Characterization of U1-70K’s liquid-liquid phase separation in vitro

U1-70K is an RNA splicing protein that is known to localize to nuclear speckles, which are membrane-less organelles known to form via liquid-liquid phase separation. Nuclear speckles are implicated in several RNA processing events, and dysfunction of either U1-70K or nuclear speckles can lead to a plethora of disease states. The connection between nuclear speckles and alternative splicing is still unclear. A detailed molecular picture of how splicing proteins like U1-70K localize to speckles could help better approach questions related to function. U1-70K is known to be extensively phosphorylated, but no detailed studies have elucidated the effect of this phenomenon on the protein's function. This study sought to understand the mechanism by which U1-70K localizes to nuclear speckles and the effect that phosphorylation has on this process. We have shown that U1-70K phase separates with itself in vitro. U1-70K’s phase separation is mediated via an interaction between its two C-terminal basic acidic dipeptide repeat (BAD) domains and its N-terminal RNA recognition motif (RRM) domain. We also show that U1-70K’s phase separation is greatly reduced when the protein is phosphorylated. These interactions may mirror the mechanism via which U1-70K localizes to the nuclear speckles, providing novel insight into speckle composition.