{"gene":"IWS1","run_date":"2026-06-10T01:55:23","timeline":{"discoveries":[{"year":2007,"finding":"Human IWS1 (hIws1) was identified as an Spt6-interacting protein that associates with the mRNA nuclear export factor REF1/Aly; depletion of hIws1 caused mRNA processing defects, reduced REF1/Aly occupancy at the c-myc gene, and nuclear retention of bulk poly(A)+ RNAs, establishing IWS1 as a cotranscriptional recruiter of mRNA export machinery downstream of Spt6 binding to Ser2-phosphorylated RNAPII CTD.","method":"Co-immunoprecipitation, siRNA knockdown, chromatin immunoprecipitation (ChIP), nuclear poly(A)+ RNA retention assay in HeLa cells","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ChIP, and functional knockdown with multiple orthogonal readouts; foundational paper replicated by subsequent studies","pmids":["17234882"],"is_preprint":false},{"year":2008,"finding":"IWS1 bridges two CTD-binding proteins, Spt6 and HYPB/Setd2, in a megacomplex on the RNAPII elongation machinery; knockdown of IWS1 abolished H3K36me3 across the transcribed regions of c-Myc, HIV-1, and PABPC1 genes and also increased H3K27me3 at the 5' end of PABPC1 and histone acetylation across coding regions, demonstrating that IWS1 recruits Setd2 to direct co-transcriptional H3K36 trimethylation.","method":"siRNA knockdown, ChIP for H3K36me3/H3K27me3/acetylation, Co-IP, in vitro Spt6-CTD binding assay with recombinant proteins","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro reconstitution of Spt6-CTD-Iws1 complex combined with multiple ChIP readouts and siRNA knockdown, replicated at multiple loci","pmids":["19141475"],"is_preprint":false},{"year":2021,"finding":"AKT-mediated phosphorylation of IWS1 promotes H3K36me3 deposition in target gene bodies, which controls LEDGF/SRSF1-dependent inclusion of exon 2 in U2AF2 pre-mRNA; the resulting exon-2-containing U2AF65 is required for proper CDCA5 pre-mRNA processing, Sororin expression, ERK phosphorylation, and G2/M progression. Loss of IWS1 phosphorylation produces an RS-domain-deficient U2AF65 that cannot support these downstream events, impairing cell proliferation.","method":"RNA-seq after IWS1 phosphorylation block, RT-PCR, ChIP for H3K36me3, functional rescue experiments, xenograft tumor growth assays, analysis of EGFR-mutant lung adenocarcinoma specimens","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multi-omics RNA-seq, ChIP, and functional rescue across multiple cell lines and in vivo models; mechanistic pathway dissected with multiple orthogonal methods","pmids":["34330897"],"is_preprint":false},{"year":2021,"finding":"The RS-domain-containing U2AF65 isoform (produced under phospho-IWS1-dependent splicing) recruits Prp19 to CAR-element-containing intronless mRNAs and promotes their nuclear export; U2AF65 loading to CAR-elements was RS-domain-independent but RNA Pol II-dependent. IWS1-phosphorylation-deficient cells express reduced IFNα1/IFNβ1 protein and show enhanced sensitivity to cytolytic virus infection.","method":"RNA immunoprecipitation, Co-IP, siRNA knockdown, viral infection assays, caspase activation assays","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and functional assays in single lab, multiple orthogonal readouts but no structural validation","pmids":["34635782"],"is_preprint":false},{"year":2019,"finding":"In mouse preimplantation embryos, IWS1 interacts with nuclear AKT, and inhibition of the PI3K/AKT pathway reduced global H3K36me3 whereas activation increased it, suggesting AKT modulates H3K36me3 through interaction with IWS1; knockdown of Iws1 or Supt6 individually blocked development at the 8/16-cell stage with defects in pre-mRNA splicing, mRNA export, and Nanog expression.","method":"siRNA microinjection in mouse embryos, Co-IP (IWS1 with nuclear AKT), immunofluorescence for H3K36me3, PI3K/AKT pathway inhibitor/activator treatment","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP for AKT interaction and pharmacological modulation of H3K36me3, single lab, embryo knockdown phenotype without full mechanistic dissection of phosphorylation site","pmids":["30846735"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM structure of the activated Pol II elongation complex shows IWS1 acts as a scaffold that positions downstream DNA within the cleft of Pol II. The intrinsically disordered C-terminal region of IWS1 contacts the Pol II cleft and, together with ELOF1, stimulates Pol II elongation velocity. Rapid depletion of IWS1 in human cells caused a global decrease in RNA synthesis and Pol II elongation velocity; the associated decrease in H3K36me3 was found to be an indirect, secondary consequence of reduced transcription rather than a direct IWS1 function.","method":"Cryo-EM structure determination, multi-omics kinetic analysis after auxin-inducible IWS1 degron, in vitro transcription assays, mutagenesis of IWS1 C-terminal disordered region","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure combined with in vitro functional assays, mutagenesis, and multi-omics in human cells; H3K36me3 secondary effect explicitly tested and excluded as