{"gene":"SRSF4","run_date":"2026-06-10T07:46:41","timeline":{"discoveries":[{"year":1993,"finding":"SRp75 (SRSF4) contains an N-terminal RNA recognition motif (RRM), a glycine-rich region, an internal region homologous to the RRM, and a long C-terminal SR domain; it can complement a splicing-deficient S100 extract, restoring pre-mRNA splicing activity. Dephosphorylation of SRp75 causes a mobility shift proportional to SR domain length, indicating serines in the SR domain are phosphorylated.","method":"cDNA cloning, S100 complementation splicing assay, dephosphorylation/mobility-shift analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro splicing reconstitution assay with biochemical dephosphorylation analysis; foundational paper replicated across the SR protein field","pmids":["8321209"],"is_preprint":false},{"year":2003,"finding":"SRp75 (SRSF4) interacts with Pinin (Pnn/DRS/memA) via Pnn's polyserine/RS motif, as well as with SRm300 and SRrp130, forming a multiprotein complex in the nucleus of corneal epithelial cells that co-localizes with components of the pre-mRNA splicing machinery in nuclear speckles.","method":"Yeast two-hybrid, co-immunoprecipitation, co-transfection with immunostaining/immunoblotting","journal":"Investigative ophthalmology & visual science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP plus yeast two-hybrid, single lab, two orthogonal methods","pmids":["14578391"],"is_preprint":false},{"year":2008,"finding":"CLK family kinases specifically hyperphosphorylate SRp75 (SRSF4) among SR kinases tested (SRPK, CLK, PRP4, DYRK). This phosphorylation alters SRp75 nuclear distribution and, together with SRp75 overexpression, promotes selection of the 12S 5' splice site in Adenovirus E1A pre-mRNA.","method":"Comparative SR kinase assay with phospho-SR antibody (mAb1H4), CLK inhibitor TG003, live-cell imaging of mRFP-SRp75, co-transfection with HA-SRp75 and kinase constructs, alternative splicing reporter","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (pharmacological inhibition, live imaging, co-transfection assay) in a single lab","pmids":["18298798"],"is_preprint":false},{"year":2010,"finding":"SRp75 (SRSF4) associates with hundreds of distinct endogenous mRNAs in cycling and neurally differentiating P19 cells; these mRNP associations are functionally relevant, as SRp75 knockdown causes up- or down-regulation of specific target transcripts, rescued by GFP-tagged SRp75.","method":"GFP-tagged SR protein immunopurification of mRNPs, genome-wide mRNA target identification, siRNA knockdown, rescue by GFP-SR transgene","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — immunopurification of endogenous mRNPs combined with genome-wide analysis, knockdown, and functional rescue; multiple orthogonal methods","pmids":["20639886"],"is_preprint":false},{"year":2010,"finding":"SRp75 (SRSF4) inhibits splicing from the 5' splice site of HIV-1 exon 3, causing accumulation of the partially unspliced 13a7 vpr mRNA. This is distinct from SRp40, which promotes splicing from exon 3 to exon 4 (tat mRNA production).","method":"Transfection-based splicing assay with SR protein expression constructs, mRNA quantification","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — functional splicing assay in cell-based system, single lab, replicated across multiple HIV-1 splice sites","pmids":["20685659"],"is_preprint":false},{"year":2011,"finding":"SRp75 (SRSF4) binds the proximal downstream intron of tau exon 10 at the FTDP-17 hotspot region and inhibits exon 10 splicing. SRp75 physically interacts with hnRNPG and hnRNPE2 (the latter activates exon 10 inclusion), forming a regulatory complex at this splice site.","method":"Co-transfection splicing assay, co-immunoprecipitation, RNAi knockdown","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional splicing assay plus RNAi, single lab, multiple methods","pmids":["21723381"],"is_preprint":false},{"year":2015,"finding":"SRSF4 is required for the majority of cisplatin-induced alternative splicing changes and for cisplatin-induced cell death in breast carcinoma cells; this process requires class I PI3K (p110β) but not ATM, ATR, or p53. siRNA depletion of SRSF4 (but not SRSF6) abrogated both the splicing alterations and apoptosis.","method":"siRNA knockdown, next-generation sequencing of transcriptome, RT-PCR, FACS apoptosis analysis, pathway-specific inhibitors and knockout cells","journal":"BMC cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockdown with transcriptome-wide readout and epistasis with PI3K pathway; single lab, multiple orthogonal methods","pmids":["25884497"],"is_preprint":false},{"year":2015,"finding":"Androgen receptor (AR) directly binds an androgen-responsive element (ARE) in the Srsf4 promoter in mouse Sertoli cells, repressing Srsf4 expression; testosterone treatment down-regulates Srsf4 in the Sertoli-cell line TM4, and SRSF4 is localized to Sertoli cell nuclei.","method":"ChIP/AR binding to ARE, promoter assay, testosterone treatment of TM4 cells, immunofluorescence localization, S-AR knockout mouse comparison","journal":"Molecular reproduction and development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct AR binding to promoter ARE with expression readout and in vivo knockout comparison; single lab","pmids":["26308373"],"is_preprint":false},{"year":2018,"finding":"SRSF4 promotes inclusion