Long-read sequencing and profiling of RNA-binding proteins reveals the pathogenic mechanism of aberrant splicing of an SCN1A poison exon in epilepsy

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 Nav1.1 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 Nav1.1 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 Nav1.1 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.

致病性功能丧失型SCN1A变异（pathogenic loss-of-function SCN1A variants）可引发一系列癫痫发作相关疾病。我们此前在SCN1A相关癫痫患者中鉴定到了位于SCN1A内含子20（20N）的毒外显子（poison exon, PE）内或其邻近区域的变异。我们推测此类变异可提升毒外显子的剪接入率，进而引入提前终止密码子，最终降低全长SCN1A转录本与Nav1.1蛋白的丰度。我们借助剪接报告基因实验，在HEK293T细胞中对毒外显子的剪接入水平进行了探究。此外，我们将患者来源的诱导多能干细胞（induced pluripotent stem cells, iPSCs）诱导分化为神经元，通过长读长测序与短读长测序定量分析20N的剪接入水平，并通过蛋白质印迹法检测Nav1.1的蛋白丰度。我们还利用RNA反义纯化联合质谱技术，鉴定了可能参与异常毒外显子剪接的RNA结合蛋白（RNA-binding proteins, RBPs）。研究结果表明，20N区域内或邻近的变异可通过长读长测序或剪接报告基因实验证实提升20N的剪接入水平，并降低Nav1.1的蛋白丰度。我们还鉴定出28种与野生型载体相比，在变异型载体中差异结合的RNA结合蛋白，其中包括SRSF1与HNRNPL。我们提出了如下调控模型：20N区域的变异会破坏RNA结合蛋白与剪接增强子（SRSF1）及剪接抑制子（HNRNPL）的结合，从而促进毒外显子的剪接入。综上，我们证实SCN1A 20N变异可导致单倍体剂量不足，并引发SCN1A相关癫痫。本研究揭示了RNA结合蛋白介导的毒外显子可变剪接的复杂调控机制，其研究成果可为其他遗传疾病中毒外显子的发现及致病性毒外显子变异的鉴定提供新的思路与参考。