{"gene":"RC3H1","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2007,"finding":"Roquin (RC3H1) limits ICOS expression by promoting degradation of ICOS mRNA; a conserved 47-bp segment in the ICOS 3' UTR is essential for Roquin-mediated regulation, and the M199R mutation in the ROQ domain abolishes this activity in vivo.","method":"Genetic mouse model (sanroque M199R mutation), mRNA decay assays, 3' UTR functional analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — foundational genetic epistasis with defined molecular target, replicated extensively across labs","pmids":["18172933"],"is_preprint":false},{"year":2010,"finding":"Roquin localizes to processing (P) bodies and directly binds ICOS mRNA via its 3' UTR; repression of ICOS requires the RNA helicase Rck and the decapping enhancer Edc4, and depends on P-body localization sequences in Roquin, but is independent of microRNAs or RISC.","method":"Co-immunoprecipitation, fluorescence microscopy (P-body localization), RNA binding assays, microRNA-independence demonstrated by Ago2/RISC decoupling","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, direct RNA binding, subcellular localization linked to function, independence from miRNA validated","pmids":["20639877"],"is_preprint":false},{"year":2010,"finding":"The ROQ domain of Roquin is necessary and sufficient for localization to stress granules and for triggering ICOS mRNA decay; gel-shift, SPR and footprinting show the ROQ domain binds RNA from the ICOS 3' UTR adjacent to the miR-101 complementarity site.","method":"Fluorescence microscopy, gel-shift, SPR, RNA footprinting, domain deletion analysis","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal biochemical methods defining domain function and RNA binding","pmids":["20412057"],"is_preprint":false},{"year":2013,"finding":"Roquin recognizes a constitutive decay element (CDE) stem-loop in the TNF-α 3' UTR and promotes mRNA degradation by recruiting the Ccr4-Caf1-Not deadenylase complex; CDE motifs are conserved in >50 vertebrate mRNAs encoding regulators of development and inflammation.","method":"RNA-protein binding assays, mRNA decay assays in macrophages, transcriptome-wide target identification, deadenylase complex recruitment assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1-2 — mechanistic reconstitution of deadenylase recruitment, genome-wide target validation, replicated","pmids":["23663784"],"is_preprint":false},{"year":2013,"finding":"Roquin-1 and Roquin-2 redundantly repress ICOS and OX40 costimulatory receptor mRNAs in T cells; the RING domain of Roquin-1 is required for stress granule localization, and its absence allows Roquin-2 to compensate, whereas the ROQ mutation disrupts mRNA regulation while preserving stress granule occupancy.","method":"Conditional gene knockout, stress granule localization (fluorescence microscopy), mRNA reporter assays, bone marrow chimeras","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with domain-specific functional readouts, two independent papers simultaneously","pmids":["23583643","23583642"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of the mouse Roquin ROQ domain in complex with CDE RNA reveals an extended winged-helix fold; the ROQ domain recognizes the RNA stem and triloop primarily through non-sequence-specific contacts, explaining broad target spectrum; cell-based assays confirm ROQ domain is sufficient for CDE-regulated mRNA decay.","method":"X-ray crystallography, NMR, binding experiments, cell-based mRNA decay assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional validation in cells","pmids":["25026077"],"is_preprint":false},{"year":2014,"finding":"Crystal structures of human Roquin ROQ domain reveal two distinct RNA-binding sites: the A-site binds stem-loop CDE RNA and the B-site binds double-stranded RNA at a separate interface; mutagenesis of either site disrupts Roquin-mediated degradation of HMGXB3 and IL6 mRNAs in human cells.","method":"X-ray crystallography (two crystal structures), site-directed mutagenesis, mRNA decay assays in human cells","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — two crystal structures plus mutagenesis with functional readout in cells","pmids":["25026078"],"is_preprint":false},{"year":2014,"finding":"Upon TCR antigen recognition, the paracaspase MALT1 cleaves both Roquin-1 and Regnase-1; this cleavage releases cooperative post-transcriptional repression of IL-6, ICOS, c-Rel, IRF4, IκBNS, and IκBζ mRNAs, thereby promoting TH17 differentiation in a signal-strength-dependent manner.","method":"Biochemical cleavage assays, T cell genetic models, mRNA reporter assays, MALT1 activity assays","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 — direct biochemical demonstration of MALT1-mediated cleavage with defined functional consequences, replicated","pmids":["25282160"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of the Roquin ROQ domain shows a winged helix-turn-helix (wHTH) motif involved in CDE stem-loop RNA binding; biochemical and mutational analyses show Roquin can bind duplex RNA and function in different oligomeric states.","method":"X-ray crystallography, biochemical binding assays, mutagenesis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with biochemical and mutational validation","pmids":["25504471"],"is_preprint":false},{"year":2015,"finding":"Regnase-1 and Roquin regulate an overlapping set of inflammatory mRNAs via a common stem-loop structure but act in distinct subcellular compartments: Regnase-1 degrades translationally active mRNAs at ribosomes/ER and requires UPF1 helicase activity, while Roquin acts on translationally inactive mRNAs at P-bodies/stress granules independently of UPF1.","method":"Subcellular fractionation, ribosome profiling, siRNA knockdown, mRNA decay assays, UPF1 requirement assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods defining spatiotemporally distinct mechanisms, high-impact journal, widely cited","pmids":["26000482"],"is_preprint":false},{"year":2015,"finding":"PAR-CLIP of human RC3H1 identifies ~3,800 mRNA targets with >16,000 binding sites; RC3H1 uses ROQ and Zn-finger domains to contact a binding site in the A20 3' UTR; knockdown of RC3H1 increases A20 protein, thereby reducing IKK and NF-κB activities.","method":"PAR-CLIP (transcriptome-wide), domain deletion/mutagenesis, NF-κB pathway assays, siRNA knockdown","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 — genome-wide binding map plus domain-specific binding and signaling pathway readout","pmids":["26170170"],"is_preprint":false},{"year":2015,"finding":"Roquin binds Argonaute2 directly and enhances Dicer-mediated processing of pre-miR-146a; in the absence of functional Roquin, mature miR-146a accumulates due to decreased 3'-end uridylation; crystallographic studies reveal a unique HEPN domain in Roquin and define the structural basis for the sanroque mutation.","method":"Co-immunoprecipitation (Roquin-Ago2, Roquin-miR-146a), miRNA processing assays, 3'-end uridylation analysis, X-ray crystallography","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 — crystal structure plus multiple orthogonal binding and functional assays","pmids":["25697406"],"is_preprint":false},{"year":2015,"finding":"The RING domain of Roquin-1 directly antagonizes the catalytic α1 subunit of AMPK, promoting Tfh cell differentiation; T cell-specific deletion of the RING domain selectively abolishes antigen-specific Tfh responses without affecting Th1, Th2, Th17, or Treg populations.","method":"Conditional RING domain knockout mice, biochemical AMPK interaction assays, T cell subset analysis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — domain-specific knockout with defined biochemical target and selective cellular phenotype","pmids":["26496200"],"is_preprint":false},{"year":2015,"finding":"The solution structure of the CDE RNA shows a unique conformation that explains sequence-dependent recognition by Roquin; a CDE mutant with impaired Roquin binding adopts a distinct structure in both the loop and stem, revealing that Roquin recognizes CDE stem-loops in a sequence-specific manner through shape recognition.","method":"NMR solution structure determination of free and mutant CDE RNA","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 — NMR structure with functional validation explaining sequence specificity","pmids":["26165594"],"is_preprint":false},{"year":2015,"finding":"Crystal structure of human Roquin-2 ROQ domain shows a winged-helix fold with a flexible wing acting as a lid for CDE stem-loop RNA binding; CDE recognition is primarily through phosphate backbone and triloop contacts, with conserved features between Roquin-1 and Roquin-2 explaining functional redundancy.","method":"X-ray crystallography (apo and RNA-bound forms)","journal":"Acta crystallographica. Section F","confidence":"High","confidence_rationale":"Tier 1 — crystal structure in two forms with structural comparison","pmids":["26249698"],"is_preprint":false},{"year":2016,"finding":"Roquin-1 recognizes a novel U-rich hexaloop alternative decay element (ADE) in the Ox40 3' UTR via its ROQ domain; crystal structures and NMR data show ADE and CDE hexaloop motifs are bound with identical binding modes; ADE-like and CDE-like motifs cooperate in Ox40 repression in cells.","method":"SELEX, X-ray crystallography, NMR, cell-based repression assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus NMR plus functional cell assays, defines a second class of Roquin RNA target","pmids":["27010430"],"is_preprint":false},{"year":2017,"finding":"Roquin inhibits the PI3K-mTOR signaling pathway by upregulating PTEN through competition with miR-17~92 binding to the Pten 3' UTR, and by downregulating Foxo1-specific E3 ubiquitin ligase Itch; loss of Roquin enhances Akt-mTOR signaling and PI3K inhibition corrects enhanced Tfh/Th17 differentiation phenotypes.","method":"Conditional T cell-specific and Treg-specific knockout, miR-17~92 binding competition assays, PI3K/mTOR inhibitor rescue experiments","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — genetic knockout with pathway-level rescue by pharmacological inhibitors, multiple T cell subset phenotypes","pmids":["29246441"],"is_preprint":false},{"year":2017,"finding":"Drosophila Roquin and human Roquin-2 recruit the CCR4-NOT deadenylase complex through C-terminal regions containing a CAF40-binding motif (CBM); crystal structure of Dm Roquin CBM bound to CAF40 shows CBM adopts an α-helical conformation binding a conserved CAF40 surface.","method":"X-ray crystallography (CBM-CAF40 complex), Co-immunoprecipitation, binding assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — crystal structure of effector-complex interaction with biochemical validation","pmids":["28165457"],"is_preprint":false},{"year":2018,"finding":"Roquin interacts not only with stem-loop structures but also with a linear sequence element present in ~half of its targets; multiple stem-loops in the Nfkbid 3' UTR cooperate for robust repression requiring binding of multiple Roquin proteins; repression involves both deadenylation/decapping and translational inhibition depending on the number of binding sites.","method":"iCLIP, reporter assays, polysome profiling, decapping/deadenylation mutant rescue, PAR-CLIP","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 — comprehensive multi-method analysis with mechanistic dissection of regulatory modes","pmids":["30232334"],"is_preprint":false},{"year":2018,"finding":"NUFIP2 directly and with high affinity binds Roquin-1 and -2, is stabilized by this interaction, and acts as a cofactor for Roquin-induced mRNA decay; NUFIP2 cooperates with Roquin to bind tandem non-canonical stem-loops in the ICOS and Ox40 3' UTRs required for post-transcriptional repression.","method":"siRNA screen (~1500 genes), Co-IP, affinity binding assays, cooperative RNA-binding experiments, reporter assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — systematic screen plus direct binding assays plus functional