{"gene":"INTS4","run_date":"2026-06-10T01:55:23","timeline":{"discoveries":[{"year":2018,"finding":"INTS4 is a specific and conserved interaction partner of INTS9 and INTS11 that binds only the INTS9/11 heterodimer, not either subunit individually. INTS4 possesses N-terminal HEAT repeats (similar to Symplekin) and a β-sheet-rich C-terminal region, both of which are required for binding INTS9/11. INTS4/9/11 form a heterotrimeric 'cleavage module' responsible for the endonucleolytic activity of the Integrator complex.","method":"Co-immunoprecipitation, domain-deletion analysis, functional assays for UsnRNA 3'-end processing, Cajal body integrity, and histone locus body formation; depletion of INTS4 abrogated snRNA processing","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP with domain mapping, multiple orthogonal functional readouts, replicated in subsequent structural studies","pmids":["29471365"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structure of the INTS4/9/11 ternary complex at 3.5 Å resolution shows that INTS4, a helical-repeat protein, stabilizes the nuclease domains of INTS11 and INTS9 via several interdependent interfaces. All three subunits form a composite electropositive groove that constitutes a putative RNA-binding path within the catalytic core.","method":"Cryo-electron microscopy at 3.5 Å resolution; biochemical reconstitution of INTS4/9/11 subcomplex","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-atomic resolution cryo-EM structure with biochemical reconstitution, independently consistent with prior Co-IP data","pmids":["33548203"],"is_preprint":false},{"year":2012,"finding":"Depletion of INTS4 abrogates snRNA 3'-end processing, causes accumulation of the Cajal body marker coilin in nucleoli, disrupts normal Cajal body composition (loss of SMN, Sm proteins, and snRNAs from foci), and induces cytoplasmic granule formation of SMN and Sm proteins, demonstrating that INTS4 is essential for snRNA maturation and Cajal body homeostasis.","method":"siRNA-mediated knockdown of INTS4 in human cells, immunofluorescence microscopy, northern blotting for snRNA processing","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KD with defined cellular and molecular phenotypes, multiple orthogonal readouts, independently replicated in Wagner lab (PMID 29471365)","pmids":["22250197"],"is_preprint":false},{"year":2022,"finding":"Cryo-EM structure of the Drosophila INTS4/9/11 (ICM) at 2.74 Å resolution reveals stable association of an inositol hexakisphosphate (IP6) molecule in a highly electropositive pocket at the interface of all three ICM subunits, 55 Å from the INTS11 active site. IP6 binding was confirmed in human ICM. Mutations in the IP6-binding site or disruption of IP6 biosynthesis significantly reduced Integrator function in snRNA 3'-end processing and mRNA transcription attenuation.","method":"Cryo-EM structure determination (2.74 Å), site-directed mutagenesis of IP6-binding residues, IP6-binding assays, snRNA processing assays, mRNA transcription attenuation assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic-resolution cryo-EM with mutagenesis and multiple functional assays; replicated across Drosophila and human ICM","pmids":["36180473"],"is_preprint":false},{"year":2024,"finding":"BRAT1 and WDR73 are biogenesis factors for the INTS4/9/11 cleavage module: they keep INTS9/11 inactive by blocking the endonuclease active site and prevent premature INTS4 association during maturation. BRAT1 also facilitates nuclear import of INTS9/11, after which INTS4 joins in the nucleus. Final BRAT1 release requires locking of the mature cleavage module conformation by IP6.","method":"Co-immunoprecipitation, assembly/maturation assays, nuclear import assays, IP6-dependence experiments; mutations in BRAT1, WDR73, and INTS11 linked to Integrator assembly defects","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP and reconstitution experiments with functional assembly readouts, multiple orthogonal methods in one study","pmids":["39032489"],"is_preprint":false},{"year":2022,"finding":"An INTS11 SUMOylation-deficient mutant retains interaction with INTS4 and INTS9 but loses interaction with other Integrator subunits, indicating that the INTS4/9/11 cleavage module interaction is independent of INTS11 SUMOylation whereas broader complex assembly is