{"gene":"SMNDC1","run_date":"2026-06-10T07:46:36","timeline":{"discoveries":[{"year":2001,"finding":"SPF30 (SMNDC1) is a nuclear protein that associates with both U4/U5/U6 tri-snRNP and U2 snRNP components. In the absence of SPF30, the preformed tri-snRNP fails to assemble into the spliceosome. GST-SPF30 pull-down from HeLa nuclear extract associated most strongly with U4/U6-90 and core Sm and tri-snRNP proteins, establishing SPF30 as an essential factor for docking the U4/U5/U6 tri-snRNP to the A complex during spliceosome assembly.","method":"Immunodepletion of SPF30 from HeLa nuclear extract (splicing assay), GST pull-down with mass spectrometric identification of co-purified proteins, nuclear localization confirmed","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal functional depletion plus GST pull-down with MS, two orthogonal methods establishing the same conclusion in a single focused study","pmids":["11331295"],"is_preprint":false},{"year":2008,"finding":"SPF30 bridges the prespliceosome and tri-snRNP: (1) the central Tudor domain of SPF30 interacts with the C-terminal tails of SmD1, SmD3, and Lsm4; (2) the N-terminal domain of SPF30 interacts with U2AF35 (a prespliceosome 3′-splice-site recognition factor); (3) the C-terminus of SPF30 interacts with the middle domain of hPrp3 (a U4/U6 di-snRNP/tri-snRNP component). The U2AF35 and hPrp3 interactions can occur simultaneously, linking 3′ splice site recognition to tri-snRNP addition.","method":"Co-immunoprecipitation and GST pull-down mapping experiments with domain-deletion constructs of SPF30 and its partners","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — domain-mapping pull-downs and co-IP with multiple partners, single lab, no in vivo reconstitution or structural validation","pmids":["18211889"],"is_preprint":false},{"year":2011,"finding":"The Tudor domain of SPF30 recognizes symmetrically and asymmetrically dimethylated arginine (DMA) through an aromatic cage that mediates cation-π interactions. Solution NMR structures of the SPF30 Tudor domain bound to DMA-containing peptides show that binding is independent of residues proximal to the dimethylarginine in the ligand, but is enhanced by cooperativity when multiple methylation marks are presented in RGG-rich peptide ligands. SPF30 is the weaker DMA binder compared with SMN.","method":"NMR solution structures of Tudor domain–DMA peptide complexes, isothermal titration calorimetry, site-directed mutagenesis","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure plus affinity measurements and mutagenesis in one rigorous study; confirmed by independent crystal structure study (PMID:22363433)","pmids":["22101937","22363433"],"is_preprint":false},{"year":2012,"finding":"Quantitative binding characterization showed that SPF30's Tudor domain is the weakest methyl-arginine binder among SMN, TDRD3, and SPF30, recognizing only GAR (glycine-arginine-rich) motif sequences, and does not efficiently bind non-GAR arginine-containing sequences. SPF30 Tudor domain binds both symmetrical and asymmetrical DMA but with lower affinity than SMN.","method":"Fluorescence polarization and ITC binding assays with a peptide library; crystal structures of TDRD3 Tudor domain used comparatively","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — quantitative in vitro binding assays with multiple orthogonal methods, single lab, but SPF30 is not the primary focus of the structural work","pmids":["22363433"],"is_preprint":false},{"year":2009,"finding":"Fission yeast Spf30 (ortholog of SMNDC1) is required for exosome-mediated heterochromatin silencing at centromeres. Spf30 colocalizes with the exosome RNase Dis3 at centromeric heterochromatin; Dis3 helps recruit Spf30. Loss of Spf30 phenocopies the dis3-54 mutant: reduced silencing and accumulation of polyadenylated centromeric transcripts without loss of siRNA production. Spf30 binds centromeric transcripts and localizes to centromeres in an RNA-dependent manner.","method":"Genetic epistasis (double mutant analysis), chromatin immunoprecipitation (ChIP), RNA immunoprecipitation, fluorescence localization studies in S. pombe","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis plus ChIP and RNA-IP in fission yeast ortholog, multiple methods, single lab","pmids":["20028739"],"is_preprint":false},{"year":2021,"finding":"SPF30 associates with the MTR4-exosome RNA-decay machinery. The interaction between SPF30 and the exosome core is mediated by MTR4 and RRP6. The N- and C-terminal regions of SPF30 (not the Tudor domain) mediate association with MTR4 and the exosome. SPF30 knockdown caused subtle delay in 12S pre-rRNA processing to mature 5.8S rRNA. Shotgun proteomics of the SPF30 interactome linked it to ribosome biogenesis, pre-mRNA splicing, and box C/D snoRNA biogenesis. The SPF30–MTR4 interaction is regulated by ATP hydrolysis of AAA-ATPase NVL2.","method":"Co-immunoprecipitation, domain-deletion mapping, shotgun proteomics (interactome), siRNA knockdown with rRNA processing