Figure 3.
Allele-specific effects on RNA binding and splicing in splicing factor mutations seen in patients harboring 2 concomitant mutations in splicing factors. (A) Pie chart of SRSF2 P95 amino acid substitutions across the entire cohort. (B) Binding affinities of WT vs P95H/L/R/A mutant SRSF2 peptides to UCCAGU RNA oligonucleotides as absolute Kd values. The column labeled “change in affinity” provides the Kd ratio of the mutant:WT peptide. (C) Comparison of the quantitative effects of SF3B1K700E and SF3B1K666N mutations on splicing, stratified by mutant allele fraction. Each point illustrates the absolute change in isoform usage (ΔPSI) for 1 of the top 20 most misspliced events associated with each mutation. For each panel, the top 20 most misspliced events were computed using only samples with SF3B1K700E or SF3B1K666N mutations. Missplicing of those 20 events was then computed for all samples, irrespective of mutation, and plotted as illustrated. SF3B1K700E and SF3B1K666N mutations cause missplicing of similar sets of genes, but SF3B1K700E mutations cause more dramatic changes. Lines, shading, and equations indicate the best-fit linear regressions and corresponding 95% confidence intervals. (D) As in panel C, but computed using the top 5 most misspliced events for each mutation (SF3B1K700E; SF3B1K666N). (E) Box plot illustrating the data from panel C and associated P-value, computed using a 2-sided Wilcoxon rank-sum test. (F) Fraction of U2AF1S34F, U2AF1Q157R, or dual U2AF1S34F/Q157R mutated cells from a patient harboring both U2AF1S34F/WT and U2AF1Q157R/WT mutations. Red bar indicates fraction of U2AF1S34F/Q157R dual mutant cells. (G) Clonal hierarchy of mutations in the patient from panel E. Each column represents a cell at the indicated scale, as in panel A. Cells with mutations and WT cells are indicated in blue and white, respectively. (H) Fish plots showing the inferred clonal hierarchy based on the single-cell genotype data from panel G. (I) Sanger sequencing electropherograms from representative single cell clones from the patient in panel F. As enumerated on the right, all colonies were either U2AF1 dual WT or U2AF1S34F/Q157R dual mutant, indicating that these mutations always occur in cis with preservation of the WT allele. (J) Heat map of percentage spliced in values of cassette exons in patients with U2AF1S34, Q157, and U2AF1S34/Q157 dual mutations displaying cassette exon splicing events specific to the U2AF1S34 or Q157 single-mutant state. Standard deviation of <0.2 among single mutants and mean(U2AF1 S34) − mean (U2AF1 Q157) < 0.32. Each row is a unique patient, and each column is a single splicing event.

Allele-specific effects on RNA binding and splicing in splicing factor mutations seen in patients harboring 2 concomitant mutations in splicing factors. (A) Pie chart of SRSF2 P95 amino acid substitutions across the entire cohort. (B) Binding affinities of WT vs P95H/L/R/A mutant SRSF2 peptides to UCCAGU RNA oligonucleotides as absolute Kd values. The column labeled “change in affinity” provides the Kd ratio of the mutant:WT peptide. (C) Comparison of the quantitative effects of SF3B1K700E and SF3B1K666N mutations on splicing, stratified by mutant allele fraction. Each point illustrates the absolute change in isoform usage (ΔPSI) for 1 of the top 20 most misspliced events associated with each mutation. For each panel, the top 20 most misspliced events were computed using only samples with SF3B1K700E or SF3B1K666N mutations. Missplicing of those 20 events was then computed for all samples, irrespective of mutation, and plotted as illustrated. SF3B1K700E and SF3B1K666N mutations cause missplicing of similar sets of genes, but SF3B1K700E mutations cause more dramatic changes. Lines, shading, and equations indicate the best-fit linear regressions and corresponding 95% confidence intervals. (D) As in panel C, but computed using the top 5 most misspliced events for each mutation (SF3B1K700E; SF3B1K666N). (E) Box plot illustrating the data from panel C and associated P-value, computed using a 2-sided Wilcoxon rank-sum test. (F) Fraction of U2AF1S34F, U2AF1Q157R, or dual U2AF1S34F/Q157R mutated cells from a patient harboring both U2AF1S34F/WT and U2AF1Q157R/WT mutations. Red bar indicates fraction of U2AF1S34F/Q157R dual mutant cells. (G) Clonal hierarchy of mutations in the patient from panel E. Each column represents a cell at the indicated scale, as in panel A. Cells with mutations and WT cells are indicated in blue and white, respectively. (H) Fish plots showing the inferred clonal hierarchy based on the single-cell genotype data from panel G. (I) Sanger sequencing electropherograms from representative single cell clones from the patient in panel F. As enumerated on the right, all colonies were either U2AF1 dual WT or U2AF1S34F/Q157R dual mutant, indicating that these mutations always occur in cis with preservation of the WT allele. (J) Heat map of percentage spliced in values of cassette exons in patients with U2AF1S34, Q157, and U2AF1S34/Q157 dual mutations displaying cassette exon splicing events specific to the U2AF1S34 or Q157 single-mutant state. Standard deviation of <0.2 among single mutants and mean(U2AF1 S34) − mean (U2AF1 Q157) < 0.32. Each row is a unique patient, and each column is a single splicing event.

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