{"gene":"CD2BP2","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":2005,"finding":"CD2BP2 (U5-52K) is a constituent of the 20S U5 snRNP but is absent from the U4/U6.U5 tri-snRNP, making it the only 20S U5-specific protein not integrated into the tri-snRNP. Its N-terminal two-thirds interact with U5-102K (hPrp6), and its C-terminal GYF domain binds U5-15K in a polyproline-independent manner, suggesting CD2BP2 may function in tri-snRNP assembly.","method":"Protein sequencing, immunofluorescence, yeast two-hybrid screening, pulldown assays, glycerol gradient sedimentation","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal two-hybrid and pulldown assays, combined with biochemical fractionation; multiple orthogonal methods in a single focused study","pmids":["15840814"],"is_preprint":false},{"year":2007,"finding":"Crystal structure of the CD2BP2 GYF domain in complex with U5-15K reveals that the interaction between U5-52K and U5-15K does not involve the canonical polyproline-binding site of the GYF domain nor the common ligand-binding cleft of the thioredoxin-like U5-15K protein, providing the structural basis for the bifunctionality of U5-52K in both spliceosomal assembly and CD2 receptor binding.","method":"X-ray crystallography (crystal structure of GYF domain–U5-15K complex)","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional validation of binding interfaces; single lab but Tier 1 method","pmids":["17467737"],"is_preprint":false},{"year":2004,"finding":"The CD2BP2 GYF domain binds proline-rich sequences with the consensus motif (R/K/G)XXPPGX(R/K). The core spliceosomal protein SmB/B', which contains multiple PPPPGMR motifs, interacts with the CD2BP2 GYF domain both in vitro and in vivo, and CD2BP2 colocalizes with SmB proteins in the nucleus of Jurkat T cells and HeLa cells.","method":"Peptide binding assays, in vitro pulldown, yeast two-hybrid, immunofluorescence colocalization","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (in vitro binding, two-hybrid, colocalization) in single focused study","pmids":["15105431"],"is_preprint":false},{"year":2005,"finding":"Novel interaction partners of the CD2BP2 GYF domain include PI31 and NPWBP; the recognition motif was refined by phage display to PPG(W/F/Y/M/L), and NMR spectroscopy revealed a conserved binding surface on the GYF domain for these peptide interactions.","method":"Phage display, SPOT analysis, NMR spectroscopy, yeast two-hybrid","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods (phage display, NMR, two-hybrid), but limited functional follow-up on novel partners","pmids":["16000308"],"is_preprint":false},{"year":2015,"finding":"CD2BP2 is essential for embryogenesis in mice; conditional knockout causes growth retardation, vascularization defects, and death at embryonic day 10.5. Ablation in macrophages affects alternative splicing of diverse mRNA transcripts. CD2BP2 recruits the phosphatase PP1 to the spliceosome via its N-terminus. Podocyte-specific depletion causes exon usage changes in VEGF and actin regulator genes, foot process effacement, proteinuria, and lethal kidney failure.","method":"Conditional gene targeting in mice (KO), RNA-seq/next-generation sequencing of exon usage, co-immunoprecipitation (PP1 interaction), histology/electron microscopy","journal":"Journal of molecular cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo conditional KO with defined molecular phenotypes, PP1 interaction by Co-IP, RNA-seq; multiple orthogonal approaches","pmids":["26082520"],"is_preprint":false},{"year":2012,"finding":"C. elegans TEG-1 (CD2BP2 ortholog) directly binds UAF-1 (U2AF65) splicing factor; human CD2BP2 also directly binds UAF-1/U2AF65, indicating that CD2BP2 functions at two distinct steps in the splicing cascade: early (U2AF interaction) and later (U4/U6.U5 tri-snRNP assembly).","method":"Genetic screens, co-immunoprecipitation (C. elegans and human cells), nuclear localization assay","journal":"Developmental dynamics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding confirmed by Co-IP in both C. elegans and human cells, but single lab","pmids":["22275078"],"is_preprint":false},{"year":2017,"finding":"TEG-1 (CD2BP2 ortholog in C. elegans) regulates miRISC stability by physically interacting with VIG-1 and complexing with mature let-7 miRNA; loss of teg-1 reduces abundance of multiple miRNA families and destabilizes miRISC effectors VIG-1 and ALG-1. In human HeLa cells, CD2BP2 (human TEG-1 ortholog), SERBP1/PAI-RBP1 (VIG-1 ortholog), and AGO2 (ALG-1 ortholog) form a complex, and knockdown of CD2BP2 results in reduced miRNA levels.","method":"Co-immunoprecipitation, RNA immunoprecipitation, genetic epistasis (C. elegans), siRNA knockdown (HeLa cells), Northern blot/quantitative PCR","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP in both model organism and human cells, RIP, and functional knockdown with miRNA level readout; single lab","pmids":["28180320"],"is_preprint":false},{"year":2007,"finding":"CD2BP2's nuclear localization is not altered when CD2 and CD2BP2 are co-expressed in HeLa cells, indicating that nuclear proline-rich sequence partners compete effectively with CD2 for GYF domain binding. CD2BP2 knockdown in primary T cells (PBMCs) did not produce a major difference in cytokine expression, suggesting CD2 signaling is at least partially independent of CD2BP2.","method":"Immunofluorescence (HeLa co-expression), siRNA knockdown in PBMCs, cytokine expression assay","journal":"International immunology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, largely negative/inconclusive results with single methods per assay","pmids":["17906334"],"is_preprint":false},{"year":2024,"finding":"T cell-specific ablation of CD2BP2/U5-52K causes increased T cell lymphopenia, T cell death, and a proliferation/differentiation imbalance. This is accompanied by a substantial increase in exon skipping, including skipping of exon 7 in Mdm4, coinciding with upregulation of pro-apoptotic gene expression. Naïve T cells show enhanced sensitivity compared to memory T cells to loss of CD2BP2/U5-52K.","method":"Conditional T cell-specific knockout (Cre-lox), RNA-seq (exon skipping analysis), crosslinking mass spectrometry, flow cytometry","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with RNA-seq and specific Mdm4 exon skipping mechanistic link, but single lab","pmids":["39308865"],"is_preprint":false}],"current_model":"CD2BP2 (U5-52K) is a bifunctional nuclear GYF domain-containing adaptor protein that serves as a pre-spliceosomal assembly factor: it integrates into the 20S U5 snRNP via GYF domain–U5-15K interaction (structurally characterized by crystallography) and N-terminal–U5-102K interaction, but is excluded from the mature U4/U6.U5 tri-snRNP; it also recruits PP1 phosphatase to the spliceosome via its N-terminus, interacts with U2AF65 (UAF-1) at an earlier splicing step, and stabilizes miRISC components (AGO2/SERBP1) to maintain miRNA levels, while its GYF domain mediates polyproline-dependent interactions with SmB/B' and CD2 as well as polyproline-independent interaction with U5-15K; conditional knockout in mice demonstrates it is essential for embryogenesis, podocyte integrity, and T cell homeostasis through its role in regulating alternative splicing."},"narrative":{"mechanistic_narrative":"CD2BP2 (U5-52K) is a nuclear GYF domain adaptor that functions as a pre-spliceosomal assembly factor regulating alternative splicing [PMID:15840814, PMID:26082520]. It is the only protein specific to the 20S U5 snRNP that is excluded from the mature U4/U6.U5 tri-snRNP, integrating into the U5 particle through an N-terminal interaction with U5-102K (hPrp6) and a C-terminal GYF domain interaction with U5-15K [PMID:15840814]. Crystallography established that the GYF domain binds U5-15K in a polyproline-independent manner, away from the canonical proline-recognition cleft, structurally explaining how the same domain can simultaneously engage proline-rich ligands such as the core spliceosomal protein SmB/B' and the CD2 receptor [PMID:17467737, PMID:15105431]. Through its N-terminus, CD2BP2 recruits the phosphatase PP1 to the spliceosome, and it engages the early splicing factor U2AF65 (UAF-1), placing it at two distinct steps of the splicing cascade [PMID:26082520, PMID:22275078]. Beyond splicing, CD2BP2 forms