{"gene":"RIOK3","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2015,"finding":"RIOK3 phosphorylates the C-terminal region of MDA5 (at S828), impairing MDA5 multimer/filament formation and attenuating MDA5-mediated type I IFN signaling. PP1α/PP1γ phosphatases counteract this by dephosphorylating MDA5. RIOK3 knockout strongly enhanced type I IFN production following measles virus infection, while phosphomimetic MDA5-S828D mutation attenuated signaling.","method":"RIOK3 knockout cells, phosphomimetic mutation (S828D), in vitro kinase assay, multimer formation assay, IFN reporter assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct kinase-substrate identification with mutagenesis (phosphomimetic), KO phenotype, and mechanistic readout of filament formation; multiple orthogonal methods in single study","pmids":["25865883"],"is_preprint":false},{"year":2021,"finding":"RIOK3 recruits and interacts with the E3 ubiquitin ligase TRIM40, leading to K48- and K27-linked ubiquitination and proteasomal degradation of both RIG-I and MDA5, thereby negatively regulating type I IFN signaling. Myeloid-specific Riok3 knockout mice showed enhanced type I IFN induction and resistance to RNA virus-induced pathogenesis.","method":"Co-immunoprecipitation, myeloid-specific knockout mice, ubiquitination assay (K48/K27 linkage), in vitro and in vivo viral infection models","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vivo KO mouse model, biochemical ubiquitination assays; multiple orthogonal methods","pmids":["34161773"],"is_preprint":false},{"year":2014,"finding":"RIOK3 functions as an adaptor protein downstream of TBK1 and upstream of IRF3 in the type I IFN pathway, physically interacting with both TBK1 and IRF3 and bridging their interaction. RIOK3 knockdown blocks cytosolic dsRNA- and dsDNA-induced IRF3 activation and IFN-β production; overexpression activates IRF3.","method":"Kinome-wide RNAi screens (two independent), Co-immunoprecipitation (RIOK3 with TBK1 and IRF3), IFN-β reporter assay, IRF3 activation assay, transcriptome analysis","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent genome-wide RNAi screens plus reciprocal Co-IP, epistasis placement downstream of TBK1 and upstream of IRF3, confirmed by gain- and loss-of-function experiments","pmids":["24807708"],"is_preprint":false},{"year":2009,"finding":"RIOK3 interacts with caspase-10 via its RIO domain (binding to each death effector domain of caspase-10) and negatively regulates NF-κB signaling. RIOK3 suppresses caspase-10-mediated NF-κB activation by competing with RIP1 and NIK for binding to caspase-10. Kinase activity of RIOK3 is required for suppression of TNFα-induced NF-κB activation but not for suppression of caspase-10-mediated NF-κB activation.","method":"Yeast two-hybrid, GST pull-down, endogenous co-immunoprecipitation, siRNA knockdown, NF-κB reporter assay, domain-mapping experiments","journal":"Molecular and cellular biochemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by GST pulldown and endogenous Co-IP, domain mapping, kinase-dead mutant, multiple orthogonal methods in single lab","pmids":["19557502"],"is_preprint":false},{"year":2025,"finding":"RIOK3 specifically recognizes ubiquitylated 40S ribosomes (ubiquitylated by E3 ligase RNF10 on uS3 and uS5 during starvation) through a unique ubiquitin-interacting motif (UIM). RIOK3 binding drives progressive 3'-to-5' decay of 18S rRNA, mediating selective 40S ribosome degradation during starvation. Cryo-EM structures of RIOK3-ubiquitylated 40S complexes and degradation intermediates were resolved.","method":"Cryo-EM structural analysis, ubiquitin-interacting motif mutagenesis, starvation assays, rRNA decay analysis, functional rescue experiments","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with functional mutagenesis of UIM, multiple degradation intermediate structures, mechanistic pathway reconstitution","pmids":["39947183"],"is_preprint":false},{"year":2025,"finding":"RIOK3 is a crucial factor in the initiation-specific ribosome-associated quality control (iRQC) pathway; it interacts with ubiquitylated 40S subunits (ubiquitylated by RNF10) to mediate their degradation. Both RNF10 and RIOK3 protein levels increase upon iRQC activation, establishing a feedforward mechanism. Amino acid starvation and 60S:40S stoichiometry imbalance (from disrupted 60S biogenesis) activate iRQC-dependent 40S decay.","method":"RIOK3 depletion, RNF10 depletion, quantitative ribosome profiling, 40S ubiquitylation assays, 60S biogenesis disruption, amino acid starvation conditions","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (RNF10/RIOK3 double depletion), feedforward mechanism via protein level measurements, multiple pathway activation conditions tested; corroborated by independent lab (PMID 39947183)","pmids":["40022732"],"is_preprint":false},{"year":2012,"finding":"Human RioK3 is a cytoplasmic protein that co-sediments with cytoplasmic pre-40S ribosomal particles and associates with pre-40S particle components hLtv1, hEnp1, and 18S-E pre-rRNA. RioK3 depletion leads to increased levels of 21S rRNA precursor, indicating a role in 21S pre-rRNA processing during 40S subunit biogenesis. RioK3 does not shuttle via Crm1-dependent nuclear export (unlike RioK2).","method":"Sucrose gradient sedimentation, co-immunoprecipitation (with hLtv1, hEnp1, 18S-E pre-rRNA), siRNA depletion (RioK3, rpS15, rpS19, RioK2), Northern blot (pre-rRNA analysis)","journal":"RNA biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — sucrose gradient co-sedimentation, reciprocal Co-IP with pre-40S components, pre-rRNA processing analysis; multiple orthogonal methods in single study","pmids":["22418843"],"is_preprint":false},{"year":2010,"finding":"Riok3 is a direct target of miR-191 in mouse erythroblasts. Knockdown of Riok3 blocks erythroid enucleation and chromatin condensation during terminal erythroid differentiation. Down-regulation of miR-191 during terminal differentiation is required to allow Riok3 upregulation, which is essential for these processes.","method":"RNA-seq, miR-191 overexpression, Riok3 knockdown (siRNA), erythroid enucleation assays, chromatin condensation analysis, luciferase reporter (miR-191 target validation)","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct miRNA target validation, gain- and loss-of-function experiments, defined cellular phenotypes (enucleation, chromatin condensation); replicated with multiple approaches","pmids":["21196494"],"is_preprint":false},{"year":2014,"finding":"RIOK3 is upregulated by hypoxia in an HIF1α-dependent manner. In normoxic cells, RIOK3 localizes to distinct cytoplasmic aggregates; under hypoxia it redistributes to the leading edge of cells with reorganization of the actin cytoskeleton. RIOK3 interacts with actin and actin-binding proteins including tropomyosins TPM3 and TPM4 and tropomodulin 3. RIOK3 depletion reduces actin filament number and organization, decreases TPM3 association with filaments (particularly during hypoxia), impairs cell migration and invasion, and reduces metastasis in zebrafish and mouse models.","method":"HIF1α manipulation, live imaging/localization experiments, proteomic interaction analysis (MS), siRNA depletion, actin staining, 2D migration assay, 3D invasion assay, zebrafish metastasis model, mouse pulmonary metastasis model","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization by live imaging with functional consequence, proteomic pulldown for interactors, in vivo metastasis models, multiple orthogonal methods","pmids":["25486436"],"is_preprint":false},{"year":1998,"finding":"The