMID:33931764], PPARγ [PMID:30323259], ERG [PMID:27626314], RIP3 (K48-linked, suppressing necrosis) [PMID:33953350], and TRAF2 (K63-linked, enhancing NF-κB signaling) [PMID:32024699]. TRIM25 activity is controlled post-translationally by USP15- and OTUD5-mediated deubiquitination that counter LUBAC-driven K48 degradation [PMID:24399297, PMID:32826889], by MAP3K13 phosphorylation at Ser12 that stabilizes the protein [PMID:31186535], and by neddylation at K117 that relieves RING steric hindrance to promote substrate engagement [PMID:38926803]. Notably, clean genetic deletion established that endogenous TRIM25 is dispensable for RIG-I-dependent IFN responses—with RIPLET being the essential ligase—reframing TRIM25's antiviral role toward RNA-directed and substrate-directed mechanisms [PMID:31335993].","teleology":[{"year":2002,"claim":"Established TRIM25's founding enzymatic identity: that it is a RING-dependent E3 ubiquitin ligase coupling estrogen signaling to cell-cycle control by degrading a growth inhibitor.","evidence":"In vitro ubiquitination, antisense knockdown in MCF7 xenografts, and MEF knockout showing 14-3-3 sigma accumulation","pmids":["12075357"],"confidence":"High","gaps":["Did not define ubiquitin linkage type on 14-3-3 sigma","Did not address non-cell-cycle functions"]},{"year":2005,"claim":"Showed TRIM25 is bifunctional as both a ubiquitin and an ISG15 E3 ligase and is interferon-inducible, linking it to the IFN response before its RIG-I role was known.","evidence":"siRNA knockdown and RING-domain mutagenesis with ISGylation readout in 293T and MCF-7 cells","pmids":["16352599"],"confidence":"High","gaps":["Did not establish physiological ISGylation targets beyond 14-3-3 sigma","Mechanism of E2 selection for ISG15 vs ubiquitin unresolved"]},{"year":2007,"claim":"Defined TRIM25 as the essential E3 ligase delivering K63-linked ubiquitin to RIG-I CARDs at Lys172, providing the molecular basis for RIG-I-driven antiviral interferon induction.","evidence":"Co-IP, in vivo ubiquitination, K172R mutagenesis, and Trim25-/- knockout with IFN-β phenotype","pmids":["17392790"],"confidence":"High","gaps":["Did not anticipate redundancy with RIPLET","Did not resolve how the SPRY-CARD interaction is spatially coordinated with MAVS handoff"]},{"year":2007,"claim":"Resolved how TRIM25 ISG15 activity is autoregulated and how its expression is wired into IFN signaling, explaining its induction kinetics.","evidence":"K117R autoISGylation mutagenesis with UbcH6/UbcH8; ISRE/STAT1 EMSA and ChIP on the first intron","pmids":["17222803","17069755"],"confidence":"Medium","gaps":["K117 was later found to also be a neddylation site, complicating interpretation","Autoregulatory loop not quantified in vivo"]},{"year":2011,"claim":"Extended TRIM25's substrate repertoire in estrogen-responsive growth control by identifying degradative targets beyond 14-3-3 sigma.","evidence":"Co-IP, ubiquitination assay, knockdown/overexpression and cycloheximide chase for KLF5, ERα, and ATBF1","pmids":["21542805","17418098","22452784"],"confidence":"Medium","gaps":["Linkage types and direct vs indirect ubiquitination not fully resolved","Physiological context restricted to overexpression/knockdown systems"]},{"year":2014,"claim":"Revealed that TRIM25 protein levels are gated by deubiquitination, defining a stability checkpoint that controls antiviral output.","evidence":"MS-based interactome, USP15-C269A catalytic mutant, knockdown with IFN and viral replication readouts","pmids":["24399297"],"confidence":"High","gaps":["Did not map the LUBAC-targeted lysines on TRIM25","Interplay with later-identified OTUD5 not addressed"]},{"year":2014,"claim":"Uncovered an RNA-cofactor role distinct from ubiquitination, showing TRIM25 directly binds pre-let-7 to activate uridylation, foreshadowing its identity as an RNA-binding protein.","evidence":"RNA pull-down with quantitative MS and in vitro uridylation assays","pmids":["25457611"],"confidence":"Medium","gaps":["RNA-binding determinants not yet mapped","Relationship of this RNA role to ligase activity unclear"]},{"year":2015,"claim":"Demonstrated that viral RNA can subvert TRIM25 by blocking its deubiquitylation, defining a host-pathogen control point linked to viral fitness.","evidence":"Sequence-dependent sfRNA-TRIM25 binding, deubiquitylation assay, and DENV clade fitness comparison","pmids":["26138103"],"confidence":"High","gaps":["TRIM25 RNA-binding interface for sfRNA not defined","Relationship to RIPLET redundancy not tested"]},{"year":2017,"claim":"Established TRIM25 as a required cofactor and ubiquitin source for ZAP-mediated translational restriction, broadening its antiviral mechanism beyond RIG-I.","evidence":"Genome-wide RNAi screen, RING and coiled-coil deletion mutants, ZAP ubiquitination and Sindbis translation inhibition assays","pmids":["28060952","28202764"],"confidence":"High","gaps":["Ubiquitin linkage specificity on ZAP (K48 vs K63) mixed","Whether TRIM25-ZAP cooperation is IFN-dependent left open"]},{"year":2017,"claim":"Identified a ligase-independent nuclear antiviral activity, showing TRIM25 directly inhibits influenza RNA elongation by binding vRNPs.","evidence":"Nuclear fractionation, vRNP Co-IP, RNA synthesis assay with ligase-dead mutant","pmids":["29107643"],"confidence":"Medium","gaps":["Structural basis of vRNP recognition not defined","Quantitative contribution relative to IFN pathway unclear"]},{"year":2017,"claim":"Mapped the spatial choreography of RIG-I activation, placing TRIM25 at stress-granule-associated cytoplasmic dots from which ubiquitinated RIG-I transfers to mitochondrial MAVS.","evidence":"BiFC, super-resolution and live-cell imaging, Co-IP showing MAVS-TRIM25 competition for RIG-I","pmids":["27807226"],"confidence":"Medium","gaps":["Did not establish whether condensation is required for activity","NS1 disruption of homocomplex not structurally resolved"]},{"year":2018,"claim":"Provided structural mechanism for viral antagonism, showing influenza NS1 binds the coiled-coil to mispositions the PRYSPRY domain and block substrate ubiquitination.","evidence":"X-ray crystallography of coiled-coil-PRYSPRY and NS1 complexes with in vitro ubiquitination validation","pmids":["29739942"],"confidence":"High","gaps":["Structure of full-length TRIM25 on a substrate not solved","RING dimerization and chain synthesis shown unaffected, leaving substrate-positioning model partly inferential"]},{"year":2018,"claim":"Defined TRIM25 as a bona fide RNA-binding protein and connected RNA binding causally to its ligase activity, localization, and antiviral function.","evidence":"In vitro RNA binding (gel shift, filter binding), in vitro ubiquitination, localization and antiviral reporter assays with domain mutagenesis","pmids":["30342007"],"confidence":"Medium","gaps":["Precise RNA-binding residues not yet resolved (addressed later)","RNA sequence/structure preference in cells undefined"]},{"year":2018,"claim":"Broadened TRIM25's oncogenic substrate network, identifying PPARγ degradation controlling adipocyte differentiation.","evidence":"Ubiquitination assay and TRIM25 knockout MEF differentiation phenotype","pmids":["30323259"],"confidence":"Medium","gaps":["Linkage type on PPARγ not defined","Single-lab, limited cellular context"]},{"year":2019,"claim":"Overturned the founding paradigm by showing endogenous TRIM25 is dispensable for RIG-I-dependent IFN responses, with RIPLET being the essential ligase.","evidence":"CRISPR knockout of Trim25, Riplet, and Rig-i in mouse and human cells, IFN reporter and viral assays, plus in vivo IAV infection","pmids":["31335993"],"confidence":"High","gaps":["Did not define the alternative antiviral mechanism explaining increased IAV susceptibility","Reconciliation with prior overexpression-based RIG-I data left to subsequent RNA-directed studies"]},{"year":2019,"claim":"Identified scaffolding and inhibitory partners (Lnczc3h7a, NDR2, NLRP12) that gate TRIM25-RIG-I complex