validation","pmids":["29352114"],"is_preprint":false},{"year":2019,"finding":"A human homozygous nonsense R688* RC3H1 mutation causes hyperinflammation; R688* Roquin-1 fails to localize to P-bodies and fails to interact with the CCR4-NOT deadenylation complex, impairing mRNA decay of immune targets including ICOS, OX40, and TNF.","method":"Patient genetic analysis, P-body localization assay, CCR4-NOT co-immunoprecipitation, mRNA decay assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — human loss-of-function with mechanistic validation of P-body localization and CCR4-NOT interaction","pmids":["31636267"],"is_preprint":false},{"year":2019,"finding":"HCMV actively induces Roquin expression; Roquin binds IRF1 mRNA via CLIP and reduces IRF1 protein expression, thereby suppressing a broad antiviral gene expression program and enabling viral replication.","method":"CLIP-seq, loss-of-function screening, transcriptome profiling, siRNA knockdown","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — CLIP-seq identification of direct target with functional antiviral readout","pmids":["31451648"],"is_preprint":false},{"year":2020,"finding":"Roquin recognizes AU-rich elements (AREs) folded into CDE-like structures in a shape-specific manner via the ROQ domain; the same ARE sequences in the UCP3 3' UTR are also bound by AUF1 in their linear form, revealing context-dependent dual recognition of the same sequence.","method":"NMR spectroscopy (free and protein-bound RNA structures), SHAPE-seq, in-line probing, protein binding assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — NMR structures of free and bound RNA with complementary biochemical assays","pmids":["32491174"],"is_preprint":false},{"year":2021,"finding":"Roquin-1 physically interacts with Regnase-1; the sanroque residue M199 is part of the Roquin-1 interaction surface for Regnase-1; mutations disrupting this interaction induce Tfh cells, germinal center B cells, and autoantibody formation, establishing direct physical cooperation as mechanistically required.","method":"Co-immunoprecipitation, interaction surface mapping, conditional KO mice, autoimmune phenotyping","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP with domain mapping plus genetic rescue experiments","pmids":["34811541"],"is_preprint":false},{"year":2021,"finding":"Roquin (RC3H1) and its paralog are major mediators of iron-regulated transferrin receptor-1 (TfR1) mRNA degradation; Roquin destabilizes TfR1 mRNA through three hairpin loops in the TfR1 3' UTR that are essential for iron-regulated instability; this activity is translation-independent.","method":"Gene knockout (HAP1, HUVEC, L-M, MEF cells), siRNA knockdown, 3' UTR mutational analysis, mRNA decay assays","journal":"iScience","confidence":"High","confidence_rationale":"Tier 2 — multiple cell types, gene KO, 3' UTR functional dissection, translation independence demonstrated","pmids":["33898949"],"is_preprint":false},{"year":2021,"finding":"RC3H1 deficiency in osteoclasts post-transcriptionally derepresses transferrin receptor 1 (Tfr1) mRNA, increasing iron uptake and mitochondrial respiration, thereby promoting osteoclast activation and bone loss; Tfr1 inhibition rescues the Rc3h1-deficient osteoclast phenotype.","method":"Conditional osteoclast-specific knockout mice, bone mass measurement, mitochondrial function assays, Tfr1 inhibition rescue","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 2 — tissue-specific KO with rescue experiment and defined molecular target","pmids":["39659568"],"is_preprint":false},{"year":2022,"finding":"NMR analysis of the complete Ox40 3' UTR reveals two decay elements (CDE and ADE) that are bound by Roquin in a 2:1 stoichiometry via the bimodal ROQ domain; differential binding is observed depending on structural embedment of decay elements within the full UTR context.","method":"NMR spectroscopy, SAXS, In-line probing, SHAPE-seq on complete 3' UTR","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — comprehensive structural analysis of full 3' UTR with binding stoichiometry determination","pmids":["35357505"],"is_preprint":false},{"year":2023,"finding":"MALT1 cleavage of Roquin-1 at a specific site (rendering it insensitive to cleavage via Mins mutation) is the primary mechanism by which TCR signaling inactivates Roquin; graded TCR signal strength leads to proportionally more Roquin cleavage and differential derepression of target mRNAs, with high-affinity targets (like IκBNS) requiring stronger TCR signals for derepression; Mins mice are protected from EAE.","method":"Knock-in mice with cleavage-resistant Roquin-1 (Rc3h1Mins/Mins), genetic epistasis with constitutively active MALT1 (TBM), EAE model, Th17 differentiation assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — elegant genetic epistasis with knock-in alleles plus disease model, defines signal-strength gradient mechanism","pmids":["37988467"],"is_preprint":false},{"year":2024,"finding":"MALT1 inhibition upregulates RC3H1 (Roquin-1), which functions as an E3 ubiquitin ligase promoting ubiquitination-mediated degradation of GPX4; pharmacological MALT1 targeting thus triggers ferroptosis in liver cancer cells through the MALT1-RC3H1-GPX4 axis.","method":"FACS-based genome-wide CRISPR screen, rescue assays, ubiquitination assays, ferroptosis readouts","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — genome-wide CRISPR screen with mechanistic rescue experiments and ubiquitination assays","pmids":["39739814"],"is_preprint":false},{"year":2024,"finding":"NMR solution structure of the Roquin-1 CCCH-type zinc finger (ZnF) shows it recognizes AU-rich RNA sequences; the ZnF and ROQ domain simultaneously bind natural Roquin target transcripts, suggesting Roquin integrates RNA shape (via ROQ) and sequence features (via ZnF) through their tandem arrangement.","method":"NMR structure determination, RBNS-NMR pipeline for RNA motif preference, simultaneous binding experiments with natural targets","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — NMR structure with biochemical characterization of dual-domain RNA binding","pmids":["38953172"],"is_preprint":false},{"year":2025,"finding":"Single-molecule force spectroscopy combined with NMR reveals the two RNA-binding sites of Roquin's ROQ domain have opposing effects on target RNA stability: the A-site strongly stabilizes the folded CDE/ADE apical stem-loop, while the B-site destabilizes the central stem of the ADE through single-strand RNA binding, suggesting RNA motif nature and A/B-site cooperation encode target specificity.","method":"Single-molecule force spectroscopy (SMFS), NMR spectroscopy, integrated microfluidics for binding kinetics","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — novel single-molecule plus NMR approach revealing mechanistic detail of dual-site RNA recognition","pmids":["40203046"],"is_preprint":false},{"year":2021,"finding":"MNSFβ physically binds RC3H1 (Roquin-1) as shown by co-immunoprecipitation; MNSFβ promotes TNFα expression by interacting with RC3H1 and increasing RC3H1 localization to stress granules, reducing RC3H1's inhibitory effect on TNFα mRNA.","method":"Co-immunoprecipitation, stress granule localization assay, siRNA knockdown, TNFα measurement","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP with functional follow-up but single lab, mechanistic detail limited","pmids":["34589082"],"is_preprint":false},{"year":2024,"finding":"MNSFβ binds RC3H1 via its 81-326 aa region interacting with MNSFβ 101-133 aa region; MNSFβ promotes stress granule formation and RC3H1 recruitment to stress granules by interacting with RC3H1 and FMR1, sequestering RC3H1 away from TNFα mRNA and thus increasing TNFα expression.","method":"Co-immunoprecipitation, domain mapping, stress granule localization assay, peptide interference experiment, mRNA stability assay","journal":"International immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 3 — detailed domain mapping and localization data but single lab","pmids":["39260307"],"is_preprint":false},{"year":2009,"finding":"Roquin acts as an E3 ubiquitin ligase; mutation of Roquin causes ICOS-dependent gain of function where CD28-mediated immune responses (Tfh cell generation, germinal center formation, T helper cell responses) become dependent on ICOS instead, demonstrating that Roquin-mediated ICOS restriction is critical for maintaining functional compartmentalization of costimulatory signals.","method":"Genetic mouse model (sanroque), immune cell subset analysis, infection challenge experiments","journal":"Immunity","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis establishing pathway position but no direct biochemical mechanism","pmids":["19217324"],"is_preprint":false},{"year":2025,"finding":"Comprehensive sRBNS (structured RNA Bind-n-Seq) mapping defines Roquin ROQ domain binding preferences at nucleotide resolution: clear preference for specific loop sizes; extended consensus motifs for CDEs and ADEs identified; new target mRNAs predicted and verified in cells.","method":"sRBNS (high-throughput structured RNA library screen), affinity measurements, cell-based reporter assays","journal":"Angewandte Chemie","confidence":"Medium","confidence_rationale":"Tier 2 — systematic high-throughput binding assay with cell validation, single lab","pmids":["39344866"],"is_preprint":false}],"current_model":"RC3H1/Roquin-1 is an RNA-binding protein that post-transcriptionally represses proinflammatory target mRNAs (including ICOS, OX40, TNF-α, IL-6, A20, IRF1, and TfR1) by recognizing constitutive decay elements (CDEs) and alternative decay elements (ADEs) in their 3' UTRs via its winged-helix ROQ domain (A-site for stem-loops, B-site for dsRNA) and CCCH-type zinc finger (for AU-rich sequences), recruiting the CCR4-NOT deadenylase complex and mRNA decapping machinery (Rck, Edc4) at processing bodies and stress granules; it physically cooperates with Regnase-1 (which acts on translationally active mRNAs at ribosomes/ER), and both are inactivated by MALT1-mediated cleavage in a TCR signal-strength-graded manner, while its RING domain also exerts E3 ubiquitin ligase functions including antagonizing AMPK to promote Tfh differentiation and promoting GPX4 ubiquitination to regulate ferroptosis."