human RIOK3 gene (mapped to 18q11.2) encodes the human homologue of Aspergillus nidulans SUDD, the founding member of a conserved protein family found in archaea through humans. SUDD was originally identified as an extragenic suppressor of the bimD6 chromosome segregation mutation in A. nidulans.","method":"cDNA cloning, sequence analysis, genetic suppressor screen (A. nidulans bimD6), chromosomal mapping","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — genetic suppressor identification and cloning; establishes evolutionary conservation but limited mechanistic detail about the human protein","pmids":["9602165"],"is_preprint":false},{"year":2022,"finding":"RIOK3 interacts with FAK (Focal Adhesion Kinase) via co-immunoprecipitation and stabilizes FAK protein expression. RIOK3 increases FAK phosphorylation at Y397 and Y925, but does not stabilize FAK-Y925F mutant protein. The pro-invasive and pro-migratory function of RIOK3 in pancreatic ductal adenocarcinoma cells is dependent on FAK activation.","method":"Co-immunoprecipitation, siRNA knockdown, Western blot (FAK protein stability and phosphorylation), transwell invasion/migration assays, transcriptome analysis","journal":"Heliyon","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single Co-IP interaction, protein stability measured by western blot, functional rescue with FAK inhibition; single lab, limited mechanistic depth","pmids":["35982848"],"is_preprint":false},{"year":2022,"finding":"RIOK3 kinase activity mediates Akt phosphorylation; wild-type but not kinase-dead RIOK3 promoted Akt phosphorylation and synergistic MDV/REV viral replication. RIOK3 was recruited to regulate Akt in co-infected cells, operating independently of the PI3K/Akt pathway.","method":"Kinase-dead RIOK3 mutant, LC/MS quantitative proteomics (tandem mass tag), Akt overexpression/inhibition, viral titer assays","journal":"Virulence","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — kinase-dead mutant establishes catalytic requirement, but single lab, limited mechanistic detail on direct vs. indirect Akt phosphorylation","pmids":["35795905"],"is_preprint":false},{"year":2024,"finding":"RIOK3 interacts with HSP90α and facilitates HSP90α binding to IDH1, upregulating IDH1 expression and enhancing NADPH production to sustain colorectal cancer cell survival under glucose deprivation. RIOK3 inhibition had no effect on NADPH levels in HSP90α-knockdown cells, confirming HSP90α dependence.","method":"Co-immunoprecipitation (RIOK3-HSP90α, HSP90α-IDH1), RIOK3 knockout, HSP90α knockdown, NADPH measurement, cell viability assay under glucose deprivation","journal":"Oncogenesis","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — Co-IP interaction, epistasis via HSP90α knockdown rescue experiment; single lab, limited mechanistic depth on how RIOK3 facilitates the interaction","pmids":["38453884"],"is_preprint":false},{"year":2023,"finding":"RIOK3 promotes arginine uptake and mTORC1 activation in pancreatic ductal adenocarcinoma cells via upregulation of the arginine transporter SLC7A2. RIOK3 knockdown significantly inhibited SLC7A2 expression, arginine uptake, and mTORC1 activation.","method":"LC-MS metabolomics, RNA-seq, Western blot (SLC7A2, mTORC1 pathway), RIOK3 knockdown, arginine uptake assay","journal":"Aging","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — metabolomics plus RNA-seq correlation with knockdown validation; single lab, indirect transcriptional regulation without direct binding shown","pmids":["36880835"],"is_preprint":false},{"year":2021,"finding":"RIOK3 is required for mounting an antiviral IFN response to RVFV infection in epithelial cells. During RVFV infection (and after stimulation with other RNA viruses or RIG-I agonists), RIOK3 mRNA is alternatively spliced to produce variants encoding premature termination codons (X2 isoform). This alternative splicing event dampens IFN expression. Forcing alternative splicing with a morpholino oligonucleotide reduced IFN expression.","method":"Transcriptome profiling, RIOK3 knockdown/overexpression, morpholino-induced alternative splicing, IFN reporter assays, RT-PCR for splice variants","journal":"Viruses","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — morpholino functional intervention establishes causal role of splicing, but single lab; splice isoform identification with functional consequence by multiple methods","pmids":["33652597"],"is_preprint":false},{"year":2022,"finding":"RIOK3 (full-length) negatively regulates NF-κB-mediated inflammation during RVFV infection, while the alternatively spliced RIOK3-X2 isoform stimulates the NF-κB inflammatory response. The two isoforms have opposing functions on both IFN and NF-κB pathways.","method":"RIOK3 and RIOK3-X2 overexpression/knockdown, NF-κB reporter assays, IFN reporter assays, RVFV infection model","journal":"Viruses","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — gain- and loss-of-function for both isoforms with reporter assays; single lab, no direct binding or structural data","pmids":["36146870"],"is_preprint":false},{"year":2025,"finding":"METTL3-mediated m6A modification of RIOK3 mRNA enhances RIOK3 expression during CVB3 (Coxsackievirus B3) enterovirus infection. RIOK3 in turn downregulates CDC42, a small GTPase, promoting NF-κB pathway activation and CVB3 replication. RIOK3 and CDC42 jointly modulate NF-κB signaling during infection.","method":"m6A-seq/METTL3 manipulation, RIOK3 overexpression/knockdown, CDC42 expression analysis, NF-κB reporter assay, viral replication assays in vitro and in vivo","journal":"International journal of biological macromolecules","confidence":"Low","confidence_rationale":"Tier 3 / Weak — indirect pathway placement, no direct biochemical interaction between RIOK3 and CDC42 shown; single lab, mechanism of CDC42 suppression by RIOK3 not established","pmids":["39961559"],"is_preprint":false},{"year":2025,"finding":"RIOK3 modulates the Jak1/STAT1 signaling pathway in macrophages during RSV infection; RIOK3 knockout in bone marrow-derived macrophages enhanced viral replication and disrupted type I IFN balance.","method":"RIOK3 knockout mice (BMM), in vitro and in vivo RSV infection models, IFN measurement, Jak1/STAT1 pathway analysis","journal":"Frontiers in microbiology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — KO phenotype in macrophages but no direct biochemical interaction with Jak1/STAT1 components shown; single lab, mechanism not established beyond pathway correlation","pmids":["40371100"],"is_preprint":false}],"current_model":"RIOK3 is an atypical serine/threonine kinase with multiple mechanistic roles: it acts as a negative regulator of innate antiviral immunity by phosphorylating MDA5 (impairing filament formation) and by recruiting TRIM40 to ubiquitinate and degrade both RIG-I and MDA5; it also functions as a positive adaptor that bridges TBK1 and IRF3 to promote type I IFN production in certain contexts; it recognizes ubiquitylated 40S ribosomes (via a ubiquitin-interacting motif) to drive their selective degradation during starvation through progressive 3'-to-5' 18S rRNA decay; it promotes actin cytoskeletal organization at the cell leading edge under hypoxia in an HIF1α-dependent manner, facilitating cell migration and metastasis; it interacts with caspase-10 to negatively regulate NF-κB signaling in a kinase-activity-dependent manner; and it is required for erythroid chromatin condensation and enucleation downstream of miR-191."