a complex with SERBP1 and AGO2 to stabilize miRISC and maintain miRNA levels [PMID:28180320]. Conditional knockout in mice shows it is essential for embryogenesis, podocyte integrity, and T cell homeostasis, where its loss drives aberrant exon skipping—including Mdm4 exon 7—and pro-apoptotic gene expression [PMID:26082520, PMID:39308865].","teleology":[{"year":2004,"claim":"Defining the binding specificity of the CD2BP2 GYF domain established how this adaptor selects proline-rich partners and linked it to the core spliceosome.","evidence":"Peptide binding, in vitro pulldown, yeast two-hybrid, and immunofluorescence colocalization identifying SmB/B' as a GYF ligand","pmids":["15105431"],"confidence":"High","gaps":["Functional consequence of the SmB/B'-GYF interaction for splicing not established","Did not resolve whether nuclear and CD2 receptor binding are mutually exclusive"]},{"year":2005,"claim":"Placing CD2BP2 within the 20S U5 snRNP but not the tri-snRNP positioned it as a candidate tri-snRNP assembly factor and mapped its two snRNP contacts.","evidence":"Protein sequencing, yeast two-hybrid, pulldown, and glycerol gradient sedimentation showing U5-102K and U5-15K interactions","pmids":["15840814"],"confidence":"High","gaps":["Direct demonstration that CD2BP2 drives tri-snRNP assembly absent","Mechanism of CD2BP2 exclusion from mature tri-snRNP unknown"]},{"year":2005,"claim":"Refining the GYF recognition motif and mapping its binding surface generalized CD2BP2's role as a modular proline-rich peptide adaptor.","evidence":"Phage display, SPOT analysis, NMR, and yeast two-hybrid identifying PI31 and NPWBP","pmids":["16000308"],"confidence":"Medium","gaps":["Biological roles of PI31 and NPWBP interactions not pursued","No structural complex with these ligands"]},{"year":2007,"claim":"The crystal structure resolved how a single GYF domain accommodates both polyproline-independent (U5-15K) and polyproline-dependent (CD2) partners, explaining CD2BP2 bifunctionality.","evidence":"X-ray crystallography of the GYF domain–U5-15K complex with binding-interface validation","pmids":["17467737"],"confidence":"High","gaps":["Structure of full-length CD2BP2 within the snRNP not determined","Dynamics of ligand switching not addressed"]},{"year":2007,"claim":"Testing whether CD2 signaling depends on CD2BP2 indicated nuclear partners outcompete CD2 and that CD2 function is largely independent of CD2BP2.","evidence":"HeLa co-expression immunofluorescence and siRNA knockdown in PBMCs with cytokine readout","pmids":["17906334"],"confidence":"Low","gaps":["Largely negative/inconclusive results from single methods per assay","No quantitative measure of competition between CD2 and nuclear ligands"]},{"year":2012,"claim":"Identifying a direct U2AF65/UAF-1 interaction extended CD2BP2's action to an early splicing step, beyond tri-snRNP assembly.","evidence":"Genetic screens and co-immunoprecipitation in C. elegans and human cells","pmids":["22275078"],"confidence":"Medium","gaps":["Functional consequence of the U2AF65 interaction for specific splicing events not shown","Single lab; no reciprocal structural mapping"]},{"year":2015,"claim":"Conditional knockouts established CD2BP2 as essential in vivo for embryogenesis and podocyte function and linked it mechanistically to PP1 recruitment and alternative exon usage.","evidence":"Conditional mouse knockouts with RNA-seq exon-usage analysis, PP1 co-immunoprecipitation, and histology/EM","pmids":["26082520"],"confidence":"High","gaps":["How PP1 recruitment modulates specific splicing events not resolved","Direct splicing targets responsible for podocyte failure not pinpointed"]},{"year":2017,"claim":"Discovery of a CD2BP2–SERBP1–AGO2 complex revealed a splicing-independent role in stabilizing miRISC and maintaining miRNA abundance.","evidence":"Co-IP, RNA immunoprecipitation, genetic epistasis in C. elegans, and siRNA knockdown in HeLa with miRNA-level readouts","pmids":["28180320"],"confidence":"Medium","gaps":["Whether miRISC stabilization is direct or a downstream consequence of splicing changes unresolved","Domain of CD2BP2 mediating the miRISC interaction not