{"gene":"CHERP","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2000,"finding":"CHERP was cloned as a ~100 kDa endoplasmic reticulum protein; antisense-mediated depletion of CHERP in HEL cells caused ~80% loss of CHERP protein, markedly decreased intracellular Ca2+ mobilization by thrombin, decreased DNA synthesis, and growth arrest, indicating a functional role in Ca2+ homeostasis and cell proliferation. CHERP co-localized with the IP3 receptor by two-colour immunofluorescence.","method":"Antisense cDNA knockdown, immunofluorescence co-localization, Ca2+ mobilization assay, DNA synthesis measurement","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — antisense KD with multiple phenotypic readouts (Ca2+ signaling, DNA synthesis, growth), single lab, two orthogonal methods","pmids":["10794731"],"is_preprint":false},{"year":2003,"finding":"Antisense knockdown of CHERP in Jurkat T-lymphocytes impaired PHA- and thrombin-stimulated cytoplasmic Ca2+ rise, reduced ER Ca2+ store content (assessed by thapsigargin response), suppressed NFAT translocation to the nucleus, decreased cyclin D1 levels by ~60%, and slowed cell proliferation. Ca2+ influx was unaffected at moderate CHERP depletion (~50%) but reduced at >70% depletion. IP3 receptor levels were unchanged, placing CHERP functionally upstream of or parallel to Ca2+ release at the ER.","method":"Antisense cDNA knockdown, fura-2 Ca2+ imaging, confocal immunofluorescence, Western blotting, NFAT translocation assay","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal functional assays in a second cell type, single lab, replicating and extending prior findings","pmids":["12656674"],"is_preprint":false},{"year":2011,"finding":"CHERP was identified as a physical interacting partner of RyR1: a soluble His-tagged cytosolic fragment of RyR1 (aa 1–4243) co-purified CHERP by metal affinity chromatography; Western blotting confirmed co-purification. Endogenous CHERP co-localizes with endogenous RyR1 in the sarcoplasmic reticulum of rat soleus muscle by immunofluorescence. siRNA-mediated suppression of CHERP in HEK-293 cells overexpressing RyR1 reduced Ca2+ release via RyR1.","method":"Metal affinity chromatography / LC-MS proteomics, Western blotting, immunofluorescence co-localization, siRNA knockdown + Ca2+ release assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-purification confirmed by Western blot, co-localization, and functional siRNA assay, single lab, multiple orthogonal methods","pmids":["21454501"],"is_preprint":false},{"year":2012,"finding":"Re-evaluation study challenged the model that CHERP acts as a direct cytoplasmic regulator of IP3Rs and RyRs; instead, the authors found CHERP localizes to the nucleus and is associated with the U2 snRNA spliceosomal complex. Effects of CHERP on cellular growth were reinterpreted as indirect consequences of altered spliceosomal function rather than direct ER Ca2+ channel regulation.","method":"Subcellular fractionation, immunofluorescence/nuclear localization, co-immunoprecipitation with U2 snRNP components, cell proliferation assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — nuclear localization and spliceosomal association established by multiple methods; directly contradicts earlier ER-focused model; single lab","pmids":["23148228"],"is_preprint":false},{"year":2013,"finding":"CHERP was identified as a Ca2+-dependent ALG-2-interacting protein in the nucleus. CHERP localizes to nuclear speckles (sites of pre-mRNA splicing factor storage/modification) and binds a phosphorylated form of RNA polymerase II by co-IP. Live cell imaging showed nuclear ALG-2 is recruited to CHERP-containing speckles upon Ca2+ mobilization. Knockdown of CHERP in HT1080 cells altered alternative splicing of IP3R1 pre-mRNA (inclusion of exons 41 and 42). RNA immunoprecipitation demonstrated direct binding of CHERP to IP3R1 RNA.","method":"Co-immunoprecipitation, immunofluorescence, live-cell time-lapse imaging, siRNA knockdown + RT-PCR splicing assay, RNA immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, RNA-IP, live imaging, KD splicing assay) in a single rigorous study establishing nuclear spliceosomal function","pmids":["24078636"],"is_preprint":false},{"year":2018,"finding":"CHERP physically interacts with RBM17 and U2SURP (spliceosomal factors); the three proteins reciprocally regulate each other's protein stability in both mouse and human cells. Individual knockdown of CHERP, RBM17, or U2SURP causes overlapping changes in alternative splicing and gene expression of transcripts enriched for RNA-processing factors, linking CHERP to regulation of downstream RNA-binding