Fig. 3. RECTAS directly interacts with CDC-like kinase 1 (CLK1), and it enhances cellular SRSF6 activity.
a Structures of RECTAS and b-RECTAS. b Extent of IKBKAP-FD exon 20 inclusion represented by GFP/RFP ratio, following treatment with biotin (50 µM), RECTAS (2 µM), b-RECTAS (2, 10, and 50 µM), or solvent only (0.1% DMSO) for 24 h in reporter-transfected HeLa cells. c, d Western blotting of pull-down products by b-RECTAS (0, 100, or 200 nmol/reaction) for HeLa cells transfected with a flag-tagged CLK1 expression vector (c) or GST-tagged recombinant CLK1 (d). RECTAS (500 nmol) was incubated with 100 nmol b-RECTAS for the competition assay in (d). e, f Western blot (e) for phosphor SRSF6 in FD patient primary fibroblasts (P1 and P2), treated with RECTAS (10 µM) or solvent only (0.1% DMSO) for 24 h. f Phosphor SRSF6 in FD fibroblasts (P1) was quantified at 0, 6, 10, 24 h after RECTAS (10 µM) treatment (columns, 0 h, 6 h, 10 h, and 24 h), as well as 10 h after washout of compound following RECTAS treatment (10 µM) for 24 h (column, wo). Lamin B1 served as a loading control in (e). g Western blot for U1-70k and SmB/B’ in pull-down products with biotin-conjugated RNA surrounding IKBKAP exon 20 donor site with (oAM154) or without (oAM153) IVS20 + 6 T > C mutation (labeled as FD and WT, respectively). Pull-down was conducted in the presence of 10 µM RECTAS with 1% DMSO, or 1% DMSO only. RNA (−), control sample without RNA bait. Input, HeLa nuclear extract of 5% input amount. Representative data from four replicates were shown in (b). Representative data from two replicates were shown in (c), (d), and (g). Data from two experiments are shown in (e, f) and three experiments are shown in (f). Columns, mean; bars, SE; n.d. ≥0.05; *P < 0.05; ***P < 0.001 for unpaired two-tailed t test in (b) (biotin, P = 0.26; RECTAS, P = 2.5 × 10−8; b-RECTAS 2 µM, 5.5 × 10−4; b-RECTAS 10 µM, 7.5 × 10−4; b-RECTAS 50 µM, 9.6 × 10−8) and one-tailed t test in (f) (vs. the 0 h control: 6 h, P = 1.6 × 10−2; 10 h, P = 2.8 × 10−5; 24 h, P = 4.8 × 10−2; wo, P = 0.18). No sample was loaded in blank lanes in (c, d).