assay","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP with domain mapping plus MS interactome and functional knockdown, single lab","pmids":["33422691"],"is_preprint":false},{"year":2022,"finding":"Knockdown of Smndc1 in a murine α-cell line triggers global repression of α-cell gene-expression programs and upregulation of β-cell markers, including the transcription factor Pdx1. Mechanistically, Smndc1 loss modulates the activities of the BAF and Atrx chromatin remodeling complexes to derepress Pdx1 expression, linking SMNDC1 function to control of both splicing and chromatin remodeling in pancreatic islet cell identity. The repressive role of SMNDC1 is conserved in human pancreatic islets.","method":"RNAi screen, siRNA knockdown, transcriptomic analysis, chromatin remodeling complex activity assays (BAF/ATRX), human islet knockdown experiments with insulin secretion and PDX1 expression readouts","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi knockdown with defined cellular phenotype and pathway placement via chromatin complex perturbation, validated in human islets, single lab","pmids":["36044849"],"is_preprint":false},{"year":2023,"finding":"SMNDC1 localizes to phase-separated membraneless organelles that partially overlap with nuclear speckles. This condensation is driven by the unstructured C-terminal region of SMNDC1, depends on RNA interaction, and can be recapitulated in vitro. Small-molecule inhibitors targeting the dimethylarginine-binding pocket of the SMNDC1 Tudor domain drastically alter protein-protein interactions and subcellular localization, and cause splicing changes for SMNDC1-dependent genes.","method":"Live-cell fluorescence imaging, in vitro phase-separation assay, small-molecule Tudor domain inhibitors, proteomics of inhibitor-treated cells, splicing assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (live imaging, in vitro reconstitution, chemical perturbation with proteomic and splicing readouts), single lab but comprehensive","pmids":["37587144"],"is_preprint":false},{"year":2024,"finding":"A poison exon in Smndc1 (within intron 2) is conserved across mammals and plants and mediates autoregulatory control of SMNDC1 protein levels via nonsense-mediated mRNA decay (NMD). Mice and A. thaliana lacking this poison exon show deregulated SMNDC1 protein levels, pervasive alterations in mRNA processing, and organismal size restriction, demonstrating that this autoregulatory mechanism is functionally important in vivo.","method":"Mouse and Arabidopsis genetic models with poison-exon deletion, RT-PCR for NMD-targeted transcripts, mRNA processing analysis, organismal growth phenotyping","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo loss-of-function in two independent organisms (mouse + plant) with molecular and organismal phenotypic readouts, convergent results","pmids":["39150991"],"is_preprint":false},{"year":2025,"finding":"SPF30 autoregulates its own expression through a negative feedback mechanism: increased SPF30 promotes inclusion of a cassette exon (intron 2) and/or generation of an exon 4a splice variant, both of which are degraded by NMD, reducing SPF30 mRNA levels. Exon 4a inclusion contributes more than cassette exon inclusion to adjusting SPF30 levels. The C-terminal region of SPF30 (including the latter part of an α-helix and a kink-like structure) is required for binding to RNA containing exon 4a and for the autoregulatory mechanism. A short sequence within exon 4 of SPF30 mRNA is required for exon 4a inclusion.","method":"In vivo splicing assays with deletion constructs, siRNA knockdown and overexpression of SPF30, RT-PCR quantification of splice variants, RNA-binding domain mapping by mutagenesis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional assays (splicing reporters, RNA-binding domain mapping, NMD readout), single lab, preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.03.04.641417"],"is_preprint":true}],"current_model":"SMNDC1 (SPF30) is an essential nuclear splicing factor whose central Tudor domain binds dimethylated arginine (DMA) on Sm/RGG-motif proteins via an aromatic cage, mediating recruitment of the U4/U5/U6 tri-snRNP to the prespliceosome by simultaneously bridging U2AF35 (via its N-terminus) and hPrp3/tri-snRNP (via its C-terminus); its unstructured C-terminal region drives phase separation into nuclear speckle-associated condensates in an RNA-dependent manner; it also associates with the MTR4-exosome machinery through its N- and C-termini to participate in rRNA processing and RNA surveillance; and it autoregulates its own expression through NMD-inducing poison exon inclusion, a mechanism that depends on the C-terminal region binding to nascent pre-mRNA."