{"gene":"INTS3","run_date":"2026-04-28T18:06:54","timeline":{"discoveries":[{"year":2009,"finding":"INTS3 forms a complex with hSSB1 (human single-stranded DNA binding protein 1) and a subset of Integrator complex subunits, along with the uncharacterized protein MISE/C9ORF80, identified by tandem affinity purification. This INTS3-MISE-hSSB1 complex plays a key role in ATM activation and RAD51 recruitment to DNA damage foci, and INTS3 controls hSSB1 levels transcriptionally.","method":"Tandem affinity purification, co-purification, ATM activation and RAD51 foci assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal purification with functional readouts (ATM activation, RAD51 recruitment), Moderate evidence from single lab with multiple orthogonal methods","pmids":["19786574"],"is_preprint":false},{"year":2021,"finding":"The C-terminus of INTS3 dimerizes and interacts with the C-terminus of INTS6 via conserved residues, as revealed by a 2.4 Å crystal structure. INTS3 dimerization is required for recognizing longer ssDNA, and perturbation of INTS3 dimerization or disruption of the INTS3/INTS6 interaction impairs double-strand break (DSB) repair.","method":"X-ray crystallography, biochemical binding assays, mutagenesis, DSB repair functional assays","journal":"Cell discovery","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with mutagenesis and in vitro functional validation, single lab but multiple orthogonal methods","pmids":["34400606"],"is_preprint":false},{"year":2020,"finding":"The C-terminal domain of INTS3 adopts a HEAT-repeat superhelical fold and forms a stable dimer. A basic groove on the dimer binds ssRNA/ssDNA (requiring dimerization), while a cluster of conserved residues on the opposite face binds INTS6. INTS6 interaction is critical for maintaining SSB1 protein levels in cells.","method":"X-ray crystallography, EMSA, mutagenesis, HEK 293T cell-based protein level assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — crystal structure combined with mutagenesis and functional in vitro/cell-based assays in a single study","pmids":["33434574"],"is_preprint":false},{"year":2018,"finding":"Recombinant INTS3 binds ssRNA with higher affinity than ssDNA and requires a minimum of 30 nucleotides for binding; it does not bind dsDNA, dsRNA, or RNA:DNA hybrids. The N-terminus of INTS3 mediates protein-protein interactions while the C-terminus is required for nucleic acid binding. INTS3 modulates the nucleic acid-binding ability of hNABP1/2 within the heterotrimeric complex, whereas C9ORF80 does not.","method":"EMSA (electrophoretic mobility shift assay), GST pulldown, recombinant protein purification, reconstituted heterotrimeric complex","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 1-2 — in vitro biochemical reconstitution with domain-mapping, single lab","pmids":["29150435"],"is_preprint":false},{"year":2015,"finding":"In the absence of RPA, hSSB1 and its partner INTS3 form sub-nuclear foci, associate with the ATR-ATRIP complex, and recruit it to sites of genomic stress. INTS3 depletion abrogates ATRIP foci formed after RPA depletion. Depletion of hSSB1/2 and INTS3 in RPA-deficient cells attenuates Chk1 phosphorylation, indicating the hSSB1/2-INTS3 complex can initiate an alternative ATR signaling pathway requiring TopBP1 and the Rad9-Rad1-Hus1 complex.","method":"siRNA knockdown, immunofluorescence foci assays, co-immunoprecipitation, Chk1 phosphorylation assays","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 — clean KD with defined cellular phenotype and epistasis, single lab with multiple readouts","pmids":["25916848"],"is_preprint":false},{"year":2024,"finding":"INTS3 acts as an RNA-binding protein that destabilizes pro-apoptotic gene transcripts, thereby promoting colorectal cancer cell survival. INTS3 deletion triggers apoptosis in CRC cells in vitro and delays tumor growth in vivo.","method":"CRISPR-Cas9 pooled screen, RNA sequencing, in vitro apoptosis assays, in vivo xenograft","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 — CRISPR KO with defined apoptotic phenotype and RNA-seq mechanistic follow-up, single lab","pmids":["38665208"],"is_preprint":false},{"year":2024,"finding":"Computational screening identified small molecules that disrupt the INTS3-hSSB1 protein-protein