Affinage

RNF216

E3 ubiquitin-protein ligase RNF216 · UniProt Q9NWF9

Length
866 aa
Mass
99.4 kDa
Annotated
2026-06-10
30 papers in source corpus 16 papers cited in narrative 16 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 5/5 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

RNF216 (TRIAD3) is an RBR-type E3 ubiquitin ligase that controls the abundance of diverse substrates and thereby regulates autophagy, synaptic plasticity, reproductive endocrine signaling, and apoptotic/ferroptotic cell fate (PMID:25484083, PMID:31087003, PMID:27995769). In vitro reconstitution with purified protein shows it is catalytically active and exclusively assembles K63-linked ubiquitin chains, with chain-type specificity dictated by the C-terminal R2-C/Zn-finger region; Arg-660 is required for ubiquitin transfer from E2 to the catalytic cysteine, and the protein also carries SUMO-interaction motifs that recognize SUMO2 chains and a central ubiquitin-binding domain (PMID:31087003). Despite this in vitro K63 preference, in cells RNF216 directs several substrates to degradation: it K48-ubiquitinates BECN1 (Beclin1) for proteasomal turnover, suppressing autophagy (PMID:25484083), and similarly drives degradation of Arc/Arg3.1 to regulate synaptic plasticity and memory (PMID:27995769), of p53 to suppress apoptosis and ferroptosis (PMID:35594003, PMID:40506397), of PRKACB via the lysosomal pathway to control meiotic progression (PMID:33724554), and of STAU2 in the hypothalamus (PMID:37439148). Through BECN1-dependent autophagy control it supports GnRH neuron migration and HPG axis function, where loss disrupts GnRH production, gonadal output, and spermatogenesis (PMID:30733708, PMID:35620441, PMID:33724554). The GHS-associated missense mutations R660C and R694C abolish ligase activity and substrate degradation, linking RNF216 loss-of-function to Gordon Holmes syndrome via disrupted ubiquitin-proteasome and autophagy regulation (PMID:27995769, PMID:31087003, PMID:41271602).

Mechanistic history

Synthesis pass · year-by-year structured walk · 16 steps
  1. 2014 High

    Established RNF216 as a substrate-specific E3 ligase coupling the ubiquitin-proteasome system to autophagy control by identifying BECN1 as a degradation target.

    Evidence Reciprocal Co-IP, K48-linkage-specific ubiquitination assay, and knockdown/overexpression with L. monocytogenes infection in macrophages

    PMID:25484083

    Open questions at the time
    • Did not define the ubiquitination site on BECN1 in structural detail
    • In-cell K48 specificity not reconciled with intrinsic enzyme chain preference
  2. 2016 High

    Connected RNF216 to neuronal function and to Gordon Holmes syndrome pathology by showing it degrades the synaptic protein Arc and that GHS mutations disrupt this activity.

    Evidence Co-IP, ubiquitination assay, hippocampal CA1 knockdown, spatial memory behavior, and rescue with R660C/R694C mutant constructs in mice

    PMID:27995769

    Open questions at the time
    • Did not resolve why distinct substrates receive different chain linkages
    • Mechanism linking Arc accumulation to specific synaptic deficits not fully detailed
  3. 2016 Medium

    Extended the RNF216-BECN1-autophagy axis to disease by showing it promotes colorectal cancer cell proliferation through autophagy suppression.

    Evidence Knockdown/overexpression in CRC lines, proliferation/migration assays, xenografts, and epistasis via BECN1 siRNA and autophagy inhibitors

    PMID:27203674

    Open questions at the time
    • Single lab
    • Did not assess whether direct ubiquitination of BECN1 occurs in CRC context
  4. 2017 Medium

    Placed RNF216 in an inflammatory signaling circuit by showing cGMP/67LR signaling upregulates it to suppress TLR4 levels in adipose tissue.