assembly, refining how its activity is set in cells.","evidence":"RNA-IP and Co-IP with ubiquitination assays, in vivo lncRNA knockdown, and conditional knockout mice for NDR2 and NLRP12","pmids":["31036902","30775439","30902577"],"confidence":"High","gaps":["These results coexist with the RIPLET-redundancy finding, leaving the in vivo weight of TRIM25-RIG-I unresolved","Stoichiometry of scaffold complexes undefined"]},{"year":2019,"claim":"Connected TRIM25 stability control to oncogenesis, showing MAP3K13 phosphorylation at Ser12 protects TRIM25 from degradation to drive Myc stabilization.","evidence":"In vitro kinase assay, ubiquitination assay, S12 mutagenesis, and xenograft tumor model (FBXW7α/Myc axis)","pmids":["31186535"],"confidence":"Medium","gaps":["Did not resolve which degradative ligase Ser12 phosphorylation counteracts","FBXW7α K412 ubiquitination linkage not characterized"]},{"year":2020,"claim":"Expanded TRIM25's pro-survival substrate axes, defining Keap1 degradation (Nrf2 activation), TRAF2 K63 ubiquitination (NF-κB), and an OTUD5 deubiquitination control node.","evidence":"Co-IP, ubiquitination assays, knockout/knockdown with ER stress, NF-κB reporter, and xenograft readouts","pmids":["31953436","32024699","32826889"],"confidence":"High","gaps":["Substrate selectivity rules among these competing targets undefined","How RNA binding influences these non-immune substrates not tested"]},{"year":2021,"claim":"Defined TRIM25's roles in cell-death and growth-signaling control through linkage-specific ubiquitination of RIP3 (K48, anti-necrosis) and PTEN (K63, AKT/mTOR activation).","evidence":"Co-IP, in vitro ubiquitination with K501R and K266 mutagenesis, knockout necrosis assay, and PTEN localization/phosphatase assays","pmids":["33953350","33931764"],"confidence":"Medium","gaps":["Tissue specificity of these substrate choices unclear","Single-lab findings without reciprocal validation"]},{"year":2022,"claim":"Demonstrated that TRIM25 restricts viruses by ligase-independent RNA destabilization and by direct ubiquitination of stress-granule/RNA-metabolism substrates, establishing RNA-directed antiviral mechanisms separate from RIG-I.","evidence":"ΔRBD/ΔRING mutants and tethering assays for IAV mRNA decay; R54P substrate-trap MS identifying G3BP1/2, UPF1, NME1, PABPC4; EBOV vRNP/NP ubiquitination","pmids":["35736141","36067236","35533151"],"confidence":"Medium","gaps":["Endonuclease(s) executing TRIM25-directed RNA decay not yet identified here","Which substrates are functionally dominant for restriction unresolved"]},{"year":2024,"claim":"Resolved the molecular determinants and condensation behavior of TRIM25 RNA binding, mapping RNA-binding residues, demonstrating viral-RNA binding by iCLIP2, and showing dsRNA-driven phase separation with G3BP1 enhances ligase activity.","evidence":"iCLIP2 and biophysical mapping with TRIM25-m9 mutant; LLPS assays, live-cell imaging, ubiquitination and viral replication assays","pmids":["39353916","38750080"],"confidence":"High","gaps":["In vivo requirement of condensation for restriction not genetically dissected","Link between RNA-binding map and specific substrate ubiquitination incomplete"]},{"year":2024,"claim":"Showed neddylation at K117 is a positive activating modification, relieving RING steric hindrance to promote substrate engagement and TFEB-driven autophagy.","evidence":"UBC12 neddylation, K117 mutagenesis, molecular dynamics, ubiquitination assay, and xenograft model in TNBC","pmids":["38926803"],"confidence":"Medium","gaps":["K117 is also an autoISGylation site, and crosstalk between these modifications is unresolved","Structural model relies partly on docking/MD rather than experimental structure"]},{"year":2025,"claim":"Established TRIM25 as a pH-sensitive sensor of exogenous RNA that triggers mRNA turnover, with direct relevance to mRNA-therapeutic evasion via nucleoside modification.","evidence":"Genome-wide CRISPR screen, pH-dependent RNA-affinity measurements, mRNA stability assays, and knockout of N4BP1/KHNYN/ZAP","pmids":["40179174"],"confidence":"High","gaps":["Structural basis of pH-dependent affinity switch not defined","Whether endogenous RNA turnover uses the same mechanism unresolved"]},{"year":null,"claim":"It remains unresolved how TRIM25 integrates its RNA-sensing, condensate, and ligase activities into a unified in vivo function, and what its dominant physiological role is given that it is dispensable for RIG-I-dependent IFN yet broadly active across antiviral and oncogenic substrate networks.","evidence":"No single study in the timeline reconciles the RIPLET-redundancy finding with the many substrate- and RNA-directed activities","pmids":[],"confidence":"Medium","gaps":["No structure of full-length TRIM25 engaging RNA and substrate together","Quantitative ranking of physiologically dominant substrates lacking","Crosstalk among K117 neddylation/ISGylation and Ser12 phosphorylation not integrated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,23,26,27,28,37]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,26,27,29]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[11,16,30,33,34]},{"term_id":"GO:0031386","term_label":"protein tag activity","supporting_discovery_ids":[2,3]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[22,36]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[18,32]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[14,4]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[18,32]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,8,19,22,36]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[9,14,29,30,35]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[11,30,31,34]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,23,26,27,28,37]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[38]}],"complexes":["RIG-I–TRIM25 complex","antiviral stress granules (with G3BP1)"],"partners":["RIG-I","USP15","G3BP1","ZAP","TRAF2","NLRP12","G3BP2","OTUD5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q14258","full_name":"E3 ubiquitin/ISG15 ligase TRIM25","aliases":["Estrogen-responsive finger protein","RING finger protein 147","RING-type E3 ubiquitin transferase","RING-type E3 ubiquitin transferase TRIM25","Tripartite motif-containing protein 25","Ubiquitin/ISG15-conjugating enzyme TRIM25","Zinc finger protein 147"],"length_aa":630,"mass_kda":71.0,"function":"Functions as a ubiquitin E3 ligase and as an ISG15 E3 ligase (PubMed:16352599). Involved in innate immune defense against viruses by mediating ubiquitination of RIGI and IFIH1 (PubMed:17392790, PubMed:29357390, PubMed:30193849, PubMed:31710640, PubMed:33849980, PubMed:36045682). Mediates 'Lys-63'-linked polyubiquitination of the RIGI N-terminal CARD-like region and may play a role in signal transduction that leads to the production of interferons in response to viral infection (PubMed:17392790, PubMed:23950712). Mediates 'Lys-63'-linked polyubiquitination of IFIH1 (PubMed:30193849). Promotes ISGylation of 14-3-3 sigma (SFN), an adapter protein implicated in the regulation of a large spectrum signaling pathway (PubMed:16352599, PubMed:17069755). Mediates estrogen action in various target organs (PubMed:22452784). Mediates the ubiquitination and subsequent proteasomal degradation of ZFHX3 (PubMed:22452784). Plays a role in promoting the restart of stalled replication forks via interaction with the KHDC3L-OOEP scaffold and subsequent ubiquitination of BLM, resulting in the recruitment and retainment of BLM at DNA replication forks (By similarity). Plays an essential role in the antiviral activity of ZAP/ZC3HAV1; an antiviral protein which inhibits the replication of certain viruses. Mechanistically, mediates 'Lys-63'-linked polyubiquitination of ZAP/ZC3HAV1 that is required for its optimal binding to target mRNA (PubMed:28060952, PubMed:28202764). Also mediates the ubiquitination of various substrates implicated in stress granule formation, nonsense-mediated mRNA