direct function","pmids":["40835814"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM mapping revealed that the intrinsically disordered C-terminal region of IWS1 contains short linear motifs (SLiMs) that contact Pol II subunits RPB1, RPB2, and RPB5, elongation factors DSIF, SPT6, and ELOF1; IWS1 recruitment to the elongation complex requires the RPB1 jaw interaction and downstream DNA binding, while transcription stimulation requires RPB2 lobe and ELOF1 contacts. IWS1 was found to protect the elongation complex from RECQL5 inhibition. Additionally, the histone reader LEDGF (an IWS1 interactor) was shown to bind a transcribed downstream nucleosome.","method":"Cryo-EM structure determination, in vitro transcription assays with IWS1 SLiM mutants, functional recruitment assays, RECQL5 competition assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with mutagenesis of individual SLiMs and multiple in vitro functional assays; published in peer-reviewed journal","pmids":["42134803"],"is_preprint":false},{"year":2025,"finding":"Molecular docking using the AlphaFold-predicted IWS1 structure identified a core Spt6-binding region (AA 545–694) of IWS1; candidate small-molecule inhibitors Ketotifen and Desloratadine were predicted to mimic Spt6 Phe217 and disrupt the IWS1/Spt6 complex, which was confirmed by co-immunoprecipitation. Disruption of this interaction increased nuclear localization of IWS1 and reduced migration, invasion, and spheroid formation in dedifferentiated liposarcoma cells.","method":"Molecular docking/virtual screening, Co-immunoprecipitation, cell migration/invasion assays, spheroid formation assay, immunofluorescence for subcellular localization","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP validation of inhibitor effect in a single lab, docking-guided; no structural or biochemical reconstitution of the interaction domain itself","pmids":["40594510"],"is_preprint":false},{"year":2022,"finding":"Genetic suppressor screen in S. cerevisiae showed that viability in the absence of Spn1/Iws1 can be achieved by mutations in FACT, Set2, Rpd3S, Rtt109, Chd1, or Sgf73, placing Spn1/Iws1 function at the intersection of histone acetylation and multiple histone chaperone pathways; this epistasis indicates Spn1 acts to overcome repressive chromatin through multiple mechanisms during transcription elongation.","method":"Suppressor genetics (bypass suppressor screen), yeast viability assays, genetic epistasis","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis by suppressor screen with multiple independent suppressor classes; yeast ortholog with well-conserved function","pmids":["35977387"],"is_preprint":false},{"year":2025,"finding":"IWS1's intrinsically disordered C-terminal region (SLiMs) is responsible for stimulation of Pol II elongation activity, as demonstrated by in vitro assays; this region acts together with ELOF1 to enhance Pol II processivity.","method":"In vitro transcription elongation assay, cryo-EM, SLiM mutagenesis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — cryo-EM and in vitro assays with mutagenesis in preprint; subsequently published as peer-reviewed paper (PMID 42134803), but preprint entry kept for completeness","pmids":["40909601"],"is_preprint":true}],"current_model":"IWS1 is an intrinsically disordered modular scaffold protein that associates with the RNA Pol II elongation complex via short linear motifs (SLiMs) in its C-terminal region contacting RPB1, RPB2, RPB5, DSIF, SPT6, and ELOF1; it positions downstream DNA within the Pol II cleft to globally stimulate elongation velocity, recruits HYPB/Setd2 to drive co-transcriptional H3K36me3 (which secondarily controls alternative splicing of targets such as U2AF2 via LEDGF/SRSF1), recruits REF1/Aly to facilitate mRNA export, and is subject to AKT-mediated phosphorylation that amplifies the H3K36me3-dependent splicing and export functions in normal development and cancer contexts."},"narrative":{"mechanistic_narrative":"IWS1 is an intrinsically disordered scaffold of the RNA Polymerase II transcription elongation machinery that couples elongation to chromatin modification, mRNA processing, and nuclear export [PMID:17234882, PMID:19141475, PMID:40835814]. Cryo-EM of the activated elongation complex shows that the disordered C-terminal region of IWS1 uses short linear motifs to contact Pol II subunits RPB1, RPB2, and RPB5 together with the elongation factors DSIF, SPT6, and ELOF1, positioning downstream DNA within the Pol II cleft; recruitment depends on the RPB1 jaw and downstream-DNA contacts while stimulation of elongation velocity depends on the RPB2 lobe and ELOF1, and IWS1 also protects the complex from RECQL5 inhibition [PMID:40835814, PMID:42134803]. Rapid IWS1 depletion globally lowers RNA synthesis and Pol II elongation velocity, establishing this elongation-stimulating activity as its core function [PMID:40835814]. IWS1 bridges the CTD-binding proteins SPT6 and HYPB/Setd2 and is required for co-transcriptional H3K36me3 deposition across transcribed regions, and it recruits the export factor REF1/Aly to license