interaction at the hSSB1-binding interface of INTS3; one compound impaired recruitment of both hSSB1 and INTS3 to chromatin following DNA damage, validated by co-immunoprecipitation and immunofluorescence.","method":"Molecular docking virtual screening, co-immunoprecipitation, immunofluorescence, molecular dynamics simulation","journal":"ACS omega","confidence":"Low","confidence_rationale":"Tier 3-4 — computational screening with limited in vitro validation, single lab, weak follow-up","pmids":["38405517"],"is_preprint":false}],"current_model":"INTS3 functions as a scaffold subunit of the SOSS1 (sensor of single-stranded DNA) complex, dimerizing via its HEAT-repeat C-terminal domain to bind ssDNA/ssRNA, interacting with hSSB1/SSBIP1 through its N-terminal domain to stabilize the complex, and engaging INTS6 through conserved C-terminal residues; together, the INTS3-hSSB1-C9ORF80 complex is recruited to DNA double-strand breaks to activate ATM and ATR signaling pathways and promote RAD51-dependent homologous recombination repair."},"narrative":{"teleology":[{"year":2009,"claim":"Identification of INTS3 as a core member of a novel ssDNA-sensing complex (SOSS1) established that it functions beyond the Integrator complex, directly linking it to ATM activation and RAD51-mediated homologous recombination repair.","evidence":"Tandem affinity purification of hSSB1 complexes followed by ATM activation and RAD51 foci assays in human cells","pmids":["19786574"],"confidence":"High","gaps":["Structural basis of INTS3–hSSB1 interaction unknown","Whether INTS3 directly contacts DNA or acts solely as a scaffold was unresolved","Contribution of C9ORF80 to complex function undefined"]},{"year":2015,"claim":"Demonstrating that INTS3 and hSSB1 can recruit ATRIP and activate ATR–Chk1 signaling independently of RPA revealed a second, parallel DNA damage checkpoint pathway mediated by the SOSS1 complex.","evidence":"siRNA knockdown of RPA, hSSB1/2, and INTS3 with epistasis analysis of Chk1 phosphorylation and ATRIP foci in human cells","pmids":["25916848"],"confidence":"Medium","gaps":["Physiological contexts where this alternative ATR pathway dominates remain unclear","Direct physical interaction between INTS3 and ATRIP not demonstrated"]},{"year":2018,"claim":"Biochemical reconstitution showed that INTS3 itself is a nucleic acid-binding protein with preference for ssRNA over ssDNA, resolving whether INTS3 contributes directly to substrate recognition or only serves as a scaffold.","evidence":"EMSA and GST pulldown with recombinant INTS3 and reconstituted heterotrimeric SOSS1 complex","pmids":["29150435"],"confidence":"Medium","gaps":["Structural basis of ssRNA preference not determined","In vivo RNA targets of INTS3 unknown","Role of INTS3 nucleic acid binding in DSB repair versus RNA metabolism not distinguished"]},{"year":2020,"claim":"Crystal structure of the INTS3 C-terminal domain revealed a HEAT-repeat dimer whose basic groove binds ssDNA/ssRNA, while a distinct conserved surface engages INTS6, providing the first structural framework for understanding SOSS1 assembly and nucleic acid recognition.","evidence":"X-ray crystallography at atomic resolution, EMSA, mutagenesis, and HEK 293T protein stability assays","pmids":["33434574"],"confidence":"High","gaps":["Full-length SOSS1 complex structure not available","How INTS6 binding stabilizes hSSB1 protein levels mechanistically is unclear"]},{"year":2021,"claim":"A higher-resolution structure of the INTS3–INTS6 interface showed that INTS3 dimerization is functionally required for recognizing longer ssDNA substrates and for proficient DSB repair, linking structural oligomerization to genome maintenance.","evidence":"2.4 Å crystal structure, mutagenesis of dimer and INTS6-binding interfaces, DSB repair functional assays","pmids":["34400606"],"confidence":"High","gaps":["Whether INTS3 dimerization status is regulated in response to DNA damage is unknown","Relationship between INTS3 dimer-mediated ssDNA binding and ATM/ATR activation not mechanistically connected"]},{"year":2024,"claim":"Discovery of an RNA-regulatory role for INTS3 — destabilizing pro-apoptotic transcripts to promote cancer cell survival — expanded its functional scope beyond DNA repair to post-transcriptional gene regulation.","evidence":"CRISPR-Cas9 