decay, nucleoside synthesis and mRNA translation and stability (PubMed:36067236)","subcellular_location":"Cytoplasm; Cytoplasm, Stress granule; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q14258/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TRIM25","classification":"Not Classified","n_dependent_lines":14,"n_total_lines":1208,"dependency_fraction":0.011589403973509934},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TRIM25","total_profiled":1310},"omim":[{"mim_id":"621451","title":"SMALL NUCLEOLAR RNA HOST GENE 12; SNHG12","url":"https://www.omim.org/entry/621451"},{"mim_id":"619579","title":"KH DOMAIN- AND NYN DOMAIN-CONTAINING PROTEIN; KHNYN","url":"https://www.omim.org/entry/619579"},{"mim_id":"618326","title":"ZINC FINGER CCHC DOMAIN-CONTAINING PROTEIN 3; ZCCHC3","url":"https://www.omim.org/entry/618326"},{"mim_id":"614371","title":"DENGUE VIRUS, SUSCEPTIBILITY TO","url":"https://www.omim.org/entry/614371"},{"mim_id":"609631","title":"RNA SENSOR RIGI; RIGI","url":"https://www.omim.org/entry/609631"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Nuclear bodies","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TRIM25"},"hgnc":{"alias_symbol":["EFP","RNF147"],"prev_symbol":["ZNF147"]},"alphafold":{"accession":"Q14258","domains":[{"cath_id":"3.30.40.10","chopping":"5-83","consensus_level":"high","plddt":87.6781,"start":5,"end":83},{"cath_id":"3.30.160.60","chopping":"118-194","consensus_level":"medium","plddt":90.8335,"start":118,"end":194},{"cath_id":"-","chopping":"195-354","consensus_level":"medium","plddt":92.9614,"start":195,"end":354},{"cath_id":"2.60.120.920","chopping":"446-628","consensus_level":"high","plddt":96.4547,"start":446,"end":628}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14258","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q14258-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q14258-F1-predicted_aligned_error_v6.png","plddt_mean":84.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TRIM25","jax_strain_url":"https://www.jax.org/strain/search?query=TRIM25"},"sequence":{"accession":"Q14258","fasta_url":"https://rest.uniprot.org/uniprotkb/Q14258.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q14258/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14258"}},"corpus_meta":[{"pmid":"17392790","id":"PMC_17392790","title":"TRIM25 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The C-terminal SPRY domain of TRIM25 interacts with the N-terminal CARDs of RIG-I to deliver the ubiquitin moiety. Gene targeting confirmed TRIM25 is essential for RIG-I ubiquitination and IFN-β production in response to RNA virus infection.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assay, site-directed mutagenesis (K172R), gene knockout (Trim25-/- cells), reporter assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, mutagenesis, gene KO with IFN phenotype), replicated across many subsequent studies\",\n      \"pmids\": [\"17392790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"EFP (TRIM25) is a RING-finger-dependent E3 ubiquitin ligase that targets 14-3-3 sigma for ubiquitin-mediated proteasomal degradation, thereby promoting G2→S cell cycle progression and breast cancer cell proliferation. Loss of EFP in MEFs leads to accumulation of 14-3-3 sigma and reduced cell growth.\",\n      \"method\": \"In vitro ubiquitination assay, antisense knockdown in MCF7 xenografts, MEF knockout analysis, cell proliferation assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro enzymatic assay plus in vivo genetic loss-of-function, replicated in subsequent studies\",\n      \"pmids\": [\"12075357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"EFP (TRIM25) functions as an ISG15 E3 ligase for 14-3-3 sigma, in addition to its ubiquitin E3 ligase activity. EFP's RING domain is required for ISGylation activity, and EFP expression is interferon-inducible. RNAi knockdown of EFP decreased ISGylation of 14-3-3 sigma in 293T cells and MCF-7 cells upon interferon treatment.\",\n      \"method\": \"siRNA knockdown, in vivo ISGylation assay, RING domain mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — RING-domain mutagenesis plus RNAi knockdown with functional ISGylation readout in two cell lines\",\n      \"pmids\": [\"16352599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EFP (TRIM25) undergoes autoISGylation at Lys117, mediated by UbcH6 and UbcH8 E2 enzymes and dependent on the RING domain. AutoISGylation of EFP negatively regulates its ISG15 E3 ligase activity toward 14-3-3 sigma (ISGylation-resistant mutant EFP-K117R shows enhanced ISGylation of 14-3-3 sigma).\",\n      \"method\": \"Site-directed mutagenesis (K117R), in vivo ISGylation assay, E2 co-expression experiments\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis plus functional assay in single lab, two orthogonal methods\",\n      \"pmids\": [\"17222803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EFP (TRIM25) mRNA and protein are upregulated by Type I IFN in HeLa and HepG2 cells; the first intron contains a functional ISRE that binds STAT1 (verified by EMSA and ChIP). EFP protein is conjugated with both ubiquitin and ISG15 in an IFN-dependent manner.\",\n      \"method\": \"Luciferase reporter assay, EMSA, chromatin immunoprecipitation (ChIP), Western blot\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — EMSA and ChIP validate STAT1-ISRE interaction; single lab with multiple methods\",\n      \"pmids\": [\"17069755\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EFP (TRIM25) mediates estrogen-induced degradation of KLF5 protein; EFP interacts with and ubiquitinates KLF5, and EFP knockdown increases KLF5 protein levels while overexpression decreases them even when protein synthesis is blocked.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, overexpression, cycloheximide chase\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and ubiquitination assay plus knockdown/OE with functional protein-level readout, single lab\",\n      \"pmids\": [\"21542805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EFP (TRIM25) interacts with ERα, ubiquitinates it, and promotes its degradation. In the presence of estrogen, EFP-ERα interaction is enhanced, leading to robust interaction with co-activator Tip60 and activation of ERα transcriptional activity. A dominant negative RING-deleted EFP prolonged ERα half-life and inhibited ERα-mediated transcription.\",\n      \"method\": \"Co-immunoprecipitation, in vitro and in vivo ubiquitination assay, dominant-negative mutagenesis, transcriptional reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ubiquitination assay plus dominant-negative mutagenesis and functional transcription readout, single lab\",\n      \"pmids\": [\"17418098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EFP (TRIM25) is the E3 ubiquitin ligase mediating oestrogen-induced degradation of the tumor suppressor ATBF1. EFP interacts with and ubiquitinates ATBF1; knockdown of EFP increases ATBF1 levels and overexpression decreases them.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, overexpression experiments\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus ubiquitination and knockdown/OE functional readout, single lab\",\n      \"pmids\": [\"22452784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"USP15 deubiquitylates TRIM25, preventing LUBAC-mediated K48-linked ubiquitination and proteasomal degradation of TRIM25. USP15 was identified as a TRIM25 interaction partner by protein purification and mass spectrometry. Catalytically inactive USP15 failed to stabilize TRIM25. USP15 knockdown enhanced TRIM25 ubiquitination, reduced IFN production, and enhanced viral replication.