bulk poly(A)+ RNA export downstream of SPT6 engagement of the Ser2-phosphorylated Pol II CTD [PMID:17234882, PMID:19141475]. AKT-mediated phosphorylation of IWS1 amplifies an H3K36me3-dependent, LEDGF/SRSF1-directed splicing program controlling inclusion of U2AF2 exon 2, which in turn governs CDCA5/Sororin expression, ERK signaling, G2/M progression, and—via the RS-domain U2AF65 isoform that recruits Prp19—the export of CAR-element intronless mRNAs including interferon transcripts [PMID:34330897, PMID:34635782]. In mouse preimplantation embryos IWS1 acts with SPT6 and nuclear AKT to support splicing, export, and Nanog expression required for development beyond the 8/16-cell stage [PMID:30846735].","teleology":[{"year":2007,"claim":"Established IWS1 as the molecular link coupling transcription elongation to mRNA nuclear export, answering how export machinery is loaded co-transcriptionally.","evidence":"Co-IP, siRNA knockdown, ChIP, and nuclear poly(A)+ retention assays in HeLa cells","pmids":["17234882"],"confidence":"High","gaps":["Direct structural basis of the IWS1–REF1/Aly contact not resolved","Whether export recruitment is separable from elongation function untested at this stage"]},{"year":2008,"claim":"Showed IWS1 is a physical bridge between SPT6 and Setd2 that directs co-transcriptional H3K36 trimethylation, defining its role in writing an elongation-coupled chromatin mark.","evidence":"siRNA knockdown, ChIP for H3K36me3/H3K27me3/acetylation, Co-IP, and in vitro Spt6-CTD binding with recombinant proteins","pmids":["19141475"],"confidence":"High","gaps":["Did not distinguish whether H3K36me3 loss is direct or secondary to reduced transcription","No structural map of the SPT6–IWS1–Setd2 megacomplex"]},{"year":2019,"claim":"Extended the IWS1/SPT6 axis to development and linked nuclear AKT to H3K36me3 control, addressing how a signaling kinase modulates this chromatin output in vivo.","evidence":"siRNA microinjection in mouse embryos, Co-IP of IWS1 with nuclear AKT, H3K36me3 immunofluorescence, PI3K/AKT modulation","pmids":["30846735"],"confidence":"Medium","gaps":["Phosphorylation site not mapped in this system","Causal chain from AKT to H3K36me3 inferred pharmacologically, not by direct enzymatic assay"]},{"year":2021,"claim":"Dissected how AKT-phosphorylated IWS1 drives an H3K36me3-dependent splicing program controlling U2AF2 exon 2 inclusion and downstream cell-cycle progression, connecting the scaffold to proliferation and cancer.","evidence":"RNA-seq after phosphorylation block, RT-PCR, ChIP, rescue experiments, xenografts, and EGFR-mutant lung adenocarcinoma specimens","pmids":["34330897"],"confidence":"High","gaps":["IWS1 phosphosite kinetics relative to elongation not defined","Whether splicing effects persist independent of bulk elongation changes untested"]},{"year":2021,"claim":"Showed the phospho-IWS1-dependent RS-domain U2AF65 isoform recruits Prp19 to export CAR-element intronless mRNAs including interferon transcripts, linking the pathway to antiviral defense.","evidence":"RNA immunoprecipitation, Co-IP, siRNA knockdown, viral infection and caspase assays","pmids":["34635782"],"confidence":"Medium","gaps":["Single-lab reciprocal Co-IP without structural validation","Direct contribution of IWS1 versus the downstream U2AF65 isoform not fully separated"]},{"year":2022,"claim":"Genetic epistasis in yeast placed Spn1/Iws1 at the intersection of multiple histone chaperone and acetylation pathways, framing its role as overcoming repressive chromatin during elongation.","evidence":"Bypass suppressor screen and genetic epistasis in S. cerevisiae","pmids":["35977387"],"confidence":"Medium","gaps":["Genetic, not biochemical, relationships","Conservation of specific suppressor interactions to human IWS1 untested"]},{"year":2025,"claim":"Cryo-EM and degron kinetics redefined the core IWS1 function as direct stimulation of Pol II elongation via downstream-DNA positioning, and showed the H3K36me3 decrease is an indirect consequence of reduced transcription.","evidence":"Cryo-EM of the activated elongation complex, auxin-inducible degron multi-omics, in vitro transcription, and C-terminal SLiM mutagenesis","pmids":["40835814"],"confidence":"High","gaps":["Reconciliation with earlier models placing H3K36me3 as a direct IWS1 output","Whether export/splicing roles are likewise downstream of elongation not fully resolved"]},{"year":2025,"claim":"Mapped the specific SLiMs by which IWS1 contacts Pol II subunits and elongation factors and defined which contacts drive recruitment versus stimulation, providing a structural mechanism for its scaffolding.","evidence":"Cryo-EM with individual SLiM mutants, in vitro transcription, recruitment assays, and RECQL5 competition assays","pmids":["42134803"],"confidence":"High","gaps":["Functional consequence of RECQL5 protection in cells not quantified","How LEDGF nucleosome binding integrates with IWS1 elongation function unresolved"]},{"year":2025,"claim":"Docking-guided screening identified the SPT6-binding region of IWS1 and candidate disruptors that alter IWS1 localization and