Systematic exploration of dynamic splicing networks reveals conserved multistage regulators of neurogenesis

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 mammalian 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 mouse 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 establish how an alternative exon in a widely expressed splicing factor orchestrates temporal control over cell differentiation.The authors thank members of the Blencowe and Moffat laboratories for helpful discussions, and the Donnelly Sequencing Centre are gratefully acknowledged for sequencing samples. We also thank Drice Challal, Mingkun Wu, and Patricia Mero for technical assistance. Our research was funded by grants from the Canadian Institutes of Health Research (B.J.B., J.M., H.H., A.-C.G., J.L.W., and J.F.G.), Canada First Research Excellence Fund Medicine by Design Program (B.J.B., J.M, and J.L.W.), and European Union's Horizon 2020 Research and Innovation Program (ERC-CoG-LS2-101002275) to M.I. H.H. was supported by the Donnelly Centre Home Fellowship and a MITACS Award. A.J.B. was supported by Postdoctoral Fellowships from EMBO and CIHR. Proteomics work was performed at the Network Biology Collaborative Centre at the Lunenfeld-Tanenbaum Research Institute, a facility supported by the Canada Foundation for Innovation funding, Government of Ontario, Genome Canada, and Ontario Genomics (OGI-139). B.J.B. holds the University of Toronto Banbury Chair in Medical Research and the Canada Research Chair in RNA Biology and Genomics