},"narrative":{"teleology":[{"year":2007,"claim":"Establishing that Roquin-1 controls immune homeostasis by limiting ICOS mRNA stability through its ROQ domain answered the foundational question of why sanroque mice develop lupus-like autoimmunity.","evidence":"Genetic analysis of M199R sanroque mice with ICOS 3′ UTR functional dissection and mRNA decay assays","pmids":["18172933"],"confidence":"High","gaps":["Precise RNA element recognized by Roquin not yet defined","Effector machinery recruited by Roquin unknown","Whether Roquin directly binds ICOS mRNA not established"]},{"year":2010,"claim":"Demonstrating that Roquin directly binds ICOS mRNA at P-bodies and requires the decapping machinery (Rck, Edc4) but not the miRNA/RISC pathway defined Roquin as a miRNA-independent post-transcriptional repressor acting through mRNA decapping.","evidence":"Co-IP, fluorescence microscopy of P-body localization, RNA binding assays, Ago2/RISC decoupling in cell-based assays","pmids":["20639877","20412057"],"confidence":"High","gaps":["Identity of the cis-regulatory RNA element beyond the ICOS 3′ UTR region not resolved","Mechanism linking P-body localization to mRNA decay not defined"]},{"year":2013,"claim":"Identification of the constitutive decay element (CDE) stem-loop as a conserved Roquin-recognition motif in >50 mRNAs, and demonstration that Roquin recruits the CCR4-CAF1-NOT deadenylase complex, established the core effector mechanism and broadened the target repertoire beyond ICOS.","evidence":"RNA-protein binding assays, mRNA decay assays in macrophages, deadenylase recruitment assays, transcriptome-wide CDE identification; concurrent genetic KO studies showing Roquin-1/2 redundancy","pmids":["23663784","23583643"],"confidence":"High","gaps":["Structural basis of CDE recognition unknown","Whether Roquin recognizes non-CDE elements not addressed","Mechanism of CCR4-NOT recruitment not defined at atomic level"]},{"year":2014,"claim":"Crystal structures of the ROQ domain bound to CDE RNA revealed a winged-helix fold with two distinct RNA-binding surfaces (A-site for stem-loops, B-site for dsRNA), explaining how Roquin achieves broad yet specific target recognition.","evidence":"X-ray crystallography of mouse and human ROQ domains (multiple structures), NMR, mutagenesis with cell-based mRNA decay readouts","pmids":["25026077","25026078","25504471"],"confidence":"High","gaps":["Role of the B-site in vivo not established","Contribution of non-ROQ domains to target selection not defined","Stoichiometry of Roquin on natural 3′ UTRs unknown"]},{"year":2014,"claim":"Discovery that MALT1 protease cleaves both Roquin-1 and Regnase-1 upon TCR stimulation answered how antigen receptor signaling releases post-transcriptional repression of inflammatory mRNAs to permit T helper differentiation.","evidence":"Biochemical cleavage assays, T cell genetic models, mRNA reporter assays","pmids":["25282160"],"confidence":"High","gaps":["Whether cleavage is graded with signal strength not yet shown","Relative contributions of Roquin vs. Regnase-1 inactivation not dissected"]},{"year":2015,"claim":"Defining that Roquin and Regnase-1 repress overlapping mRNAs but in distinct subcellular compartments—Roquin on translationally inactive mRNAs at P-bodies/stress granules, Regnase-1 on ribosome-associated mRNAs—resolved how two seemingly redundant repressors achieve complementary surveillance.","evidence":"Subcellular fractionation, ribosome profiling, UPF1 requirement assays, mRNA decay assays","pmids":["26000482"],"confidence":"High","gaps":["How targets are partitioned between compartments not explained","Whether Roquin-Regnase-1 physical interaction is required for this compartmentalization unknown"]},{"year":2015,"claim":"PAR-CLIP revealed ~3,800 RC3H1 mRNA targets genome-wide including A20, and showed that both the ROQ and zinc finger domains contribute to RNA binding, while the RING domain was shown to antagonize AMPK to specifically promote Tfh differentiation, establishing a second, non-RNA-decay function for Roquin.","evidence":"PAR-CLIP transcriptome-wide, domain mutagenesis with NF-κB pathway assays; conditional RING-domain KO mice with AMPK interaction assays","pmids":["26170170","26496200"],"confidence":"High","gaps":["RING domain substrates beyond AMPK unknown","How RNA-binding and E3 ligase functions are coordinated unclear"]},{"year":2016,"claim":"Discovery of the alternative decay element (ADE) hexaloop as a second class of Roquin target motif, bound via the same ROQ domain interface as CDEs, expanded the Roquin target code and explained cooperative repression of Ox40 through tandem CDE+ADE elements.","evidence":"SELEX, X-ray crystallography of ROQ-ADE complex, NMR, cell-based repression assays","pmids":["27010430"],"confidence":"High","gaps":["Genome-wide prevalence of ADE motifs not mapped","How CDE and ADE elements cooperate quantitatively in full-length 3′ UTRs not defined"]},{"year":2017,"claim":"Structural characterization of how Roquin recruits the CCR4-NOT complex via a C-terminal CAF40-binding motif (CBM) provided the atomic-level mechanism for deadenylation-dependent mRNA decay.","evidence":"X-ray crystallography of Drosophila Roquin CBM–CAF40 complex, Co-IP, binding assays","pmids":["28165457"],"confidence":"High","gaps":["Whether human Roquin-1 uses an identical CBM interface not structurally confirmed","Contribution of CBM vs. other CCR4-NOT contacts not dissected in vivo"]},{"year":2018,"claim":"iCLIP and reporter studies showed that Roquin binds both structured and linear sequence elements, and that multiple Roquin molecules cooperatively occupy tandem sites on a single 3′ UTR to achieve both deadenylation/decapping and translational repression, revealing dose-dependent regulatory modes.","evidence":"iCLIP, polysome profiling, deadenylation/decapping mutant rescue assays","pmids":["30232334","29352114"],"confidence":"High","gaps":["Structural basis of NUFIP2 cofactor cooperation with Roquin not resolved","In vivo stoichiometry on endogenous targets not measured"]},{"year":2019,"claim":"A human homozygous R688* RC3H1 mutation causing hyperinflammation established that Roquin-1 is essential for immune homeostasis in humans, and that the C-terminal region is required for P-body localization and CCR4-NOT interaction.","evidence":"Patient genetic analysis, P-body localization assays, CCR4-NOT Co-IP, mRNA decay assays","pmids":["31636267"],"confidence":"High","gaps":["Additional human disease-causing alleles not reported","Whether Roquin-2 partially compensates in the patient not assessed"]},{"year":2021,"claim":"Mapping the direct Roquin-1–Regnase-1 physical interaction surface (including M199) demonstrated that their cooperation is not merely functional overlap but requires direct protein–protein contact, explaining why the sanroque mutation disrupts both partners' function.","evidence":"Co-IP, interaction surface mapping, conditional KO mice with autoimmune phenotyping","pmids":["34811541"],"confidence":"High","gaps":["Structural model of the Roquin–Regnase-1 complex not available","Whether interaction is RNA-dependent not resolved"]},{"year":2021,"claim":"Identification of transferrin receptor 1 (TfR1) mRNA as a Roquin target connected Roquin to iron homeostasis and metabolism beyond inflammation, with osteoclast-specific KO revealing bone loss due to increased iron uptake and mitochondrial respiration.","evidence":"Gene KO in multiple cell types, 3′ UTR mutational analysis, osteoclast-specific conditional KO with Tfr1 rescue","pmids":["33898949","39659568"],"confidence":"High","gaps":["Whether iron status feeds back to regulate Roquin expression unknown","Relative contribution of Roquin vs. IRP/IRE system to TfR1 regulation in vivo not quantified"]},{"year":2023,"claim":"Knock-in cleavage-resistant Roquin-1 (Mins) mice proved that MALT1-mediated cleavage is the dominant mechanism inactivating Roquin downstream of TCR, and that graded cleavage creates a signal-strength decoder for differential target mRNA derepression.","evidence":"Rc3h1Mins/Mins knock-in mice, genetic epistasis with constitutively active MALT1, EAE disease model, Th17 assays","pmids":["37988467"],"confidence":"High","gaps":["Whether other proteases contribute to Roquin inactivation in non-T cells not addressed","Quantitative relationship between cleavage fraction and individual target derepression not modeled"]},{"year":2024,"claim":"Discovery that Roquin-1 functions as an E3 ubiquitin ligase targeting GPX4 for degradation, triggered by MALT1 inhibition, connected Roquin to ferroptosis regulation in cancer and revealed a non-RNA-decay effector axis.","evidence":"Genome-wide CRISPR screen, ubiquitination assays, ferroptosis readouts in liver cancer cells","pmids":["39739814"],"confidence":"High","gaps":["Whether GPX4 ubiquitination depends on the RING domain specifically not shown","Breadth of E3 ligase substrates beyond AMPK and GPX4 unknown","In vivo relevance of the MALT1-Roquin-GPX4 axis in tumors not established"]},{"year":2024,"claim":"NMR structure of the CCCH zinc finger revealed it recognizes AU-rich linear RNA sequences, and simultaneous binding experiments showed that the ROQ and ZnF domains engage natural targets concurrently, establishing that Roquin integrates RNA shape and sequence through tandem domains.","evidence":"NMR structure, RBNS-NMR pipeline, simultaneous dual-domain binding on natural target RNAs","pmids":["38953172"],"confidence":"High","gaps":["Whether ZnF binding is required for in vivo target selection genome-wide not tested","Structural model of full-length Roquin on a natural 3′ UTR not available"]},{"year":2025,"claim":"Single-molecule force spectroscopy showed that the ROQ domain A-site stabilizes while the B-site destabilizes target RNA structure, revealing that the two binding sites have opposing mechanical effects that together encode target specificity.","evidence":"SMFS with integrated NMR and microfluidics on CDE/ADE RNA elements","pmids":["40203046"],"confidence":"High","gaps":["Whether opposing A/B-site effects operate on all target classes not tested","How these mechanical effects translate into differential decay kinetics in cells unknown"]},{"year":null,"claim":"Key unresolved questions include the full substrate range of Roquin's RING-domain E3 ligase activity, the structural basis of the Roquin–Regnase-1 complex, how Roquin's RNA-decay and ubiquitin-ligase functions are coordinated on the same protein, and whether Roquin plays roles in non-immune tissues beyond bone metabolism.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structure of the full-length Roquin–Regnase-1 complex","Complete E3 ligase substrate repertoire undefined","Coordination between RNA-decay and ubiquitin-ligase activities unknown","Non-immune physiological roles largely unexplored"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,1,2,3,5,6,10,15,18,22,29]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[28,33]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[3,9,18,24]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[12,16]}],"localization":[{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[1,2,4,20,31,32]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[9]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,4,7,12,16,27]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[10,16,12]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[28]}],"complexes":["CCR4-NOT deadenylase complex (recruited effector)"],"partners":["RC3H2","ZC3H12A","NUFIP2","CNOT9","MALT1","PRKAA1","GPX4","AGO2"],"other_free_text":[]},"mechanistic_narrative":"RC3H1 (Roquin-1) is an RNA-binding E3 ubiquitin ligase that post-transcriptionally represses proinflammatory and costimulatory mRNAs—including ICOS, OX40, TNF-α, IL-6, TfR1, and IRF1—by recognizing constitutive and alternative decay element (CDE/ADE) stem-loops and AU-rich sequences in 3′ UTRs via its bimodal ROQ domain (A-site for apical stem-loops, B-site for double-stranded RNA) and CCCH-type zinc finger, then recruiting the CCR4-NOT deadenylase complex and decapping machinery at P-bodies to trigger mRNA degradation [PMID:18172933, PMID:23663784, PMID:25026078, PMID:38953172, PMID:27010430]. Roquin-1 physically cooperates with Regnase-1 on overlapping target mRNAs but acts on translationally inactive transcripts at P-bodies and stress granules, whereas Regnase-1 degrades ribosome-associated mRNAs; both are inactivated by MALT1-mediated proteolytic cleavage in a TCR signal-strength-graded manner that tunes T helper cell differentiation [PMID:26000482, PMID:25282160, PMID:37988467, PMID:34811541]. The RING domain confers E3 ubiquitin ligase activity that antagonizes AMPK to promote T follicular helper cell differentiation and mediates GPX4 ubiquitination to regulate ferroptosis [PMID:26496200, PMID:39739814]. A homozygous human loss-of-function RC3H1 mutation (R688*) causes hyperinflammation due to impaired P-body localization and CCR4-NOT recruitment [PMID:31636267]."