\",\n      \"method\": \"Protein purification, mass spectrometry, Co-IP, siRNA knockdown, catalytic mutant (USP15-C269A), IFN reporter assay\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mass spectrometry-based interactome, catalytic mutant validation, functional phenotype (IFN and viral replication), single lab with multiple orthogonal methods\",\n      \"pmids\": [\"24399297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Dengue virus subgenomic flavivirus RNA (sfRNA) binds TRIM25 in a sequence-dependent manner and prevents its deubiquitylation, which is critical for sustained and amplified RIG-I-induced type I IFN expression. PR-2B DENV-2 produced more sfRNA relative to genomic RNA and showed greater epidemiological fitness via this mechanism.\",\n      \"method\": \"RNA-protein binding assay, ubiquitination/deubiquitylation assay, viral fitness comparison between DENV clades\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct RNA-protein binding demonstrated, functional consequence on TRIM25 deubiquitylation and IFN expression established, single lab with multiple methods\",\n      \"pmids\": [\"26138103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRIM25 has a dual role in the p53/Mdm2 circuit: TRIM25 inhibits ubiquitination and proteasomal degradation of both p53 and Mdm2, increasing their abundance. Despite increased p53 levels, TRIM25 inhibits p53 transcriptional activity by interfering with p300-Mdm2 interaction (a critical step for p53 polyubiquitination). TRIM25 knockdown increases p53 acetylation and p53-dependent cell death. In medaka, TRIM25 knockdown-induced apoptosis was rescued by co-knockdown of p53.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, genetic epistasis (medaka knockdown rescue), p53 transcriptional activity assay, apoptosis assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, and in vivo epistasis in medaka; single lab with multiple methods\",\n      \"pmids\": [\"25728675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TRIM25 acts as an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation: it binds to the conserved terminal loop (CTL) of pre-let-7 and activates TuT4, allowing more efficient Lin28a-mediated uridylation. This function was identified via RNA pull-downs coupled with quantitative mass spectrometry.\",\n      \"method\": \"RNA pull-down, quantitative mass spectrometry, uridylation assay, RNA binding experiments\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro uridylation assay plus RNA pull-down and MS identification, single lab\",\n      \"pmids\": [\"25457611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRIM25 enhances ZAP's antiviral activity by interacting with ZAP through the SPRY domain. Both TRIM25's RING domain (ligase activity) and coiled-coil domain (oligomerization) are required. TRIM25 increases K48- and K63-linked polyubiquitination of ZAP and is critical for ZAP's ability to inhibit translation of incoming Sindbis virus genome.\",\n      \"method\": \"Co-immunoprecipitation, genome-wide RNAi screen, RING and coiled-coil domain deletion mutants, viral replication assay, translation inhibition assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — genome-wide screen with stringent confirmation, domain mutagenesis, and functional translation inhibition readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"28060952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRIM25 is required for the antiviral activity of ZAP; TRIM25 E3 ligase activity is required for this function. TRIM25 mediates ZAP ubiquitination and modulates ZAP's target RNA-binding activity. Downregulation of endogenous ubiquitin or overexpression of OTUB1 deubiquitinase impaired ZAP's antiviral activity.\",\n      \"method\": \"siRNA knockdown, E3 ligase mutant, ubiquitination assay, RNA-binding assay, viral replication assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown plus mutant E3 ligase and RNA-binding assay, single lab\",\n      \"pmids\": [\"28202764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Nuclear TRIM25 specifically binds to influenza A virus ribonucleoproteins (vRNPs) and inhibits viral RNA synthesis independently of its ubiquitin ligase activity and the interferon pathway. TRIM25 does not block initiation of viral mRNA synthesis but prevents movement of RNA into the polymerase complex (blocks RNA chain elongation). NS1 can inhibit this nuclear TRIM25 function.\",\n      \"method\": \"Nuclear fractionation, Co-IP with vRNPs, RNA synthesis assay, ubiquitin ligase-dead mutant, viral replication assay\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct vRNP binding shown, ligase-independent mechanism established by mutant analysis, single lab\",\n      \"pmids\": [\"29107643\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Crystal structure of the human TRIM25 coiled-coil–PRYSPRY module and of complexes with influenza NS1 revealed that NS1 binding to the coiled-coil domain interferes with correct positioning of the PRYSPRY domain required for substrate ubiquitination, explaining how NS1 suppresses RIG-I ubiquitination. NS1 binding does not affect RING dimerization or unanchored K63-linked poly-Ub chain synthesis.\",\n      \"method\": \"X-ray crystallography, in vitro ubiquitination assay, structural mutagenesis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional validation by in vitro ubiquitination assay and mutagenesis, single lab\",\n      \"pmids\": [\"29739942\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TRIM25 binds both single-stranded and double-stranded RNA. Multiple regions including the C-terminal SPRY domain and a lysine-rich linker motif contribute to RNA binding. RNA binding modulates TRIM25's ubiquitination activity in vitro, its subcellular localization in cells, and its antiviral activity.\",\n      \"method\": \"In vitro RNA binding assay (gel shift, filter binding), in vitro ubiquitination assay, subcellular fractionation/localization, antiviral reporter assay, domain mutagenesis\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro assays with mutagenesis plus localization and antiviral function, single lab\",\n      \"pmids\": [\"30342007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TRIM25 interacts with G3BP2 and modulates p53 via the G3BP2/RanBP2-mediated p53 nuclear export mechanism in prostate cancer. TRIM25 knockdown activates p53 downstream cell cycle inhibition and apoptosis, while overexpression promotes proliferation. TRIM25 is required for G3BP2/RanBP2-mediated sumoylation of p53 leading to its cytoplasmic localization.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, overexpression, apoptosis/cell cycle assays, xenograft model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus KD/OE with functional phenotype and in vivo model, single lab\",\n      \"pmids\": [\"29379164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Upon viral infection, TRIM25 is redistributed into cytoplasmic dots associated with stress granules, while RIG-I associates with TRIM25/stress granules and then with mitochondrial MAVS. MAVS competes with TRIM25 for RIG-I binding, suggesting that upon TRIM25-mediated K63-ubiquitination, RIG-I moves from TRIM25 to MAVS at mitochondria. Influenza NS1 inhibits TRIM25 homocomplex formation but not RIG-I/TRIM25 heterocomplex formation.\",\n      \"method\": \"Bimolecular fluorescence complementation (BiFC), super-resolution microscopy, live-cell imaging, Co-IP\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BiFC and super-resolution imaging with endogenous and overexpressed proteins, functional context established, single lab\",\n      \"pmids\": [\"27807226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NLRP12 binds TRIM25 through its nucleotide-binding domain, preventing TRIM25-mediated K63-linked ubiquitination and activation of RIG-I. NLRP12 also enhances RNF125-mediated K48-linked degradative ubiquitination of RIG-I. VSV infection downregulates NLRP12 to allow RIG-I activation. Myeloid-specific Nlrp12-deficient mice show heightened IFN and TNF responses and are more resistant to VSV.