{"gene":"CRNKL1","run_date":"2026-06-09T22:57:19","timeline":{"discoveries":[{"year":1999,"finding":"Yeast CLF1 (ortholog of Drosophila crooked neck / human CRNKL1) encodes an essential TPR-repeat protein required for pre-mRNA 5' splice site cleavage (first step of splicing). Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 tri-snRNP association, establishing Clf1p as a scaffolding factor required for the prespliceosome-to-spliceosome transition.","method":"In vitro splicing assays with Clf1p-depleted extracts; spliceosome assembly analysis; yeast two-hybrid and in vitro binding studies","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with depletion + spliceosome assembly arrest phenotype, complemented by binding assays; single focused study with multiple orthogonal methods","pmids":["10445879"],"is_preprint":false},{"year":1999,"finding":"Clf1p interacts specifically with the U1 snRNP protein Prp40p and the yeast U2AF65 homolog Mud2p, as shown by yeast two-hybrid and in vitro binding assays, suggesting Clf1p bridges U1 and U2 snRNP components during spliceosome assembly.","method":"Yeast two-hybrid; in vitro binding studies","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal two-hybrid and in vitro binding in single study, two orthogonal methods","pmids":["10445879"],"is_preprint":false},{"year":2000,"finding":"Yeast Syf3p (same protein as Clf1p/CRNKL1 ortholog) and Syf1p are spliceosome-associated proteins that associate weakly or transiently with U6 and U5 snRNAs; depletion of Syf1p or Syf3p blocks pre-mRNA splicing and causes cell cycle arrest at the G2/M transition, demonstrating dual roles in splicing and cell cycle progression.","method":"Spliceosome association assays (snRNA co-precipitation); in vivo depletion with splicing and cell cycle phenotype readouts","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — replicated independently from PMID:11102353, two orthogonal methods (RNA association + cell cycle phenotype), and consistent with PMID:10445879","pmids":["11105756"],"is_preprint":false},{"year":2000,"finding":"Genetic screen in S. cerevisiae identified SYF1, SYF2, and SYF3 (CLF1) as synthetically lethal with prp17/cdc40Δ; two-hybrid and biochemical analyses showed SYF gene products interact with each other and with Isy1p, Cef1p, Prp22p, and Ntc20p, placing them in a protein complex that assembles into spliceosomes.","method":"Synthetic lethality screen; yeast two-hybrid; biochemical co-immunoprecipitation","journal":"Genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis + reciprocal biochemical interactions, replicated across two independent labs (PMID:11102353 and PMID:11105756)","pmids":["11102353"],"is_preprint":false},{"year":2002,"finding":"Clf1p (CRNKL1 ortholog) plays a direct role in the initiation of DNA replication in S. cerevisiae: temperature-sensitive clf1 mutants show delayed S-phase entry that cannot be suppressed by deletion of the CDK inhibitor SIC1; Clf1p interacts with the DNA replication initiation protein Orc2p by two-hybrid and co-immunoprecipitation; and Clf1p preferentially associates with origins of DNA replication in an Orc2p-dependent manner.","method":"Temperature-sensitive mutant analysis; cell cycle block/release; yeast two-hybrid; co-immunoprecipitation; chromatin association assays at replication origins","journal":"Genetics","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (two-hybrid, Co-IP, chromatin association, genetic epistasis with SIC1) in a single focused study","pmids":["11973290"],"is_preprint":false},{"year":2005,"finding":"The NMR solution structure of the first FF domain of yeast Prp40 revealed a novel ligand-binding site that directly contacts the N-terminal crooked-neck TPR (crn-TPR1) motif of Clf1p; this binding site is structurally distinct from the CTD-RNAPII binding pocket of the related FF domain of FBP11. No interaction was detected between Prp40 FF1 and CTD-RNAPII peptides, indicating domain-specific and protein-specific binding of the Clf1p–Prp40 interaction.","method":"NMR structure determination; chemical shift perturbation assays mapping the binding interface","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with direct binding site mapping by chemical shift assays; single lab but rigorous structural method","pmids":["16253993"],"is_preprint":false},{"year":2009,"finding":"CLF1/SYF3 genetically interacts with the spliceosomal NTC component Prp45/SNW1: a synthetic lethality screen showed that the prp45(1-169) allele is lethal in combination with clf1/syf3 alleles, and Cwc2-associated spliceosomal complexes from prp45 mutant cells show altered Prp22 stoichiometry, placing Clf1p/Syf3p in the same functional network as Prp45 and the second-step helicase Prp22.","method":"Synthetic lethality screen; spliceosomal complex purification and stoichiometry analysis by Cwc2-pulldown","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis plus biochemical complex analysis, single lab, two orthogonal methods","pmids":["19016306"],"is_preprint":false},{"year":2020,"finding":"Molecular dynamics simulations and community network analysis of the yeast spliceosome C complex identified Clf1p (CRNKL1 ortholog) as a critical signal conveyor in cross-communication pathways across the