{"gene":"PF3D7_0318600","run_date":"2026-09-07T10:55:51","timeline":{"discoveries":[{"year":2017,"finding":"PfCPSF3 (PF3D7_0318600), a homologue of mammalian CPSF subunit 3 (CPSF-73), is the direct target of the antimalarial benzoxaborole AN3661: in vitro resistance selection and CRISPR-Cas9 introduction of pfcpsf3 point mutations into parental lines both recapitulated AN3661 resistance, and homology modelling placed the resistance-conferring mutations in the active site where AN3661 is predicted to bind. AN3661 treatment caused loss of trophozoite-expressed transcripts, consistent with inhibition of pre-mRNA 3′-end processing, and this effect was absent in resistant parasites, establishing PfCPSF3 as the functional target.","method":"In vitro resistance selection, CRISPR-Cas9 active-site mutagenesis, homology modelling, transcript analysis in wild-type vs. resistant parasites","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — CRISPR-Cas9 mutagenesis directly recapitulated resistance phenotype, combined with homology modelling and functional transcript readout; multiple orthogonal methods in a single rigorous study","pmids":["28262680"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2024,"finding":"PF3D7_0318600 (cleavage and polyadenylation specificity factor subunit_A) was identified as a component of the PfCRL4 complex in P. falciparum by immunoprecipitation and sequence analysis; the complex also contains PfCullin-2, PfRbx1, and WD40 repeat proteins.","method":"Immunoprecipitation, sequence analysis","journal":"PLoS Pathogens","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP experiment identifying PF3D7_0318600 as a complex member; no direct functional follow-up on this subunit itself","pmids":["38416790"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PfCPSF3 (PF3D7_0318600) is a pre-mRNA 3′-end processing endonuclease (CPSF-73 homologue) whose active site is the direct molecular target of the benzoxaborole AN3661, and it additionally participates as a subunit of a CRL4-like ubiquitin E3 ligase complex (PfCRL4) in P. falciparum."},"narrative":{"mechanistic_narrative":"PfCPSF3 (PF3D7_0318600) is a homologue of the mammalian CPSF-73 subunit that functions in pre-mRNA 3′-end processing in Plasmodium falciparum, and it is the direct molecular target of the antimalarial benzoxaborole AN3661 [PMID:28262680]. In vitro resistance selection and CRISPR-Cas9 introduction of point mutations into pfcpsf3 both recapitulated AN3661 resistance, and homology modelling placed the resistance-conferring residues in the predicted AN3661-binding active site; AN3661 treatment caused loss of trophozoite-expressed transcripts consistent with inhibition of 3′-end processing, an effect abolished in resistant parasites [PMID:28262680]. Beyond its role as an AN3661 target in pre-mRNA processing, the available corpus establishes only that PF3D7_0318600 was identified as a component of the PfCRL4 ubiquitin E3 ligase complex (which also contains PfCullin-2, PfRbx1, and WD40 repeat proteins) by immunoprecipitation, without direct functional characterization of this subunit within that complex [PMID:38416790].","teleology":[{"year":2017,"claim":"It was unknown what protein mediated the antimalarial activity of the benzoxaborole AN3661; identifying PfCPSF3 as the direct target established both the drug's mechanism and a functional role for this protein in pre-mRNA 3′-end processing.","evidence":"In vitro resistance selection with CRISPR-Cas9 active-site mutagenesis, homology modelling, and transcript analysis in wild-type versus resistant parasites","pmids":["28262680"],"confidence":"High","gaps":["No direct enzymatic reconstitution of PfCPSF3 endonuclease activity","Interaction with other CPSF subunits or the broader 3′-end processing machinery not mapped","AN3661 binding mode inferred from homology modelling rather than an experimental structure"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2024,"claim":"Whether PF3D7_0318600 participates in complexes beyond RNA processing was unaddressed; it was identified as a member of the PfCRL4 E3 ubiquitin ligase complex, hinting at a role outside canonical 3′-end processing.","evidence":"Immunoprecipitation and sequence analysis in P. falciparum","pmids":["38416790"],"confidence":"Low","gaps":["Single Co-IP without reciprocal validation or functional follow-up on this subunit","The functional contribution of PF3D7_0318600 within PfCRL4 is undefined","Relationship between the PfCRL4 association and the 3′-end processing role is unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"How PfCPSF3 integrates a canonical pre-mRNA 3′-end processing function with membership in a CRL4-like ubiquitin ligase complex, and its direct catalytic mechanism, remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No experimental structure of PfCPSF3 or its AN3661 complex","Endonuclease activity not biochemically reconstituted","Functional role within PfCRL4 uncharacterized"],"attribution":[]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["PfCRL4 CRL4-like ubiquitin E3 ligase complex"],"partners":["PFCULLIN-2","PFRBX1","WD40 REPEAT PROTEINS"],"other_free_text":["Direct molecular target of the antimalarial benzoxaborole AN3661; active-site point mutations confer AN3661 resistance [#0]","Homologue of mammalian CPSF subunit 3 (CPSF-73) [#0]","PfCRL4 complex membership established by a single Co-IP without functional characterization of this subunit [#1]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"28262680","id":"PMC_28262680","title":"A potent antimalarial benzoxaborole targets a Plasmodium falciparum cleavage and polyadenylation specificity factor homologue.","date":"2017","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/28262680","citation_count":104,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["cleavage and polyadenylation specificity factor"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"38416790","id":"PMC_38416790","title":"Plasmodium falciparum contains functional SCF and CRL4 ubiquitin E3 ligases, and CRL4 is critical for cell division and membrane integrity.","date":"2024","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/38416790","citation_count":12,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["cleavage and polyadenylation specificity factor"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":4112,"output_tokens":783,"usd":0.012041,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":5367,"output_tokens":1615,"usd":0.033605,"stage2_stop_reason":"end_turn"},"total_usd":0.045646,"stage1_batch_id":"msgbatch_01DbiymDmubkPpYvdbAu5a7V","stage2_batch_id":"msgbatch_012TbrWvMRH129T7PQ4SmvrJ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"PfCPSF3 (PF3D7_0318600), a homologue of mammalian CPSF subunit 3 (CPSF-73), is the direct target of the antimalarial benzoxaborole AN3661: in vitro resistance selection and CRISPR-Cas9 introduction of pfcpsf3 point mutations into parental lines both recapitulated AN3661 resistance, and homology modelling placed the resistance-conferring mutations in the active site where AN3661 is predicted to bind. AN3661 treatment caused loss of trophozoite-expressed transcripts, consistent with inhibition of pre-mRNA 3′-end processing, and this effect was absent in resistant parasites, establishing PfCPSF3 as the functional target.