of exon 6 in Fas pre-mRNA by binding a novel exonic splicing enhancer on exon 6; a weaker 5' splice site abrogates this effect. Reduced SRSF4 promotes exon 6 exclusion (soluble anti-apoptotic isoform), while increased SRSF4 promotes exon 6 inclusion (membrane-bound pro-apoptotic isoform).","method":"SRSF4 overexpression and siRNA knockdown, 5' splice-site mutation analysis, RNA binding/functional enhancer mapping","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with splice-site mutagenesis and physical binding to enhancer; single lab","pmids":["30376989"],"is_preprint":false},{"year":2020,"finding":"SRSF4 nuclear localization is mediated by multiple nuclear localization signals (NLSs): the RS-rich domain confers nuclear localization activity, but not all RS-rich sub-regions are sufficient; additional classical-type NLS-like basic amino acid stretches were identified, indicating SRSF4 uses at least two distinct nuclear import pathways.","method":"Pyruvate kinase (PK) fusion nuclear localization assay with serial deletions and domain mapping","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct domain-mapping by PK fusion localization assay; single lab, systematic deletion analysis","pmids":["32050040"],"is_preprint":false},{"year":2021,"finding":"SRSF4 regulates ventricular hypertrophy through an axis involving GAS5 lncRNA and the glucocorticoid receptor.","method":"Not fully described in available abstract","journal":"Circulation research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — abstract is figure-only with no experimental detail extractable; single citation of a pathway axis without method description","pmids":["34333993"],"is_preprint":false},{"year":2023,"finding":"SRSF4 promotes temozolomide resistance in glioma by positively regulating MDC1 expression, thereby accelerating DNA double-strand break repair.","method":"Colony formation assay, flow cytometry, western blot, immunofluorescence, bioinformatic analysis, orthotopic xenograft model with SRSF4 manipulation","journal":"Journal of molecular neuroscience : MN","confidence":"Low","confidence_rationale":"Tier 3 / Weak — functional assays with phenotypic readout and MDC1 association, but molecular mechanism linking SRSF4 to MDC1 regulation is not biochemically defined in the abstract","pmids":["37014544"],"is_preprint":false},{"year":2024,"finding":"A missense variant of SRSF4 (p.R235W) causes reduced SRSF4 protein expression, leading to mitochondrial dysfunction and altered energetic metabolism in patient lymphocytes; transfection with wild-type SRSF4 restored mitochondrial function. The mitochondrial defect was associated with low mTOR phosphorylation and imbalance of CLUH, DRP1, and OPA1.","method":"Whole genome sequencing, primary patient cell analysis (lymphocytes, EBV-immortalized lymphoblasts), western blot, mitochondrial function assays, wtSRSF4 rescue transfection","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient variant with functional rescue by wild-type transfection; multiple orthogonal methods; single lab case study","pmids":["38396760"],"is_preprint":false}],"current_model":"SRSF4 (SRp75) is a phosphorylated SR-family splicing factor that, through its RRM domains and RS domain (which harbors multiple nuclear localization signals and is hyperphosphorylated specifically by CLK kinases), associates with distinct endogenous mRNPs and directly regulates both constitutive and alternative pre-mRNA splicing—promoting or inhibiting specific exon inclusion events (e.g., Fas exon 6, tau exon 10, HIV-1 exon 3, E1A 12S)—by binding exonic/intronic enhancers and interacting with partners such as hnRNPG, hnRNPE2, and Pinin; beyond splicing, SRSF4 also influences cisplatin-induced apoptosis (via a PI3K-dependent pathway), DNA damage repair (via MDC1), mitochondrial function, and cardiac hypertrophy (via a GAS5/glucocorticoid receptor axis), and its expression in Sertoli cells is directly repressed by androgen receptor binding to the SRSF4 promoter."},"narrative":{"mechanistic_narrative":"SRSF4 (SRp75) is a phosphorylated SR-family pre-mRNA splicing factor built from an N-terminal RRM, a glycine-rich region, an internal RRM-homologous region, and a long C-terminal SR domain whose serines are phosphorylated, and it restores splicing activity to S100-depleted extracts [PMID:8321209]. Nuclear import of SRSF4 depends on the RS-rich domain together with additional basic NLS-like stretches, indicating use of at least two distinct import pathways [PMID:32050040], and the SR domain is selectively hyperphosphorylated by CLK-family kinases, which reshapes its nuclear distribution and modulates 5' splice-site choice [PMID:18298798]. Within the nucleus, SRSF4 associates with hundreds of distinct endogenous mRNPs and regulates their levels [PMID:20639886], operating as both a positive and negative regulator of alternative splicing depending on context: it inhibits tau exon 10 inclusion by binding the downstream intronic FTDP-17 hotspot while interacting with hnRNPG and hnRNPE2 [PMID:21723381], inhibits splicing at HIV-1 exon 3 [PMID:20685659], and promotes Fas exon 6 inclusion by binding an exonic splicing enhancer, thereby biasing toward the pro-apoptotic membrane-bound isoform [PMID:30376989]. Consistent with a role in cell-fate decisions, SRSF4 is required for most cisplatin-induced alternative splicing changes and