},"prefetch_data":{"uniprot":{"accession":"Q5TC82","full_name":"Roquin-1","aliases":["RING finger and C3H zinc finger protein 1","RING finger and CCCH-type zinc finger domain-containing protein 1","RING finger protein 198"],"length_aa":1133,"mass_kda":125.7,"function":"Post-transcriptional repressor of mRNAs containing a conserved stem loop motif, called constitutive decay element (CDE), which is often located in the 3'-UTR, as in HMGXB3, ICOS, IER3, NFKBID, NFKBIZ, PPP1R10, TNF, TNFRSF4 and in many more mRNAs (PubMed:25026078, PubMed:31636267). Cleaves translationally inactive mRNAs harboring a stem-loop (SL), often located in their 3'-UTRs, during the early phase of inflammation in a helicase UPF1-independent manner (By similarity). Binds to CDE and promotes mRNA deadenylation and degradation. This process does not involve miRNAs (By similarity). In follicular helper T (Tfh) cells, represses of ICOS and TNFRSF4 expression, thus preventing spontaneous Tfh cell differentiation, germinal center B-cell differentiation in the absence of immunization and autoimmunity (By similarity). In resting or LPS-stimulated macrophages, controls inflammation by suppressing TNF expression (By similarity). Also recognizes CDE in its own mRNA and in that of paralogous RC3H2, possibly leading to feedback loop regulation (By similarity). Recognizes and binds mRNAs containing a hexaloop stem-loop motif, called alternative decay element (ADE) (By similarity). Together with ZC3H12A, destabilizes TNFRSF4/OX40 mRNA by binding to the conserved stem loop structure in its 3'UTR (By similarity). Able to interact with double-stranded RNA (dsRNA) (PubMed:25026078, PubMed:25504471). miRNA-binding protein that regulates microRNA homeostasis. Enhances DICER-mediated processing of pre-MIR146a but reduces mature MIR146a levels through an increase of 3' end uridylation. Both inhibits ICOS mRNA expression and they may act together to exert the suppression (PubMed:25697406, PubMed:31636267). Acts as a ubiquitin E3 ligase. Pairs with E2 enzymes UBE2A, UBE2B, UBE2D2, UBE2F, UBE2G1, UBE2G2 and UBE2L3 and produces polyubiquitin chains (PubMed:26489670). Shows the strongest activity when paired with UBE2N:UBE2V1 or UBE2N:UBE2V2 E2 complexes and generate both short and long polyubiquitin chains (PubMed:26489670)","subcellular_location":"Cytoplasm, P-body; Cytoplasmic granule","url":"https://www.uniprot.org/uniprotkb/Q5TC82/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RC3H1","classification":"Not Classified","n_dependent_lines":60,"n_total_lines":1208,"dependency_fraction":0.04966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RC3H1","total_profiled":1310},"omim":[{"mim_id":"618998","title":"IMMUNE DYSREGULATION AND SYSTEMIC HYPERINFLAMMATION SYNDROME; IMDYSHI","url":"https://www.omim.org/entry/618998"},{"mim_id":"617026","title":"PONTOCEREBELLAR HYPOPLASIA, TYPE 2F; PCH2F","url":"https://www.omim.org/entry/617026"},{"mim_id":"615231","title":"RING FINGER AND CCCH-TYPE ZINC FINGER DOMAINS-CONTAINING PROTEIN 2; RC3H2","url":"https://www.omim.org/entry/615231"},{"mim_id":"615001","title":"ZINC FINGER CCCH DOMAIN-CONTAINING PROTEIN 12C; ZC3H12C","url":"https://www.omim.org/entry/615001"},{"mim_id":"611106","title":"ZINC FINGER CCCH DOMAIN-CONTAINING PROTEIN 12D; ZC3H12D","url":"https://www.omim.org/entry/611106"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Golgi apparatus","reliability":"Additional"},{"location":"Plasma membrane","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RC3H1"},"hgnc":{"alias_symbol":["KIAA2025","roquin","RP5-1198E17.5","RNF198"],"prev_symbol":[]},"alphafold":{"accession":"Q5TC82","domains":[{"cath_id":"1.20.120.1790","chopping":"176-269","consensus_level":"medium","plddt":96.5719,"start":176,"end":269},{"cath_id":"-","chopping":"421-449","consensus_level":"high","plddt":85.8003,"start":421,"end":449}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5TC82","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5TC82-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5TC82-F1-predicted_aligned_error_v6.png","plddt_mean":61.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RC3H1","jax_strain_url":"https://www.jax.org/strain/search?query=RC3H1"},"sequence":{"accession":"Q5TC82","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5TC82.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5TC82/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5TC82"}},"corpus_meta":[{"pmid":"18172933","id":"PMC_18172933","title":"Roquin 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cell death by ubiquitination-dependent degradation of TAK1.","date":"2024","source":"Free radical biology & medicine","url":"https://pubmed.ncbi.nlm.nih.gov/38729452","citation_count":4,"is_preprint":false},{"pmid":"25201726","id":"PMC_25201726","title":"Over-expression of Roquin aggravates T cell mediated hepatitis in transgenic mice using T cell specific promoter.","date":"2014","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/25201726","citation_count":4,"is_preprint":false},{"pmid":"31745085","id":"PMC_31745085","title":"Author Correction: A human immune dysregulation syndrome characterized by severe hyperinflammation with a homozygous nonsense Roquin-1 mutation.","date":"2019","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/31745085","citation_count":4,"is_preprint":false},{"pmid":"33509784","id":"PMC_33509784","title":"RNA-binding protein Roquin negatively regulates STING-dependent innate immune response in Drosophila.","date":"2020","source":"Yi chuan = Hereditas","url":"https://pubmed.ncbi.nlm.nih.gov/33509784","citation_count":3,"is_preprint":false},{"pmid":"40261659","id":"PMC_40261659","title":"Enhanced Therapeutic Effect of IL-10-ADSCs on Rabbit Autoimmune Dacryoadenitis By Suppressing T Follicular Helper Cell Responses Via miR-142-5p/RC3H1 Axis.","date":"2025","source":"Investigative ophthalmology & visual science","url":"https://pubmed.ncbi.nlm.nih.gov/40261659","citation_count":3,"is_preprint":false},{"pmid":"35819231","id":"PMC_35819231","title":"The silencing of ets-4 mRNA relies on the functional cooperation between REGE-1/Regnase-1 and RLE-1/Roquin-1.","date":"2022","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/35819231","citation_count":3,"is_preprint":false},{"pmid":"40097661","id":"PMC_40097661","title":"Roquin-1 interaction with Regnase-1 inhibits the progression of rheumatoid arthritis via suppressing FGF2 expression and NF-κB pathway.","date":"2025","source":"Inflammation research : official journal of the European Histamine Research Society ... 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alternative decay cis element into a high-affinity target for the immunomodulatory protein Roquin.","date":"2025","source":"RNA biology","url":"https://pubmed.ncbi.nlm.nih.gov/39801464","citation_count":1,"is_preprint":false},{"pmid":"40769319","id":"PMC_40769319","title":"Autosomal-dominant Roquin-1 immunodeficiency and hyperinflammation.","date":"2025","source":"The Journal of allergy and clinical immunology","url":"https://pubmed.ncbi.nlm.nih.gov/40769319","citation_count":0,"is_preprint":false},{"pmid":"40203046","id":"PMC_40203046","title":"Roquin exhibits opposing effects on RNA stem-loop stability through its two ROQ domain binding sites.","date":"2025","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/40203046","citation_count":0,"is_preprint":false},{"pmid":"41416953","id":"PMC_41416953","title":"Serous Cavity Mast Cells Depend on the ROQUIN Paralogs.","date":"2025","source":"European journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/41416953","citation_count":0,"is_preprint":false},{"pmid":"41943150","id":"PMC_41943150","title":"miR-3613-3p Is a New Diagnostic Indicator of Acute Coronary Syndrome (ACS) That Reduces Endothelial Damage by Targeting Ring Finger and CCCH-Type Domains 1 (RC3H1).","date":"2026","source":"Journal of biochemical and molecular toxicology","url":"https://pubmed.ncbi.nlm.nih.gov/41943150","citation_count":0,"is_preprint":false},{"pmid":"41294801","id":"PMC_41294801","title":"Roquin Modulates Cardiac Post-Infarct Remodeling via microRNA Stability Control.","date":"2025","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/41294801","citation_count":0,"is_preprint":false},{"pmid":"40287742","id":"PMC_40287742","title":"Identification of RC3H1 as antiviral host factor binding to the non-structural protein 1 of Influenza A virus via a 3-stage computational pipeline and cell-based analysis.","date":"2025","source":"Virology 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myeloma","date":"2024-11-19","source":"bioRxiv","url":"https://doi.org/10.1101/2024.11.19.624352","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":39804,"output_tokens":7742,"usd":0.117771},"stage2":{"model":"claude-opus-4-6","input_tokens":11579,"output_tokens":8884,"usd":0.419992},"total_usd":0.537763,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"Roquin (RC3H1) limits ICOS expression by promoting degradation of ICOS mRNA; a conserved 47-bp segment in the ICOS 3' UTR is essential for Roquin-mediated regulation, and the M199R mutation in the ROQ domain abolishes this activity in vivo.\",\n      \"method\": \"Genetic mouse model (sanroque M199R mutation), mRNA decay assays, 3' UTR functional analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — foundational genetic epistasis with defined molecular target, replicated extensively across labs\",\n      \"pmids\": [\"18172933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Roquin localizes to processing (P) bodies and directly binds ICOS mRNA via its 3' UTR; repression of ICOS requires the RNA helicase Rck and the decapping enhancer Edc4, and depends on P-body localization sequences in Roquin, but is independent of microRNAs or RISC.\",\n      \"method\": \"Co-immunoprecipitation, fluorescence microscopy (P-body localization), RNA binding assays, microRNA-independence demonstrated by Ago2/RISC decoupling\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, direct RNA binding, subcellular localization linked to function, independence from miRNA validated\",\n      \"pmids\": [\"20639877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The ROQ domain of Roquin is necessary and sufficient for localization to stress granules and for triggering ICOS mRNA decay; gel-shift, SPR and footprinting show the ROQ domain binds RNA from the ICOS 3' UTR adjacent to the miR-101 complementarity site.\",\n      \"method\": \"Fluorescence microscopy, gel-shift, SPR, RNA footprinting, domain deletion analysis\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal biochemical methods defining domain function and RNA binding\",\n      \"pmids\": [\"20412057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Roquin recognizes a constitutive decay element (CDE) stem-loop in the TNF-α 3' UTR and promotes mRNA degradation by recruiting the Ccr4-Caf1-Not deadenylase complex; CDE motifs are conserved in >50 vertebrate mRNAs encoding regulators of development and inflammation.\",\n      \"method\": \"RNA-protein binding assays, mRNA decay assays in macrophages, transcriptome-wide target identification, deadenylase complex recruitment assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mechanistic reconstitution of deadenylase recruitment, genome-wide target validation, replicated\",\n      \"pmids\": [\"23663784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Roquin-1 and Roquin-2 redundantly repress ICOS and OX40 costimulatory receptor mRNAs in T cells; the RING domain of Roquin-1 is required for stress granule localization, and its absence allows Roquin-2 to compensate, whereas the ROQ mutation disrupts mRNA regulation while preserving stress granule occupancy.\",\n      \"method\": \"Conditional gene knockout, stress granule localization (fluorescence microscopy), mRNA reporter assays, bone marrow chimeras\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with domain-specific functional readouts, two independent papers simultaneously\",\n      \"pmids\": [\"23583643\", \"23583642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of the mouse Roquin ROQ domain in complex with CDE RNA reveals an extended winged-helix fold; the ROQ domain recognizes the RNA stem and triloop primarily through non-sequence-specific contacts, explaining broad target spectrum; cell-based assays confirm ROQ domain is sufficient for CDE-regulated mRNA decay.\",\n      \"method\": \"X-ray crystallography, NMR, binding experiments, cell-based mRNA decay assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional validation in cells\",\n      \"pmids\": [\"25026077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structures of human Roquin ROQ domain reveal two distinct RNA-binding sites: the A-site binds stem-loop CDE RNA and the B-site binds double-stranded RNA at a separate interface; mutagenesis of either site disrupts Roquin-mediated degradation of HMGXB3 and IL6 mRNAs in human cells.