mapped"]},{"year":2024,"claim":"T cell-specific ablation tied CD2BP2 loss to defined exon-skipping events (Mdm4 exon 7) and apoptosis, explaining its requirement for T cell homeostasis.","evidence":"Conditional T cell knockout with RNA-seq exon-skipping analysis, crosslinking mass spectrometry, and flow cytometry","pmids":["39308865"],"confidence":"Medium","gaps":["Causal contribution of Mdm4 mis-splicing to T cell death not isolated from other targets","Why naive T cells are more sensitive than memory T cells unexplained"]},{"year":null,"claim":"It remains unresolved how CD2BP2 integrates its multiple roles—tri-snRNP assembly, U2AF/early splicing, PP1 recruitment, and miRISC stabilization—into a unified mechanism governing splice-site selection.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of CD2BP2 within an assembling spliceosome","Rules governing which alternative exons depend on CD2BP2 not defined","Mechanistic connection between splicing and miRISC functions unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[6]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,2]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,4,5]}],"complexes":["20S U5 snRNP","miRISC (CD2BP2-SERBP1-AGO2)"],"partners":["U5-15K","U5-102K","SMB/B'","U2AF65","PP1","SERBP1","AGO2","CD2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O95400","full_name":"CD2 antigen cytoplasmic tail-binding protein 2","aliases":["U5 snRNP 52K protein","U5-52K"],"length_aa":341,"mass_kda":37.6,"function":"Involved in pre-mRNA splicing as component of the U5 snRNP complex that is involved in spliceosome assembly","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/O95400/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CD2BP2","classification":"Common Essential","n_dependent_lines":479,"n_total_lines":1208,"dependency_fraction":0.396523178807947},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000169217","cell_line_id":"CID001623","localizations":[{"compartment":"nucleoplasm","grade":3},{"compartment":"nuclear_punctae","grade":2}],"interactors":[{"gene":"PRPF8","stoichiometry":10.0},{"gene":"DDX23","stoichiometry":4.0},{"gene":"SNRPE","stoichiometry":4.0},{"gene":"PRPF6","stoichiometry":4.0},{"gene":"SNRNP200","stoichiometry":4.0},{"gene":"C9ORF78","stoichiometry":0.2},{"gene":"SMU1","stoichiometry":0.2},{"gene":"EAPP","stoichiometry":0.2},{"gene":"DDX42","stoichiometry":0.2},{"gene":"TXNL4A","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001623","total_profiled":1310},"omim":[{"mim_id":"604470","title":"CD2 ANTIGEN-BINDING PROTEIN 2; CD2BP2","url":"https://www.omim.org/entry/604470"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nuclear speckles","reliability":"Enhanced"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CD2BP2"},"hgnc":{"alias_symbol":["LIN1","Snu40","PPP1R59","U5-52K"],"prev_symbol":[]},"alphafold":{"accession":"O95400","domains":[{"cath_id":"-","chopping":"64-128","consensus_level":"medium","plddt":81.4275,"start":64,"end":128},{"cath_id":"-","chopping":"153-179_195-235","consensus_level":"high","plddt":86.3869,"start":153,"end":235},{"cath_id":"3.30.1490.40","chopping":"281-338","consensus_level":"high","plddt":86.9657,"start":281,"end":338}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O95400","model_url":"https://alphafold.ebi.ac.uk/files/AF-O95400-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O95400-F1-predicted_aligned_error_v6.png","plddt_mean":69.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CD2BP2","jax_strain_url":"https://www.jax.org/strain/search?query=CD2BP2"},"sequence":{"accession":"O95400","fasta_url":"https://rest.uniprot.org/uniprotkb/O95400.