    Evidence RNF216 overexpression, cGMP pathway modulation, and TLR4 protein measurement in high-fat/high-sugar-diet mice

    PMID:28154178

    Open questions at the time
    • Limited direct ubiquitination assay detail for TLR4
    • Single lab
  5. 2019 High

    Defined the biochemical identity of RNF216 as an RBR ligase that intrinsically builds K63 chains and mapped the determinants of chain specificity and catalysis.

    Evidence In vitro reconstitution with purified protein, K63-linkage-specific assay, domain mapping, active-site mutagenesis, and chemical ubiquitin-loading rescue of R660C

    PMID:31087003

    Open questions at the time
    • In vitro K63 preference is not reconciled with K48-linked substrate degradation seen in cells
    • Functional role of SUMO-interaction motifs not established
  6. 2019 Medium

    Revealed a sex-specific physiological requirement for RNF216 in male germ cell development.

    Evidence Germline knockout mouse, testis histology, and fertility assessment

    PMID:30649198

    Open questions at the time
    • Did not identify the responsible substrate at the time
    • Mechanism of female sparing unexplained
  7. 2019 Medium

    Linked RNF216 to reproductive neuroendocrine development by showing it enables GnRH neuron migration through BECN1-dependent autophagy suppression.

    Evidence siRNA knockdown in GN11 cells, migration and autophagy-flux assays, and pharmacological/genetic rescue (Beclin1 siRNA, CQ, 3-MA)

    PMID:30733708

    Open questions at the time
    • Arc knockdown did not rescue migration, leaving its role in this context unclear
    • Single lab
  8. 2020 Medium

    Demonstrated that RNF216 loss is neuroprotective after hemorrhagic brain injury via Arc-mediated AMPAR/Ca2+ modulation.

    Evidence siRNA knockdown in cortical neurons, Ca2+ imaging, AMPAR western blots, and intraventricular siRNA in a subarachnoid hemorrhage model

    PMID:32918771

    Open questions at the time
    • Did not establish direct ubiquitination of Arc in this injury model
    • Single lab
  9. 2021 High

    Identified PRKACB as a lysosomally degraded substrate, providing the molecular basis for the spermatogenic phenotype via PKA hyperactivity.

    Evidence CRISPR-Cas9 KO mice, RNF216-PRKACB Co-IP, PKA activity assay, lysosome inhibitor experiments, and staged spermatogenesis histology

    PMID:33724554

    Open questions at the time
    • Lysosomal route for PRKACB contrasts with proteasomal routes of other substrates without a unifying mechanism
    • Chain linkage on PRKACB not defined
  10. 2021 Low

    Reported a candidate physical partner, Filamin B, expanding the potential interactome.

    Evidence Yeast two-hybrid screen with human cDNA library, verified in yeast

    PMID:34247365

    Open questions at the time
    • Single yeast two-hybrid method without mammalian-cell confirmation
    • No functional consequence demonstrated
    • No reciprocal validation
  11. 2022 High

    Established RNF216 as a regulator of the HPG axis and sex-specific microglial activation through GnRH-producing neurons.

    Evidence KO cell line and neural-stem-cell conditional KO mice, GnRH measurement, calcium imaging, FSH/inhibin B ELISA, testicular morphometry, estrous monitoring, and microglial immunofluorescence

    PMID:35620441

    Open questions at the time
    • Substrate driving microglial phenotype not identified
    • Mechanism linking GnRH neuron defects to hormonal output incompletely defined
  12. 2022 Medium

    Identified p53 as a degradation target, positioning RNF216 as a suppressor of apoptosis and a determinant of radioresistance in glioblastoma.

    Evidence Overexpression/knockdown in GBM lines, p53 ubiquitination assay, apoptosis assay, and radiosensitized xenografts

    PMID:35594003

    Open questions at the time
    • p53 ubiquitination site/linkage not mapped
    • Single lab
  13. 2023 Medium

    Defined a catalysis-independent structural role for the R2-C domain in shaping neuronal morphology, distinct from RNF216 ligase output.