tumor-cell behavior, opening a therapeutic targeting hypothesis.","evidence":"Molecular docking, Co-IP validation, migration/invasion/spheroid assays in dedifferentiated liposarcoma cells","pmids":["40594510"],"confidence":"Low","gaps":["No structural or biochemical reconstitution of the interaction domain","Inhibitor specificity and on-target mechanism not established"]},{"year":null,"claim":"It remains unresolved which IWS1 outputs (H3K36me3, splicing, export, antiviral signaling, cancer phenotypes) are direct functions versus indirect consequences of its core elongation-stimulating activity.","evidence":"","pmids":[],"confidence":"High","gaps":["Causal hierarchy between elongation velocity and chromatin/splicing/export outputs not disentangled","AKT phosphosite role in the structural elongation mechanism unmapped","In vivo relevance of the RECQL5-protection function unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,5,6]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[5,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,6]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,7]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,1,5,6]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,2,3]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[1,8]}],"complexes":["RNA Pol II elongation complex"],"partners":["SPT6","SETD2","REF1/ALY","ELOF1","RPB1","RPB2","LEDGF","AKT"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96ST2","full_name":"Protein IWS1 homolog","aliases":["IWS1-like protein"],"length_aa":819,"mass_kda":92.0,"function":"Transcription factor which plays a key role in defining the composition of the RNA polymerase II (RNAPII) elongation complex and in modulating the production of mature mRNA transcripts. Acts as an assembly factor to recruit various factors to the RNAPII elongation complex and is recruited to the complex via binding to the transcription elongation factor SUPT6H bound to the C-terminal domain (CTD) of the RNAPII subunit RPB1 (POLR2A). The SUPT6H:IWS1:CTD complex recruits mRNA export factors (ALYREF/THOC4, EXOSC10) as well as histone modifying enzymes (such as SETD2) to ensure proper mRNA splicing, efficient mRNA export and elongation-coupled H3K36 methylation, a signature chromatin mark of active transcription","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q96ST2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/IWS1","classification":"Not Classified","n_dependent_lines":110,"n_total_lines":1208,"dependency_fraction":0.09105960264900662},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CPSF6","stoichiometry":0.2},{"gene":"HIST2H2BE","stoichiometry":0.2},{"gene":"HMGA1","stoichiometry":0.2},{"gene":"SNRPA","stoichiometry":0.2},{"gene":"SNRPC","stoichiometry":0.2},{"gene":"SNRPF","stoichiometry":0.2},{"gene":"SRP9","stoichiometry":0.2},{"gene":"SSRP1","stoichiometry":0.2},{"gene":"SUPT5H","stoichiometry":0.2},{"gene":"TOP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/IWS1","total_profiled":1310},"omim":[],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/IWS1"},"hgnc":{"alias_symbol":["DKFZp761G0123","FLJ10006","FLJ14655","FLJ32319"],"prev_symbol":[]},"alphafold":{"accession":"Q96ST2","domains":[{"cath_id":"1.20.930.10","chopping":"557-710","consensus_level":"high","plddt":89.2438,"start":557,"end":710}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96ST2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96ST2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96ST2-F1-predicted_aligned_error_v6.png","plddt_mean":54.53},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=IWS1","jax_strain_url":"https://www.jax.org/strain/search?query=IWS1"},"sequence":{"accession":"Q96ST2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96ST2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96ST2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96ST2"}},"corpus_meta":[{"pmid":"17234882","id":"PMC_17234882","title":"The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicing and export.","date":"2007","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/17234882","citation_count":214,"is_preprint":false},{"pmid":"19141475","id":"PMC_19141475","title":"The Iws1:Spt6:CTD complex controls cotranscriptional mRNA biosynthesis and HYPB/Setd2-mediated histone H3K36 methylation.","date":"2008","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/19141475","citation_count":200,"is_preprint":false},{"pmid":"30846735","id":"PMC_30846735","title":"Iws1 and Spt6 Regulate Trimethylation of Histone H3 on Lysine 36 through Akt Signaling and are Essential for Mouse Embryonic Genome Activation.","date":"2019","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/30846735","citation_count":24,"is_preprint":false},{"pmid":"30208029","id":"PMC_30208029","title":"Genetic ablation of interacting with Spt6 (Iws1) causes early embryonic lethality.","date":"2018","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/30208029","citation_count":10,"is_preprint":false},{"pmid":"35977387","id":"PMC_35977387","title":"Suppressor mutations that make the essential transcription factor Spn1/Iws1 dispensable in Saccharomyces cerevisiae.","date":"2022","source":"Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/35977387","citation_count":10,"is_preprint":false},{"pmid":"34330897","id":"PMC_34330897","title":"AKT3-mediated IWS1 phosphorylation promotes the proliferation of EGFR-mutant lung adenocarcinomas through cell cycle-regulated U2AF2 RNA splicing.