{"gene":"C9ORF78","run_date":"2026-04-28T17:12:38","timeline":{"discoveries":[{"year":2022,"finding":"C9ORF78 tightly interacts with the spliceosome remodeling factor BRR2 in vitro, and cryo-EM structures reveal that C9ORF78 and the spliceosomal B complex protein FBP21 wrap around the C-terminal helicase cassette of BRR2 in a mutually exclusive manner. Knockdown of C9ORF78 leads to alternative NAGNAG 3'-splice site usage and exon skipping, the latter dependent on BRR2. C9ORF78 could also contact PRPF22 when bound to BRR2, establishing C9ORF78 as a late-stage splicing regulatory protein that uses a multi-factor trafficking site on BRR2.","method":"Cryo-EM structure determination, in vitro binding assay, affinity purification/mass spectrometry, RNA UV-crosslinking, siRNA knockdown with alternative splicing readout","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure plus reconstituted in vitro interaction plus functional knockdown with multiple orthogonal methods in a single study","pmids":["35241646"],"is_preprint":false},{"year":2014,"finding":"The fission yeast homologue of C9ORF78, Tls1, associates with the spliceosome subunit Brr2 and regulates splicing of shelterin components rap1+ and poz1+; missplicing of these mRNAs causes telomeric heterochromatin and telomere length defects. The human homologue C9ORF78 was also shown to associate with the spliceosome, suggesting conserved function.","method":"Affinity purification/mass spectrometry of Tls1 interactors, RNA sequencing, genetic rescue with cDNA, human spliceosome association data","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal purification + epistasis in yeast ortholog with supporting human spliceosome association data","pmids":["25245948"],"is_preprint":false},{"year":2022,"finding":"C9ORF78 localizes to the nucleus in interphase cells and a subpopulation co-localizes with ACA, Mad1, and Ndc80 at kinetochores/centromeres in mitotic cells; centromeric levels increase upon mitotic checkpoint activation. Knockdown of C9ORF78 causes mitotic defects, indicating a role in chromosome segregation. Expression of C9ORF78 is cell-cycle regulated, induced by serum stimulation, and elevated by E2F1 or N-Myc overexpression.","method":"GFP tagging and immunofluorescence, co-localization with kinetochore markers (ACA, Mad1, Ndc80), siRNA knockdown with mitotic phenotype readout, overexpression of E2F1/N-Myc","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2-3 — direct localization with functional KD phenotype; single lab, multiple markers","pmids":["35167828"],"is_preprint":false},{"year":2025,"finding":"FAM50A forms a complex with C9ORF78, specifically at the S121 residue of C9ORF78, to enhance ASNS (asparagine synthetase) transcription and promote asparagine biosynthesis, facilitating breast cancer brain metastasis.","method":"Co-immunoprecipitation, site-directed mutagenesis (S121 residue), genetic knockdown/overexpression with ASNS transcription and asparagine synthesis readouts, in vivo brain metastasis model","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with mutagenesis and functional transcription/metabolic readout; single lab","pmids":["40531994"],"is_preprint":false},{"year":2024,"finding":"C9ORF78 is a direct target of both miR-34c and miR-449a in sinonasal cancers, as identified by AGO2-RIP-seq, placing C9ORF78 under post-transcriptional regulation by these miRNAs.","method":"AGO2 RNA immunoprecipitation followed by high-throughput sequencing (RIP-seq)","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 — single immunoprecipitation method, no functional validation of C9ORF78 regulation downstream","pmids":["38215077"],"is_preprint":false}],"current_model":"C9ORF78 is an intrinsically unstructured protein that associates with the spliceosome remodeling helicase BRR2 at a multi-factor trafficking site (mutually exclusive with FBP21), where it regulates alternative splicing including NAGNAG 3'-splice site usage and BRR2-dependent exon skipping; it also partially localizes to centromeres/kinetochores during mitosis and is required for proper chromosome segregation, and can interact with FAM50A at its S121 residue to enhance ASNS transcription and asparagine biosynthesis."