for cisplatin-induced apoptosis via a class I PI3K (p110β)-dependent, ATM/ATR/p53-independent pathway [PMID:25884497]. A patient-derived p.R235W missense variant that lowers SRSF4 protein causes mitochondrial dysfunction rescuable by wild-type SRSF4, linking the factor to energetic metabolism [PMID:38396760].","teleology":[{"year":1993,"claim":"Established SRSF4 as a bona fide SR splicing factor by defining its modular RRM–glycine–RRM-homology–SR domain architecture and showing it can functionally complement splicing activity.","evidence":"cDNA cloning with S100 complementation splicing assay and dephosphorylation/mobility-shift analysis","pmids":["8321209"],"confidence":"High","gaps":["Did not identify endogenous target transcripts","Functional role of SR-domain phosphorylation not established"]},{"year":2003,"claim":"Placed SRSF4 within a defined nuclear-speckle protein complex by identifying physical partners, addressing how it is organized with the splicing machinery.","evidence":"Yeast two-hybrid and reciprocal co-immunoprecipitation in corneal epithelial cells","pmids":["14578391"],"confidence":"Medium","gaps":["Functional consequence of Pinin interaction on splicing not tested","Single cell type"]},{"year":2008,"claim":"Identified CLK kinases as the specific regulators of SRSF4 hyperphosphorylation and linked this modification to nuclear distribution and 5' splice-site selection.","evidence":"Comparative SR kinase assay, CLK inhibitor TG003, live-cell imaging, and E1A alternative splicing reporter","pmids":["18298798"],"confidence":"Medium","gaps":["Phosphosites on SRSF4 not mapped","Effect generality across targets beyond E1A not shown"]},{"year":2010,"claim":"Demonstrated genome-wide that SRSF4 binds hundreds of endogenous mRNPs and that these associations functionally control transcript levels, establishing its role beyond single reporters.","evidence":"GFP-SR immunopurification of mRNPs with genome-wide target ID, siRNA knockdown, and GFP-SR rescue in P19 cells","pmids":["20639886"],"confidence":"High","gaps":["Direct binding sites not resolved at nucleotide level","Splicing vs. stability contributions not separated"]},{"year":2010,"claim":"Showed SRSF4 acts as a splicing inhibitor at a viral splice site, distinguishing its activity from related SR proteins.","evidence":"Transfection-based HIV-1 splicing assay with SR protein expression constructs","pmids":["20685659"],"confidence":"Medium","gaps":["RNA element bound by SRSF4 not mapped","Single viral context"]},{"year":2011,"claim":"Defined SRSF4 as a context-dependent splicing repressor at a disease-relevant exon and identified hnRNP cofactors, clarifying its regulatory mechanism at tau exon 10.","evidence":"Co-transfection splicing assay, co-IP, and RNAi knockdown","pmids":["21723381"],"confidence":"Medium","gaps":["Stoichiometry/order of complex assembly unknown","In vivo relevance to tauopathy not tested"]},{"year":2015,"claim":"Connected SRSF4 to a stress-response apoptotic program, showing it drives cisplatin-induced splicing changes and cell death through PI3K rather than canonical DNA-damage kinases.","evidence":"siRNA knockdown with transcriptome-wide sequencing, RT-PCR, FACS apoptosis, and pathway-specific inhibitors/knockout cells","pmids":["25884497"],"confidence":"Medium","gaps":["Direct SRSF4 targets mediating apoptosis not isolated","Mechanistic link to PI3K not biochemically defined"]},{"year":2015,"claim":"Identified upstream transcriptional control of SRSF4 by androgen receptor, showing its expression is hormonally repressed in Sertoli cells.","evidence":"AR ChIP/ARE binding, promoter assay, testosterone treatment of TM4 cells, immunofluorescence, and S-AR knockout comparison","pmids":["26308373"],"confidence":"Medium","gaps":["Functional splicing consequence in Sertoli cells not defined","Single regulatory element"]},{"year":2018,"claim":"Resolved SRSF4 as a positive splicing regulator that binds an exonic enhancer to control an apoptotic isoform switch, demonstrating bidirectional splicing activity.","evidence":"SRSF4 overexpression/knockdown, 5' splice-site mutagenesis, and enhancer mapping on Fas pre-mRNA","pmids":["30376989"],"confidence":"Medium","gaps":["Enhancer sequence consensus not generalized","Endogenous physiological context not tested"]},{"year":2020,"claim":"Dissected the determinants of SRSF4 nuclear import, showing reliance on the RS domain plus additional basic NLS motifs and multiple import routes.","evidence":"Pyruvate kinase fusion nuclear localization assay with serial deletion/domain mapping","pmids":["32050040"],"confidence":"Medium","gaps":["Import receptors not identified","Link between import route and function not established"]},{"year":2021,"claim":"Implicated SRSF4 in cardiac hypertrophy through a GAS5 lncRNA/glucocorticoid receptor axis.","evidence":"Not fully described in available abstract","pmids":["34333993"],"confidence":"Low","gaps":["Experimental detail not extractable from abstract","Direct molecular role of SRSF4 in the axis undefined"]},{"year":2023,"claim":"Linked SRSF4 to DNA double-strand break repair and chemoresistance via positive regulation of MDC1 in glioma.","evidence":"Colony formation, flow cytometry, western blot, immunofluorescence, and orthotopic xenograft with SRSF4 manipulation","pmids":["37014544"],"confidence":"Low","gaps":["Biochemical mechanism linking