Systematic exploration of dynamic splicing networks reveals conserved multi-stage regulators of neurogenesis [SPARseq]

Alternative splicing (AS) is a critical regulatory layer, yet factors controlling networks of functionally coordinated splicing events during developmental transitions remain poorly understood. Here, we employ a multifaceted screening strategy to define factors that control dynamically regulated splicing events associated with neurogenesis. Among numerous previously unknown regulators, Rbm38 acts widely to negatively impact neural AS through Ptbp1-dependent and -independent mechanisms. Puf60, a ubiquitous splicing factor, is surprisingly found to promote neural splicing patterns. This activity is determined by a vertebrate-conserved, neural-differential exon that remodels Puf60 co-factor interactions. Ablation of this exon rewires distinct AS networks in embryonic stem cells and at multiple stages of neural Rbm38etc. Single-cell transcriptome analyses further reveal critical, multi-stage roles for Rbm38 and Puf60 isoforms in establishing neuronal identity. Our results thus reveal key new regulators of neurogenesis and establish how a single exon in a widely expressed splicing factor orchestrates temporal control over cell Rbm38etc. Overall design: Systematic Parallel Analysis of endogenous RNA regulation coupled to barcode Sequencing (SPAR-seq) of amplicons in 108 genes upon a total of 1536 knockdowns and controls in mouse N2A cells, in duplicate

可变剪接（Alternative Splicing, AS）是一类关键的基因调控层级，但在发育转变过程中，调控功能协同剪接事件网络的相关因子仍未被充分阐明。本研究采用多维度筛选策略，鉴定出与神经发生相关的动态调控剪接事件的调控因子。在众多此前未被报道的调控因子中，Rbm38可通过依赖Ptbp1与不依赖Ptbp1的两种机制，广泛负向调控神经源性可变剪接事件。普遍表达的剪接因子Puf60则被意外发现可促进神经剪接模式的形成。该活性由一个脊椎动物保守且具有神经特异性的外显子决定，该外显子可重塑Puf60的辅因子互作模式。敲除该外显子可重塑胚胎干细胞以及神经发生多个阶段中独特的可变剪接网络（涉及Rbm38等通路）。单细胞转录组分析进一步揭示了Rbm38与Puf60的剪接变体在确立神经元身份过程中发挥的关键多阶段调控作用。本研究结果不仅鉴定出神经发生的关键新型调控因子，还阐明了广泛表达的剪接因子中的单个外显子如何介导细胞相关过程的时间维度调控（如Rbm38等相关通路）。整体实验设计：本研究在小鼠N2A细胞中对108个基因开展共计1536次基因敲降实验与对照实验，并设置生物学重复，采用结合条形码测序的内源RNA调控系统平行分析（Systematic Parallel Analysis of endogenous RNA regulation coupled to barcode Sequencing, SPAR-seq）技术对扩增子进行检测。