},"narrative":{"teleology":[{"year":2014,"claim":"Identification of the fission yeast orthologue Tls1 as a Brr2-associated splicing factor established that C9ORF78 family proteins regulate pre-mRNA splicing with downstream consequences for chromatin and telomere homeostasis.","evidence":"Affinity purification/mass spectrometry of Tls1 interactors, RNA-seq of splicing changes, and genetic rescue in S. pombe, with supporting human spliceosome association data","pmids":["25245948"],"confidence":"Medium","gaps":["Mechanism of C9ORF78–BRR2 interaction in human cells was unknown","No structural information on how C9ORF78 contacts BRR2","Whether C9ORF78 regulates the same splicing targets as Tls1 in human cells was untested"]},{"year":2022,"claim":"Cryo-EM structures and functional studies resolved how C9ORF78 binds BRR2 at a multi-factor trafficking site in a mutually exclusive manner with FBP21, directly linking this interaction to regulation of alternative splicing including NAGNAG 3′-splice site choice and exon skipping.","evidence":"Cryo-EM structure determination, in vitro binding assays, RNA UV-crosslinking, affinity purification/mass spectrometry, and siRNA knockdown with splicing readout in human cells","pmids":["35241646"],"confidence":"High","gaps":["How C9ORF78-to-FBP21 exchange is temporally regulated during the splicing cycle is unknown","Whether C9ORF78 catalytically modulates BRR2 helicase activity or only serves as a scaffold is unresolved","Full repertoire of C9ORF78-dependent splicing events genome-wide not defined"]},{"year":2022,"claim":"Discovery of C9ORF78 at centromeres/kinetochores during mitosis revealed a second, non-spliceosomal function in chromosome segregation, with expression under cell-cycle and oncogenic transcription factor control.","evidence":"GFP tagging, immunofluorescence co-localization with ACA/Mad1/Ndc80, siRNA knockdown with mitotic phenotype scoring, E2F1/N-Myc overexpression","pmids":["35167828"],"confidence":"Medium","gaps":["Molecular mechanism by which C9ORF78 promotes faithful chromosome segregation is unknown","Whether the kinetochore function is independent of splicing regulation or secondary to it has not been dissected","Direct kinetochore-binding partners of C9ORF78 have not been identified"]},{"year":2025,"claim":"Identification of the FAM50A–C9ORF78 interaction at residue S121 revealed an unexpected role in transcriptional activation of ASNS and asparagine biosynthesis, linking C9ORF78 to metabolic reprogramming.","evidence":"Co-immunoprecipitation, S121 site-directed mutagenesis, ASNS transcription and asparagine synthesis assays, in vivo brain metastasis model","pmids":["40531994"],"confidence":"Medium","gaps":["Whether S121 is a phospho-regulatory site that controls the FAM50A interaction is unknown","How a spliceosome-associated protein activates transcription of a specific gene (ASNS) mechanistically is unresolved","Whether this function operates in normal physiology or only in cancer contexts is untested"]},{"year":null,"claim":"It remains unknown how C9ORF78's spliceosomal, kinetochore, and transcriptional functions are coordinated across the cell cycle, and whether they share a common structural or regulatory basis.","evidence":"","pmids":[],"confidence":"Low","gaps":["No separation-of-function mutants distinguishing splicing, kinetochore, and transcription roles","No in vivo animal model for C9ORF78 loss-of-function phenotype","Whether BRR2 helicase activity is modulated by C9ORF78 binding has not been measured biochemically"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[2]}],"complexes":[],"partners":["BRR2","FBP21","PRPF22","FAM50A"],"other_free_text":[]},"mechanistic_narrative":"C9ORF78 is an intrinsically unstructured nuclear protein that functions as a spliceosome-associated regulator of alternative splicing and participates in mitotic chromosome segregation. Cryo-EM structures show that C9ORF78 wraps around the C-terminal helicase cassette of the spliceosome remodeling factor BRR2 in a manner mutually exclusive with FBP21, and can simultaneously contact PRPF22, establishing it as a late-stage splicing regulator at a multi-factor trafficking site; its depletion alters NAGNAG 3′-splice site usage and causes BRR2-dependent exon skipping [PMID:35241646]. This spliceosomal role is conserved: the fission yeast orthologue Tls1 associates with Brr2 and regulates splicing of shelterin component mRNAs, with consequent effects on telomere integrity [PMID:25245948]. Beyond splicing, C9ORF78 partially localizes to centromeres/kinetochores during mitosis and is required for proper chromosome segregation [PMID:35167828], and it interacts with FAM50A via its S121 residue to enhance ASNS transcription and asparagine biosynthesis [PMID:40531994]."},"prefetch_data":{"uniprot":{"accession":"Q9NZ63","full_name":"Splicing factor C9orf78","aliases":["Hepatocellular carcinoma-associated antigen 59"],"length_aa":289,"mass_kda":33.7,"function":"Plays a role in pre-mRNA splicing by promoting usage of the upstream 3'-splice site at alternative NAGNAG splice sites; these are sites featuring alternative acceptor motifs separated by only a few nucleotides (PubMed:35241646). May also modulate exon inclusion events (PubMed:35241646). Plays a role in spliceosomal remodeling by displacing WBP4 from SNRNP200 and may act to inhibit SNRNP200 helicase activity (PubMed:35241646). Binds U5 snRNA (PubMed:35241646). Required for proper chromosome segregation (PubMed:35167828). Not required for splicing of shelterin components (PubMed:35167828)","subcellular_location":"Nucleus; Chromosome, centromere","url":"https://www.uniprot.org/uniprotkb/Q9NZ63/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/C9ORF78"},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000136819","cell_line_id":"CID000961","localizations":[{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"CD2BP2","stoichiometry":0.2},{"gene":"FAM50B","stoichiometry":0.2},{"gene":"FAM50A","stoichiometry":0.2},{"gene":"EFTUD2","stoichiometry":0.2},{"gene":"PRPF8","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000961","total_profiled":1310},"omim":[{"mim_id":"619569","title":"CHROMOSOME 9 OPEN READING FRAME 78; C9ORF78","url":"https://www.omim.org/entry/619569"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/C9ORF78"},"hgnc":{"alias_symbol":["HSPC220","HCA59","CSU2"],"prev_symbol":[]},"alphafold":{"accession":"Q9NZ63","domains":[{"cath_id":"-","chopping":"22-64","consensus_level":"medium","plddt":80.7702,"start":22,"end":64}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NZ63","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NZ63-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NZ63-F1-predicted_aligned_error_v6.png","plddt_mean":68.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=C9ORF78","jax_strain_url":"https://www.jax.org/strain/search?query=C9ORF78"},"sequence":{"accession":"Q9NZ63","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NZ63.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NZ63/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NZ63"}},"corpus_meta":[{"pmid":"12097419","id":"PMC_12097419","title":"Large scale identification of human hepatocellular carcinoma-associated antigens by autoantibodies.","date":"2002","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/12097419","citation_count":160,"is_preprint":false},{"pmid":"20065082","id":"PMC_20065082","title":"Combined interphase fluorescence in situ hybridization elucidates the genetic heterogeneity of T-cell acute lymphoblastic leukemia in adults.","date":"2010","source":"Haematologica","url":"https://pubmed.ncbi.nlm.nih.gov/20065082","citation_count":35,"is_preprint":false},{"pmid":"26714275","id":"PMC_26714275","title":"Arabidopsis COP1 SUPPRESSOR 2 Represses COP1 E3 Ubiquitin Ligase Activity through Their Coiled-Coil Domains Association.","date":"2015","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/26714275","citation_count":25,"is_preprint":false},{"pmid":"30871600","id":"PMC_30871600","title":"Hepatocellular carcinoma-associated antigen 59 of Haemonchus contortus modulates the functions of PBMCs and the differentiation and maturation of monocyte-derived dendritic cells of goats in vitro.","date":"2019","source":"Parasites & vectors","url":"https://pubmed.ncbi.nlm.nih.gov/30871600","citation_count":20,"is_preprint":false},{"pmid":"25245948","id":"PMC_25245948","title":"Tls1 regulates splicing of shelterin components to control telomeric heterochromatin assembly and telomere length.","date":"2014","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/25245948","citation_count":16,"is_preprint":false},{"pmid":"31412573","id":"PMC_31412573","title":"Combined Use of Indirect ELISA and Western Blotting with Recombinant Hepatocellular Carcinoma-Associated Antigen 59 Is a Potential Immunodiagnostic Tool for the Detection of Prepatent Haemonchus contortus Infection in Goat.","date":"2019","source":"Animals : an open access journal from MDPI","url":"https://pubmed.ncbi.nlm.nih.gov/31412573","citation_count":14,"is_preprint":false},{"pmid":"35241646","id":"PMC_35241646","title":"A multi-factor trafficking site on the spliceosome remodeling enzyme BRR2 recruits C9ORF78 to regulate alternative splicing.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/35241646","citation_count":10,"is_preprint":false},{"pmid":"33677347","id":"PMC_33677347","title":"Haemonchus contortus hepatocellular carcinoma-associated antigen 59 with poly (lactic-co-glycolic acid): A promising nanovaccine candidate against Haemonchus contortus infection.","date":"2021","source":"Veterinary parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/33677347","citation_count":10,"is_preprint":false},{"pmid":"33981142","id":"PMC_33981142","title":"Nanoparticles of Chitosan/Poly(D,L-Lactide-Co-Glycolide) Enhanced the Immune Responses of Haemonchus contortus HCA59 Antigen in Model Mice.","date":"2021","source":"International journal of nanomedicine","url":"https://pubmed.ncbi.nlm.nih.gov/33981142","citation_count":9,"is_preprint":false},{"pmid":"38215077","id":"PMC_38215077","title":"AGO2-RIP-Seq reveals miR-34/miR-449 cluster targetome in sinonasal cancers.","date":"2024","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/38215077","citation_count":5,"is_preprint":false},{"pmid":"35167828","id":"PMC_35167828","title":"C9ORF78 partially localizes to centromeres and plays a role in chromosome segregation.","date":"2022","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/35167828","citation_count":2,"is_preprint":false},{"pmid":"40531994","id":"PMC_40531994","title":"FAM50A drives breast