screen in colorectal cancer cells, RNA-seq, apoptosis assays, and xenograft models","pmids":["38665208"],"confidence":"Medium","gaps":["Specific RNA targets and degradation pathway through which INTS3 acts are not fully defined","Whether this RNA-regulatory function depends on the SOSS1 complex or on INTS3 alone is unresolved","Generalizability beyond colorectal cancer not tested"]},{"year":null,"claim":"Key open questions include the structure of the full SOSS1 holocomplex, the mechanism by which INTS3 selectively destabilizes RNA transcripts, whether INTS3 dimerization is dynamically regulated during the DNA damage response, and how its dual DNA repair and RNA regulatory functions are coordinated.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length SOSS1 complex structure available","Mechanism of INTS3-mediated mRNA destabilization unknown","Regulation of INTS3 dimerization in vivo not characterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[2,3,5]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[1,2,3]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,4]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,4]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,4,6]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[0,1,4]}],"complexes":["SOSS1 (INTS3–hSSB1–C9ORF80)"],"partners":["NABP2","INTS6","C9ORF80","ATRIP"],"other_free_text":[]},"mechanistic_narrative":"INTS3 is a scaffold subunit of the SOSS1 complex (INTS3–hSSB1–C9ORF80) that coordinates single-stranded nucleic acid recognition with DNA damage signaling and repair. Its C-terminal HEAT-repeat domain dimerizes to form a basic groove that binds ssDNA and ssRNA (preferring ssRNA, minimum ~30 nt), while its N-terminal domain mediates interaction with hSSB1/NABP2, and a conserved surface on the C-terminal dimer engages INTS6 to maintain hSSB1 protein levels [PMID:33434574, PMID:34400606, PMID:29150435]. The INTS3-containing SOSS1 complex is recruited to DNA double-strand breaks where it activates ATM signaling, promotes RAD51-dependent homologous recombination, and can also initiate an alternative ATR–Chk1 pathway in the absence of RPA [PMID:19786574, PMID:25916848]. INTS3 additionally functions as an RNA-binding protein that destabilizes pro-apoptotic transcripts, and its deletion triggers apoptosis in colorectal cancer cells and suppresses tumor growth in vivo [PMID:38665208]."},"prefetch_data":{"uniprot":{"accession":"Q68E01","full_name":"Integrator complex subunit 3","aliases":["SOSS complex subunit A","Sensor of single-strand DNA complex subunit A","SOSS-A","Sensor of ssDNA subunit A"],"length_aa":1043,"mass_kda":118.1,"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: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: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, INTS3 is involved in the post-termination step: INTS3 binds INTS7 in the open conformation of integrator complex and prevents the rebinding of Pol II to the integrator after termination cycle (PubMed:38570683). Mediates recruitment of cytoplasmic dynein to the nuclear envelope, probably as component of the integrator complex (PubMed:23904267) Component of the SOSS complex, a multiprotein complex that functions downstream of the MRN complex to promote DNA repair and G2/M checkpoint. The SOSS complex associates with single-stranded DNA at DNA lesions and influences diverse endpoints in the cellular DNA damage response including cell-cycle checkpoint activation, recombinational repair and maintenance of genomic stability. The SOSS complex is required for efficient homologous recombination-dependent repair of double-strand breaks (DSBs) and ATM-dependent signaling pathways. In the SOSS complex, it is required for the assembly of the complex and for stabilization of the complex at DNA damage sites","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q68E01/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/INTS3","classification":"Common Essential","n_dependent_lines":1205,"n_total_lines":1208,"dependency_fraction":0.9975165562913907},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"INTS9","stoichiometry":10.0},{"gene":"INTS14","stoichiometry":4.0},{"gene":"CDS2","stoichiometry":0.2},{"gene":"HIST2H2BE","stoichiometry":0.2},{"gene":"HMGA1","stoichiometry":0.2},{"gene":"HMGN5","stoichiometry":0.2},{"gene":"NUCKS1","stoichiometry":0.2},{"gene":"NUMA1","stoichiometry":0.2},{"gene":"POLR2B","stoichiometry":0.2},{"gene":"POLR2E","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/INTS3","total_profiled":1310},"omim":[{"mim_id":"613273","title":"INST3- AND