proteins.","method":"Co-immunoprecipitation, siRNA knockdown, RNA-seq splicing analysis, Western blotting across mouse and human cells","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP and cross-species KD with transcriptome-wide splicing readout, replicated in two cell types/species","pmids":["30332651"],"is_preprint":false},{"year":2019,"finding":"CHERP forms a protein complex with SR140 (U2SURP) that stabilizes both proteins. The complex binds specifically to the regulated exon 4 of UPF3A pre-mRNA and controls its alternative splicing. Knockdown of CHERP or SR140 induces double-stranded DNA breaks and cell death; overexpression of UPF3A partially rescues the proliferation defect of CHERP/SR140-depleted cells, placing UPF3A as a key downstream splicing target.","method":"Co-immunoprecipitation, siRNA knockdown, RT-PCR splicing assay, rescue overexpression experiment, in vivo (mouse) tumor model","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, functional KD with rescue, single lab but multiple orthogonal methods","pmids":["30977118"],"is_preprint":false},{"year":2021,"finding":"SPF45 (RBM17), SR140 (U2SURP), and CHERP form a tight physical complex that represses short alternative exons flanked by suboptimal 3' splice sites. Regulated targets include cell-cycle genes FOXM1 and SPDL1. Knockdown of any of the three factors causes G2/M arrest and enhanced apoptosis in HeLa cells; forced changes in FOXM1 or SPDL1 splicing (mimicking complex knockdown) partially recapitulate cell growth defects, placing these splicing events downstream of the complex.","method":"Co-immunoprecipitation, siRNA knockdown, RNA-seq splicing analysis, cell cycle analysis, minigene splicing assay, genetic epistasis via splicing isoform overexpression","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — physical complex demonstrated by Co-IP, functional KD in multiple factors, epistasis via splicing isoform rescue, RNA-seq genome-wide, independent replication of SPF45/SR140/CHERP complex concept","pmids":["34544891"],"is_preprint":false},{"year":2022,"finding":"CHERP depletion in U2OS cells caused accumulation of poly(A)+ RNAs in the nucleus. Global analysis revealed CHERP regulates alternative mRNA splicing (particularly intron retention) through interaction with U2 snRNPs and U2 snRNP-related proteins. Intron retention frequency was influenced by 5'/3' splice site strength, branch point, GC content, and intron length. CHERP depletion also induced cell cycle defects at M phase and abnormal cell division.","method":"siRNA knockdown, RNA-FISH for poly(A)+ RNA localization, RNA-seq alternative splicing analysis, cell cycle analysis (FACS), live-cell imaging","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — nuclear RNA accumulation and global splicing changes by RNA-seq with KD, cell cycle phenotype, single lab, multiple methods","pmids":["35269695"],"is_preprint":false}],"current_model":"CHERP is a nuclear spliceosomal protein that forms a tight complex with SR140 (U2SURP) and SPF45 (RBM17) within the U2 snRNP, where it regulates alternative splicing—especially repression of short exons at suboptimal 3' splice sites and intron retention—of targets including FOXM1, SPDL1, UPF3A, and IP3R1 pre-mRNAs; disruption of CHERP causes nuclear poly(A)+ RNA accumulation, G2/M arrest, and impaired cell proliferation, while the earlier model of CHERP as a direct ER/cytoplasmic regulator of IP3R and RyR1 Ca2+ channels has been substantially re-evaluated in favour of these splicing-based mechanisms."},"narrative":{"mechanistic_narrative":"CHERP is a nuclear spliceosomal protein that regulates alternative pre-mRNA splicing as part of the U2 snRNP machinery [PMID:23148228, PMID:35269695]. It localizes to the nucleus and nuclear speckles, associates with the U2 snRNA spliceosomal complex, and binds a phosphorylated form of RNA polymerase II [PMID:23148228, PMID:24078636]. CHERP forms a tight, mutually stabilizing complex with SR140 (U2SURP) and SPF45 (RBM17) that represses short alternative exons flanked by suboptimal 3' splice sites and controls intron retention, acting on targets including FOXM1, SPDL1, UPF3A, and IP3R1 pre-mRNAs [PMID:24078636, PMID:30332651, PMID:30977118, PMID:34544891]. Through these splicing events, CHERP is required for cell-cycle progression: its depletion drives nuclear accumulation of poly(A)+ RNA, G2/M arrest, DNA damage, and impaired proliferation, with the growth defect partially rescued by restoring UPF3A or by mimicking the FOXM1/SPDL1 splicing outcomes [PMID:30977118, PMID:34544891, PMID:35269695]. An earlier model in which CHERP acted as a direct ER/cytoplasmic regulator of IP3R and