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, conditional knockout mice, viral infection assay\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay with in vivo mouse KO validation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"30902577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRIM25 deletion does not affect the IFN response to influenza A/B, Sendai virus, or RIG-I agonists in mouse or human cell lines, and does not affect RIG-I ubiquitination endogenously, in contrast to RIPLET deletion which completely abrogates RIG-I-dependent IFN responses. Despite this, TRIM25 loss increases susceptibility to IAV infection in vivo, suggesting an alternative antiviral role.\",\n      \"method\": \"CRISPR knockout (Trim25, Riplet, Rig-i), IFN reporter assay, viral replication assay, in vivo mouse infection\",\n      \"journal\": \"Immunology and cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean CRISPR KO in multiple cell lines (mouse and human) plus in vivo, replicating negative result for TRIM25 in endogenous RIG-I ubiquitination; challenges prior paradigm\",\n      \"pmids\": [\"31335993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MAP3K13 phosphorylates TRIM25 at Ser12, decreasing its polyubiquitination and proteasomal degradation. Stabilized TRIM25 then ubiquitinates FBXW7α at Lys412, preventing Myc ubiquitination and promoting Myc protein stability and tumor development.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, ubiquitination assay, phosphorylation site mutagenesis, xenograft tumor model\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase assay and ubiquitination assay with site mutagenesis and in vivo tumor model, single lab\",\n      \"pmids\": [\"31186535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The lncRNA Lnczc3h7a binds to both TRIM25 and activated RIG-I, serving as a molecular scaffold that stabilizes the RIG-I–TRIM25 complex at early stages of viral infection. This facilitates TRIM25-mediated K63-linked ubiquitination of RIG-I and promotes downstream antiviral signaling. Depletion of Lnczc3h7a impairs RIG-I signaling in vitro and in vivo.\",\n      \"method\": \"RNA immunoprecipitation, Co-IP, in vitro ubiquitination assay, lncRNA knockdown in vivo\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA-protein Co-IP plus ubiquitination and in vivo knockdown, single lab; the lncRNA as scaffold is a new interaction mode for TRIM25\",\n      \"pmids\": [\"31036902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TRIM25 directly ubiquitinates Keap1, leading to its proteasomal degradation and consequent Nrf2 activation, which bolsters antioxidant defense and promotes ER-associated degradation and cell survival in hepatocellular carcinoma. Depletion of TRIM25 causes ER stress and attenuates tumor growth in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, TRIM25 knockout/knockdown, xenograft model, ER stress assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct ubiquitination assay plus KO with ER stress and in vivo tumor phenotype, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"31953436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OTUD5 deubiquitinase deubiquitinates TRIM25, altering its ubiquitination level. OTUD5 depletion leads to enhanced TRIM25 transcriptional activity and inhibited PML expression, promoting tumor growth.\",\n      \"method\": \"RNAi screen, Co-immunoprecipitation, ubiquitination assay, xenograft model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — RNAi screen plus Co-IP and ubiquitination assay with in vivo tumor model, single lab\",\n      \"pmids\": [\"32826889\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TRIM25 interacts with TRAF2 and promotes K63-linked polyubiquitination of TRAF2, enhancing TNF-α-induced NF-κB activation. TRIM25 bridges the interaction between TRAF2 and TAK1 or IKKβ. Knockdown of TRIM25 reduces NF-κB signaling.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, NF-κB reporter assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus ubiquitination assay and functional NF-κB reporter, single lab\",\n      \"pmids\": [\"32024699\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRIM25 directly interacts with RIP3 through its SPRY domain and mediates K48-linked polyubiquitination of RIP3 at Lys501 via its RING domain, promoting proteasomal degradation of RIP3 and inhibiting TNF-induced cell necrosis. TRIM25 deficiency inhibits RIP3 ubiquitination and promotes necrosis.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, site-directed mutagenesis (K501R), TRIM25 knockout, cell necrosis assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and in vitro ubiquitination with mutagenesis and KO functional phenotype, single lab\",\n      \"pmids\": [\"33953350\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRIM25 activates AKT/mTOR signaling in NSCLC by binding PTEN and mediating K63-linked ubiquitination at K266, which prevents PTEN plasma membrane translocation and reduces its phosphatase activity, thereby accumulating PI(3,4,5)P3.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K266), PTEN localization assay, phosphatase activity assay\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination with mutagenesis, and functional localization/phosphatase assay, single lab\",\n      \"pmids\": [\"33931764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TRIM25 is a novel E3 ubiquitin ligase of PPARγ that directly ubiquitinates PPARγ, leading to its proteasome-dependent degradation. Stable TRIM25 expression suppresses adipocyte differentiation in 3T3-L1 cells; TRIM25 knockout MEFs show increased adipocyte differentiation capability.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, TRIM25 knockout MEFs, adipocyte differentiation assay\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct ubiquitination assay with KO MEF phenotype, single lab\",\n      \"pmids\": [\"30323259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRIM25 interacts with the EBOV vRNP, undergoes autoubiquitination, and ubiquitinates viral nucleoprotein (NP). TRIM25 is recruited to incoming vRNPs shortly after cell entry and causes dissociation of NP from vRNA. TRIM25 antiviral activity against EBOV requires ZAP and is modulated by CpG content of viral genome.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, confocal microscopy (TRIM25 recruitment to vRNPs), TRIM25 knockout, ISG screen\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay with localization and KO functional readout, single lab\",\n      \"pmids\": [\"35533151\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRIM25 binds and destabilizes influenza A virus mRNAs independently of its E3 ubiquitin ligase activity and independently of the RIG-I/IFN pathway. Direct tethering of TRIM25 to RNA is sufficient to downregulate the targeted RNA. TRIM25 RNA-binding-deficient mutant (ΔRBD) and RING-dead mutant still inhibited IAV replication. TRIM25 is not required for RIG-I pathway activation by IAV-derived 5'-triphosphate RNA.\",\n      \"method\": \"TRIM25 mutant analysis (ΔRBD, ΔRING), tethering assay, viral replication assay, IFN pathway reporter assay, RNA stability assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple domain mutants with viral replication and RNA stability readout, single lab\",\n      \"pmids\": [\"35736141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRIM25 ubiquitinates multiple substrates identified by a substrate-trapping R54P catalytic mutant, including G3BP1/2 (stress granule formation), UPF1 (NMD), NME1 (nucleoside synthesis), and PABPC4 (mRNA translation/stability). The R54P mutation abolishes TRIM25 inhibition of alphaviruses independently of the host IFN response, indicating direct ubiquitination-dependent antiviral activity.