Extended Data Fig. 7: YC-1 covalently binds RNA processing factors and influences RNA splicing.

a, Immunoblot from streptavidin affinity purification validating YC-1 binding proteins. RBE cells were treated with YC-1 biotin or DH-YC-1 biotin control in the presence of excess non-biotinylated YC-1 or DH-YC-1 as indicated. Left: Expression of candidate YC-1 binding proteins in whole cell lysates. Right: Immunoblot after Streptavidin capture, showing dose-dependent competition by parent YC-1. b, Immunoblot from TARDBP immunoprecipitation validating direct YC-1 binding. RBE cells were treated as in (a). The immunoblots (a, b) were performed two times with similar results. c, Scatter plot of genes with altered intron retention identified from RNA-seq analysis of RBE and SNU1079 cells treated with YC-1 or vehicle for 6 and 16 hours. n = 3 biological replicates per condition. ΔRI is the intron retention score calculated by the SALMON software package. d, Left, a TARDBP splicing efficiency assay assessing SULT1A1 dependent YC-1 impact on TARDBP RNA splicing activity. 293 T cells exogenously expressing SULT1A1 or empty vector were transiently transfected with the reporter module containing plasmid and treated with YC-1 or DMSO vehicle and analyzed by fluorescent confocal microscope with GFP (G) and mCherry (R) laser. Statistical significance annotated between individual conditions (Welch unpaired t-test). n = 3 biologically independent experiments with cells from two independent images per experiment included (>500 cells in total). ‘n.s.’ denotes not significant. Right, YC-1 sensitivity assay confirming stable SULT1A1 expression. Two biologically independent experiments are shown. e, siRNA targeting TARDBP (left) or DDX42 (right) reduced target protein expression and cell number monitored for 5–6 d post transfection. Error bars in left panel are mean ± s.d. Data shown were from one of the two performed experiments with similar results.

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