    Evidence Domain deletion constructs, ubiquitin chain assembly assay, live imaging of localization, Sholl/spine analysis, and catalytically inactive mutant control in hippocampal neurons (preprint)

    PMID:37905043

    Open questions at the time
    • Preprint, single lab
    • Molecular partner mediating the localization/morphology effect unknown
  14. 2023 Medium

    Added STAU2 as a hypothalamic proteasomal substrate, connecting RNF216 to GnRH and prolactin signaling pathways.

    Evidence RNF216-STAU2 Co-IP, proteasome inhibitor experiments, western blot in Rnf216-/- hypothalamus, and RNA-seq of KO vs WT

    PMID:37439148

    Open questions at the time
    • Direct ubiquitination of STAU2 not shown with a ubiquitination assay
    • Single lab
  15. 2025 Medium

    Generalized the RNF216-p53 axis to ferroptosis control, linking p53 stabilization upon RNF216 loss to lipid peroxidation and iron accumulation in lung adenocarcinoma.

    Evidence siRNA knockdown in LUAD lines, p53 ubiquitination assay, ROS/LPO/Fe2+ measurement, and xenografts

    PMID:40506397

    Open questions at the time
    • Overlapping claim with GBM study not independently cross-validated
    • Single lab
  16. 2026 Medium

    Tied GHS-associated loss-of-function variants to an autophagy-mediated, caspase-independent cell death mechanism.

    Evidence In vitro analysis of RNF216 variants including p.Arg686*, autophagy flux and viability assays, and 3-MA versus Z-VAD pharmacological epistasis

    PMID:41271602

    Open questions at the time
    • Single lab
    • Did not trace the death pathway back to a specific accumulated substrate

Open questions

Synthesis pass · forward-looking unresolved questions
  • It remains unresolved how RNF216 reconciles its intrinsic in vitro K63-chain specificity with the K48-linked, proteasomal and lysosomal degradation it produces on cellular substrates, and what determines substrate-specific routing.
  • No structural model of substrate engagement
  • Mechanism switching chain linkage in cells unknown
  • Determinants of proteasomal versus lysosomal targeting unidentified

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016874 ligase activity 3 GO:0140096 catalytic activity, acting on a protein 3 GO:0031386 protein tag activity 2
Pathway
R-HSA-392499 Metabolism of proteins 4 R-HSA-9612973 Autophagy 4 R-HSA-5357801 Programmed Cell Death 2
Partners
ARCBECN1FLNBPRKACBSTAU2TLR4TP53

