{"gene":"RC3H1","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2007,"finding":"Roquin (RC3H1) promotes degradation of ICOS mRNA via a conserved 47-bp segment in the ICOS 3' UTR; the M199R sanroque mutation in the ROQ domain abolishes this repression, leading to increased ICOS expression on T cells and lupus-like autoimmunity.","method":"Genetic mouse model (sanroque M199R point mutation), mRNA stability assays, 3' UTR reporter assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (genetic model, reporter assays, mRNA decay), replicated across subsequent studies","pmids":["18172933"],"is_preprint":false},{"year":2010,"finding":"Roquin localizes to processing (P) bodies in helper T cells and represses ICOS expression by directly binding the ICOS mRNA 3' UTR (independent of microRNAs/RISC); repression requires the RNA helicase Rck and the decapping enhancer Edc4, and depends on sequences conferring P-body localization.","method":"Fluorescence microscopy (P-body localization), co-immunoprecipitation (Rck, Edc4 interactions), RNA binding assays, dominant-negative and deletion mutant analysis","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, direct RNA-binding demonstrated, P-body localization linked to function, single lab with multiple orthogonal methods","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; Roquin and its orthologs (Drosophila, C. elegans) all concentrate in stress granules via the ROQ domain.","method":"Fluorescence microscopy, deletion mutant analysis, gel-shift/SPR/footprinting RNA-binding assays","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — ROQ domain sufficiency shown by deletion mapping, RNA binding confirmed by SPR and footprinting, replicated across species","pmids":["20412057"],"is_preprint":false},{"year":2013,"finding":"Roquin recognizes a conserved stem-loop motif (constitutive decay element, CDE) in the 3' UTR of TNF-α mRNA and initiates its degradation by recruiting the Ccr4-Caf1-Not deadenylase complex; CDE sequences are present in >50 vertebrate mRNAs encoding inflammatory and developmental regulators.","method":"In vitro RNA-binding assays, mRNA stability assays in macrophages, co-immunoprecipitation of Ccr4-Caf1-Not, transcriptome-wide target identification (RNA-seq)","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct RNA binding, deadenylase complex recruitment by Co-IP, functional decay assays, genome-wide target identification, replicated by subsequent structural studies","pmids":["23663784"],"is_preprint":false},{"year":2013,"finding":"Roquin-1 and Roquin-2 redundantly repress ICOS and Ox40 costimulatory receptor mRNAs in T cells; combined loss of both paralogs (but not either alone) causes accumulation of follicular helper T cells and effector T cells.","method":"Conditional gene knockout (T-cell-specific deletion), flow cytometry, mRNA stability assays","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal single and double KO genetic epistasis, independently replicated by two labs (PMID 23583643 and 23583642)","pmids":["23583643","23583642"],"is_preprint":false},{"year":2013,"finding":"The RING domain of Roquin-1 is required for its localization to stress granules; RING-deficient Roquin-1 fails to localize to stress granules and permits Roquin-2 to compensate for ICOS and Tfh repression, whereas the ROQ (sanroque) mutant retains stress granule localization.","method":"Conditional knock-in/knockout mouse models, fluorescence microscopy, flow cytometry","journal":"Immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-specific mouse models with cellular phenotype readout, single lab","pmids":["23583642"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of the mouse Roquin ROQ domain in complex with a CDE RNA stem-loop reveals that the ROQ domain adopts an extended winged-helix (WH) fold and recognizes mainly via non-sequence-specific contacts to the RNA stem and triloop.","method":"X-ray crystallography, NMR, binding assays, cell-based reporter assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation by binding and cell-based assays, independently confirmed by contemporaneous structure (PMID 25026078)","pmids":["25026077"],"is_preprint":false},{"year":2014,"finding":"Crystal structures of human Roquin ROQ domain reveal two separate RNA-binding sites: the A site binds stem-loop RNA (CDE) and the B site binds double-stranded RNA; mutagenesis of either site impairs Roquin-mediated degradation of HMGXB3 and IL6 mRNAs in human cells.","method":"X-ray crystallography, site-directed mutagenesis, mRNA decay assays in human cells","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures plus mutagenesis with functional readout, independent of PMID 25026077","pmids":["25026078"],"is_preprint":false},{"year":2014,"finding":"Upon TCR antigen recognition, the paracaspase MALT1 cleaves both roquin-1 and regnase-1, inactivating their cooperative post-transcriptional repression of target mRNAs (IL-6, ICOS, c-Rel, IRF4, IκBNS, IκBζ) and thereby promoting Th17 differentiation; this derepression requires RNA binding by roquin and nuclease activity of regnase-1.","method":"MALT1 cleavage assays, T cell-specific deletion/reconstitution, mRNA decay assays, Th17 differentiation assays","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct cleavage assay for MALT1 substrate, genetic rescue experiments, functional Th17 readout, replicated by subsequent study (PMID 37988467)","pmids":["25282160"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of the Roquin ROQ domain shows a winged helix-turn-helix motif responsible for CDE stem-loop binding; Roquin proteins can also 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 / Strong — crystal structure with biochemical validation, consistent with other structural studies","pmids":["25504471"],"is_preprint":false},{"year":2015,"finding":"Regnase-1 and Roquin regulate an overlapping set of mRNAs via a common stem-loop structure but operate in distinct subcellular compartments: Regnase-1 degrades translationally active mRNAs at the ribosome/ER (requiring UPF1 helicase), whereas Roquin controls translationally inactive mRNAs at P-bodies/stress granules (independent of UPF1).","method":"Subcellular fractionation, ribosome profiling, mRNA decay assays, UPF1 knockdown, fluorescence microscopy","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (fractionation, ribosome profiling, genetic knockdown), single lab with comprehensive mechanistic dissection","pmids":["26000482"],"is_preprint":false},{"year":2015,"finding":"RC3H1/Roquin binds ~3,800 mRNA targets via CDE motifs and a novel U-rich sequence embedded in hairpins (identified by PAR-CLIP); RC3H1 uses its ROQ and Zn-finger domains to contact a binding site in the A20 3' UTR, leading to A20 mRNA decay; RC3H1 knockdown increases A20 protein expression, thereby attenuating IKK and NF-κB activity.","method":"PAR-CLIP (transcriptome-wide binding), siRNA knockdown, western blotting, NF-κB reporter assays, domain mutagenesis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — PAR-CLIP genome-wide plus domain mutagenesis, functional IKK/NF-κB pathway readout, multiple orthogonal methods","pmids":["26170170"],"is_preprint":false},{"year":2015,"finding":"Roquin directly binds Argonaute2 (Ago2) and miR-146a; Roquin enhances Dicer-mediated processing of pre-miR-146a; in the absence of functional Roquin, mature miR-146a is stabilized due to decreased 3'-end uridylation. Crystallography reveals a unique HEPN domain in Roquin and the structural basis of the sanroque mutation.","method":"Co-immunoprecipitation (Ago2, miR-146a), Dicer processing assays, miRNA uridylation assays, X-ray crystallography","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — crystal structure, direct binding by Co-IP, biochemical processing assays, single lab with multiple orthogonal methods","pmids":["25697406"],"is_preprint":false},{"year":2015,"finding":"Crystal structure of the human Roquin-2 ROQ domain bound to CDE RNA shows a winged-helix fold with a flexible wing region acting as a lid for RNA binding; CDE stem-loop is recognized mainly via backbone contacts, consistent with Roquin-1 binding mode.","method":"X-ray crystallography (ligand-free and RNA-bound forms)","journal":"Acta crystallographica. Section F, Structural biology communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure in two forms, consistent with multiple independent structural studies","pmids":["26249698"],"is_preprint":false},{"year":2015,"finding":"The ROQUIN RING domain directly antagonizes the catalytic α1 subunit of AMPK, promoting Tfh cell differentiation; T cell-specific deletion of the RING domain selectively abolishes Tfh responses without affecting Th1, Th2, Th17, or Treg populations.","method":"Conditional RING domain deletion in T cells, genetic interaction/rescue experiments, AMPK activity assays","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional domain-specific KO with selective phenotype, AMPK interaction assay, single lab","pmids":["26496200"],"is_preprint":false},{"year":2016,"finding":"Roquin