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O95400/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O95400"}},"corpus_meta":[{"pmid":"9604932","id":"PMC_9604932","title":"MAP kinase signaling specificity mediated by the LIN-1 Ets/LIN-31 WH transcription factor complex during C. elegans vulval induction.","date":"1998","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/9604932","citation_count":171,"is_preprint":false},{"pmid":"8543158","id":"PMC_8543158","title":"The Caenorhabditis elegans gene lin-1 encodes an ETS-domain protein and defines a branch of the vulval induction pathway.","date":"1995","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/8543158","citation_count":141,"is_preprint":false},{"pmid":"9691039","id":"PMC_9691039","title":"Gain-of-function mutations in the Caenorhabditis elegans lin-1 ETS gene identify a C-terminal regulatory domain phosphorylated by ERK MAP kinase.","date":"1998","source":"Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9691039","citation_count":95,"is_preprint":false},{"pmid":"15689373","id":"PMC_15689373","title":"Sumoylation of LIN-1 promotes transcriptional repression and inhibition of vulval cell fates.","date":"2005","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/15689373","citation_count":55,"is_preprint":false},{"pmid":"15840814","id":"PMC_15840814","title":"The human U5 snRNP 52K protein (CD2BP2) interacts with U5-102K (hPrp6), a U4/U6.U5 tri-snRNP bridging protein, but dissociates upon tri-snRNP formation.","date":"2005","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/15840814","citation_count":50,"is_preprint":false},{"pmid":"25020258","id":"PMC_25020258","title":"Molecular characterization and expression of CD2BP2 in Nile tilapia (Oreochromis niloticus) in response to Streptococcus agalactiae stimulus.","date":"2014","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/25020258","citation_count":35,"is_preprint":false},{"pmid":"15105431","id":"PMC_15105431","title":"Recognition sequences for the GYF domain reveal a possible spliceosomal function of CD2BP2.","date":"2004","source":"The Journal of biological 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B","url":"https://pubmed.ncbi.nlm.nih.gov/22624583","citation_count":4,"is_preprint":false},{"pmid":"39308865","id":"PMC_39308865","title":"The nuclear GYF protein CD2BP2/U5-52K is required for T cell homeostasis.","date":"2024","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/39308865","citation_count":3,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11315,"output_tokens":2578,"usd":0.036307,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9736,"output_tokens":3069,"usd":0.062702,"stage2_stop_reason":"end_turn"},"total_usd":0.099009,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"CD2BP2 (U5-52K) is a constituent of the 20S U5 snRNP but is absent from the U4/U6.U5 tri-snRNP, making it the only 20S U5-specific protein not integrated into the tri-snRNP. Its N-terminal two-thirds interact with U5-102K (hPrp6), and its C-terminal GYF domain binds U5-15K in a polyproline-independent manner, suggesting CD2BP2 may function in tri-snRNP assembly.\",\n      \"method\": \"Protein sequencing, immunofluorescence, yeast two-hybrid screening, pulldown assays, glycerol gradient sedimentation\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal two-hybrid and pulldown assays, combined with biochemical fractionation; multiple orthogonal methods in a single focused study\",\n      \"pmids\": [\"15840814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structure of the CD2BP2 GYF domain in complex with U5-15K reveals that the interaction between U5-52K and U5-15K does not involve the canonical polyproline-binding site of the GYF domain nor the common ligand-binding cleft of the thioredoxin-like U5-15K protein, providing the structural basis for the bifunctionality of U5-52K in both spliceosomal assembly and CD2 receptor binding.\",\n      \"method\": \"X-ray crystallography (crystal structure of GYF domain–U5-15K complex)\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional validation of binding interfaces; single lab but Tier 1 method\",\n      \"pmids\": [\"17467737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The CD2BP2 GYF domain binds proline-rich sequences with the consensus motif (R/K/G)XXPPGX(R/K). The core spliceosomal protein SmB/B', which contains multiple PPPPGMR motifs, interacts with the CD2BP2 GYF domain both in vitro and in vivo, and CD2BP2 colocalizes with SmB proteins in the nucleus of Jurkat T cells and HeLa cells.