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/34330897","citation_count":9,"is_preprint":false},{"pmid":"37237004","id":"PMC_37237004","title":"Phosphorylation of IWS1 by AKT maintains liposarcoma tumor heterogeneity through preservation of cancer stem cell phenotypes and mesenchymal-epithelial plasticity.","date":"2023","source":"Oncogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/37237004","citation_count":7,"is_preprint":false},{"pmid":"36715543","id":"PMC_36715543","title":"Toxoplasma IWS1 Determines Fitness in Interferon-γ-Activated Host Cells and Mice by Indirectly Regulating ROP18 mRNA Expression.","date":"2023","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/36715543","citation_count":5,"is_preprint":false},{"pmid":"40835814","id":"PMC_40835814","title":"IWS1 positions downstream DNA to globally stimulate Pol II elongation.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/40835814","citation_count":3,"is_preprint":false},{"pmid":"20124725","id":"PMC_20124725","title":"Crystallization and preliminary crystallographic analysis of eukaryotic transcription and mRNA export factor Iws1 from Encephalitozoon cuniculi.","date":"2010","source":"Acta crystallographica. Section F, Structural biology and crystallization communications","url":"https://pubmed.ncbi.nlm.nih.gov/20124725","citation_count":3,"is_preprint":false},{"pmid":"40909601","id":"PMC_40909601","title":"Structure and function of IWS1 in transcription elongation.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/40909601","citation_count":1,"is_preprint":false},{"pmid":"34635782","id":"PMC_34635782","title":"Phosphor-IWS1-dependent U2AF2 splicing regulates trafficking of CAR-E-positive intronless gene mRNAs and sensitivity to viral infection.","date":"2021","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/34635782","citation_count":1,"is_preprint":false},{"pmid":"40594510","id":"PMC_40594510","title":"Molecular docking and biological evaluation of a novel IWS1 inhibitor for the treatment of human retroperitoneal liposarcoma.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/40594510","citation_count":0,"is_preprint":false},{"pmid":"42134803","id":"PMC_42134803","title":"Structure and function of IWS1 in transcription elongation.","date":"2026","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/42134803","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8852,"output_tokens":3134,"usd":0.036783,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10487,"output_tokens":3517,"usd":0.07018,"stage2_stop_reason":"end_turn"},"total_usd":0.106963,"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\": 2007,\n      \"finding\": \"Human IWS1 (hIws1) was identified as an Spt6-interacting protein that associates with the mRNA nuclear export factor REF1/Aly; depletion of hIws1 caused mRNA processing defects, reduced REF1/Aly occupancy at the c-myc gene, and nuclear retention of bulk poly(A)+ RNAs, establishing IWS1 as a cotranscriptional recruiter of mRNA export machinery downstream of Spt6 binding to Ser2-phosphorylated RNAPII CTD.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, chromatin immunoprecipitation (ChIP), nuclear poly(A)+ RNA retention assay in HeLa cells\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ChIP, and functional knockdown with multiple orthogonal readouts; foundational paper replicated by subsequent studies\",\n      \"pmids\": [\"17234882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"IWS1 bridges two CTD-binding proteins, Spt6 and HYPB/Setd2, in a megacomplex on the RNAPII elongation machinery; knockdown of IWS1 abolished H3K36me3 across the transcribed regions of c-Myc, HIV-1, and PABPC1 genes and also increased H3K27me3 at the 5' end of PABPC1 and histone acetylation across coding regions, demonstrating that IWS1 recruits Setd2 to direct co-transcriptional H3K36 trimethylation.\",\n      \"method\": \"siRNA knockdown, ChIP for H3K36me3/H3K27me3/acetylation, Co-IP, in vitro Spt6-CTD binding assay with recombinant proteins\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro reconstitution of Spt6-CTD-Iws1 complex combined with multiple ChIP readouts and siRNA knockdown, replicated at multiple loci\",\n      \"pmids\": [\"19141475\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"AKT-mediated phosphorylation of IWS1 promotes H3K36me3 deposition in target gene bodies, which controls LEDGF/SRSF1-dependent inclusion of exon 2 in U2AF2 pre-mRNA; the resulting exon-2-containing U2AF65 is required for proper CDCA5 pre-mRNA processing, Sororin expression, ERK phosphorylation, and G2/M progression. Loss of IWS1 phosphorylation produces an RS-domain-deficient U2AF65 that cannot support these downstream events, impairing cell proliferation.