recognizes a novel U-rich hexaloop motif (alternative decay element, ADE) in addition to the triloop CDE; crystal structures and NMR show the ROQ domain binds ADE hexaloops from SELEX-derived sequences and from the Ox40 3' UTR with identical binding modes; ADE-like and CDE-like motifs cooperate in Ox40 repression.","method":"SELEX, X-ray crystallography, NMR, cell-based reporter assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures with SELEX identification and functional cell-based validation, single lab with multiple orthogonal methods","pmids":["27010430"],"is_preprint":false},{"year":2017,"finding":"Drosophila Roquin (and human Roquin-2) recruits the CCR4-NOT deadenylase complex via a CAF40-binding motif (CBM) in their C-terminal regions; crystal structure of Dm Roquin CBM bound to CAF40 shows the CBM adopts an α-helical conformation binding a conserved CAF40 surface.","method":"X-ray crystallography, co-immunoprecipitation, pulldown assays, mRNA decay reporter assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus Co-IP and functional decay assays, conserved mechanism across species","pmids":["28165457"],"is_preprint":false},{"year":2017,"finding":"Roquin inhibits the PI3K-mTOR signaling pathway by upregulating PTEN (by interfering with miR-17~92 binding to the PTEN 3' UTR) and by downregulating the Foxo1-specific E3 ubiquitin ligase Itch; loss of Roquin in T cells enhances Akt-mTOR signaling and protein synthesis.","method":"Conditional Roquin knockout in Treg cells, PI3K/mTOR inhibitor rescue experiments, luciferase 3' UTR reporter assays, western blotting","journal":"Immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with pharmacological rescue and reporter assays, single lab with multiple methods","pmids":["29246441"],"is_preprint":false},{"year":2018,"finding":"Roquin not only interacts with stem-loop structures but also with a linear sequence element in ~half of its target mRNAs; multiple Roquin proteins bound to several stem-loops in the Nfkbid 3' UTR exert full repression redundantly via deadenylation, decapping, and translational inhibition.","method":"PAR-CLIP, luciferase reporter assays with stem-loop mutations, polysome profiling, CRISPR-mediated deletion of 3' UTR elements","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — PAR-CLIP with multiple functional readouts (decay, translation), CRISPR deletions, multiple orthogonal methods","pmids":["30232334"],"is_preprint":false},{"year":2018,"finding":"NUFIP2 directly binds Roquin with high affinity and stabilizes NUFIP2 in cells; NUFIP2 acts as a cofactor required for Roquin-mediated repression of ICOS mRNA, cooperatively binding with Roquin to non-canonical tandem stem-loops in the ICOS 3' UTR.","method":"siRNA screen (~1500 genes), co-immunoprecipitation, in vitro binding assays, reporter assays, electrophoretic mobility shift assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic RNAi screen followed by direct binding assays and functional reporter validation, multiple orthogonal methods","pmids":["29352114"],"is_preprint":false},{"year":2019,"finding":"A human homozygous nonsense R688* RC3H1 mutation causes Roquin-1 loss that leads to hyperinflammation (HLH-like); mechanistically, R688* Roquin-1 fails to localize to P-bodies and interact with the CCR4-NOT deadenylation complex, impairing mRNA decay of ICOS, OX40, and TNF targets.","method":"Patient-derived cells, immunofluorescence (P-body localization), co-immunoprecipitation (CCR4-NOT), mRNA stability assays, flow cytometry","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — human genetic variant with mechanistic dissection by Co-IP, localization assays, and mRNA decay measurements; unique patient case with multiple orthogonal methods","pmids":["31636267"],"is_preprint":false},{"year":2019,"finding":"HCMV exploits Roquin by upregulating its expression; Roquin binds directly to IRF1 mRNA (identified by CLIP-seq) and reduces IRF1 expression, thereby suppressing IRF1-dependent antiviral gene activation during HCMV infection.","method":"CLIP-seq, loss-of-function screening, transcriptome profiling, mRNA stability assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CLIP-seq for direct binding plus functional transcriptome and loss-of-function data, single lab","pmids":["31451648"],"is_preprint":false},{"year":2020,"finding":"The Roquin ROQ domain recognizes AU-rich elements (AREs) in a CDE-like stem-loop conformation, while AUF1 recognizes the same AREs in a linear form; NMR structures of CDE-like folded AREs (free and Roquin-bound) demonstrate shape-specific recognition by Roquin.","method":"NMR spectroscopy (solution structures of free and protein-bound AREs), RNA binding assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structures in free and bound forms with biochemical binding data, single lab","pmids":["32491174"],"is_preprint":false},{"year":2021,"finding":"RC3H1/Roquin is a major mediator of iron-regulated TfR1 mRNA stability; Roquin destabilizes TfR1 mRNA in a manner fully dependent on three hairpin loops in the TfR1 3' UTR; TfR1 mRNA degradation by Roquin does not require ongoing translation.","method":"Gene knockout (HAP1, HUVEC, L-M, MEF cells), siRNA knockdown, 3' UTR hairpin loop mutagenesis, mRNA stability assays","journal":"iScience","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple cell-type knockouts, hairpin mutagenesis, translation-independence demonstrated, multiple orthogonal methods","pmids":["33898949"],"is_preprint":false},{"year":2021,"finding":"Physical interaction between Roquin-1 and Regnase-1 is mediated by a defined interaction surface in Roquin-1 that includes the sanroque residue (M199); mutations disrupting this interaction cause Tfh cell accumulation, germinal center B cells, and autoantibody formation.","method":"Co-immunoprecipitation, mutagenesis (interaction surface mapping), conditional triple KO (Roquin-1/2 and Regnase-1), flow cytometry","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP with mutagenesis defining interaction surface, genetic triple-KO epistasis, multiple orthogonal methods","pmids":["34811541"],"is_preprint":false},{"year":2021,"finding":"Roquin is the strongest repressor of CD8 T cell expansion identified in genome-wide CRISPR screens; Roquin ablation boosts T cell proliferation by enhancing cell-cycle progression and upregulating IRF4.","method":"In vivo and in vitro CRISPR screens, KO validation, flow cytometry, gene expression analysis","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo CRISPR screen with functional validation, single lab","pmids":["34879274"],"is_preprint":false},{"year":2022,"finding":"NMR analysis of the full-length Ox40 mRNA 3' UTR reveals that Roquin uses its extended bimodal ROQ domain to sequentially engage decay elements in a 2:1 (protein:RNA) stoichiometry; the binding of Roquin to individual decay elements depends on their structural embedment within the full 3' UTR.","method":"NMR spectroscopy, SAXS, In-line probing, SHAPE-seq, binding assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR plus SAXS and multiple RNA probing methods provide structural and binding data on the full 3' UTR, single lab","pmids":["35357505"],"is_preprint":false},{"year":2023,"finding":"TCR signal strength is translated by MALT1 into graded cleavage of Roquin-1; only high TCR strength enables derepression of high-affinity Roquin targets (e.g., IκBNS) and promotes Th17 differentiation; rendering Roquin-1 insensitive to MALT1 cleavage (Rc3h1Mins/Mins) prevents spontaneous T cell activation caused by constitutively active MALT1.","method":"Knock-in mice (MALT1-cleavage-resistant Roquin-1), genetic epistasis (Rc3h1Mins × Malt1TBM), EAE model, Th17 differentiation assays, western blotting for Roquin cleavage","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knock-in genetic epistasis with specific cleavage-resistant allele, multiple in vivo and in vitro readouts","pmids":["37988467"],"is_preprint":false},{"year":2024,"finding":"RC3H1 acts as an E3 ubiquitin ligase promoting GPX4 ubiquitination-mediated degradation; MALT1 inhibition upregulates RC3H1, which then enhances GPX4 ubiquitination and degradation to induce ferroptosis in liver cancer cells.","method":"Genome-wide CRISPR screen (FACS-based), rescue assays, ubiquitination assays, western blotting","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen validated by rescue and ubiquitination assays, single lab","pmids":["39739814"],"is_preprint":false},{"year":2024,"finding":"Rc3h1 post-transcriptionally represses transferrin receptor 1 (Tfr1) mRNA in osteoclasts, restricting iron absorption and mitochondrial respiration; Rc3h1-deficient osteoclasts show enhanced Tfr1 expression, increased mitochondrial respiration and osteoclast activation, and mice exhibit low bone mass.","method":"Conditional Rc3h1 knockout in osteoclasts, mRNA stability assays, iron content measurement, mitochondrial respiration assays, Tfr1 inhibitor rescue","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with