\",\n      \"method\": \"Peptide binding assays, in vitro pulldown, yeast two-hybrid, immunofluorescence colocalization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (in vitro binding, two-hybrid, colocalization) in single focused study\",\n      \"pmids\": [\"15105431\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Novel interaction partners of the CD2BP2 GYF domain include PI31 and NPWBP; the recognition motif was refined by phage display to PPG(W/F/Y/M/L), and NMR spectroscopy revealed a conserved binding surface on the GYF domain for these peptide interactions.\",\n      \"method\": \"Phage display, SPOT analysis, NMR spectroscopy, yeast two-hybrid\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods (phage display, NMR, two-hybrid), but limited functional follow-up on novel partners\",\n      \"pmids\": [\"16000308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CD2BP2 is essential for embryogenesis in mice; conditional knockout causes growth retardation, vascularization defects, and death at embryonic day 10.5. Ablation in macrophages affects alternative splicing of diverse mRNA transcripts. CD2BP2 recruits the phosphatase PP1 to the spliceosome via its N-terminus. Podocyte-specific depletion causes exon usage changes in VEGF and actin regulator genes, foot process effacement, proteinuria, and lethal kidney failure.\",\n      \"method\": \"Conditional gene targeting in mice (KO), RNA-seq/next-generation sequencing of exon usage, co-immunoprecipitation (PP1 interaction), histology/electron microscopy\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo conditional KO with defined molecular phenotypes, PP1 interaction by Co-IP, RNA-seq; multiple orthogonal approaches\",\n      \"pmids\": [\"26082520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"C. elegans TEG-1 (CD2BP2 ortholog) directly binds UAF-1 (U2AF65) splicing factor; human CD2BP2 also directly binds UAF-1/U2AF65, indicating that CD2BP2 functions at two distinct steps in the splicing cascade: early (U2AF interaction) and later (U4/U6.U5 tri-snRNP assembly).\",\n      \"method\": \"Genetic screens, co-immunoprecipitation (C. elegans and human cells), nuclear localization assay\",\n      \"journal\": \"Developmental dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding confirmed by Co-IP in both C. elegans and human cells, but single lab\",\n      \"pmids\": [\"22275078\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TEG-1 (CD2BP2 ortholog in C. elegans) regulates miRISC stability by physically interacting with VIG-1 and complexing with mature let-7 miRNA; loss of teg-1 reduces abundance of multiple miRNA families and destabilizes miRISC effectors VIG-1 and ALG-1. In human HeLa cells, CD2BP2 (human TEG-1 ortholog), SERBP1/PAI-RBP1 (VIG-1 ortholog), and AGO2 (ALG-1 ortholog) form a complex, and knockdown of CD2BP2 results in reduced miRNA levels.\",\n      \"method\": \"Co-immunoprecipitation, RNA immunoprecipitation, genetic epistasis (C. elegans), siRNA knockdown (HeLa cells), Northern blot/quantitative PCR\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP in both model organism and human cells, RIP, and functional knockdown with miRNA level readout; single lab\",\n      \"pmids\": [\"28180320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CD2BP2's nuclear localization is not altered when CD2 and CD2BP2 are co-expressed in HeLa cells, indicating that nuclear proline-rich sequence partners compete effectively with CD2 for GYF domain binding. CD2BP2 knockdown in primary T cells (PBMCs) did not produce a major difference in cytokine expression, suggesting CD2 signaling is at least partially independent of CD2BP2.\",\n      \"method\": \"Immunofluorescence (HeLa co-expression), siRNA knockdown in PBMCs, cytokine expression assay\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, largely negative/inconclusive results with single methods per assay\",\n      \"pmids\": [\"17906334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"T cell-specific ablation of CD2BP2/U5-52K causes increased T cell lymphopenia, T cell death, and a proliferation/differentiation imbalance. This is accompanied by a substantial increase in exon skipping, including skipping of exon 7 in Mdm4, coinciding with upregulation of pro-apoptotic gene expression. Naïve T cells show enhanced sensitivity compared to memory T cells to loss of CD2BP2/U5-52K.