\",\n      \"method\": \"X-ray crystallography (two crystal structures), site-directed mutagenesis, mRNA decay assays in human cells\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — two crystal structures plus mutagenesis with functional readout in cells\",\n      \"pmids\": [\"25026078\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Upon TCR antigen recognition, the paracaspase MALT1 cleaves both Roquin-1 and Regnase-1; this cleavage releases cooperative post-transcriptional repression of IL-6, ICOS, c-Rel, IRF4, IκBNS, and IκBζ mRNAs, thereby promoting TH17 differentiation in a signal-strength-dependent manner.\",\n      \"method\": \"Biochemical cleavage assays, T cell genetic models, mRNA reporter assays, MALT1 activity assays\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct biochemical demonstration of MALT1-mediated cleavage with defined functional consequences, replicated\",\n      \"pmids\": [\"25282160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of the Roquin ROQ domain shows a winged helix-turn-helix (wHTH) motif involved in CDE stem-loop RNA binding; biochemical and mutational analyses show Roquin can bind duplex RNA and function in different oligomeric states.\",\n      \"method\": \"X-ray crystallography, biochemical binding assays, mutagenesis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with biochemical and mutational validation\",\n      \"pmids\": [\"25504471\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Regnase-1 and Roquin regulate an overlapping set of inflammatory mRNAs via a common stem-loop structure but act in distinct subcellular compartments: Regnase-1 degrades translationally active mRNAs at ribosomes/ER and requires UPF1 helicase activity, while Roquin acts on translationally inactive mRNAs at P-bodies/stress granules independently of UPF1.\",\n      \"method\": \"Subcellular fractionation, ribosome profiling, siRNA knockdown, mRNA decay assays, UPF1 requirement assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods defining spatiotemporally distinct mechanisms, high-impact journal, widely cited\",\n      \"pmids\": [\"26000482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PAR-CLIP of human RC3H1 identifies ~3,800 mRNA targets with >16,000 binding sites; RC3H1 uses ROQ and Zn-finger domains to contact a binding site in the A20 3' UTR; knockdown of RC3H1 increases A20 protein, thereby reducing IKK and NF-κB activities.\",\n      \"method\": \"PAR-CLIP (transcriptome-wide), domain deletion/mutagenesis, NF-κB pathway assays, siRNA knockdown\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — genome-wide binding map plus domain-specific binding and signaling pathway readout\",\n      \"pmids\": [\"26170170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Roquin binds Argonaute2 directly and enhances Dicer-mediated processing of pre-miR-146a; in the absence of functional Roquin, mature miR-146a accumulates due to decreased 3'-end uridylation; crystallographic studies reveal a unique HEPN domain in Roquin and define the structural basis for the sanroque mutation.\",\n      \"method\": \"Co-immunoprecipitation (Roquin-Ago2, Roquin-miR-146a), miRNA processing assays, 3'-end uridylation analysis, X-ray crystallography\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — crystal structure plus multiple orthogonal binding and functional assays\",\n      \"pmids\": [\"25697406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The RING domain of Roquin-1 directly antagonizes the catalytic α1 subunit of AMPK, promoting Tfh cell differentiation; T cell-specific deletion of the RING domain selectively abolishes antigen-specific Tfh responses without affecting Th1, Th2, Th17, or Treg populations.\",\n      \"method\": \"Conditional RING domain knockout mice, biochemical AMPK interaction assays, T cell subset analysis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain-specific knockout with defined biochemical target and selective cellular phenotype\",\n      \"pmids\": [\"26496200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The solution structure of the CDE RNA shows a unique conformation that explains sequence-dependent recognition by Roquin; a CDE mutant with impaired Roquin binding adopts a distinct structure in both the loop and stem, revealing that Roquin recognizes CDE stem-loops in a sequence-specific manner through shape recognition.\",\n      \"method\": \"NMR solution structure determination of free and mutant CDE RNA\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with functional validation explaining sequence specificity\",\n      \"pmids\": [\"26165594\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structure of human Roquin-2 ROQ domain shows a winged-helix fold with a flexible wing acting as a lid for CDE stem-loop RNA binding; CDE recognition is primarily through phosphate backbone and triloop contacts, with conserved features between Roquin-1 and Roquin-2 explaining functional redundancy.\",\n      \"method\": \"X-ray crystallography (apo and RNA-bound forms)\",\n      \"journal\": \"Acta crystallographica. Section F\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure in two forms with structural comparison\",\n      \"pmids\": [\"26249698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Roquin-1 recognizes a novel U-rich hexaloop alternative decay element (ADE) in the Ox40 3' UTR via its ROQ domain; crystal structures and NMR data show ADE and CDE hexaloop motifs are bound with identical binding modes; ADE-like and CDE-like motifs cooperate in Ox40 repression in cells.\",\n      \"method\": \"SELEX, X-ray crystallography, NMR, cell-based repression assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus NMR plus functional cell assays, defines a second class of Roquin RNA target\",\n      \"pmids\": [\"27010430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Roquin inhibits the PI3K-mTOR signaling pathway by upregulating PTEN through competition with miR-17~92 binding to the Pten 3' UTR, and by downregulating Foxo1-specific E3 ubiquitin ligase Itch; loss of Roquin enhances Akt-mTOR signaling and PI3K inhibition corrects enhanced Tfh/Th17 differentiation phenotypes.\",\n      \"method\": \"Conditional T cell-specific and Treg-specific knockout, miR-17~92 binding competition assays, PI3K/mTOR inhibitor rescue experiments\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic knockout with pathway-level rescue by pharmacological inhibitors, multiple T cell subset phenotypes\",\n      \"pmids\": [\"29246441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Drosophila Roquin and human Roquin-2 recruit the CCR4-NOT deadenylase complex through C-terminal regions containing a CAF40-binding motif (CBM); crystal structure of Dm Roquin CBM bound to CAF40 shows CBM adopts an α-helical conformation binding a conserved CAF40 surface.\",\n      \"method\": \"X-ray crystallography (CBM-CAF40 complex), Co-immunoprecipitation, binding assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure of effector-complex interaction with biochemical validation\",\n      \"pmids\": [\"28165457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Roquin interacts not only with stem-loop structures but also with a linear sequence element present in ~half of its targets; multiple stem-loops in the Nfkbid 3' UTR cooperate for robust repression requiring binding of multiple Roquin proteins; repression involves both deadenylation/decapping and translational inhibition depending on the number of binding sites.\",\n      \"method\": \"iCLIP, reporter assays, polysome profiling, decapping/deadenylation mutant rescue, PAR-CLIP\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — comprehensive multi-method analysis with mechanistic dissection of regulatory modes\",\n      \"pmids\": [\"30232334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"NUFIP2 directly and with high affinity binds Roquin-1 and -2, is stabilized by this interaction, and acts as a cofactor for Roquin-induced mRNA decay; NUFIP2 cooperates with Roquin to bind tandem non-canonical stem-loops in the ICOS and Ox40 3' UTRs required for post-transcriptional repression.\",\n      \"method\": \"siRNA screen (~1500 genes), Co-IP, affinity binding assays, cooperative RNA-binding experiments, reporter assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic screen plus direct binding assays plus functional validation\",\n      \"pmids\": [\"29352114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"A human homozygous nonsense R688* RC3H1 mutation causes hyperinflammation; R688* Roquin-1 fails to localize to P-bodies and fails to interact with the CCR4-NOT deadenylation complex, impairing mRNA decay of immune targets including ICOS, OX40, and TNF.\",\n      \"method\": \"Patient genetic analysis, P-body localization assay, CCR4-NOT co-immunoprecipitation, mRNA decay assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — human loss-of-function with mechanistic validation of P-body localization and CCR4-NOT interaction\",\n      \"pmids\": [\"31636267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HCMV actively induces Roquin expression; Roquin binds IRF1 mRNA via CLIP and reduces IRF1 protein expression, thereby suppressing a broad antiviral gene expression program and enabling viral replication.\",\n      \"method\": \"CLIP-seq, loss-of-function screening, transcriptome profiling, siRNA knockdown\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — CLIP-seq identification of direct target with functional antiviral readout\",\n      \"pmids\": [\"31451648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Roquin recognizes AU-rich elements (AREs) folded into CDE-like structures in a shape-specific manner via the ROQ domain; the same ARE sequences in the UCP3 3' UTR are also bound by AUF1 in their linear form, revealing context-dependent dual recognition of the same sequence.\",\n      \"method\": \"NMR spectroscopy (free and protein-bound RNA structures), SHAPE-seq, in-line probing, protein binding assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structures of free and bound RNA with complementary biochemical assays\",\n      \"pmids\": [\"32491174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Roquin-1 physically interacts with Regnase-1; the sanroque residue M199 is part of the Roquin-1 interaction surface for Regnase-1; mutations disrupting this interaction induce Tfh cells, germinal center B cells, and autoantibody formation, establishing direct physical cooperation as mechanistically required.\",\n      \"method\": \"Co-immunoprecipitation, interaction surface mapping, conditional KO mice, autoimmune phenotyping\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with domain mapping plus genetic rescue experiments\",\n      \"pmids\": [\"34811541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Roquin (RC3H1) and its paralog are major mediators of iron-regulated transferrin receptor-1 (TfR1) mRNA degradation; Roquin destabilizes TfR1 mRNA through three hairpin loops in the TfR1 3' UTR that are essential for iron-regulated instability; this activity is translation-independent.\",\n      \"method\": \"Gene knockout (HAP1, HUVEC, L-M, MEF cells), siRNA knockdown, 3' UTR mutational analysis, mRNA decay assays\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple cell types, gene KO, 3' UTR functional dissection, translation independence demonstrated\",\n      \"pmids\": [\"33898949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RC3H1 deficiency in osteoclasts post-transcriptionally derepresses transferrin receptor 1 (Tfr1) mRNA, increasing iron uptake and mitochondrial respiration, thereby promoting osteoclast activation and bone loss; Tfr1 inhibition rescues the Rc3h1-deficient osteoclast phenotype.