cancer brain metastasis through interaction with C9ORF78 to enhance ʟ-asparagine production.","date":"2025","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/40531994","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7945,"output_tokens":1448,"usd":0.022777},"stage2":{"model":"claude-opus-4-6","input_tokens":4667,"output_tokens":1715,"usd":0.099315},"total_usd":0.122092,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2022,\n      \"finding\": \"C9ORF78 tightly interacts with the spliceosome remodeling factor BRR2 in vitro, and cryo-EM structures reveal that C9ORF78 and the spliceosomal B complex protein FBP21 wrap around the C-terminal helicase cassette of BRR2 in a mutually exclusive manner. Knockdown of C9ORF78 leads to alternative NAGNAG 3'-splice site usage and exon skipping, the latter dependent on BRR2. C9ORF78 could also contact PRPF22 when bound to BRR2, establishing C9ORF78 as a late-stage splicing regulatory protein that uses a multi-factor trafficking site on BRR2.\",\n      \"method\": \"Cryo-EM structure determination, in vitro binding assay, affinity purification/mass spectrometry, RNA UV-crosslinking, siRNA knockdown with alternative splicing readout\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure plus reconstituted in vitro interaction plus functional knockdown with multiple orthogonal methods in a single study\",\n      \"pmids\": [\"35241646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The fission yeast homologue of C9ORF78, Tls1, associates with the spliceosome subunit Brr2 and regulates splicing of shelterin components rap1+ and poz1+; missplicing of these mRNAs causes telomeric heterochromatin and telomere length defects. The human homologue C9ORF78 was also shown to associate with the spliceosome, suggesting conserved function.\",\n      \"method\": \"Affinity purification/mass spectrometry of Tls1 interactors, RNA sequencing, genetic rescue with cDNA, human spliceosome association data\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal purification + epistasis in yeast ortholog with supporting human spliceosome association data\",\n      \"pmids\": [\"25245948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"C9ORF78 localizes to the nucleus in interphase cells and a subpopulation co-localizes with ACA, Mad1, and Ndc80 at kinetochores/centromeres in mitotic cells; centromeric levels increase upon mitotic checkpoint activation. Knockdown of C9ORF78 causes mitotic defects, indicating a role in chromosome segregation. Expression of C9ORF78 is cell-cycle regulated, induced by serum stimulation, and elevated by E2F1 or N-Myc overexpression.\",\n      \"method\": \"GFP tagging and immunofluorescence, co-localization with kinetochore markers (ACA, Mad1, Ndc80), siRNA knockdown with mitotic phenotype readout, overexpression of E2F1/N-Myc\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — direct localization with functional KD phenotype; single lab, multiple markers\",\n      \"pmids\": [\"35167828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FAM50A forms a complex with C9ORF78, specifically at the S121 residue of C9ORF78, to enhance ASNS (asparagine synthetase) transcription and promote asparagine biosynthesis, facilitating breast cancer brain metastasis.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (S121 residue), genetic knockdown/overexpression with ASNS transcription and asparagine synthesis readouts, in vivo brain metastasis model\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with mutagenesis and functional transcription/metabolic readout; single lab\",\n      \"pmids\": [\"40531994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"C9ORF78 is a direct target of both miR-34c and miR-449a in sinonasal cancers, as identified by AGO2-RIP-seq, placing C9ORF78 under post-transcriptional regulation by these miRNAs.\",\n      \"method\": \"AGO2 RNA immunoprecipitation followed by high-throughput sequencing (RIP-seq)\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single immunoprecipitation method, no functional validation of C9ORF78 regulation downstream\",\n      \"pmids\": [\"38215077\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"C9ORF78 is an intrinsically unstructured protein that associates with the spliceosome remodeling helicase BRR2 at a multi-factor trafficking site (mutually exclusive with FBP21), where it regulates alternative splicing including NAGNAG 3'-splice site usage and BRR2-dependent exon skipping; it also partially localizes to centromeres/kinetochores during mitosis and is required for proper chromosome segregation, and can interact with FAM50A at its S121 residue to enhance ASNS transcription and asparagine biosynthesis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"C9ORF78 is an intrinsically unstructured nuclear protein that functions as a spliceosome-associated regulator of alternative splicing and participates in mitotic chromosome segregation. Cryo-EM