},"narrative":{"mechanistic_narrative":"RIOK3 is an atypical RIO-family serine/threonine kinase that operates at the intersection of ribosome biogenesis, ribosome quality control, and innate antiviral signaling [PMID:25865883, PMID:22418843, PMID:39947183]. In 40S subunit maturation, it is a cytoplasmic component of pre-40S particles, associating with hLtv1, hEnp1, and 18S-E pre-rRNA, and its depletion stalls 21S pre-rRNA processing [PMID:22418843]. Building on this ribosome association, RIOK3 executes initiation-specific ribosome-associated quality control: through a dedicated ubiquitin-interacting motif it recognizes 40S subunits ubiquitylated by RNF10 on uS3/uS5 during starvation and drives progressive 3'-to-5' decay of 18S rRNA to eliminate defective small subunits, with RNF10 and RIOK3 forming a feedforward circuit [PMID:39947183, PMID:40022732]. In antiviral immunity RIOK3 is predominantly a negative regulator of type I IFN: it phosphorylates MDA5 at S828 to block filament assembly, an event reversed by PP1α/PP1γ, and it recruits the E3 ligase TRIM40 to drive K48/K27-linked ubiquitination and proteasomal degradation of both RIG-I and MDA5 [PMID:25865883, PMID:34161773]. In a distinct context it bridges TBK1 and IRF3 as an adaptor to promote IFN-β induction, and alternative splicing produces an X2 isoform with opposing effects on IFN and NF-κB outputs [PMID:24807708, PMID:33652597, PMID:36146870]. Independently, RIOK3 binds caspase-10 via its RIO domain and restrains NF-κB signaling by competing with RIP1 and NIK, with kinase activity required for suppression of TNFα-induced NF-κB [PMID:19557502]. RIOK3 also supports cellular remodeling and migration: it is induced by hypoxia in an HIF1α-dependent manner, redistributes to the leading edge, and organizes the actin cytoskeleton through interactions with tropomyosins TPM3/TPM4 and tropomodulin 3 to promote migration, invasion, and metastasis [PMID:25486436]. Finally, it is a miR-191 target required for chromatin condensation and enucleation during terminal erythroid differentiation [PMID:21196494].","teleology":[{"year":1998,"claim":"Established the human RIOK3 gene as a conserved member of an ancient protein family, providing the molecular identity that later mechanistic work would build upon.","evidence":"cDNA cloning, sequence analysis, and genetic suppressor screen of the A. nidulans bimD6 chromosome-segregation mutation","pmids":["9602165"],"confidence":"Medium","gaps":["No biochemical function assigned to the human protein","Connection between the SUDD suppressor phenotype and human RIOK3 activity unresolved"]},{"year":2009,"claim":"Identified the first defined molecular partner and signaling role, showing RIOK3 binds caspase-10 through its RIO domain to dampen NF-κB activation.","evidence":"Yeast two-hybrid, GST pull-down, endogenous Co-IP, domain mapping, and kinase-dead mutant in NF-κB reporter assays","pmids":["19557502"],"confidence":"High","gaps":["Direct kinase substrate not identified","Why kinase activity is required for TNFα but not caspase-10-mediated suppression unexplained"]},{"year":2010,"claim":"Placed RIOK3 in a developmental program, demonstrating it is a miR-191 target essential for erythroid chromatin condensation and enucleation.","evidence":"miR-191 target validation by luciferase reporter, RIOK3 knockdown, and enucleation/chromatin condensation assays in mouse erythroblasts","pmids":["21196494"],"confidence":"High","gaps":["Molecular mechanism linking RIOK3 to chromatin condensation unknown","No substrate or partner identified in the erythroid context"]},{"year":2012,"claim":"Defined RIOK3 as a cytoplasmic pre-40S ribosome biogenesis factor, anchoring its molecular activity to 18S rRNA maturation.","evidence":"Sucrose gradient co-sedimentation, Co-IP with hLtv1/hEnp1/18S-E pre-rRNA, and siRNA depletion with Northern blot pre-rRNA analysis","pmids":["22418843"],"confidence":"High","gaps":["Whether kinase activity is required for pre-rRNA processing not resolved","Direct catalytic substrate within the pre-40S particle unidentified"]},{"year":2014,"claim":"Revealed two distinct cytoplasmic roles: a positive adaptor function bridging TBK1 and IRF3 in IFN induction, and a hypoxia-induced actin-organizing function driving migration and metastasis.","evidence":"Two kinome-wide RNAi screens with reciprocal Co-IP and IRF3/IFN-β assays; and HIF1α manipulation, proteomic interactor identification, and zebrafish/mouse metastasis models","pmids":["24807708","25486436"],"confidence":"High","gaps":["Reconciliation of positive (adaptor) versus negative IFN regulation context-dependence unresolved","Whether RIOK3 phosphorylates TBK1, IRF3, or actin partners not established"]},{"year":2015,"claim":"Established RIOK3 as a direct negative regulator of MDA5 by identifying it as a kinase that phosphorylates S828 to block filament formation.","evidence":"RIOK3 knockout cells, phosphomimetic S828D mutation, in vitro kinase assay, multimer formation assay, and IFN reporter readout after measles virus infection","pmids":["25865883"],"confidence":"High","gaps":["Upstream signals controlling RIOK3 kinase activity unknown","Structural basis of MDA5 recognition not resolved"]},{"year":2021,"claim":"Uncovered a degradative arm of RIOK3 antiviral regulation, showing it recruits TRIM40 to ubiquitinate and destroy RIG-I and MDA5, with in vivo confirmation.","evidence":"Reciprocal Co-IP, K48/K27 ubiquitination assays, and myeloid-specific Riok3 knockout mice in RNA virus infection models","pmids":["34161773"],"confidence":"High","gaps":["Whether kinase activity is required for TRIM40 recruitment not established","Interplay between phosphorylation-based and degradation-based MDA5 control unresolved"]},{"year":2021,"claim":"Demonstrated that alternative splicing of RIOK3 mRNA toward a premature-termination X2 variant tunes the IFN response during RNA virus infection.","evidence":"Transcriptome profiling, morpholino-induced splicing, and IFN reporter assays in RVFV-infected epithelial cells","pmids":["33652597"],"confidence":"Medium","gaps":["Single-lab functional data","Protein product and stability of the X2 isoform not characterized"]},{"year":2022,"claim":"Extended the splicing model by showing full-length and X2 isoforms exert opposing effects on both IFN and NF-κB pathways, and linked RIOK3 to FAK and Akt in cancer migration and viral replication.","evidence":"Isoform overexpression/knockdown with reporter assays in RVFV infection; Co-IP and FAK Y397/Y925 phosphorylation in PDAC cells; kinase-dead mutant and proteomics for Akt phosphorylation in co-infection","pmids":["36146870","35982848","35795905"],"confidence":"Medium","gaps":["Whether Akt and FAK are direct kinase substrates not established","Single-lab observations without reciprocal structural validation"]},{"year":2023,"claim":"Connected RIOK3 to cancer metabolism, linking it to arginine uptake/mTORC1 activation via SLC7A2 and to NADPH production via HSP90α-IDH1.","evidence":"Metabolomics, RNA-seq, knockdown/knockout with SLC7A2 and arginine uptake assays; Co-IP and HSP90α knockdown rescue of NADPH levels under glucose deprivation","pmids":["36880835","38453884"],"confidence":"Medium","gaps":["Direct binding of RIOK3 to transporters/metabolic enzymes not shown","Mechanism by which RIOK3 facilitates HSP90α-IDH1 interaction undefined"]},{"year":2025,"claim":"Defined the structural and mechanistic basis for RIOK3-driven 40S ribosome decay, establishing it as the effector of initiation-specific ribosome-associated quality control.","evidence":"Cryo-EM of RIOK3-ubiquitylated 40S complexes with UIM mutagenesis and 18S rRNA decay analysis; genetic epistasis with RNF10 depletion and ribosome profiling under starvation","pmids":["39947183","40022732"],"confidence":"High","gaps":["Whether RIOK3 kinase activity contributes to 40S decay versus its UIM-based recognition unresolved","Nuclease responsible for 18S rRNA decay not defined"]},{"year":2025,"claim":"Broadened RIOK3's antiviral roles to additional pathways via m6A-regulated expression and Jak1/STAT1 modulation in different infection contexts.","evidence":"METTL3/m6A manipulation, RIOK3 and CDC42 expression analysis with NF-κB assays in CVB3 infection; RIOK3 knockout BMMs in RSV infection with Jak1/STAT1 analysis","pmids":["39961559","40371100"],"confidence":"Low","gaps":["No direct biochemical interaction between RIOK3 and CDC42 or Jak1/STAT1 shown","Single-lab pathway-correlation evidence only"]},{"year":null,"claim":"How RIOK3's atypical kinase activity is regulated and which of its many roles depend on catalysis versus scaffolding/UIM-based recognition remains the central unresolved question.