targeted rescue, multiple functional readouts, single lab","pmids":["39659568"],"is_preprint":false},{"year":2024,"finding":"Solution NMR structure of the Roquin-1 CCCH zinc finger domain reveals it recognizes AU-rich RNA sequences; the ZnF and ROQ domains simultaneously bind a natural Roquin target transcript, suggesting Roquin integrates RNA shape (ROQ) and sequence (ZnF) features through a tandem domain mechanism.","method":"NMR spectroscopy (solution structure), RBNS-NMR pipeline for RNA binding preferences, in vitro RNA binding assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR solution structure with systematic RNA binding characterization, demonstrates cooperative domain engagement","pmids":["38953172"],"is_preprint":false},{"year":2021,"finding":"MNSFβ binds directly to RC3H1 (demonstrated by co-immunoprecipitation), and elevated MNSFβ promotes TNFα production by interacting with RC3H1, which normally suppresses TNFα mRNA.","method":"Co-immunoprecipitation, siRNA knockdown in THP1-derived macrophages, ELISA","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP interaction confirmed and functional rescue by RC3H1 knockdown, single lab","pmids":["34589082"],"is_preprint":false},{"year":2024,"finding":"MNSFβ promotes LPS-induced TNFα expression by facilitating stress granule formation and translocation of RC3H1 to stress granules (via interaction with RC3H1 and FMR1), thereby inactivating RC3H1-mediated TNFα mRNA degradation; the RC3H1 81-326 aa region binds MNSFβ 101-133 aa region.","method":"Co-immunoprecipitation, domain mapping, stress granule imaging, mRNA stability assays, peptide inhibitor (HEPN2) rescue experiments","journal":"International immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mapping by Co-IP, functional mRNA decay assays, stress granule imaging, single lab with multiple methods","pmids":["39260307"],"is_preprint":false},{"year":2025,"finding":"Single-molecule force spectroscopy (SMFS) combined with NMR reveals opposing effects of the two Roquin ROQ domain RNA-binding sites on ADE stem stability: the A-site strongly stabilizes the apical ADE stem-loop, while the B-site destabilizes the ADE central stem through single-strand RNA binding, suggesting both sites jointly direct mRNA decay with context-dependent 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 / Moderate — two orthogonal biophysical methods (SMFS and NMR) with kinetic measurements, mechanistically novel finding","pmids":["40203046"],"is_preprint":false},{"year":2025,"finding":"A heterozygous missense F225S variant in the ROQ domain of RC3H1 causes autosomal-dominant primary immunodeficiency; F225S Roquin-1 fails to repress ICOS1, IL6, and other target mRNAs (increased message stability), demonstrating that ROQ domain integrity is required for post-transcriptional repression.","method":"Whole-exome sequencing, patient primary cells, transfection of F225S variant, mRNA stability assays, flow cytometry","journal":"The Journal of allergy and clinical immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived cells with single-variant transfection and mRNA stability readout, single lab","pmids":["40769319"],"is_preprint":false}],"current_model":"RC3H1/Roquin-1 is an RNA-binding E3 ubiquitin ligase that post-transcriptionally silences inflammatory and co-stimulatory mRNAs (including ICOS, Ox40, TNF, IL-6, A20, IRF1, and TfR1) by recognizing stem-loop decay elements (CDEs and ADEs) in their 3' UTRs via its ROQ domain (winged-helix fold with two RNA-binding sites) and a flanking CCCH zinc finger domain; it recruits the CCR4-NOT deadenylase complex (and decapping factors Rck/Edc4) to drive mRNA decay of translationally inactive mRNAs in P-bodies/stress granules, cooperates physically with Regnase-1 (which degrades translationally active mRNAs at the ribosome), and is inactivated by MALT1-mediated cleavage downstream of TCR signaling in a signal-strength-dependent manner to allow graded derepression of target mRNAs during T cell activation and Th17 differentiation."},"narrative":{"mechanistic_narrative":"RC3H1 (Roquin-1) is an RNA-binding post-transcriptional repressor that silences inflammatory and co-stimulatory mRNAs by recognizing structured decay elements in their 3' UTRs and targeting them for degradation [PMID:18172933, PMID:23663784]. Its central ROQ domain adopts an extended winged-helix fold that recognizes stem-loop constitutive decay elements (CDEs) largely through shape-specific backbone contacts to the stem and triloop [PMID:25026077, PMID:25504471], and contains two distinct RNA-binding surfaces — an A site that engages folded stem-loops and a B site that binds duplex/single-stranded RNA — both of which are required for efficient target decay [PMID:25026078, PMID:40203046]. Beyond the canonical CDE triloop, the ROQ domain also recognizes U-rich hexaloop alternative decay elements (ADEs) and CDE-folded AU-rich elements with the same binding mode, while the flanking CCCH zinc finger reads AU-rich sequence so that shape and sequence recognition are integrated across tandem domains; multiple decay elements within a single 3' UTR are engaged cooperatively [PMID:27010430, PMID:32491174, PMID:38953172, PMID:35357505]. Roquin drives decay by recruiting the CCR4-NOT deadenylase complex via a CAF40-binding motif and by acting through decapping factors (Rck/Edc4) at P-bodies and stress granules, where it controls translationally inactive mRNAs in cooperation with the ribosome-associated endonuclease Regnase-1 that handles translationally active transcripts [PMID:20639877, PMID:23663784, PMID:28165457, PMID:26000482, PMID:34811541]. Validated targets include ICOS, Ox40, TNF, IL-6, A20, IRF1 and transferrin receptor 1 (TfR1) mRNAs, linking Roquin to control of follicular helper and effector T-cell responses, NF-κB signaling, antiviral IRF1 responses, and iron homeostasis [PMID:18172933, PMID:23583643, PMID:23583642, PMID:26170170, PMID:31451648, PMID:33898949, PMID:39659568]. Roquin activity is set by T-cell receptor signal strength: the paracaspase MALT1 cleaves Roquin-1 in a graded, signal-strength-dependent manner to derepress target mRNAs and license Th17 differentiation [PMID:25282160, PMID:37988467]. Loss-of-function and missense ROQ-domain mutations in humans cause immune disease — a homozygous R688* nonsense allele produces HLH-like hyperinflammation through failed P-body localization and CCR4-NOT recruitment, and a heterozygous F225S ROQ variant causes autosomal-dominant primary immunodeficiency through loss of mRNA repression [PMID:31636267, PMID:40769319]. Separately, Roquin functions as an E3 ubiquitin ligase promoting GPX4 ubiquitination and ferroptosis [PMID:39739814].","teleology":[{"year":2007,"claim":"Established that Roquin is a physiological repressor of an immune mRNA, defining its core function before any molecular mechanism was known.","evidence":"sanroque M199R point-mutant mouse, ICOS 3' UTR reporter and mRNA stability assays","pmids":["18172933"],"confidence":"High","gaps":["Did not identify the RNA element recognized or the decay machinery engaged","Mechanism of the M199R defect unresolved at the structural level"]},{"year":2010,"claim":"Localized Roquin function to RNA-decay foci and identified decapping machinery, showing it acts directly on mRNA independent of the RISC pathway.","evidence":"P-body/stress-granule fluorescence microscopy, Rck/Edc4 Co-IP, RNA-binding and deletion-mutant analysis in helper T cells","pmids":["20639877","20412057"],"confidence":"High","gaps":["The precise RNA structural motif recognized was not defined","Whether deadenylation precedes decapping was not established"]},{"year":2013,"claim":"Defined the constitutive decay element (CDE) stem-loop as the recognition motif and CCR4-NOT recruitment as the decay mechanism, generalizing Roquin to a regulon of inflammatory mRNAs.","evidence":"In vitro RNA binding, macrophage decay assays, CCR4-NOT Co-IP, transcriptome-wide target identification","pmids":["23663784"],"confidence":"High","gaps":["Structural basis of CDE recognition not yet solved","Did not explain redundancy with the Roquin-2 paralog"]},{"year":2013,"claim":"Demonstrated functional redundancy between Roquin-1 and Roquin-2 and dissected RING-domain contribution to subcellular targeting, clarifying genetic interpretation of single-paralog studies.","evidence":"Single and double conditional T-cell knockouts, RING-domain mouse models, flow cytometry, mRNA stability assays","pmids":["23583643","23583642"],"confidence":"High","gaps":["Molecular role of the RING domain in stress-granule localization left mechanistically open","Did not resolve whether RING has catalytic ligase activity in this context"]},{"year":2014,"claim":"Provided crystal structures of the ROQ domain bound to CDE RNA, revealing a winged-helix fold with two RNA-binding sites that read RNA shape rather than sequence.","evidence":"X-ray crystallography of mouse and human ROQ domains, NMR, mutagenesis, cell-based decay