Abstract
Phase separation (PS) 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 C. elegans, that contains two structured domains (D1-D2), an internal IDR, and a C-terminal IDR rich in RGG motifs. Theoretical and in vitro studies implicated the internal IDR and RGG motifs in driving PGL-3 PS via self-interactions and binding to RNA. Studies in cells, however, implicated the D1 and D2 domains. Here, we investigate the molecular basis of PGL-3 PS in vitro using microscopy, crosslinking mass spectrometry, and biophysical measurements. We find that D1-D2 forms oligomers and is necessary and sufficient for PS. The terminal RGG region interacts with D1-D2 in a manner that enhances PS even in the absence of RNA. In contrast, the internal IDR is neither necessary nor sufficient for PS. These findings support an alternative model for PGL-3 PS that does not require RNA and is driven by oligomerization of structured domains that interact with RGG repeats.