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

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 differentiation. 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 differentiation. Overall design: RNA-seq of CGR8 mouse embryonic stem cells with mutations of the splicing factors Puf60 and Rbm38, and their parental wild type, as well as embryoid bodies and glutamatergic neurons differentiated from them, from two independent clones.