\",\n      \"method\": \"RNA-seq after IWS1 phosphorylation block, RT-PCR, ChIP for H3K36me3, functional rescue experiments, xenograft tumor growth assays, analysis of EGFR-mutant lung adenocarcinoma specimens\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multi-omics RNA-seq, ChIP, and functional rescue across multiple cell lines and in vivo models; mechanistic pathway dissected with multiple orthogonal methods\",\n      \"pmids\": [\"34330897\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The RS-domain-containing U2AF65 isoform (produced under phospho-IWS1-dependent splicing) recruits Prp19 to CAR-element-containing intronless mRNAs and promotes their nuclear export; U2AF65 loading to CAR-elements was RS-domain-independent but RNA Pol II-dependent. IWS1-phosphorylation-deficient cells express reduced IFNα1/IFNβ1 protein and show enhanced sensitivity to cytolytic virus infection.\",\n      \"method\": \"RNA immunoprecipitation, Co-IP, siRNA knockdown, viral infection assays, caspase activation assays\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and functional assays in single lab, multiple orthogonal readouts but no structural validation\",\n      \"pmids\": [\"34635782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In mouse preimplantation embryos, IWS1 interacts with nuclear AKT, and inhibition of the PI3K/AKT pathway reduced global H3K36me3 whereas activation increased it, suggesting AKT modulates H3K36me3 through interaction with IWS1; knockdown of Iws1 or Supt6 individually blocked development at the 8/16-cell stage with defects in pre-mRNA splicing, mRNA export, and Nanog expression.\",\n      \"method\": \"siRNA microinjection in mouse embryos, Co-IP (IWS1 with nuclear AKT), immunofluorescence for H3K36me3, PI3K/AKT pathway inhibitor/activator treatment\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP for AKT interaction and pharmacological modulation of H3K36me3, single lab, embryo knockdown phenotype without full mechanistic dissection of phosphorylation site\",\n      \"pmids\": [\"30846735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structure of the activated Pol II elongation complex shows IWS1 acts as a scaffold that positions downstream DNA within the cleft of Pol II. The intrinsically disordered C-terminal region of IWS1 contacts the Pol II cleft and, together with ELOF1, stimulates Pol II elongation velocity. Rapid depletion of IWS1 in human cells caused a global decrease in RNA synthesis and Pol II elongation velocity; the associated decrease in H3K36me3 was found to be an indirect, secondary consequence of reduced transcription rather than a direct IWS1 function.\",\n      \"method\": \"Cryo-EM structure determination, multi-omics kinetic analysis after auxin-inducible IWS1 degron, in vitro transcription assays, mutagenesis of IWS1 C-terminal disordered region\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure combined with in vitro functional assays, mutagenesis, and multi-omics in human cells; H3K36me3 secondary effect explicitly tested and excluded as direct function\",\n      \"pmids\": [\"40835814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM mapping revealed that the intrinsically disordered C-terminal region of IWS1 contains short linear motifs (SLiMs) that contact Pol II subunits RPB1, RPB2, and RPB5, elongation factors DSIF, SPT6, and ELOF1; IWS1 recruitment to the elongation complex requires the RPB1 jaw interaction and downstream DNA binding, while transcription stimulation requires RPB2 lobe and ELOF1 contacts. IWS1 was found to protect the elongation complex from RECQL5 inhibition. Additionally, the histone reader LEDGF (an IWS1 interactor) was shown to bind a transcribed downstream nucleosome.\",\n      \"method\": \"Cryo-EM structure determination, in vitro transcription assays with IWS1 SLiM mutants, functional recruitment assays, RECQL5 competition assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with mutagenesis of individual SLiMs and multiple in vitro functional assays; published in peer-reviewed journal\",\n      \"pmids\": [\"42134803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Molecular docking using the AlphaFold-predicted IWS1 structure identified a core Spt6-binding region (AA 545–694) of IWS1; candidate small-molecule inhibitors Ketotifen and Desloratadine were predicted to mimic Spt6 Phe217 and disrupt the IWS1/Spt6 complex, which was confirmed by co-immunoprecipitation. Disruption of this interaction increased nuclear localization of IWS1 and reduced migration, invasion, and spheroid formation in dedifferentiated liposarcoma cells.\",\n      \"method\": \"Molecular docking/virtual screening, Co-immunoprecipitation, cell migration/invasion assays, spheroid formation assay, immunofluorescence for subcellular localization\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP validation of inhibitor effect in a single lab, docking-guided; no structural or biochemical reconstitution of the interaction domain itself\",\n      \"pmids\": [\"40594510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Genetic suppressor screen in S. cerevisiae showed that viability in the absence of Spn1/Iws1 can be achieved by mutations in FACT, Set2, Rpd3S, Rtt109, Chd1, or Sgf73, placing Spn1/Iws1 function at the intersection of histone acetylation and multiple histone chaperone pathways; this epistasis indicates Spn1 acts to overcome repressive chromatin through multiple mechanisms during transcription elongation.