},"narrative":{"mechanistic_narrative":"SMNDC1 (SPF30) is an essential nuclear splicing factor that drives recruitment of the U4/U5/U6 tri-snRNP to the prespliceosome during spliceosome assembly; in its absence the preformed tri-snRNP fails to dock onto the A complex [PMID:11331295]. It bridges 3′ splice site recognition to tri-snRNP addition by binding U2AF35 through its N-terminus and hPrp3 through its C-terminus, two interactions that can occur simultaneously, while its central Tudor domain engages the C-terminal tails of Sm proteins SmD1, SmD3, and Lsm4 [PMID:18211889]. This Tudor domain recognizes symmetrically and asymmetrically dimethylated arginine via an aromatic cage that mediates cation-π interactions, with binding enhanced by cooperative recognition of multiple methyl marks on RGG/GAR-rich ligands [PMID:22101937, PMID:22363433]. The unstructured C-terminal region drives RNA-dependent phase separation into condensates that partially overlap nuclear speckles, and chemical occupation of the Tudor methylarginine pocket reorganizes SMNDC1 interactions, localization, and splicing of SMNDC1-dependent genes [PMID:37587144]. Beyond splicing, SMNDC1 associates through its N- and C-termini (not the Tudor domain) with the MTR4-exosome RNA-decay machinery to participate in rRNA processing and RNA surveillance [PMID:33422691], and it autoregulates its own abundance through inclusion of NMD-targeted poison/cassette exons in an evolutionarily conserved, in vivo-essential feedback loop [PMID:39150991].","teleology":[{"year":2001,"claim":"Established SMNDC1/SPF30 as a non-redundant assembly factor for the spliceosome, answering whether it acts before or during catalytic complex formation.","evidence":"Immunodepletion from HeLa nuclear extract with splicing assay plus GST pull-down/MS, in human cells","pmids":["11331295"],"confidence":"High","gaps":["Did not resolve which molecular surfaces contact the snRNPs","Mechanism of bridging prespliceosome to tri-snRNP undefined"]},{"year":2008,"claim":"Defined the modular architecture by which SPF30 physically links 3′ splice site recognition to tri-snRNP recruitment, explaining how it docks the tri-snRNP onto the A complex.","evidence":"Co-IP and GST pull-down domain mapping with deletion constructs of SPF30, U2AF35, hPrp3, and Sm proteins","pmids":["18211889"],"confidence":"Medium","gaps":["No in vivo reconstitution of the bridged complex","No structural validation of the simultaneous U2AF35/hPrp3 contacts","Single lab"]},{"year":2011,"claim":"Resolved the structural basis of SPF30's ligand recognition, showing the Tudor domain reads dimethylarginine through an aromatic cage and ranks as a comparatively weak DMA binder.","evidence":"NMR solution structures of Tudor–DMA peptide complexes with ITC and mutagenesis; corroborated by an independent crystal/binding study","pmids":["22101937","22363433"],"confidence":"High","gaps":["Functional consequence of weak affinity for substrate selection unclear","In-cell methylarginine targets not enumerated"]},{"year":2009,"claim":"Extended SPF30 function beyond splicing by linking the fission yeast ortholog to exosome-mediated heterochromatin silencing, raising the possibility of an RNA-surveillance role.","evidence":"Genetic epistasis, ChIP, RNA-IP, and fluorescence localization in S. pombe","pmids":["20028739"],"confidence":"Medium","gaps":["Conservation of the heterochromatin role to human SMNDC1 not established here","Direct vs indirect exosome coupling unresolved"]},{"year":2021,"claim":"Connected human SPF30 to the MTR4-exosome machinery and rRNA processing, showing termini rather than the Tudor domain mediate this RNA-decay association.","evidence":"Co-IP with domain mapping, shotgun interactome proteomics, and siRNA knockdown rRNA processing assay in human cells","pmids":["33422691"],"confidence":"Medium","gaps":["Only a subtle rRNA processing delay observed on knockdown","Functional separation of splicing vs exosome roles unresolved","Single lab"]},{"year":2022,"claim":"Placed SMNDC1 in cell-identity control, showing its loss reprograms pancreatic α-cells toward β-cell programs via chromatin remodeling, linking splicing factor activity to chromatin output.","evidence":"RNAi/siRNA knockdown, transcriptomics, BAF/ATRX activity assays, validated in human islets","pmids":["36044849"],"confidence":"Medium","gaps":["Whether chromatin effects are direct or secondary to splicing changes unclear","No mechanistic link between Tudor binding and BAF/ATRX modulation"]},{"year":2023,"claim":"Revealed that SMNDC1 forms RNA-dependent phase-separated condensates at nuclear speckles and that its Tudor pocket is chemically druggable with functional splicing consequences.","evidence":"Live-cell imaging, in vitro phase-separation reconstitution, small-molecule Tudor inhibitors with proteomic and splicing readouts in human cells","pmids":["37587144"],"confidence":"High","gaps":["Role of condensation in catalytic splicing not isolated","Which RNA species nucleate condensation undefined"]},{"year":2024,"claim":"Demonstrated that an evolutionarily conserved poison exon autoregulates SMNDC1 levels via NMD and is functionally essential in vivo across mammals and plants.","evidence":"Poison-exon deletion in mouse and Arabidopsis, RT-PCR of NMD transcripts, mRNA processing and growth