SRSF4 to MDC1 not defined","Whether effect is splicing-mediated unknown"]},{"year":2024,"claim":"Provided human genetic and functional evidence tying SRSF4 dosage to mitochondrial function via a rescuable missense variant.","evidence":"Whole genome sequencing, patient lymphocyte/lymphoblast analysis, mitochondrial function assays, and wild-type SRSF4 rescue transfection","pmids":["38396760"],"confidence":"Medium","gaps":["Splicing targets mediating mitochondrial phenotype not identified","Single-patient case study"]},{"year":null,"claim":"How SRSF4's diverse non-splicing phenotypes (apoptosis, DNA repair, mitochondrial and cardiac function) connect to its core RNA-binding/splicing activity remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unifying set of direct RNA targets links the disparate phenotypes","Phosphorylation-state-specific target maps lacking","Structural basis of enhancer vs. silencer recognition unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,3,5,8]}],"localization":[{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[1,9]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[7,9]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,3,8]}],"complexes":[],"partners":["PNN","SRRM2","HNRNPG","HNRNPE2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q08170","full_name":"Serine/arginine-rich splicing factor 4","aliases":["Pre-mRNA-splicing factor SRP75","SRP001LB","Splicing factor, arginine/serine-rich 4"],"length_aa":494,"mass_kda":56.7,"function":"Plays a role in alternative splice site selection during pre-mRNA splicing. Represses the splicing of MAPT/Tau exon 10","subcellular_location":"Nucleus speckle","url":"https://www.uniprot.org/uniprotkb/Q08170/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SRSF4","classification":"Not Classified","n_dependent_lines":33,"n_total_lines":1208,"dependency_fraction":0.027317880794701987},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SNRPA","stoichiometry":0.2},{"gene":"SNRPB","stoichiometry":0.2},{"gene":"SNRPC","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/SRSF4","total_profiled":1310},"omim":[{"mim_id":"617031","title":"PRE-mRNA-PROCESSING FACTOR 38A; PRPF38A","url":"https://www.omim.org/entry/617031"},{"mim_id":"616653","title":"PNN-INTERACTING SERINE/ARGININE-RICH PROTEIN; PNISR","url":"https://www.omim.org/entry/616653"},{"mim_id":"606032","title":"SERINE/ARGININE REPETITIVE MATRIX PROTEIN 2; SRRM2","url":"https://www.omim.org/entry/606032"},{"mim_id":"603154","title":"PININ; PNN","url":"https://www.omim.org/entry/603154"},{"mim_id":"601940","title":"SPLICING FACTOR, SERINE/ARGININE-RICH, 4; SRSF4","url":"https://www.omim.org/entry/601940"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nuclear speckles","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SRSF4"},"hgnc":{"alias_symbol":["SRP75"],"prev_symbol":["SFRS4"]},"alphafold":{"accession":"Q08170","domains":[{"cath_id":"3.30.70.330","chopping":"4-70","consensus_level":"high","plddt":85.6407,"start":4,"end":70},{"cath_id":"3.30.70.330","chopping":"106-175","consensus_level":"high","plddt":85.2897,"start":106,"end":175}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q08170","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q08170-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q08170-F1-predicted_aligned_error_v6.png","plddt_mean":56.16},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SRSF4","jax_strain_url":"https://www.jax.org/strain/search?query=SRSF4"},"sequence":{"accession":"Q08170","fasta_url":"https://rest.uniprot.org/uniprotkb/Q08170.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q08170/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q08170"}},"corpus_meta":[{"pmid":"8321209","id":"PMC_8321209","title":"Human SR proteins and isolation of a cDNA encoding SRp75.","date":"1993","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/8321209","citation_count":106,"is_preprint":false},{"pmid":"14578391","id":"PMC_14578391","title":"Pinin/DRS/memA interacts with SRp75, SRm300 and SRrp130 in corneal epithelial cells.","date":"2003","source":"Investigative ophthalmology & visual science","url":"https://pubmed.ncbi.nlm.nih.gov/14578391","citation_count":54,"is_preprint":false},{"pmid":"20639886","id":"PMC_20639886","title":"Global analysis reveals SRp20- and SRp75-specific mRNPs in cycling and neural cells.","date":"2010","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/20639886","citation_count":53,"is_preprint":false},{"pmid":"18298798","id":"PMC_18298798","title":"Combination of Clk family kinase and SRp75 modulates alternative splicing of Adenovirus E1A.","date":"2008","source":"Genes to cells : devoted to molecular & cellular mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/18298798","citation_count":41,"is_preprint":false},{"pmid":"25884497","id":"PMC_25884497","title":"Role of the splicing factor SRSF4 in cisplatin-induced modifications of pre-mRNA splicing and apoptosis.","date":"2015","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/25884497","citation_count":41,"is_preprint":false},{"pmid":"21723381","id":"PMC_21723381","title":"An SRp75/hnRNPG complex interacting with hnRNPE2 regulates the 5' splice site of tau exon 10, whose misregulation causes frontotemporal dementia.","date":"2011","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/21723381","citation_count":35,"is_preprint":false},{"pmid":"20685659","id":"PMC_20685659","title":"Serine- and arginine-rich proteins 55 and 75 (SRp55 and SRp75) induce production of HIV-1 vpr mRNA by inhibiting the 5'-splice site of exon 3.