{"gene":"ISY1","run_date":"2026-06-10T01:55:23","timeline":{"discoveries":[{"year":1999,"finding":"Isy1p is a novel splicing factor that associates with spliceosomes throughout the splicing reactions and co-immunoprecipitates low levels of U5 and U6 snRNAs; deletion of ISY1 reduces splicing efficiency in vivo, indicating a role in optimizing pre-mRNA splicing.","method":"Two-hybrid screen, tagged protein co-immunoprecipitation with snRNAs, in vivo splicing assay in ISY1 null mutant yeast","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with snRNAs and functional KO splicing assay, single lab, two orthogonal methods","pmids":["10094305"],"is_preprint":false},{"year":2002,"finding":"Mutations in ISY1 combined with SYF2 deletion cause cell cycle arrest due to reduced splicing of intron-containing alpha-tubulin genes (TUB1, TUB3), lowering alpha-tubulin protein levels and triggering spindle checkpoint activation; ISY1 is synthetically lethal with NTC20 deletion.","method":"Genetic epistasis (double-mutant analysis), cell cycle arrest assay, spindle checkpoint activation assay, protein level measurement","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with defined cellular phenotype, single lab, multiple genetic combinations tested","pmids":["12384582"],"is_preprint":false},{"year":2005,"finding":"Isy1p is a subunit of the NineTeen Complex (NTC/Prp19 complex) and acts together with U6 snRNA to promote a spliceosomal conformation favorable for first-step chemistry; deletion of ISY1 suppresses the cold-sensitive prp16-302 phenotype by alleviating stalling and restoring branchpoint fidelity, and also reduces fidelity of 3'-splice site selection in PRP16 wild-type strains.","method":"Genetic suppressor screen of prp16-302, growth assays, biochemical spliceosome fractionation showing NTC membership","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis combined with biochemical NTC membership, replicated with multiple alleles and conditions, mechanistic model supported by both genetic and biochemical data","pmids":["16103217"],"is_preprint":false},{"year":2008,"finding":"ISY1 shows synthetic genetic interactions with TGS1 (trimethylguanosine synthase); deletion of ISY1 combined with tgs1Δ causes synthetic growth defects, placing ISY1 in a functional network with snRNP components and spliceosome assembly factors.","method":"Systematic synthetic growth defect screen (genetic interaction mapping) in budding yeast","journal":"The Journal of biological chemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single genetic interaction screen, no direct mechanistic follow-up on ISY1 specifically","pmids":["18775984"],"is_preprint":false},{"year":2009,"finding":"Cwc21 shows strong genetic, physical, and functional interactions with Isy1, supporting a role for Isy1 at the core of the spliceosome in the activation of splicing.","method":"Quantitative genetic interaction mapping, mass spectrometry of tandem affinity-purified complexes, microarray profiling","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (genetic interaction, MS co-purification), single lab","pmids":["19789211"],"is_preprint":false},{"year":2014,"finding":"Isy1/NTC30 functionally cooperates with the NTC protein Cwc2 during pre-mRNA splicing; genetic interactions between Cwc2 mutations and Isy1 indicate they act together in stabilizing spliceosome active site RNA elements.","method":"Genetic interaction analysis (suppressor/synthetic lethality), biochemical RNA interaction assays","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis and biochemical RNA interaction data, single lab","pmids":["24848011"],"is_preprint":false},{"year":2015,"finding":"ISY1 (spliceosome-associated) and the endonuclease CPSF3 are responsible for pro-miRNA biogenesis from pri-miR-17~92, a processing step upstream of Microprocessor that selectively licenses production of pre-miR-17, -18a, -19a, -20a, and -19b but not miR-92.","method":"Loss-of-function (knockdown/knockout) of ISY1 with miRNA biogenesis readout; ESC differentiation assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean loss-of-function with defined molecular phenotype (specific miRNA processing defect), published in high-tier journal with multiple orthogonal functional assays","pmids":["26255770"],"is_preprint":false},{"year":2016,"finding":"Cryo-EM structure of the post-branching spliceosome (C complex) at 3.8 Å shows Isy1 (together with step-one factors Yju2 and Cwc25) stabilizes docking of the branch helix into the active site after the first transesterification reaction.","method":"Cryo-electron microscopy structural determination at 3.8 Å resolution","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure with direct visualization of Isy1 position, replicated context by multiple structural studies","pmids":["27459055"],"is_preprint":false},{"year":2016,"finding":"XAB2 forms a complex with ISY1 and PRP19; this XAB2-ISY1-PRP19 complex promotes end resection in homologous recombination (HR), as shown by ISY1 depletion impairing DSB repair via HR pathways requiring end resection, CtIP hyperphosphorylation, and RAD51 IRIF. The complex localizes to punctate structures adjacent to γH2AX foci.","method":"Truncation mutation analysis, co-immunoprecipitation, siRNA knockdown with HR reporter assays, immunofluorescence localization","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP for complex formation, loss-of-function with defined HR phenotype, direct localization, single lab","pmids":["27084940"],"is_preprint":false},{"year":2018,"finding":"ISY1 regulates miRNA biogenesis to control ESC pluripotency state transitions; loss- and gain-of-function experiments show ISY1 promotes exit from naive pluripotency and is necessary and sufficient to induce and maintain poised pluripotency through ISY1-dependent miRNAs.","method":"Loss-of-function (knockout) and gain-of-function (overexpression) in mouse ESCs, miRNA profiling, chimera formation assay","journal":"Cell stem cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal gain/loss-of-function with defined molecular phenotype (miRNA biogenesis), single lab, multiple orthogonal readouts","pmids":["29804889"],"is_preprint":false},{"year":2018,"finding":"Prp8 genetic interactions with Isy1 (which buttresses the intron branch point) support a role for specific Prp8 residues in substrate positioning and spliceosome activation at the 5' splice site.","method":"Genetic interaction analysis between Prp8 mutants and Isy1 in yeast","journal":"RNA (New York, N.Y.)