phenotyping","pmids":["39150991"],"confidence":"High","gaps":["RNA element directing poison-exon inclusion not mapped here","Trigger sensing excess SMNDC1 protein undefined"]},{"year":2025,"claim":"Mapped the autoregulatory feedback to specific splice events and the C-terminal RNA-binding region, refining how SMNDC1 senses and tunes its own abundance.","evidence":"In vivo splicing reporters, SPF30 knockdown/overexpression, RT-PCR of variants, RNA-binding domain mutagenesis (preprint)","pmids":["bio_10.1101_2025.03.04.641417"],"confidence":"Medium","gaps":["Preprint not yet peer-reviewed","Direct binding of C-terminal region to exon 4a RNA not structurally resolved","Relative contribution of exon 4a vs cassette exon in other tissues untested"]},{"year":null,"claim":"How SMNDC1's splicing, exosome-surveillance, chromatin-remodeling, and condensate-forming activities are coordinated by a single protein, and which depend on Tudor-mediated methylarginine reading versus terminal interactions, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated structural model of SMNDC1 within the assembling spliceosome","Causal hierarchy among splicing, chromatin, and exosome roles undefined","No human disease linkage established in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[4,7,9]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[6]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,1,5]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[6]}],"complexes":["spliceosome","U4/U5/U6 tri-snRNP","MTR4-exosome"],"partners":["U2AF35","PRPF3","SNRPD1","SNRPD3","LSM4","MTR4","EXOSC10","NVL"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75940","full_name":"Survival of motor neuron-related-splicing factor 30","aliases":["30 kDa splicing factor SMNrp","SMN-related protein","Survival motor neuron domain-containing protein 1"],"length_aa":238,"mass_kda":26.7,"function":"Involved in spliceosome assembly","subcellular_location":"Nucleus speckle; Nucleus, Cajal body","url":"https://www.uniprot.org/uniprotkb/O75940/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/SMNDC1","classification":"Common Essential","n_dependent_lines":1192,"n_total_lines":1208,"dependency_fraction":0.9867549668874173},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PRPF4B","stoichiometry":4.0},{"gene":"ATG13","stoichiometry":0.2},{"gene":"RBM39","stoichiometry":0.2},{"gene":"SF3A1","stoichiometry":0.2},{"gene":"SF3B1","stoichiometry":0.2},{"gene":"SNRPA","stoichiometry":0.2},{"gene":"SNRPB","stoichiometry":0.2},{"gene":"SNRPC","stoichiometry":0.2},{"gene":"SNRPF","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/SMNDC1","total_profiled":1310},"omim":[{"mim_id":"603519","title":"SURVIVAL MOTOR NEURON DOMAIN-CONTAINING PROTEIN 1; SMNDC1","url":"https://www.omim.org/entry/603519"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"},{"location":"Nuclear bodies","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SMNDC1"},"hgnc":{"alias_symbol":["SPF30","SMNR","TDRD16C"],"prev_symbol":[]},"alphafold":{"accession":"O75940","domains":[{"cath_id":"2.30.30.140","chopping":"78-125","consensus_level":"high","plddt":90.9798,"start":78,"end":125},{"cath_id":"1.20.5","chopping":"2-52","consensus_level":"high","plddt":87.2602,"start":2,"end":52}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75940","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75940-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75940-F1-predicted_aligned_error_v6.png","plddt_mean":77.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SMNDC1","jax_strain_url":"https://www.jax.org/strain/search?query=SMNDC1"},"sequence":{"accession":"O75940","fasta_url":"https://rest.uniprot.org/uniprotkb/O75940.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75940/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75940"}},"corpus_meta":[{"pmid":"22101937","id":"PMC_22101937","title":"Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins.","date":"2011","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/22101937","citation_count":160,"is_preprint":false},{"pmid":"22363433","id":"PMC_22363433","title":"Crystal structure of TDRD3 and methyl-arginine binding characterization of TDRD3, SMN and SPF30.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/22363433","citation_count":72,"is_preprint":false},{"pmid":"11331295","id":"PMC_11331295","title":"SPF30 is an essential human splicing factor required for assembly of the U4/U5/U6 tri-small nuclear ribonucleoprotein into the spliceosome.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11331295","citation_count":46,"is_preprint":false},{"pmid":"20028739","id":"PMC_20028739","title":"Splicing factor Spf30 assists exosome-mediated gene silencing in fission yeast.","date":"2009","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/20028739","citation_count":23,"is_preprint":false},{"pmid":"18211889","id":"PMC_18211889","title":"Splicing factor SPF30 bridges an interaction between the prespliceosome protein U2AF35 and tri-small nuclear ribonucleoprotein protein hPrp3.