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unified model reconciling positive and negative IFN regulation","Direct in vivo kinase substrates beyond MDA5 not catalogued","Catalytic requirement for ribosome decay, migration, and metabolic roles undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,1]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[8]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[4,6]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6,8]},{"term_id":"GO:0005840","term_label":"ribosome","supporting_discovery_ids":[4,6]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[4,5,6]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[8]}],"complexes":["pre-40S ribosomal particle","RIOK3-ubiquitylated 40S complex"],"partners":["MDA5","TRIM40","RIG-I","TBK1","IRF3","CASP10","TPM3","HSP90AA1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O14730","full_name":"Serine/threonine-protein kinase RIO3","aliases":["RIO kinase 3","sudD homolog"],"length_aa":519,"mass_kda":59.1,"function":"Serine/threonine-protein kinase involved in a ribosome quality control that takes place when ribosomes have stalled, leading to 18S non-functional rRNA decay and degradation of the 40S ribosomal subunit (PubMed:39947182, PubMed:39947183, PubMed:40022732). Acts downstream of RNF10: specifically recognizes and binds RPS2/us5 and RPS3/us3 monoubiquitinated by RNF10, promoting degradation of the 40S ribosomal subunit in a kinase-dependent manner (PubMed:39947182, PubMed:39947183, PubMed:40022732). The RNF10-RIOK3 ribosome quality control takes place in response to ribosome subunit imbalance or downstream the EIF2AK4/GCN2-mediated integrated stress response (ISR) (PubMed:39947182, PubMed:39947183, PubMed:40022732). Also involved in regulation of type I interferon (IFN)-dependent immune response, possibly by acting as an adapter protein essential for the recruitment of TBK1 to IRF3 (PubMed:24807708). Phosphorylates IFIH1 on 'Ser-828' interfering with IFIH1 filament assembly on long dsRNA and resulting in attenuated IFIH1-signaling (PubMed:25865883). Can inhibit CASP10 isoform 7-mediated activation of the NF-kappa-B signaling pathway (PubMed:19557502)","subcellular_location":"Cytoplasm, cytosol","url":"https://www.uniprot.org/uniprotkb/O14730/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RIOK3","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000101782","cell_line_id":"CID001258","localizations":[{"compartment":"cytoplasmic","grade":3}],"interactors":[{"gene":"LTV1","stoichiometry":10.0},{"gene":"TSR1","stoichiometry":4.0},{"gene":"BYSL","stoichiometry":4.0},{"gene":"NOB1","stoichiometry":4.0},{"gene":"RACK1","stoichiometry":0.2},{"gene":"ASCC3","stoichiometry":0.2},{"gene":"RPS27A","stoichiometry":0.2},{"gene":"GNB2L1","stoichiometry":0.2},{"gene":"RPS20","stoichiometry":0.2},{"gene":"RPS11","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001258","total_profiled":1310},"omim":[{"mim_id":"603579","title":"RIO KINASE 3; RIOK3","url":"https://www.omim.org/entry/603579"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Golgi apparatus","reliability":"Additional"},{"location":"Centriolar satellite","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":164.5}],"url":"https://www.proteinatlas.org/search/RIOK3"},"hgnc":{"alias_symbol":[],"prev_symbol":["SUDD"]},"alphafold":{"accession":"O14730","domains":[{"cath_id":"-","chopping":"154-224","consensus_level":"high","plddt":72.2151,"start":154,"end":224},{"cath_id":"3.30.200.20","chopping":"232-368","consensus_level":"medium","plddt":82.565,"start":232,"end":368},{"cath_id":"1.10.510.10","chopping":"370-505","consensus_level":"medium","plddt":89.448,"start":370,"end":505}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O14730","model_url":"https://alphafold.ebi.ac.uk/files/AF-O14730-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O14730-F1-predicted_aligned_error_v6.png","plddt_mean":74.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RIOK3","jax_strain_url":"https://www.jax.org/strain/search?query=RIOK3"},"sequence":{"accession":"O14730","fasta_url":"https://rest.uniprot.org/uniprotkb/O14730.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O14730/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O14730"}},"corpus_meta":[{"pmid":"21196494","id":"PMC_21196494","title":"miR-191 regulates mouse erythroblast enucleation by down-regulating Riok3 and Mxi1.","date":"2010","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/21196494","citation_count":103,"is_preprint":false},{"pmid":"25865883","id":"PMC_25865883","title":"RIOK3-mediated phosphorylation of MDA5 interferes with its assembly and attenuates the innate immune response.","date":"2015","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/25865883","citation_count":72,"is_preprint":false},{"pmid":"29233656","id":"PMC_29233656","title":"The atypical protein kinase RIOK3 contributes to glioma cell proliferation/survival, migration/invasion and the AKT/mTOR signaling pathway.","date":"2017","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/29233656","citation_count":48,"is_preprint":false},{"pmid":"34161773","id":"PMC_34161773","title":"Riok3 inhibits the antiviral immune response by facilitating TRIM40-mediated RIG-I and MDA5 degradation.","date":"2021","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/34161773","citation_count":45,"is_preprint":false},{"pmid":"25486436","id":"PMC_25486436","title":"Hypoxic regulation of RIOK3 is a major mechanism for cancer cell invasion and metastasis.","date":"2014","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/25486436","citation_count":45,"is_preprint":false},{"pmid":"22418843","id":"PMC_22418843","title":"Human RioK3 is a novel component of cytoplasmic pre-40S pre-ribosomal particles.","date":"2012","source":"RNA biology","url":"https://pubmed.ncbi.nlm.nih.gov/22418843","citation_count":45,"is_preprint":false},{"pmid":"24807708","id":"PMC_24807708","title":"RIOK3 is an adaptor protein required for IRF3-mediated antiviral type I interferon production.","date":"2014","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/24807708","citation_count":40,"is_preprint":false},{"pmid":"19557502","id":"PMC_19557502","title":"RIOK3 interacts with caspase-10 and negatively regulates the NF-kappaB signaling pathway.","date":"2009","source":"Molecular and cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19557502","citation_count":34,"is_preprint":false},{"pmid":"9602165","id":"PMC_9602165","title":"Isolation of the Aspergillus nidulans