not.","method":"Co-immunoprecipitation with SUMOylation-deficient INTS11 mutant (Lys381/462/475Arg), comparison of interactions with INTS4, INTS9, and other subunits","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP with defined mutant, single lab, findings specifically about INTS4 interaction are a subsidiary result of a study focused on INTS11","pmids":["36454007"],"is_preprint":false},{"year":2025,"finding":"In C. elegans, auxin-inducible degradation of INTS-4 (the INTS4 ortholog) demonstrates that Integrator activity is critical for development at the L1 larval stage but becomes dispensable after L2/L3; germline-specific INTS-4 degradation causes accumulation of misprocessed snRNA transcripts without impairing development or lifespan.","method":"Auxin-inducible degron (AID) system for temporal depletion of C. elegans INTS-4; snRNA processing assays, developmental and lifespan phenotyping","journal":"Biology open","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean conditional KO with defined molecular (snRNA misprocessing) and developmental phenotypes in C. elegans, single lab","pmids":["40071568"],"is_preprint":false}],"current_model":"INTS4 is a helical-repeat scaffold protein that, together with INTS9 and INTS11, forms the heterotrimeric Integrator cleavage module (ICM): INTS4 binds only the pre-assembled INTS9/11 heterodimer (not either subunit alone), stabilizes the nuclease domains via multiple interfaces, and contributes to an electropositive RNA-binding groove; ICM assembly in the nucleus is chaperoned by BRAT1 and WDR73 (which block premature INTS4 association and the endonuclease active site), and is completed by incorporation of inositol hexakisphosphate (IP6) as an essential co-factor that locks the mature conformation and is required for both snRNA 3'-end processing and mRNA transcription attenuation."},"narrative":{"mechanistic_narrative":"INTS4 is a helical-repeat scaffold subunit of the Integrator complex that, together with INTS9 and INTS11, forms the heterotrimeric cleavage module responsible for the complex's endonucleolytic activity in small nuclear RNA (snRNA) 3'-end processing [PMID:29471365, PMID:22250197]. INTS4 binds specifically to the pre-assembled INTS9/11 heterodimer—not either subunit alone—using both N-terminal HEAT repeats and a β-sheet-rich C-terminal region, and cryo-EM shows it stabilizes the INTS9/INTS11 nuclease domains through multiple interdependent interfaces while contributing to a composite electropositive groove that forms the catalytic core's RNA-binding path [PMID:29471365, PMID:33548203]. Maturation of the cleavage module is templated: BRAT1 and WDR73 keep INTS9/11 inactive and block premature INTS4 association, BRAT1 mediates nuclear import of INTS9/11, INTS4 joins in the nucleus, and incorporation of inositol hexakisphosphate (IP6) into an electropositive pocket at the three-subunit interface locks the mature conformation required for both snRNA 3'-end processing and mRNA transcription attenuation [PMID:36180473, PMID:39032489]. Loss of INTS4 abrogates snRNA maturation and disrupts Cajal body homeostasis, causing mislocalization of coilin, SMN, and Sm proteins [PMID:22250197].","teleology":[{"year":2012,"claim":"Established that INTS4 is functionally essential for snRNA maturation, answering whether this subunit contributes to Integrator output at the cellular level.","evidence":"siRNA knockdown of INTS4 in human cells with immunofluorescence and northern blotting","pmids":["22250197"],"confidence":"High","gaps":["Did not define INTS4's biochemical role within the complex","Did not identify direct binding partners of INTS4"]},{"year":2018,"claim":"Defined INTS4 as a dedicated scaffold of a heterotrimeric cleavage module, showing it binds the INTS9/11 heterodimer but neither subunit alone and mapping the required domains.","evidence":"Reciprocal Co-IP with domain-deletion analysis and snRNA processing/Cajal body readouts in human cells","pmids":["29471365"],"confidence":"High","gaps":["No structural basis for the INTS4/9/11 interaction","Did not address how the module is assembled in vivo"]},{"year":2021,"claim":"Provided the structural mechanism by which INTS4 organizes the catalytic core, showing it stabilizes the INTS9/INTS11 nuclease domains and helps form a composite RNA-binding groove.","evidence":"3.5 Å cryo-EM structure with