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18211889","citation_count":15,"is_preprint":false},{"pmid":"36044849","id":"PMC_36044849","title":"SMNDC1 links chromatin remodeling and splicing to regulate pancreatic hormone expression.","date":"2022","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/36044849","citation_count":12,"is_preprint":false},{"pmid":"37587144","id":"PMC_37587144","title":"Pharmacological perturbation of the phase-separating protein SMNDC1.","date":"2023","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/37587144","citation_count":11,"is_preprint":false},{"pmid":"22020225","id":"PMC_22020225","title":"Fungal Smn and Spf30 homologues are mainly present in filamentous fungi and genomes with many introns: implications for spinal muscular atrophy.","date":"2011","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/22020225","citation_count":8,"is_preprint":false},{"pmid":"30904945","id":"PMC_30904945","title":"Identification, evolution and alternative splicing profile analysis of the splicing factor 30 (SPF30) in plant species.","date":"2019","source":"Planta","url":"https://pubmed.ncbi.nlm.nih.gov/30904945","citation_count":7,"is_preprint":false},{"pmid":"33422691","id":"PMC_33422691","title":"Interactome analysis of the Tudor domain-containing protein SPF30 which associates with the MTR4-exosome RNA-decay machinery under the regulation of AAA-ATPase NVL2.","date":"2021","source":"The international journal of biochemistry & cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/33422691","citation_count":7,"is_preprint":false},{"pmid":"39150991","id":"PMC_39150991","title":"An autoregulatory poison exon in Smndc1 is conserved across kingdoms and influences organism growth.","date":"2024","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/39150991","citation_count":5,"is_preprint":false},{"pmid":"42118219","id":"PMC_42118219","title":"Phylogenetic Comparison and Splice Site Conservation of the Animal SMNDC1 Gene Family.","date":"2026","source":"Genesis (New York, N.Y. : 2000)","url":"https://pubmed.ncbi.nlm.nih.gov/42118219","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.03.04.641417","title":"Molecular basis of the autoregulatory mechanism of motor neuron-related splicing factor 30","date":"2025-03-10","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.04.641417","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7559,"output_tokens":3218,"usd":0.035473,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10701,"output_tokens":3259,"usd":0.06749,"stage2_stop_reason":"end_turn"},"total_usd":0.102963,"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\": 2001,\n      \"finding\": \"SPF30 (SMNDC1) is a nuclear protein that associates with both U4/U5/U6 tri-snRNP and U2 snRNP components. In the absence of SPF30, the preformed tri-snRNP fails to assemble into the spliceosome. GST-SPF30 pull-down from HeLa nuclear extract associated most strongly with U4/U6-90 and core Sm and tri-snRNP proteins, establishing SPF30 as an essential factor for docking the U4/U5/U6 tri-snRNP to the A complex during spliceosome assembly.\",\n      \"method\": \"Immunodepletion of SPF30 from HeLa nuclear extract (splicing assay), GST pull-down with mass spectrometric identification of co-purified proteins, nuclear localization confirmed\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal functional depletion plus GST pull-down with MS, two orthogonal methods establishing the same conclusion in a single focused study\",\n      \"pmids\": [\"11331295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"SPF30 bridges the prespliceosome and tri-snRNP: (1) the central Tudor domain of SPF30 interacts with the C-terminal tails of SmD1, SmD3, and Lsm4; (2) the N-terminal domain of SPF30 interacts with U2AF35 (a prespliceosome 3′-splice-site recognition factor); (3) the C-terminus of SPF30 interacts with the middle domain of hPrp3 (a U4/U6 di-snRNP/tri-snRNP component). The U2AF35 and hPrp3 interactions can occur simultaneously, linking 3′ splice site recognition to tri-snRNP addition.\",\n      \"method\": \"Co-immunoprecipitation and GST pull-down mapping experiments with domain-deletion constructs of SPF30 and its partners\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — domain-mapping pull-downs and co-IP with multiple partners, single lab, no in vivo reconstitution or structural validation\",\n      \"pmids\": [\"18211889\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The Tudor domain of SPF30 recognizes symmetrically and asymmetrically dimethylated arginine (DMA) through an aromatic cage that mediates cation-π interactions. Solution NMR structures of the SPF30 Tudor domain bound to DMA-containing peptides show that binding is independent of residues proximal to the dimethylarginine in the ligand, but is enhanced by cooperativity when multiple methylation marks are presented in RGG-rich peptide ligands. SPF30 is the weaker DMA binder compared with SMN.