sudD gene and its human homologue.","date":"1998","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/9602165","citation_count":21,"is_preprint":false},{"pmid":"39947183","id":"PMC_39947183","title":"RIOK3 mediates the degradation of 40S ribosomes.","date":"2025","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/39947183","citation_count":18,"is_preprint":false},{"pmid":"40022732","id":"PMC_40022732","title":"RNF10 and RIOK3 facilitate 40S ribosomal subunit degradation upon 60S biogenesis disruption or amino acid starvation.","date":"2025","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/40022732","citation_count":13,"is_preprint":false},{"pmid":"36880835","id":"PMC_36880835","title":"RIOK3 promotes mTORC1 activation by facilitating SLC7A2-mediated arginine uptake in pancreatic ductal adenocarcinoma.","date":"2023","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/36880835","citation_count":11,"is_preprint":false},{"pmid":"33652597","id":"PMC_33652597","title":"The Atypical Kinase RIOK3 Limits RVFV Propagation and Is Regulated by Alternative Splicing.","date":"2021","source":"Viruses","url":"https://pubmed.ncbi.nlm.nih.gov/33652597","citation_count":9,"is_preprint":false},{"pmid":"36146870","id":"PMC_36146870","title":"RIOK3 and Its Alternatively Spliced Isoform Have Disparate Roles in the Innate Immune Response to Rift Valley Fever Virus (MP12) Infection.","date":"2022","source":"Viruses","url":"https://pubmed.ncbi.nlm.nih.gov/36146870","citation_count":9,"is_preprint":false},{"pmid":"37515252","id":"PMC_37515252","title":"Alternative Splicing of RIOK3 Engages the Noncanonical NFκB Pathway during Rift Valley Fever Virus Infection.","date":"2023","source":"Viruses","url":"https://pubmed.ncbi.nlm.nih.gov/37515252","citation_count":6,"is_preprint":false},{"pmid":"35982848","id":"PMC_35982848","title":"RIOK3 promotes pancreatic ductal adenocarcinoma cell invasion and metastasis by stabilizing FAK.","date":"2022","source":"Heliyon","url":"https://pubmed.ncbi.nlm.nih.gov/35982848","citation_count":5,"is_preprint":false},{"pmid":"39961559","id":"PMC_39961559","title":"m6A-modified RIOK3 activated the NF-κB-signaling pathway by CDC42, promoting the replication and proliferation of enterovirus.","date":"2025","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/39961559","citation_count":5,"is_preprint":false},{"pmid":"35795905","id":"PMC_35795905","title":"RIOK3-Mediated Akt phosphorylation facilitates synergistic replication of Marek's disease and reticuloendotheliosis viruses.","date":"2022","source":"Virulence","url":"https://pubmed.ncbi.nlm.nih.gov/35795905","citation_count":5,"is_preprint":false},{"pmid":"37866658","id":"PMC_37866658","title":"RIOK3 potentially regulates osteogenesis-related pathways in ankylosing spondylitis and the differentiation of bone marrow mesenchymal stem cells.","date":"2023","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/37866658","citation_count":4,"is_preprint":false},{"pmid":"38453884","id":"PMC_38453884","title":"RIOK3 sustains colorectal cancer cell survival under glucose deprivation via an HSP90α-dependent pathway.","date":"2024","source":"Oncogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/38453884","citation_count":2,"is_preprint":false},{"pmid":"37722630","id":"PMC_37722630","title":"Black carp RIOK3 suppresses MDA5-mediated IFN signaling in the antiviral innate immunity.","date":"2023","source":"Developmental and comparative immunology","url":"https://pubmed.ncbi.nlm.nih.gov/37722630","citation_count":0,"is_preprint":false},{"pmid":"34758896","id":"PMC_34758896","title":"Induction of right open reading frame kinase 3 (RIOK3) during ovulation and luteinisation in rat ovary.","date":"2021","source":"Reproduction, fertility, and development","url":"https://pubmed.ncbi.nlm.nih.gov/34758896","citation_count":0,"is_preprint":false},{"pmid":"40371100","id":"PMC_40371100","title":"The economical role of RIOK3 in modulating the Jak1/STAT1 pathway and antiviral immunity against respiratory syncytial virus infection in macrophages: implications for therapeutic potential.","date":"2025","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/40371100","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12439,"output_tokens":5186,"usd":0.057554,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13446,"output_tokens":4588,"usd":0.090965,"stage2_stop_reason":"end_turn"},"total_usd":0.148519,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2015,\n      \"finding\": \"RIOK3 phosphorylates the C-terminal region of MDA5 (at S828), impairing MDA5 multimer/filament formation and attenuating MDA5-mediated type I IFN signaling. PP1α/PP1γ phosphatases counteract this by dephosphorylating MDA5. RIOK3 knockout strongly enhanced type I IFN production following measles virus infection, while phosphomimetic MDA5-S828D mutation attenuated signaling.\",\n      \"method\": \"RIOK3 knockout cells, phosphomimetic mutation (S828D), in vitro kinase assay, multimer formation assay, IFN reporter assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct kinase-substrate identification with mutagenesis (phosphomimetic), KO phenotype, and mechanistic readout of filament formation; multiple orthogonal methods in single study\",\n      \"pmids\": [\"25865883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RIOK3 recruits and interacts with the E3 ubiquitin ligase TRIM40, leading to K48- and K27-linked ubiquitination and proteasomal degradation of both RIG-I and MDA5, thereby negatively regulating type I IFN signaling. Myeloid-specific Riok3 knockout mice showed enhanced type I IFN induction and resistance to RNA virus-induced pathogenesis.\",\n      \"method\": \"Co-immunoprecipitation, myeloid-specific knockout mice, ubiquitination assay (K48/K27 linkage), in vitro and in vivo viral infection models\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vivo KO mouse model, biochemical ubiquitination assays; multiple orthogonal methods\",\n      \"pmids\": [\"34161773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RIOK3 functions as an adaptor protein downstream of TBK1 and upstream of IRF3 in the type I IFN pathway, physically interacting with both TBK1 and IRF3 and bridging their interaction. RIOK3 knockdown blocks cytosolic dsRNA- and dsDNA-induced IRF3 activation and IFN-β production; overexpression activates IRF3.\",\n      \"method\": \"Kinome-wide RNAi screens (two independent), Co-immunoprecipitation (RIOK3 with TBK1 and IRF3), IFN-β reporter assay, IRF3 activation assay, transcriptome analysis\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent genome-wide RNAi screens plus reciprocal Co-IP, epistasis placement downstream of TBK1 and upstream of IRF3, confirmed by gain- and loss-of-function experiments\",\n      \"pmids\": [\"24807708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"RIOK3 interacts with caspase-10 via its RIO domain (binding to each death effector domain of caspase-10) and negatively regulates NF-κB signaling. RIOK3 suppresses caspase-10-mediated NF-κB activation by competing with RIP1 and NIK for binding to caspase-10. Kinase activity of RIOK3 is required for suppression of TNFα-induced NF-κB activation but not for suppression of caspase-10-mediated NF-κB activation.