U2AF⁶⁵ assemblies drive sequence-specific splice site recognition

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.