spliceosome, functioning alongside Cwc2 and specific Prp8 domains to direct conformational rearrangements required for splicing fidelity.","method":"Molecular dynamics simulations (800,000-atom model); community network analysis","journal":"Journal of the American Chemical Society","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational simulation only, no direct experimental validation of Clf1 function in this study","pmids":["32275149"],"is_preprint":false},{"year":2021,"finding":"CRNKL1 (human ortholog) suppresses cytoplasmic levels of unspliced HIV-1 RNA and a subset of intron-retaining cellular mRNAs; CRISPR/Cas knockdown of CRNKL1 causes more than 20-fold enhancement of unspliced HIV-1 RNA cytoplasmic levels, predominantly by affecting nuclear export efficiency rather than splicing per se, identifying CRNKL1-dependent nuclear retention as a cellular mechanism regulating intron-retaining RNAs.","method":"Genome-wide CRISPR/Cas screen; targeted CRNKL1 depletion; cytoplasmic/nuclear RNA fractionation; transcriptomic analysis (RNA-seq)","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide screen plus targeted validation with multiple orthogonal methods (CRISPR KD, RNA fractionation, transcriptomics) in a single rigorous study","pmids":["33468685"],"is_preprint":false},{"year":2021,"finding":"In fission yeast, the C-terminal region of Nrl1 interacts with the spliceosome component Syf3 (CRNKL1 ortholog), as demonstrated by bacterial two-hybrid assay; however, Syf3 did not co-purify with the N-terminal region of Nrl1 by tandem affinity purification, indicating the interaction is domain-specific.","method":"Bacterial two-hybrid (BACTH); tandem affinity purification","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single method (BACTH) for positive result; TAP did not confirm; fission yeast ortholog context","pmids":["34209806"],"is_preprint":false},{"year":2021,"finding":"In budding yeast, Rat1 (5'→3' exoribonuclease) co-immunoprecipitates and was identified by mass spectrometry to interact with Clf1p among other splicing factors; recruitment of splicing factors to introns was compromised in Rat1 mutants, placing Clf1p in the cotranscriptional splicing recruitment pathway.","method":"Mass spectrometry; co-immunoprecipitation; ChIP-Seq; splicing assays in Rat1 mutants","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP + MS identification + ChIP-Seq functional context, single lab","pmids":["33978753"],"is_preprint":false},{"year":2021,"finding":"In trypanosomes, SYF3 (CRNKL1 ortholog) forms a distinct PRP19-related complex (PRC) together with SYF1, ISY1, and trypanosomatid-specific proteins PRC3 and PRC5; PRC co-precipitates U4 snRNA, indicating it enters the spliceosome prior to the PRP19 complex, establishing a unique pre-organization of these factors.","method":"Gene silencing; affinity purification; sucrose gradient sedimentation; snRNA co-precipitation","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — purification plus snRNA association plus sedimentation analysis, single lab, multiple orthogonal methods in non-mammalian organism","pmids":["34850936"],"is_preprint":false},{"year":2025,"finding":"Recurrent de novo missense variants in human CRNKL1 (specifically at Arg267) cause severe microcephaly with pontocerebellar hypoplasia and seizures. Microinjection of mRNA encoding mutant Crnkl1 into zebrafish caused severe lack of brain development, significant reduction of proliferating cells, widespread cellular stress (p53 staining), and broad transcriptomic changes including altered expression of neuronal and cell cycle genes, establishing CRNKL1 as required for brain development.","method":"Human genetics (de novo variant cohort); zebrafish mRNA microinjection; immunostaining (p53, proliferation markers); RNA sequencing","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — human variant cohort (10 families) with in vivo zebrafish functional validation using multiple orthogonal methods (imaging, RNA-seq, immunostaining)","pmids":["40857589"],"is_preprint":false},{"year":2025,"finding":"ASFV protein pI73R overexpression causes downregulation of CRNKL1 (a core spliceosome component) in host cells, suggesting a mechanism by which the viral protein modulates host pre-mRNA splicing through suppression of CRNKL1.","method":"Overexpression of viral pI73R; mass spectrometry interactome; Western blot/protein abundance measurement","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single method for CRNKL1 downregulation observation, no direct mechanistic follow-up on CRNKL1 function itself","pmids":["41465200"],"is_preprint":false},{"year":2025,"finding":"CRNKL1 was validated as a repressor of unspliced HIV-1 RNA nuclear export using a dual-fluorescent HIV-1 reporter system; targeted depletion of CRNKL1 enhanced Rev-independent (unspliced) transcript levels, confirming its role in nuclear retention of unspliced RNA.","method":"Dual-fluorescent HIV-1 reporter system; flow cytometry; targeted CRNKL1 depletion","journal":"Microbiology spectrum","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — orthogonal reporter validation of PMID:33468685 finding using independent reporter system, single lab","pmids":["39902983"],"is_preprint":false}],"current_model":"CRNKL1 (also known as SYF3/CLF1) is an essential TPR-repeat scaffolding protein of the spliceosome that is required for U4/U6.U5 tri-snRNP addition during spliceosome assembly, interacts with Prp40p (via its crn-TPR1 motif), Mud2p, and multiple NTC components (Cef1p, Isy1p, Ntc20p, Prp22p), participates in DNA replication initiation through interaction with Orc2p at replication origins, is required for G2/M cell cycle progression, and in human cells functions as a nuclear retention factor that suppresses cytoplasmic accumulation of intron-retaining RNAs (including unspliced HIV-1 transcripts); loss-of-function or dominant missense variants at Arg267 cause severe microcephaly with pontocerebellar hypoplasia in humans, establishing a critical role in brain development."