RyR1 Ca2+ channels [PMID:10794731, PMID:21454501] was re-evaluated, with its effects on Ca2+ signaling and growth reinterpreted as indirect consequences of altered spliceosomal function [PMID:23148228]. CHERP additionally binds ALG-2 in a Ca2+-dependent manner, providing a candidate link between Ca2+ signaling and splicing regulation [PMID:24078636].","teleology":[{"year":2000,"claim":"Established CHERP as a functional protein affecting Ca2+ homeostasis and proliferation, the founding observation that defined the gene's phenotypic footprint.","evidence":"Antisense knockdown in HEL cells with Ca2+ mobilization, DNA synthesis assays, and IP3R co-localization by immunofluorescence","pmids":["10794731"],"confidence":"Medium","gaps":["Co-localization does not demonstrate direct molecular interaction with IP3R","Cannot distinguish direct Ca2+ channel regulation from indirect effects","Mechanism of the growth arrest unresolved"]},{"year":2003,"claim":"Extended the Ca2+/proliferation phenotype to a second cell type and placed CHERP upstream of or parallel to ER Ca2+ release rather than altering IP3R levels.","evidence":"Antisense knockdown in Jurkat cells with fura-2 imaging, NFAT translocation, and cyclin D1 Western blotting","pmids":["12656674"],"confidence":"Medium","gaps":["Did not identify the molecular target through which CHERP affects Ca2+ release","Indirect versus direct mechanism not resolved"]},{"year":2011,"claim":"Reported a physical and functional link to the RyR1 Ca2+ channel, reinforcing the ER/SR-centric model of CHERP function.","evidence":"Co-purification of CHERP with a soluble cytosolic RyR1 fragment, SR co-localization, and siRNA + Ca2+ release assay in HEK-293 cells","pmids":["21454501"],"confidence":"Medium","gaps":["Co-purification with an overexpressed fragment may not reflect endogenous direct binding","Did not exclude indirect contribution via splicing later proposed for CHERP"]},{"year":2012,"claim":"Reassigned CHERP from a cytoplasmic Ca2+ channel regulator to a nuclear spliceosome-associated protein, redirecting the entire mechanistic interpretation of the earlier growth phenotypes.","evidence":"Subcellular fractionation, nuclear immunofluorescence, and co-IP with U2 snRNP components plus proliferation assays","pmids":["23148228"],"confidence":"Medium","gaps":["Did not define which splicing targets account for the growth defect","Did not formally exclude a residual direct Ca2+ channel role"]},{"year":2013,"claim":"Provided direct molecular evidence for nuclear splicing function and linked CHERP back to Ca2+ signaling via ALG-2, showing it binds pre-mRNA and regulates IP3R1 splicing.","evidence":"Co-IP with phospho-RNA Pol II and ALG-2, nuclear speckle imaging, live-cell recruitment imaging, RNA-IP, and siRNA + RT-PCR splicing assay in HT1080 cells","pmids":["24078636"],"confidence":"High","gaps":["Functional consequence of Ca2+-dependent ALG-2 recruitment on splicing output not established","Genome-wide target spectrum not yet defined"]},{"year":2018,"claim":"Defined a mutually stabilizing module of CHERP with RBM17 and U2SURP and showed it controls splicing of RNA-processing factors, establishing CHERP as part of a coordinated splicing regulatory complex.","evidence":"Reciprocal co-IP, siRNA knockdown, and RNA-seq splicing analysis across mouse and human cells with Western blotting","pmids":["30332651"],"confidence":"High","gaps":["Stoichiometry and architecture of the CHERP-RBM17-U2SURP complex not resolved","Direct versus indirect splicing targets not fully separated"]},{"year":2019,"claim":"Identified UPF3A exon 4 as a specific regulated target of the CHERP-SR140 complex and tied the proliferation defect causally to splicing output via rescue.","evidence":"Co-IP, siRNA knockdown, RT-PCR splicing assay, UPF3A overexpression rescue, and an in vivo mouse tumor model","pmids":["30977118"],"confidence":"Medium","gaps":["Rescue was only partial, implying additional downstream targets","Mechanism linking the splicing defect to DNA breaks not detailed"]},{"year":2021,"claim":"Defined the catalytic logic of the SPF45-SR140-CHERP complex as repression of short exons at suboptimal 3' splice sites and connected it to specific cell-cycle gene splicing controlling G2/M progression.","evidence":"Co-IP, siRNA knockdown of all three factors, RNA-seq, minigene assays, cell cycle analysis, and splicing-isoform epistasis in HeLa cells","pmids":["34544891"],"confidence":"High","gaps":["Structural basis for recognition of suboptimal 3' splice sites unknown","Individual contribution of CHERP within the trimeric complex not isolated"]},{"year":2022,"claim":"Demonstrated that