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20685659","citation_count":22,"is_preprint":false},{"pmid":"34333993","id":"PMC_34333993","title":"The SRSF4-GAS5-Glucocorticoid Receptor Axis Regulates Ventricular Hypertrophy.","date":"2021","source":"Circulation research","url":"https://pubmed.ncbi.nlm.nih.gov/34333993","citation_count":20,"is_preprint":false},{"pmid":"30376989","id":"PMC_30376989","title":"Binding of SRSF4 to a novel enhancer modulates splicing of exon 6 of Fas pre-mRNA.","date":"2018","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/30376989","citation_count":13,"is_preprint":false},{"pmid":"39973750","id":"PMC_39973750","title":"SRSF4-Associated ca-circFOXP1 Regulates Hypoxia-Induced PASMC Proliferation by the Formation of R Loop With Host Gene.","date":"2025","source":"Arteriosclerosis, thrombosis, and vascular biology","url":"https://pubmed.ncbi.nlm.nih.gov/39973750","citation_count":6,"is_preprint":false},{"pmid":"32050040","id":"PMC_32050040","title":"Multiple nuclear localization sequences in SRSF4 protein.","date":"2020","source":"Genes to cells : devoted to molecular & cellular mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/32050040","citation_count":4,"is_preprint":false},{"pmid":"37014544","id":"PMC_37014544","title":"SRSF4 Confers Temozolomide Resistance of Glioma via Accelerating Double Strand Break Repair.","date":"2023","source":"Journal of molecular neuroscience : MN","url":"https://pubmed.ncbi.nlm.nih.gov/37014544","citation_count":4,"is_preprint":false},{"pmid":"26308373","id":"PMC_26308373","title":"Androgen receptor binding to an androgen-responsive element in the promoter of the Srsf4 gene inhibits its expression in mouse Sertoli cells.","date":"2015","source":"Molecular reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/26308373","citation_count":4,"is_preprint":false},{"pmid":"38396760","id":"PMC_38396760","title":"Impaired Mitochondrial Function and Marrow Failure in Patients Carrying a Variant of the SRSF4 Gene.","date":"2024","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/38396760","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.11.08.24316999","title":"Gene-diet interaction analysis in UK Biobank identified genetic loci that modify the association between fish oil supplementation and the incidence of dementia","date":"2024-11-10","source":"bioRxiv","url":"https://doi.org/10.1101/2024.11.08.24316999","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8949,"output_tokens":3308,"usd":0.038233,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10772,"output_tokens":3678,"usd":0.072905,"stage2_stop_reason":"end_turn"},"total_usd":0.111138,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1993,\n      \"finding\": \"SRp75 (SRSF4) contains an N-terminal RNA recognition motif (RRM), a glycine-rich region, an internal region homologous to the RRM, and a long C-terminal SR domain; it can complement a splicing-deficient S100 extract, restoring pre-mRNA splicing activity. Dephosphorylation of SRp75 causes a mobility shift proportional to SR domain length, indicating serines in the SR domain are phosphorylated.\",\n      \"method\": \"cDNA cloning, S100 complementation splicing assay, dephosphorylation/mobility-shift analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro splicing reconstitution assay with biochemical dephosphorylation analysis; foundational paper replicated across the SR protein field\",\n      \"pmids\": [\"8321209\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"SRp75 (SRSF4) interacts with Pinin (Pnn/DRS/memA) via Pnn's polyserine/RS motif, as well as with SRm300 and SRrp130, forming a multiprotein complex in the nucleus of corneal epithelial cells that co-localizes with components of the pre-mRNA splicing machinery in nuclear speckles.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, co-transfection with immunostaining/immunoblotting\",\n      \"journal\": \"Investigative ophthalmology & visual science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP plus yeast two-hybrid, single lab, two orthogonal methods\",\n      \"pmids\": [\"14578391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CLK family kinases specifically hyperphosphorylate SRp75 (SRSF4) among SR kinases tested (SRPK, CLK, PRP4, DYRK). This phosphorylation alters SRp75 nuclear distribution and, together with SRp75 overexpression, promotes selection of the 12S 5' splice site in Adenovirus E1A pre-mRNA.\",\n      \"method\": \"Comparative SR kinase assay with phospho-SR antibody (mAb1H4), CLK inhibitor TG003, live-cell imaging of mRFP-SRp75, co-transfection with HA-SRp75 and kinase constructs, alternative splicing reporter\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (pharmacological inhibition, live imaging, co-transfection assay) in a single lab\",\n      \"pmids\": [\"18298798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"SRp75 (SRSF4) associates with hundreds of distinct endogenous mRNAs in cycling and neurally differentiating P19 cells; these mRNP associations are functionally relevant, as SRp75 knockdown causes up- or down-regulation of specific target transcripts, rescued by GFP-tagged SRp75.