\",\n      \"method\": \"NMR solution structures of Tudor domain–DMA peptide complexes, isothermal titration calorimetry, site-directed mutagenesis\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure plus affinity measurements and mutagenesis in one rigorous study; confirmed by independent crystal structure study (PMID:22363433)\",\n      \"pmids\": [\"22101937\", \"22363433\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Quantitative binding characterization showed that SPF30's Tudor domain is the weakest methyl-arginine binder among SMN, TDRD3, and SPF30, recognizing only GAR (glycine-arginine-rich) motif sequences, and does not efficiently bind non-GAR arginine-containing sequences. SPF30 Tudor domain binds both symmetrical and asymmetrical DMA but with lower affinity than SMN.\",\n      \"method\": \"Fluorescence polarization and ITC binding assays with a peptide library; crystal structures of TDRD3 Tudor domain used comparatively\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative in vitro binding assays with multiple orthogonal methods, single lab, but SPF30 is not the primary focus of the structural work\",\n      \"pmids\": [\"22363433\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Fission yeast Spf30 (ortholog of SMNDC1) is required for exosome-mediated heterochromatin silencing at centromeres. Spf30 colocalizes with the exosome RNase Dis3 at centromeric heterochromatin; Dis3 helps recruit Spf30. Loss of Spf30 phenocopies the dis3-54 mutant: reduced silencing and accumulation of polyadenylated centromeric transcripts without loss of siRNA production. Spf30 binds centromeric transcripts and localizes to centromeres in an RNA-dependent manner.\",\n      \"method\": \"Genetic epistasis (double mutant analysis), chromatin immunoprecipitation (ChIP), RNA immunoprecipitation, fluorescence localization studies in S. pombe\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis plus ChIP and RNA-IP in fission yeast ortholog, multiple methods, single lab\",\n      \"pmids\": [\"20028739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPF30 associates with the MTR4-exosome RNA-decay machinery. The interaction between SPF30 and the exosome core is mediated by MTR4 and RRP6. The N- and C-terminal regions of SPF30 (not the Tudor domain) mediate association with MTR4 and the exosome. SPF30 knockdown caused subtle delay in 12S pre-rRNA processing to mature 5.8S rRNA. Shotgun proteomics of the SPF30 interactome linked it to ribosome biogenesis, pre-mRNA splicing, and box C/D snoRNA biogenesis. The SPF30–MTR4 interaction is regulated by ATP hydrolysis of AAA-ATPase NVL2.\",\n      \"method\": \"Co-immunoprecipitation, domain-deletion mapping, shotgun proteomics (interactome), siRNA knockdown with rRNA processing assay\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP with domain mapping plus MS interactome and functional knockdown, single lab\",\n      \"pmids\": [\"33422691\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Knockdown of Smndc1 in a murine α-cell line triggers global repression of α-cell gene-expression programs and upregulation of β-cell markers, including the transcription factor Pdx1. Mechanistically, Smndc1 loss modulates the activities of the BAF and Atrx chromatin remodeling complexes to derepress Pdx1 expression, linking SMNDC1 function to control of both splicing and chromatin remodeling in pancreatic islet cell identity. The repressive role of SMNDC1 is conserved in human pancreatic islets.\",\n      \"method\": \"RNAi screen, siRNA knockdown, transcriptomic analysis, chromatin remodeling complex activity assays (BAF/ATRX), human islet knockdown experiments with insulin secretion and PDX1 expression readouts\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi knockdown with defined cellular phenotype and pathway placement via chromatin complex perturbation, validated in human islets, single lab\",\n      \"pmids\": [\"36044849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SMNDC1 localizes to phase-separated membraneless organelles that partially overlap with nuclear speckles. This condensation is driven by the unstructured C-terminal region of SMNDC1, depends on RNA interaction, and can be recapitulated in vitro. Small-molecule inhibitors targeting the dimethylarginine-binding pocket of the SMNDC1 Tudor domain drastically alter protein-protein interactions and subcellular localization, and cause splicing changes for SMNDC1-dependent genes.\",\n      \"method\": \"Live-cell fluorescence imaging, in vitro phase-separation assay, small-molecule Tudor domain inhibitors, proteomics of inhibitor-treated cells, splicing assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (live imaging, in vitro reconstitution, chemical perturbation with proteomic and splicing readouts), single lab but comprehensive\",\n      \"pmids\": [\"37587144\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A poison exon in Smndc1 (within intron 2) is conserved across mammals and plants and mediates autoregulatory control of SMNDC1 protein levels via nonsense-mediated mRNA decay (NMD). Mice and A. thaliana lacking this poison exon show deregulated SMNDC1 protein levels, pervasive alterations in mRNA processing, and organismal size restriction, demonstrating that this autoregulatory mechanism is functionally important in vivo.