},"narrative":{"mechanistic_narrative":"CRNKL1 (yeast CLF1/SYF3) is an essential TPR-repeat scaffolding protein of the spliceosome required for the prespliceosome-to-spliceosome transition during pre-mRNA splicing [PMID:10445879]. In yeast, depletion of the protein arrests spliceosome assembly after U2 snRNP addition but before productive U4/U6.U5 tri-snRNP association, blocking the first step of splicing [PMID:10445879], and the protein associates transiently with U6 and U5 snRNAs [PMID:11105756]. It operates as a hub within the NTC/Prp19-associated network, interacting with Syf1p, Isy1p, Cef1p, Prp22p, and Ntc20p and functioning in the same genetic pathway as Prp45 and the second-step helicase Prp22 [PMID:11102353, PMID:19016306]; structurally, its N-terminal crooked-neck TPR (crn-TPR1) motif is directly contacted by the first FF domain of Prp40, and it additionally interacts with the U2AF65 homolog Mud2p, consistent with a bridging role between snRNP and NTC components during assembly [PMID:10445879, PMID:16253993]. Beyond splicing, yeast Clf1p has a direct role in DNA replication initiation, interacting with Orc2p and associating with replication origins in an Orc2p-dependent manner, and its loss causes G2/M cell cycle arrest [PMID:11105756, PMID:11973290]. In human cells CRNKL1 acts as a nuclear retention factor that suppresses cytoplasmic accumulation of intron-retaining RNAs, including unspliced HIV-1 transcripts, with depletion enhancing cytoplasmic levels of unspliced RNA primarily by affecting nuclear export rather than splicing itself [PMID:33468685, PMID:39902983]. Recurrent de novo missense variants at Arg267 cause severe microcephaly with pontocerebellar hypoplasia and seizures, and mutant Crnkl1 disrupts brain development, cell proliferation, and cell cycle gene expression in zebrafish, establishing CRNKL1 as essential for human brain development [PMID:40857589].","teleology":[{"year":1999,"claim":"Established the core function of the protein by showing it is essential for the first catalytic step of splicing and acts at a defined point in spliceosome assembly, answering what stage of splicing requires this factor.","evidence":"In vitro splicing with Clf1p-depleted yeast extracts and spliceosome assembly analysis, plus two-hybrid/binding assays mapping interactions with Prp40p and Mud2p","pmids":["10445879"],"confidence":"High","gaps":["Whether the Prp40p/Mud2p interactions are direct in a reconstituted system was not resolved","No structural model of Clf1p within the assembling spliceosome"]},{"year":2000,"claim":"Linked the splicing factor to cell cycle control and placed it within a defined protein complex, showing the protein has a dual role and operates in a network of NTC-associated factors.","evidence":"snRNA co-precipitation and in vivo depletion with cell cycle readouts; synthetic lethality screen with prp17/cdc40Δ plus two-hybrid and Co-IP mapping interactions with Isy1p, Cef1p, Prp22p, Ntc20p","pmids":["11105756","11102353"],"confidence":"High","gaps":["Whether G2/M arrest is a direct consequence of splicing defects or a separate function was not separated","Stoichiometry and architecture of the complex not defined"]},{"year":2002,"claim":"Demonstrated a splicing-independent role in DNA replication initiation, answering whether the cell cycle phenotype reflects a direct function at replication origins.","evidence":"Temperature-sensitive clf1 mutants, SIC1-deletion epistasis, two-hybrid and Co-IP with Orc2p, and origin chromatin association assays in S. cerevisiae","pmids":["11973290"],"confidence":"High","gaps":["Mechanism by which a TPR splicing scaffold acts at origins is unknown","Not tested whether the human ortholog retains a replication role"]},{"year":2005,"claim":"Provided structural definition of the Clf1p–Prp40 interaction, showing the crn-TPR1 motif is recognized by a distinct FF-domain binding site, clarifying interaction specificity.","evidence":"NMR solution structure of Prp40 FF1 and chemical shift perturbation mapping of the crn-TPR1 binding interface","pmids":["16253993"],"confidence":"High","gaps":["No structure of full-length Clf1p or the complete TPR array","Functional consequence of disrupting this interface in vivo not tested"]},{"year":2009,"claim":"Extended the functional network by genetically connecting the protein to Prp45 and the second-step helicase Prp22, indicating involvement beyond initial assembly.","evidence":"Synthetic lethality screen with prp45(1-169) and Cwc2-pulldown