\",\n      \"method\": \"GFP-tagged SR protein immunopurification of mRNPs, genome-wide mRNA target identification, siRNA knockdown, rescue by GFP-SR transgene\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — immunopurification of endogenous mRNPs combined with genome-wide analysis, knockdown, and functional rescue; multiple orthogonal methods\",\n      \"pmids\": [\"20639886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"SRp75 (SRSF4) inhibits splicing from the 5' splice site of HIV-1 exon 3, causing accumulation of the partially unspliced 13a7 vpr mRNA. This is distinct from SRp40, which promotes splicing from exon 3 to exon 4 (tat mRNA production).\",\n      \"method\": \"Transfection-based splicing assay with SR protein expression constructs, mRNA quantification\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — functional splicing assay in cell-based system, single lab, replicated across multiple HIV-1 splice sites\",\n      \"pmids\": [\"20685659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SRp75 (SRSF4) binds the proximal downstream intron of tau exon 10 at the FTDP-17 hotspot region and inhibits exon 10 splicing. SRp75 physically interacts with hnRNPG and hnRNPE2 (the latter activates exon 10 inclusion), forming a regulatory complex at this splice site.\",\n      \"method\": \"Co-transfection splicing assay, co-immunoprecipitation, RNAi knockdown\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional splicing assay plus RNAi, single lab, multiple methods\",\n      \"pmids\": [\"21723381\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SRSF4 is required for the majority of cisplatin-induced alternative splicing changes and for cisplatin-induced cell death in breast carcinoma cells; this process requires class I PI3K (p110β) but not ATM, ATR, or p53. siRNA depletion of SRSF4 (but not SRSF6) abrogated both the splicing alterations and apoptosis.\",\n      \"method\": \"siRNA knockdown, next-generation sequencing of transcriptome, RT-PCR, FACS apoptosis analysis, pathway-specific inhibitors and knockout cells\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockdown with transcriptome-wide readout and epistasis with PI3K pathway; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"25884497\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Androgen receptor (AR) directly binds an androgen-responsive element (ARE) in the Srsf4 promoter in mouse Sertoli cells, repressing Srsf4 expression; testosterone treatment down-regulates Srsf4 in the Sertoli-cell line TM4, and SRSF4 is localized to Sertoli cell nuclei.\",\n      \"method\": \"ChIP/AR binding to ARE, promoter assay, testosterone treatment of TM4 cells, immunofluorescence localization, S-AR knockout mouse comparison\",\n      \"journal\": \"Molecular reproduction and development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct AR binding to promoter ARE with expression readout and in vivo knockout comparison; single lab\",\n      \"pmids\": [\"26308373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SRSF4 promotes inclusion of exon 6 in Fas pre-mRNA by binding a novel exonic splicing enhancer on exon 6; a weaker 5' splice site abrogates this effect. Reduced SRSF4 promotes exon 6 exclusion (soluble anti-apoptotic isoform), while increased SRSF4 promotes exon 6 inclusion (membrane-bound pro-apoptotic isoform).\",\n      \"method\": \"SRSF4 overexpression and siRNA knockdown, 5' splice-site mutation analysis, RNA binding/functional enhancer mapping\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with splice-site mutagenesis and physical binding to enhancer; single lab\",\n      \"pmids\": [\"30376989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SRSF4 nuclear localization is mediated by multiple nuclear localization signals (NLSs): the RS-rich domain confers nuclear localization activity, but not all RS-rich sub-regions are sufficient; additional classical-type NLS-like basic amino acid stretches were identified, indicating SRSF4 uses at least two distinct nuclear import pathways.\",\n      \"method\": \"Pyruvate kinase (PK) fusion nuclear localization assay with serial deletions and domain mapping\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct domain-mapping by PK fusion localization assay; single lab, systematic deletion analysis\",\n      \"pmids\": [\"32050040\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SRSF4 regulates ventricular hypertrophy through an axis involving GAS5 lncRNA and the glucocorticoid receptor.\",\n      \"method\": \"Not fully described in available abstract\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — abstract is figure-only with no experimental detail extractable; single citation of a pathway axis without method description\",\n      \"pmids\": [\"34333993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SRSF4 promotes temozolomide resistance in glioma by positively regulating MDC1 expression, thereby accelerating DNA double-strand break repair.