\",\n      \"method\": \"Mouse and Arabidopsis genetic models with poison-exon deletion, RT-PCR for NMD-targeted transcripts, mRNA processing analysis, organismal growth phenotyping\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo loss-of-function in two independent organisms (mouse + plant) with molecular and organismal phenotypic readouts, convergent results\",\n      \"pmids\": [\"39150991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SPF30 autoregulates its own expression through a negative feedback mechanism: increased SPF30 promotes inclusion of a cassette exon (intron 2) and/or generation of an exon 4a splice variant, both of which are degraded by NMD, reducing SPF30 mRNA levels. Exon 4a inclusion contributes more than cassette exon inclusion to adjusting SPF30 levels. The C-terminal region of SPF30 (including the latter part of an α-helix and a kink-like structure) is required for binding to RNA containing exon 4a and for the autoregulatory mechanism. A short sequence within exon 4 of SPF30 mRNA is required for exon 4a inclusion.\",\n      \"method\": \"In vivo splicing assays with deletion constructs, siRNA knockdown and overexpression of SPF30, RT-PCR quantification of splice variants, RNA-binding domain mapping by mutagenesis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional assays (splicing reporters, RNA-binding domain mapping, NMD readout), single lab, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.03.04.641417\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"SMNDC1 (SPF30) is an essential nuclear splicing factor whose central Tudor domain binds dimethylated arginine (DMA) on Sm/RGG-motif proteins via an aromatic cage, mediating recruitment of the U4/U5/U6 tri-snRNP to the prespliceosome by simultaneously bridging U2AF35 (via its N-terminus) and hPrp3/tri-snRNP (via its C-terminus); its unstructured C-terminal region drives phase separation into nuclear speckle-associated condensates in an RNA-dependent manner; it also associates with the MTR4-exosome machinery through its N- and C-termini to participate in rRNA processing and RNA surveillance; and it autoregulates its own expression through NMD-inducing poison exon inclusion, a mechanism that depends on the C-terminal region binding to nascent pre-mRNA.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SMNDC1 (SPF30) is an essential nuclear splicing factor that drives recruitment of the U4/U5/U6 tri-snRNP to the prespliceosome during spliceosome assembly; in its absence the preformed tri-snRNP fails to dock onto the A complex [#0]. It bridges 3′ splice site recognition to tri-snRNP addition by binding U2AF35 through its N-terminus and hPrp3 through its C-terminus, two interactions that can occur simultaneously, while its central Tudor domain engages the C-terminal tails of Sm proteins SmD1, SmD3, and Lsm4 [#1]. This Tudor domain recognizes symmetrically and asymmetrically dimethylated arginine via an aromatic cage that mediates cation-π interactions, with binding enhanced by cooperative recognition of multiple methyl marks on RGG/GAR-rich ligands [#2]. The unstructured C-terminal region drives RNA-dependent phase separation into condensates that partially overlap nuclear speckles, and chemical occupation of the Tudor methylarginine pocket reorganizes SMNDC1 interactions, localization, and splicing of SMNDC1-dependent genes [#7]. Beyond splicing, SMNDC1 associates through its N- and C-termini (not the Tudor domain) with the MTR4-exosome RNA-decay machinery to participate in rRNA processing and RNA surveillance [#5], and it autoregulates its own abundance through inclusion of NMD-targeted poison/cassette exons in an evolutionarily conserved, in vivo-essential feedback loop [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established SMNDC1/SPF30 as a non-redundant assembly factor for the spliceosome, answering whether it acts before or during catalytic complex formation.\",\n      \"evidence\": \"Immunodepletion from HeLa nuclear extract with splicing assay plus GST pull-down/MS, in human cells\",\n      \"pmids\": [\"11331295\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which molecular surfaces contact the snRNPs\", \"Mechanism of bridging prespliceosome to tri-snRNP undefined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined the modular architecture by which SPF30 physically links 3′ splice site recognition to tri-snRNP recruitment, explaining how it docks the tri-snRNP onto the A complex.\",\n      \"evidence\": \"Co-IP and GST pull-down domain mapping with deletion constructs of SPF30, U2AF35, hPrp3, and Sm proteins\",\n      \"pmids\": [\"18211889\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo reconstitution of the bridged complex\", \"No structural validation of the simultaneous U2AF35/hPrp3 contacts\", \"Single lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Resolved the structural basis of SPF30's ligand recognition, showing the Tudor domain reads dimethylarginine through an aromatic cage and ranks as a comparatively weak DMA binder.