{"doi":"10.1101/2023.05.04.538282","title":"Long-read sequencing and profiling of RNA-binding proteins reveals the pathogenic mechanism of aberrant splicing of an <i>SCN1A</i> poison exon in epilepsy","abstract":"Abstract Pathogenic loss-of-function SCN1A variants cause a spectrum of seizure disorders. We previously identified variants in individuals with SCN1A -related epilepsy that fall in or near a poison exon (PE) in SCN1A intron 20 (20N). We hypothesized these variants lead to increased PE inclusion, which introduces a premature stop codon, and, therefore, reduced abundance of the full-length SCN1A transcript and Na v 1.1 protein. We used a splicing reporter assay to interrogate PE inclusion in HEK293T cells. In addition, we used patient-specific induced pluripotent stem cells (iPSCs) differentiated into neurons to quantify 20N inclusion by long and short-read sequencing and Na v 1.1 abundance by western blot. We performed RNA-antisense purification with mass spectrometry to identify RNA-binding proteins (RBPs) that could account for the aberrant PE splicing. We demonstrate that variants in/near 20N lead to increased 20N inclusion by long-read sequencing or splicing reporter assay and decreased Na v 1.1 abundance. We also identified 28 RBPs that differentially interact with variant constructs compared to wild-type, including SRSF1 and HNRNPL. We propose a model whereby 20N variants disrupt RBP binding to splicing enhancers (SRSF1) and suppressors (HNRNPL), to favor PE inclusion. Overall, we demonstrate that SCN1A 20N variants cause haploinsufficiency and SCN1A -related epilepsies. This work provides insights into the complex control of RBP-mediated PE alternative splicing, with broader implications for PE discovery and identification of pathogenic PE variants in other genetic conditions.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":396335,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9572,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1171862,"name":"Patricia N. Schneider","orcid":"0000-0002-9745-0694","position":1,"is_corresponding":false},{"id":687598,"name":"Jung Hwa Hong","orcid":"0000-0002-6819-9242","position":2,"is_corresponding":false},{"id":1172232,"name":"Eleanor Goes","orcid":null,"position":3,"is_corresponding":false},{"id":1172233,"name":"Masha D Bandouil","orcid":null,"position":4,"is_corresponding":false},{"id":1171863,"name":"Carina G Biar","orcid":"0000-0002-0647-3752","position":5,"is_corresponding":false},{"id":938255,"name":"Aishwarya Ramamurthy","orcid":"0000-0003-3965-5082","position":6,"is_corresponding":false},{"id":479159,"name":"Fairlie Reese","orcid":"0000-0002-9240-0102","position":7,"is_corresponding":false},{"id":278700,"name":"Krysta L. Engel","orcid":"0000-0003-3132-1491","position":8,"is_corresponding":false},{"id":65647,"name":"Sarah Weckhuysen","orcid":"0000-0003-2878-1147","position":9,"is_corresponding":false},{"id":154477,"name":"Ingrid E Scheffer","orcid":"0000-0002-2311-2174","position":10,"is_corresponding":false},{"id":320041,"name":"Heather C. Mefford","orcid":"0000-0001-7188-522X","position":11,"is_corresponding":false},{"id":741878,"name":"Jeffrey D. Calhoun","orcid":"0000-0003-0554-2231","position":12,"is_corresponding":false},{"id":320120,"name":"Gemma L. Carvill","orcid":"0000-0003-4945-3628","position":13,"is_corresponding":false},{"id":741880,"name":"Hannah C. Happ","orcid":"0000-0002-8266-266X","position":0,"is_corresponding":true}],"reference_count":29,"raw_metadata":null,"created_at":"2026-07-19T01:19:31.472087Z","pmid":"37205386","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}