RNF25

RNF25 is a RING finger E3 ubiquitin ligase that operates through both ligase-dependent and ligase-independent mechanisms to regulate transcription, signaling, DNA replication fork integrity, and ribosome quality control. Its RING domain engages the E2 enzyme UbcH5B via a structurally unique high-affinity clamp involving a dedicated UbcH5B-binding region (U5BR) tethered by a short linker, and its catalytic activity drives proteasomal degradation of substrates including Naked2, E-cadherin, and FKBP8, as well as non-degradative ubiquitylation of TRIP4 and ribosomal protein eS31 (PMID:26475854, PMID:18757723, PMID:38286671, PMID:39921520, PMID:40765826, PMID:41875887). RNF25 activates NF-κB-dependent transcription by binding the p65 transactivation domain and ubiquitylating TRIP4 to liberate p65, and it modulates Wnt signaling independently of its ligase activity by disrupting the inhibitory Nkd1–Axin complex (PMID:12748188, PMID:40765826, PMID:27007149). Separately from ubiquitin conjugation, RNF25 protects reversed DNA replication forks from MRE11/CtIP-mediated degradation by recruiting the fork-protection factor REV7 to nascent DNA, and it ubiquitylates ribosomal protein eS31 to suppress GCN2-dependent integrated stress response activation upon encounter with damaged-mRNA-stalled ribosomes (PMID:40764480, PMID:41875887).

1. 2003 High

   Establishing that RNF25 is a nuclear RING finger protein whose ubiquitin ligase activity is required for NF-κB-dependent transcription answered the question of whether this uncharacterized RING protein functions in gene regulation.

   Evidence Yeast two-hybrid screen identifying p65 TAD interaction, Co-IP, luciferase reporter, and dominant-negative RING mutant in mammalian cells

   PMID:12748188

   Open questions at the time

   • Direct ubiquitylation substrate for NF-κB activation was not identified
   • Whether RNF25 modifies p65 itself or a co-regulator was unknown
   • In vivo relevance beyond reporter assays not tested
2. 2008 High

   Identification of Naked2 as a direct ubiquitylation substrate targeted for proteasomal degradation established RNF25 as a bona fide E3 ligase with a defined substrate and revealed its role in modulating Wnt-pathway-related signaling molecules.

   Evidence Overexpression/knockdown, protein half-life assays, Co-IP, and proteasome inhibitor treatment in mammalian cells

   PMID:18757723

   Open questions at the time

   • Specific ubiquitylation sites on Naked2 were not mapped
   • Whether TGF-α-mediated protection of Naked2 occurs in vivo was not addressed
   • Chain type preference of RNF25-catalyzed ubiquitylation remained unknown
3. 2015 High

   Crystal structure of the RNF25–UbcH5B complex revealed a unique clamp mechanism whereby a dedicated U5BR contacts the E2 backside, explaining unusually high-affinity E2 engagement and showing that this paradoxically attenuates catalytic rate by occluding non-covalent ubiquitin binding.

   Evidence X-ray co-crystallography, in vitro ubiquitination assays, and site-directed mutagenesis

   PMID:26475854

   Open questions at the time

   • Physiological consequence of the auto-inhibitory clamp mechanism in cells was not tested
   • Whether RNF25 uses E2 enzymes other than UbcH5B in vivo was not determined
   • How the clamp is relieved to permit productive ubiquitylation remained unclear
4. 2016 Medium

   Demonstration that RNF25 disrupts the Nkd1–Axin complex to promote Wnt signaling independently of its RING ligase activity revealed a non-catalytic scaffolding function, broadening the mechanistic repertoire of RNF25.

   Evidence Co-IP with ligase-dead mutant, RNAi knockdown in zebrafish embryos, and Wnt reporter assays

   PMID:27007149

   Open questions at the time

   • Structural basis for Nkd1 and Axin binding was not resolved
   • Whether ligase-independent and ligase-dependent functions compete or cooperate was not examined
   • Single-lab finding without independent replication
5. 2018 Medium

   A genome-wide RNAi screen linked RNF25 to gefitinib resistance in NSCLC by showing it activates NF-κB, which induces IL-6 to reactivate ERK signaling, connecting RNF25's transcriptional function to a clinically relevant drug-resistance pathway.

   Evidence Genome-wide RNAi screen, siRNA/overexpression, NF-κB reporter, cytokine measurement, and ERK phosphorylation analysis in NSCLC cells

   PMID:29789542

   Open questions at the time

   • Direct ubiquitylation target mediating NF-κB activation in this context was not identified
   • In vivo drug-resistance model was not included
   • Generalizability beyond NSCLC cell lines not tested
6. 2024 High

   Discovery that PKA phosphorylates RNF25 at Ser450 in response to oxidative stress to activate its E3 ligase activity toward E-cadherin established a redox-regulated post-translational switch controlling RNF25 substrate selection and EMT-related metastasis.

   Evidence In vitro ubiquitination, phosphosite and Cys mutagenesis, Co-IP, protein stability assays, and in vivo xenograft metastasis model in hepatocellular carcinoma

   PMID:38286671

   Open questions at the time

   • Whether Ser450 phosphorylation affects other RNF25 substrates was not tested
   • Structural mechanism by which phosphorylation activates ligase activity unknown
   • Contribution relative to other E-cadherin-targeting E3 ligases not assessed
7. 2025 High

   Three independent studies simultaneously expanded the substrate repertoire and revealed a ligase-independent genome-protection role: RNF25 ubiquitylates FKBP8 (scaffolded by circSATB1) to relieve mTOR inhibition, non-degradatively ubiquitylates TRIP4 at K135 to liberate p65 for NF-κB activation, and recruits REV7 to reversed replication forks to protect nascent DNA from MRE11/CtIP degradation without requiring catalytic activity.

   Evidence Co-IP, ubiquitination assays, site-directed mutagenesis, single-molecule DNA fiber analysis, proximity ligation, ligase-dead separation-of-function mutant, drug-binding assay, and in vivo metastasis models

   PMID:39921520 PMID:40764480 PMID:40765826

   Open questions at the time

   • How REV7 recruitment is coordinated with other fork-protection factors is unknown
   • Structural basis for RNF25–REV7 interaction not resolved
   • Whether the circSATB1-scaffolded mechanism operates outside colorectal cancer is untested
8. 2026 High

   Identification of ribosomal protein eS31 as an RNF25 substrate linked the E3 ligase to ribosome quality control by showing that eS31 ubiquitylation suppresses GCN2-dependent integrated stress response activation at damaged-mRNA-stalled ribosomes, conferring mRNA damage tolerance.

   Evidence Genetic screens, ubiquitination and ribosome stalling assays, GCN2 pathway reporters, cell viability assays, and mass spectrometry identification of eS31 ubiquitylation site

   PMID:41875887

   Open questions at the time

   • Ubiquitin chain type on eS31 not determined
   • Whether RNF25's ribosome quality-control function intersects with its NF-κB or fork-protection roles is unknown
   • Structural context of eS31 ubiquitylation on the ribosome not resolved

• A unifying model explaining how RNF25 partitions between its diverse catalytic and non-catalytic roles — NF-κB activation, substrate-specific proteasomal degradation, replication fork protection, and ribosome quality control — and whether distinct regulatory inputs (phosphorylation, scaffold RNAs, protein partners) route RNF25 to specific substrates and cellular compartments remains to be established.
• No knockout mouse or organismal loss-of-function phenotype reported
• Ubiquitin chain-type specificity for most substrates undetermined
• How the auto-inhibitory U5BR clamp is relieved in vivo is not known