A requirement of BuGZ and Bub3 for RNA splicing. (A) Proteins with known functions in RNA splicing (highlighted in orange) were found as candidate BuGZ-interacting proteins. (B) FLAG-tagged BuGZ or Bub3, but not FLAG-luciferase (Luci.), immunoprecipitated the spliceosome components SF3a3 and U2AF65 in HEK293T cells. (C) BuGZ and Bub3 interacted with SF3a3 and U2AF65 in vivo. Reciprocal immunoprecipitations of endogenous proteins were performed using HT29 cell lysates with the indicated mouse monoclonal (mAb) or rabbit antibodies (rAb). h.c., antibody heavy chain. Experiments were performed three times. (D) Analyses of splicing defects by RNA-sequencing of HFFs and TOV21G cells treated by the splicing inhibitor pladienolide B or RNAi of BuGZ or Bub3. The numbers of genes exhibiting the depicted splicing defects are shown as pie charts. RNA-Seq was from two biological repeats. (E) Two representative genes, BRD2 and WAC, exhibiting intron retention upon treatment with pladienolide B or RNAi of BuGZ or Bub3. The red boxes highlight examples of intron retention. The fold increases of the intron retention are plotted to the right. The p-values were generated by simulation (MATS: a Bayesian framework for flexible detection of differential alternative splicing from RNA-Seq data).