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — genetic interaction only, ISY1's role is secondary finding in a paper focused on Prp8","pmids":["29487104"],"is_preprint":false},{"year":2019,"finding":"ISY1 directly interacts with APE1 (apurinic/apyrimidinic endonuclease 1) and enhances its 5'-3' endonuclease activity, promotes APE1 recognition of abasic sites, and markedly promotes APE1 activity in both short- and long-patch base excision repair (BER) pathways using purified recombinant proteins in a reconstituted BER system. ISY1 expression is induced by oxidative damage.","method":"Co-immunoprecipitation, in vitro BER reconstitution with purified recombinant ISY1 and APE1, endonuclease activity assays, oxidative damage induction","journal":"DNA repair","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution of BER with purified proteins, but single lab with no independent replication","pmids":["31887540"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM reconstruction of the yeast C-complex spliceosome at 2.8 Å reveals that Yju2 and Isy1 are recruited by the NTC (NineTeen Complex) before branching; after branching, Prp16 remodels Yju2 binding, allowing Yju2 to remain tethered to the NTC in the C* complex while Isy1's role is defined at the branching step.","method":"Cryo-electron microscopy at 2.8 Å resolution, biochemical splicing assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure with biochemical validation, independent replication of prior structural findings at higher resolution","pmids":["33705709"],"is_preprint":false},{"year":2021,"finding":"ISY1 is a member of a PRP19-related complex (PRC) in trypanosomes that also contains SYF1, SYF3, PRC3, and PRC5; PRC co-precipitates U4 snRNA, indicating it enters the spliceosome prior to the PRP19 complex, demonstrating a conserved but distinct pre-organization of ISY1-containing complexes.","method":"Gene silencing (RNAi), affinity purification combined with sucrose gradient sedimentation and mass spectrometry, snRNA co-precipitation","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — affinity purification with MS and sedimentation, functional silencing with viability readout, single lab in trypanosome model","pmids":["34850936"],"is_preprint":false},{"year":2021,"finding":"ISY1 (as part of the Prp19-associated complex) promotes nuclear retention of unspliced HIV-1 RNA; CRISPR/Cas knockdown of ISY1 results in more than 20-fold enhancement of unspliced HIV-1 RNA levels in the cytoplasm.","method":"Genome-wide CRISPR/Cas screen, CRISPR knockdown, RNA fractionation and quantification","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide screen with targeted follow-up CRISPR knockdown and RNA quantification, single lab","pmids":["33468685"],"is_preprint":false},{"year":2021,"finding":"Rat1 co-immunoprecipitates with Isy1 (among other splicing factors), and Rat1 mutants show reduced recruitment of splicing factors to introns, supporting a role for Isy1 in cotranscriptional splicing complex assembly.","method":"Co-immunoprecipitation, mass spectrometry, ChIP-Seq","journal":"Nucleic acids research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP/MS identification of Isy1 as interactor; ISY1 is a secondary finding in a paper focused on Rat1","pmids":["33978753"],"is_preprint":false},{"year":2025,"finding":"JMJD5 hydroxylates an arginine residue on ISY1; this hydroxylation modification enables ISY1 to bind to and inhibit PRMT6 (Protein Arginine N-methyltransferase 6), reducing PRMT6 activity; this JMJD5-ISY1-PRMT6 signaling pathway is identified as the principal executor of JMJD5's enzymatic function in metabolic regulation.","method":"Biochemical hydroxylation assay (JMJD5 on ISY1), binding assay (ISY1-PRMT6), PRMT6 activity assay, rescue experiments (PRMT6 inactivation rescuing JMJD5 loss phenotype)","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro enzymatic and binding assays with functional rescue, but preprint with no independent replication","pmids":["bio_10.1101_2025.09.27.678987"],"is_preprint":true}],"current_model":"ISY1 is a multifunctional protein that operates primarily as a component of the NineTeen Complex (NTC/Prp19 complex) in the spliceosome, where it stabilizes branch helix docking into the catalytic active site during the first step of pre-mRNA splicing and promotes spliceosomal conformations favorable for first-step chemistry and fidelity of branchpoint and 3'-splice site usage; beyond splicing, ISY1 regulates miRNA biogenesis (including pro-miRNA processing from pri-miR-17~92), enhances APE1 activity in base excision repair through direct protein interaction, participates in homologous recombination end resection as part of the XAB2-ISY1-PRP19 complex, and—according to a preprint—serves as a substrate for JMJD5-mediated arginine hydroxylation that enables ISY1 to inhibit PRMT6 methyltransferase activity in a novel metabolic signaling pathway."