structures show that C9ORF78 wraps around the C-terminal helicase cassette of the spliceosome remodeling factor BRR2 in a manner mutually exclusive with FBP21, and can simultaneously contact PRPF22, establishing it as a late-stage splicing regulator at a multi-factor trafficking site; its depletion alters NAGNAG 3′-splice site usage and causes BRR2-dependent exon skipping [PMID:35241646]. This spliceosomal role is conserved: the fission yeast orthologue Tls1 associates with Brr2 and regulates splicing of shelterin component mRNAs, with consequent effects on telomere integrity [PMID:25245948]. Beyond splicing, C9ORF78 partially localizes to centromeres/kinetochores during mitosis and is required for proper chromosome segregation [PMID:35167828], and it interacts with FAM50A via its S121 residue to enhance ASNS transcription and asparagine biosynthesis [PMID:40531994].\",\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"Identification of the fission yeast orthologue Tls1 as a Brr2-associated splicing factor established that C9ORF78 family proteins regulate pre-mRNA splicing with downstream consequences for chromatin and telomere homeostasis.\",\n      \"evidence\": \"Affinity purification/mass spectrometry of Tls1 interactors, RNA-seq of splicing changes, and genetic rescue in S. pombe, with supporting human spliceosome association data\",\n      \"pmids\": [\"25245948\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism of C9ORF78–BRR2 interaction in human cells was unknown\",\n        \"No structural information on how C9ORF78 contacts BRR2\",\n        \"Whether C9ORF78 regulates the same splicing targets as Tls1 in human cells was untested\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Cryo-EM structures and functional studies resolved how C9ORF78 binds BRR2 at a multi-factor trafficking site in a mutually exclusive manner with FBP21, directly linking this interaction to regulation of alternative splicing including NAGNAG 3′-splice site choice and exon skipping.\",\n      \"evidence\": \"Cryo-EM structure determination, in vitro binding assays, RNA UV-crosslinking, affinity purification/mass spectrometry, and siRNA knockdown with splicing readout in human cells\",\n      \"pmids\": [\"35241646\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How C9ORF78-to-FBP21 exchange is temporally regulated during the splicing cycle is unknown\",\n        \"Whether C9ORF78 catalytically modulates BRR2 helicase activity or only serves as a scaffold is unresolved\",\n        \"Full repertoire of C9ORF78-dependent splicing events genome-wide not defined\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Discovery of C9ORF78 at centromeres/kinetochores during mitosis revealed a second, non-spliceosomal function in chromosome segregation, with expression under cell-cycle and oncogenic transcription factor control.\",\n      \"evidence\": \"GFP tagging, immunofluorescence co-localization with ACA/Mad1/Ndc80, siRNA knockdown with mitotic phenotype scoring, E2F1/N-Myc overexpression\",\n      \"pmids\": [\"35167828\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Molecular mechanism by which C9ORF78 promotes faithful chromosome segregation is unknown\",\n        \"Whether the kinetochore function is independent of splicing regulation or secondary to it has not been dissected\",\n        \"Direct kinetochore-binding partners of C9ORF78 have not been identified\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identification of the FAM50A–C9ORF78 interaction at residue S121 revealed an unexpected role in transcriptional activation of ASNS and asparagine biosynthesis, linking C9ORF78 to metabolic reprogramming.\",\n      \"evidence\": \"Co-immunoprecipitation, S121 site-directed mutagenesis, ASNS transcription and asparagine synthesis assays, in vivo brain metastasis model\",\n      \"pmids\": [\"40531994\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether S121 is a phospho-regulatory site that controls the FAM50A interaction is unknown\",\n        \"How a spliceosome-associated protein activates transcription of a specific gene (ASNS) mechanistically is unresolved\",\n        \"Whether this function operates in normal physiology or only in cancer contexts is untested\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how C9ORF78's spliceosomal, kinetochore, and transcriptional functions are coordinated across the cell cycle, and whether they share a common structural or regulatory basis.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No separation-of-function mutants distinguishing splicing, kinetochore, and transcription roles\",\n        \"No in vivo animal model for C9ORF78 loss-of-function phenotype\",\n        \"Whether BRR2 helicase activity is modulated by C9ORF78 binding has not been measured biochemically\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"BRR2\", \"FBP21\", \"PRPF22\", \"FAM50A\"],\n    \"other_free_text\": []\n  }\n}\n```"}