stoichiometry analysis of spliceosomal complexes","pmids":["19016306"],"confidence":"Medium","gaps":["Direct physical contact with Prp45 not demonstrated","Mechanistic basis of altered Prp22 stoichiometry unclear"]},{"year":2021,"claim":"Identified a conserved human function as a nuclear retention factor for intron-retaining RNAs, answering what the mammalian ortholog does beyond canonical splicing.","evidence":"Genome-wide CRISPR screen, targeted depletion, nuclear/cytoplasmic RNA fractionation and RNA-seq, with later validation via a dual-fluorescent HIV-1 reporter","pmids":["33468685","39902983"],"confidence":"High","gaps":["Molecular mechanism by which CRNKL1 enforces nuclear retention/export control not defined","Whether retention is a direct activity or a consequence of altered spliceosome states unresolved"]},{"year":2021,"claim":"Refined the cotranscriptional and complex context of the protein across organisms, connecting it to splicing-factor recruitment and a pre-organized PRP19-related complex.","evidence":"Co-IP/MS and ChIP-Seq with Rat1 in budding yeast; affinity purification, sedimentation and snRNA co-precipitation of the trypanosome PRC; bacterial two-hybrid with fission yeast Nrl1","pmids":["33978753","34850936","34209806"],"confidence":"Medium","gaps":["Nrl1 interaction rests on a single positive method not confirmed by TAP","Generality of the PRC pre-organization to mammalian spliceosomes untested"]},{"year":2025,"claim":"Established CRNKL1 as a human disease gene required for brain development, connecting its molecular function to a Mendelian neurodevelopmental disorder.","evidence":"De novo variant cohort identifying recurrent Arg267 mutations; zebrafish mRNA microinjection with imaging, proliferation/p53 immunostaining, and RNA-seq","pmids":["40857589"],"confidence":"High","gaps":["How Arg267 substitutions impair CRNKL1 molecular function (dominant-negative vs loss-of-function) not resolved","Link between splicing/nuclear-retention activity and the neurodevelopmental phenotype not mechanistically established"]},{"year":null,"claim":"How CRNKL1's spliceosomal scaffolding activity mechanistically produces nuclear retention of intron-retaining RNAs and brain-specific developmental requirements remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural or biochemical model linking splicing function to export control in human cells","No demonstration that the disease variants alter specific splicing or retention events in neural lineages"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[2]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,5]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,5]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,2,8]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[2,4]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[4]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[12]}],"complexes":["spliceosome (U4/U6.U5 tri-snRNP-containing)","NTC/Prp19-associated complex","trypanosome PRP19-related complex (PRC)"],"partners":["PRP40","MUD2","SYF1","ISY1","CEF1","PRP22","NTC20","ORC2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BZJ0","full_name":"Crooked neck-like protein 1","aliases":["Crooked neck homolog","hCrn"],"length_aa":848,"mass_kda":100.5,"function":"Involved in pre-mRNA splicing process (PubMed:11991638, PubMed:12084575, PubMed:28076346, PubMed:28502770). As a component of the minor spliceosome, involved in the splicing of U12-type introns in pre-mRNAs (Probable)","subcellular_location":"Nucleus; Nucleus speckle","url":"https://www.uniprot.org/uniprotkb/Q9BZJ0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CRNKL1","classification":"Common Essential","n_dependent_lines":1204,"n_total_lines":1208,"dependency_fraction":0.9966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CAPZB","stoichiometry":0.2},{"gene":"CPSF6","stoichiometry":0.2},{"gene":"CTNNBL1","stoichiometry":0.2},{"gene":"DDX21","stoichiometry":0.2},{"gene":"DNAJC17","stoichiometry":0.2},{"gene":"NPM1","stoichiometry":0.2},{"gene":"PRPF19","stoichiometry":0.2},{"gene":"PRPF4B","stoichiometry":0.2},{"gene":"PSPC1","stoichiometry":0.2},{"gene":"RBM39","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CRNKL1","total_profiled":1310},"omim":[{"mim_id":"621436","title":"MICROCEPHALY, PROGRESSIVE, WITH SIMPLIFIED GYRAL PATTERN AND CEREBELLAR HYPOPLASIA; MGCH","url":"https://www.omim.org/entry/621436"},{"mim_id":"610952","title":"CROOKED NECK PRE-mRNA SPLICING FACTOR-LIKE 1; CRNKL1","url":"https://www.omim.org/entry/610952"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CRNKL1"},"hgnc":{"alias_symbol":["CRN","CLF","SYF3","Clf1"],"prev_symbol":[]},"alphafold":{"accession":"Q9BZJ0","domains":[{"cath_id":"-","chopping":"782-824","consensus_level":"medium","plddt":72.4367,"start":782,"end":824}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BZJ0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BZJ0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BZJ0-F1-predicted_aligned_error_v6.png","plddt_mean":74.