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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 1

Characterization of sftb-1, the C. elegans ortholog of SF3B1, and different sftb-1 mutations.

(A) Number of SF3B1 missense mutations annotated in COSMIC database [75] at each amino acid position, with labels placed on the relevant residues for this study. A schematic structure of the SF3B1 protein is shown below, where purple, yellow, and blue boxes represent the U2AF ligand motifs (ULMs), the p14-interacting region, and the 20 HEAT repeats, respectively. The bars at the bottom line indicate residues that are conserved in the C. elegans protein. (B) Representative image of an embryo, L2, and L4 larvae with mCherry::SFTB-1 endogenous expression. The fluorescence image (top panel) is merged with the corresponding differential interference contrast (DIC) image (bottom panel). (C) Fluorescence images of dissected gonads from adult worms expressing mCherry::SFTB-1. Gonads were stained with DAPI to visualize nuclei. Insets show differentiated sperm nuclei, where mCherry::SFTB-1 is not expressed. (D) Left: diagram depicting how the cer6 deletion allele is maintained in heterozygosis. The WT sftb-1 copy is genetically linked to a red fluorescent marker (oxTi615). Right: worm body length of WT and heterozygotes (red, n = 475), and cer6 homozygotes (gray, n = 134) grown for 48 h at 20°C (N = 3). (E) Representative image of synchronized red and non-red siblings after growing for 48 h at 20°C. (F) Incidence of different sftb-1[Q552P, R643C, K718E] phenotypes at 25°C. Dots represent percentages observed in each replicate (n = 119, 116; N = 2). (G) Brood size of WT or sftb-1[Q552P, R643C, K718E] fertile worms at 25°C (n = 22, 35; N = 2). (H) sftb-1[Q552P, R643C, K718E] animals display developmental delay at 25°C. The proportion of the population at each stage is represented by the size of the dots (n = 119, 116; N = 2). In (D) and (G), dots represent measures in individual worms, and overlaid Tukey-style boxplots represent the median and interquartile range (IQR). Whiskers extend to the minimum and maximum values or at a maximum distance of 1.5xIQR from the box limits. Statistics: (D) and (G), Mann-Whitney’s test; (F), Fisher’s exact test. n.s., no significant difference, **** p<0.0001. Scale bars: 100 μm (B and E), 10 μm (C).

Fig 1

doi: https://doi.org/10.1371/journal.pgen.1008464.g001