{"gene":"NSRP1","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2011,"finding":"NSrp70 (NSRP1/CCDC55) localizes to nuclear speckles and physically interacts with splicing factors SC35 and ASF/SF2; it modulates alternative splice site selection in vivo using CD44, Tra2β1, and Fas minigene reporters. The region spanning amino acids 290–471 is critical for speckle localization and SC35/ASF/SF2 binding, the N-terminal region (107–161) is essential for pre-mRNA splicing activity, and the C-terminal 10 amino acids (531–540), including RD at positions 536–537, constitute a novel nuclear localization signal.","method":"Co-immunoprecipitation, co-localization by confocal microscopy, minigene splicing reporter assays, deletion mutagenesis, NSrp70 knockout mice","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, reporter assay, mutagenesis, KO mouse) in a single rigorous study","pmids":["21296756"],"is_preprint":false},{"year":2011,"finding":"Knockout of the Nsrp1 (NSrp70) gene in mice results in a complete lack of progeny including fetal embryos, establishing that NSrp70 is essentially required for early embryonic development.","method":"Gene knockout in mice (loss-of-function)","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined developmental phenotype, part of a multi-method study","pmids":["21296756"],"is_preprint":false},{"year":2015,"finding":"NSRP1 (CCDC55) physically interacts with RanBP9 and DISC1, and co-localizes with RanBP9 in the nucleus of human neuronal cells. NSRP1 also interacts with the cannabinoid receptor CNR1 and cannabinoid receptor-interacting protein CNRIP1a, as demonstrated by yeast two-hybrid screening and GST pull-down assay.","method":"Yeast two-hybrid screening, GST pull-down assay, confocal laser scanning microscopy","journal":"Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — two orthogonal binding assays (Y2H + GST pull-down) plus co-localization, single lab","pmids":["26475744"],"is_preprint":false},{"year":2016,"finding":"Knockdown of NSrp70 in Xenopus embryos blocks gastrulation and convergent extension, and animal cap assays with activin A treatment show NSrp70 is required for dorsal mesoderm induction, with loss causing downregulation of dorsal mesoderm-specific genes.","method":"Morpholino knockdown in Xenopus, animal cap assay with activin A treatment, phenotypic readout of gastrulation and mesoderm markers","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with specific molecular phenotype in two assay systems, single lab","pmids":["27638308"],"is_preprint":false},{"year":2021,"finding":"NSrp70 is selectively expressed in developing thymocytes and is highest at the double-positive (DP) stage. Conditional knockout (CD4Cre-driven Nsrp1 cKO) causes severe defects in T cell maturation to single-positive thymocytes due to insufficient TCR signaling and dysregulated cell growth/death. NSrp70 controls thymocyte cell cycle and survival by regulating alternative splicing of various RNA splicing factors including the oncogenic splicing factor SRSF1.","method":"Conditional knockout mouse (CD4Cre), RNA-seq and splicing profiling, flow cytometry of thymic subsets","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with defined cellular and molecular phenotype, global splicing profiling, replicated across multiple analyses in one rigorous study","pmids":["34037780"],"is_preprint":false},{"year":2021,"finding":"Biallelic loss-of-function frameshift variants in NSRP1 causing premature termination in the last exon are predicted to escape NMD and result in loss of the C-terminal nuclear localization signal required for NSRP1 function, establishing that NLS loss is a pathogenic mechanism in NSRP1-associated neurodevelopmental disorder.","method":"Exome sequencing, molecular analysis of mutant transcripts, prediction of NMD escape and NLS loss","journal":"Genetics in medicine","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — molecular characterization of variants and prediction of NLS loss supported by prior functional data on NLS; no in-cell validation in this paper","pmids":["34385670"],"is_preprint":false},{"year":2022,"finding":"NSrp70 suppresses breast cancer metastasis by promoting inclusion of