reporters","pmids":["25026077","25026078","25504471"],"confidence":"High","gaps":["Did not address how full 3' UTRs with multiple elements are engaged","Role of the zinc finger in RNA recognition unresolved"]},{"year":2014,"claim":"Identified MALT1 cleavage of Roquin downstream of TCR signaling as the switch that derepresses targets and drives Th17 differentiation, linking Roquin to signal-dependent control.","evidence":"MALT1 cleavage assays, T-cell deletion/reconstitution, decay assays, Th17 differentiation readouts","pmids":["25282160"],"confidence":"High","gaps":["Did not establish how cleavage extent is tuned to signal strength","Cooperative mechanism with Regnase-1 not fully resolved"]},{"year":2015,"claim":"Separated Roquin and Regnase-1 into distinct subcellular decay pathways acting on overlapping targets, explaining their non-redundant cooperation.","evidence":"Subcellular fractionation, ribosome profiling, UPF1 knockdown, fluorescence microscopy, decay assays","pmids":["26000482"],"confidence":"High","gaps":["Physical basis of Roquin–Regnase-1 cooperation not yet mapped","How targets are partitioned between compartments unclear"]},{"year":2015,"claim":"Mapped the transcriptome-wide binding landscape and extended the binding repertoire to U-rich and linear motifs, while linking Roquin to A20/NF-κB control and miRNA biogenesis.","evidence":"PAR-CLIP, domain mutagenesis, NF-κB reporters, Ago2/miR-146a Co-IP, Dicer processing and uridylation assays, crystallography","pmids":["26170170","25697406"],"confidence":"High","gaps":["Functional weight of linear versus structured motifs not quantified","Relationship between mRNA decay and miRNA-processing roles unresolved"]},{"year":2016,"claim":"Defined the alternative decay element (ADE), a U-rich hexaloop, as a second class of structured motif recognized by the same ROQ surface, broadening the structural code for target recognition.","evidence":"SELEX, X-ray crystallography, NMR, cell-based reporter assays of Ox40 elements","pmids":["27010430"],"confidence":"High","gaps":["How CDE and ADE elements cooperate within a UTR was not structurally resolved","Did not address stoichiometry on full-length transcripts"]},{"year":2017,"claim":"Identified a CAF40-binding motif as the direct molecular link recruiting the CCR4-NOT complex, defining the conserved effector step of Roquin-mediated decay.","evidence":"Crystal structure of Roquin CBM–CAF40, Co-IP, pulldowns, decay reporter assays","pmids":["28165457"],"confidence":"High","gaps":["Did not resolve coordination of deadenylation with decapping in vivo","Contribution of CBM versus P-body localization to net decay unclear"]},{"year":2018,"claim":"Showed that multiple Roquin molecules engage several stem-loops redundantly to enforce repression through deadenylation, decapping and translational inhibition, and identified NUFIP2 as a required cofactor.","evidence":"PAR-CLIP, polysome profiling, CRISPR 3' UTR deletions, siRNA screen, in vitro binding and reporter assays","pmids":["30232334","29352114"],"confidence":"High","gaps":["Generality of NUFIP2 dependence across the target set not defined","Stoichiometry of multi-element engagement not structurally resolved"]},{"year":2019,"claim":"Connected Roquin to human disease and to antiviral and metabolic signaling, showing loss of function causes hyperinflammation and that viruses co-opt Roquin to blunt IRF1 responses.","evidence":"Patient cells with R688* (IF, CCR4-NOT Co-IP, decay assays), CLIP-seq and loss-of-function screening in HCMV infection, PI3K-mTOR rescue and reporter assays","pmids":["31636267","31451648","29246441"],"confidence":"High","gaps":["PI3K-mTOR regulation rests on single-lab Medium evidence","Causality of the HCMV-Roquin axis in vivo not established"]},{"year":2021,"claim":"Mapped the physical Roquin-1–Regnase-1 interaction surface (including the sanroque residue) and extended Roquin function to iron homeostasis via TfR1 mRNA, while genome-wide screens flagged it as a dominant brake on CD8 T-cell expansion.","evidence":"Co-IP and interaction mapping, conditional triple-KO epistasis, TfR1 3' UTR hairpin mutagenesis and decay assays in multiple cell types, CRISPR screens, MNSFβ Co-IP","pmids":["34811541","33898949","34879274","34589082"],"confidence":"High","gaps":["MNSFβ interaction and CD8 expansion roles rest on single-lab Medium evidence","How the same residue serves both RNA recognition and Regnase-1 binding not fully separated"]},{"year":2022,"claim":"Resolved how Roquin engages an intact 3' UTR, showing sequential bimodal engagement of decay elements with defined stoichiometry dependent on RNA structural context.","evidence":"NMR, SAXS, in-line probing and SHAPE-seq on full-length Ox40 3' UTR, binding assays","pmids":["35357505"],"confidence":"High","gaps":["Did not link binding order to in-cell decay kinetics","Role of cofactors in shaping engagement not addressed"]},{"year":2023,"claim":"Demonstrated that MALT1 converts TCR signal strength into graded Roquin-1 cleavage, establishing an analog-to-threshold mechanism for selective target derepression.","evidence":"Cleavage-resistant knock-in mice, genetic epistasis with constitutively active MALT1, EAE and Th17 assays, cleavage western blots","pmids":["37988467"],"confidence":"High","gaps":["Affinity hierarchy that sets target thresholds not biochemically mapped for all targets","Whether Regnase-1 cleavage is similarly graded not addressed here"]},{"year":2024,"claim":"Defined the zinc-finger contribution to RNA recognition and revealed a distinct E3 ligase function promoting GPX4 degradation and ferroptosis, broadening Roquin beyond mRNA decay.","evidence":"NMR solution structure of the CCCH ZnF with RBNS-NMR binding, CRISPR screen with ubiquitination and rescue assays, osteoclast conditional KO with TfR1 rescue, MNSFβ stress-granule imaging","pmids":["38953172","39739814","39659568","39260307"],"confidence":"High","gaps":["E3 ligase activity toward GPX4 rests on single-lab Medium evidence","Relationship between RNA-decay and ubiquitin-ligase functions not reconciled"]},{"year":2025,"claim":"Provided biophysical insight into how the two ROQ RNA-binding sites exert opposing structural effects on a target stem-loop, and added a second human disease allele confirming ROQ-domain integrity as essential for repression.","evidence":"Single-molecule force spectroscopy and NMR on ADE stem-loops; whole-exome sequencing and F225S patient-cell mRNA stability assays","pmids":["40203046","40769319"],"confidence":"High","gaps":["F225S mechanism rests on single-variant transfection (Medium)","How A-/B-site cooperation selects targets in cells not established"]},{"year":null,"claim":"How Roquin's mRNA-decay activity and its E3 ubiquitin-ligase/GPX4-ferroptosis activity are coordinated within a cell, and whether they share regulatory inputs, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural or genetic dissection separating the two activities","Substrate scope of the E3 ligase function beyond GPX4 unknown","Whether MALT1 cleavage regulates the ligase activity untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,3,6,7,11,15,22,26,30]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[3,10,16,23]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[16,19]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[28]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,2,10]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[1,2,5,10,20,32]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[3,10,11,18,23]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,4,8,11,21,27]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8,11,17,27]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[28]}],"complexes":["P-body","stress granule"],"partners":["CNOT1/CCR4-NOT","CAF40","REGNASE-1 (ZC3H12A)","NUFIP2","AGO2","MALT1","MNSFΒ","GPX4"],"other_free_text":[]}},"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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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":"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 ... [et al.]","url":"https://pubmed.ncbi.nlm.nih.gov/40097661","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":"23601678","id":"PMC_23601678","title":"Roquin paralogs add a new dimension to ICOS 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trout","date":"2025-04-24","source":"bioRxiv","url":"https://doi.org/10.1101/2025.04.19.649654","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":40619,"output_tokens":8119,"usd":0.121821,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":17714,"output_tokens":5766,"usd":0.11636,"stage2_stop_reason":"end_turn"},"total_usd":0.238181,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"Roquin (RC3H1) promotes degradation of ICOS mRNA via a conserved 47-bp segment in the ICOS 3' UTR; the M199R sanroque mutation in the ROQ domain abolishes this repression, leading to increased ICOS expression on T cells and lupus-like autoimmunity.