Precision analysis of mutant U2AF1 activity reveals deployment of stress granules in myeloid malignancies

Abstract

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.

Keywords: AML; MDS; RNA; RNA binding; RNA granules; U2AF1; freCLIP; splicing; stress granules; stress response.

Figure 1.. Distinct targets of U2AF subunits achieved through fractionated eCLIP-seq.

(A) Schematic representation of the U2AF complex on the intronic 3’SS. The presumed locations of U2AF1 and U2AF2, their relevant domains, the approximate position of U2AF1 pathological mutations and the consensus sequence of the human 3’SS are displayed. RRM, RNA recognition motif; ULM, U2AF ligand motif; UHM, U2AF homology motif; ZnF, zinc finger domain. (B) Schematic overview of eCLIP-seq vs freCLIP-seq protocol. Light Fraction: 37–65 kD, Heavy Fraction: 65–110 kD. (C) Alluvial plot showing different classes of intron-exon junctions bound by U2AF1, U2AF2 or both (color-coded), based on eCLIP-seq. Junction classes are defined by the presence of canonical AG at the intronic 3’SS, constitutive vs non-constitutive exon, and the position within the transcript. (D) Binding metaprofile (y-axis, mean±SEM of the percentage of crosslinking events) and 3’SS sequence logo for U2AF1 (top panel, n=3) and U2AF2 (bottom panel, n=4) eCLIP-seq. N, number of intron-exon junctions. (E) Binding metaprofile (mean±SEM) and 3’SS sequence logo for heavy (top panel, n=4) and light (bottom panel, n=3) fractions of U2AF1 WT freCLIP-seq. N, number of intron-exon junctions. See also Figure S1 and Table S1.

Figure 2.. U2AF1 mutations determine the position of 3`SS contacts.

(A) Number of differentially spliced events in S34F and Q157R compared with WT (absolute delta PSI>10%; FDR<0.05). SE, skipped exons; A5SS, alternative 5’SS; A3SS, alternative 3’SS, MXE, mutually exclusive exons; RI, retained introns. (B) 3’SS sequence logos for differential SE events in U2AF1 S34F (top panel) and Q157R (bottom panel) conditions. Sequence-specific positions between more included and less included exons are highlighted (position −3 for S34F mutant, +1 for Q157R mutant) N, number of differentially spliced events. (C) Binding metaprofiles (y-axis, mean±SEM of the percentage of crosslinking events) and 3’SS sequence logo for U2AF1 freCLIP-seq fractions, comparing WT with S34F (n=3) and Q157R (n=2) mutations. Arrows indicate the de novo binding peaks in position −3 for S34F mutant and +1 for Q157R mutant, respectively. N, number of intron-exon junctions. See also Figure S2 and Tables S2–S3–S4.

Figure 3.. Mutant U2AF1 binding strength and position influence splicing outcome.

(A) Right panel: scatter plot representing intron-exon junctions significantly affected by both differential binding (y-axis, log2 FC based on freCLIP-seq) and differential splicing (x-axis, delta PSI of SE events based on RNA-seq) in S34F vs WT. Significantly affected junctions (P-value<0.05, Fisher’s method) in each binding-splicing class (“<binding;<inclusion”, “>binding;>inclusion”, “>binding;<inclusion”, “<binding;>inclusion”) are color-coded. N, number of significantly affected junctions in each class. Left panel: binding metaprofiles (y-axis, mean±SEM of the number of crosslinking events per million reads) of U2AF subunits, based on freCLIP-seq fractions, considering junctions belonging to the most represented binding-splicing class. Positions characterized by a significant change in mutant vs WT U2AF1 binding are starred (P-value<0.05, one-tailed t-test). (B) Binding-splicing analysis in Q157R vs WT, displayed as in (A). See also Figure S3 and Tables S5–S6.

Figure 4.. Mutant U2AF1 binding-splicing alterations affect transcripts enriched in stress granules.