Evidence

Reading pass · 16 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2014 RNF216 interacts with BECN1 (Beclin1) via its TRIAD domain and ubiquitinates BECN1 at lysine 48, leading to BECN1 proteasomal degradation and inhibition of autophagy in macrophages. Co-immunoprecipitation, ubiquitination assay, K48 linkage-specific analysis, overexpression and knockdown in macrophages, mouse model of L. monocytogenes infection Autophagy High 25484083
2016 TRIAD3A (RNF216) interacts with Arc/Arg3.1 and facilitates its K48-linked ubiquitination and proteasomal degradation; GHS-associated missense mutations R660C and R694C abolish this interaction and Arc degradation, leading to synaptic deficits and spatial learning/memory impairment in mice. Co-immunoprecipitation, ubiquitination assay, loss-of-function knockdown in mouse hippocampal CA1, spatial memory behavioral testing, rescue experiments with mutant constructs Aging cell High 27995769
2016 RNF216 promotes colorectal cancer cell proliferation and migration by ubiquitinating BECN1 and enhancing its proteasomal degradation, thereby suppressing autophagy; BECN1 knockdown or autophagy inhibition rescues the proliferation/migration deficit caused by RNF216 knockdown. Knockdown and overexpression in CRC cell lines, in vitro proliferation/migration assays, in vivo xenograft, epistasis via BECN1 siRNA and autophagy inhibitors Oncotarget Medium 27203674
2017 RNF216 ubiquitinates TLR4, targeting it for degradation; cyclic GMP signaling downstream of the 67-kDa laminin receptor (67LR) upregulates RNF216 expression, thereby suppressing TLR4 levels in adipose tissue. RNF216 overexpression studies, cGMP pathway modulation, TLR4 protein level measurement, in vivo adipose tissue analysis in HF/HS-diet mice The Journal of biological chemistry Medium 28154178
2019 Purified RNF216 is an active RBR-type E3 ubiquitin ligase that exclusively forms K63-linked ubiquitin chains in vitro; the chain-type specificity is determined by the last two Zn-fingers of the RBR triad plus a short C-terminal extension (R2-C domain). RNF216 also contains SUMO-interaction motifs (SIMs) that recognize SUMO2 chains without ubiquitinating them, and a central ubiquitin-binding domain. GHS-associated missense mutations R660C and R694C completely abolish ubiquitin ligase activity; R660C activity can be restored by a chemical ubiquitin-loading protocol that bypasses E2 enzymes, identifying Arg-660 as required for ubiquitin transfer from E2 to the catalytic cysteine. In vitro reconstitution with purified protein, K63-linkage-specific ubiquitin chain assay, domain deletion/mapping, active-site mutagenesis, chemical ubiquitin-loading rescue assay, bioinformatic SIM identification confirmed biochemically Human molecular genetics High 31087003
2019 Targeted deletion of Rnf216 in mice disrupts spermatogenesis and causes male infertility, but does not affect female fertility, establishing an essential and sex-specific role for RNF216 in spermatogenesis. Germline knockout mouse model, histological analysis of testes, fertility assessment Biology of reproduction Medium 30649198
2019 RNF216 regulates GnRH neuron migration by suppressing Beclin1-mediated autophagy; RNF216 knockdown increases Beclin1 and Arc protein levels and enhances autophagy, inhibiting GN11 cell migration. Beclin1 (but not Arc) knockdown, and pharmacological autophagy inhibitors (chloroquine, 3-MA) rescue the migration deficit. siRNA knockdown in GN11 neuronal cells, migration assay, autophagy flux measurement, pharmacological rescue with CQ/3-MA/rapamycin, epistasis via Beclin1 siRNA Frontiers in endocrinology Medium 30733708
2020 RNF216 knockdown protects cortical neurons from oxyhemoglobin-induced cytotoxicity and apoptosis in vitro and reduces brain injury after subarachnoid hemorrhage in vivo; these protective effects are mediated via upregulation of Arc protein, which modulates the AMPAR pathway and intracellular Ca2+ dynamics. siRNA knockdown in primary cortical neurons, Ca2+ imaging, western blot for Arc and AMPAR subunits, intraventricular siRNA injection in SAH animal model, neurological scoring FASEB journal Medium 32918771