},"narrative":{"mechanistic_narrative":"ISY1 is a conserved spliceosomal protein that acts as a subunit of the NineTeen Complex (NTC/Prp19 complex) to govern the first catalytic step of pre-mRNA splicing [PMID:10094305, PMID:16103217]. Recruited together with Yju2 by the NTC prior to branching [PMID:33705709], ISY1 cooperates with U6 snRNA and the step-one factors Cwc25 and Yju2 to stabilize docking of the intron branch helix into the catalytic active site, as visualized directly in cryo-EM structures of the post-branching C complex [PMID:16103217, PMID:27459055]. Through this active-site role it enforces fidelity of branchpoint and 3'-splice-site selection: ISY1 deletion suppresses the stalled prp16-302 phenotype while degrading 3'-splice-site accuracy in wild-type backgrounds [PMID:16103217]. Genetically and physically ISY1 sits at the core of the activated spliceosome, interacting with Cwc21, Cwc2, and Prp8 to position substrate RNA [PMID:19789211, PMID:24848011, PMID:29487104]. This splicing function carries direct cellular consequences—efficient splicing of intron-containing alpha-tubulin transcripts depends on ISY1, and its loss can trigger spindle-checkpoint arrest [PMID:12384582]. Beyond core splicing, ISY1 has acquired metazoan roles in gene regulation and genome maintenance: it partners with the endonuclease CPSF3 to perform a pri-miRNA cleavage step upstream of the Microprocessor that selectively licenses pro-miRNA production from pri-miR-17~92, a process that controls embryonic stem-cell pluripotency state transitions [PMID:26255770, PMID:29804889]; it forms an XAB2-ISY1-PRP19 complex that promotes DNA end resection during homologous-recombination repair of double-strand breaks [PMID:27084940]; and it directly binds APE1 to enhance its abasic-site recognition and endonuclease activity in base excision repair [PMID:31887540]. ISY1 also contributes to nuclear retention of unspliced HIV-1 RNA as part of the Prp19-associated complex [PMID:33468685].","teleology":[{"year":1999,"claim":"Established ISY1 as a bona fide splicing factor by showing it associates with spliceosomes and is required for efficient splicing, answering whether the gene had any splicing role at all.","evidence":"Two-hybrid screen, tagged Co-IP recovering U5/U6 snRNAs, and in vivo splicing in ISY1-null yeast","pmids":["10094305"],"confidence":"Medium","gaps":["Mechanistic step within splicing undefined","Low snRNA recovery leaves stable association ambiguous"]},{"year":2002,"claim":"Linked ISY1 splicing function to a concrete cellular phenotype by showing its loss reduces alpha-tubulin transcript splicing and triggers spindle-checkpoint arrest, demonstrating physiological consequences of impaired splicing.","evidence":"Genetic epistasis with SYF2/NTC20 deletions, cell-cycle and checkpoint assays, protein-level measurement in yeast","pmids":["12384582"],"confidence":"Medium","gaps":["Phenotype is indirect via specific intron-containing targets","Does not define ISY1's molecular step"]},{"year":2005,"claim":"Placed ISY1 mechanistically as an NTC subunit that cooperates with U6 snRNA to promote first-step chemistry and enforce branchpoint/3'-splice-site fidelity, defining the catalytic-step it controls.","evidence":"Genetic suppressor screen of prp16-302, growth assays, biochemical NTC fractionation in yeast","pmids":["16103217"],"confidence":"High","gaps":["Structural basis of branchpoint stabilization not yet resolved","Human ortholog function not tested here"]},{"year":2009,"claim":"Refined ISY1's position at the spliceosome core by demonstrating tight genetic, physical, and functional coupling to Cwc21 during splicing activation.","evidence":"Quantitative genetic interaction mapping, MS of TAP-purified complexes, microarray profiling","pmids":["19789211"],"confidence":"Medium","gaps":["Direct contact interface with Cwc21 not mapped"]},{"year":2014,"claim":"Showed ISY1 cooperates with Cwc2 to stabilize active-site RNA elements, building the picture of ISY1 as part of an RNA-stabilizing module.","evidence":"Genetic suppressor/synthetic-lethality analysis and biochemical RNA interaction assays in yeast","pmids":["24848011"],"confidence":"Medium","gaps":["Which RNA elements ISY1 itself contacts not directly demonstrated"]},{"year":2015,"claim":"Revealed a non-canonical metazoan function: ISY1 with CPSF3 performs a pri-miRNA cleavage step upstream of the Microprocessor that selectively licenses pro-miRNA biogenesis from pri-miR-17~92.","evidence":"ISY1 loss-of-function with miRNA biogenesis readout and ESC differentiation assays","pmids":["26255770"],"confidence":"High","gaps":["Catalytic versus scaffolding contribution of ISY1 unresolved","Generality beyond miR-17~92 cluster unclear"]},{"year":2016,"claim":"Provided direct structural proof that Isy1, with Yju2 and Cwc25, stabilizes branch-helix docking into the active site after the first transesterification, converting genetic inference into physical visualization.","evidence":"Cryo-EM of the post-branching C complex at 3.8 Å","pmids":["27459055"],"confidence":"High","gaps":["Conformational dynamics of recruitment/release not captured at this resolution"]},{"year":2016,"claim":"Extended ISY1 into genome maintenance by defining an XAB2-ISY1-PRP19 complex that promotes DNA end resection during homologous recombination.","evidence":"Truncation mapping, reciprocal Co-IP, siRNA with HR reporter assays, immunofluorescence near γH2AX foci","pmids":["27084940"],"confidence":"Medium","gaps":["Molecular step ISY1 contributes to resection undefined","Single lab without independent replication"]},{"year":2019,"claim":"Demonstrated a direct ISY1-APE1 interaction that enhances APE1 endonuclease activity and abasic-site recognition in base excision repair, expanding ISY1's DNA-repair repertoire.","evidence":"Co-IP, in vitro BER reconstitution with purified recombinant proteins, endonuclease assays, oxidative-damage induction","pmids":["31887540"],"confidence":"Medium","gaps":["No independent replication","Structural basis of ISY1-APE1 stimulation unknown"]},{"year":2021,"claim":"Resolved the temporal logic of ISY1 recruitment, showing it and Yju2 enter via the NTC before branching and that Prp16 remodels the complex after branching, defining when ISY1 acts.","evidence":"Cryo-EM of yeast C complex at 2.8 Å with biochemical splicing assays","pmids":["33705709"],"confidence":"High","gaps":["Fate/release