Figure 1. Esrp1^Triaka leads to altered mRNA splicing and reduced epithelial cell proliferation.

To quantify exon splicing, HEK-293 cells were co-transfected with a vector encoding Esrp1^WT or Esrp1^Triaka or an empty control (CTRL) vector and with an exon trap construct containing (A) Cd44 variant 5 (Cd44v5) or (B) Ffgr2-IIIb variable exon. Upper panels in (A) and (B) show schemes of the respective exon trap constructs. Inclusion of exon Cd44v5 or Ffgr2-IIIb results in luciferase (Luc) expression. Luciferase activity normalized to control vector-transfected cells is shown in the lower panels. Relative expression of (C) Esrp1 and (D) Cd44v4/5 transcripts were measured in CMT-93 cells transduced with inducible vectors encoding Esrp1^WT, Esrp1^Triaka, or a control construct, after treatment with 4-hydroxytamoxifen. (E) Proliferation of Esrp1^WT- and Esrp1^Triaka-expressing CMT-93 cells was measured using a WST-1 assay and normalized to control vector-transduced cells. (F) Relative expression of Esrp1 and Cd44v4/5 transcripts were measured in CMT-93 cells transduced with a vector encoding Esrp1^WT, after induction with 4-hydroxytamoxifen, and correlated. Data represent: Pooled means ± standard error of the mean from (A and B) five or (C–E) three independent experiments performed in biological triplicates. (F) Means measured in technical duplicates (n = 22). Statistics: (A–D) One-way ANOVA with Bonferroni post-test. (E) Two-way ANOVA with Bonferroni post-test. (F) Spearman correlation. **p<0.01; ***p<0.001; ****p<0.0001.

Figure 1—figure supplement 1. Mapping of the Triaka mutation.

(A) The Triaka mutation was mapped based on the hyperactivity and circling phenotype of the strain to proximal Chromosome 4, with a peak LOD score of 2.71 at D4Mit235. (B) The Triaka mutation causes a methionine to valine substitution at the amino acid position 161 (M161V) of the gene Esrp1. The mutated residue is conserved in several species and is indicated by a red box.