\",\n      \"method\": \"Colony formation assay, flow cytometry, western blot, immunofluorescence, bioinformatic analysis, orthotopic xenograft model with SRSF4 manipulation\",\n      \"journal\": \"Journal of molecular neuroscience : MN\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — functional assays with phenotypic readout and MDC1 association, but molecular mechanism linking SRSF4 to MDC1 regulation is not biochemically defined in the abstract\",\n      \"pmids\": [\"37014544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A missense variant of SRSF4 (p.R235W) causes reduced SRSF4 protein expression, leading to mitochondrial dysfunction and altered energetic metabolism in patient lymphocytes; transfection with wild-type SRSF4 restored mitochondrial function. The mitochondrial defect was associated with low mTOR phosphorylation and imbalance of CLUH, DRP1, and OPA1.\",\n      \"method\": \"Whole genome sequencing, primary patient cell analysis (lymphocytes, EBV-immortalized lymphoblasts), western blot, mitochondrial function assays, wtSRSF4 rescue transfection\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient variant with functional rescue by wild-type transfection; multiple orthogonal methods; single lab case study\",\n      \"pmids\": [\"38396760\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SRSF4 (SRp75) is a phosphorylated SR-family splicing factor that, through its RRM domains and RS domain (which harbors multiple nuclear localization signals and is hyperphosphorylated specifically by CLK kinases), associates with distinct endogenous mRNPs and directly regulates both constitutive and alternative pre-mRNA splicing—promoting or inhibiting specific exon inclusion events (e.g., Fas exon 6, tau exon 10, HIV-1 exon 3, E1A 12S)—by binding exonic/intronic enhancers and interacting with partners such as hnRNPG, hnRNPE2, and Pinin; beyond splicing, SRSF4 also influences cisplatin-induced apoptosis (via a PI3K-dependent pathway), DNA damage repair (via MDC1), mitochondrial function, and cardiac hypertrophy (via a GAS5/glucocorticoid receptor axis), and its expression in Sertoli cells is directly repressed by androgen receptor binding to the SRSF4 promoter.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"SRSF4 (SRp75) is a phosphorylated SR-family pre-mRNA splicing factor built from an N-terminal RRM, a glycine-rich region, an internal RRM-homologous region, and a long C-terminal SR domain whose serines are phosphorylated, and it restores splicing activity to S100-depleted extracts [#0]. Nuclear import of SRSF4 depends on the RS-rich domain together with additional basic NLS-like stretches, indicating use of at least two distinct import pathways [#9], and the SR domain is selectively hyperphosphorylated by CLK-family kinases, which reshapes its nuclear distribution and modulates 5' splice-site choice [#2]. Within the nucleus, SRSF4 associates with hundreds of distinct endogenous mRNPs and regulates their levels [#3], operating as both a positive and negative regulator of alternative splicing depending on context: it inhibits tau exon 10 inclusion by binding the downstream intronic FTDP-17 hotspot while interacting with hnRNPG and hnRNPE2 [#5], inhibits splicing at HIV-1 exon 3 [#4], and promotes Fas exon 6 inclusion by binding an exonic splicing enhancer, thereby biasing toward the pro-apoptotic membrane-bound isoform [#8]. Consistent with a role in cell-fate decisions, SRSF4 is required for most cisplatin-induced alternative splicing changes and for cisplatin-induced apoptosis via a class I PI3K (p110\\u03b2)-dependent, ATM/ATR/p53-independent pathway [#6]. A patient-derived p.R235W missense variant that lowers SRSF4 protein causes mitochondrial dysfunction rescuable by wild-type SRSF4, linking the factor to energetic metabolism [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 1993,\n      \"claim\": \"Established SRSF4 as a bona fide SR splicing factor by defining its modular RRM\\u2013glycine\\u2013RRM-homology\\u2013SR domain architecture and showing it can functionally complement splicing activity.\",\n      \"evidence\": \"cDNA cloning with S100 complementation splicing assay and dephosphorylation/mobility-shift analysis\",\n      \"pmids\": [\"8321209\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify endogenous target transcripts\", \"Functional role of SR-domain phosphorylation not established\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Placed SRSF4 within a defined nuclear-speckle protein complex by identifying physical partners, addressing how it is organized with the splicing machinery.\",\n      \"evidence\": \"Yeast two-hybrid and reciprocal co-immunoprecipitation in corneal epithelial cells\",\n      \"pmids\": [\"14578391\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of Pinin interaction on splicing not tested\", \"Single cell type\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified CLK kinases as the specific regulators of SRSF4 hyperphosphorylation and linked this modification to nuclear distribution and 5' splice-site selection.\",\n      \"evidence\": \"Comparative SR kinase assay, CLK inhibitor TG003, live-cell imaging, and E1A alternative splicing reporter\",\n      \"pmids\": [\"18298798\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphosites on SRSF4 not mapped\", \"Effect generality across targets beyond E1A not shown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated genome-wide that SRSF4 binds hundreds of endogenous mRNPs and that these associations functionally control transcript levels, establishing its role beyond single reporters.