NABP-INTERACTING PROTEIN; INIP","url":"https://www.omim.org/entry/613273"},{"mim_id":"611479","title":"GPN-LOOP GTPase 1; GPN1","url":"https://www.omim.org/entry/611479"},{"mim_id":"611477","title":"RNA POLYMERASE II-ASSOCIATED PROTEIN 3; RPAP3","url":"https://www.omim.org/entry/611477"},{"mim_id":"611476","title":"RNA POLYMERASE II-ASSOCIATED PROTEIN 2; RPAP2","url":"https://www.omim.org/entry/611476"},{"mim_id":"611475","title":"RNA POLYMERASE II-ASSOCIATED PROTEIN 1; RPAP1","url":"https://www.omim.org/entry/611475"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoli","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/INTS3"},"hgnc":{"alias_symbol":["FLJ21919","INT3","SOSS-A"],"prev_symbol":["C1orf60"]},"alphafold":{"accession":"Q68E01","domains":[{"cath_id":"-","chopping":"44-132","consensus_level":"medium","plddt":95.597,"start":44,"end":132},{"cath_id":"-","chopping":"204-306","consensus_level":"medium","plddt":96.1454,"start":204,"end":306},{"cath_id":"-","chopping":"562-720","consensus_level":"medium","plddt":90.6922,"start":562,"end":720},{"cath_id":"1.25.40","chopping":"307-498","consensus_level":"high","plddt":94.5725,"start":307,"end":498},{"cath_id":"1.25.40","chopping":"853-901_920-976","consensus_level":"high","plddt":89.4217,"start":853,"end":976}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q68E01","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q68E01-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q68E01-F1-predicted_aligned_error_v6.png","plddt_mean":83.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=INTS3","jax_strain_url":"https://www.jax.org/strain/search?query=INTS3"},"sequence":{"accession":"Q68E01","fasta_url":"https://rest.uniprot.org/uniprotkb/Q68E01.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q68E01/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q68E01"}},"corpus_meta":[{"pmid":"1372276","id":"PMC_1372276","title":"Expression of an activated Notch-related int-3 transgene interferes with cell differentiation and induces neoplastic transformation in mammary and salivary glands.","date":"1992","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/1372276","citation_count":320,"is_preprint":false},{"pmid":"1312643","id":"PMC_1312643","title":"Mouse mammary tumor gene int-3: a member of the notch gene family transforms mammary epithelial cells.","date":"1992","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/1312643","citation_count":237,"is_preprint":false},{"pmid":"7835890","id":"PMC_7835890","title":"Three genes in the human MHC class III region near the junction with the class II: gene for receptor of advanced glycosylation end products, PBX2 homeobox gene and a notch homolog, human counterpart of mouse mammary tumor gene int-3.","date":"1994","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/7835890","citation_count":210,"is_preprint":false},{"pmid":"9150355","id":"PMC_9150355","title":"The mouse mammary tumor associated gene INT3 is a unique member of the NOTCH gene family (NOTCH4).","date":"1997","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/9150355","citation_count":166,"is_preprint":false},{"pmid":"8620493","id":"PMC_8620493","title":"Expression of a truncated Int3 gene in developing secretory mammary epithelium specifically retards lobular differentiation resulting in tumorigenesis.","date":"1996","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/8620493","citation_count":155,"is_preprint":false},{"pmid":"3023699","id":"PMC_3023699","title":"A new common integration region (int-3) for mouse mammary tumor virus on mouse chromosome 17.","date":"1987","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/3023699","citation_count":118,"is_preprint":false},{"pmid":"7544153","id":"PMC_7544153","title":"Constitutive expression of a truncated INT3 gene in mouse mammary epithelium impairs differentiation and functional development.","date":"1995","source":"Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/7544153","citation_count":90,"is_preprint":false},{"pmid":"19786574","id":"PMC_19786574","title":"INTS3 