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CRNKL1","jax_strain_url":"https://www.jax.org/strain/search?query=CRNKL1"},"sequence":{"accession":"Q9BZJ0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BZJ0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BZJ0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BZJ0"}},"corpus_meta":[{"pmid":"23774526","id":"PMC_23774526","title":"Mutational landscape of basal cell carcinomas by whole-exome sequencing.","date":"2013","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/23774526","citation_count":171,"is_preprint":false},{"pmid":"18413325","id":"PMC_18413325","title":"Variation in WNT genes is associated with non-syndromic cleft lip with or without cleft palate.","date":"2008","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/18413325","citation_count":140,"is_preprint":false},{"pmid":"11102353","id":"PMC_11102353","title":"Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.","date":"2000","source":"Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/11102353","citation_count":105,"is_preprint":false},{"pmid":"15917337","id":"PMC_15917337","title":"Novel regulators of kidney development from the tips of the ureteric bud.","date":"2005","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/15917337","citation_count":100,"is_preprint":false},{"pmid":"16998816","id":"PMC_16998816","title":"Wnt9b is the mutated gene involved in multifactorial nonsyndromic cleft lip with or without cleft palate in A/WySn mice, as confirmed by a genetic complementation test.","date":"2006","source":"Birth defects research. 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Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 tri-snRNP association, establishing Clf1p as a scaffolding factor required for the prespliceosome-to-spliceosome transition.\",\n      \"method\": \"In vitro splicing assays with Clf1p-depleted extracts; spliceosome assembly analysis; yeast two-hybrid and in vitro binding studies\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with depletion + spliceosome assembly arrest phenotype, complemented by binding assays; single focused study with multiple orthogonal methods\",\n      \"pmids\": [\"10445879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Clf1p interacts specifically with the U1 snRNP protein Prp40p and the yeast U2AF65 homolog Mud2p, as shown by yeast two-hybrid and in vitro binding assays, suggesting Clf1p bridges U1 and U2 snRNP components during spliceosome assembly.\",\n      \"method\": \"Yeast two-hybrid; in vitro binding studies\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal two-hybrid and in vitro binding in single study, two orthogonal methods\",\n      \"pmids\": [\"10445879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Yeast Syf3p (same protein as Clf1p/CRNKL1 ortholog) and Syf1p are spliceosome-associated proteins that associate weakly or transiently with U6 and U5 snRNAs; depletion of Syf1p or Syf3p blocks pre-mRNA splicing and causes cell cycle arrest at the G2/M transition, demonstrating dual roles in splicing and cell cycle progression.\",\n      \"method\": \"Spliceosome association assays (snRNA co-precipitation); in vivo depletion with splicing and cell cycle phenotype readouts\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — replicated independently from PMID:11102353, two orthogonal methods (RNA association + cell cycle phenotype), and consistent with PMID:10445879\",\n      \"pmids\": [\"11105756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Genetic screen in S. cerevisiae identified SYF1, SYF2, and SYF3 (CLF1) as synthetically lethal with prp17/cdc40Δ; two-hybrid and biochemical analyses showed SYF gene products interact with each other and with Isy1p, Cef1p, Prp22p, and Ntc20p, placing them in a protein complex that assembles into spliceosomes.\",\n      \"method\": \"Synthetic lethality screen; yeast two-hybrid; biochemical co-immunoprecipitation\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis + reciprocal biochemical interactions, replicated across two independent labs (PMID:11102353 and PMID:11105756)\",\n      \"pmids\": [\"11102353\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Clf1p (CRNKL1 ortholog) plays a direct role in the initiation of DNA replication in S. cerevisiae: temperature-sensitive clf1 mutants show delayed S-phase entry that cannot be suppressed by deletion of the CDK inhibitor SIC1; Clf1p interacts with the DNA replication initiation protein Orc2p by two-hybrid and co-immunoprecipitation; and Clf1p preferentially associates with origins of DNA replication in an Orc2p-dependent manner.\",\n      \"method\": \"Temperature-sensitive mutant analysis; cell cycle block/release; yeast two-hybrid; co-immunoprecipitation; chromatin association assays at replication origins\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (two-hybrid, Co-IP, chromatin association, genetic epistasis with SIC1) in a single focused study\",\n      \"pmids\": [\"11973290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The NMR solution structure of the first FF domain of yeast Prp40 revealed a novel ligand-binding site that directly contacts the N-terminal crooked-neck TPR (crn-TPR1) motif of Clf1p; this binding site is structurally distinct from the CTD-RNAPII binding pocket of the related FF domain of FBP11. No interaction was detected between Prp40 FF1 and CTD-RNAPII peptides, indicating domain-specific and protein-specific binding of the Clf1p–Prp40 interaction.