\",\n      \"method\": \"Conditional osteoclast-specific knockout mice, bone mass measurement, mitochondrial function assays, Tfr1 inhibition rescue\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — tissue-specific KO with rescue experiment and defined molecular target\",\n      \"pmids\": [\"39659568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NMR analysis of the complete Ox40 3' UTR reveals two decay elements (CDE and ADE) that are bound by Roquin in a 2:1 stoichiometry via the bimodal ROQ domain; differential binding is observed depending on structural embedment of decay elements within the full UTR context.\",\n      \"method\": \"NMR spectroscopy, SAXS, In-line probing, SHAPE-seq on complete 3' UTR\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — comprehensive structural analysis of full 3' UTR with binding stoichiometry determination\",\n      \"pmids\": [\"35357505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MALT1 cleavage of Roquin-1 at a specific site (rendering it insensitive to cleavage via Mins mutation) is the primary mechanism by which TCR signaling inactivates Roquin; graded TCR signal strength leads to proportionally more Roquin cleavage and differential derepression of target mRNAs, with high-affinity targets (like IκBNS) requiring stronger TCR signals for derepression; Mins mice are protected from EAE.\",\n      \"method\": \"Knock-in mice with cleavage-resistant Roquin-1 (Rc3h1Mins/Mins), genetic epistasis with constitutively active MALT1 (TBM), EAE model, Th17 differentiation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — elegant genetic epistasis with knock-in alleles plus disease model, defines signal-strength gradient mechanism\",\n      \"pmids\": [\"37988467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MALT1 inhibition upregulates RC3H1 (Roquin-1), which functions as an E3 ubiquitin ligase promoting ubiquitination-mediated degradation of GPX4; pharmacological MALT1 targeting thus triggers ferroptosis in liver cancer cells through the MALT1-RC3H1-GPX4 axis.\",\n      \"method\": \"FACS-based genome-wide CRISPR screen, rescue assays, ubiquitination assays, ferroptosis readouts\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide CRISPR screen with mechanistic rescue experiments and ubiquitination assays\",\n      \"pmids\": [\"39739814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NMR solution structure of the Roquin-1 CCCH-type zinc finger (ZnF) shows it recognizes AU-rich RNA sequences; the ZnF and ROQ domain simultaneously bind natural Roquin target transcripts, suggesting Roquin integrates RNA shape (via ROQ) and sequence features (via ZnF) through their tandem arrangement.\",\n      \"method\": \"NMR structure determination, RBNS-NMR pipeline for RNA motif preference, simultaneous binding experiments with natural targets\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with biochemical characterization of dual-domain RNA binding\",\n      \"pmids\": [\"38953172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Single-molecule force spectroscopy combined with NMR reveals the two RNA-binding sites of Roquin's ROQ domain have opposing effects on target RNA stability: the A-site strongly stabilizes the folded CDE/ADE apical stem-loop, while the B-site destabilizes the central stem of the ADE through single-strand RNA binding, suggesting RNA motif nature and A/B-site cooperation encode target specificity.\",\n      \"method\": \"Single-molecule force spectroscopy (SMFS), NMR spectroscopy, integrated microfluidics for binding kinetics\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — novel single-molecule plus NMR approach revealing mechanistic detail of dual-site RNA recognition\",\n      \"pmids\": [\"40203046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MNSFβ physically binds RC3H1 (Roquin-1) as shown by co-immunoprecipitation; MNSFβ promotes TNFα expression by interacting with RC3H1 and increasing RC3H1 localization to stress granules, reducing RC3H1's inhibitory effect on TNFα mRNA.\",\n      \"method\": \"Co-immunoprecipitation, stress granule localization assay, siRNA knockdown, TNFα measurement\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP with functional follow-up but single lab, mechanistic detail limited\",\n      \"pmids\": [\"34589082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MNSFβ binds RC3H1 via its 81-326 aa region interacting with MNSFβ 101-133 aa region; MNSFβ promotes stress granule formation and RC3H1 recruitment to stress granules by interacting with RC3H1 and FMR1, sequestering RC3H1 away from TNFα mRNA and thus increasing TNFα expression.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, stress granule localization assay, peptide interference experiment, mRNA stability assay\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — detailed domain mapping and localization data but single lab\",\n      \"pmids\": [\"39260307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Roquin acts as an E3 ubiquitin ligase; mutation of Roquin causes ICOS-dependent gain of function where CD28-mediated immune responses (Tfh cell generation, germinal center formation, T helper cell responses) become dependent on ICOS instead, demonstrating that Roquin-mediated ICOS restriction is critical for maintaining functional compartmentalization of costimulatory signals.\",\n      \"method\": \"Genetic mouse model (sanroque), immune cell subset analysis, infection challenge experiments\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis establishing pathway position but no direct biochemical mechanism\",\n      \"pmids\": [\"19217324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Comprehensive sRBNS (structured RNA Bind-n-Seq) mapping defines Roquin ROQ domain binding preferences at nucleotide resolution: clear preference for specific loop sizes; extended consensus motifs for CDEs and ADEs identified; new target mRNAs predicted and verified in cells.\",\n      \"method\": \"sRBNS (high-throughput structured RNA library screen), affinity measurements, cell-based reporter assays\",\n      \"journal\": \"Angewandte Chemie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic high-throughput binding assay with cell validation, single lab\",\n      \"pmids\": [\"39344866\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RC3H1/Roquin-1 is an RNA-binding protein that post-transcriptionally represses proinflammatory target mRNAs (including ICOS, OX40, TNF-α, IL-6, A20, IRF1, and TfR1) by recognizing constitutive decay elements (CDEs) and alternative decay elements (ADEs) in their 3' UTRs via its winged-helix ROQ domain (A-site for stem-loops, B-site for dsRNA) and CCCH-type zinc finger (for AU-rich sequences), recruiting the CCR4-NOT deadenylase complex and mRNA decapping machinery (Rck, Edc4) at processing bodies and stress granules; it physically cooperates with Regnase-1 (which acts on translationally active mRNAs at ribosomes/ER), and both are inactivated by MALT1-mediated cleavage in a TCR signal-strength-graded manner, while its RING domain also exerts E3 ubiquitin ligase functions including antagonizing AMPK to promote Tfh differentiation and promoting GPX4 ubiquitination to regulate ferroptosis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RC3H1 (Roquin-1) is an RNA-binding E3 ubiquitin ligase that post-transcriptionally represses proinflammatory and costimulatory mRNAs—including ICOS, OX40, TNF-α, IL-6, TfR1, and IRF1—by recognizing constitutive and alternative decay element (CDE/ADE) stem-loops and AU-rich sequences in 3′ UTRs via its bimodal ROQ domain (A-site for apical stem-loops, B-site for double-stranded RNA) and CCCH-type zinc finger, then recruiting the CCR4-NOT deadenylase complex and decapping machinery at P-bodies to trigger mRNA degradation [PMID:18172933, PMID:23663784, PMID:25026078, PMID:38953172, PMID:27010430]. Roquin-1 physically cooperates with Regnase-1 on overlapping target mRNAs but acts on translationally inactive transcripts at P-bodies and stress granules, whereas Regnase-1 degrades ribosome-associated mRNAs; both are inactivated by MALT1-mediated proteolytic cleavage in a TCR signal-strength-graded manner that tunes T helper cell differentiation [PMID:26000482, PMID:25282160, PMID:37988467, PMID:34811541]. The RING domain confers E3 ubiquitin ligase activity that antagonizes AMPK to promote T follicular helper cell differentiation and mediates GPX4 ubiquitination to regulate ferroptosis [PMID:26496200, PMID:39739814]. A homozygous human loss-of-function RC3H1 mutation (R688*) causes hyperinflammation due to impaired P-body localization and CCR4-NOT recruitment [PMID:31636267].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Establishing that Roquin-1 controls immune homeostasis by limiting ICOS mRNA stability through its ROQ domain answered the foundational question of why sanroque mice develop lupus-like autoimmunity.\",\n      \"evidence\": \"Genetic analysis of M199R sanroque mice with ICOS 3′ UTR functional dissection and mRNA decay assays\",\n      \"pmids\": [\"18172933\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise RNA element recognized by Roquin not yet defined\", \"Effector machinery recruited by Roquin unknown\", \"Whether Roquin directly binds ICOS mRNA not established\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrating that Roquin directly binds ICOS mRNA at P-bodies and requires the decapping machinery (Rck, Edc4) but not the miRNA/RISC pathway defined Roquin as a miRNA-independent post-transcriptional repressor acting through mRNA decapping.\",\n      \"evidence\": \"Co-IP, fluorescence microscopy of P-body localization, RNA binding assays, Ago2/RISC decoupling in cell-based assays\",\n      \"pmids\": [\"20639877\", \"20412057\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the cis-regulatory RNA element beyond the ICOS 3′ UTR region not resolved\", \"Mechanism linking P-body localization to mRNA decay not defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identification of the constitutive decay element (CDE) stem-loop as a conserved Roquin-recognition motif in >50 mRNAs, and demonstration that Roquin recruits the CCR4-CAF1-NOT deadenylase complex, established the core effector mechanism and broadened the target repertoire beyond ICOS.\",\n      \"evidence\": \"RNA-protein binding assays, mRNA decay assays in macrophages, deadenylase recruitment assays, transcriptome-wide CDE identification; concurrent genetic KO studies showing Roquin-1/2 redundancy\",\n      \"pmids\": [\"23663784\", \"23583643\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of CDE recognition unknown\", \"Whether Roquin recognizes non-CDE elements not addressed\", \"Mechanism of CCR4-NOT recruitment not defined at atomic level\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Crystal structures of the ROQ domain bound to CDE RNA revealed a winged-helix fold with two distinct RNA-binding surfaces (A-site for stem-loops, B-site for dsRNA), explaining how Roquin achieves broad yet specific target recognition.\",\n      \"evidence\": \"X-ray crystallography of mouse and human ROQ domains (multiple structures), NMR, mutagenesis with cell-based mRNA decay readouts\",\n      \"pmids\": [\"25026077\", \"25026078\", \"25504471\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Role of the B-site in vivo not established\", \"Contribution of non-ROQ domains to target selection not defined\", \"Stoichiometry of Roquin on natural 3′ UTRs unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovery that MALT1 protease cleaves both Roquin-1 and Regnase-1 upon TCR stimulation answered how antigen receptor signaling releases post-transcriptional repression of inflammatory mRNAs to permit T helper differentiation.