Natural selection has preserved and enhanced the phase-separation properties of FUS during 160 million years of mammalian evolution

Protein phase separation is essential for the self-assembly of non-membraneous organelles. However, we know little about its ability to change in evolution. Here we studied the evolution of the mammalian RNA binding protein FUS, a protein whose prion-like domain (PLD) is essential for the formation of stress granules through liquid-liquid phase separation. Although the prion-like domain evolves three times as rapidly as the remainder of FUS, it harbors absolutely conserved tyrosine residues that are crucial for phase separation. Ancestral reconstruction shows that the phosphorylation sites within the PLD are subject to stabilizing selection. They toggle among a small number of amino acid states. One exception to this pattern are primates, where the number of such phosphosites has increased through positive selection. In addition, we find frequent glutamine to proline changes that help maintain the unstructured state of FUS that is necessary for phase separation. In summary, natural selection has stabilized the liquid-forming potential of FUS and minimized the propensity of cytotoxic liquid-to-solid phase transitions during 160 million years of mammalian evolution.