controls the hSSB1-mediated DNA damage response.","date":"2009","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/19786574","citation_count":79,"is_preprint":false},{"pmid":"9189758","id":"PMC_9189758","title":"Proto-oncogene of int-3, a mouse Notch homologue, is expressed in endothelial cells during early embryogenesis.","date":"1997","source":"Genes to cells : devoted to molecular & cellular mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/9189758","citation_count":60,"is_preprint":false},{"pmid":"18836481","id":"PMC_18836481","title":"Rbpj conditional knockout reveals distinct functions of Notch4/Int3 in mammary gland development and tumorigenesis.","date":"2008","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/18836481","citation_count":44,"is_preprint":false},{"pmid":"18068530","id":"PMC_18068530","title":"CREB3L4, INTS3, and SNAPAP are targets for the 1q21 amplicon frequently detected in hepatocellular carcinoma.","date":"2008","source":"Cancer genetics and cytogenetics","url":"https://pubmed.ncbi.nlm.nih.gov/18068530","citation_count":43,"is_preprint":false},{"pmid":"15531924","id":"PMC_15531924","title":"Mammary development and tumorigenesis in mice expressing a truncated human Notch4/Int3 intracellular domain (h-Int3sh).","date":"2004","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/15531924","citation_count":41,"is_preprint":false},{"pmid":"14961038","id":"PMC_14961038","title":"Expression of constitutively active Notch4 (Int-3) modulates myeloid proliferation and differentiation and promotes expansion of hematopoietic progenitors.","date":"2004","source":"Leukemia","url":"https://pubmed.ncbi.nlm.nih.gov/14961038","citation_count":24,"is_preprint":false},{"pmid":"9168133","id":"PMC_9168133","title":"Gene organization of human NOTCH4 and (CTG)n polymorphism in this human counterpart gene of mouse proto-oncogene Int3.","date":"1997","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/9168133","citation_count":23,"is_preprint":false},{"pmid":"8030284","id":"PMC_8030284","title":"Insertional mutation of int protooncogenes in the mammary tumors of a new strain of mice derived from the wild in China: normal- and tumor-tissue-specific expression of int-3 transcripts.","date":"1994","source":"Virology","url":"https://pubmed.ncbi.nlm.nih.gov/8030284","citation_count":23,"is_preprint":false},{"pmid":"34400606","id":"PMC_34400606","title":"Crystal structure of the INTS3/INTS6 complex reveals the functional importance of INTS3 dimerization in DSB repair.","date":"2021","source":"Cell discovery","url":"https://pubmed.ncbi.nlm.nih.gov/34400606","citation_count":22,"is_preprint":false},{"pmid":"10233982","id":"PMC_10233982","title":"Intracisternal type A particle-mediated activation of the Notch4/int3 gene in a mouse mammary tumor: generation of truncated Notch4/int3 mRNAs by retroviral splicing events.","date":"1999","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/10233982","citation_count":18,"is_preprint":false},{"pmid":"7494323","id":"PMC_7494323","title":"A novel non-mouse mammary tumor virus activation of the Int-3 gene in a spontaneous mouse mammary tumor.","date":"1995","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/7494323","citation_count":17,"is_preprint":false},{"pmid":"33434574","id":"PMC_33434574","title":"Structural basis for multifunctional roles of human Ints3 C-terminal domain.","date":"2020","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/33434574","citation_count":16,"is_preprint":false},{"pmid":"29150435","id":"PMC_29150435","title":"Biochemical characterization of INTS3 and C9ORF80, two subunits of hNABP1/2 heterotrimeric complex in nucleic acid binding.","date":"2018","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/29150435","citation_count":14,"is_preprint":false},{"pmid":"16878155","id":"PMC_16878155","title":"Kit and PDGFR-alpha activities are necessary for Notch4/Int3-induced tumorigenesis.","date":"2006","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/16878155","citation_count":14,"is_preprint":false},{"pmid":"29057904","id":"PMC_29057904","title":"The ANK repeats of Notch-4/Int3 activate NF-κB canonical pathway in the absence of Rbpj and causes mammary tumorigenesis.