biochemical reconstitution of the INTS4/9/11 subcomplex","pmids":["33548203"],"confidence":"High","gaps":["RNA-binding path inferred from electrostatics, not RNA-bound structure","Did not reveal cofactor requirements"]},{"year":2022,"claim":"Identified IP6 as an essential structural cofactor locking the mature cleavage module, linking a small-molecule ligand to both snRNA processing and transcription attenuation.","evidence":"2.74 Å cryo-EM of Drosophila ICM plus mutagenesis, IP6-binding assays, and functional assays, confirmed in human ICM","pmids":["36180473"],"confidence":"High","gaps":["IP6 site is 55 Å from the active site; the allosteric coupling mechanism is not detailed","Did not establish the assembly order of IP6 incorporation"]},{"year":2022,"claim":"Distinguished cleavage-module assembly from broader complex assembly, showing the INTS4/9/11 interaction is independent of INTS11 SUMOylation.","evidence":"Co-IP with a SUMOylation-deficient INTS11 mutant comparing interactions across subunits","pmids":["36454007"],"confidence":"Medium","gaps":["Subsidiary result of an INTS11-focused study","Single lab; functional consequence for INTS4 not directly assayed"]},{"year":2024,"claim":"Revealed a chaperoned biogenesis pathway, showing BRAT1/WDR73 keep INTS9/11 inactive and block premature INTS4 binding, with INTS4 joining in the nucleus and IP6-dependent BRAT1 release completing maturation.","evidence":"Co-IP, assembly/maturation and nuclear import assays with IP6-dependence experiments","pmids":["39032489"],"confidence":"High","gaps":["Precise trigger for INTS4 recruitment in the nucleus not fully resolved","How disease mutations in BRAT1/WDR73 perturb the pathway not detailed"]},{"year":2025,"claim":"Demonstrated temporal and tissue-specific requirements for INTS4 in a whole organism, separating developmental essentiality from germline snRNA processing.","evidence":"Auxin-inducible degron depletion of C. elegans INTS-4 with snRNA processing and developmental/lifespan phenotyping","pmids":["40071568"],"confidence":"Medium","gaps":["Single lab in C. elegans; mammalian stage-specificity not addressed","Molecular basis for stage-dependent dispensability unknown"]},{"year":null,"claim":"How IP6 binding at a site 55 Å from the active site allosterically licenses catalysis, and how RNA substrate is engaged by the electropositive groove, remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No RNA-bound structure of the cleavage module","Allosteric pathway from the IP6 pocket to the active site uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,2,3]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[1]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,4]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,2,3]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[3]}],"complexes":["Integrator complex","Integrator cleavage module (INTS4/9/11)"],"partners":["INTS9","INTS11","BRAT1","WDR73"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96HW7","full_name":"Integrator complex subunit 4","aliases":[],"length_aa":963,"mass_kda":108.2,"function":"Component of the integrator complex, a multiprotein complex that terminates RNA polymerase II (Pol II) transcription in the promoter-proximal region of genes (PubMed:29471365, PubMed:33243860, PubMed:33548203, PubMed:38570683). The integrator complex provides a quality checkpoint during transcription elongation by driving premature transcription termination of transcripts that are unfavorably configured for transcriptional elongation: the complex terminates transcription by (1) catalyzing dephosphorylation of the C-terminal domain (CTD) of Pol II subunit POLR2A/RPB1 and SUPT5H/SPT5, (2) degrading the exiting nascent RNA transcript via endonuclease activity and (3) promoting the release of Pol II from bound DNA (PubMed:33243860, PubMed:38570683). The integrator complex is also involved in terminating the synthesis of non-coding Pol II transcripts, such as enhancer RNAs (eRNAs), small nuclear RNAs (snRNAs), telomerase RNAs and long non-coding RNAs (lncRNAs) (PubMed:16239144). Within the integrator complex, INTS4 acts as an scaffold that links INTS9 and INTS11 (PubMed:29471365, PubMed:33548203). Mediates recruitment of cytoplasmic dynein to the nuclear envelope, probably as component of the integrator complex (PubMed:23904267)","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q96HW7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/INTS4","classification":"Common Essential","n_dependent_lines":381,"n_total_lines":383,"dependency_fraction":0.9947780678851175},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"POLR2B","stoichiometry":0.2},{"gene":"POLR2E","stoichiometry":0.2},{"gene":"POLR2F","stoichiometry":0.2},{"gene":"POLR2K","stoichiometry":0.2},{"gene":"PPP2CA","stoichiometry":0.2},{"gene":"SEM1","stoichiometry":0.2},{"gene":"SSRP1","stoichiometry":0.2},{"gene":"SUPT5H","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/INTS4","total_profiled":1310},"omim":[{"mim_id":"611675","title":"KIAA0513 GENE; KIAA0513","url":"https://www.omim.org/entry/611675"},{"mim_id":"611348","title":"INTEGRATOR COMPLEX SUBUNIT 4; INTS4","url":"https://www.omim.org/entry/611348"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoli rim","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/INTS4"},"hgnc":{"alias_symbol":["INT4","MGC16733","MST093"],"prev_symbol":[]},"alphafold":{"accession":"Q96HW7","domains":[{"cath_id":"1.25.10.10","chopping":"42-182_193-262","consensus_level":"medium","plddt":90.5569,"start":42,"end":262},{"cath_id":"1.25.10.10","chopping":"264-323_371-435","consensus_level":"medium","plddt":89.4332,"start":264,"end":435},{"cath_id":"1.25.10","chopping":"443-588","consensus_level":"medium","plddt":91.8753,"start":443,"end":588},{"cath_id":"-","chopping":"667-804","consensus_level":"high","plddt":84.8228,"start":667,"end":804},{"cath_id":"2.60.40.10","chopping":"820-926_940-958","consensus_level":"high","plddt":84.3349,"start":820,"end":958}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96HW7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96HW7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96HW7-F1-predicted_aligned_error_v6.png","plddt_mean":83.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=INTS4","jax_strain_url":"https://www.jax.org/strain/search?query=INTS4"},"sequence":{"accession":"Q96HW7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96HW7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96HW7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96HW7"}},"corpus_meta":[{"pmid":"29471365","id":"PMC_29471365","title":"Integrator subunit 4 is a 'Symplekin-like' scaffold that associates with INTS9/11 to form the Integrator cleavage module.","date":"2018","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/29471365","citation_count":67,"is_preprint":false},{"pmid":"33548203","id":"PMC_33548203","title":"Structure of the catalytic core of the Integrator complex.","date":"2021","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/33548203","citation_count":55,"is_preprint":false},{"pmid":"22250197","id":"PMC_22250197","title":"The integrator complex is required for integrity of Cajal bodies.","date":"2012","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/22250197","citation_count":37,"is_preprint":false},{"pmid":"17010949","id":"PMC_17010949","title":"Characterization of KIAA0513, a novel signaling molecule that interacts with modulators of neuroplasticity, apoptosis, and the cytoskeleton.","date":"2006","source":"Brain research","url":"https://pubmed.ncbi.nlm.nih.gov/17010949","citation_count":23,"is_preprint":false},{"pmid":"36180473","id":"PMC_36180473","title":"Inositol hexakisphosphate is required for Integrator function.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/36180473","citation_count":17,"is_preprint":false},{"pmid":"36454007","id":"PMC_36454007","title":"SUMO conjugation regulates the activity of the Integrator complex.","date":"2022","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/36454007","citation_count":10,"is_preprint":false},{"pmid":"39032489","id":"PMC_39032489","title":"Assembly mechanism of Integrator's RNA cleavage module.","date":"2024","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/39032489","citation_count":9,"is_preprint":false},{"pmid":"38074468","id":"PMC_38074468","title":"Coevolution combined with molecular dynamics simulations provides structural and mechanistic insights into the interactions between the integrator complex subunits.","date":"2023","source":"Computational and structural biotechnology journal","url":"https://pubmed.ncbi.nlm.nih.gov/38074468","citation_count":3,"is_preprint":false},{"pmid":"40071568","id":"PMC_40071568","title":"Differential