U2AF1 mutations cause sequence-dependent changes in 3′ splice site recognition. (A) Schematic of ATR minigene (top) and inclusion of ATR cassette exon transcribed from minigenes with A/C/G/T at the −3 position of the 3′ splice site in K562 cells expressing WT or S34Y U2AF1 (bottom). (Error bars) Standard deviation from biological triplicates. (B) Schematic of EPB49 minigene (top) and inclusion of EPB49 cassette exon transcribed from minigenes with A/C/G/T at the +1 position of the 3′ splice site in K562 cells expressing WT or Q157R U2AF1 (bottom). (C) Schematic of AdML pre-mRNA substrate used for in vitro splicing (top) and in vitro splicing of AdML substrate incubated with nuclear extract from K562 cells expressing WT or S34Y U2AF1 (bottom). Percentages are the fraction of second step products (spliced mRNA and lariat intron) relative to all RNA species after 60 min of incubation. (RNA) Input radiolabeled RNA; (GG) pre-mRNA with the AG dinucleotide replaced by GG to illustrate the first step product of splicing; (black dot) exonucleolytic “chew back” product of the lariat intermediate.