CHERP is required for nuclear mRNA export competence and controls intron retention through U2 snRNP interaction, broadening its role beyond exon repression.","evidence":"siRNA knockdown, RNA-FISH for poly(A)+ RNA, RNA-seq intron-retention analysis, FACS cell cycle, and live-cell imaging in U2OS cells","pmids":["35269695"],"confidence":"Medium","gaps":["Whether poly(A)+ RNA accumulation is a direct splicing consequence or a secondary effect not resolved","Sequence determinants of intron retention correlative rather than mechanistic"]},{"year":null,"claim":"How Ca2+/ALG-2 signaling functionally tunes CHERP-dependent splicing, and the structural basis by which the CHERP-SR140-SPF45 complex recognizes suboptimal splice sites, remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No structural model of the CHERP-containing U2 snRNP subcomplex","Functional output of Ca2+-dependent ALG-2 recruitment to CHERP speckles undefined","Complete catalogue of physiologically critical splicing targets not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[4,7,8]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[4,5,7,8]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3,4]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[3,4,5,7,8]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[6,7,8]}],"complexes":["U2 snRNP","CHERP-SR140(U2SURP)-SPF45(RBM17) splicing complex"],"partners":["U2SURP","RBM17","ALG-2","RNA POLYMERASE II","RYR1","ITPR1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8IWX8","full_name":"Calcium homeostasis endoplasmic reticulum protein","aliases":["ERPROT 213-21","SR-related CTD-associated factor 6"],"length_aa":916,"mass_kda":103.7,"function":"Involved in calcium homeostasis, growth and proliferation","subcellular_location":"Cytoplasm; Cytoplasm, perinuclear region; Endoplasmic reticulum","url":"https://www.uniprot.org/uniprotkb/Q8IWX8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CHERP","classification":"Common Essential","n_dependent_lines":1207,"n_total_lines":1208,"dependency_fraction":0.9991721854304636},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"COMMD2","stoichiometry":10.0},{"gene":"COMMD4","stoichiometry":10.0},{"gene":"COMMD6","stoichiometry":10.0},{"gene":"GNL3","stoichiometry":10.0},{"gene":"PIP4K2C","stoichiometry":10.0},{"gene":"RANBP2","stoichiometry":10.0},{"gene":"RBM17","stoichiometry":10.0},{"gene":"RSL1D1","stoichiometry":10.0},{"gene":"U2SURP","stoichiometry":10.0},{"gene":"CSNK2B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CHERP","total_profiled":1310},"omim":[{"mim_id":"618539","title":"CALCIUM HOMEOSTASIS ENDOPLASMIC RETICULUM PROTEIN; CHERP","url":"https://www.omim.org/entry/618539"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CHERP"},"hgnc":{"alias_symbol":["ERPROT213-21","DAN16"],"prev_symbol":[]},"alphafold":{"accession":"Q8IWX8","domains":[{"cath_id":"1.10.10.790","chopping":"11-71","consensus_level":"high","plddt":85.3579,"start":11,"end":71},{"cath_id":"1.25.40.90","chopping":"137-303","consensus_level":"high","plddt":94.0136,"start":137,"end":303}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IWX8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IWX8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IWX8-F1-predicted_aligned_error_v6.png","plddt_mean":63.22},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CHERP","jax_strain_url":"https://www.jax.org/strain/search?query=CHERP"},"sequence":{"accession":"Q8IWX8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8IWX8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8IWX8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IWX8"}},"corpus_meta":[{"pmid":"23148228","id":"PMC_23148228","title":"Re-evaluation of the role of calcium homeostasis endoplasmic reticulum protein (CHERP) in cellular calcium signaling.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23148228","citation_count":72,"is_preprint":false},{"pmid":"30332651","id":"PMC_30332651","title":"RBM17 Interacts with U2SURP and CHERP to Regulate Expression and Splicing of RNA-Processing Proteins.","date":"2018","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/30332651","citation_count":53,"is_preprint":false},{"pmid":"24078636","id":"PMC_24078636","title":"Nuclear ALG-2 protein interacts with Ca2+ homeostasis endoplasmic reticulum protein (CHERP) Ca2+-dependently and participates in regulation of alternative splicing of inositol trisphosphate receptor type 1 (IP3R1) pre-mRNA.