\",\n      \"method\": \"In vitro resistance selection, CRISPR-Cas9 active-site mutagenesis, homology modelling, transcript analysis in wild-type vs. resistant parasites\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — CRISPR-Cas9 mutagenesis directly recapitulated resistance phenotype, combined with homology modelling and functional transcript readout; multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"28262680\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PF3D7_0318600 (cleavage and polyadenylation specificity factor subunit_A) was identified as a component of the PfCRL4 complex in P. falciparum by immunoprecipitation and sequence analysis; the complex also contains PfCullin-2, PfRbx1, and WD40 repeat proteins.\",\n      \"method\": \"Immunoprecipitation, sequence analysis\",\n      \"journal\": \"PLoS Pathogens\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP experiment identifying PF3D7_0318600 as a complex member; no direct functional follow-up on this subunit itself\",\n      \"pmids\": [\"38416790\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    }\n  ],\n  \"current_model\": \"PfCPSF3 (PF3D7_0318600) is a pre-mRNA 3′-end processing endonuclease (CPSF-73 homologue) whose active site is the direct molecular target of the benzoxaborole AN3661, and it additionally participates as a subunit of a CRL4-like ubiquitin E3 ligase complex (PfCRL4) in P. falciparum.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PfCPSF3 (PF3D7_0318600) is a homologue of the mammalian CPSF-73 subunit that functions in pre-mRNA 3′-end processing in Plasmodium falciparum, and it is the direct molecular target of the antimalarial benzoxaborole AN3661 [#0]. In vitro resistance selection and CRISPR-Cas9 introduction of point mutations into pfcpsf3 both recapitulated AN3661 resistance, and homology modelling placed the resistance-conferring residues in the predicted AN3661-binding active site; AN3661 treatment caused loss of trophozoite-expressed transcripts consistent with inhibition of 3′-end processing, an effect abolished in resistant parasites [#0]. Beyond its role as an AN3661 target in pre-mRNA processing, the available corpus establishes only that PF3D7_0318600 was identified as a component of the PfCRL4 ubiquitin E3 ligase complex (which also contains PfCullin-2, PfRbx1, and WD40 repeat proteins) by immunoprecipitation, without direct functional characterization of this subunit within that complex [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 2017,\n      \"claim\": \"It was unknown what protein mediated the antimalarial activity of the benzoxaborole AN3661; identifying PfCPSF3 as the direct target established both the drug's mechanism and a functional role for this protein in pre-mRNA 3′-end processing.\",\n      \"evidence\": \"In vitro resistance selection with CRISPR-Cas9 active-site mutagenesis, homology modelling, and transcript analysis in wild-type versus resistant parasites\",\n      \"pmids\": [\"28262680\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No direct enzymatic reconstitution of PfCPSF3 endonuclease activity\", \"Interaction with other CPSF subunits or the broader 3′-end processing machinery not mapped\", \"AN3661 binding mode inferred from homology modelling rather than an experimental structure\"],\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Whether PF3D7_0318600 participates in complexes beyond RNA processing was unaddressed; it was identified as a member of the PfCRL4 E3 ubiquitin ligase complex, hinting at a role outside canonical 3′-end processing.\",\n      \"evidence\": \"Immunoprecipitation and sequence analysis in P. falciparum\",\n      \"pmids\": [\"38416790\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP without reciprocal validation or functional follow-up on this subunit\", \"The functional contribution of PF3D7_0318600 within PfCRL4 is undefined\", \"Relationship between the PfCRL4 association and the 3′-end processing role is unknown\"],\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PfCPSF3 integrates a canonical pre-mRNA 3′-end processing function with membership in a CRL4-like ubiquitin ligase complex, and its direct catalytic mechanism, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No experimental structure of PfCPSF3 or its AN3661 complex\", \"Endonuclease activity not biochemically reconstituted\", \"Functional role within PfCRL4 uncharacterized\"],\n      \"attribution\": []\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004521\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"GO:0031124\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [\"PfCRL4 CRL4-like ubiquitin E3 ligase complex\"],\n    \"partners\": [\"PfCullin-2\", \"PfRbx1\", \"WD40 repeat proteins\"],\n    \"other_free_text\": [\n      \"Direct molecular target of the antimalarial benzoxaborole AN3661; active-site point mutations confer AN3661 resistance [#0]\",\n      \"Homologue of mammalian CPSF subunit 3 (CPSF-73) [#0]\",\n      \"PfCRL4 complex membership established by a single Co-IP without functional characterization of this subunit [#1]\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0318600","name":null,"product":"cleavage and polyadenylation specificity factor, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_0806600","name":null,"product":"cleavage and polyadenylation specificity factor, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_0822800","name":null,"product":"cleavage and polyadenylation specificity factor, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"","evaluation":null}