Determination of Trunk Neural Crest Cell Fate and Susceptibility to Splicing Perturbation by the DLC1-SF3B1-PHF5A splicing complex

How the ubiquitously expressed splicing factors specifically regulate neural crest (NC) development and enhance their vulnerability to splicing perturbations remain poorly understood. Here, we show that NC-specific DLC1, partnering with SF3B1-PHF5A splicing complex, are crucial for determining avian trunk NC cell fate by regulating the splicing of NC specifiers SOX9 and SNAI2 pre-mRNAs rather than their upstream regulators BMP4, WNT1, and PAX7. Mechanistically, SF3B1-PHF5A binds to the intronic branch site (BS) sequences of all factors, while DLC1 interacts with a specific motif near the BS sequences of SOX9 and SNAI2, thereby determining their functional specificity in NC specification. Moreover, DLC1 increases NC cells’ vulnerability to splicing modulator pladienolide B by reducing the binding capacity of the SF3B1-PHF5A splicing complex to the shorter length of both SOX9 intron 2 and SNAI2 intron 1, which possess weaker polypyrimidine tract 3’ of the BS sequence, resulting in intron retention and loss of NC progenitors. Conversely, somite specific SLU7-SF3B1-PHF5A splicing complex regulates SOX9 and SNAI2 expression and imparts resistance to PB. Our data reveal the cell-type specific splicing complexes with distinct vulnerabilities to PB, highlighting the critical role of the DLC1-SF3B1-PHF5A in determining trunk NC cell fate and enhancing its susceptibility to splicing perturbation. RNA-seq profiling of GFP+ cells following CRISPR-Cas9-mediated knockout of DLC1, PHF5A, and SF3B1 compared to scrambled gRNA control samples.