The cancer-associated U2AF35 470A>G (Q157R) mutation creates an in-frame alternative 5′ splice site that impacts splicing regulation in Q157R patients

The U2AF35 Q157R and Q157P alleles create three proteins that affect different target exons. (A) Western blot of endogenous U2AF35 in the presence and absence of siU2AF35 and rescue with ectopically expressed Flag-tagged U2AF35wt, Q157R, Q157Rdel, and Q157P variants in HEK293T cells. hnRNPL was used as the loading control. (B) Example of a splicing-sensitive radioactive RT-PCR for CHEK2 in HEK293T cells with a knockdown of endogenous U2AF35 and rescue with ectopically expressed U2AF35 mutants. (C) Venn diagram showing overlaps of Q157P, Q157R, and Q157Rdel rescued U2AF35-dependent splicing targets analyzed via radioactive RT-PCR. In total, splicing of 16 alternative exons was analyzed. (D,E) Quantifications of splicing-sensitive radioactive RT-PCRs as in B for targets where Q157Rdel can (D) or cannot (E) rescue U2AF35 knockdown. n ≥ 3; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.