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24078636","citation_count":28,"is_preprint":false},{"pmid":"34544891","id":"PMC_34544891","title":"Alternative splicing regulation of cell-cycle genes by SPF45/SR140/CHERP complex controls cell proliferation.","date":"2021","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/34544891","citation_count":24,"is_preprint":false},{"pmid":"30977118","id":"PMC_30977118","title":"U2-related proteins CHERP and SR140 contribute to colorectal tumorigenesis via alternative splicing regulation.","date":"2019","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/30977118","citation_count":21,"is_preprint":false},{"pmid":"10794731","id":"PMC_10794731","title":"Cloning of human Ca2+ homoeostasis endoplasmic reticulum protein (CHERP): regulated expression of antisense cDNA depletes CHERP, inhibits intracellular Ca2+ mobilization and decreases cell proliferation.","date":"2000","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/10794731","citation_count":19,"is_preprint":false},{"pmid":"12656674","id":"PMC_12656674","title":"Antisense-mediated loss of calcium homoeostasis endoplasmic reticulum protein (CHERP; ERPROT213-21) impairs Ca2+ mobilization, nuclear factor of activated T-cells (NFAT) activation and cell proliferation in Jurkat T-lymphocytes.","date":"2003","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/12656674","citation_count":17,"is_preprint":false},{"pmid":"21454501","id":"PMC_21454501","title":"Identification of novel ryanodine receptor 1 (RyR1) protein interaction with calcium homeostasis endoplasmic reticulum protein (CHERP).","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21454501","citation_count":17,"is_preprint":false},{"pmid":"29113358","id":"PMC_29113358","title":"Down-regulation of CHERP inhibits neuroblastoma cell proliferation and induces apoptosis through ER stress induction.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/29113358","citation_count":14,"is_preprint":false},{"pmid":"35269695","id":"PMC_35269695","title":"CHERP Regulates the Alternative Splicing of pre-mRNAs in the Nucleus.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35269695","citation_count":5,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7096,"output_tokens":2747,"usd":0.031246,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10087,"output_tokens":3471,"usd":0.068605,"stage2_stop_reason":"end_turn"},"total_usd":0.099851,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"CHERP was cloned as a ~100 kDa endoplasmic reticulum protein; antisense-mediated depletion of CHERP in HEL cells caused ~80% loss of CHERP protein, markedly decreased intracellular Ca2+ mobilization by thrombin, decreased DNA synthesis, and growth arrest, indicating a functional role in Ca2+ homeostasis and cell proliferation. CHERP co-localized with the IP3 receptor by two-colour immunofluorescence.\",\n      \"method\": \"Antisense cDNA knockdown, immunofluorescence co-localization, Ca2+ mobilization assay, DNA synthesis measurement\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — antisense KD with multiple phenotypic readouts (Ca2+ signaling, DNA synthesis, growth), single lab, two orthogonal methods\",\n      \"pmids\": [\"10794731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Antisense knockdown of CHERP in Jurkat T-lymphocytes impaired PHA- and thrombin-stimulated cytoplasmic Ca2+ rise, reduced ER Ca2+ store content (assessed by thapsigargin response), suppressed NFAT translocation to the nucleus, decreased cyclin D1 levels by ~60%, and slowed cell proliferation. Ca2+ influx was unaffected at moderate CHERP depletion (~50%) but reduced at >70% depletion. IP3 receptor levels were unchanged, placing CHERP functionally upstream of or parallel to Ca2+ release at the ER.\",\n      \"method\": \"Antisense cDNA knockdown, fura-2 Ca2+ imaging, confocal immunofluorescence, Western blotting, NFAT translocation assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal functional assays in a second cell type, single lab, replicating and extending prior findings\",\n      \"pmids\": [\"12656674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CHERP was identified as a physical interacting partner of RyR1: a soluble His-tagged cytosolic fragment of RyR1 (aa 1–4243) co-purified CHERP by metal affinity chromatography; Western blotting confirmed co-purification. Endogenous CHERP co-localizes with endogenous RyR1 in the sarcoplasmic reticulum of rat soleus muscle by immunofluorescence. siRNA-mediated suppression of CHERP in HEK-293 cells overexpressing RyR1 reduced Ca2+ release via RyR1.