Strategy for classification of rare, non-hotspot SRSF2 and U2AF1 mutations. (A) Hotspot (bold) and select rare and private mutations affecting SRSF2 and U2AF1. RRM, RNA recognition motif; RS, arginine/serine-rich domain; UHM, U2AF homology motif; Zn, zinc finger domain. (B) Numbers of reported mutations in SRSF2 and U2AF1 in the Catalogue of Somatic Mutations in Cancer (COSMIC) database as of 17 September 2018. SRSF2S54A was identified in a patient sample, but is not present in COSMIC. (C) Schematic of our strategy for transgenically expressing individual mutations in cell culture and performing subsequent transcriptome analyses. (D) Western blot for FLAG, SRSF2, and Histone H3 (H3), using lysate from untransduced K562 cells or K562 cells that stably expressed FLAG-tagged WT or mutant SRSF2 (mutation indicated earlier). H3 is a loading control. FLAG and SRSF2 band intensities were quantified using ImageJ and normalized to the respective band intensity for H3. (E) As in panel D, but for U2AF1. (F-G) Heat map and associated dendrogram representing an unsupervised cluster analysis based on cassette exon inclusion levels computed from the transcriptomes of K562 cells stably expressing the indicated alleles of SRSF2 (F) or U2AF1 (G). Exon inclusion values were z-score normalized.