Neu-SPAR-Seq

Alternative splicing (AS) is a critical regulatory layer, yet factors controlling functionally coordinated splicing programs during developmental transitions are poorly understood. Here, we employ a screening strategy to identify factors controlling dynamic splicing events important for neurogenesis. Among previously unknown regulators, Rbm38 acts widely to negatively control neural AS, in part through interactions mediated by the established repressor of splicing, Ptbp1. Puf60, a ubiquitous factor, is surprisingly found to promote neural splicing patterns. This activity requires a conserved, neural-differential exon that remodels Puf60 co-factor interactions. Ablation of this exon rewires distinct AS networks in embryonic stem cells and at different stages of neurogenesis. Single-cell transcriptome analyses further reveal distinct roles for Rbm38 and Puf60 isoforms in establishing neuronal identity. Our results describe important roles for previously unknown regulators of neurogenesis and further establish how an alternative exon in a widely expressed splicing factor orchestrates temporal control over cell differentiation.可变剪接（Alternative splicing, AS）是关键的基因调控层面，然而在发育转变过程中调控功能协同性剪接程序的因子尚未得到充分解析。本研究采用筛选策略，旨在鉴定调控对神经发生至关重要的动态剪接事件的因子。在此前未被发现的调控因子中，Rbm38可广泛负向调控神经源性可变剪接，其部分作用通过与经典剪接抑制因子Ptbp1介导的相互作用实现。普遍表达的因子Puf60则意外被发现可促进神经剪接模式的建立。该活性依赖于一个保守的神经特异性差异外显子，该外显子可重塑Puf60的辅因子相互作用网络。敲除该外显子可重塑胚胎干细胞以及不同神经发生阶段的特异性可变剪接网络。单细胞转录组（single-cell transcriptome）分析进一步揭示了Rbm38与Puf60异构体在确立神经元身份过程中的差异化功能。本研究结果阐明了此前未被发现的神经发生调控因子的重要功能，并进一步揭示了广泛表达的剪接因子中的可变外显子如何协调细胞分化的时序调控过程。