\",\n      \"method\": \"Yeast two-hybrid, GST pull-down, endogenous co-immunoprecipitation, siRNA knockdown, NF-κB reporter assay, domain-mapping experiments\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by GST pulldown and endogenous Co-IP, domain mapping, kinase-dead mutant, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"19557502\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RIOK3 specifically recognizes ubiquitylated 40S ribosomes (ubiquitylated by E3 ligase RNF10 on uS3 and uS5 during starvation) through a unique ubiquitin-interacting motif (UIM). RIOK3 binding drives progressive 3'-to-5' decay of 18S rRNA, mediating selective 40S ribosome degradation during starvation. Cryo-EM structures of RIOK3-ubiquitylated 40S complexes and degradation intermediates were resolved.\",\n      \"method\": \"Cryo-EM structural analysis, ubiquitin-interacting motif mutagenesis, starvation assays, rRNA decay analysis, functional rescue experiments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with functional mutagenesis of UIM, multiple degradation intermediate structures, mechanistic pathway reconstitution\",\n      \"pmids\": [\"39947183\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RIOK3 is a crucial factor in the initiation-specific ribosome-associated quality control (iRQC) pathway; it interacts with ubiquitylated 40S subunits (ubiquitylated by RNF10) to mediate their degradation. Both RNF10 and RIOK3 protein levels increase upon iRQC activation, establishing a feedforward mechanism. Amino acid starvation and 60S:40S stoichiometry imbalance (from disrupted 60S biogenesis) activate iRQC-dependent 40S decay.\",\n      \"method\": \"RIOK3 depletion, RNF10 depletion, quantitative ribosome profiling, 40S ubiquitylation assays, 60S biogenesis disruption, amino acid starvation conditions\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (RNF10/RIOK3 double depletion), feedforward mechanism via protein level measurements, multiple pathway activation conditions tested; corroborated by independent lab (PMID 39947183)\",\n      \"pmids\": [\"40022732\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Human RioK3 is a cytoplasmic protein that co-sediments with cytoplasmic pre-40S ribosomal particles and associates with pre-40S particle components hLtv1, hEnp1, and 18S-E pre-rRNA. RioK3 depletion leads to increased levels of 21S rRNA precursor, indicating a role in 21S pre-rRNA processing during 40S subunit biogenesis. RioK3 does not shuttle via Crm1-dependent nuclear export (unlike RioK2).\",\n      \"method\": \"Sucrose gradient sedimentation, co-immunoprecipitation (with hLtv1, hEnp1, 18S-E pre-rRNA), siRNA depletion (RioK3, rpS15, rpS19, RioK2), Northern blot (pre-rRNA analysis)\",\n      \"journal\": \"RNA biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — sucrose gradient co-sedimentation, reciprocal Co-IP with pre-40S components, pre-rRNA processing analysis; multiple orthogonal methods in single study\",\n      \"pmids\": [\"22418843\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Riok3 is a direct target of miR-191 in mouse erythroblasts. Knockdown of Riok3 blocks erythroid enucleation and chromatin condensation during terminal erythroid differentiation. Down-regulation of miR-191 during terminal differentiation is required to allow Riok3 upregulation, which is essential for these processes.\",\n      \"method\": \"RNA-seq, miR-191 overexpression, Riok3 knockdown (siRNA), erythroid enucleation assays, chromatin condensation analysis, luciferase reporter (miR-191 target validation)\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct miRNA target validation, gain- and loss-of-function experiments, defined cellular phenotypes (enucleation, chromatin condensation); replicated with multiple approaches\",\n      \"pmids\": [\"21196494\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RIOK3 is upregulated by hypoxia in an HIF1α-dependent manner. In normoxic cells, RIOK3 localizes to distinct cytoplasmic aggregates; under hypoxia it redistributes to the leading edge of cells with reorganization of the actin cytoskeleton. RIOK3 interacts with actin and actin-binding proteins including tropomyosins TPM3 and TPM4 and tropomodulin 3. RIOK3 depletion reduces actin filament number and organization, decreases TPM3 association with filaments (particularly during hypoxia), impairs cell migration and invasion, and reduces metastasis in zebrafish and mouse models.\",\n      \"method\": \"HIF1α manipulation, live imaging/localization experiments, proteomic interaction analysis (MS), siRNA depletion, actin staining, 2D migration assay, 3D invasion assay, zebrafish metastasis model, mouse pulmonary metastasis model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization by live imaging with functional consequence, proteomic pulldown for interactors, in vivo metastasis models, multiple orthogonal methods\",\n      \"pmids\": [\"25486436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The human RIOK3 gene (mapped to 18q11.2) encodes the human homologue of Aspergillus nidulans SUDD, the founding member of a conserved protein family found in archaea through humans. SUDD was originally identified as an extragenic suppressor of the bimD6 chromosome segregation mutation in A. nidulans.\",\n      \"method\": \"cDNA cloning, sequence analysis, genetic suppressor screen (A. nidulans bimD6), chromosomal mapping\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — genetic suppressor identification and cloning; establishes evolutionary conservation but limited mechanistic detail about the human protein\",\n      \"pmids\": [\"9602165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RIOK3 interacts with FAK (Focal Adhesion Kinase) via co-immunoprecipitation and stabilizes FAK protein expression. RIOK3 increases FAK phosphorylation at Y397 and Y925, but does not stabilize FAK-Y925F mutant protein. The pro-invasive and pro-migratory function of RIOK3 in pancreatic ductal adenocarcinoma cells is dependent on FAK activation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, Western blot (FAK protein stability and phosphorylation), transwell invasion/migration assays, transcriptome analysis\",\n      \"journal\": \"Heliyon\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP interaction, protein stability measured by western blot, functional rescue with FAK inhibition; single lab, limited mechanistic depth\",\n      \"pmids\": [\"35982848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RIOK3 kinase activity mediates Akt phosphorylation; wild-type but not kinase-dead RIOK3 promoted Akt phosphorylation and synergistic MDV/REV viral replication. RIOK3 was recruited to regulate Akt in co-infected cells, operating independently of the PI3K/Akt pathway.\",\n      \"method\": \"Kinase-dead RIOK3 mutant, LC/MS quantitative proteomics (tandem mass tag), Akt overexpression/inhibition, viral titer assays\",\n      \"journal\": \"Virulence\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — kinase-dead mutant establishes catalytic requirement, but single lab, limited mechanistic detail on direct vs. indirect Akt phosphorylation\",\n      \"pmids\": [\"35795905\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RIOK3 interacts with HSP90α and facilitates HSP90α binding to IDH1, upregulating IDH1 expression and enhancing NADPH production to sustain colorectal cancer cell survival under glucose deprivation. RIOK3 inhibition had no effect on NADPH levels in HSP90α-knockdown cells, confirming HSP90α dependence.