2021 RNF216 interacts with the PKA catalytic subunit β (PRKACB) and promotes its degradation through the ubiquitin-lysosome pathway; Rnf216 knockout mice show elevated PRKACB and increased PKA activity in testes, accompanied by arrest at the zygotene stage of meiosis I and spermatocyte apoptosis at the pachytene stage. CRISPR-Cas9 Rnf216-knockout mice, Co-immunoprecipitation (RNF216-PRKACB), PKA activity assay, lysosome inhibitor experiments, histological staging of spermatogenesis FASEB journal High 33724554
2022 RNF216/TRIAD3 knockout in a GnRH hypothalamic cell line decreases steady-state GnRH levels and calcium transients; KO adult mice have reduced GnRH neuron soma size and GnRH production, smaller testicular volumes, abnormal inhibin B and FSH release, and irregular estrous cycling in females. Conditional deletion in neural stem cells causes abnormal microglial activation in males without affecting reproduction, implicating RNF216 in HPG axis function and sex-specific microglial regulation. Knockout cell line, conditional KO mouse (neural stem cell Cre), GnRH measurement, calcium imaging, FSH/inhibin B ELISA, testicular morphometry, estrous cycle monitoring, microglial immunofluorescence iScience High 35620441
2022 RNF216 promotes ubiquitination and proteasomal degradation of p53, reducing p53 stability and suppressing p53-dependent apoptosis and DNA damage responses; RNF216 knockdown stabilizes p53 and sensitizes glioblastoma cells to radiation. Overexpression and siRNA knockdown in GBM cell lines, ubiquitination assay for p53, western blot, apoptosis assay, xenograft mouse model with radiosensitivity assessment Molecular neurobiology Medium 35594003
2023 The R2-C domain of RNF216 (homologous to the LDD of HOIP) promotes self-assembly of ubiquitin chains with a dominance for K63-linkages. Deletion of the R2-C domain alters RNF216 subcellular localization and reduces dendritic complexity, and changes apical dendritic spine morphology type distribution in primary hippocampal neurons. These structural effects are independent of RNF216's RBR catalytic activity, as a catalytically inactive RNF216 had no such effect. Domain deletion constructs, ubiquitin chain assembly assay, live imaging of localization in neurons, dendritic morphology analysis (Sholl analysis, spine classification), catalytically inactive mutant comparison bioRxivpreprint Medium 37905043
2023 RNF216 interacts with STAU2 (Staufen2, a double-stranded RNA-binding protein) and promotes its degradation via the ubiquitin-proteasome pathway; STAU2 protein levels are increased in the hypothalamus of Rnf216-/- mice, accompanied by decreased activity of the GnRH signaling pathway, prolactin signaling, and neuroactive ligand-receptor interaction pathways. Co-immunoprecipitation (RNF216-STAU2), proteasome inhibitor experiments, western blot in Rnf216-/- hypothalamus, RNA sequencing of hypothalamus from KO vs WT mice Development, growth & differentiation Medium 37439148
2021 RNF216 interacts with Filamin B (FLNB) as identified by yeast two-hybrid screening with a human cDNA library, with interaction verified in yeast. Yeast two-hybrid screen with pGBKT7-RNF216 bait against human cDNA library, interaction verification in Y2HGold Chinese journal of medical genetics Low 34247365
2026 RNF216 loss-of-function (via a homozygous p.Arg686* nonsense variant or other GHS-associated variants) disrupts autophagy regulation in cells; inhibiting RNF216 expression decreases cell viability through an autophagy-mediated (not caspase-dependent) cell death mechanism, as autophagy inhibitor 3-MA prevented cell death while pan-caspase inhibitor Z-VAD did not. In vitro functional analysis of RNF216 variants, autophagy flux assay, cell viability assay, pharmacological rescue with 3-MA and Z-VAD The Journal of clinical endocrinology and metabolism Medium 41271602
2025 RNF216 promotes ubiquitination and degradation of p53 in lung adenocarcinoma cells; RNF216 knockdown stabilizes p53, increases reactive oxygen species, lipid peroxidation, and intracellular Fe2+, thereby inducing ferroptosis. siRNA knockdown in LUAD cell lines, ubiquitination assay for p53, ROS/LPO/Fe2+ measurement, xenograft mouse model Human & experimental toxicology Medium 40506397

