Two components of the early ASH1 mRNA transport machinery undergo PUN motif-dependent liquid-liquid-phase separation
In eukaryotes, mRNA localization is a widespread mechanism of spatial gene regulation. Amongst the best-studied examples is the directional transport of ASH1 mRNA during mitosis of the budding yeast Saccharomyces cerevisiae. ASH1 mRNA is co-transcriptionally bound by the two RNA-binding proteins She2p and Loc1p and subsequently exported. In the cytoplasm, the adapter She3p binds to She2p forming an active transport complex with the myosin motor Myo4p to mediate actin-dependent transport into the daughter cell. Loc1p stays in the nucleus where it has a second function in ribosome biogenesis. In this study, we map the interaction surface of Loc1p on She2p and observe that it does not interfere with the RNA-binding interface of She2p. Analogous to a previous report on Loc1p and RNA, we could show that also Loc1p and She2p undergo liquid-liquid phase separation (LLPS). This event is caused by electrostatic interactions and can be regulated by the phosphorylation-driven alteration of She2ps oligomeric state. Furthermore, we observed that LLPS formation only requires the PUN motifs of Loc1p and that a ternary Loc1p-RNA-She2p complex also undergoes LLPS. In summary, our findings indicate that Loc1p co-transcriptionally recruits the nuclear ASH1 mRNA-She2p complex to LLPS, while the cytoplasmic Loc1p-lacking She2p-She3p-RNA transport complex does not form such LLPS.