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. 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