effect of ubiquitous and germline depletion of Integrator complex function on C. elegans physiology.","date":"2025","source":"Biology open","url":"https://pubmed.ncbi.nlm.nih.gov/40071568","citation_count":2,"is_preprint":false},{"pmid":"41562477","id":"PMC_41562477","title":"Blood-Based Immunoglobulin-Bound Neoantigen Signatures Associated with MEN Type 1-Related Duodenopancreatic Neuroendocrine Tumor Progression.","date":"2026","source":"Journal of the American College of Surgeons","url":"https://pubmed.ncbi.nlm.nih.gov/41562477","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6506,"output_tokens":2109,"usd":0.025576,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9002,"output_tokens":2554,"usd":0.05443,"stage2_stop_reason":"end_turn"},"total_usd":0.080006,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2018,\n      \"finding\": \"INTS4 is a specific and conserved interaction partner of INTS9 and INTS11 that binds only the INTS9/11 heterodimer, not either subunit individually. INTS4 possesses N-terminal HEAT repeats (similar to Symplekin) and a β-sheet-rich C-terminal region, both of which are required for binding INTS9/11. INTS4/9/11 form a heterotrimeric 'cleavage module' responsible for the endonucleolytic activity of the Integrator complex.\",\n      \"method\": \"Co-immunoprecipitation, domain-deletion analysis, functional assays for UsnRNA 3'-end processing, Cajal body integrity, and histone locus body formation; depletion of INTS4 abrogated snRNA processing\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP with domain mapping, multiple orthogonal functional readouts, replicated in subsequent structural studies\",\n      \"pmids\": [\"29471365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structure of the INTS4/9/11 ternary complex at 3.5 Å resolution shows that INTS4, a helical-repeat protein, stabilizes the nuclease domains of INTS11 and INTS9 via several interdependent interfaces. All three subunits form a composite electropositive groove that constitutes a putative RNA-binding path within the catalytic core.\",\n      \"method\": \"Cryo-electron microscopy at 3.5 Å resolution; biochemical reconstitution of INTS4/9/11 subcomplex\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-atomic resolution cryo-EM structure with biochemical reconstitution, independently consistent with prior Co-IP data\",\n      \"pmids\": [\"33548203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Depletion of INTS4 abrogates snRNA 3'-end processing, causes accumulation of the Cajal body marker coilin in nucleoli, disrupts normal Cajal body composition (loss of SMN, Sm proteins, and snRNAs from foci), and induces cytoplasmic granule formation of SMN and Sm proteins, demonstrating that INTS4 is essential for snRNA maturation and Cajal body homeostasis.\",\n      \"method\": \"siRNA-mediated knockdown of INTS4 in human cells, immunofluorescence microscopy, northern blotting for snRNA processing\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KD with defined cellular and molecular phenotypes, multiple orthogonal readouts, independently replicated in Wagner lab (PMID 29471365)\",\n      \"pmids\": [\"22250197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cryo-EM structure of the Drosophila INTS4/9/11 (ICM) at 2.74 Å resolution reveals stable association of an inositol hexakisphosphate (IP6) molecule in a highly electropositive pocket at the interface of all three ICM subunits, 55 Å from the INTS11 active site. IP6 binding was confirmed in human ICM. Mutations in the IP6-binding site or disruption of IP6 biosynthesis significantly reduced Integrator function in snRNA 3'-end processing and mRNA transcription attenuation.\",\n      \"method\": \"Cryo-EM structure determination (2.74 Å), site-directed mutagenesis of IP6-binding residues, IP6-binding assays, snRNA processing assays, mRNA transcription attenuation assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic-resolution cryo-EM with mutagenesis and multiple functional assays; replicated across Drosophila and human ICM\",\n      \"pmids\": [\"36180473\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"BRAT1 and WDR73 are biogenesis factors for the INTS4/9/11 cleavage module: they keep INTS9/11 inactive by blocking the endonuclease active site and prevent premature INTS4 association during maturation. BRAT1 also facilitates nuclear import of INTS9/11, after which INTS4 joins in the nucleus. Final BRAT1 release requires locking of the mature cleavage module conformation by IP6.