skipped exons in NUMB pre-mRNA (inhibiting skipped-exon alternative splicing of NUMB), leading to increased TGFβ receptor 1 (TβR1) degradation via the lysosome pathway and suppression of TGFβ/SMAD-mediated EMT. Direct binding between NSrp70 and NUMB pre-mRNA was confirmed by RNA pull-down and RNA immunoprecipitation.","method":"RNA-seq with AS bioinformatics, in vitro splicing assays, RNA pull-down, RNA immunoprecipitation, in vitro and in vivo functional assays (migration/invasion), proteomic screen","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (in vitro splicing assay, RIP, RNA pulldown, functional in vivo assays) in one study establishing mechanism","pmids":["35568738"],"is_preprint":false},{"year":2024,"finding":"A missense variant p.Val532Glu in the nuclear localization signal of NSRP1 causes mislocalization of NSRP1 to the cytosol in HEK293T cells, confirming that the NLS (around Val532) is required for correct nuclear targeting of NSRP1.","method":"HEK293T transfection with GFP-tagged wild-type or mutant NSRP1, confocal microscopy for subcellular localization","journal":"American journal of medical genetics. Part A","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with GFP-tagged mutant vs WT, functional consequence (loss of nuclear targeting) clearly demonstrated, single lab","pmids":["38808951"],"is_preprint":false},{"year":2024,"finding":"NSRP1 knockdown in MCF7 breast cancer cells activates the IFN signaling pathway and elevates IRPS gene expression; mechanistically, NSRP1 controls alternative splicing of NSD2 such that its knockdown increases inclusion of NSD2 exon 2, which elevates NSD2 protein expression and thereby activates the IFN signaling pathway, conferring CDK4/6 inhibitor resistance.","method":"NSRP1 knockdown and overexpression in MCF7 cells, RNA-seq, alternative splicing analysis, CDK4/6 inhibitor sensitivity assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD/OE with defined molecular pathway (NSD2 exon 2 inclusion → NSD2 upregulation → IFN activation), single lab, two complementary perturbations","pmids":["39667501"],"is_preprint":false}],"current_model":"NSRP1 (NSrp70) is a nuclear speckle-resident splicing regulator that physically associates with SR proteins SC35 and ASF/SF2 via its 290–471 aa region, uses its N-terminal RRM-containing region (107–161 aa) to modulate alternative splice site selection in vivo, and requires a C-terminal nuclear localization signal (aa 531–540, RD motif; critical residue Val532) for nuclear targeting; it regulates alternative splicing of diverse targets including NUMB, SRSF1, and NSD2 to control EMT, thymocyte development, and IFN signaling, and is essential for early embryogenesis in mice and Xenopus."},"narrative":{"mechanistic_narrative":"NSRP1 (NSrp70/CCDC55) is a nuclear speckle-resident regulator of alternative splicing that controls splice-site selection of diverse pre-mRNA targets to govern cell fate, development, and signaling [PMID:21296756, PMID:34037780]. It localizes to nuclear speckles and physically associates with the SR proteins SC35 and ASF/SF2 through its 290–471 aa region, while its N-terminal RRM-containing region (107–161 aa) is required for splicing activity, as demonstrated on CD44, Tra2β1, and Fas minigene reporters [PMID:21296756]. Correct nuclear targeting depends on a C-terminal nuclear localization signal (aa 531–540, including critical residue Val532), and disruption of this NLS mislocalizes the protein to the cytosol [PMID:21296756, PMID:38808951]. Through target-specific splicing control, NSRP1 promotes inclusion of skipped exons in NUMB pre-mRNA to drive TGFβ receptor 1 degradation and suppress TGFβ/SMAD-mediated EMT and breast cancer metastasis [PMID:35568738], and it regulates NSD2 exon 2 inclusion to restrain IFN signaling and CDK4/6 inhibitor resistance [PMID:39667501]. NSRP1 is essential for early embryogenesis, with mouse knockout abolishing progeny and Xenopus knockdown blocking gastrulation and dorsal mesoderm induction [PMID:21296756, PMID:27638308], and conditional deletion in thymocytes impairs T cell maturation by dysregulating splicing of factors including SRSF1 [PMID:34037780]. Biallelic loss-of-function variants that ablate