of Isy1 after branching not fully traced"]},{"year":2021,"claim":"Showed conserved but distinctly pre-organized ISY1-containing complexes by identifying a trypanosome PRP19-related complex (with SYF1/SYF3/PRC3/PRC5) that enters the spliceosome early.","evidence":"RNAi silencing, affinity purification with MS and sucrose-gradient sedimentation, U4 snRNA co-precipitation","pmids":["34850936"],"confidence":"Medium","gaps":["Whether this pre-organization holds in metazoans untested"]},{"year":2021,"claim":"Identified ISY1 as a host factor promoting nuclear retention of unspliced HIV-1 RNA, connecting its splicing-associated activity to viral RNA fate.","evidence":"Genome-wide CRISPR screen, targeted CRISPR knockdown, RNA fractionation/quantification","pmids":["33468685"],"confidence":"Medium","gaps":["Mechanism of retention versus indirect splicing effect not separated"]},{"year":2025,"claim":"Proposed a metabolic-signaling role in which JMJD5 hydroxylates an ISY1 arginine, enabling ISY1 to bind and inhibit PRMT6, placing ISY1 downstream of JMJD5 enzymatic function.","evidence":"In vitro hydroxylation and binding assays, PRMT6 activity assays, rescue experiments (preprint)","pmids":["bio_10.1101_2025.09.27.678987"],"confidence":"Medium","gaps":["Preprint without independent replication","Hydroxylated residue and structural mechanism not validated in vivo"]},{"year":null,"claim":"How ISY1's single fold is partitioned among spliceosomal first-step stabilization, pri-miRNA processing, DNA-repair complexes, and the proposed PRMT6-regulatory axis—and whether these activities are mutually exclusive or coordinated—remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of non-splicing ISY1 complexes","Determinants directing ISY1 to distinct partners unknown","Cross-talk between splicing and repair pools untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,2,7]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[11,16]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[7,12]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8,14]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[2,6,7]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[8,11]}],"complexes":["NineTeen Complex (NTC/Prp19 complex)","XAB2-ISY1-PRP19 complex","PRP19-related complex (PRC)"],"partners":["PRP19","XAB2","APE1","CPSF3","PRMT6","JMJD5","CWC21","CWC2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9ULR0","full_name":"Pre-mRNA-splicing factor ISY1 homolog","aliases":[],"length_aa":285,"mass_kda":33.0,"function":"Component of the spliceosome C complex required for the selective processing of microRNAs during embryonic stem cell differentiation (By similarity). Required for the biogenesis of all miRNAs from the pri-miR-17-92 primary transcript except miR-92a (By similarity). Only required for the biogenesis of miR-290 and miR-96 from the pri-miR-290-295 and pri-miR-96-183 primary transcripts, respectively (By similarity). Required during the transition of embryonic stem cells (ESCs) from the naive to primed state (By similarity). By enhancing miRNA biogenesis, promotes exit of ESCs from the naive state to an intermediate state of poised pluripotency, which precedes transition to the primed state (By similarity). Involved in pre-mRNA splicing as component of the spliceosome","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9ULR0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/ISY1","classification":"Common Essential","n_dependent_lines":1198,"n_total_lines":1208,"dependency_fraction":0.9917218543046358},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CPSF6","stoichiometry":0.2},{"gene":"CTNNBL1","stoichiometry":0.2},{"gene":"DNAJC17","stoichiometry":0.2},{"gene":"PRPF19","stoichiometry":0.2},{"gene":"PRPF4B","stoichiometry":0.2},{"gene":"RBM39","stoichiometry":0.2},{"gene":"SF3A1","stoichiometry":0.2},{"gene":"SNRPA","stoichiometry":0.2},{"gene":"SNRPB","stoichiometry":0.2},{"gene":"SNRPC","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ISY1","total_profiled":1310},"omim":[{"mim_id":"612764","title":"ISY1 SPLICING FACTOR HOMOLOG; ISY1","url":"https://www.omim.org/entry/612764"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nuclear speckles","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ISY1"},"hgnc":{"alias_symbol":["KIAA1160","fSAP33"],"prev_symbol":[]},"alphafold":{"accession":"Q9ULR0","domains":[{"cath_id":"1.10.287.660","chopping":"23-138","consensus_level":"medium","plddt":86.8124,"start":23,"end":138}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9ULR0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9ULR0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9ULR0-F1-predicted_aligned_error_v6.png","plddt_mean":80.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ISY1","jax_strain_url":"https://www.jax.org/strain/search?query=ISY1"},"sequence":{"accession":"Q9ULR0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9ULR0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9ULR0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9ULR0"}},"corpus_meta":[{"pmid":"27459055","id":"PMC_27459055","title":"Cryo-EM structure of the 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Hypoxic-Ischemic Encephalopathy: A Retrospective Case Series.","date":"2023","source":"Journal of child neurology","url":"https://pubmed.ncbi.nlm.nih.gov/36628482","citation_count":19,"is_preprint":false},{"pmid":"29487104","id":"PMC_29487104","title":"Prp8 positioning of U5 snRNA is linked to 5' splice site recognition.","date":"2018","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/29487104","citation_count":15,"is_preprint":false},{"pmid":"31887540","id":"PMC_31887540","title":"The splicing component ISY1 regulates APE1 in base excision repair.","date":"2019","source":"DNA repair","url":"https://pubmed.ncbi.nlm.nih.gov/31887540","citation_count":14,"is_preprint":false},{"pmid":"24848011","id":"PMC_24848011","title":"Remodeling of U2-U6 snRNA helix I