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29057904","citation_count":10,"is_preprint":false},{"pmid":"25916848","id":"PMC_25916848","title":"RPA70 depletion induces hSSB1/2-INTS3 complex to initiate ATR signaling.","date":"2015","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/25916848","citation_count":9,"is_preprint":false},{"pmid":"38665208","id":"PMC_38665208","title":"CRISPR-Cas9 screening identifies INTS3 as an anti-apoptotic RNA-binding protein and therapeutic target for colorectal cancer.","date":"2024","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/38665208","citation_count":4,"is_preprint":false},{"pmid":"38405517","id":"PMC_38405517","title":"Targeting the hSSB1-INTS3 Interface: A Computational Screening Driven Approach to Identify Potential Modulators.","date":"2024","source":"ACS omega","url":"https://pubmed.ncbi.nlm.nih.gov/38405517","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13369,"output_tokens":1849,"usd":0.033921},"stage2":{"model":"claude-opus-4-6","input_tokens":5106,"output_tokens":2009,"usd":0.113632},"total_usd":0.147553,"stage1_batch_id":"msgbatch_01DzrsgVgYngY1hvM3DU1yF2","stage2_batch_id":"msgbatch_01KA9b7iRzKFdhoBUT7vCvSe","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"INTS3 forms a complex with hSSB1 (human single-stranded DNA binding protein 1) and a subset of Integrator complex subunits, along with the uncharacterized protein MISE/C9ORF80, identified by tandem affinity purification. This INTS3-MISE-hSSB1 complex plays a key role in ATM activation and RAD51 recruitment to DNA damage foci, and INTS3 controls hSSB1 levels transcriptionally.\",\n      \"method\": \"Tandem affinity purification, co-purification, ATM activation and RAD51 foci assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal purification with functional readouts (ATM activation, RAD51 recruitment), Moderate evidence from single lab with multiple orthogonal methods\",\n      \"pmids\": [\"19786574\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The C-terminus of INTS3 dimerizes and interacts with the C-terminus of INTS6 via conserved residues, as revealed by a 2.4 Å crystal structure. INTS3 dimerization is required for recognizing longer ssDNA, and perturbation of INTS3 dimerization or disruption of the INTS3/INTS6 interaction impairs double-strand break (DSB) repair.\",\n      \"method\": \"X-ray crystallography, biochemical binding assays, mutagenesis, DSB repair functional assays\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with mutagenesis and in vitro functional validation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"34400606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The C-terminal domain of INTS3 adopts a HEAT-repeat superhelical fold and forms a stable dimer. A basic groove on the dimer binds ssRNA/ssDNA (requiring dimerization), while a cluster of conserved residues on the opposite face binds INTS6. INTS6 interaction is critical for maintaining SSB1 protein levels in cells.\",\n      \"method\": \"X-ray crystallography, EMSA, mutagenesis, HEK 293T cell-based protein level assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure combined with mutagenesis and functional in vitro/cell-based assays in a single study\",\n      \"pmids\": [\"33434574\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Recombinant INTS3 binds ssRNA with higher affinity than ssDNA and requires a minimum of 30 nucleotides for binding; it does not bind dsDNA, dsRNA, or RNA:DNA hybrids. The N-terminus of INTS3 mediates protein-protein interactions while the C-terminus is required for nucleic acid binding. INTS3 modulates the nucleic acid-binding ability of hNABP1/2 within the heterotrimeric complex, whereas C9ORF80 does not.\",\n      \"method\": \"EMSA (electrophoretic mobility shift assay), GST pulldown, recombinant protein purification, reconstituted heterotrimeric complex\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro biochemical reconstitution with domain-mapping, single lab\",\n      \"pmids\": [\"29150435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In the absence of RPA, hSSB1 and its partner INTS3 form sub-nuclear foci, associate with the ATR-ATRIP complex, and recruit it to sites of genomic stress. INTS3 depletion abrogates ATRIP foci formed after RPA depletion. Depletion of hSSB1/2 and INTS3 in RPA-deficient cells attenuates Chk1 phosphorylation, indicating the hSSB1/2-INTS3 complex can initiate an alternative ATR signaling pathway requiring TopBP1 and the Rad9-Rad1-Hus1 complex.