\",\n      \"method\": \"Metal affinity chromatography / LC-MS proteomics, Western blotting, immunofluorescence co-localization, siRNA knockdown + Ca2+ release assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-purification confirmed by Western blot, co-localization, and functional siRNA assay, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"21454501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Re-evaluation study challenged the model that CHERP acts as a direct cytoplasmic regulator of IP3Rs and RyRs; instead, the authors found CHERP localizes to the nucleus and is associated with the U2 snRNA spliceosomal complex. Effects of CHERP on cellular growth were reinterpreted as indirect consequences of altered spliceosomal function rather than direct ER Ca2+ channel regulation.\",\n      \"method\": \"Subcellular fractionation, immunofluorescence/nuclear localization, co-immunoprecipitation with U2 snRNP components, cell proliferation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — nuclear localization and spliceosomal association established by multiple methods; directly contradicts earlier ER-focused model; single lab\",\n      \"pmids\": [\"23148228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CHERP was identified as a Ca2+-dependent ALG-2-interacting protein in the nucleus. CHERP localizes to nuclear speckles (sites of pre-mRNA splicing factor storage/modification) and binds a phosphorylated form of RNA polymerase II by co-IP. Live cell imaging showed nuclear ALG-2 is recruited to CHERP-containing speckles upon Ca2+ mobilization. Knockdown of CHERP in HT1080 cells altered alternative splicing of IP3R1 pre-mRNA (inclusion of exons 41 and 42). RNA immunoprecipitation demonstrated direct binding of CHERP to IP3R1 RNA.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, live-cell time-lapse imaging, siRNA knockdown + RT-PCR splicing assay, RNA immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, RNA-IP, live imaging, KD splicing assay) in a single rigorous study establishing nuclear spliceosomal function\",\n      \"pmids\": [\"24078636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CHERP physically interacts with RBM17 and U2SURP (spliceosomal factors); the three proteins reciprocally regulate each other's protein stability in both mouse and human cells. Individual knockdown of CHERP, RBM17, or U2SURP causes overlapping changes in alternative splicing and gene expression of transcripts enriched for RNA-processing factors, linking CHERP to regulation of downstream RNA-binding proteins.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, RNA-seq splicing analysis, Western blotting across mouse and human cells\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP and cross-species KD with transcriptome-wide splicing readout, replicated in two cell types/species\",\n      \"pmids\": [\"30332651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CHERP forms a protein complex with SR140 (U2SURP) that stabilizes both proteins. The complex binds specifically to the regulated exon 4 of UPF3A pre-mRNA and controls its alternative splicing. Knockdown of CHERP or SR140 induces double-stranded DNA breaks and cell death; overexpression of UPF3A partially rescues the proliferation defect of CHERP/SR140-depleted cells, placing UPF3A as a key downstream splicing target.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, RT-PCR splicing assay, rescue overexpression experiment, in vivo (mouse) tumor model\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, functional KD with rescue, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"30977118\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPF45 (RBM17), SR140 (U2SURP), and CHERP form a tight physical complex that represses short alternative exons flanked by suboptimal 3' splice sites. Regulated targets include cell-cycle genes FOXM1 and SPDL1. Knockdown of any of the three factors causes G2/M arrest and enhanced apoptosis in HeLa cells; forced changes in FOXM1 or SPDL1 splicing (mimicking complex knockdown) partially recapitulate cell growth defects, placing these splicing events downstream of the complex.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, RNA-seq splicing analysis, cell cycle analysis, minigene splicing assay, genetic epistasis via splicing isoform overexpression\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — physical complex demonstrated by Co-IP, functional KD in multiple factors, epistasis via splicing isoform rescue, RNA-seq genome-wide, independent replication of SPF45/SR140/CHERP complex concept\",\n      \"pmids\": [\"34544891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CHERP depletion in U2OS cells caused accumulation of poly(A)+ RNAs in the nucleus. Global analysis revealed CHERP regulates alternative mRNA splicing (particularly intron retention) through interaction with U2 snRNPs and U2 snRNP-related proteins. Intron retention frequency was influenced by 5'/3' splice site strength, branch point, GC content, and intron length. CHERP depletion also induced cell cycle defects at M phase and abnormal cell division.