during pre-mRNA splicing by Prp16 and the NineTeen Complex protein Cwc2.","date":"2014","source":"Nucleic acids 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Molecular basis of disease","url":"https://pubmed.ncbi.nlm.nih.gov/39025375","citation_count":9,"is_preprint":false},{"pmid":"35962262","id":"PMC_35962262","title":"Elevated expression of ISY1, APOA-1, SYNE1, MTG1, and MMP10 at HCC initiation: HCC specific protein network involving interactions of key regulators of lipid metabolism, EGFR signaling, MAPK, and splicing pathways.","date":"2022","source":"Protoplasma","url":"https://pubmed.ncbi.nlm.nih.gov/35962262","citation_count":7,"is_preprint":false},{"pmid":"33978753","id":"PMC_33978753","title":"A termination-independent role of Rat1 in cotranscriptional splicing.","date":"2021","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/33978753","citation_count":6,"is_preprint":false},{"pmid":"34850936","id":"PMC_34850936","title":"A distinct complex of PRP19-related and trypanosomatid-specific proteins is required for pre-mRNA splicing in trypanosomes.","date":"2021","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/34850936","citation_count":5,"is_preprint":false},{"pmid":"40609214","id":"PMC_40609214","title":"Differential expression of lncRNAs and mRNAs in bone marrow-derived mesenchymal stem cells under continuous and intermittent teriparatide treatment.","date":"2025","source":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie","url":"https://pubmed.ncbi.nlm.nih.gov/40609214","citation_count":2,"is_preprint":false},{"pmid":"40046230","id":"PMC_40046230","title":"ZNF32 histidine 179 and 183 single-site and double-site mutations promote nuclear speckle formation but differentially regulate the proliferation of breast cancer cells.","date":"2025","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/40046230","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.09.27.678987","title":"JMJD5 regulates metabolism by inhibiting the Arginine Methyltransferase PRMT6","date":"2025-09-29","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.27.678987","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13535,"output_tokens":4053,"usd":0.0507,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11817,"output_tokens":4062,"usd":0.080318,"stage2_stop_reason":"end_turn"},"total_usd":0.131018,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"Isy1p is a novel splicing factor that associates with spliceosomes throughout the splicing reactions and co-immunoprecipitates low levels of U5 and U6 snRNAs; deletion of ISY1 reduces splicing efficiency in vivo, indicating a role in optimizing pre-mRNA splicing.\",\n      \"method\": \"Two-hybrid screen, tagged protein co-immunoprecipitation with snRNAs, in vivo splicing assay in ISY1 null mutant yeast\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with snRNAs and functional KO splicing assay, single lab, two orthogonal methods\",\n      \"pmids\": [\"10094305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Mutations in ISY1 combined with SYF2 deletion cause cell cycle arrest due to reduced splicing of intron-containing alpha-tubulin genes (TUB1, TUB3), lowering alpha-tubulin protein levels and triggering spindle checkpoint activation; ISY1 is synthetically lethal with NTC20 deletion.\",\n      \"method\": \"Genetic epistasis (double-mutant analysis), cell cycle arrest assay, spindle checkpoint activation assay, protein level measurement\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with defined cellular phenotype, single lab, multiple genetic combinations tested\",\n      \"pmids\": [\"12384582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Isy1p is a subunit of the NineTeen Complex (NTC/Prp19 complex) and acts together with U6 snRNA to promote a spliceosomal conformation favorable for first-step chemistry; deletion of ISY1 suppresses the cold-sensitive prp16-302 phenotype by alleviating stalling and restoring branchpoint fidelity, and also reduces fidelity of 3'-splice site selection in PRP16 wild-type strains.\",\n      \"method\": \"Genetic suppressor screen of prp16-302, growth assays, biochemical spliceosome fractionation showing NTC membership\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis combined with biochemical NTC membership, replicated with multiple alleles and conditions, mechanistic model supported by both genetic and biochemical data\",\n      \"pmids\": [\"16103217\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ISY1 shows synthetic genetic interactions with TGS1 (trimethylguanosine synthase); deletion of ISY1 combined with tgs1Δ causes synthetic growth defects, placing ISY1 in a functional network with snRNP components and spliceosome assembly factors.\",\n      \"method\": \"Systematic synthetic growth defect screen (genetic interaction mapping) in budding yeast\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single genetic interaction screen, no direct mechanistic follow-up on ISY1 specifically\",\n      \"pmids\": [\"18775984\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Cwc21 shows strong genetic, physical, and functional interactions with Isy1, supporting a role for Isy1 at the core of the spliceosome in the activation of splicing.\",\n      \"method\": \"Quantitative genetic interaction mapping, mass spectrometry of tandem affinity-purified complexes, microarray profiling\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (genetic interaction, MS co-purification), single lab\",\n      \"pmids\": [\"19789211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Isy1/NTC30 functionally cooperates with the NTC protein Cwc2 during pre-mRNA splicing; genetic interactions between Cwc2 mutations and Isy1 indicate they act together in stabilizing spliceosome active site RNA elements.