\",\n      \"evidence\": \"GFP-SR immunopurification of mRNPs with genome-wide target ID, siRNA knockdown, and GFP-SR rescue in P19 cells\",\n      \"pmids\": [\"20639886\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct binding sites not resolved at nucleotide level\", \"Splicing vs. stability contributions not separated\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed SRSF4 acts as a splicing inhibitor at a viral splice site, distinguishing its activity from related SR proteins.\",\n      \"evidence\": \"Transfection-based HIV-1 splicing assay with SR protein expression constructs\",\n      \"pmids\": [\"20685659\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"RNA element bound by SRSF4 not mapped\", \"Single viral context\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined SRSF4 as a context-dependent splicing repressor at a disease-relevant exon and identified hnRNP cofactors, clarifying its regulatory mechanism at tau exon 10.\",\n      \"evidence\": \"Co-transfection splicing assay, co-IP, and RNAi knockdown\",\n      \"pmids\": [\"21723381\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry/order of complex assembly unknown\", \"In vivo relevance to tauopathy not tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected SRSF4 to a stress-response apoptotic program, showing it drives cisplatin-induced splicing changes and cell death through PI3K rather than canonical DNA-damage kinases.\",\n      \"evidence\": \"siRNA knockdown with transcriptome-wide sequencing, RT-PCR, FACS apoptosis, and pathway-specific inhibitors/knockout cells\",\n      \"pmids\": [\"25884497\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct SRSF4 targets mediating apoptosis not isolated\", \"Mechanistic link to PI3K not biochemically defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified upstream transcriptional control of SRSF4 by androgen receptor, showing its expression is hormonally repressed in Sertoli cells.\",\n      \"evidence\": \"AR ChIP/ARE binding, promoter assay, testosterone treatment of TM4 cells, immunofluorescence, and S-AR knockout comparison\",\n      \"pmids\": [\"26308373\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional splicing consequence in Sertoli cells not defined\", \"Single regulatory element\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Resolved SRSF4 as a positive splicing regulator that binds an exonic enhancer to control an apoptotic isoform switch, demonstrating bidirectional splicing activity.\",\n      \"evidence\": \"SRSF4 overexpression/knockdown, 5' splice-site mutagenesis, and enhancer mapping on Fas pre-mRNA\",\n      \"pmids\": [\"30376989\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Enhancer sequence consensus not generalized\", \"Endogenous physiological context not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Dissected the determinants of SRSF4 nuclear import, showing reliance on the RS domain plus additional basic NLS motifs and multiple import routes.\",\n      \"evidence\": \"Pyruvate kinase fusion nuclear localization assay with serial deletion/domain mapping\",\n      \"pmids\": [\"32050040\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Import receptors not identified\", \"Link between import route and function not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Implicated SRSF4 in cardiac hypertrophy through a GAS5 lncRNA/glucocorticoid receptor axis.\",\n      \"evidence\": \"Not fully described in available abstract\",\n      \"pmids\": [\"34333993\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Experimental detail not extractable from abstract\", \"Direct molecular role of SRSF4 in the axis undefined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Linked SRSF4 to DNA double-strand break repair and chemoresistance via positive regulation of MDC1 in glioma.\",\n      \"evidence\": \"Colony formation, flow cytometry, western blot, immunofluorescence, and orthotopic xenograft with SRSF4 manipulation\",\n      \"pmids\": [\"37014544\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Biochemical mechanism linking SRSF4 to MDC1 not defined\", \"Whether effect is splicing-mediated unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided human genetic and functional evidence tying SRSF4 dosage to mitochondrial function via a rescuable missense variant.\",\n      \"evidence\": \"Whole genome sequencing, patient lymphocyte/lymphoblast analysis, mitochondrial function assays, and wild-type SRSF4 rescue transfection\",\n      \"pmids\": [\"38396760\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Splicing targets mediating mitochondrial phenotype not identified\", \"Single-patient case study\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SRSF4's diverse non-splicing phenotypes (apoptosis, DNA repair, mitochondrial and cardiac function) connect to its core RNA-binding/splicing activity remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unifying set of direct RNA targets links the disparate phenotypes\", \"Phosphorylation-state-specific target maps lacking\", \"Structural basis of enhancer vs. silencer recognition unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 3, 5, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [1, 9]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [7, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 3, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PNN\", \"SRRM2\", \"HNRNPG\", \"HNRNPE2\"],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}