\",\n      \"evidence\": \"Biochemical cleavage assays, T cell genetic models, mRNA reporter assays\",\n      \"pmids\": [\"25282160\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether cleavage is graded with signal strength not yet shown\", \"Relative contributions of Roquin vs. Regnase-1 inactivation not dissected\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defining that Roquin and Regnase-1 repress overlapping mRNAs but in distinct subcellular compartments—Roquin on translationally inactive mRNAs at P-bodies/stress granules, Regnase-1 on ribosome-associated mRNAs—resolved how two seemingly redundant repressors achieve complementary surveillance.\",\n      \"evidence\": \"Subcellular fractionation, ribosome profiling, UPF1 requirement assays, mRNA decay assays\",\n      \"pmids\": [\"26000482\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How targets are partitioned between compartments not explained\", \"Whether Roquin-Regnase-1 physical interaction is required for this compartmentalization unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"PAR-CLIP revealed ~3,800 RC3H1 mRNA targets genome-wide including A20, and showed that both the ROQ and zinc finger domains contribute to RNA binding, while the RING domain was shown to antagonize AMPK to specifically promote Tfh differentiation, establishing a second, non-RNA-decay function for Roquin.\",\n      \"evidence\": \"PAR-CLIP transcriptome-wide, domain mutagenesis with NF-κB pathway assays; conditional RING-domain KO mice with AMPK interaction assays\",\n      \"pmids\": [\"26170170\", \"26496200\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RING domain substrates beyond AMPK unknown\", \"How RNA-binding and E3 ligase functions are coordinated unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Discovery of the alternative decay element (ADE) hexaloop as a second class of Roquin target motif, bound via the same ROQ domain interface as CDEs, expanded the Roquin target code and explained cooperative repression of Ox40 through tandem CDE+ADE elements.\",\n      \"evidence\": \"SELEX, X-ray crystallography of ROQ-ADE complex, NMR, cell-based repression assays\",\n      \"pmids\": [\"27010430\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genome-wide prevalence of ADE motifs not mapped\", \"How CDE and ADE elements cooperate quantitatively in full-length 3′ UTRs not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Structural characterization of how Roquin recruits the CCR4-NOT complex via a C-terminal CAF40-binding motif (CBM) provided the atomic-level mechanism for deadenylation-dependent mRNA decay.\",\n      \"evidence\": \"X-ray crystallography of Drosophila Roquin CBM–CAF40 complex, Co-IP, binding assays\",\n      \"pmids\": [\"28165457\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether human Roquin-1 uses an identical CBM interface not structurally confirmed\", \"Contribution of CBM vs. other CCR4-NOT contacts not dissected in vivo\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"iCLIP and reporter studies showed that Roquin binds both structured and linear sequence elements, and that multiple Roquin molecules cooperatively occupy tandem sites on a single 3′ UTR to achieve both deadenylation/decapping and translational repression, revealing dose-dependent regulatory modes.\",\n      \"evidence\": \"iCLIP, polysome profiling, deadenylation/decapping mutant rescue assays\",\n      \"pmids\": [\"30232334\", \"29352114\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of NUFIP2 cofactor cooperation with Roquin not resolved\", \"In vivo stoichiometry on endogenous targets not measured\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"A human homozygous R688* RC3H1 mutation causing hyperinflammation established that Roquin-1 is essential for immune homeostasis in humans, and that the C-terminal region is required for P-body localization and CCR4-NOT interaction.\",\n      \"evidence\": \"Patient genetic analysis, P-body localization assays, CCR4-NOT Co-IP, mRNA decay assays\",\n      \"pmids\": [\"31636267\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Additional human disease-causing alleles not reported\", \"Whether Roquin-2 partially compensates in the patient not assessed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mapping the direct Roquin-1–Regnase-1 physical interaction surface (including M199) demonstrated that their cooperation is not merely functional overlap but requires direct protein–protein contact, explaining why the sanroque mutation disrupts both partners' function.\",\n      \"evidence\": \"Co-IP, interaction surface mapping, conditional KO mice with autoimmune phenotyping\",\n      \"pmids\": [\"34811541\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural model of the Roquin–Regnase-1 complex not available\", \"Whether interaction is RNA-dependent not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of transferrin receptor 1 (TfR1) mRNA as a Roquin target connected Roquin to iron homeostasis and metabolism beyond inflammation, with osteoclast-specific KO revealing bone loss due to increased iron uptake and mitochondrial respiration.\",\n      \"evidence\": \"Gene KO in multiple cell types, 3′ UTR mutational analysis, osteoclast-specific conditional KO with Tfr1 rescue\",\n      \"pmids\": [\"33898949\", \"39659568\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether iron status feeds back to regulate Roquin expression unknown\", \"Relative contribution of Roquin vs. IRP/IRE system to TfR1 regulation in vivo not quantified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Knock-in cleavage-resistant Roquin-1 (Mins) mice proved that MALT1-mediated cleavage is the dominant mechanism inactivating Roquin downstream of TCR, and that graded cleavage creates a signal-strength decoder for differential target mRNA derepression.\",\n      \"evidence\": \"Rc3h1Mins/Mins knock-in mice, genetic epistasis with constitutively active MALT1, EAE disease model, Th17 assays\",\n      \"pmids\": [\"37988467\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other proteases contribute to Roquin inactivation in non-T cells not addressed\", \"Quantitative relationship between cleavage fraction and individual target derepression not modeled\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery that Roquin-1 functions as an E3 ubiquitin ligase targeting GPX4 for degradation, triggered by MALT1 inhibition, connected Roquin to ferroptosis regulation in cancer and revealed a non-RNA-decay effector axis.\",\n      \"evidence\": \"Genome-wide CRISPR screen, ubiquitination assays, ferroptosis readouts in liver cancer cells\",\n      \"pmids\": [\"39739814\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether GPX4 ubiquitination depends on the RING domain specifically not shown\", \"Breadth of E3 ligase substrates beyond AMPK and GPX4 unknown\", \"In vivo relevance of the MALT1-Roquin-GPX4 axis in tumors not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"NMR structure of the CCCH zinc finger revealed it recognizes AU-rich linear RNA sequences, and simultaneous binding experiments showed that the ROQ and ZnF domains engage natural targets concurrently, establishing that Roquin integrates RNA shape and sequence through tandem domains.\",\n      \"evidence\": \"NMR structure, RBNS-NMR pipeline, simultaneous dual-domain binding on natural target RNAs\",\n      \"pmids\": [\"38953172\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ZnF binding is required for in vivo target selection genome-wide not tested\", \"Structural model of full-length Roquin on a natural 3′ UTR not available\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Single-molecule force spectroscopy showed that the ROQ domain A-site stabilizes while the B-site destabilizes target RNA structure, revealing that the two binding sites have opposing mechanical effects that together encode target specificity.\",\n      \"evidence\": \"SMFS with integrated NMR and microfluidics on CDE/ADE RNA elements\",\n      \"pmids\": [\"40203046\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether opposing A/B-site effects operate on all target classes not tested\", \"How these mechanical effects translate into differential decay kinetics in cells unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the full substrate range of Roquin's RING-domain E3 ligase activity, the structural basis of the Roquin–Regnase-1 complex, how Roquin's RNA-decay and ubiquitin-ligase functions are coordinated on the same protein, and whether Roquin plays roles in non-immune tissues beyond bone metabolism.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of the full-length Roquin–Regnase-1 complex\", \"Complete E3 ligase substrate repertoire undefined\", \"Coordination between RNA-decay and ubiquitin-ligase activities unknown\", \"Non-immune physiological roles largely unexplored\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5, 6, 10, 15, 18, 22, 29]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [28, 33]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [3, 9, 18, 24]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [12, 16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [1, 2, 4, 20, 31, 32]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0008953854\", \"supporting_discovery_ids\": [0, 3, 18, 24]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 4, 7, 12, 16, 27]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [10, 16, 12]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [28]}\n    ],\n    \"complexes\": [\n      \"CCR4-NOT deadenylase complex (recruited effector)\"\n    ],\n    \"partners\": [\n      \"RC3H2\",\n      \"ZC3H12A\",\n      \"NUFIP2\",\n      \"CNOT9\",\n      \"MALT1\",\n      \"PRKAA1\",\n      \"GPX4\",\n      \"AGO2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\nWait, I made an error in the pathway term_ids. Let me fix the Metabolism of RNA entry - it should be `R-HSA-8953854` not `GO:0008953854`. Let me provide the corrected JSON:\n\n```json\n{\n  \"mechanistic_narrative\": \"RC3H1 (Roquin-1) is an RNA-binding E3 ubiquitin ligase that post-transcriptionally represses proinflammatory and costimulatory mRNAs—including ICOS, OX40, TNF-α, IL-6, TfR1, and IRF1—by recognizing constitutive and alternative decay element (CDE/ADE) stem-loops and AU-rich sequences in 3′ UTRs via its bimodal ROQ domain (A-site for apical stem-loops, B-site for double-stranded RNA) and CCCH-type zinc finger, then recruiting the CCR4-NOT deadenylase complex and decapping machinery at P-bodies to trigger mRNA degradation [PMID:18172933, PMID:23663784, PMID:25026078, PMID:38953172, PMID:27010430]. Roquin-1 physically cooperates with Regnase-1 on overlapping target mRNAs but acts on translationally inactive transcripts at P-bodies and stress granules, whereas Regnase-1 degrades ribosome-associated mRNAs; both are inactivated by MALT1-mediated proteolytic cleavage in a TCR signal-strength-graded manner that tunes T helper cell differentiation [PMID:26000482, PMID:25282160, PMID:37988467, PMID:34811541]. The RING domain confers E3 ubiquitin ligase activity that antagonizes AMPK to promote T follicular helper cell differentiation and mediates GPX4 ubiquitination to regulate ferroptosis [PMID:26496200, PMID:39739814]. A homozygous human loss-of-function RC3H1 mutation (R688*) causes hyperinflammation due to impaired P-body localization and CCR4-NOT recruitment [PMID:31636267].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Establishing that Roquin-1 controls immune homeostasis by limiting ICOS mRNA stability through its ROQ domain answered the foundational question of why sanroque mice develop lupus-like autoimmunity.\",\n      \"evidence\": \"Genetic analysis of M199R sanroque mice with ICOS 3′ UTR functional dissection and mRNA decay assays\",\n      \"pmids\": [\"18172933\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise RNA element recognized by Roquin not yet defined\", \"Effector machinery recruited by Roquin unknown\", \"Whether Roquin directly binds ICOS mRNA not established\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrating that Roquin directly binds ICOS mRNA at P-bodies and requires the decapping machinery (Rck, Edc4) but not the miRNA/RISC pathway defined Roquin as a miRNA-independent post-transcriptional repressor acting through mRNA decapping.