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

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: iCLIP-seq of Rbm38, Puf60 including exon 5, and Puf60 lacking exon 5, each in triplicate with size-matched input