Multi-omics analysis of RNA binding, splicing and turnover alterations induced by mutant U2AF1 in myeloid malignancies.

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA binding motifs; yet how they affect splicing and promote cancer remains 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 detecting contacts between U2AF1 and the 3’SS AG at single-nucleotide resolution (fractionated eCLIP-seq or freCLIP-seq). Our data reveal that 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 (eCLIP-seq and freCLIP-seq), splicing (RNA-seq) and turnover (TimeLapse-seq or TL-seq) data, we predicted that U2AF1 mutations directly affect stress granule components. 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: splicing, RNA binding, U2AF1, U2AF2, S34F, Q157R, hotspot mutations, 3'SS, 3' splice site, myeloid malignancies, eCLIP, freCLIP, RNA-seq, RNA turnover, TimeLapse-seq, TL-seq, RNA granules, stress granules, stress response, biomolecular condensates, MDS, AML In this study, we performed high-throughput sequencing of RNA isolated by enhanced crosslinking immunoprecipitation (eCLIP-seq) to determine the function of WT U2AF1 alongside two cancer-associated U2AF1 mutants (S34F, Q157R) in the context of a human hematopoietic cell line. U2AF1 and U2AF2 eCLIP-seq were performed in 2 biological replicates, with also 1-2 technical replicates. Human erythroid leukemia (HEL) cell lines were stably transduced with either wild type U2AF1 (WT) or mutant U2AF1 (S34F, Q157R). Central to this work was the development of (fr)eCLIP-seq – for fractionated eCLIP-seq, a modification of eCLIP that enabled us to separate a light and a heavy fraction, dissecting U2AF1 from U2AF2 RNA binding signals within the 3’SS region in vivo. U2AF1 freCLIP-seq was performed in 2-4 biological replicates. RNA-seq and TimeLapse-seq (TL-seq, providing RNA turnover data) were performed in biological duplicates. Uninduced cells were used as controls in RNA-seq. Unlabeled and uninduced cell samples were used as controls in TL-seq.