(A) Enrichment in GO terms related to RNA granule biology for genes differentially bound and spliced in U2AF1 S34F vs WT and Q157R vs WT HEL cells. Node size: number of affected genes belonging to a specific term. P-value based on Fisher’s exact test. (B) Enrichment analysis of mutant U2AF1 differentially bound-spliced genes among SG-related experimental datasets. Top panel: SG-enriched proteins; Bottom panel: SG-enriched (white-highlighted) and SG-depleted (grey-highlighted) transcripts. Dot size is proportional to the overlap, measured by odds ratio (Fisher’s exact test). (C) Network visualization of differentially bound-spliced transcripts in U2AF1 mutants that are also enriched in stress granules, according to multiple SG experimental datasets. See also Figure S4 and Tables S7–S8–S9–S10.

Figure 5.. Mutant U2AF1 cells show increased capability to form stress granules.

(A) Representative IF images (scale bars, 10 μm) and quantification of stress granules in mutant and WT U2AF1 HEL cells. SGs were identified by IMARIS (see Methods). The plot displays the mean±SEM G3BP1 field intensity, normalized to the relative controls (ctrl, uninduced HEL cells without arsenite treatment; dox, doxycycline-induced HEL cells without arsenite treatment; dox+ars, doxycycline-induced HEL cells treated with 500 μM arsenite for 1 hour). G3BP1 field intensity is the mean intensity of all the single SGs identified in the field. For each condition, 6 fields were acquired (3 fields per replicate), containing on average 74 cells each. Differences between S34F or Q157R and WT were tested with two-tailed t-test. (B, D) Scatter plot of gene expression changes (x-axis) and relative stability/degradation contributions (y-axis) in S34F (B) or Q157R (D) vs WT, measured by TL-seq (2 replicates per condition). Transcripts enriched (left panel) or depleted (right panel) in stress granules are highlighted. N, number of transcripts in each TL-seq class (stabilized, induced, destabilized, shutdown) (C, E) Fraction of SG enriched vs depleted transcripts in each TL-seq class in S34F (C) or Q157R (E) vs WT. Differences in fractions within each class were tested with a proportion test. (F) Percentage of 7-AAD negative viable cells detected by flow cytometry under arsenite stress (500 μM for 24 hours). Statistical differences between mutant (S34F, n=3; Q157R, n=3) vs WT (n=3) U2AF1 cells were calculated by t-test. (G) Difference in cell viability upon arsenite stress and ISRIB treatment (20 nM for 24 hours), compared with arsenite stress alone. P-values between mutant (S34F, n=3; Q157R, n=3) vs WT (n=3) U2AF1 cells were calculated by t-test. See also Figure S5, Table S11 and Files S1–S2.

Figure 6.. U2AF1-mutant MDS and AML patient cells display increased stress granule response.

(A) UMAP representation of 4271 CD34⁺ cells isolated from MDS patients, based on single-cell RNA-seq. WT and S34F cells from each patient are color-coded. (B) Distributions of SG-expression scores in WT and S34F cells from each MDS patient. The SG-expression score for each cell is based on the average expression of 149 genes enriched in stress granules and differentially bound and spliced by U2AF1 mutants. Differences in the distributions were tested with two-tailed Wilcoxon rank-sum test. (C, D) Representative IF images (scale bars, 10 μm) and quantification of stress granules in AML patients with WT (n=3) or S34F/Y (n=3) U2AF1. SGs were identified by IMARIS (see Methods). The plot displays the mean±SEM G3BP1 field intensity, normalized to the relative controls (ctrl, primary cells without arsenite treatment; ars, primary cells treated with 500 μM arsenite for 1 hour). G3BP1 field intensity is the mean intensity of all the single SGs identified in the field. For each patient, 6 fields per condition were acquired, containing on average 25 cells each. Differences between S34F/Y and WT patients were tested with two-tailed t-test. (E) Enrichment analysis of genes differentially spliced in 2 published cohorts of U2AF1-S34 AML patients among SG experimental datasets (Fisher’s exact test). See also Figure S6 and Tables S12–S13.

Figure 7.. Proposed model connecting cancer-associated U2AF1 mutations to enhanced stress adaptation.