\",\n      \"method\": \"Substrate-trapping mutagenesis (R54P), mass spectrometry, knockdown of interactors, viral replication assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — substrate-trap MS approach with functional confirmation by knockdown and viral assay, single lab\",\n      \"pmids\": [\"36067236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the stress granule core protein G3BP1 in a dsRNA-dependent manner. This co-condensation into antiviral stress granules significantly enhances TRIM25's ubiquitination activity toward multiple antiviral proteins localized in SGs, and is critical for activating the RIG-I signaling pathway to restrain RNA virus infection.\",\n      \"method\": \"LLPS assay, co-immunoprecipitation, live-cell imaging, ubiquitination assay, TRIM25 knockout, viral replication assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — LLPS demonstrated in vitro and in cells, ubiquitination enhanced in condensates shown functionally, single lab\",\n      \"pmids\": [\"38750080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TRIM25's RNA-binding residues were comprehensively mapped using biophysical techniques, identifying key residues required for RNA interaction. A RNA-binding-deficient mutant (TRIM25-m9) was developed. iCLIP2 in virus-infected and uninfected cells showed TRIM25 binds specifically to viral RNA, and RNA binding is critical for TRIM25 antiviral activity.\",\n      \"method\": \"iCLIP2, biophysical RNA-binding assays, mutagenesis (TRIM25-m9), antiviral activity assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple biophysical methods plus iCLIP2 transcriptome-wide mapping and functional mutagenesis, single lab\",\n      \"pmids\": [\"39353916\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TRIM25 is a key suppressor of exogenous (LNP-delivered) mRNA turnover; it is activated by acidic pH (protons released from ruptured endosomes), increasing its RNA affinity and inducing turnover of both linear and circular exogenous mRNAs. The endoribonucleases N4BP1 and KHNYN and antiviral protein ZAP act redundantly downstream of TRIM25 in this surveillance pathway. N1-methylpseudouridine modification reduces TRIM25's RNA binding, enabling RNAs to evade suppression.\",\n      \"method\": \"Genome-wide CRISPR screen, RNA binding assay at varying pH, mRNA stability assay, KO of pathway components\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — genome-wide CRISPR screen with functional validation, pH-dependent binding mechanism biochemically demonstrated, multiple orthogonal approaches, single lab\",\n      \"pmids\": [\"40179174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRIM25 inhibits IBDV replication by specifically interacting with and mediating K27-linked polyubiquitination of viral structural protein VP3 at Lys854, promoting its proteasomal degradation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K854R), viral replication assay in vitro and in vivo\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay with mutagenesis and in vivo confirmation, single lab\",\n      \"pmids\": [\"34516573\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NDR2 kinase directly associates with both RIG-I and TRIM25, facilitating the RIG-I–TRIM25 complex and enhancing TRIM25-mediated K63-linked polyubiquitination of RIG-I. Both wild-type and kinase-inactive NDR2 potentiate antiviral IFN production, indicating the scaffolding rather than kinase activity mediates this effect.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, conditional knockout mice (Lysm+NDR2f/f), viral replication assay\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay with conditional KO mouse model, single lab\",\n      \"pmids\": [\"30775439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TRIM25 (EFP) ubiquitinates and promotes degradation of ERG transcription factor in prostate cancer. TRIM25 binds full-length ERG and N-terminally truncated TMPRSS2-ERG fusion variants. TRIM25 polyubiquitinates ERG in vitro; inactivation of TRIM25 stabilizes ERG. ERG upregulates TRIM25 expression, creating a regulatory feedback.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, TRIM25 inactivation, ERG protein stability assay\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro ubiquitination plus Co-IP and genetic inactivation, single lab\",\n      \"pmids\": [\"27626314\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UBC12 transfers NEDD8 to TRIM25 at K117, and this neddylation modification reduces steric hindrance in the TRIM25 RING domain, facilitating TRIM25 binding to ubiquitylated substrates. Neddylated TRIM25 then promotes K63-polyubiquitination of TFEB, increasing TFEB nuclear translocation and autophagy gene transcription, thereby reducing TNBC sensitivity to paclitaxel.\",\n      \"method\": \"Co-IP, Western blot, molecular docking and dynamics simulation, ubiquitination assay, site mutagenesis (K117), xenograft model\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and ubiquitination assay with site mutagenesis and in vivo model; molecular dynamics as supporting but not definitive evidence\",\n      \"pmids\": [\"38926803\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TRIM25 is a multi-functional RING-type E3 ubiquitin ligase (and ISG15 E3 ligase) that primarily acts by mediating K63-linked ubiquitination of RIG-I CARDs (via SPRY domain–CARD interaction) to activate antiviral innate immunity, but also ubiquitinates a broad range of substrates including 14-3-3 sigma (K48, proteasomal), Keap1/Nrf2 axis, PTEN, RIP3, PPARγ, ERG, TRAF2, and viral proteins; its activity is regulated by RNA binding (which enhances ubiquitination activity and is required for antiviral function), by pH-dependent RNA affinity, by liquid-liquid phase separation with G3BP1 at antiviral stress granules, by USP15-mediated deubiquitination that prevents LUBAC/K48-linked degradation, by OTUD5-mediated deubiquitination, by neddylation at K117, and by MAP3K13-mediated phosphorylation at Ser12; TRIM25 is also inhibited by viral proteins (influenza NS1, SARS-CoV-2 N, dengue sfRNA, paramyxovirus V proteins) that disrupt SPRY–CARD interactions or TRIM25 deubiquitylation, and—contrary to the original paradigm—endogenous TRIM25 is dispensable for RIG-I-dependent IFN responses, where RIPLET is the essential E3 ligase.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TRIM25 (EFP) is a RING-type E3 ubiquitin ligase that operates at the interface of antiviral innate immunity, RNA surveillance, and cell-growth control, exerting its effects both through substrate ubiquitination and through ligase-independent RNA binding [#0, #1, #30]. In the canonical antiviral arm, TRIM25 mediates K63-linked ubiquitination of the RIG-I CARDs at Lys172 via a SPRY domain–CARD interaction, a modification originally defined as required for MAVS engagement and IFN-\\u03b2 production [#0]; this reaction is scaffolded and tuned by accessory factors including the lncRNA Lnczc3h7a, which stabilizes the RIG-I–TRIM25 complex, and the NDR2 kinase acting as a non-catalytic scaffold [#22, #36], and is restrained by NLRP12 binding [#19]. A substantial body of work establishes that TRIM25 is itself a sequence-specific RNA-binding protein: it binds single- and double-stranded RNA through its SPRY domain and a lysine-rich linker, binds viral RNA in cells, and this RNA engagement modulates its ubiquitination activity, localization, and antiviral function [#16, #33]. RNA-dependent regulation extends to liquid–liquid phase separation with G3BP1 in antiviral stress granules, which concentrates and enhances TRIM25 ligase activity [#32], and to a pH-sensitive RNA-surveillance pathway in which acidic conditions raise TRIM25 RNA affinity to trigger turnover of exogenous mRNAs through the redundant nucleases N4BP1, KHNYN, and ZAP [#34]. TRIM25 also restricts viruses by ligase-independent mechanisms\\u2014binding influenza vRNPs to block viral RNA synthesis and destabilizing viral mRNAs\\u2014and partners with ZAP to enforce antiviral activity [#14, #30, #12]. Beyond immunity, TRIM25 was first characterized as an estrogen-responsive ligase driving proteasomal degradation of 14-3-3 sigma to promote cell-cycle progression and also functions as an ISG15 E3 ligase [#1, #2]. It ubiquitinates a broad substrate set spanning oncogenic and tumor-suppressor pathways, including Keap1 (activating Nrf2 antioxidant defense) [#23], PTEN (K63 ubiquitination restraining its membrane localization to activate AKT/mTOR) [#27], PPAR\\u03b3 [#28], ERG [#37], RIP3 (K48-linked, suppressing necrosis) [#26], and TRAF2 (K63-linked, enhancing NF-\\u03baB signaling) [#25]. TRIM25 activity is controlled post-translationally by USP15- and OTUD5-mediated deubiquitination that counter LUBAC-driven K48 degradation [#8, #24], by MAP3K13 phosphorylation at Ser12 that stabilizes the protein [#21], and by neddylation at K117 that relieves RING steric hindrance to promote substrate engagement [#38]. Notably, clean genetic deletion established that endogenous TRIM25 is dispensable for RIG-I-dependent IFN responses\\u2014with RIPLET being the essential ligase\\u2014reframing TRIM25's antiviral role toward RNA-directed and substrate-directed mechanisms [#20].