Fig. 7: The DLC1-SF3B1-PHF5A complex binding to shorter introns of NC specifier genes increases their vulnerability to splicing perturbation by PB, whereas SLU7-SF3B1-PHF5A regulates the splicing of SOX9 and SNAI2 pre-mRNAs and confers resistance to PB in somites.

Overexpression of DLC1 in somites of embryos treated with PB resulted in reduced binding capacity of PHF5A to SOX9 intron 2 (a) and SNAI2 intron 1 (b) compared to PBS Ctrl. Overexpression of DLC1 in somites of embryos treated with PB led to a reduction in the binding capacity of SF3B1 to SOX9 intron 2 (c) and SNAI2 intron 1 (d) compared to PBS Ctrl. n = 3 embryos per treatment. Interactomes of splicing factors associated with PHF5A (e) and SF3B1 (f). PHF5A, SF3B1, and SLU7 are highlighted in green. g IP validation of the association of endogenous SLU7 with PHF5A and SF3B1. h Co-localization of SLU7, SOX9 and SNAI2 in somites. Open arrowheads indicate the expression of genes in the dorsal neural tube, somites, and notochord. i RT-qPCR demonstrated reduced expression levels of SLU7 in gRNA treatment compared to Ctrl gRNA, n = 5 embryos per treatment. HCR (j) and RT-qPCR (k) analysis revealed the loss of SOX9 and SNAI2 in SLU7-depleted somites, n = 5 embryos per treatment. IR-qPCR analysis showed increased levels of SOX9 introns 1/2 (l) and SNAI2 introns 1/3 (m) in embryos treated with SLU7 gRNA1 compared to Ctrl gRNA, n = 5 embryos per treatment. n HCR showing unaltered levels of SOX9 and SNAI2 expression in the dorsal neural tubes transfected with SLU7 followed by PB or PBS treatment. Insets indicate the right side of neural tube with SLU7 overexpression. Scale bar, 50 µm. The exact p-values are listed in the Source data file. Mean ± SD. ns: no significant difference. **P < 0.01, ***P < 0.001, ****P < 0.0001.