\",\n      \"method\": \"Co-immunoprecipitation (RIOK3-HSP90α, HSP90α-IDH1), RIOK3 knockout, HSP90α knockdown, NADPH measurement, cell viability assay under glucose deprivation\",\n      \"journal\": \"Oncogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP interaction, epistasis via HSP90α knockdown rescue experiment; single lab, limited mechanistic depth on how RIOK3 facilitates the interaction\",\n      \"pmids\": [\"38453884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RIOK3 promotes arginine uptake and mTORC1 activation in pancreatic ductal adenocarcinoma cells via upregulation of the arginine transporter SLC7A2. RIOK3 knockdown significantly inhibited SLC7A2 expression, arginine uptake, and mTORC1 activation.\",\n      \"method\": \"LC-MS metabolomics, RNA-seq, Western blot (SLC7A2, mTORC1 pathway), RIOK3 knockdown, arginine uptake assay\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — metabolomics plus RNA-seq correlation with knockdown validation; single lab, indirect transcriptional regulation without direct binding shown\",\n      \"pmids\": [\"36880835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RIOK3 is required for mounting an antiviral IFN response to RVFV infection in epithelial cells. During RVFV infection (and after stimulation with other RNA viruses or RIG-I agonists), RIOK3 mRNA is alternatively spliced to produce variants encoding premature termination codons (X2 isoform). This alternative splicing event dampens IFN expression. Forcing alternative splicing with a morpholino oligonucleotide reduced IFN expression.\",\n      \"method\": \"Transcriptome profiling, RIOK3 knockdown/overexpression, morpholino-induced alternative splicing, IFN reporter assays, RT-PCR for splice variants\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — morpholino functional intervention establishes causal role of splicing, but single lab; splice isoform identification with functional consequence by multiple methods\",\n      \"pmids\": [\"33652597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RIOK3 (full-length) negatively regulates NF-κB-mediated inflammation during RVFV infection, while the alternatively spliced RIOK3-X2 isoform stimulates the NF-κB inflammatory response. The two isoforms have opposing functions on both IFN and NF-κB pathways.\",\n      \"method\": \"RIOK3 and RIOK3-X2 overexpression/knockdown, NF-κB reporter assays, IFN reporter assays, RVFV infection model\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — gain- and loss-of-function for both isoforms with reporter assays; single lab, no direct binding or structural data\",\n      \"pmids\": [\"36146870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"METTL3-mediated m6A modification of RIOK3 mRNA enhances RIOK3 expression during CVB3 (Coxsackievirus B3) enterovirus infection. RIOK3 in turn downregulates CDC42, a small GTPase, promoting NF-κB pathway activation and CVB3 replication. RIOK3 and CDC42 jointly modulate NF-κB signaling during infection.\",\n      \"method\": \"m6A-seq/METTL3 manipulation, RIOK3 overexpression/knockdown, CDC42 expression analysis, NF-κB reporter assay, viral replication assays in vitro and in vivo\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — indirect pathway placement, no direct biochemical interaction between RIOK3 and CDC42 shown; single lab, mechanism of CDC42 suppression by RIOK3 not established\",\n      \"pmids\": [\"39961559\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RIOK3 modulates the Jak1/STAT1 signaling pathway in macrophages during RSV infection; RIOK3 knockout in bone marrow-derived macrophages enhanced viral replication and disrupted type I IFN balance.\",\n      \"method\": \"RIOK3 knockout mice (BMM), in vitro and in vivo RSV infection models, IFN measurement, Jak1/STAT1 pathway analysis\",\n      \"journal\": \"Frontiers in microbiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — KO phenotype in macrophages but no direct biochemical interaction with Jak1/STAT1 components shown; single lab, mechanism not established beyond pathway correlation\",\n      \"pmids\": [\"40371100\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RIOK3 is an atypical serine/threonine kinase with multiple mechanistic roles: it acts as a negative regulator of innate antiviral immunity by phosphorylating MDA5 (impairing filament formation) and by recruiting TRIM40 to ubiquitinate and degrade both RIG-I and MDA5; it also functions as a positive adaptor that bridges TBK1 and IRF3 to promote type I IFN production in certain contexts; it recognizes ubiquitylated 40S ribosomes (via a ubiquitin-interacting motif) to drive their selective degradation during starvation through progressive 3'-to-5' 18S rRNA decay; it promotes actin cytoskeletal organization at the cell leading edge under hypoxia in an HIF1α-dependent manner, facilitating cell migration and metastasis; it interacts with caspase-10 to negatively regulate NF-κB signaling in a kinase-activity-dependent manner; and it is required for erythroid chromatin condensation and enucleation downstream of miR-191.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RIOK3 is an atypical RIO-family serine/threonine kinase that operates at the intersection of ribosome biogenesis, ribosome quality control, and innate antiviral signaling [#0, #6, #4]. In 40S subunit maturation, it is a cytoplasmic component of pre-40S particles, associating with hLtv1, hEnp1, and 18S-E pre-rRNA, and its depletion stalls 21S pre-rRNA processing [#6]. Building on this ribosome association, RIOK3 executes initiation-specific ribosome-associated quality control: through a dedicated ubiquitin-interacting motif it recognizes 40S subunits ubiquitylated by RNF10 on uS3/uS5 during starvation and drives progressive 3'-to-5' decay of 18S rRNA to eliminate defective small subunits, with RNF10 and RIOK3 forming a feedforward circuit [#4, #5]. In antiviral immunity RIOK3 is predominantly a negative regulator of type I IFN: it phosphorylates MDA5 at S828 to block filament assembly, an event reversed by PP1\\u03b1/PP1\\u03b3, and it recruits the E3 ligase TRIM40 to drive K48/K27-linked ubiquitination and proteasomal degradation of both RIG-I and MDA5 [#0, #1]. In a distinct context it bridges TBK1 and IRF3 as an adaptor to promote IFN-\\u03b2 induction, and alternative splicing produces an X2 isoform with opposing effects on IFN and NF-\\u03baB outputs [#2, #14, #15]. Independently, RIOK3 binds caspase-10 via its RIO domain and restrains NF-\\u03baB signaling by competing with RIP1 and NIK, with kinase activity required for suppression of TNF\\u03b1-induced NF-\\u03baB [#3]. RIOK3 also supports cellular remodeling and migration: it is induced by hypoxia in an HIF1\\u03b1-dependent manner, redistributes to the leading edge, and organizes the actin cytoskeleton through interactions with tropomyosins TPM3/TPM4 and tropomodulin 3 to promote migration, invasion, and metastasis [#8]. Finally, it is a miR-191 target required for chromatin condensation and enucleation during terminal erythroid differentiation [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established the human RIOK3 gene as a conserved member of an ancient protein family, providing the molecular identity that later mechanistic work would build upon.\",\n      \"evidence\": \"cDNA cloning, sequence analysis, and genetic suppressor screen of the A. nidulans bimD6 chromosome-segregation mutation\",\n      \"pmids\": [\"9602165\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No biochemical function assigned to the human protein\", \"Connection between the SUDD suppressor phenotype and human RIOK3 activity unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified the first defined molecular partner and signaling role, showing RIOK3 binds caspase-10 through its RIO domain to dampen NF-\\u03baB activation.