Phase separation of PGL-3 driven by structured domains that oligomerize and interact with terminal RGG motifs.

Phase separation of biomolecular condensates is often assumed to be driven by interactions involving nucleic acids and intrinsically disordered regions (IDRs) of proteins. PGL-3 is a component of P granules, biomolecular condensates in the germline, that contains two structured domains in tandem (D1-D2), an internal IDR, and a C-terminal IDR rich with RGG motifs. Theoretical and studies have implicated the internal IDR and RGG motifs in driving PGL-3 phase separation via self-interactions and binding to RNA. Studies in cells, however, have implicated the D1 and D2 domains. Here, we investigate the molecular basis of PGL-3 phase separation using microscopy, crosslinking mass spectrometry and biophysical measurements. We find that D1-D2 is oligomeric and necessary and sufficient for phase separation independent of RNA. D1-D2 also interacts with the terminal RGG domain in a manner that correlates with phase separation. In contrast, the internal IDR is neither necessary nor sufficient for phase separation. These findings support a new model for PGL-3 phase separation driven by oligomerization of structured domains and enhanced by RGG repeats independent of RNA.