\",\n      \"method\": \"NMR structure determination; chemical shift perturbation assays mapping the binding interface\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with direct binding site mapping by chemical shift assays; single lab but rigorous structural method\",\n      \"pmids\": [\"16253993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CLF1/SYF3 genetically interacts with the spliceosomal NTC component Prp45/SNW1: a synthetic lethality screen showed that the prp45(1-169) allele is lethal in combination with clf1/syf3 alleles, and Cwc2-associated spliceosomal complexes from prp45 mutant cells show altered Prp22 stoichiometry, placing Clf1p/Syf3p in the same functional network as Prp45 and the second-step helicase Prp22.\",\n      \"method\": \"Synthetic lethality screen; spliceosomal complex purification and stoichiometry analysis by Cwc2-pulldown\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis plus biochemical complex analysis, single lab, two orthogonal methods\",\n      \"pmids\": [\"19016306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Molecular dynamics simulations and community network analysis of the yeast spliceosome C complex identified Clf1p (CRNKL1 ortholog) as a critical signal conveyor in cross-communication pathways across the spliceosome, functioning alongside Cwc2 and specific Prp8 domains to direct conformational rearrangements required for splicing fidelity.\",\n      \"method\": \"Molecular dynamics simulations (800,000-atom model); community network analysis\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational simulation only, no direct experimental validation of Clf1 function in this study\",\n      \"pmids\": [\"32275149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CRNKL1 (human ortholog) suppresses cytoplasmic levels of unspliced HIV-1 RNA and a subset of intron-retaining cellular mRNAs; CRISPR/Cas knockdown of CRNKL1 causes more than 20-fold enhancement of unspliced HIV-1 RNA cytoplasmic levels, predominantly by affecting nuclear export efficiency rather than splicing per se, identifying CRNKL1-dependent nuclear retention as a cellular mechanism regulating intron-retaining RNAs.\",\n      \"method\": \"Genome-wide CRISPR/Cas screen; targeted CRNKL1 depletion; cytoplasmic/nuclear RNA fractionation; transcriptomic analysis (RNA-seq)\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide screen plus targeted validation with multiple orthogonal methods (CRISPR KD, RNA fractionation, transcriptomics) in a single rigorous study\",\n      \"pmids\": [\"33468685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In fission yeast, the C-terminal region of Nrl1 interacts with the spliceosome component Syf3 (CRNKL1 ortholog), as demonstrated by bacterial two-hybrid assay; however, Syf3 did not co-purify with the N-terminal region of Nrl1 by tandem affinity purification, indicating the interaction is domain-specific.\",\n      \"method\": \"Bacterial two-hybrid (BACTH); tandem affinity purification\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single method (BACTH) for positive result; TAP did not confirm; fission yeast ortholog context\",\n      \"pmids\": [\"34209806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In budding yeast, Rat1 (5'→3' exoribonuclease) co-immunoprecipitates and was identified by mass spectrometry to interact with Clf1p among other splicing factors; recruitment of splicing factors to introns was compromised in Rat1 mutants, placing Clf1p in the cotranscriptional splicing recruitment pathway.\",\n      \"method\": \"Mass spectrometry; co-immunoprecipitation; ChIP-Seq; splicing assays in Rat1 mutants\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP + MS identification + ChIP-Seq functional context, single lab\",\n      \"pmids\": [\"33978753\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In trypanosomes, SYF3 (CRNKL1 ortholog) forms a distinct PRP19-related complex (PRC) together with SYF1, ISY1, and trypanosomatid-specific proteins PRC3 and PRC5; PRC co-precipitates U4 snRNA, indicating it enters the spliceosome prior to the PRP19 complex, establishing a unique pre-organization of these factors.\",\n      \"method\": \"Gene silencing; affinity purification; sucrose gradient sedimentation; snRNA co-precipitation\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — purification plus snRNA association plus sedimentation analysis, single lab, multiple orthogonal methods in non-mammalian organism\",\n      \"pmids\": [\"34850936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Recurrent de novo missense variants in human CRNKL1 (specifically at Arg267) cause severe microcephaly with pontocerebellar hypoplasia and seizures. Microinjection of mRNA encoding mutant Crnkl1 into zebrafish caused severe lack of brain development, significant reduction of proliferating cells, widespread cellular stress (p53 staining), and broad transcriptomic changes including altered expression of neuronal and cell cycle genes, establishing CRNKL1 as required for brain development.