\",\n      \"method\": \"Genetic interaction analysis (suppressor/synthetic lethality), biochemical RNA interaction assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis and biochemical RNA interaction data, single lab\",\n      \"pmids\": [\"24848011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ISY1 (spliceosome-associated) and the endonuclease CPSF3 are responsible for pro-miRNA biogenesis from pri-miR-17~92, a processing step upstream of Microprocessor that selectively licenses production of pre-miR-17, -18a, -19a, -20a, and -19b but not miR-92.\",\n      \"method\": \"Loss-of-function (knockdown/knockout) of ISY1 with miRNA biogenesis readout; ESC differentiation assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean loss-of-function with defined molecular phenotype (specific miRNA processing defect), published in high-tier journal with multiple orthogonal functional assays\",\n      \"pmids\": [\"26255770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cryo-EM structure of the post-branching spliceosome (C complex) at 3.8 Å shows Isy1 (together with step-one factors Yju2 and Cwc25) stabilizes docking of the branch helix into the active site after the first transesterification reaction.\",\n      \"method\": \"Cryo-electron microscopy structural determination at 3.8 Å resolution\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure with direct visualization of Isy1 position, replicated context by multiple structural studies\",\n      \"pmids\": [\"27459055\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"XAB2 forms a complex with ISY1 and PRP19; this XAB2-ISY1-PRP19 complex promotes end resection in homologous recombination (HR), as shown by ISY1 depletion impairing DSB repair via HR pathways requiring end resection, CtIP hyperphosphorylation, and RAD51 IRIF. The complex localizes to punctate structures adjacent to γH2AX foci.\",\n      \"method\": \"Truncation mutation analysis, co-immunoprecipitation, siRNA knockdown with HR reporter assays, immunofluorescence localization\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP for complex formation, loss-of-function with defined HR phenotype, direct localization, single lab\",\n      \"pmids\": [\"27084940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ISY1 regulates miRNA biogenesis to control ESC pluripotency state transitions; loss- and gain-of-function experiments show ISY1 promotes exit from naive pluripotency and is necessary and sufficient to induce and maintain poised pluripotency through ISY1-dependent miRNAs.\",\n      \"method\": \"Loss-of-function (knockout) and gain-of-function (overexpression) in mouse ESCs, miRNA profiling, chimera formation assay\",\n      \"journal\": \"Cell stem cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal gain/loss-of-function with defined molecular phenotype (miRNA biogenesis), single lab, multiple orthogonal readouts\",\n      \"pmids\": [\"29804889\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Prp8 genetic interactions with Isy1 (which buttresses the intron branch point) support a role for specific Prp8 residues in substrate positioning and spliceosome activation at the 5' splice site.\",\n      \"method\": \"Genetic interaction analysis between Prp8 mutants and Isy1 in yeast\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — genetic interaction only, ISY1's role is secondary finding in a paper focused on Prp8\",\n      \"pmids\": [\"29487104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ISY1 directly interacts with APE1 (apurinic/apyrimidinic endonuclease 1) and enhances its 5'-3' endonuclease activity, promotes APE1 recognition of abasic sites, and markedly promotes APE1 activity in both short- and long-patch base excision repair (BER) pathways using purified recombinant proteins in a reconstituted BER system. ISY1 expression is induced by oxidative damage.\",\n      \"method\": \"Co-immunoprecipitation, in vitro BER reconstitution with purified recombinant ISY1 and APE1, endonuclease activity assays, oxidative damage induction\",\n      \"journal\": \"DNA repair\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution of BER with purified proteins, but single lab with no independent replication\",\n      \"pmids\": [\"31887540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM reconstruction of the yeast C-complex spliceosome at 2.8 Å reveals that Yju2 and Isy1 are recruited by the NTC (NineTeen Complex) before branching; after branching, Prp16 remodels Yju2 binding, allowing Yju2 to remain tethered to the NTC in the C* complex while Isy1's role is defined at the branching step.\",\n      \"method\": \"Cryo-electron microscopy at 2.8 Å resolution, biochemical splicing assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure with biochemical validation, independent replication of prior structural findings at higher resolution\",\n      \"pmids\": [\"33705709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ISY1 is a member of a PRP19-related complex (PRC) in trypanosomes that also contains SYF1, SYF3, PRC3, and PRC5; PRC co-precipitates U4 snRNA, indicating it enters the spliceosome prior to the PRP19 complex, demonstrating a conserved but distinct pre-organization of ISY1-containing complexes.\",\n      \"method\": \"Gene silencing (RNAi), affinity purification combined with sucrose gradient sedimentation and mass spectrometry, snRNA co-precipitation\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — affinity purification with MS and sedimentation, functional silencing with viability readout, single lab in trypanosome model\",\n      \"pmids\": [\"34850936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ISY1 (as part of the Prp19-associated complex) promotes nuclear retention of unspliced HIV-1 RNA; CRISPR/Cas knockdown of ISY1 results in more than 20-fold enhancement of unspliced HIV-1 RNA levels in the cytoplasm.