\",\n      \"method\": \"Co-immunoprecipitation, assembly/maturation assays, nuclear import assays, IP6-dependence experiments; mutations in BRAT1, WDR73, and INTS11 linked to Integrator assembly defects\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP and reconstitution experiments with functional assembly readouts, multiple orthogonal methods in one study\",\n      \"pmids\": [\"39032489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"An INTS11 SUMOylation-deficient mutant retains interaction with INTS4 and INTS9 but loses interaction with other Integrator subunits, indicating that the INTS4/9/11 cleavage module interaction is independent of INTS11 SUMOylation whereas broader complex assembly is not.\",\n      \"method\": \"Co-immunoprecipitation with SUMOylation-deficient INTS11 mutant (Lys381/462/475Arg), comparison of interactions with INTS4, INTS9, and other subunits\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP with defined mutant, single lab, findings specifically about INTS4 interaction are a subsidiary result of a study focused on INTS11\",\n      \"pmids\": [\"36454007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In C. elegans, auxin-inducible degradation of INTS-4 (the INTS4 ortholog) demonstrates that Integrator activity is critical for development at the L1 larval stage but becomes dispensable after L2/L3; germline-specific INTS-4 degradation causes accumulation of misprocessed snRNA transcripts without impairing development or lifespan.\",\n      \"method\": \"Auxin-inducible degron (AID) system for temporal depletion of C. elegans INTS-4; snRNA processing assays, developmental and lifespan phenotyping\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean conditional KO with defined molecular (snRNA misprocessing) and developmental phenotypes in C. elegans, single lab\",\n      \"pmids\": [\"40071568\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"INTS4 is a helical-repeat scaffold protein that, together with INTS9 and INTS11, forms the heterotrimeric Integrator cleavage module (ICM): INTS4 binds only the pre-assembled INTS9/11 heterodimer (not either subunit alone), stabilizes the nuclease domains via multiple interfaces, and contributes to an electropositive RNA-binding groove; ICM assembly in the nucleus is chaperoned by BRAT1 and WDR73 (which block premature INTS4 association and the endonuclease active site), and is completed by incorporation of inositol hexakisphosphate (IP6) as an essential co-factor that locks the mature conformation and is required for both snRNA 3'-end processing and mRNA transcription attenuation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"INTS4 is a helical-repeat scaffold subunit of the Integrator complex that, together with INTS9 and INTS11, forms the heterotrimeric cleavage module responsible for the complex's endonucleolytic activity in small nuclear RNA (snRNA) 3'-end processing [#0, #2]. INTS4 binds specifically to the pre-assembled INTS9/11 heterodimer—not either subunit alone—using both N-terminal HEAT repeats and a β-sheet-rich C-terminal region, and cryo-EM shows it stabilizes the INTS9/INTS11 nuclease domains through multiple interdependent interfaces while contributing to a composite electropositive groove that forms the catalytic core's RNA-binding path [#0, #1]. Maturation of the cleavage module is templated: BRAT1 and WDR73 keep INTS9/11 inactive and block premature INTS4 association, BRAT1 mediates nuclear import of INTS9/11, INTS4 joins in the nucleus, and incorporation of inositol hexakisphosphate (IP6) into an electropositive pocket at the three-subunit interface locks the mature conformation required for both snRNA 3'-end processing and mRNA transcription attenuation [#3, #4]. Loss of INTS4 abrogates snRNA maturation and disrupts Cajal body homeostasis, causing mislocalization of coilin, SMN, and Sm proteins [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Established that INTS4 is functionally essential for snRNA maturation, answering whether this subunit contributes to Integrator output at the cellular level.