\",\n      \"method\": \"siRNA knockdown, RNA-FISH for poly(A)+ RNA localization, RNA-seq alternative splicing analysis, cell cycle analysis (FACS), live-cell imaging\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — nuclear RNA accumulation and global splicing changes by RNA-seq with KD, cell cycle phenotype, single lab, multiple methods\",\n      \"pmids\": [\"35269695\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CHERP is a nuclear spliceosomal protein that forms a tight complex with SR140 (U2SURP) and SPF45 (RBM17) within the U2 snRNP, where it regulates alternative splicing—especially repression of short exons at suboptimal 3' splice sites and intron retention—of targets including FOXM1, SPDL1, UPF3A, and IP3R1 pre-mRNAs; disruption of CHERP causes nuclear poly(A)+ RNA accumulation, G2/M arrest, and impaired cell proliferation, while the earlier model of CHERP as a direct ER/cytoplasmic regulator of IP3R and RyR1 Ca2+ channels has been substantially re-evaluated in favour of these splicing-based mechanisms.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CHERP is a nuclear spliceosomal protein that regulates alternative pre-mRNA splicing as part of the U2 snRNP machinery [#3, #8]. It localizes to the nucleus and nuclear speckles, associates with the U2 snRNA spliceosomal complex, and binds a phosphorylated form of RNA polymerase II [#3, #4]. CHERP forms a tight, mutually stabilizing complex with SR140 (U2SURP) and SPF45 (RBM17) that represses short alternative exons flanked by suboptimal 3' splice sites and controls intron retention, acting on targets including FOXM1, SPDL1, UPF3A, and IP3R1 pre-mRNAs [#4, #5, #6, #7]. Through these splicing events, CHERP is required for cell-cycle progression: its depletion drives nuclear accumulation of poly(A)+ RNA, G2/M arrest, DNA damage, and impaired proliferation, with the growth defect partially rescued by restoring UPF3A or by mimicking the FOXM1/SPDL1 splicing outcomes [#6, #7, #8]. An earlier model in which CHERP acted as a direct ER/cytoplasmic regulator of IP3R and RyR1 Ca2+ channels [#0, #2] was re-evaluated, with its effects on Ca2+ signaling and growth reinterpreted as indirect consequences of altered spliceosomal function [#3]. CHERP additionally binds ALG-2 in a Ca2+-dependent manner, providing a candidate link between Ca2+ signaling and splicing regulation [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established CHERP as a functional protein affecting Ca2+ homeostasis and proliferation, the founding observation that defined the gene's phenotypic footprint.\",\n      \"evidence\": \"Antisense knockdown in HEL cells with Ca2+ mobilization, DNA synthesis assays, and IP3R co-localization by immunofluorescence\",\n      \"pmids\": [\"10794731\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Co-localization does not demonstrate direct molecular interaction with IP3R\",\n        \"Cannot distinguish direct Ca2+ channel regulation from indirect effects\",\n        \"Mechanism of the growth arrest unresolved\"\n      ]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Extended the Ca2+/proliferation phenotype to a second cell type and placed CHERP upstream of or parallel to ER Ca2+ release rather than altering IP3R levels.\",\n      \"evidence\": \"Antisense knockdown in Jurkat cells with fura-2 imaging, NFAT translocation, and cyclin D1 Western blotting\",\n      \"pmids\": [\"12656674\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Did not identify the molecular target through which CHERP affects Ca2+ release\",\n        \"Indirect versus direct mechanism not resolved\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Reported a physical and functional link to the RyR1 Ca2+ channel, reinforcing the ER/SR-centric model of CHERP function.\",\n      \"evidence\": \"Co-purification of CHERP with a soluble cytosolic RyR1 fragment, SR co-localization, and siRNA + Ca2+ release assay in HEK-293 cells\",\n      \"pmids\": [\"21454501\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Co-purification with an overexpressed fragment may not reflect endogenous direct binding\",\n        \"Did not exclude indirect contribution via splicing later proposed for CHERP\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Reassigned CHERP from a cytoplasmic Ca2+ channel regulator to a nuclear spliceosome-associated protein, redirecting the entire mechanistic interpretation of the earlier growth phenotypes.