The essential splicing factor U2AF65 is known to help anchoring U2 snRNP at the branch site. Its C‐terminal UHM domain interacts with ULM motifs of SF3b155, an U2 snRNP protein. Here, we report a cooperative binding of U2AF65 and the related protein CAPERα to the multi‐ULM domain of SF3b155. In addition, we show that the RS domain of U2AF65 drives a liquid–liquid phase separation that is amplified by intronic RNA with repeated pyrimidine tracts. In cells, knockdown of either U2AF65 or CAPERα improves the inclusion of cassette exons that are preceded by such repeated pyrimidine‐rich motifs. These results support a model in which liquid‐like assemblies of U2AF65 and CAPERα on repetitive pyrimidine‐rich RNA sequences are driven by their RS domains, and facilitate the recruitment of the multi‐ULM domain of SF3b155. We anticipate that posttranslational modifications and proteins recruited in dynamical U2AF65 and CAPERα condensates may further contribute to the complex mechanisms leading to specific splice site choice that occurs in cells.

Splicing factor mutations are frequent in myeloid neoplasms, blood cancers, and solid tumors. Cancer cells harboring these mutations present a particular vulnerability to drugs that target splicing factors such as SF3b155 or CAPERα. Still, the arsenal of chemical probes that target the spliceosome is very limited. U2AF homology motifs (UHMs) are common protein interaction domains among splicing factors. They present a hydrophobic pocket ideally suited to anchor small molecules with the aim to inhibit protein-protein interaction. Here, we combined a virtual screening of a small molecules database and an in vitro competition assay and identified a small molecule, we named UHMCP1 that prevents the SF3b155/U2AF 65 interaction. NMR analyses and molecular dynamics simulations confirmed the binding of this molecule in the hydrophobic pocket of the U2AF 65 UHM domain. We further provide evidence that UHMCP1 impacts RNA splicing and cell viability and is therefore an interesting novel compound targeting an UHM domain with potential anticancer properties.