Fig 2: U2AF1 mutations alter 3′ splice site consensus sequences. (A) Consensus 3′ splice sites of cassette exons with increased or decreased inclusion in U2AF1 mutant relative to WT AML transcriptomes. Boxes highlight sequence preferences at the −3 and +1 positions that differ from the normal 3′ splice site consensus. (Vertical axis) Information content in bits; (N) number of cassette exons with increased or decreased inclusion in each sample. Data for all U2AF1 mutant samples is shown in Supplemental Figure S6. (B) As in A, but for K562 cells expressing the indicated mutation versus WT. (C) As in A, but for K562 cells following U2AF1 KD or control KD. (D) Overlap between cassette exons that are promoted or repressed by mutant versus WT expression (rows) and U2AF1 versus control KD (columns) in K562 cells. The third column indicates the enrichment for U2AF1 dependence, defined as the overlap between exons affected by mutant U2AF1 expression and exons repressed versus promoted by U2AF1 KD.

Fig 3: U2AF1 mutations affect genes involved in disease-relevant cellular processes. (A) Overlap between mutation-dependent differential splicing in AML S34F/Y patients, K562 S34F/Y cells, and K562 Q157P/R cells. Overlap taken at the level of specific events (left) or genes containing differentially spliced events (right). (Percentages) The fraction of differentially spliced events (left) or genes containing differentially spliced events (right) in S34F/Y AML transcriptomes that are similarly differentially spliced in K562 cells expressing S34F/Y or Q157P/R U2AF1. (B) DNMT3B gene structure and protein domains (The UniProt Consortium 2012). Upstream 5′ UTR not shown. (PWWP) Pro-Trp-Trp-Pro domain; (ADD) ATRX-DNMT3-DNMT3L domain; (red stop sign) stop codon. (C,D) Inclusion of DNMT3B cassette exons. (Error bars) 95% confidence intervals as estimated from read coverage levels by MISO (Katz et al. 2010). (E) Inclusion of H2AFY cassette exon. (F) Cassette exon at 3′ end of ATR. Conservation is phastCons (Siepel et al. 2005) track from UCSC (Meyer et al. 2013). (G) Inclusion of cassette exon in ATR. (H) Usage of intron-proximal 3′ splice site of CASP8. (I) Inclusion of cassette exon in FANCA.

Fig 4: Theoretical model of the U2AF1:RNA complex. (A) Overview, with the zinc finger domains colored cyan, the RNA in salmon, the UHM beta sheet in blue, and alpha helices in red. The frequently mutated positions S34 and Q157 are shown in stick representation. (ZF) Zinc finger. (B–D) Interactions with individual bases characteristic of the 3′ splice site consensus. Green dotted lines indicate hydrogen bonds and favorable electrostatic interactions; RNA and selected side chains are shown in stick representation.

Fig 5: U2AF1 mutations contribute to splicing programs in AML. (A) U2AF1 domain structure (Kielkopf et al. 2001; The UniProt Consortium 2012) and common mutations. (CCCH) CCCH zinc finger. (B) Schematic of U2AF1 interaction with the 3′ splice site of a cassette exon (black). (C) Heat map illustrating similarity of alternative splicing programs in AML transcriptomes. Dendrogram is from an unsupervised cluster analysis based on cassette exon inclusion levels. (Blue) Samples with U2AF1 mutations. (D) U2AF1 mutant allele expression as a percentage of total U2AF1 mRNA in AML transcriptomes. Numbers above bars indicate the ratio of mutant to WT allele expression.