Long-read sequencing and profiling of RNA-binding proteins reveals the pathogenic mechanism of aberrant splicing of an SCN1A poison exon in epilepsy
Year: 2023
Type: preprint
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.
Institutions: Northwestern University, Louisiana State University, University of California, Irvine, University of Colorado Anschutz, University of Colorado Denver, University of Antwerp, Antwerp University Hospital, VIB-UAntwerp Center for Molecular Neurology, Royal Children's Hospital, The University of Melbourne, Florey Institute of Neuroscience and Mental Health, Murdoch Children's Research Institute, Austin Health, St. Jude Children's Research Hospital
Language: English
Topic: RNA Research and Splicing
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Awards: G1004323642, G1284175734, G1382277591, G1697802790, G2124791380, G2323657099, National Resource for Translational and Developmental Proteomics, G2873586948, G317886719, G6329083223, G6544658276, Acquisition of a Thermo Fisher Scientific Q Exactive UHMR mass spectrometer, G777488164, G9000046704, G9000150115, G9000243610, G9000345326, The Robert H. Lurie Comprehensive Cancer Center
OpenAlex ID: W4372319081