\",\n      \"method\": \"Genetic mouse model (sanroque M199R point mutation), mRNA stability assays, 3' UTR reporter assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (genetic model, reporter assays, mRNA decay), replicated across subsequent studies\",\n      \"pmids\": [\"18172933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Roquin localizes to processing (P) bodies in helper T cells and represses ICOS expression by directly binding the ICOS mRNA 3' UTR (independent of microRNAs/RISC); repression requires the RNA helicase Rck and the decapping enhancer Edc4, and depends on sequences conferring P-body localization.\",\n      \"method\": \"Fluorescence microscopy (P-body localization), co-immunoprecipitation (Rck, Edc4 interactions), RNA binding assays, dominant-negative and deletion mutant analysis\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, direct RNA-binding demonstrated, P-body localization linked to function, single lab with multiple orthogonal methods\",\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; Roquin and its orthologs (Drosophila, C. elegans) all concentrate in stress granules via the ROQ domain.\",\n      \"method\": \"Fluorescence microscopy, deletion mutant analysis, gel-shift/SPR/footprinting RNA-binding assays\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ROQ domain sufficiency shown by deletion mapping, RNA binding confirmed by SPR and footprinting, replicated across species\",\n      \"pmids\": [\"20412057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Roquin recognizes a conserved stem-loop motif (constitutive decay element, CDE) in the 3' UTR of TNF-α mRNA and initiates its degradation by recruiting the Ccr4-Caf1-Not deadenylase complex; CDE sequences are present in >50 vertebrate mRNAs encoding inflammatory and developmental regulators.\",\n      \"method\": \"In vitro RNA-binding assays, mRNA stability assays in macrophages, co-immunoprecipitation of Ccr4-Caf1-Not, transcriptome-wide target identification (RNA-seq)\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct RNA binding, deadenylase complex recruitment by Co-IP, functional decay assays, genome-wide target identification, replicated by subsequent structural studies\",\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; combined loss of both paralogs (but not either alone) causes accumulation of follicular helper T cells and effector T cells.\",\n      \"method\": \"Conditional gene knockout (T-cell-specific deletion), flow cytometry, mRNA stability assays\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal single and double KO genetic epistasis, independently replicated by two labs (PMID 23583643 and 23583642)\",\n      \"pmids\": [\"23583643\", \"23583642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The RING domain of Roquin-1 is required for its localization to stress granules; RING-deficient Roquin-1 fails to localize to stress granules and permits Roquin-2 to compensate for ICOS and Tfh repression, whereas the ROQ (sanroque) mutant retains stress granule localization.\",\n      \"method\": \"Conditional knock-in/knockout mouse models, fluorescence microscopy, flow cytometry\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-specific mouse models with cellular phenotype readout, single lab\",\n      \"pmids\": [\"23583642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of the mouse Roquin ROQ domain in complex with a CDE RNA stem-loop reveals that the ROQ domain adopts an extended winged-helix (WH) fold and recognizes mainly via non-sequence-specific contacts to the RNA stem and triloop.\",\n      \"method\": \"X-ray crystallography, NMR, binding assays, cell-based reporter assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation by binding and cell-based assays, independently confirmed by contemporaneous structure (PMID 25026078)\",\n      \"pmids\": [\"25026077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structures of human Roquin ROQ domain reveal two separate RNA-binding sites: the A site binds stem-loop RNA (CDE) and the B site binds double-stranded RNA; mutagenesis of either site impairs Roquin-mediated degradation of HMGXB3 and IL6 mRNAs in human cells.\",\n      \"method\": \"X-ray crystallography, site-directed mutagenesis, mRNA decay assays in human cells\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures plus mutagenesis with functional readout, independent of PMID 25026077\",\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, inactivating their cooperative post-transcriptional repression of target mRNAs (IL-6, ICOS, c-Rel, IRF4, IκBNS, IκBζ) and thereby promoting Th17 differentiation; this derepression requires RNA binding by roquin and nuclease activity of regnase-1.\",\n      \"method\": \"MALT1 cleavage assays, T cell-specific deletion/reconstitution, mRNA decay assays, Th17 differentiation assays\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct cleavage assay for MALT1 substrate, genetic rescue experiments, functional Th17 readout, replicated by subsequent study (PMID 37988467)\",\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 motif responsible for CDE stem-loop binding; Roquin proteins can also 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 / Strong — crystal structure with biochemical validation, consistent with other structural studies\",\n      \"pmids\": [\"25504471\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Regnase-1 and Roquin regulate an overlapping set of mRNAs via a common stem-loop structure but operate in distinct subcellular compartments: Regnase-1 degrades translationally active mRNAs at the ribosome/ER (requiring UPF1 helicase), whereas Roquin controls translationally inactive mRNAs at P-bodies/stress granules (independent of UPF1).\",\n      \"method\": \"Subcellular fractionation, ribosome profiling, mRNA decay assays, UPF1 knockdown, fluorescence microscopy\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (fractionation, ribosome profiling, genetic knockdown), single lab with comprehensive mechanistic dissection\",\n      \"pmids\": [\"26000482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RC3H1/Roquin binds ~3,800 mRNA targets via CDE motifs and a novel U-rich sequence embedded in hairpins (identified by PAR-CLIP); RC3H1 uses its ROQ and Zn-finger domains to contact a binding site in the A20 3' UTR, leading to A20 mRNA decay; RC3H1 knockdown increases A20 protein expression, thereby attenuating IKK and NF-κB activity.\",\n      \"method\": \"PAR-CLIP (transcriptome-wide binding), siRNA knockdown, western blotting, NF-κB reporter assays, domain mutagenesis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — PAR-CLIP genome-wide plus domain mutagenesis, functional IKK/NF-κB pathway readout, multiple orthogonal methods\",\n      \"pmids\": [\"26170170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Roquin directly binds Argonaute2 (Ago2) and miR-146a; Roquin enhances Dicer-mediated processing of pre-miR-146a; in the absence of functional Roquin, mature miR-146a is stabilized due to decreased 3'-end uridylation. Crystallography reveals a unique HEPN domain in Roquin and the structural basis of the sanroque mutation.\",\n      \"method\": \"Co-immunoprecipitation (Ago2, miR-146a), Dicer processing assays, miRNA uridylation assays, X-ray crystallography\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — crystal structure, direct binding by Co-IP, biochemical processing assays, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"25697406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structure of the human Roquin-2 ROQ domain bound to CDE RNA shows a winged-helix fold with a flexible wing region acting as a lid for RNA binding; CDE stem-loop is recognized mainly via backbone contacts, consistent with Roquin-1 binding mode.\",\n      \"method\": \"X-ray crystallography (ligand-free and RNA-bound forms)\",\n      \"journal\": \"Acta crystallographica. Section F, Structural biology communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure in two forms, consistent with multiple independent structural studies\",\n      \"pmids\": [\"26249698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The ROQUIN RING domain directly antagonizes the catalytic α1 subunit of AMPK, promoting Tfh cell differentiation; T cell-specific deletion of the RING domain selectively abolishes Tfh responses without affecting Th1, Th2, Th17, or Treg populations.\",\n      \"method\": \"Conditional RING domain deletion in T cells, genetic interaction/rescue experiments, AMPK activity assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional domain-specific KO with selective phenotype, AMPK interaction assay, single lab\",\n      \"pmids\": [\"26496200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Roquin recognizes a novel U-rich hexaloop motif (alternative decay element, ADE) in addition to the triloop CDE; crystal structures and NMR show the ROQ domain binds ADE hexaloops from SELEX-derived sequences and from the Ox40 3' UTR with identical binding modes; ADE-like and CDE-like motifs cooperate in Ox40 repression.