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established TRIM25's founding enzymatic identity: that it is a RING-dependent E3 ubiquitin ligase coupling estrogen signaling to cell-cycle control by degrading a growth inhibitor.\",\n      \"evidence\": \"In vitro ubiquitination, antisense knockdown in MCF7 xenografts, and MEF knockout showing 14-3-3 sigma accumulation\",\n      \"pmids\": [\"12075357\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define ubiquitin linkage type on 14-3-3 sigma\", \"Did not address non-cell-cycle functions\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed TRIM25 is bifunctional as both a ubiquitin and an ISG15 E3 ligase and is interferon-inducible, linking it to the IFN response before its RIG-I role was known.\",\n      \"evidence\": \"siRNA knockdown and RING-domain mutagenesis with ISGylation readout in 293T and MCF-7 cells\",\n      \"pmids\": [\"16352599\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish physiological ISGylation targets beyond 14-3-3 sigma\", \"Mechanism of E2 selection for ISG15 vs ubiquitin unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined TRIM25 as the essential E3 ligase delivering K63-linked ubiquitin to RIG-I CARDs at Lys172, providing the molecular basis for RIG-I-driven antiviral interferon induction.\",\n      \"evidence\": \"Co-IP, in vivo ubiquitination, K172R mutagenesis, and Trim25-/- knockout with IFN-\\u03b2 phenotype\",\n      \"pmids\": [\"17392790\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not anticipate redundancy with RIPLET\", \"Did not resolve how the SPRY-CARD interaction is spatially coordinated with MAVS handoff\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Resolved how TRIM25 ISG15 activity is autoregulated and how its expression is wired into IFN signaling, explaining its induction kinetics.\",\n      \"evidence\": \"K117R autoISGylation mutagenesis with UbcH6/UbcH8; ISRE/STAT1 EMSA and ChIP on the first intron\",\n      \"pmids\": [\"17222803\", \"17069755\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"K117 was later found to also be a neddylation site, complicating interpretation\", \"Autoregulatory loop not quantified in vivo\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Extended TRIM25's substrate repertoire in estrogen-responsive growth control by identifying degradative targets beyond 14-3-3 sigma.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, knockdown/overexpression and cycloheximide chase for KLF5, ER\\u03b1, and ATBF1\",\n      \"pmids\": [\"21542805\", \"17418098\", \"22452784\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Linkage types and direct vs indirect ubiquitination not fully resolved\", \"Physiological context restricted to overexpression/knockdown systems\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revealed that TRIM25 protein levels are gated by deubiquitination, defining a stability checkpoint that controls antiviral output.\",\n      \"evidence\": \"MS-based interactome, USP15-C269A catalytic mutant, knockdown with IFN and viral replication readouts\",\n      \"pmids\": [\"24399297\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map the LUBAC-targeted lysines on TRIM25\", \"Interplay with later-identified OTUD5 not addressed\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Uncovered an RNA-cofactor role distinct from ubiquitination, showing TRIM25 directly binds pre-let-7 to activate uridylation, foreshadowing its identity as an RNA-binding protein.\",\n      \"evidence\": \"RNA pull-down with quantitative MS and in vitro uridylation assays\",\n      \"pmids\": [\"25457611\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"RNA-binding determinants not yet mapped\", \"Relationship of this RNA role to ligase activity unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated that viral RNA can subvert TRIM25 by blocking its deubiquitylation, defining a host-pathogen control point linked to viral fitness.\",\n      \"evidence\": \"Sequence-dependent sfRNA-TRIM25 binding, deubiquitylation assay, and DENV clade fitness comparison\",\n      \"pmids\": [\"26138103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"TRIM25 RNA-binding interface for sfRNA not defined\", \"Relationship to RIPLET redundancy not tested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established TRIM25 as a required cofactor and ubiquitin source for ZAP-mediated translational restriction, broadening its antiviral mechanism beyond RIG-I.\",\n      \"evidence\": \"Genome-wide RNAi screen, RING and coiled-coil deletion mutants, ZAP ubiquitination and Sindbis translation inhibition assays\",\n      \"pmids\": [\"28060952\", \"28202764\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin linkage specificity on ZAP (K48 vs K63) mixed\", \"Whether TRIM25-ZAP cooperation is IFN-dependent left open\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified a ligase-independent nuclear antiviral activity, showing TRIM25 directly inhibits influenza RNA elongation by binding vRNPs.\",\n      \"evidence\": \"Nuclear fractionation, vRNP Co-IP, RNA synthesis assay with ligase-dead mutant\",\n      \"pmids\": [\"29107643\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of vRNP recognition not defined\", \"Quantitative contribution relative to IFN pathway unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Mapped the spatial choreography of RIG-I activation, placing TRIM25 at stress-granule-associated cytoplasmic dots from which ubiquitinated RIG-I transfers to mitochondrial MAVS.\",\n      \"evidence\": \"BiFC, super-resolution and live-cell imaging, Co-IP showing MAVS-TRIM25 competition for RIG-I\",\n      \"pmids\": [\"27807226\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not establish whether condensation is required for activity\", \"NS1 disruption of homocomplex not structurally resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Provided structural mechanism for viral antagonism, showing influenza NS1 binds the coiled-coil to mispositions the PRYSPRY domain and block substrate ubiquitination.\",\n      \"evidence\": \"X-ray crystallography of coiled-coil-PRYSPRY and NS1 complexes with in vitro ubiquitination validation\",\n      \"pmids\": [\"29739942\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of full-length TRIM25 on a substrate not solved\", \"RING dimerization and chain synthesis shown unaffected, leaving substrate-positioning model partly inferential\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined TRIM25 as a bona fide RNA-binding protein and connected RNA binding causally to its ligase activity, localization, and antiviral function.\",\n      \"evidence\": \"In vitro RNA binding (gel shift, filter binding), in vitro ubiquitination, localization and antiviral reporter assays with domain mutagenesis\",\n      \"pmids\": [\"30342007\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Precise RNA-binding residues not yet resolved (addressed later)\", \"RNA sequence/structure preference in cells undefined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Broadened TRIM25's oncogenic substrate network, identifying PPAR\\u03b3 degradation controlling adipocyte differentiation.