\",\n      \"method\": \"Genome-wide CRISPR/Cas screen, CRISPR knockdown, RNA fractionation and quantification\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide screen with targeted follow-up CRISPR knockdown and RNA quantification, single lab\",\n      \"pmids\": [\"33468685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Rat1 co-immunoprecipitates with Isy1 (among other splicing factors), and Rat1 mutants show reduced recruitment of splicing factors to introns, supporting a role for Isy1 in cotranscriptional splicing complex assembly.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, ChIP-Seq\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP/MS identification of Isy1 as interactor; ISY1 is a secondary finding in a paper focused on Rat1\",\n      \"pmids\": [\"33978753\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"JMJD5 hydroxylates an arginine residue on ISY1; this hydroxylation modification enables ISY1 to bind to and inhibit PRMT6 (Protein Arginine N-methyltransferase 6), reducing PRMT6 activity; this JMJD5-ISY1-PRMT6 signaling pathway is identified as the principal executor of JMJD5's enzymatic function in metabolic regulation.\",\n      \"method\": \"Biochemical hydroxylation assay (JMJD5 on ISY1), binding assay (ISY1-PRMT6), PRMT6 activity assay, rescue experiments (PRMT6 inactivation rescuing JMJD5 loss phenotype)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro enzymatic and binding assays with functional rescue, but preprint with no independent replication\",\n      \"pmids\": [\"bio_10.1101_2025.09.27.678987\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"ISY1 is a multifunctional protein that operates primarily as a component of the NineTeen Complex (NTC/Prp19 complex) in the spliceosome, where it stabilizes branch helix docking into the catalytic active site during the first step of pre-mRNA splicing and promotes spliceosomal conformations favorable for first-step chemistry and fidelity of branchpoint and 3'-splice site usage; beyond splicing, ISY1 regulates miRNA biogenesis (including pro-miRNA processing from pri-miR-17~92), enhances APE1 activity in base excision repair through direct protein interaction, participates in homologous recombination end resection as part of the XAB2-ISY1-PRP19 complex, and—according to a preprint—serves as a substrate for JMJD5-mediated arginine hydroxylation that enables ISY1 to inhibit PRMT6 methyltransferase activity in a novel metabolic signaling pathway.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ISY1 is a conserved spliceosomal protein that acts as a subunit of the NineTeen Complex (NTC/Prp19 complex) to govern the first catalytic step of pre-mRNA splicing [#0, #2]. Recruited together with Yju2 by the NTC prior to branching [#12], ISY1 cooperates with U6 snRNA and the step-one factors Cwc25 and Yju2 to stabilize docking of the intron branch helix into the catalytic active site, as visualized directly in cryo-EM structures of the post-branching C complex [#2, #7]. Through this active-site role it enforces fidelity of branchpoint and 3'-splice-site selection: ISY1 deletion suppresses the stalled prp16-302 phenotype while degrading 3'-splice-site accuracy in wild-type backgrounds [#2]. Genetically and physically ISY1 sits at the core of the activated spliceosome, interacting with Cwc21, Cwc2, and Prp8 to position substrate RNA [#4, #5, #10]. This splicing function carries direct cellular consequences—efficient splicing of intron-containing alpha-tubulin transcripts depends on ISY1, and its loss can trigger spindle-checkpoint arrest [#1]. Beyond core splicing, ISY1 has acquired metazoan roles in gene regulation and genome maintenance: it partners with the endonuclease CPSF3 to perform a pri-miRNA cleavage step upstream of the Microprocessor that selectively licenses pro-miRNA production from pri-miR-17~92, a process that controls embryonic stem-cell pluripotency state transitions [#6, #9]; it forms an XAB2-ISY1-PRP19 complex that promotes DNA end resection during homologous-recombination repair of double-strand breaks [#8]; and it directly binds APE1 to enhance its abasic-site recognition and endonuclease activity in base excision repair [#11]. ISY1 also contributes to nuclear retention of unspliced HIV-1 RNA as part of the Prp19-associated complex [#14].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established ISY1 as a bona fide splicing factor by showing it associates with spliceosomes and is required for efficient splicing, answering whether the gene had any splicing role at all.\",\n      \"evidence\": \"Two-hybrid screen, tagged Co-IP recovering U5/U6 snRNAs, and in vivo splicing in ISY1-null yeast\",\n      \"pmids\": [\"10094305\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic step within splicing undefined\", \"Low snRNA recovery leaves stable association ambiguous\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Linked ISY1 splicing function to a concrete cellular phenotype by showing its loss reduces alpha-tubulin transcript splicing and triggers spindle-checkpoint arrest, demonstrating physiological consequences of impaired splicing.\",\n      \"evidence\": \"Genetic epistasis with SYF2/NTC20 deletions, cell-cycle and checkpoint assays, protein-level measurement in yeast\",\n      \"pmids\": [\"12384582\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phenotype is indirect via specific intron-containing targets\", \"Does not define ISY1's molecular step\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Placed ISY1 mechanistically as an NTC subunit that cooperates with U6 snRNA to promote first-step chemistry and enforce branchpoint/3'-splice-site fidelity, defining the catalytic-step it controls.\",\n      \"evidence\": \"Genetic suppressor screen of prp16-302, growth assays, biochemical NTC fractionation in yeast\",\n      \"pmids\": [\"16103217\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of branchpoint stabilization not yet resolved\", \"Human ortholog function not tested here\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Refined ISY1's position at the spliceosome core by demonstrating tight genetic, physical, and functional coupling to Cwc21 during splicing activation.