Source papers

Stage 0 corpus · 30 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2014 Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination. Autophagy 101 25484083
2015 RNF216 mutations as a novel cause of autosomal recessive Huntington-like disorder. Neurology 54 25841028
2017 Green Tea Polyphenol Epigallocatechin-3-gallate Suppresses Toll-like Receptor 4 Expression via Up-regulation of E3 Ubiquitin-protein Ligase RNF216. The Journal of biological chemistry 48 28154178
2016 TRIAD3/RNF216 mutations associated with Gordon Holmes syndrome lead to synaptic and cognitive impairments via Arc misregulation. Aging cell 39 27995769
2016 RNF216 contributes to proliferation and migration of colorectal cancer via suppressing BECN1-dependent autophagy. Oncotarget 38 27203674
2019 RNF216 Regulates the Migration of Immortalized GnRH Neurons by Suppressing Beclin1-Mediated Autophagy. Frontiers in endocrinology 36 30733708
2016 Ataxia and Hypogonadotropic Hypogonadism with Intrafamilial Variability Caused by RNF216 Mutation. Neurology international 29 27441066
2020 GnRH Deficient Patients With Congenital Hypogonadotropic Hypogonadism: Novel Genetic Findings in ANOS1, RNF216, WDR11, FGFR1, CHD7, and POLR3A Genes in a Case Series and Review of the Literature. Frontiers in endocrinology 22 32982993
2019 RNF216 is essential for spermatogenesis and male fertility†. Biology of reproduction 21 30649198
2020 RNF216 mediates neuronal injury following experimental subarachnoid hemorrhage through the Arc/Arg3.1-AMPAR pathway. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 19 32918771
2019 Mechanism and chain specificity of RNF216/TRIAD3, the ubiquitin ligase mutated in Gordon Holmes syndrome. Human molecular genetics 17 31087003
2021 RNF216 regulates meiosis and PKA stability in the testes. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 14 33724554
2009 Expression of two inflammation-related genes (RIPK3 and RNF216) in mononuclear cells is associated with weight-loss regain in obese subjects. Journal of nutrigenetics and nutrigenomics 12 19690434
2022 Whole-Exome Sequencing Identified a Novel Mutation in RNF216 in a Family with Gordon Holmes Syndrome. Journal of molecular neuroscience : MN 10 35088240
2022 The E3 ubiquitin ligase RNF216/TRIAD3 is a key coordinator of the hypothalamic-pituitary-gonadal axis. iScience 10 35620441
2020 A novel de novo RNF216 mutation associated with autosomal recessive Huntington-like disorder. Annals of clinical and translational neurology 10 32358900
2023 A novel mutation in RNF216 gene in a Turkish case with Gordon Holmes syndrome. BMC medical genomics 8 37161390
2022 RNF216 Alleviates Radiation-Induced Apoptosis and DNA Damage Through Regulating Ubiquitination-Mediated Degradation of p53 in Glioblastoma. Molecular neurobiology 6 35594003
2024 RNF216 Inhibits the Replication of H5N1 Avian Influenza Virus and Regulates the RIG-I Signaling Pathway in Ducks. Journal of immunology (Baltimore, Md. : 1950) 4 38829131
2024 Copy number variations in RNF216 and postsynaptic membrane-associated genes are associated with bipolar disorder: a case-control study in the Japanese population. Psychiatry and clinical neurosciences 4 39403837
2023 A novel mutation in RNF216 gene in an Indian case with Gordon Holmes syndrome. BMJ case reports 4 37977846
2025 Identification of a large homozygous RNF216 deletion in a Chinese patient with gordon holmes syndrome. Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology 3 40237971
2015 Mutations in RNF216 do not cause 4H syndrome. Parkinsonism & related disorders 3 26365775
2023 The E3 ubiquitin ligase RNF216 contains a linear ubiquitin chain-determining-like domain that functions to regulate dendritic arborization and dendritic spine type in hippocampal neurons. bioRxiv : the preprint server for biology 2 37905043
2026 RNF216 Variants Found in Patients With Dementia, Hypogonadotropic Hypogonadism, and Severe Ataxia Deregulate Autophagy. The Journal of clinical endocrinology and metabolism 1 41271602
2025 RNF216 inhibits ferroptosis in lung adenocarcinoma by promoting p53 ubiquitination. Human & experimental toxicology 1 40506397
2024 Establishment of FDHSi003-A, a human induced pluripotent stem cell (hiPSC) line with a mutation of RNF216 c.1948G > T. Stem cell research 1 38377650
2025 Identifying Circ-RNF216 as a Regulator of Renal Tubular Epithelial Cell Proliferation Via the TGF-Β1/Smad3-Mediated Pathway in Chronic Kidney Disease. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 0 41340590
2023 RNF216 affects the stability of STAU2 in the hypothalamus. Development, growth & differentiation 0 37439148
2021 [Screening of interacting proteins of idiopathic gonadotropin-releasing hormone deficiency pathogenic gene RNF216]. Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 0 34247365

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