ABSTRACT: 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