\",\n      \"method\": \"Suppressor genetics (bypass suppressor screen), yeast viability assays, genetic epistasis\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis by suppressor screen with multiple independent suppressor classes; yeast ortholog with well-conserved function\",\n      \"pmids\": [\"35977387\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"IWS1's intrinsically disordered C-terminal region (SLiMs) is responsible for stimulation of Pol II elongation activity, as demonstrated by in vitro assays; this region acts together with ELOF1 to enhance Pol II processivity.\",\n      \"method\": \"In vitro transcription elongation assay, cryo-EM, SLiM mutagenesis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM and in vitro assays with mutagenesis in preprint; subsequently published as peer-reviewed paper (PMID 42134803), but preprint entry kept for completeness\",\n      \"pmids\": [\"40909601\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"IWS1 is an intrinsically disordered modular scaffold protein that associates with the RNA Pol II elongation complex via short linear motifs (SLiMs) in its C-terminal region contacting RPB1, RPB2, RPB5, DSIF, SPT6, and ELOF1; it positions downstream DNA within the Pol II cleft to globally stimulate elongation velocity, recruits HYPB/Setd2 to drive co-transcriptional H3K36me3 (which secondarily controls alternative splicing of targets such as U2AF2 via LEDGF/SRSF1), recruits REF1/Aly to facilitate mRNA export, and is subject to AKT-mediated phosphorylation that amplifies the H3K36me3-dependent splicing and export functions in normal development and cancer contexts.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"IWS1 is an intrinsically disordered scaffold of the RNA Polymerase II transcription elongation machinery that couples elongation to chromatin modification, mRNA processing, and nuclear export [#0, #1, #5]. Cryo-EM of the activated elongation complex shows that the disordered C-terminal region of IWS1 uses short linear motifs to contact Pol II subunits RPB1, RPB2, and RPB5 together with the elongation factors DSIF, SPT6, and ELOF1, positioning downstream DNA within the Pol II cleft; recruitment depends on the RPB1 jaw and downstream-DNA contacts while stimulation of elongation velocity depends on the RPB2 lobe and ELOF1, and IWS1 also protects the complex from RECQL5 inhibition [#5, #6]. Rapid IWS1 depletion globally lowers RNA synthesis and Pol II elongation velocity, establishing this elongation-stimulating activity as its core function [#5]. IWS1 bridges the CTD-binding proteins SPT6 and HYPB/Setd2 and is required for co-transcriptional H3K36me3 deposition across transcribed regions, and it recruits the export factor REF1/Aly to license bulk poly(A)+ RNA export downstream of SPT6 engagement of the Ser2-phosphorylated Pol II CTD [#0, #1]. AKT-mediated phosphorylation of IWS1 amplifies an H3K36me3-dependent, LEDGF/SRSF1-directed splicing program controlling inclusion of U2AF2 exon 2, which in turn governs CDCA5/Sororin expression, ERK signaling, G2/M progression, and—via the RS-domain U2AF65 isoform that recruits Prp19—the export of CAR-element intronless mRNAs including interferon transcripts [#2, #3]. In mouse preimplantation embryos IWS1 acts with SPT6 and nuclear AKT to support splicing, export, and Nanog expression required for development beyond the 8/16-cell stage [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established IWS1 as the molecular link coupling transcription elongation to mRNA nuclear export, answering how export machinery is loaded co-transcriptionally.\",\n      \"evidence\": \"Co-IP, siRNA knockdown, ChIP, and nuclear poly(A)+ retention assays in HeLa cells\",\n      \"pmids\": [\"17234882\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct structural basis of the IWS1–REF1/Aly contact not resolved\", \"Whether export recruitment is separable from elongation function untested at this stage\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showed IWS1 is a physical bridge between SPT6 and Setd2 that directs co-transcriptional H3K36 trimethylation, defining its role in writing an elongation-coupled chromatin mark.\",\n      \"evidence\": \"siRNA knockdown, ChIP for H3K36me3/H3K27me3/acetylation, Co-IP, and in vitro Spt6-CTD binding with recombinant proteins\",\n      \"pmids\": [\"19141475\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not distinguish whether H3K36me3 loss is direct or secondary to reduced transcription\", \"No structural map of the SPT6–IWS1–Setd2 megacomplex\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended the IWS1/SPT6 axis to development and linked nuclear AKT to H3K36me3 control, addressing how a signaling kinase modulates this chromatin output in vivo.