\",\n      \"evidence\": \"siRNA knockdown of INTS4 in human cells with immunofluorescence and northern blotting\",\n      \"pmids\": [\"22250197\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define INTS4's biochemical role within the complex\", \"Did not identify direct binding partners of INTS4\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined INTS4 as a dedicated scaffold of a heterotrimeric cleavage module, showing it binds the INTS9/11 heterodimer but neither subunit alone and mapping the required domains.\",\n      \"evidence\": \"Reciprocal Co-IP with domain-deletion analysis and snRNA processing/Cajal body readouts in human cells\",\n      \"pmids\": [\"29471365\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural basis for the INTS4/9/11 interaction\", \"Did not address how the module is assembled in vivo\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided the structural mechanism by which INTS4 organizes the catalytic core, showing it stabilizes the INTS9/INTS11 nuclease domains and helps form a composite RNA-binding groove.\",\n      \"evidence\": \"3.5 Å cryo-EM structure with biochemical reconstitution of the INTS4/9/11 subcomplex\",\n      \"pmids\": [\"33548203\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RNA-binding path inferred from electrostatics, not RNA-bound structure\", \"Did not reveal cofactor requirements\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified IP6 as an essential structural cofactor locking the mature cleavage module, linking a small-molecule ligand to both snRNA processing and transcription attenuation.\",\n      \"evidence\": \"2.74 Å cryo-EM of Drosophila ICM plus mutagenesis, IP6-binding assays, and functional assays, confirmed in human ICM\",\n      \"pmids\": [\"36180473\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"IP6 site is 55 Å from the active site; the allosteric coupling mechanism is not detailed\", \"Did not establish the assembly order of IP6 incorporation\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Distinguished cleavage-module assembly from broader complex assembly, showing the INTS4/9/11 interaction is independent of INTS11 SUMOylation.\",\n      \"evidence\": \"Co-IP with a SUMOylation-deficient INTS11 mutant comparing interactions across subunits\",\n      \"pmids\": [\"36454007\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Subsidiary result of an INTS11-focused study\", \"Single lab; functional consequence for INTS4 not directly assayed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed a chaperoned biogenesis pathway, showing BRAT1/WDR73 keep INTS9/11 inactive and block premature INTS4 binding, with INTS4 joining in the nucleus and IP6-dependent BRAT1 release completing maturation.\",\n      \"evidence\": \"Co-IP, assembly/maturation and nuclear import assays with IP6-dependence experiments\",\n      \"pmids\": [\"39032489\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise trigger for INTS4 recruitment in the nucleus not fully resolved\", \"How disease mutations in BRAT1/WDR73 perturb the pathway not detailed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated temporal and tissue-specific requirements for INTS4 in a whole organism, separating developmental essentiality from germline snRNA processing.\",\n      \"evidence\": \"Auxin-inducible degron depletion of C. elegans INTS-4 with snRNA processing and developmental/lifespan phenotyping\",\n      \"pmids\": [\"40071568\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab in C. elegans; mammalian stage-specificity not addressed\", \"Molecular basis for stage-dependent dispensability unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How IP6 binding at a site 55 Å from the active site allosterically licenses catalysis, and how RNA substrate is engaged by the electropositive groove, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No RNA-bound structure of the cleavage module\", \"Allosteric pathway from the IP6 pocket to the active site uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 2, 3]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 2, 3]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [\n      \"Integrator complex\",\n      \"Integrator cleavage module (INTS4/9/11)\"\n    ],\n    \"partners\": [\n      \"INTS9\",\n      \"INTS11\",\n      \"BRAT1\",\n      \"WDR73\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}