\",\n      \"method\": \"Human genetics (de novo variant cohort); zebrafish mRNA microinjection; immunostaining (p53, proliferation markers); RNA sequencing\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — human variant cohort (10 families) with in vivo zebrafish functional validation using multiple orthogonal methods (imaging, RNA-seq, immunostaining)\",\n      \"pmids\": [\"40857589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ASFV protein pI73R overexpression causes downregulation of CRNKL1 (a core spliceosome component) in host cells, suggesting a mechanism by which the viral protein modulates host pre-mRNA splicing through suppression of CRNKL1.\",\n      \"method\": \"Overexpression of viral pI73R; mass spectrometry interactome; Western blot/protein abundance measurement\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single method for CRNKL1 downregulation observation, no direct mechanistic follow-up on CRNKL1 function itself\",\n      \"pmids\": [\"41465200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CRNKL1 was validated as a repressor of unspliced HIV-1 RNA nuclear export using a dual-fluorescent HIV-1 reporter system; targeted depletion of CRNKL1 enhanced Rev-independent (unspliced) transcript levels, confirming its role in nuclear retention of unspliced RNA.\",\n      \"method\": \"Dual-fluorescent HIV-1 reporter system; flow cytometry; targeted CRNKL1 depletion\",\n      \"journal\": \"Microbiology spectrum\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — orthogonal reporter validation of PMID:33468685 finding using independent reporter system, single lab\",\n      \"pmids\": [\"39902983\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CRNKL1 (also known as SYF3/CLF1) is an essential TPR-repeat scaffolding protein of the spliceosome that is required for U4/U6.U5 tri-snRNP addition during spliceosome assembly, interacts with Prp40p (via its crn-TPR1 motif), Mud2p, and multiple NTC components (Cef1p, Isy1p, Ntc20p, Prp22p), participates in DNA replication initiation through interaction with Orc2p at replication origins, is required for G2/M cell cycle progression, and in human cells functions as a nuclear retention factor that suppresses cytoplasmic accumulation of intron-retaining RNAs (including unspliced HIV-1 transcripts); loss-of-function or dominant missense variants at Arg267 cause severe microcephaly with pontocerebellar hypoplasia in humans, establishing a critical role in brain development.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CRNKL1 (yeast CLF1/SYF3) is an essential TPR-repeat scaffolding protein of the spliceosome required for the prespliceosome-to-spliceosome transition during pre-mRNA splicing [#0]. In yeast, depletion of the protein arrests spliceosome assembly after U2 snRNP addition but before productive U4/U6.U5 tri-snRNP association, blocking the first step of splicing [#0], and the protein associates transiently with U6 and U5 snRNAs [#2]. It operates as a hub within the NTC/Prp19-associated network, interacting with Syf1p, Isy1p, Cef1p, Prp22p, and Ntc20p and functioning in the same genetic pathway as Prp45 and the second-step helicase Prp22 [#3, #6]; structurally, its N-terminal crooked-neck TPR (crn-TPR1) motif is directly contacted by the first FF domain of Prp40, and it additionally interacts with the U2AF65 homolog Mud2p, consistent with a bridging role between snRNP and NTC components during assembly [#1, #5]. Beyond splicing, yeast Clf1p has a direct role in DNA replication initiation, interacting with Orc2p and associating with replication origins in an Orc2p-dependent manner, and its loss causes G2/M cell cycle arrest [#2, #4]. In human cells CRNKL1 acts as a nuclear retention factor that suppresses cytoplasmic accumulation of intron-retaining RNAs, including unspliced HIV-1 transcripts, with depletion enhancing cytoplasmic levels of unspliced RNA primarily by affecting nuclear export rather than splicing itself [#8, #14]. Recurrent de novo missense variants at Arg267 cause severe microcephaly with pontocerebellar hypoplasia and seizures, and mutant Crnkl1 disrupts brain development, cell proliferation, and cell cycle gene expression in zebrafish, establishing CRNKL1 as essential for human brain development [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established the core function of the protein by showing it is essential for the first catalytic step of splicing and acts at a defined point in spliceosome assembly, answering what stage of splicing requires this factor.\",\n      \"evidence\": \"In vitro splicing with Clf1p-depleted yeast extracts and spliceosome assembly analysis, plus two-hybrid/binding assays mapping interactions with Prp40p and Mud2p\",\n      \"pmids\": [\"10445879\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether the Prp40p/Mud2p interactions are direct in a reconstituted system was not resolved\",\n        \"No structural model of Clf1p within the assembling spliceosome\"\n      ]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Linked the splicing factor to cell cycle control and placed it within a defined protein complex, showing the protein has a dual role and operates in a network of NTC-associated factors.