Fig. 5: Eftud2 promotes the SHH signaling pathway activation via regulating Kif3a alternative splicing in vivo.

a Bar chart depicting the percentage of alternative splicing events altered in cerebellar tissue from SmoM2^fl/+ mice and tumor tissue from SmoM2^Atoh1 mice, as identified in RNA-seq data (Wilcoxon rank-sum test). b Pie chart illustrating the distribution of significantly changed alternative splicing event types in SmoM2^Atoh1 mouse tumors, as determined by Iso-seq. c KEGG enrichment analysis of alternative splicing genes in the Iso-seq data occurring in SmoM2^Atoh1 mouse tumors. d Wayne diagram showing Kif3a and Btrc co-existing in the Iso-seq gene set of SmoM2^Atoh1 mouse tumors, the RIP-seq (Eftud2-IP) gene set of SmoM2^Atoh1 mouse tumors, and the RNA-seq gene set (SHH signaling pathway) of siEFTUD2 treated Daoy cells. e Immunoprecipitation and RNA-binding protein precipitation verified that Eftud2 binds to both mRNA and protein of Kif3a in SmoM2^Atoh1 mouse tumors. Western blot validation f and quantification g of Kif3a in SmoM2^fl/+ mice cerebellum, SmoM2^Atoh1 mice and Eftud2^Atoh1; SmoM2^Atoh1 mice tumors (n = 6 mice, 3 males, 3 females, one-way ANOVA and Bonferroni’s multiple comparisons test). h Kif3a exons 10–11 skipped in SmoM2^Atoh1 mouse tumors. RT-PCR validation i–k and quantification l–n of Kif3a exon 9 i, l exons 10–11 j, m and exon 12 k, n in cerebellum of SmoM2^fl/+ and SmoM2^Atoh1 mouse tumors (n = 6 mice, 3 males, 3 females, one-way ANOVA and Bonferroni’s multiple comparisons test). The data are presented as the mean ± SD (bar plots). ****p < 0.0001, n.s. no significant. SE Skipped exon, A5SS Alternative 5′ splice site, A3SS Alternative 3′ splice site, MXE mutually exclusive exon, RI Retained intron.