the C-terminal NLS cause an NSRP1-associated neurodevelopmental disorder [PMID:34385670, PMID:38808951].","teleology":[{"year":2011,"claim":"Established NSRP1 as a bona fide splicing regulator by defining where it acts (nuclear speckles), which factors it partners with, and which protein regions drive localization, binding, and splicing activity.","evidence":"Co-IP, confocal co-localization, minigene splicing reporters, deletion mutagenesis in cells","pmids":["21296756"],"confidence":"High","gaps":["Endogenous physiological splicing targets not yet identified","Direct RNA binding by the N-terminal region not demonstrated","Structural basis of SC35/ASF-SF2 interaction unresolved"]},{"year":2011,"claim":"Demonstrated organismal necessity of NSRP1 by showing knockout mice fail to produce embryos, placing it among genes essential for early development.","evidence":"Constitutive gene knockout in mice","pmids":["21296756"],"confidence":"High","gaps":["Developmental stage of lethality not pinpointed","Splicing targets responsible for the embryonic requirement unknown"]},{"year":2015,"claim":"Expanded the interactome beyond splicing factors to nuclear and neuronal partners, raising the possibility of roles outside canonical spliceosome assembly.","evidence":"Yeast two-hybrid screen, GST pull-down, confocal co-localization in neuronal cells","pmids":["26475744"],"confidence":"Medium","gaps":["No functional consequence assigned to RanBP9/DISC1/CNR1 interactions","Single lab without reciprocal endogenous validation","Relevance to splicing function unclear"]},{"year":2016,"claim":"Showed the developmental requirement is conserved and mechanistically tied to mesoderm patterning, linking NSRP1 loss to defective gastrulation and dorsal mesoderm gene expression.","evidence":"Morpholino knockdown and activin A animal cap assays in Xenopus","pmids":["27638308"],"confidence":"Medium","gaps":["Splicing targets driving the gastrulation phenotype not identified","Morpholino specificity not cross-validated genetically"]},{"year":2021,"claim":"Connected NSRP1 splicing activity to a defined cellular program by showing thymocyte-stage-specific function in T cell maturation via splicing of factors including SRSF1.","evidence":"CD4Cre conditional knockout mouse, RNA-seq splicing profiling, flow cytometry","pmids":["34037780"],"confidence":"High","gaps":["Direct binding to SRSF1 transcript not shown","Causal splicing event linking to TCR signaling defect not isolated"]},{"year":2021,"claim":"Tied NSRP1 to human disease by showing biallelic last-exon frameshift variants escape NMD and remove the NLS, defining NLS loss as the pathogenic mechanism of an NSRP1 neurodevelopmental disorder.","evidence":"Exome sequencing, mutant transcript analysis, NMD-escape and NLS-loss prediction","pmids":["34385670"],"confidence":"Medium","gaps":["No in-cell validation of mislocalization in this study","Genotype-phenotype relationship across patients not established"]},{"year":2022,"claim":"Provided a complete molecular mechanism in cancer by showing NSRP1 directly binds NUMB pre-mRNA to promote exon inclusion, driving TβR1 degradation and suppressing TGFβ-driven EMT and metastasis.","evidence":"RNA-seq, in vitro splicing assays, RNA pull-down, RIP, migration/invasion and in vivo assays, proteomics","pmids":["35568738"],"confidence":"High","gaps":["RNA sequence element bound by NSRP1 not mapped","Generality of TβR1 lysosomal degradation across cell types untested"]},{"year":2024,"claim":"Validated the disease NLS mechanism experimentally by showing the p.Val532Glu missense variant mislocalizes NSRP1 to the cytosol, confirming Val532 is required for nuclear targeting.","evidence":"GFP-tagged WT vs mutant NSRP1 transfection in HEK293T, confocal microscopy","pmids":["38808951"],"confidence":"Medium","gaps":["Effect of mislocalization on splicing output not measured","Single cell-line localization assay"]},{"year":2024,"claim":"Extended target-specific splicing control to immune signaling and drug resistance by showing NSRP1 regulates NSD2 exon 2 inclusion to restrain IFN signaling and CDK4/6 inhibitor resistance.","evidence":"NSRP1 knockdown/overexpression in MCF7, RNA-seq, splicing analysis, CDK4/6 