\",\n      \"method\": \"Conditional T cell-specific knockout (Cre-lox), RNA-seq (exon skipping analysis), crosslinking mass spectrometry, flow cytometry\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with RNA-seq and specific Mdm4 exon skipping mechanistic link, but single lab\",\n      \"pmids\": [\"39308865\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CD2BP2 (U5-52K) is a bifunctional nuclear GYF domain-containing adaptor protein that serves as a pre-spliceosomal assembly factor: it integrates into the 20S U5 snRNP via GYF domain–U5-15K interaction (structurally characterized by crystallography) and N-terminal–U5-102K interaction, but is excluded from the mature U4/U6.U5 tri-snRNP; it also recruits PP1 phosphatase to the spliceosome via its N-terminus, interacts with U2AF65 (UAF-1) at an earlier splicing step, and stabilizes miRISC components (AGO2/SERBP1) to maintain miRNA levels, while its GYF domain mediates polyproline-dependent interactions with SmB/B' and CD2 as well as polyproline-independent interaction with U5-15K; conditional knockout in mice demonstrates it is essential for embryogenesis, podocyte integrity, and T cell homeostasis through its role in regulating alternative splicing.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CD2BP2 (U5-52K) is a nuclear GYF domain adaptor that functions as a pre-spliceosomal assembly factor regulating alternative splicing [#0, #4]. It is the only protein specific to the 20S U5 snRNP that is excluded from the mature U4/U6.U5 tri-snRNP, integrating into the U5 particle through an N-terminal interaction with U5-102K (hPrp6) and a C-terminal GYF domain interaction with U5-15K [#0]. Crystallography established that the GYF domain binds U5-15K in a polyproline-independent manner, away from the canonical proline-recognition cleft, structurally explaining how the same domain can simultaneously engage proline-rich ligands such as the core spliceosomal protein SmB/B' and the CD2 receptor [#1, #2]. Through its N-terminus, CD2BP2 recruits the phosphatase PP1 to the spliceosome, and it engages the early splicing factor U2AF65 (UAF-1), placing it at two distinct steps of the splicing cascade [#4, #5]. Beyond splicing, CD2BP2 forms a complex with SERBP1 and AGO2 to stabilize miRISC and maintain miRNA levels [#6]. Conditional knockout in mice shows it is essential for embryogenesis, podocyte integrity, and T cell homeostasis, where its loss drives aberrant exon skipping—including Mdm4 exon 7—and pro-apoptotic gene expression [#4, #8].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Defining the binding specificity of the CD2BP2 GYF domain established how this adaptor selects proline-rich partners and linked it to the core spliceosome.\",\n      \"evidence\": \"Peptide binding, in vitro pulldown, yeast two-hybrid, and immunofluorescence colocalization identifying SmB/B' as a GYF ligand\",\n      \"pmids\": [\"15105431\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of the SmB/B'-GYF interaction for splicing not established\", \"Did not resolve whether nuclear and CD2 receptor binding are mutually exclusive\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Placing CD2BP2 within the 20S U5 snRNP but not the tri-snRNP positioned it as a candidate tri-snRNP assembly factor and mapped its two snRNP contacts.\",\n      \"evidence\": \"Protein sequencing, yeast two-hybrid, pulldown, and glycerol gradient sedimentation showing U5-102K and U5-15K interactions\",\n      \"pmids\": [\"15840814\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct demonstration that CD2BP2 drives tri-snRNP assembly absent\", \"Mechanism of CD2BP2 exclusion from mature tri-snRNP unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Refining the GYF recognition motif and mapping its binding surface generalized CD2BP2's role as a modular proline-rich peptide adaptor.