\",\n      \"evidence\": \"Yeast two-hybrid, GST pull-down, endogenous Co-IP, domain mapping, and kinase-dead mutant in NF-\\u03baB reporter assays\",\n      \"pmids\": [\"19557502\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct kinase substrate not identified\", \"Why kinase activity is required for TNF\\u03b1 but not caspase-10-mediated suppression unexplained\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Placed RIOK3 in a developmental program, demonstrating it is a miR-191 target essential for erythroid chromatin condensation and enucleation.\",\n      \"evidence\": \"miR-191 target validation by luciferase reporter, RIOK3 knockdown, and enucleation/chromatin condensation assays in mouse erythroblasts\",\n      \"pmids\": [\"21196494\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism linking RIOK3 to chromatin condensation unknown\", \"No substrate or partner identified in the erythroid context\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined RIOK3 as a cytoplasmic pre-40S ribosome biogenesis factor, anchoring its molecular activity to 18S rRNA maturation.\",\n      \"evidence\": \"Sucrose gradient co-sedimentation, Co-IP with hLtv1/hEnp1/18S-E pre-rRNA, and siRNA depletion with Northern blot pre-rRNA analysis\",\n      \"pmids\": [\"22418843\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether kinase activity is required for pre-rRNA processing not resolved\", \"Direct catalytic substrate within the pre-40S particle unidentified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revealed two distinct cytoplasmic roles: a positive adaptor function bridging TBK1 and IRF3 in IFN induction, and a hypoxia-induced actin-organizing function driving migration and metastasis.\",\n      \"evidence\": \"Two kinome-wide RNAi screens with reciprocal Co-IP and IRF3/IFN-\\u03b2 assays; and HIF1\\u03b1 manipulation, proteomic interactor identification, and zebrafish/mouse metastasis models\",\n      \"pmids\": [\"24807708\", \"25486436\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation of positive (adaptor) versus negative IFN regulation context-dependence unresolved\", \"Whether RIOK3 phosphorylates TBK1, IRF3, or actin partners not established\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established RIOK3 as a direct negative regulator of MDA5 by identifying it as a kinase that phosphorylates S828 to block filament formation.\",\n      \"evidence\": \"RIOK3 knockout cells, phosphomimetic S828D mutation, in vitro kinase assay, multimer formation assay, and IFN reporter readout after measles virus infection\",\n      \"pmids\": [\"25865883\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals controlling RIOK3 kinase activity unknown\", \"Structural basis of MDA5 recognition not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Uncovered a degradative arm of RIOK3 antiviral regulation, showing it recruits TRIM40 to ubiquitinate and destroy RIG-I and MDA5, with in vivo confirmation.\",\n      \"evidence\": \"Reciprocal Co-IP, K48/K27 ubiquitination assays, and myeloid-specific Riok3 knockout mice in RNA virus infection models\",\n      \"pmids\": [\"34161773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether kinase activity is required for TRIM40 recruitment not established\", \"Interplay between phosphorylation-based and degradation-based MDA5 control unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated that alternative splicing of RIOK3 mRNA toward a premature-termination X2 variant tunes the IFN response during RNA virus infection.\",\n      \"evidence\": \"Transcriptome profiling, morpholino-induced splicing, and IFN reporter assays in RVFV-infected epithelial cells\",\n      \"pmids\": [\"33652597\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab functional data\", \"Protein product and stability of the X2 isoform not characterized\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended the splicing model by showing full-length and X2 isoforms exert opposing effects on both IFN and NF-\\u03baB pathways, and linked RIOK3 to FAK and Akt in cancer migration and viral replication.\",\n      \"evidence\": \"Isoform overexpression/knockdown with reporter assays in RVFV infection; Co-IP and FAK Y397/Y925 phosphorylation in PDAC cells; kinase-dead mutant and proteomics for Akt phosphorylation in co-infection\",\n      \"pmids\": [\"36146870\", \"35982848\", \"35795905\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Akt and FAK are direct kinase substrates not established\", \"Single-lab observations without reciprocal structural validation\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected RIOK3 to cancer metabolism, linking it to arginine uptake/mTORC1 activation via SLC7A2 and to NADPH production via HSP90\\u03b1-IDH1.\",\n      \"evidence\": \"Metabolomics, RNA-seq, knockdown/knockout with SLC7A2 and arginine uptake assays; Co-IP and HSP90\\u03b1 knockdown rescue of NADPH levels under glucose deprivation\",\n      \"pmids\": [\"36880835\", \"38453884\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding of RIOK3 to transporters/metabolic enzymes not shown\", \"Mechanism by which RIOK3 facilitates HSP90\\u03b1-IDH1 interaction undefined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined the structural and mechanistic basis for RIOK3-driven 40S ribosome decay, establishing it as the effector of initiation-specific ribosome-associated quality control.\",\n      \"evidence\": \"Cryo-EM of RIOK3-ubiquitylated 40S complexes with UIM mutagenesis and 18S rRNA decay analysis; genetic epistasis with RNF10 depletion and ribosome profiling under starvation\",\n      \"pmids\": [\"39947183\", \"40022732\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RIOK3 kinase activity contributes to 40S decay versus its UIM-based recognition unresolved\", \"Nuclease responsible for 18S rRNA decay not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Broadened RIOK3's antiviral roles to additional pathways via m6A-regulated expression and Jak1/STAT1 modulation in different infection contexts.\",\n      \"evidence\": \"METTL3/m6A manipulation, RIOK3 and CDC42 expression analysis with NF-\\u03baB assays in CVB3 infection; RIOK3 knockout BMMs in RSV infection with Jak1/STAT1 analysis\",\n      \"pmids\": [\"39961559\", \"40371100\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct biochemical interaction between RIOK3 and CDC42 or Jak1/STAT1 shown\", \"Single-lab pathway-correlation evidence only\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RIOK3's atypical kinase activity is regulated and which of its many roles depend on catalysis versus scaffolding/UIM-based recognition remains the central unresolved question.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified model reconciling positive and negative IFN regulation\", \"Direct in vivo kinase substrates beyond MDA5 not catalogued\", \"Catalytic requirement for ribosome decay, migration, and metabolic roles undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 1]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [4, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6, 8]},\n      {\"term_id\": \"GO:0005840\", \"supporting_discovery_ids\": [4, 6]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [4, 5, 6]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [\n      \"pre-40S ribosomal particle\",\n      \"RIOK3-ubiquitylated 40S complex\"\n    ],\n    \"partners\": [\n      \"MDA5\",\n      \"TRIM40\",\n      \"RIG-I\",\n      \"TBK1\",\n      \"IRF3\",\n      \"CASP10\",\n      \"TPM3\",\n      \"HSP90AA1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":8,"faith_pct":87.5}}