inhibitor sensitivity assays","pmids":["39667501"],"confidence":"Medium","gaps":["Direct binding to NSD2 pre-mRNA not demonstrated","Single cell line; clinical relevance untested"]},{"year":null,"claim":"The RNA sequence determinants and global rules by which NSRP1 selects splice sites, and how its diverse target programs (NUMB, SRSF1, NSD2) are coordinated across tissues, remain undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No consensus binding motif defined","No structural model of NSRP1 on RNA or with SR proteins","Tissue-specific target selection mechanism unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,4,6,8]}],"localization":[{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[0]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,7]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,4,6,8]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[1,3,4]}],"complexes":[],"partners":["SRSF2","SRSF1","RANBP9","DISC1","CNR1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9H0G5","full_name":"Nuclear speckle splicing regulatory protein 1","aliases":["Coiled-coil domain-containing protein 55","Nuclear speckle-related protein 70","NSrp70"],"length_aa":558,"mass_kda":66.4,"function":"RNA-binding protein that mediates pre-mRNA alternative splicing regulation (PubMed:21296756). Through CCDC118 regulation, may promote pre-adipocyte differentiation (By similarity)","subcellular_location":"Nucleus; Nucleus speckle","url":"https://www.uniprot.org/uniprotkb/Q9H0G5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NSRP1","classification":"Not Classified","n_dependent_lines":394,"n_total_lines":1208,"dependency_fraction":0.326158940397351},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NSRP1","total_profiled":1310},"omim":[{"mim_id":"621137","title":"COILED-COIL DOMAIN-CONTAINING PROTEIN 18; CCDC18","url":"https://www.omim.org/entry/621137"},{"mim_id":"620001","title":"NEURODEVELOPMENTAL DISORDER WITH SPASTICITY, SEIZURES, AND BRAIN ABNORMALITIES; NEDSSBA","url":"https://www.omim.org/entry/620001"},{"mim_id":"616173","title":"NUCLEAR SPECKLE SPLICING REGULATORY PROTEIN 1; NSRP1","url":"https://www.omim.org/entry/616173"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NSRP1"},"hgnc":{"alias_symbol":["DKFZP434K1421","NSrp70"],"prev_symbol":["CCDC55"]},"alphafold":{"accession":"Q9H0G5","domains":[{"cath_id":"-","chopping":"43-90","consensus_level":"high","plddt":87.3337,"start":43,"end":90},{"cath_id":"1.20.5","chopping":"104-136","consensus_level":"medium","plddt":88.4361,"start":104,"end":136},{"cath_id":"1.20.5","chopping":"363-403","consensus_level":"medium","plddt":74.5646,"start":363,"end":403}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H0G5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H0G5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H0G5-F1-predicted_aligned_error_v6.png","plddt_mean":62.78},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NSRP1","jax_strain_url":"https://www.jax.org/strain/search?query=NSRP1"},"sequence":{"accession":"Q9H0G5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H0G5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H0G5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H0G5"}},"corpus_meta":[{"pmid":"21296756","id":"PMC_21296756","title":"NSrp70 is a novel nuclear speckle-related protein that modulates alternative pre-mRNA splicing in vivo.","date":"2011","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/21296756","citation_count":33,"is_preprint":false},{"pmid":"34385670","id":"PMC_34385670","title":"Biallelic loss-of-function variants in the splicing regulator NSRP1 cause a severe neurodevelopmental disorder with spastic cerebral palsy and epilepsy.","date":"2021","source":"Genetics in medicine : official journal of the American College of Medical Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34385670","citation_count":16,"is_preprint":false},{"pmid":"22285439","id":"PMC_22285439","title":"CCDC-55 is required for larval development and distal tip cell migration in Caenorhabditis elegans.","date":"2012","source":"Mechanisms of development","url":"https://pubmed.ncbi.nlm.nih.gov/22285439","citation_count":11,"is_preprint":false},{"pmid":"34037780","id":"PMC_34037780","title":"NSrp70 