\",\n      \"evidence\": \"NMR solution structures of Tudor–DMA peptide complexes with ITC and mutagenesis; corroborated by an independent crystal/binding study\",\n      \"pmids\": [\"22101937\", \"22363433\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of weak affinity for substrate selection unclear\", \"In-cell methylarginine targets not enumerated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended SPF30 function beyond splicing by linking the fission yeast ortholog to exosome-mediated heterochromatin silencing, raising the possibility of an RNA-surveillance role.\",\n      \"evidence\": \"Genetic epistasis, ChIP, RNA-IP, and fluorescence localization in S. pombe\",\n      \"pmids\": [\"20028739\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Conservation of the heterochromatin role to human SMNDC1 not established here\", \"Direct vs indirect exosome coupling unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected human SPF30 to the MTR4-exosome machinery and rRNA processing, showing termini rather than the Tudor domain mediate this RNA-decay association.\",\n      \"evidence\": \"Co-IP with domain mapping, shotgun interactome proteomics, and siRNA knockdown rRNA processing assay in human cells\",\n      \"pmids\": [\"33422691\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Only a subtle rRNA processing delay observed on knockdown\", \"Functional separation of splicing vs exosome roles unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed SMNDC1 in cell-identity control, showing its loss reprograms pancreatic α-cells toward β-cell programs via chromatin remodeling, linking splicing factor activity to chromatin output.\",\n      \"evidence\": \"RNAi/siRNA knockdown, transcriptomics, BAF/ATRX activity assays, validated in human islets\",\n      \"pmids\": [\"36044849\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether chromatin effects are direct or secondary to splicing changes unclear\", \"No mechanistic link between Tudor binding and BAF/ATRX modulation\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed that SMNDC1 forms RNA-dependent phase-separated condensates at nuclear speckles and that its Tudor pocket is chemically druggable with functional splicing consequences.\",\n      \"evidence\": \"Live-cell imaging, in vitro phase-separation reconstitution, small-molecule Tudor inhibitors with proteomic and splicing readouts in human cells\",\n      \"pmids\": [\"37587144\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Role of condensation in catalytic splicing not isolated\", \"Which RNA species nucleate condensation undefined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated that an evolutionarily conserved poison exon autoregulates SMNDC1 levels via NMD and is functionally essential in vivo across mammals and plants.\",\n      \"evidence\": \"Poison-exon deletion in mouse and Arabidopsis, RT-PCR of NMD transcripts, mRNA processing and growth phenotyping\",\n      \"pmids\": [\"39150991\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RNA element directing poison-exon inclusion not mapped here\", \"Trigger sensing excess SMNDC1 protein undefined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mapped the autoregulatory feedback to specific splice events and the C-terminal RNA-binding region, refining how SMNDC1 senses and tunes its own abundance.\",\n      \"evidence\": \"In vivo splicing reporters, SPF30 knockdown/overexpression, RT-PCR of variants, RNA-binding domain mutagenesis (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.03.04.641417\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint not yet peer-reviewed\", \"Direct binding of C-terminal region to exon 4a RNA not structurally resolved\", \"Relative contribution of exon 4a vs cassette exon in other tissues untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SMNDC1's splicing, exosome-surveillance, chromatin-remodeling, and condensate-forming activities are coordinated by a single protein, and which depend on Tudor-mediated methylarginine reading versus terminal interactions, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated structural model of SMNDC1 within the assembling spliceosome\", \"Causal hierarchy among splicing, chromatin, and exosome roles undefined\", \"No human disease linkage established in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [4, 7, 9]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 1, 5]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [\"spliceosome\", \"U4/U5/U6 tri-snRNP\", \"MTR4-exosome\"],\n    \"partners\": [\"U2AF35\", \"PRPF3\", \"SNRPD1\", \"SNRPD3\", \"LSM4\", \"MTR4\", \"EXOSC10\", \"NVL\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":5,"faith_total":5,"faith_pct":100.0}}