CRISPR editing of sftb-1/SF3B1 in Caenorhabditis elegans allows the identification of synthetic interactions with cancer-related mutations and the chemical inhibition of splicing
Fig 3
Synthetic interaction between sftb-1 mutant strains and knockdown of U2 snRNP and U2 snRNP-related components.
(A) Schematics of the RNAi screen. In brief, WT or mutant worms bearing any of the three sftb-1 single mutations were fed with RNAi clones against a total of 104 splicing factors. 77 clones had the same effect in all genetic backgrounds. In contrast, we identified 27 clones that caused a stronger phenotype in at least one of the three single mutant strains compared to WT, which was indicative of a synthetic interaction. Listed are the 3 U2 snRNP and 2 U2 snRNP-related candidates from the screen that were selected for further validation. (B) sftb-1[K718E] and sftb-1[Q552P] synthetically interact with teg-4(oz210). The number of hatched larvae (orange) and dead embryos (gray) laid by worms of the indicated genotype at 20°C is represented (n≥28; N = 3). m+ z-, homozygous mutant progeny of heterozygous mothers. No significant differences were observed in the number of hatched larvae laid by sftb-1[K718E], sftb-1[R643C], and sftb-1[Q552P] compared to WT. (C) Worm length after 48h of RNAi treatment against gfp (black) or sftb-1 (blue) at 25°C (n≥150; N = 2). (D) Worm length after 48h of RNAi treatment against gfp (black) or uaf-2 (blue) at 25°C (n≥150; N = 2). In both (C) and (D), worm length was not significantly different between sftb-1 mutants and WT upon gfp(RNAi). (E-G) Mean percentage of sterile worms observed upon RNAi of mog-2 (E; n≥30), smr-1 (F; n≥27) and teg-4 (G; n≥34) at 25°C. Red dots indicate percent sterility observed in each replicate (N = 2). Gray bars, P0-treated worms giving rise to <5 F1 larvae and some dead embryos (‘dead progeny’ category); black bars, P0-treated worms that laid neither larvae nor dead embryos (‘no progeny’ category). In (B), (C), and (D), data are shown as Tukey-style boxplots and overlaid dots representing measures in individual animals. Statistics: (B), (C), and (D), Kruskal-Wallis test with Dunn’s multiple comparisons test. Data were compared to teg-4(oz210) (B), or to WT animals fed with the same RNAi clone (C) and (D). (E-G), Fisher’s exact test with Bonferroni correction for multiple comparison. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.