is a lymphocyte-essential splicing factor that controls thymocyte development.","date":"2021","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/34037780","citation_count":9,"is_preprint":false},{"pmid":"39667501","id":"PMC_39667501","title":"Downregulation of the splicing regulator NSRP1 confers resistance to CDK4/6 inhibitors via activation of interferon signaling in breast cancer.","date":"2024","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/39667501","citation_count":8,"is_preprint":false},{"pmid":"26475744","id":"PMC_26475744","title":"The CCDC55 couples cannabinoid receptor CNR1 to a putative DISC1 schizophrenia pathway.","date":"2015","source":"Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/26475744","citation_count":7,"is_preprint":false},{"pmid":"35568738","id":"PMC_35568738","title":"NSrp70 suppresses metastasis in triple-negative breast cancer by modulating Numb/TβR1/EMT axis.","date":"2022","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/35568738","citation_count":7,"is_preprint":false},{"pmid":"27638308","id":"PMC_27638308","title":"NSrp70 is significant for embryonic growth and development, being a crucial factor for gastrulation and mesoderm induction.","date":"2016","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/27638308","citation_count":5,"is_preprint":false},{"pmid":"25176346","id":"PMC_25176346","title":"Aberrant proteomic expression of NSRP70 and its clinical implications and connection to the transcriptional level in adult acute leukemia.","date":"2014","source":"Leukemia research","url":"https://pubmed.ncbi.nlm.nih.gov/25176346","citation_count":4,"is_preprint":false},{"pmid":"38474122","id":"PMC_38474122","title":"Comprehensive Atlas of Alternative Splicing Reveals NSRP1 Promoting Adipogenesis through CCDC18.","date":"2024","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/38474122","citation_count":2,"is_preprint":false},{"pmid":"38808951","id":"PMC_38808951","title":"A milder form of NSRP1-associated neurodevelopmental disorder, caused by a missense variant in the nuclear localization signal.","date":"2024","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/38808951","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7352,"output_tokens":2393,"usd":0.028976,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9534,"output_tokens":3099,"usd":0.062573,"stage2_stop_reason":"end_turn"},"total_usd":0.091549,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"NSrp70 (NSRP1/CCDC55) localizes to nuclear speckles and physically interacts with splicing factors SC35 and ASF/SF2; it modulates alternative splice site selection in vivo using CD44, Tra2β1, and Fas minigene reporters. The region spanning amino acids 290–471 is critical for speckle localization and SC35/ASF/SF2 binding, the N-terminal region (107–161) is essential for pre-mRNA splicing activity, and the C-terminal 10 amino acids (531–540), including RD at positions 536–537, constitute a novel nuclear localization signal.\",\n      \"method\": \"Co-immunoprecipitation, co-localization by confocal microscopy, minigene splicing reporter assays, deletion mutagenesis, NSrp70 knockout mice\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, reporter assay, mutagenesis, KO mouse) in a single rigorous study\",\n      \"pmids\": [\"21296756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Knockout of the Nsrp1 (NSrp70) gene in mice results in a complete lack of progeny including fetal embryos, establishing that NSrp70 is essentially required for early embryonic development.\",\n      \"method\": \"Gene knockout in mice (loss-of-function)\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined developmental phenotype, part of a multi-method study\",\n      \"pmids\": [\"21296756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NSRP1 (CCDC55) physically interacts with RanBP9 and DISC1, and co-localizes with RanBP9 in the nucleus of human neuronal cells. NSRP1 also interacts with the cannabinoid receptor CNR1 and cannabinoid receptor-interacting protein CNRIP1a, as demonstrated by yeast two-hybrid screening and GST pull-down assay.