\",\n      \"evidence\": \"Ubiquitination assay and TRIM25 knockout MEF differentiation phenotype\",\n      \"pmids\": [\"30323259\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Linkage type on PPAR\\u03b3 not defined\", \"Single-lab, limited cellular context\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Overturned the founding paradigm by showing endogenous TRIM25 is dispensable for RIG-I-dependent IFN responses, with RIPLET being the essential ligase.\",\n      \"evidence\": \"CRISPR knockout of Trim25, Riplet, and Rig-i in mouse and human cells, IFN reporter and viral assays, plus in vivo IAV infection\",\n      \"pmids\": [\"31335993\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the alternative antiviral mechanism explaining increased IAV susceptibility\", \"Reconciliation with prior overexpression-based RIG-I data left to subsequent RNA-directed studies\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified scaffolding and inhibitory partners (Lnczc3h7a, NDR2, NLRP12) that gate TRIM25-RIG-I complex assembly, refining how its activity is set in cells.\",\n      \"evidence\": \"RNA-IP and Co-IP with ubiquitination assays, in vivo lncRNA knockdown, and conditional knockout mice for NDR2 and NLRP12\",\n      \"pmids\": [\"31036902\", \"30775439\", \"30902577\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"These results coexist with the RIPLET-redundancy finding, leaving the in vivo weight of TRIM25-RIG-I unresolved\", \"Stoichiometry of scaffold complexes undefined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected TRIM25 stability control to oncogenesis, showing MAP3K13 phosphorylation at Ser12 protects TRIM25 from degradation to drive Myc stabilization.\",\n      \"evidence\": \"In vitro kinase assay, ubiquitination assay, S12 mutagenesis, and xenograft tumor model (FBXW7\\u03b1/Myc axis)\",\n      \"pmids\": [\"31186535\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not resolve which degradative ligase Ser12 phosphorylation counteracts\", \"FBXW7\\u03b1 K412 ubiquitination linkage not characterized\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Expanded TRIM25's pro-survival substrate axes, defining Keap1 degradation (Nrf2 activation), TRAF2 K63 ubiquitination (NF-\\u03baB), and an OTUD5 deubiquitination control node.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, knockout/knockdown with ER stress, NF-\\u03baB reporter, and xenograft readouts\",\n      \"pmids\": [\"31953436\", \"32024699\", \"32826889\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate selectivity rules among these competing targets undefined\", \"How RNA binding influences these non-immune substrates not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined TRIM25's roles in cell-death and growth-signaling control through linkage-specific ubiquitination of RIP3 (K48, anti-necrosis) and PTEN (K63, AKT/mTOR activation).\",\n      \"evidence\": \"Co-IP, in vitro ubiquitination with K501R and K266 mutagenesis, knockout necrosis assay, and PTEN localization/phosphatase assays\",\n      \"pmids\": [\"33953350\", \"33931764\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tissue specificity of these substrate choices unclear\", \"Single-lab findings without reciprocal validation\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated that TRIM25 restricts viruses by ligase-independent RNA destabilization and by direct ubiquitination of stress-granule/RNA-metabolism substrates, establishing RNA-directed antiviral mechanisms separate from RIG-I.\",\n      \"evidence\": \"\\u0394RBD/\\u0394RING mutants and tethering assays for IAV mRNA decay; R54P substrate-trap MS identifying G3BP1/2, UPF1, NME1, PABPC4; EBOV vRNP/NP ubiquitination\",\n      \"pmids\": [\"35736141\", \"36067236\", \"35533151\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endonuclease(s) executing TRIM25-directed RNA decay not yet identified here\", \"Which substrates are functionally dominant for restriction unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolved the molecular determinants and condensation behavior of TRIM25 RNA binding, mapping RNA-binding residues, demonstrating viral-RNA binding by iCLIP2, and showing dsRNA-driven phase separation with G3BP1 enhances ligase activity.\",\n      \"evidence\": \"iCLIP2 and biophysical mapping with TRIM25-m9 mutant; LLPS assays, live-cell imaging, ubiquitination and viral replication assays\",\n      \"pmids\": [\"39353916\", \"38750080\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo requirement of condensation for restriction not genetically dissected\", \"Link between RNA-binding map and specific substrate ubiquitination incomplete\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed neddylation at K117 is a positive activating modification, relieving RING steric hindrance to promote substrate engagement and TFEB-driven autophagy.\",\n      \"evidence\": \"UBC12 neddylation, K117 mutagenesis, molecular dynamics, ubiquitination assay, and xenograft model in TNBC\",\n      \"pmids\": [\"38926803\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"K117 is also an autoISGylation site, and crosstalk between these modifications is unresolved\", \"Structural model relies partly on docking/MD rather than experimental structure\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established TRIM25 as a pH-sensitive sensor of exogenous RNA that triggers mRNA turnover, with direct relevance to mRNA-therapeutic evasion via nucleoside modification.\",\n      \"evidence\": \"Genome-wide CRISPR screen, pH-dependent RNA-affinity measurements, mRNA stability assays, and knockout of N4BP1/KHNYN/ZAP\",\n      \"pmids\": [\"40179174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of pH-dependent affinity switch not defined\", \"Whether endogenous RNA turnover uses the same mechanism unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how TRIM25 integrates its RNA-sensing, condensate, and ligase activities into a unified in vivo function, and what its dominant physiological role is given that it is dispensable for RIG-I-dependent IFN yet broadly active across antiviral and oncogenic substrate networks.\",\n      \"evidence\": \"No single study in the timeline reconciles the RIPLET-redundancy finding with the many substrate- and RNA-directed activities\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of full-length TRIM25 engaging RNA and substrate together\", \"Quantitative ranking of physiologically dominant substrates lacking\", \"Crosstalk among K117 neddylation/ISGylation and Ser12 phosphorylation not integrated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 23, 26, 27, 28, 37]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 26, 27, 29]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [11, 16, 30, 33, 34]},\n      {\"term_id\": \"GO:0031386\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [22, 36]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [18, 32]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [14, 4]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [18, 32]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 8, 19, 22, 36]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [9, 14, 29, 30, 35]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [11, 30, 31, 34]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 23, 26, 27, 28, 37]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [38]}\n    ],\n    \"complexes\": [\n      \"RIG-I\\u2013TRIM25 complex\",\n      \"antiviral stress granules (with G3BP1)\"\n    ],\n    \"partners\": [\n      \"RIG-I\",\n      \"USP15\",\n      \"G3BP1\",\n      \"ZAP\",\n      \"TRAF2\",\n      \"NLRP12\",\n      \"G3BP2\",\n      \"OTUD5\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}