\",\n      \"method\": \"siRNA knockdown, immunofluorescence foci assays, co-immunoprecipitation, Chk1 phosphorylation assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with defined cellular phenotype and epistasis, single lab with multiple readouts\",\n      \"pmids\": [\"25916848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"INTS3 acts as an RNA-binding protein that destabilizes pro-apoptotic gene transcripts, thereby promoting colorectal cancer cell survival. INTS3 deletion triggers apoptosis in CRC cells in vitro and delays tumor growth in vivo.\",\n      \"method\": \"CRISPR-Cas9 pooled screen, RNA sequencing, in vitro apoptosis assays, in vivo xenograft\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO with defined apoptotic phenotype and RNA-seq mechanistic follow-up, single lab\",\n      \"pmids\": [\"38665208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Computational screening identified small molecules that disrupt the INTS3-hSSB1 protein-protein interaction at the hSSB1-binding interface of INTS3; one compound impaired recruitment of both hSSB1 and INTS3 to chromatin following DNA damage, validated by co-immunoprecipitation and immunofluorescence.\",\n      \"method\": \"Molecular docking virtual screening, co-immunoprecipitation, immunofluorescence, molecular dynamics simulation\",\n      \"journal\": \"ACS omega\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3-4 — computational screening with limited in vitro validation, single lab, weak follow-up\",\n      \"pmids\": [\"38405517\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"INTS3 functions as a scaffold subunit of the SOSS1 (sensor of single-stranded DNA) complex, dimerizing via its HEAT-repeat C-terminal domain to bind ssDNA/ssRNA, interacting with hSSB1/SSBIP1 through its N-terminal domain to stabilize the complex, and engaging INTS6 through conserved C-terminal residues; together, the INTS3-hSSB1-C9ORF80 complex is recruited to DNA double-strand breaks to activate ATM and ATR signaling pathways and promote RAD51-dependent homologous recombination repair.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"INTS3 is a scaffold subunit of the SOSS1 complex (INTS3–hSSB1–C9ORF80) that coordinates single-stranded nucleic acid recognition with DNA damage signaling and repair. Its C-terminal HEAT-repeat domain dimerizes to form a basic groove that binds ssDNA and ssRNA (preferring ssRNA, minimum ~30 nt), while its N-terminal domain mediates interaction with hSSB1/NABP2, and a conserved surface on the C-terminal dimer engages INTS6 to maintain hSSB1 protein levels [PMID:33434574, PMID:34400606, PMID:29150435]. The INTS3-containing SOSS1 complex is recruited to DNA double-strand breaks where it activates ATM signaling, promotes RAD51-dependent homologous recombination, and can also initiate an alternative ATR–Chk1 pathway in the absence of RPA [PMID:19786574, PMID:25916848]. INTS3 additionally functions as an RNA-binding protein that destabilizes pro-apoptotic transcripts, and its deletion triggers apoptosis in colorectal cancer cells and suppresses tumor growth in vivo [PMID:38665208].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Identification of INTS3 as a core member of a novel ssDNA-sensing complex (SOSS1) established that it functions beyond the Integrator complex, directly linking it to ATM activation and RAD51-mediated homologous recombination repair.\",\n      \"evidence\": \"Tandem affinity purification of hSSB1 complexes followed by ATM activation and RAD51 foci assays in human cells\",\n      \"pmids\": [\"19786574\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of INTS3–hSSB1 interaction unknown\",\n        \"Whether INTS3 directly contacts DNA or acts solely as a scaffold was unresolved\",\n        \"Contribution of C9ORF80 to complex function undefined\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrating that INTS3 and hSSB1 can recruit ATRIP and activate ATR–Chk1 signaling independently of RPA revealed a second, parallel DNA damage checkpoint pathway mediated by the SOSS1 complex.