\",\n      \"evidence\": \"Subcellular fractionation, nuclear immunofluorescence, and co-IP with U2 snRNP components plus proliferation assays\",\n      \"pmids\": [\"23148228\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Did not define which splicing targets account for the growth defect\",\n        \"Did not formally exclude a residual direct Ca2+ channel role\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Provided direct molecular evidence for nuclear splicing function and linked CHERP back to Ca2+ signaling via ALG-2, showing it binds pre-mRNA and regulates IP3R1 splicing.\",\n      \"evidence\": \"Co-IP with phospho-RNA Pol II and ALG-2, nuclear speckle imaging, live-cell recruitment imaging, RNA-IP, and siRNA + RT-PCR splicing assay in HT1080 cells\",\n      \"pmids\": [\"24078636\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Functional consequence of Ca2+-dependent ALG-2 recruitment on splicing output not established\",\n        \"Genome-wide target spectrum not yet defined\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined a mutually stabilizing module of CHERP with RBM17 and U2SURP and showed it controls splicing of RNA-processing factors, establishing CHERP as part of a coordinated splicing regulatory complex.\",\n      \"evidence\": \"Reciprocal co-IP, siRNA knockdown, and RNA-seq splicing analysis across mouse and human cells with Western blotting\",\n      \"pmids\": [\"30332651\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Stoichiometry and architecture of the CHERP-RBM17-U2SURP complex not resolved\",\n        \"Direct versus indirect splicing targets not fully separated\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified UPF3A exon 4 as a specific regulated target of the CHERP-SR140 complex and tied the proliferation defect causally to splicing output via rescue.\",\n      \"evidence\": \"Co-IP, siRNA knockdown, RT-PCR splicing assay, UPF3A overexpression rescue, and an in vivo mouse tumor model\",\n      \"pmids\": [\"30977118\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Rescue was only partial, implying additional downstream targets\",\n        \"Mechanism linking the splicing defect to DNA breaks not detailed\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined the catalytic logic of the SPF45-SR140-CHERP complex as repression of short exons at suboptimal 3' splice sites and connected it to specific cell-cycle gene splicing controlling G2/M progression.\",\n      \"evidence\": \"Co-IP, siRNA knockdown of all three factors, RNA-seq, minigene assays, cell cycle analysis, and splicing-isoform epistasis in HeLa cells\",\n      \"pmids\": [\"34544891\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for recognition of suboptimal 3' splice sites unknown\",\n        \"Individual contribution of CHERP within the trimeric complex not isolated\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated that CHERP is required for nuclear mRNA export competence and controls intron retention through U2 snRNP interaction, broadening its role beyond exon repression.\",\n      \"evidence\": \"siRNA knockdown, RNA-FISH for poly(A)+ RNA, RNA-seq intron-retention analysis, FACS cell cycle, and live-cell imaging in U2OS cells\",\n      \"pmids\": [\"35269695\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether poly(A)+ RNA accumulation is a direct splicing consequence or a secondary effect not resolved\",\n        \"Sequence determinants of intron retention correlative rather than mechanistic\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How Ca2+/ALG-2 signaling functionally tunes CHERP-dependent splicing, and the structural basis by which the CHERP-SR140-SPF45 complex recognizes suboptimal splice sites, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structural model of the CHERP-containing U2 snRNP subcomplex\",\n        \"Functional output of Ca2+-dependent ALG-2 recruitment to CHERP speckles undefined\",\n        \"Complete catalogue of physiologically critical splicing targets not established\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [4, 7, 8]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [4, 5, 7, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [3, 4, 5, 7, 8]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [6, 7, 8]}\n    ],\n    \"complexes\": [\n      \"U2 snRNP\",\n      \"CHERP-SR140(U2SURP)-SPF45(RBM17) splicing complex\"\n    ],\n    \"partners\": [\n      \"U2SURP\",\n      \"RBM17\",\n      \"ALG-2\",\n      \"RNA polymerase II\",\n      \"RYR1\",\n      \"ITPR1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":6,"faith_total":6,"faith_pct":100.0}}