\",\n      \"evidence\": \"Co-IP, fluorescence microscopy of P-body localization, RNA binding assays, Ago2/RISC decoupling in cell-based assays\",\n      \"pmids\": [\"20639877\", \"20412057\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the cis-regulatory RNA element beyond the ICOS 3′ UTR region not resolved\", \"Mechanism linking P-body localization to mRNA decay not defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identification of the constitutive decay element (CDE) stem-loop as a conserved Roquin-recognition motif in >50 mRNAs, and demonstration that Roquin recruits the CCR4-CAF1-NOT deadenylase complex, established the core effector mechanism and broadened the target repertoire beyond ICOS.\",\n      \"evidence\": \"RNA-protein binding assays, mRNA decay assays in macrophages, deadenylase recruitment assays, transcriptome-wide CDE identification; concurrent genetic KO studies showing Roquin-1/2 redundancy\",\n      \"pmids\": [\"23663784\", \"23583643\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of CDE recognition unknown\", \"Whether Roquin recognizes non-CDE elements not addressed\", \"Mechanism of CCR4-NOT recruitment not defined at atomic level\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Crystal structures of the ROQ domain bound to CDE RNA revealed a winged-helix fold with two distinct RNA-binding surfaces (A-site for stem-loops, B-site for dsRNA), explaining how Roquin achieves broad yet specific target recognition.\",\n      \"evidence\": \"X-ray crystallography of mouse and human ROQ domains (multiple structures), NMR, mutagenesis with cell-based mRNA decay readouts\",\n      \"pmids\": [\"25026077\", \"25026078\", \"25504471\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Role of the B-site in vivo not established\", \"Contribution of non-ROQ domains to target selection not defined\", \"Stoichiometry of Roquin on natural 3′ UTRs unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovery that MALT1 protease cleaves both Roquin-1 and Regnase-1 upon TCR stimulation answered how antigen receptor signaling releases post-transcriptional repression of inflammatory mRNAs to permit T helper differentiation.\",\n      \"evidence\": \"Biochemical cleavage assays, T cell genetic models, mRNA reporter assays\",\n      \"pmids\": [\"25282160\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether cleavage is graded with signal strength not yet shown\", \"Relative contributions of Roquin vs. Regnase-1 inactivation not dissected\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defining that Roquin and Regnase-1 repress overlapping mRNAs but in distinct subcellular compartments—Roquin on translationally inactive mRNAs at P-bodies/stress granules, Regnase-1 on ribosome-associated mRNAs—resolved how two seemingly redundant repressors achieve complementary surveillance.\",\n      \"evidence\": \"Subcellular fractionation, ribosome profiling, UPF1 requirement assays, mRNA decay assays\",\n      \"pmids\": [\"26000482\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How targets are partitioned between compartments not explained\", \"Whether Roquin-Regnase-1 physical interaction is required for this compartmentalization unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"PAR-CLIP revealed ~3,800 RC3H1 mRNA targets genome-wide including A20, and showed that both the ROQ and zinc finger domains contribute to RNA binding, while the RING domain was shown to antagonize AMPK to specifically promote Tfh differentiation, establishing a second, non-RNA-decay function for Roquin.\",\n      \"evidence\": \"PAR-CLIP transcriptome-wide, domain mutagenesis with NF-κB pathway assays; conditional RING-domain KO mice with AMPK interaction assays\",\n      \"pmids\": [\"26170170\", \"26496200\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RING domain substrates beyond AMPK unknown\", \"How RNA-binding and E3 ligase functions are coordinated unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Discovery of the alternative decay element (ADE) hexaloop as a second class of Roquin target motif, bound via the same ROQ domain interface as CDEs, expanded the Roquin target code and explained cooperative repression of Ox40 through tandem CDE+ADE elements.\",\n      \"evidence\": \"SELEX, X-ray crystallography of ROQ-ADE complex, NMR, cell-based repression assays\",\n      \"pmids\": [\"27010430\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genome-wide prevalence of ADE motifs not mapped\", \"How CDE and ADE elements cooperate quantitatively in full-length 3′ UTRs not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Structural characterization of how Roquin recruits the CCR4-NOT complex via a C-terminal CAF40-binding motif (CBM) provided the atomic-level mechanism for deadenylation-dependent mRNA decay.\",\n      \"evidence\": \"X-ray crystallography of Drosophila Roquin CBM–CAF40 complex, Co-IP, binding assays\",\n      \"pmids\": [\"28165457\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether human Roquin-1 uses an identical CBM interface not structurally confirmed\", \"Contribution of CBM vs. other CCR4-NOT contacts not dissected in vivo\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"iCLIP and reporter studies showed that Roquin binds both structured and linear sequence elements, and that multiple Roquin molecules cooperatively occupy tandem sites on a single 3′ UTR to achieve both deadenylation/decapping and translational repression, revealing dose-dependent regulatory modes.\",\n      \"evidence\": \"iCLIP, polysome profiling, deadenylation/decapping mutant rescue assays; NUFIP2 identified as essential cofactor by siRNA screen\",\n      \"pmids\": [\"30232334\", \"29352114\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of NUFIP2 cofactor cooperation with Roquin not resolved\", \"In vivo stoichiometry on endogenous targets not measured\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"A human homozygous R688* RC3H1 mutation causing hyperinflammation established that Roquin-1 is essential for immune homeostasis in humans, and that the C-terminal region is required for P-body localization and CCR4-NOT interaction.\",\n      \"evidence\": \"Patient genetic analysis, P-body localization assays, CCR4-NOT Co-IP, mRNA decay assays\",\n      \"pmids\": [\"31636267\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Additional human disease-causing alleles not reported\", \"Whether Roquin-2 partially compensates in the patient not assessed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mapping the direct Roquin-1–Regnase-1 physical interaction surface (including M199) demonstrated that their cooperation is not merely functional overlap but requires direct protein–protein contact, explaining why the sanroque mutation disrupts both partners' function.\",\n      \"evidence\": \"Co-IP, interaction surface mapping, conditional KO mice with autoimmune phenotyping\",\n      \"pmids\": [\"34811541\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural model of the Roquin–Regnase-1 complex not available\", \"Whether interaction is RNA-dependent not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of transferrin receptor 1 (TfR1) mRNA as a Roquin target connected Roquin to iron homeostasis and metabolism beyond inflammation, with osteoclast-specific KO revealing bone loss due to increased iron uptake and mitochondrial respiration.\",\n      \"evidence\": \"Gene KO in multiple cell types, 3′ UTR mutational analysis, osteoclast-specific conditional KO with Tfr1 rescue\",\n      \"pmids\": [\"33898949\", \"39659568\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether iron status feeds back to regulate Roquin expression unknown\", \"Relative contribution of Roquin vs. IRP/IRE system to TfR1 regulation in vivo not quantified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Knock-in cleavage-resistant Roquin-1 (Mins) mice proved that MALT1-mediated cleavage is the dominant mechanism inactivating Roquin downstream of TCR, and that graded cleavage creates a signal-strength decoder for differential target mRNA derepression.\",\n      \"evidence\": \"Rc3h1Mins/Mins knock-in mice, genetic epistasis with constitutively active MALT1, EAE disease model, Th17 assays\",\n      \"pmids\": [\"37988467\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other proteases contribute to Roquin inactivation in non-T cells not addressed\", \"Quantitative relationship between cleavage fraction and individual target derepression not modeled\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery that Roquin-1 functions as an E3 ubiquitin ligase targeting GPX4 for degradation, triggered by MALT1 inhibition, connected Roquin to ferroptosis regulation in cancer and revealed a non-RNA-decay effector axis.\",\n      \"evidence\": \"Genome-wide CRISPR screen, ubiquitination assays, ferroptosis readouts in liver cancer cells\",\n      \"pmids\": [\"39739814\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether GPX4 ubiquitination depends on the RING domain specifically not shown\", \"Breadth of E3 ligase substrates beyond AMPK and GPX4 unknown\", \"In vivo relevance of the MALT1-Roquin-GPX4 axis in tumors not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"NMR structure of the CCCH zinc finger revealed it recognizes AU-rich linear RNA sequences, and simultaneous binding experiments showed that the ROQ and ZnF domains engage natural targets concurrently, establishing that Roquin integrates RNA shape and sequence through tandem domains.\",\n      \"evidence\": \"NMR structure, RBNS-NMR pipeline, simultaneous dual-domain binding on natural target RNAs\",\n      \"pmids\": [\"38953172\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ZnF binding is required for in vivo target selection genome-wide not tested\", \"Structural model of full-length Roquin on a natural 3′ UTR not available\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Single-molecule force spectroscopy showed that the ROQ domain A-site stabilizes while the B-site destabilizes target RNA structure, revealing that the two binding sites have opposing mechanical effects that together encode target specificity.\",\n      \"evidence\": \"SMFS with integrated NMR and microfluidics on CDE/ADE RNA elements\",\n      \"pmids\": [\"40203046\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether opposing A/B-site effects operate on all target classes not tested\", \"How these mechanical effects translate into differential decay kinetics in cells unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the full substrate range of Roquin's RING-domain E3 ligase activity, the structural basis of the Roquin–Regnase-1 complex, how Roquin's RNA-decay and ubiquitin-ligase functions are coordinated on the same protein, and whether Roquin plays roles in non-immune tissues beyond bone metabolism.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of the full-length Roquin–Regnase-1 complex\", \"Complete E3 ligase substrate repertoire undefined\", \"Coordination between RNA-decay and ubiquitin-ligase activities unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5, 6, 10, 15, 18, 22, 29]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [12, 28, 33]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [3, 9, 18, 24]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [12, 16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [1, 2, 4, 20, 31, 32]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 3, 18, 24]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [4, 7, 12, 16, 27]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [10, 16]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [28]}\n    ],\n    \"complexes\": [\n      \"CCR4-NOT deadenylase complex (recruited effector)\"\n    ],\n    \"partners\": [\n      \"RC3H2\",\n      \"ZC3H12A\",\n      \"NUFIP2\",\n      \"CNOT9\",\n      \"MALT1\",\n      \"PRKAA1\",\n      \"GPX4\",\n      \"AGO2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}