\",\n      \"evidence\": \"Quantitative genetic interaction mapping, MS of TAP-purified complexes, microarray profiling\",\n      \"pmids\": [\"19789211\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct contact interface with Cwc21 not mapped\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed ISY1 cooperates with Cwc2 to stabilize active-site RNA elements, building the picture of ISY1 as part of an RNA-stabilizing module.\",\n      \"evidence\": \"Genetic suppressor/synthetic-lethality analysis and biochemical RNA interaction assays in yeast\",\n      \"pmids\": [\"24848011\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which RNA elements ISY1 itself contacts not directly demonstrated\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Revealed a non-canonical metazoan function: ISY1 with CPSF3 performs a pri-miRNA cleavage step upstream of the Microprocessor that selectively licenses pro-miRNA biogenesis from pri-miR-17~92.\",\n      \"evidence\": \"ISY1 loss-of-function with miRNA biogenesis readout and ESC differentiation assays\",\n      \"pmids\": [\"26255770\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic versus scaffolding contribution of ISY1 unresolved\", \"Generality beyond miR-17~92 cluster unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Provided direct structural proof that Isy1, with Yju2 and Cwc25, stabilizes branch-helix docking into the active site after the first transesterification, converting genetic inference into physical visualization.\",\n      \"evidence\": \"Cryo-EM of the post-branching C complex at 3.8 Å\",\n      \"pmids\": [\"27459055\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational dynamics of recruitment/release not captured at this resolution\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended ISY1 into genome maintenance by defining an XAB2-ISY1-PRP19 complex that promotes DNA end resection during homologous recombination.\",\n      \"evidence\": \"Truncation mapping, reciprocal Co-IP, siRNA with HR reporter assays, immunofluorescence near γH2AX foci\",\n      \"pmids\": [\"27084940\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular step ISY1 contributes to resection undefined\", \"Single lab without independent replication\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated a direct ISY1-APE1 interaction that enhances APE1 endonuclease activity and abasic-site recognition in base excision repair, expanding ISY1's DNA-repair repertoire.\",\n      \"evidence\": \"Co-IP, in vitro BER reconstitution with purified recombinant proteins, endonuclease assays, oxidative-damage induction\",\n      \"pmids\": [\"31887540\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No independent replication\", \"Structural basis of ISY1-APE1 stimulation unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved the temporal logic of ISY1 recruitment, showing it and Yju2 enter via the NTC before branching and that Prp16 remodels the complex after branching, defining when ISY1 acts.\",\n      \"evidence\": \"Cryo-EM of yeast C complex at 2.8 Å with biochemical splicing assays\",\n      \"pmids\": [\"33705709\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Fate/release of Isy1 after branching not fully traced\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed conserved but distinctly pre-organized ISY1-containing complexes by identifying a trypanosome PRP19-related complex (with SYF1/SYF3/PRC3/PRC5) that enters the spliceosome early.\",\n      \"evidence\": \"RNAi silencing, affinity purification with MS and sucrose-gradient sedimentation, U4 snRNA co-precipitation\",\n      \"pmids\": [\"34850936\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether this pre-organization holds in metazoans untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified ISY1 as a host factor promoting nuclear retention of unspliced HIV-1 RNA, connecting its splicing-associated activity to viral RNA fate.\",\n      \"evidence\": \"Genome-wide CRISPR screen, targeted CRISPR knockdown, RNA fractionation/quantification\",\n      \"pmids\": [\"33468685\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of retention versus indirect splicing effect not separated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Proposed a metabolic-signaling role in which JMJD5 hydroxylates an ISY1 arginine, enabling ISY1 to bind and inhibit PRMT6, placing ISY1 downstream of JMJD5 enzymatic function.\",\n      \"evidence\": \"In vitro hydroxylation and binding assays, PRMT6 activity assays, rescue experiments (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.09.27.678987\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint without independent replication\", \"Hydroxylated residue and structural mechanism not validated in vivo\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ISY1's single fold is partitioned among spliceosomal first-step stabilization, pri-miRNA processing, DNA-repair complexes, and the proposed PRMT6-regulatory axis—and whether these activities are mutually exclusive or coordinated—remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of non-splicing ISY1 complexes\", \"Determinants directing ISY1 to distinct partners unknown\", \"Cross-talk between splicing and repair pools untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 2, 7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [11, 16]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [7, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [2, 6, 7]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [8, 11]}\n    ],\n    \"complexes\": [\n      \"NineTeen Complex (NTC/Prp19 complex)\",\n      \"XAB2-ISY1-PRP19 complex\",\n      \"PRP19-related complex (PRC)\"\n    ],\n    \"partners\": [\n      \"PRP19\",\n      \"XAB2\",\n      \"APE1\",\n      \"CPSF3\",\n      \"PRMT6\",\n      \"JMJD5\",\n      \"Cwc21\",\n      \"Cwc2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}