\",\n      \"evidence\": \"Phage display, SPOT analysis, NMR, and yeast two-hybrid identifying PI31 and NPWBP\",\n      \"pmids\": [\"16000308\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Biological roles of PI31 and NPWBP interactions not pursued\", \"No structural complex with these ligands\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"The crystal structure resolved how a single GYF domain accommodates both polyproline-independent (U5-15K) and polyproline-dependent (CD2) partners, explaining CD2BP2 bifunctionality.\",\n      \"evidence\": \"X-ray crystallography of the GYF domain–U5-15K complex with binding-interface validation\",\n      \"pmids\": [\"17467737\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of full-length CD2BP2 within the snRNP not determined\", \"Dynamics of ligand switching not addressed\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Testing whether CD2 signaling depends on CD2BP2 indicated nuclear partners outcompete CD2 and that CD2 function is largely independent of CD2BP2.\",\n      \"evidence\": \"HeLa co-expression immunofluorescence and siRNA knockdown in PBMCs with cytokine readout\",\n      \"pmids\": [\"17906334\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Largely negative/inconclusive results from single methods per assay\", \"No quantitative measure of competition between CD2 and nuclear ligands\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identifying a direct U2AF65/UAF-1 interaction extended CD2BP2's action to an early splicing step, beyond tri-snRNP assembly.\",\n      \"evidence\": \"Genetic screens and co-immunoprecipitation in C. elegans and human cells\",\n      \"pmids\": [\"22275078\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of the U2AF65 interaction for specific splicing events not shown\", \"Single lab; no reciprocal structural mapping\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Conditional knockouts established CD2BP2 as essential in vivo for embryogenesis and podocyte function and linked it mechanistically to PP1 recruitment and alternative exon usage.\",\n      \"evidence\": \"Conditional mouse knockouts with RNA-seq exon-usage analysis, PP1 co-immunoprecipitation, and histology/EM\",\n      \"pmids\": [\"26082520\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How PP1 recruitment modulates specific splicing events not resolved\", \"Direct splicing targets responsible for podocyte failure not pinpointed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Discovery of a CD2BP2–SERBP1–AGO2 complex revealed a splicing-independent role in stabilizing miRISC and maintaining miRNA abundance.\",\n      \"evidence\": \"Co-IP, RNA immunoprecipitation, genetic epistasis in C. elegans, and siRNA knockdown in HeLa with miRNA-level readouts\",\n      \"pmids\": [\"28180320\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether miRISC stabilization is direct or a downstream consequence of splicing changes unresolved\", \"Domain of CD2BP2 mediating the miRISC interaction not mapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"T cell-specific ablation tied CD2BP2 loss to defined exon-skipping events (Mdm4 exon 7) and apoptosis, explaining its requirement for T cell homeostasis.\",\n      \"evidence\": \"Conditional T cell knockout with RNA-seq exon-skipping analysis, crosslinking mass spectrometry, and flow cytometry\",\n      \"pmids\": [\"39308865\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal contribution of Mdm4 mis-splicing to T cell death not isolated from other targets\", \"Why naive T cells are more sensitive than memory T cells unexplained\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how CD2BP2 integrates its multiple roles—tri-snRNP assembly, U2AF/early splicing, PP1 recruitment, and miRISC stabilization—into a unified mechanism governing splice-site selection.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of CD2BP2 within an assembling spliceosome\", \"Rules governing which alternative exons depend on CD2BP2 not defined\", \"Mechanistic connection between splicing and miRISC functions unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 4, 5]}\n    ],\n    \"complexes\": [\"20S U5 snRNP\", \"miRISC (CD2BP2-SERBP1-AGO2)\"],\n    \"partners\": [\"U5-15K\", \"U5-102K\", \"SmB/B'\", \"U2AF65\", \"PP1\", \"SERBP1\", \"AGO2\", \"CD2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}