\",\n      \"method\": \"SELEX, X-ray crystallography, NMR, cell-based reporter assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures with SELEX identification and functional cell-based validation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"27010430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Drosophila Roquin (and human Roquin-2) recruits the CCR4-NOT deadenylase complex via a CAF40-binding motif (CBM) in their C-terminal regions; crystal structure of Dm Roquin CBM bound to CAF40 shows the CBM adopts an α-helical conformation binding a conserved CAF40 surface.\",\n      \"method\": \"X-ray crystallography, co-immunoprecipitation, pulldown assays, mRNA decay reporter assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus Co-IP and functional decay assays, conserved mechanism across species\",\n      \"pmids\": [\"28165457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Roquin inhibits the PI3K-mTOR signaling pathway by upregulating PTEN (by interfering with miR-17~92 binding to the PTEN 3' UTR) and by downregulating the Foxo1-specific E3 ubiquitin ligase Itch; loss of Roquin in T cells enhances Akt-mTOR signaling and protein synthesis.\",\n      \"method\": \"Conditional Roquin knockout in Treg cells, PI3K/mTOR inhibitor rescue experiments, luciferase 3' UTR reporter assays, western blotting\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with pharmacological rescue and reporter assays, single lab with multiple methods\",\n      \"pmids\": [\"29246441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Roquin not only interacts with stem-loop structures but also with a linear sequence element in ~half of its target mRNAs; multiple Roquin proteins bound to several stem-loops in the Nfkbid 3' UTR exert full repression redundantly via deadenylation, decapping, and translational inhibition.\",\n      \"method\": \"PAR-CLIP, luciferase reporter assays with stem-loop mutations, polysome profiling, CRISPR-mediated deletion of 3' UTR elements\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — PAR-CLIP with multiple functional readouts (decay, translation), CRISPR deletions, multiple orthogonal methods\",\n      \"pmids\": [\"30232334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"NUFIP2 directly binds Roquin with high affinity and stabilizes NUFIP2 in cells; NUFIP2 acts as a cofactor required for Roquin-mediated repression of ICOS mRNA, cooperatively binding with Roquin to non-canonical tandem stem-loops in the ICOS 3' UTR.\",\n      \"method\": \"siRNA screen (~1500 genes), co-immunoprecipitation, in vitro binding assays, reporter assays, electrophoretic mobility shift assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic RNAi screen followed by direct binding assays and functional reporter validation, multiple orthogonal methods\",\n      \"pmids\": [\"29352114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"A human homozygous nonsense R688* RC3H1 mutation causes Roquin-1 loss that leads to hyperinflammation (HLH-like); mechanistically, R688* Roquin-1 fails to localize to P-bodies and interact with the CCR4-NOT deadenylation complex, impairing mRNA decay of ICOS, OX40, and TNF targets.\",\n      \"method\": \"Patient-derived cells, immunofluorescence (P-body localization), co-immunoprecipitation (CCR4-NOT), mRNA stability assays, flow cytometry\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — human genetic variant with mechanistic dissection by Co-IP, localization assays, and mRNA decay measurements; unique patient case with multiple orthogonal methods\",\n      \"pmids\": [\"31636267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HCMV exploits Roquin by upregulating its expression; Roquin binds directly to IRF1 mRNA (identified by CLIP-seq) and reduces IRF1 expression, thereby suppressing IRF1-dependent antiviral gene activation during HCMV infection.\",\n      \"method\": \"CLIP-seq, loss-of-function screening, transcriptome profiling, mRNA stability assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CLIP-seq for direct binding plus functional transcriptome and loss-of-function data, single lab\",\n      \"pmids\": [\"31451648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The Roquin ROQ domain recognizes AU-rich elements (AREs) in a CDE-like stem-loop conformation, while AUF1 recognizes the same AREs in a linear form; NMR structures of CDE-like folded AREs (free and Roquin-bound) demonstrate shape-specific recognition by Roquin.\",\n      \"method\": \"NMR spectroscopy (solution structures of free and protein-bound AREs), RNA binding assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structures in free and bound forms with biochemical binding data, single lab\",\n      \"pmids\": [\"32491174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RC3H1/Roquin is a major mediator of iron-regulated TfR1 mRNA stability; Roquin destabilizes TfR1 mRNA in a manner fully dependent on three hairpin loops in the TfR1 3' UTR; TfR1 mRNA degradation by Roquin does not require ongoing translation.\",\n      \"method\": \"Gene knockout (HAP1, HUVEC, L-M, MEF cells), siRNA knockdown, 3' UTR hairpin loop mutagenesis, mRNA stability assays\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple cell-type knockouts, hairpin mutagenesis, translation-independence demonstrated, multiple orthogonal methods\",\n      \"pmids\": [\"33898949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Physical interaction between Roquin-1 and Regnase-1 is mediated by a defined interaction surface in Roquin-1 that includes the sanroque residue (M199); mutations disrupting this interaction cause Tfh cell accumulation, germinal center B cells, and autoantibody formation.\",\n      \"method\": \"Co-immunoprecipitation, mutagenesis (interaction surface mapping), conditional triple KO (Roquin-1/2 and Regnase-1), flow cytometry\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP with mutagenesis defining interaction surface, genetic triple-KO epistasis, multiple orthogonal methods\",\n      \"pmids\": [\"34811541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Roquin is the strongest repressor of CD8 T cell expansion identified in genome-wide CRISPR screens; Roquin ablation boosts T cell proliferation by enhancing cell-cycle progression and upregulating IRF4.\",\n      \"method\": \"In vivo and in vitro CRISPR screens, KO validation, flow cytometry, gene expression analysis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo CRISPR screen with functional validation, single lab\",\n      \"pmids\": [\"34879274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NMR analysis of the full-length Ox40 mRNA 3' UTR reveals that Roquin uses its extended bimodal ROQ domain to sequentially engage decay elements in a 2:1 (protein:RNA) stoichiometry; the binding of Roquin to individual decay elements depends on their structural embedment within the full 3' UTR.\",\n      \"method\": \"NMR spectroscopy, SAXS, In-line probing, SHAPE-seq, binding assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR plus SAXS and multiple RNA probing methods provide structural and binding data on the full 3' UTR, single lab\",\n      \"pmids\": [\"35357505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TCR signal strength is translated by MALT1 into graded cleavage of Roquin-1; only high TCR strength enables derepression of high-affinity Roquin targets (e.g., IκBNS) and promotes Th17 differentiation; rendering Roquin-1 insensitive to MALT1 cleavage (Rc3h1Mins/Mins) prevents spontaneous T cell activation caused by constitutively active MALT1.\",\n      \"method\": \"Knock-in mice (MALT1-cleavage-resistant Roquin-1), genetic epistasis (Rc3h1Mins × Malt1TBM), EAE model, Th17 differentiation assays, western blotting for Roquin cleavage\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knock-in genetic epistasis with specific cleavage-resistant allele, multiple in vivo and in vitro readouts\",\n      \"pmids\": [\"37988467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RC3H1 acts as an E3 ubiquitin ligase promoting GPX4 ubiquitination-mediated degradation; MALT1 inhibition upregulates RC3H1, which then enhances GPX4 ubiquitination and degradation to induce ferroptosis in liver cancer cells.\",\n      \"method\": \"Genome-wide CRISPR screen (FACS-based), rescue assays, ubiquitination assays, western blotting\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen validated by rescue and ubiquitination assays, single lab\",\n      \"pmids\": [\"39739814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Rc3h1 post-transcriptionally represses transferrin receptor 1 (Tfr1) mRNA in osteoclasts, restricting iron absorption and mitochondrial respiration; Rc3h1-deficient osteoclasts show enhanced Tfr1 expression, increased mitochondrial respiration and osteoclast activation, and mice exhibit low bone mass.