(a) Missense mutations were detected in codons 34 and 157 of U2AF1. The ZnF1 (zinc finger 1), UHM (U2AF homology motif), ZnF2 (zinc finger 2), and RS (arginine-serine rich) domains are shown. The amino acid sequence of the ZnF1 domain is highly conserved (shaded). The zinc coordinating and mutated residue are shown in blue (asterisks) and red (arrow), respectively. (b) Deep sequencing of U2AF1 using DNA collected from paired normal, MDS, or secondary AML (sAML) samples. Mutant allele frequencies represent the proportion of sequencing reads supporting the mutant allele reads/total reads. Total read counts are shown below (mean 5,651 reads/sample). The mutation is present in the majority of cells (mutant allele frequency 31.4–48.2%) in all cases. (c) Deep sequencing of cDNA from MDS or sAML samples. The mutant allele is expressed in all cases tested. UPN, unique patient number.

(a) Transient coexpression of the pTN24 double-reporter splicing construct with or without a splicing enhancer (Tra2α), splicing inhibitor (hnRNPG), wild-type U2AF1, or mutant (S34F) U2AF1 cDNA in 293T cells. The double-reporter construct constitutively expresses β-galactosidase, while luciferase is expressed only if an upstream intron which contains multiple stop codons is spliced out. In the absence of the Tra2α splicing enhancer or hnRNPG splicing inhibitor, expression of the mutant U2AF1 increases splicing of the pTN24 construct resulting in an increase in the luciferase/β-galactosidase expression compared to expression of wild-type U2AF1 (P<0.001). Tra2α (positive control) and hnRNPG (negative control) cause increased or decreased splice efficiency, respectively. A representative Western blot of U2AF1 levels in the same cells used for luciferase assays is shown below each combination of plasmids. (b) Transient coexpression of the pTN24 double-reporter splicing construct with a control plasmid (vector) or mutant (S34F) U2AF1 cDNA in the presence of a control siRNA or siRNA targeting the endogenous U2AF1 in 293T cells. Expression of the S34F mutant U2AF1 results in an increase in splicing of the pTN24 construct with an increase in the luciferase/β-galactosidase expression compared to a control plasmid (P<0.001). The result is independent of endogenous U2AF1 expression levels. A representative Western blot of U2AF1 is shown below each condition. (c) The GH1 minigene was transiently transfected into 293T cells with a control plasmid (pcDNA3.1-YFP), wild-type U2AF1 cDNA, or mutant (S34F) U2AF1 cDNA in the presence of a control siRNA or siRNA targeting the endogenous U2AF1. RNA was harvested 48 hours later and a reverse transcriptase (RT) reaction was performed to create cDNA. PCR using the indicated primers resulted in a fully spliced 505 base pair amplicon or a 386 base pair amplicon that skips the middle exon shaded in black (exon skipping). A representative PCR gel image is shown and the ratio of the lower band (exon skipping = amplicon b) relative to the fully spliced upper band (amplicon a) is shown above each condition. Expression of the S34F mutant U2AF1 results in an increase in exon skipping compared to control or wild-type U2AF1 (P<0.02). bp, base pair; WT, wild-type; Mut, mutant; T7, T7 primer. Error bars, s.d.

(a) Overall and (b) Event-free survival are not impacted by U2AF1 genotype. (c) The probability of secondary AML (sAML) progression is increased in patients with U2AF1 mutations (P=0.03).