\",\n      \"method\": \"Yeast two-hybrid screening, GST pull-down assay, confocal laser scanning microscopy\",\n      \"journal\": \"Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — two orthogonal binding assays (Y2H + GST pull-down) plus co-localization, single lab\",\n      \"pmids\": [\"26475744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Knockdown of NSrp70 in Xenopus embryos blocks gastrulation and convergent extension, and animal cap assays with activin A treatment show NSrp70 is required for dorsal mesoderm induction, with loss causing downregulation of dorsal mesoderm-specific genes.\",\n      \"method\": \"Morpholino knockdown in Xenopus, animal cap assay with activin A treatment, phenotypic readout of gastrulation and mesoderm markers\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with specific molecular phenotype in two assay systems, single lab\",\n      \"pmids\": [\"27638308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NSrp70 is selectively expressed in developing thymocytes and is highest at the double-positive (DP) stage. Conditional knockout (CD4Cre-driven Nsrp1 cKO) causes severe defects in T cell maturation to single-positive thymocytes due to insufficient TCR signaling and dysregulated cell growth/death. NSrp70 controls thymocyte cell cycle and survival by regulating alternative splicing of various RNA splicing factors including the oncogenic splicing factor SRSF1.\",\n      \"method\": \"Conditional knockout mouse (CD4Cre), RNA-seq and splicing profiling, flow cytometry of thymic subsets\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with defined cellular and molecular phenotype, global splicing profiling, replicated across multiple analyses in one rigorous study\",\n      \"pmids\": [\"34037780\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Biallelic loss-of-function frameshift variants in NSRP1 causing premature termination in the last exon are predicted to escape NMD and result in loss of the C-terminal nuclear localization signal required for NSRP1 function, establishing that NLS loss is a pathogenic mechanism in NSRP1-associated neurodevelopmental disorder.\",\n      \"method\": \"Exome sequencing, molecular analysis of mutant transcripts, prediction of NMD escape and NLS loss\",\n      \"journal\": \"Genetics in medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — molecular characterization of variants and prediction of NLS loss supported by prior functional data on NLS; no in-cell validation in this paper\",\n      \"pmids\": [\"34385670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NSrp70 suppresses breast cancer metastasis by promoting inclusion of skipped exons in NUMB pre-mRNA (inhibiting skipped-exon alternative splicing of NUMB), leading to increased TGFβ receptor 1 (TβR1) degradation via the lysosome pathway and suppression of TGFβ/SMAD-mediated EMT. Direct binding between NSrp70 and NUMB pre-mRNA was confirmed by RNA pull-down and RNA immunoprecipitation.\",\n      \"method\": \"RNA-seq with AS bioinformatics, in vitro splicing assays, RNA pull-down, RNA immunoprecipitation, in vitro and in vivo functional assays (migration/invasion), proteomic screen\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (in vitro splicing assay, RIP, RNA pulldown, functional in vivo assays) in one study establishing mechanism\",\n      \"pmids\": [\"35568738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A missense variant p.Val532Glu in the nuclear localization signal of NSRP1 causes mislocalization of NSRP1 to the cytosol in HEK293T cells, confirming that the NLS (around Val532) is required for correct nuclear targeting of NSRP1.\",\n      \"method\": \"HEK293T transfection with GFP-tagged wild-type or mutant NSRP1, confocal microscopy for subcellular localization\",\n      \"journal\": \"American journal of medical genetics. Part A\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with GFP-tagged mutant vs WT, functional consequence (loss of nuclear targeting) clearly demonstrated, single lab\",\n      \"pmids\": [\"38808951\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NSRP1 knockdown in MCF7 breast cancer cells activates the IFN signaling pathway and elevates IRPS gene expression; mechanistically, NSRP1 controls alternative splicing of NSD2 such that its knockdown increases inclusion of NSD2 exon 2, which elevates NSD2 protein expression and thereby activates the IFN signaling pathway, conferring CDK4/6 inhibitor resistance.