\",\n      \"method\": \"Conditional Rc3h1 knockout in osteoclasts, mRNA stability assays, iron content measurement, mitochondrial respiration assays, Tfr1 inhibitor rescue\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with targeted rescue, multiple functional readouts, single lab\",\n      \"pmids\": [\"39659568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Solution NMR structure of the Roquin-1 CCCH zinc finger domain reveals it recognizes AU-rich RNA sequences; the ZnF and ROQ domains simultaneously bind a natural Roquin target transcript, suggesting Roquin integrates RNA shape (ROQ) and sequence (ZnF) features through a tandem domain mechanism.\",\n      \"method\": \"NMR spectroscopy (solution structure), RBNS-NMR pipeline for RNA binding preferences, in vitro RNA binding assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR solution structure with systematic RNA binding characterization, demonstrates cooperative domain engagement\",\n      \"pmids\": [\"38953172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MNSFβ binds directly to RC3H1 (demonstrated by co-immunoprecipitation), and elevated MNSFβ promotes TNFα production by interacting with RC3H1, which normally suppresses TNFα mRNA.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown in THP1-derived macrophages, ELISA\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP interaction confirmed and functional rescue by RC3H1 knockdown, single lab\",\n      \"pmids\": [\"34589082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MNSFβ promotes LPS-induced TNFα expression by facilitating stress granule formation and translocation of RC3H1 to stress granules (via interaction with RC3H1 and FMR1), thereby inactivating RC3H1-mediated TNFα mRNA degradation; the RC3H1 81-326 aa region binds MNSFβ 101-133 aa region.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, stress granule imaging, mRNA stability assays, peptide inhibitor (HEPN2) rescue experiments\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mapping by Co-IP, functional mRNA decay assays, stress granule imaging, single lab with multiple methods\",\n      \"pmids\": [\"39260307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Single-molecule force spectroscopy (SMFS) combined with NMR reveals opposing effects of the two Roquin ROQ domain RNA-binding sites on ADE stem stability: the A-site strongly stabilizes the apical ADE stem-loop, while the B-site destabilizes the ADE central stem through single-strand RNA binding, suggesting both sites jointly direct mRNA decay with context-dependent 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 / Moderate — two orthogonal biophysical methods (SMFS and NMR) with kinetic measurements, mechanistically novel finding\",\n      \"pmids\": [\"40203046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A heterozygous missense F225S variant in the ROQ domain of RC3H1 causes autosomal-dominant primary immunodeficiency; F225S Roquin-1 fails to repress ICOS1, IL6, and other target mRNAs (increased message stability), demonstrating that ROQ domain integrity is required for post-transcriptional repression.\",\n      \"method\": \"Whole-exome sequencing, patient primary cells, transfection of F225S variant, mRNA stability assays, flow cytometry\",\n      \"journal\": \"The Journal of allergy and clinical immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived cells with single-variant transfection and mRNA stability readout, single lab\",\n      \"pmids\": [\"40769319\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RC3H1/Roquin-1 is an RNA-binding E3 ubiquitin ligase that post-transcriptionally silences inflammatory and co-stimulatory mRNAs (including ICOS, Ox40, TNF, IL-6, A20, IRF1, and TfR1) by recognizing stem-loop decay elements (CDEs and ADEs) in their 3' UTRs via its ROQ domain (winged-helix fold with two RNA-binding sites) and a flanking CCCH zinc finger domain; it recruits the CCR4-NOT deadenylase complex (and decapping factors Rck/Edc4) to drive mRNA decay of translationally inactive mRNAs in P-bodies/stress granules, cooperates physically with Regnase-1 (which degrades translationally active mRNAs at the ribosome), and is inactivated by MALT1-mediated cleavage downstream of TCR signaling in a signal-strength-dependent manner to allow graded derepression of target mRNAs during T cell activation and Th17 differentiation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RC3H1 (Roquin-1) is an RNA-binding post-transcriptional repressor that silences inflammatory and co-stimulatory mRNAs by recognizing structured decay elements in their 3' UTRs and targeting them for degradation [#0, #3]. Its central ROQ domain adopts an extended winged-helix fold that recognizes stem-loop constitutive decay elements (CDEs) largely through shape-specific backbone contacts to the stem and triloop [#6, #9], and contains two distinct RNA-binding surfaces — an A site that engages folded stem-loops and a B site that binds duplex/single-stranded RNA — both of which are required for efficient target decay [#7, #33]. Beyond the canonical CDE triloop, the ROQ domain also recognizes U-rich hexaloop alternative decay elements (ADEs) and CDE-folded AU-rich elements with the same binding mode, while the flanking CCCH zinc finger reads AU-rich sequence so that shape and sequence recognition are integrated across tandem domains; multiple decay elements within a single 3' UTR are engaged cooperatively [#15, #22, #30, #26]. Roquin drives decay by recruiting the CCR4-NOT deadenylase complex via a CAF40-binding motif and by acting through decapping factors (Rck/Edc4) at P-bodies and stress granules, where it controls translationally inactive mRNAs in cooperation with the ribosome-associated endonuclease Regnase-1 that handles translationally active transcripts [#1, #3, #16, #10, #24]. Validated targets include ICOS, Ox40, TNF, IL-6, A20, IRF1 and transferrin receptor 1 (TfR1) mRNAs, linking Roquin to control of follicular helper and effector T-cell responses, NF-\\u03baB signaling, antiviral IRF1 responses, and iron homeostasis [#0, #4, #11, #21, #23, #29]. Roquin activity is set by T-cell receptor signal strength: the paracaspase MALT1 cleaves Roquin-1 in a graded, signal-strength-dependent manner to derepress target mRNAs and license Th17 differentiation [#8, #27]. Loss-of-function and missense ROQ-domain mutations in humans cause immune disease — a homozygous R688* nonsense allele produces HLH-like hyperinflammation through failed P-body localization and CCR4-NOT recruitment, and a heterozygous F225S ROQ variant causes autosomal-dominant primary immunodeficiency through loss of mRNA repression [#20, #34]. Separately, Roquin functions as an E3 ubiquitin ligase promoting GPX4 ubiquitination and ferroptosis [#28].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established that Roquin is a physiological repressor of an immune mRNA, defining its core function before any molecular mechanism was known.\",\n      \"evidence\": \"sanroque M199R point-mutant mouse, ICOS 3' UTR reporter and mRNA stability assays\",\n      \"pmids\": [\"18172933\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the RNA element recognized or the decay machinery engaged\", \"Mechanism of the M199R defect unresolved at the structural level\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Localized Roquin function to RNA-decay foci and identified decapping machinery, showing it acts directly on mRNA independent of the RISC pathway.\",\n      \"evidence\": \"P-body/stress-granule fluorescence microscopy, Rck/Edc4 Co-IP, RNA-binding and deletion-mutant analysis in helper T cells\",\n      \"pmids\": [\"20639877\", \"20412057\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The precise RNA structural motif recognized was not defined\", \"Whether deadenylation precedes decapping was not established\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the constitutive decay element (CDE) stem-loop as the recognition motif and CCR4-NOT recruitment as the decay mechanism, generalizing Roquin to a regulon of inflammatory mRNAs.\",\n      \"evidence\": \"In vitro RNA binding, macrophage decay assays, CCR4-NOT Co-IP, transcriptome-wide target identification\",\n      \"pmids\": [\"23663784\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of CDE recognition not yet solved\", \"Did not explain redundancy with the Roquin-2 paralog\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated functional redundancy between Roquin-1 and Roquin-2 and dissected RING-domain contribution to subcellular targeting, clarifying genetic interpretation of single-paralog studies.\",\n      \"evidence\": \"Single and double conditional T-cell knockouts, RING-domain mouse models, flow cytometry, mRNA stability assays\",\n      \"pmids\": [\"23583643\", \"23583642\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular role of the RING domain in stress-granule localization left mechanistically open\", \"Did not resolve whether RING has catalytic ligase activity in this context\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Provided crystal structures of the ROQ domain bound to CDE RNA, revealing a winged-helix fold with two RNA-binding sites that read RNA shape rather than sequence.