Figure 3.
Rare mutations in U2AF1 alter 3′ splice site recognition. (A) Differentially spliced events identified in K562 cells expressing each indicated U2AF1 mutant allele relative to WT-expressing control cells. Percentages represent the distribution of differentially spliced events among the indicated event types for each mutation. (B) Sequence logos representing consensus 3′ splice sites of cassette exons that are differentially spliced in K562 cells expressing mutant versus WT U2AF1. Gray boxes highlight sequence preferences at the −3 and +1 positions that are similar to those observed in cells expressing the U2AF1S34F/Y or U2AF1Q157P/R hotspot mutations. RNA-seq data from patient samples bearing U2AF1I24T (adrenocortical carcinoma), U2AF1S34F (AML), U2AF1R156H (myelodysplastic syndromes), U2AF1Q157R (AML), and U2AF1E159_M160insYE (AML) were previously published.34-37 (C) RNA-seq read coverage illustrating increased cassette exon inclusion in RHBDD2 in K562 cells expressing either a hotspot (S34F) or rare (I24T) U2AF1 mutation (top). Log2 (fold-change) illustrates log2 (exon inclusion in mutant- vs WT-expressing cells). RT-PCR validation of RNA-seq results in technical triplicate (bottom). Log fold-changes for RT-PCR computed with respect to the mean signal for WT. (D) As panel C, but for mutually exclusive exons in H2AFY. The upstream (orange) exon is the exon for which inclusion is calculated. (E) Relative inclusion of the upstream vs downstream exon for 2 mutually exclusive exons within H2AFY expressed from its endogenous locus in K562 cells expressing mutant versus WT U2AF1 as estimated by RNA-seq. Error bars represent 95% confidence intervals for the relative inclusion ratio, computed by propagating the 95% confidence intervals for the 2 isoforms to the ratio for mutant vs WT SRSF2 by standard rules for error propagation during division of quantities with individual errors. (F) As in panel (E), but where the H2AFY mutually exclusive exons are expressed from a minigene transfected into K562 cells and contain 3′ splice sites with the indicated sequences. AG is the AG dinucleotide of the 3′ splice site. Bars represent the mean ratio of inclusion of the upstream:downstream exons ± standard deviation, estimated by quantitative RT-PCR and computed over 3 biological replicates.

Rare mutations in U2AF1 alter 3′ splice site recognition. (A) Differentially spliced events identified in K562 cells expressing each indicated U2AF1 mutant allele relative to WT-expressing control cells. Percentages represent the distribution of differentially spliced events among the indicated event types for each mutation. (B) Sequence logos representing consensus 3′ splice sites of cassette exons that are differentially spliced in K562 cells expressing mutant versus WT U2AF1. Gray boxes highlight sequence preferences at the −3 and +1 positions that are similar to those observed in cells expressing the U2AF1S34F/Y or U2AF1Q157P/R hotspot mutations. RNA-seq data from patient samples bearing U2AF1I24T (adrenocortical carcinoma), U2AF1S34F (AML), U2AF1R156H (myelodysplastic syndromes), U2AF1Q157R (AML), and U2AF1E159_M160insYE (AML) were previously published.34-37  (C) RNA-seq read coverage illustrating increased cassette exon inclusion in RHBDD2 in K562 cells expressing either a hotspot (S34F) or rare (I24T) U2AF1 mutation (top). Log2 (fold-change) illustrates log2 (exon inclusion in mutant- vs WT-expressing cells). RT-PCR validation of RNA-seq results in technical triplicate (bottom). Log fold-changes for RT-PCR computed with respect to the mean signal for WT. (D) As panel C, but for mutually exclusive exons in H2AFY. The upstream (orange) exon is the exon for which inclusion is calculated. (E) Relative inclusion of the upstream vs downstream exon for 2 mutually exclusive exons within H2AFY expressed from its endogenous locus in K562 cells expressing mutant versus WT U2AF1 as estimated by RNA-seq. Error bars represent 95% confidence intervals for the relative inclusion ratio, computed by propagating the 95% confidence intervals for the 2 isoforms to the ratio for mutant vs WT SRSF2 by standard rules for error propagation during division of quantities with individual errors. (F) As in panel (E), but where the H2AFY mutually exclusive exons are expressed from a minigene transfected into K562 cells and contain 3′ splice sites with the indicated sequences. AG is the AG dinucleotide of the 3′ splice site. Bars represent the mean ratio of inclusion of the upstream:downstream exons ± standard deviation, estimated by quantitative RT-PCR and computed over 3 biological replicates.

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