\",\n      \"method\": \"NSRP1 knockdown and overexpression in MCF7 cells, RNA-seq, alternative splicing analysis, CDK4/6 inhibitor sensitivity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD/OE with defined molecular pathway (NSD2 exon 2 inclusion → NSD2 upregulation → IFN activation), single lab, two complementary perturbations\",\n      \"pmids\": [\"39667501\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NSRP1 (NSrp70) is a nuclear speckle-resident splicing regulator that physically associates with SR proteins SC35 and ASF/SF2 via its 290–471 aa region, uses its N-terminal RRM-containing region (107–161 aa) to modulate alternative splice site selection in vivo, and requires a C-terminal nuclear localization signal (aa 531–540, RD motif; critical residue Val532) for nuclear targeting; it regulates alternative splicing of diverse targets including NUMB, SRSF1, and NSD2 to control EMT, thymocyte development, and IFN signaling, and is essential for early embryogenesis in mice and Xenopus.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NSRP1 (NSrp70/CCDC55) is a nuclear speckle-resident regulator of alternative splicing that controls splice-site selection of diverse pre-mRNA targets to govern cell fate, development, and signaling [#0, #4]. It localizes to nuclear speckles and physically associates with the SR proteins SC35 and ASF/SF2 through its 290\\u2013471 aa region, while its N-terminal RRM-containing region (107\\u2013161 aa) is required for splicing activity, as demonstrated on CD44, Tra2\\u03b21, and Fas minigene reporters [#0]. Correct nuclear targeting depends on a C-terminal nuclear localization signal (aa 531\\u2013540, including critical residue Val532), and disruption of this NLS mislocalizes the protein to the cytosol [#0, #7]. Through target-specific splicing control, NSRP1 promotes inclusion of skipped exons in NUMB pre-mRNA to drive TGF\\u03b2 receptor 1 degradation and suppress TGF\\u03b2/SMAD-mediated EMT and breast cancer metastasis [#6], and it regulates NSD2 exon 2 inclusion to restrain IFN signaling and CDK4/6 inhibitor resistance [#8]. NSRP1 is essential for early embryogenesis, with mouse knockout abolishing progeny and Xenopus knockdown blocking gastrulation and dorsal mesoderm induction [#1, #3], and conditional deletion in thymocytes impairs T cell maturation by dysregulating splicing of factors including SRSF1 [#4]. Biallelic loss-of-function variants that ablate the C-terminal NLS cause an NSRP1-associated neurodevelopmental disorder [#5, #7].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Established NSRP1 as a bona fide splicing regulator by defining where it acts (nuclear speckles), which factors it partners with, and which protein regions drive localization, binding, and splicing activity.\",\n      \"evidence\": \"Co-IP, confocal co-localization, minigene splicing reporters, deletion mutagenesis in cells\",\n      \"pmids\": [\"21296756\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous physiological splicing targets not yet identified\", \"Direct RNA binding by the N-terminal region not demonstrated\", \"Structural basis of SC35/ASF-SF2 interaction unresolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated organismal necessity of NSRP1 by showing knockout mice fail to produce embryos, placing it among genes essential for early development.\",\n      \"evidence\": \"Constitutive gene knockout in mice\",\n      \"pmids\": [\"21296756\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Developmental stage of lethality not pinpointed\", \"Splicing targets responsible for the embryonic requirement unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Expanded the interactome beyond splicing factors to nuclear and neuronal partners, raising the possibility of roles outside canonical spliceosome assembly.