\",\n      \"evidence\": \"X-ray crystallography of mouse and human ROQ domains, NMR, mutagenesis, cell-based decay reporters\",\n      \"pmids\": [\"25026077\", \"25026078\", \"25504471\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address how full 3' UTRs with multiple elements are engaged\", \"Role of the zinc finger in RNA recognition unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified MALT1 cleavage of Roquin downstream of TCR signaling as the switch that derepresses targets and drives Th17 differentiation, linking Roquin to signal-dependent control.\",\n      \"evidence\": \"MALT1 cleavage assays, T-cell deletion/reconstitution, decay assays, Th17 differentiation readouts\",\n      \"pmids\": [\"25282160\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish how cleavage extent is tuned to signal strength\", \"Cooperative mechanism with Regnase-1 not fully resolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Separated Roquin and Regnase-1 into distinct subcellular decay pathways acting on overlapping targets, explaining their non-redundant cooperation.\",\n      \"evidence\": \"Subcellular fractionation, ribosome profiling, UPF1 knockdown, fluorescence microscopy, decay assays\",\n      \"pmids\": [\"26000482\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physical basis of Roquin\\u2013Regnase-1 cooperation not yet mapped\", \"How targets are partitioned between compartments unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Mapped the transcriptome-wide binding landscape and extended the binding repertoire to U-rich and linear motifs, while linking Roquin to A20/NF-\\u03baB control and miRNA biogenesis.\",\n      \"evidence\": \"PAR-CLIP, domain mutagenesis, NF-\\u03baB reporters, Ago2/miR-146a Co-IP, Dicer processing and uridylation assays, crystallography\",\n      \"pmids\": [\"26170170\", \"25697406\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional weight of linear versus structured motifs not quantified\", \"Relationship between mRNA decay and miRNA-processing roles unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined the alternative decay element (ADE), a U-rich hexaloop, as a second class of structured motif recognized by the same ROQ surface, broadening the structural code for target recognition.\",\n      \"evidence\": \"SELEX, X-ray crystallography, NMR, cell-based reporter assays of Ox40 elements\",\n      \"pmids\": [\"27010430\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CDE and ADE elements cooperate within a UTR was not structurally resolved\", \"Did not address stoichiometry on full-length transcripts\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified a CAF40-binding motif as the direct molecular link recruiting the CCR4-NOT complex, defining the conserved effector step of Roquin-mediated decay.\",\n      \"evidence\": \"Crystal structure of Roquin CBM\\u2013CAF40, Co-IP, pulldowns, decay reporter assays\",\n      \"pmids\": [\"28165457\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve coordination of deadenylation with decapping in vivo\", \"Contribution of CBM versus P-body localization to net decay unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed that multiple Roquin molecules engage several stem-loops redundantly to enforce repression through deadenylation, decapping and translational inhibition, and identified NUFIP2 as a required cofactor.\",\n      \"evidence\": \"PAR-CLIP, polysome profiling, CRISPR 3' UTR deletions, siRNA screen, in vitro binding and reporter assays\",\n      \"pmids\": [\"30232334\", \"29352114\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of NUFIP2 dependence across the target set not defined\", \"Stoichiometry of multi-element engagement not structurally resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected Roquin to human disease and to antiviral and metabolic signaling, showing loss of function causes hyperinflammation and that viruses co-opt Roquin to blunt IRF1 responses.\",\n      \"evidence\": \"Patient cells with R688* (IF, CCR4-NOT Co-IP, decay assays), CLIP-seq and loss-of-function screening in HCMV infection, PI3K-mTOR rescue and reporter assays\",\n      \"pmids\": [\"31636267\", \"31451648\", \"29246441\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PI3K-mTOR regulation rests on single-lab Medium evidence\", \"Causality of the HCMV-Roquin axis in vivo not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mapped the physical Roquin-1\\u2013Regnase-1 interaction surface (including the sanroque residue) and extended Roquin function to iron homeostasis via TfR1 mRNA, while genome-wide screens flagged it as a dominant brake on CD8 T-cell expansion.\",\n      \"evidence\": \"Co-IP and interaction mapping, conditional triple-KO epistasis, TfR1 3' UTR hairpin mutagenesis and decay assays in multiple cell types, CRISPR screens, MNSF\\u03b2 Co-IP\",\n      \"pmids\": [\"34811541\", \"33898949\", \"34879274\", \"34589082\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"MNSF\\u03b2 interaction and CD8 expansion roles rest on single-lab Medium evidence\", \"How the same residue serves both RNA recognition and Regnase-1 binding not fully separated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved how Roquin engages an intact 3' UTR, showing sequential bimodal engagement of decay elements with defined stoichiometry dependent on RNA structural context.\",\n      \"evidence\": \"NMR, SAXS, in-line probing and SHAPE-seq on full-length Ox40 3' UTR, binding assays\",\n      \"pmids\": [\"35357505\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not link binding order to in-cell decay kinetics\", \"Role of cofactors in shaping engagement not addressed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated that MALT1 converts TCR signal strength into graded Roquin-1 cleavage, establishing an analog-to-threshold mechanism for selective target derepression.\",\n      \"evidence\": \"Cleavage-resistant knock-in mice, genetic epistasis with constitutively active MALT1, EAE and Th17 assays, cleavage western blots\",\n      \"pmids\": [\"37988467\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Affinity hierarchy that sets target thresholds not biochemically mapped for all targets\", \"Whether Regnase-1 cleavage is similarly graded not addressed here\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the zinc-finger contribution to RNA recognition and revealed a distinct E3 ligase function promoting GPX4 degradation and ferroptosis, broadening Roquin beyond mRNA decay.\",\n      \"evidence\": \"NMR solution structure of the CCCH ZnF with RBNS-NMR binding, CRISPR screen with ubiquitination and rescue assays, osteoclast conditional KO with TfR1 rescue, MNSF\\u03b2 stress-granule imaging\",\n      \"pmids\": [\"38953172\", \"39739814\", \"39659568\", \"39260307\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase activity toward GPX4 rests on single-lab Medium evidence\", \"Relationship between RNA-decay and ubiquitin-ligase functions not reconciled\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided biophysical insight into how the two ROQ RNA-binding sites exert opposing structural effects on a target stem-loop, and added a second human disease allele confirming ROQ-domain integrity as essential for repression.\",\n      \"evidence\": \"Single-molecule force spectroscopy and NMR on ADE stem-loops; whole-exome sequencing and F225S patient-cell mRNA stability assays\",\n      \"pmids\": [\"40203046\", \"40769319\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"F225S mechanism rests on single-variant transfection (Medium)\", \"How A-/B-site cooperation selects targets in cells not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How Roquin's mRNA-decay activity and its E3 ubiquitin-ligase/GPX4-ferroptosis activity are coordinated within a cell, and whether they share regulatory inputs, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural or genetic dissection separating the two activities\", \"Substrate scope of the E3 ligase function beyond GPX4 unknown\", \"Whether MALT1 cleavage regulates the ligase activity untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 3, 6, 7, 11, 15, 22, 26, 30]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [3, 10, 16, 23]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [16, 19]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [28]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 2, 10]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [1, 2, 5, 10, 20, 32]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [3, 10, 11, 18, 23]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 4, 8, 11, 21, 27]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8, 11, 17, 27]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [28]}\n    ],\n    \"complexes\": [\"P-body\", \"stress granule\"],\n    \"partners\": [\"CNOT1/CCR4-NOT\", \"CAF40\", \"Regnase-1 (ZC3H12A)\", \"NUFIP2\", \"AGO2\", \"MALT1\", \"MNSFβ\", \"GPX4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}