\",\n      \"evidence\": \"snRNA co-precipitation and in vivo depletion with cell cycle readouts; synthetic lethality screen with prp17/cdc40Δ plus two-hybrid and Co-IP mapping interactions with Isy1p, Cef1p, Prp22p, Ntc20p\",\n      \"pmids\": [\"11105756\", \"11102353\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether G2/M arrest is a direct consequence of splicing defects or a separate function was not separated\",\n        \"Stoichiometry and architecture of the complex not defined\"\n      ]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Demonstrated a splicing-independent role in DNA replication initiation, answering whether the cell cycle phenotype reflects a direct function at replication origins.\",\n      \"evidence\": \"Temperature-sensitive clf1 mutants, SIC1-deletion epistasis, two-hybrid and Co-IP with Orc2p, and origin chromatin association assays in S. cerevisiae\",\n      \"pmids\": [\"11973290\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism by which a TPR splicing scaffold acts at origins is unknown\",\n        \"Not tested whether the human ortholog retains a replication role\"\n      ]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Provided structural definition of the Clf1p–Prp40 interaction, showing the crn-TPR1 motif is recognized by a distinct FF-domain binding site, clarifying interaction specificity.\",\n      \"evidence\": \"NMR solution structure of Prp40 FF1 and chemical shift perturbation mapping of the crn-TPR1 binding interface\",\n      \"pmids\": [\"16253993\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structure of full-length Clf1p or the complete TPR array\",\n        \"Functional consequence of disrupting this interface in vivo not tested\"\n      ]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended the functional network by genetically connecting the protein to Prp45 and the second-step helicase Prp22, indicating involvement beyond initial assembly.\",\n      \"evidence\": \"Synthetic lethality screen with prp45(1-169) and Cwc2-pulldown stoichiometry analysis of spliceosomal complexes\",\n      \"pmids\": [\"19016306\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct physical contact with Prp45 not demonstrated\",\n        \"Mechanistic basis of altered Prp22 stoichiometry unclear\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified a conserved human function as a nuclear retention factor for intron-retaining RNAs, answering what the mammalian ortholog does beyond canonical splicing.\",\n      \"evidence\": \"Genome-wide CRISPR screen, targeted depletion, nuclear/cytoplasmic RNA fractionation and RNA-seq, with later validation via a dual-fluorescent HIV-1 reporter\",\n      \"pmids\": [\"33468685\", \"39902983\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Molecular mechanism by which CRNKL1 enforces nuclear retention/export control not defined\",\n        \"Whether retention is a direct activity or a consequence of altered spliceosome states unresolved\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Refined the cotranscriptional and complex context of the protein across organisms, connecting it to splicing-factor recruitment and a pre-organized PRP19-related complex.\",\n      \"evidence\": \"Co-IP/MS and ChIP-Seq with Rat1 in budding yeast; affinity purification, sedimentation and snRNA co-precipitation of the trypanosome PRC; bacterial two-hybrid with fission yeast Nrl1\",\n      \"pmids\": [\"33978753\", \"34850936\", \"34209806\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Nrl1 interaction rests on a single positive method not confirmed by TAP\",\n        \"Generality of the PRC pre-organization to mammalian spliceosomes untested\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established CRNKL1 as a human disease gene required for brain development, connecting its molecular function to a Mendelian neurodevelopmental disorder.\",\n      \"evidence\": \"De novo variant cohort identifying recurrent Arg267 mutations; zebrafish mRNA microinjection with imaging, proliferation/p53 immunostaining, and RNA-seq\",\n      \"pmids\": [\"40857589\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How Arg267 substitutions impair CRNKL1 molecular function (dominant-negative vs loss-of-function) not resolved\",\n        \"Link between splicing/nuclear-retention activity and the neurodevelopmental phenotype not mechanistically established\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CRNKL1's spliceosomal scaffolding activity mechanistically produces nuclear retention of intron-retaining RNAs and brain-specific developmental requirements remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No structural or biochemical model linking splicing function to export control in human cells\",\n        \"No demonstration that the disease variants alter specific splicing or retention events in neural lineages\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 5]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 2, 8]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 4]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"complexes\": [\n      \"spliceosome (U4/U6.U5 tri-snRNP-containing)\",\n      \"NTC/Prp19-associated complex\",\n      \"trypanosome PRP19-related complex (PRC)\"\n    ],\n    \"partners\": [\n      \"PRP40\",\n      \"MUD2\",\n      \"SYF1\",\n      \"ISY1\",\n      \"CEF1\",\n      \"PRP22\",\n      \"NTC20\",\n      \"ORC2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}