\",\n      \"evidence\": \"siRNA knockdown of RPA, hSSB1/2, and INTS3 with epistasis analysis of Chk1 phosphorylation and ATRIP foci in human cells\",\n      \"pmids\": [\"25916848\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Physiological contexts where this alternative ATR pathway dominates remain unclear\",\n        \"Direct physical interaction between INTS3 and ATRIP not demonstrated\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Biochemical reconstitution showed that INTS3 itself is a nucleic acid-binding protein with preference for ssRNA over ssDNA, resolving whether INTS3 contributes directly to substrate recognition or only serves as a scaffold.\",\n      \"evidence\": \"EMSA and GST pulldown with recombinant INTS3 and reconstituted heterotrimeric SOSS1 complex\",\n      \"pmids\": [\"29150435\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Structural basis of ssRNA preference not determined\",\n        \"In vivo RNA targets of INTS3 unknown\",\n        \"Role of INTS3 nucleic acid binding in DSB repair versus RNA metabolism not distinguished\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Crystal structure of the INTS3 C-terminal domain revealed a HEAT-repeat dimer whose basic groove binds ssDNA/ssRNA, while a distinct conserved surface engages INTS6, providing the first structural framework for understanding SOSS1 assembly and nucleic acid recognition.\",\n      \"evidence\": \"X-ray crystallography at atomic resolution, EMSA, mutagenesis, and HEK 293T protein stability assays\",\n      \"pmids\": [\"33434574\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Full-length SOSS1 complex structure not available\",\n        \"How INTS6 binding stabilizes hSSB1 protein levels mechanistically is unclear\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A higher-resolution structure of the INTS3–INTS6 interface showed that INTS3 dimerization is functionally required for recognizing longer ssDNA substrates and for proficient DSB repair, linking structural oligomerization to genome maintenance.\",\n      \"evidence\": \"2.4 Å crystal structure, mutagenesis of dimer and INTS6-binding interfaces, DSB repair functional assays\",\n      \"pmids\": [\"34400606\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether INTS3 dimerization status is regulated in response to DNA damage is unknown\",\n        \"Relationship between INTS3 dimer-mediated ssDNA binding and ATM/ATR activation not mechanistically connected\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery of an RNA-regulatory role for INTS3 — destabilizing pro-apoptotic transcripts to promote cancer cell survival — expanded its functional scope beyond DNA repair to post-transcriptional gene regulation.\",\n      \"evidence\": \"CRISPR-Cas9 screen in colorectal cancer cells, RNA-seq, apoptosis assays, and xenograft models\",\n      \"pmids\": [\"38665208\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Specific RNA targets and degradation pathway through which INTS3 acts are not fully defined\",\n        \"Whether this RNA-regulatory function depends on the SOSS1 complex or on INTS3 alone is unresolved\",\n        \"Generalizability beyond colorectal cancer not tested\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include the structure of the full SOSS1 holocomplex, the mechanism by which INTS3 selectively destabilizes RNA transcripts, whether INTS3 dimerization is dynamically regulated during the DNA damage response, and how its dual DNA repair and RNA regulatory functions are coordinated.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No full-length SOSS1 complex structure available\",\n        \"Mechanism of INTS3-mediated mRNA destabilization unknown\",\n        \"Regulation of INTS3 dimerization in vivo not characterized\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [2, 3, 5]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [1, 2, 3]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 4, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [0, 1, 4]}\n    ],\n    \"complexes\": [\n      \"SOSS1 (INTS3–hSSB1–C9ORF80)\"\n    ],\n    \"partners\": [\n      \"NABP2\",\n      \"INTS6\",\n      \"C9ORF80\",\n      \"ATRIP\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}