\",\n      \"evidence\": \"Yeast two-hybrid screen, GST pull-down, confocal co-localization in neuronal cells\",\n      \"pmids\": [\"26475744\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional consequence assigned to RanBP9/DISC1/CNR1 interactions\", \"Single lab without reciprocal endogenous validation\", \"Relevance to splicing function unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed the developmental requirement is conserved and mechanistically tied to mesoderm patterning, linking NSRP1 loss to defective gastrulation and dorsal mesoderm gene expression.\",\n      \"evidence\": \"Morpholino knockdown and activin A animal cap assays in Xenopus\",\n      \"pmids\": [\"27638308\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Splicing targets driving the gastrulation phenotype not identified\", \"Morpholino specificity not cross-validated genetically\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected NSRP1 splicing activity to a defined cellular program by showing thymocyte-stage-specific function in T cell maturation via splicing of factors including SRSF1.\",\n      \"evidence\": \"CD4Cre conditional knockout mouse, RNA-seq splicing profiling, flow cytometry\",\n      \"pmids\": [\"34037780\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct binding to SRSF1 transcript not shown\", \"Causal splicing event linking to TCR signaling defect not isolated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Tied NSRP1 to human disease by showing biallelic last-exon frameshift variants escape NMD and remove the NLS, defining NLS loss as the pathogenic mechanism of an NSRP1 neurodevelopmental disorder.\",\n      \"evidence\": \"Exome sequencing, mutant transcript analysis, NMD-escape and NLS-loss prediction\",\n      \"pmids\": [\"34385670\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in-cell validation of mislocalization in this study\", \"Genotype-phenotype relationship across patients not established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Provided a complete molecular mechanism in cancer by showing NSRP1 directly binds NUMB pre-mRNA to promote exon inclusion, driving T\\u03b2R1 degradation and suppressing TGF\\u03b2-driven EMT and metastasis.\",\n      \"evidence\": \"RNA-seq, in vitro splicing assays, RNA pull-down, RIP, migration/invasion and in vivo assays, proteomics\",\n      \"pmids\": [\"35568738\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RNA sequence element bound by NSRP1 not mapped\", \"Generality of T\\u03b2R1 lysosomal degradation across cell types untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Validated the disease NLS mechanism experimentally by showing the p.Val532Glu missense variant mislocalizes NSRP1 to the cytosol, confirming Val532 is required for nuclear targeting.\",\n      \"evidence\": \"GFP-tagged WT vs mutant NSRP1 transfection in HEK293T, confocal microscopy\",\n      \"pmids\": [\"38808951\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Effect of mislocalization on splicing output not measured\", \"Single cell-line localization assay\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended target-specific splicing control to immune signaling and drug resistance by showing NSRP1 regulates NSD2 exon 2 inclusion to restrain IFN signaling and CDK4/6 inhibitor resistance.\",\n      \"evidence\": \"NSRP1 knockdown/overexpression in MCF7, RNA-seq, splicing analysis, CDK4/6 inhibitor sensitivity assays\",\n      \"pmids\": [\"39667501\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding to NSD2 pre-mRNA not demonstrated\", \"Single cell line; clinical relevance untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The RNA sequence determinants and global rules by which NSRP1 selects splice sites, and how its diverse target programs (NUMB, SRSF1, NSD2) are coordinated across tissues, remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No consensus binding motif defined\", \"No structural model of NSRP1 on RNA or with SR proteins\", \"Tissue-specific target selection mechanism unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 4, 6, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 4, 6, 8]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [1, 3, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SRSF2\", \"SRSF1\", \"RanBP9\", \"DISC1\", \"CNR1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}