\",\n      \"evidence\": \"siRNA microinjection in mouse embryos, Co-IP of IWS1 with nuclear AKT, H3K36me3 immunofluorescence, PI3K/AKT modulation\",\n      \"pmids\": [\"30846735\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphorylation site not mapped in this system\", \"Causal chain from AKT to H3K36me3 inferred pharmacologically, not by direct enzymatic assay\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Dissected how AKT-phosphorylated IWS1 drives an H3K36me3-dependent splicing program controlling U2AF2 exon 2 inclusion and downstream cell-cycle progression, connecting the scaffold to proliferation and cancer.\",\n      \"evidence\": \"RNA-seq after phosphorylation block, RT-PCR, ChIP, rescue experiments, xenografts, and EGFR-mutant lung adenocarcinoma specimens\",\n      \"pmids\": [\"34330897\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"IWS1 phosphosite kinetics relative to elongation not defined\", \"Whether splicing effects persist independent of bulk elongation changes untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed the phospho-IWS1-dependent RS-domain U2AF65 isoform recruits Prp19 to export CAR-element intronless mRNAs including interferon transcripts, linking the pathway to antiviral defense.\",\n      \"evidence\": \"RNA immunoprecipitation, Co-IP, siRNA knockdown, viral infection and caspase assays\",\n      \"pmids\": [\"34635782\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab reciprocal Co-IP without structural validation\", \"Direct contribution of IWS1 versus the downstream U2AF65 isoform not fully separated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Genetic epistasis in yeast placed Spn1/Iws1 at the intersection of multiple histone chaperone and acetylation pathways, framing its role as overcoming repressive chromatin during elongation.\",\n      \"evidence\": \"Bypass suppressor screen and genetic epistasis in S. cerevisiae\",\n      \"pmids\": [\"35977387\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Genetic, not biochemical, relationships\", \"Conservation of specific suppressor interactions to human IWS1 untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cryo-EM and degron kinetics redefined the core IWS1 function as direct stimulation of Pol II elongation via downstream-DNA positioning, and showed the H3K36me3 decrease is an indirect consequence of reduced transcription.\",\n      \"evidence\": \"Cryo-EM of the activated elongation complex, auxin-inducible degron multi-omics, in vitro transcription, and C-terminal SLiM mutagenesis\",\n      \"pmids\": [\"40835814\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation with earlier models placing H3K36me3 as a direct IWS1 output\", \"Whether export/splicing roles are likewise downstream of elongation not fully resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mapped the specific SLiMs by which IWS1 contacts Pol II subunits and elongation factors and defined which contacts drive recruitment versus stimulation, providing a structural mechanism for its scaffolding.\",\n      \"evidence\": \"Cryo-EM with individual SLiM mutants, in vitro transcription, recruitment assays, and RECQL5 competition assays\",\n      \"pmids\": [\"42134803\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of RECQL5 protection in cells not quantified\", \"How LEDGF nucleosome binding integrates with IWS1 elongation function unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Docking-guided screening identified the SPT6-binding region of IWS1 and candidate disruptors that alter IWS1 localization and tumor-cell behavior, opening a therapeutic targeting hypothesis.\",\n      \"evidence\": \"Molecular docking, Co-IP validation, migration/invasion/spheroid assays in dedifferentiated liposarcoma cells\",\n      \"pmids\": [\"40594510\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural or biochemical reconstitution of the interaction domain\", \"Inhibitor specificity and on-target mechanism not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved which IWS1 outputs (H3K36me3, splicing, export, antiviral signaling, cancer phenotypes) are direct functions versus indirect consequences of its core elongation-stimulating activity.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Causal hierarchy between elongation velocity and chromatin/splicing/export outputs not disentangled\", \"AKT phosphosite role in the structural elongation mechanism unmapped\", \"In vivo relevance of the RECQL5-protection function unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 5, 6]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [5, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 1, 5, 6]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 2, 3]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [1, 8]}\n    ],\n    \"complexes\": [\"RNA Pol II elongation complex\"],\n    \"partners\": [\"SPT6\", \"SETD2\", \"REF1/ALY\", \"ELOF1\", \"RPB1\", \"RPB2\", \"LEDGF\", \"AKT\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}