Recent work has revealed the importance of liquid-liquid phase separation (LLPS) as a mechanism to organize cells through the formation of non-membrane-bound cellular compartments such as nucleoli, Cajal bodies, and stress granules. These compartments concentrate specific proteins and nucleic acids through weak, noncovalent interactions. I investigated phase separation as an organizing principle for both eukaryotic cells and viruses. In particular, I have studied the shelterin protein complex, which binds and protects human telomeres. Telomeres form unique nuclear compartments that prevent degradation and fusion of chromosome ends by recruiting shelterin proteins and regulating access of DNA damage repair factors. To understand how these dynamic components protect chromosome ends, I combine in vivo biophysical interrogation and in vitro reconstitution of human shelterin. I show that shelterin components form multicomponent liquid condensates with selective biomolecular partitioning on telomeric DNA. Tethering and anomalous diffusion prevent multiple telomeres from coalescing into a single condensate in mammalian cells. However, telomeres can coalesce when brought into contact via an optogenetic approach. TRF1 and TRF2 subunits of shelterin drive phase separation, and their N-terminal domains specify interactions with telomeric DNA in vitro. Telomeric condensates selectively recruit telomere-associated factors and regulate access of DNA damage repair factors. I propose that shelterin mediates phase separation of telomeric chromatin, which underlies the dynamic yet persistent nature of the end-protection mechanism.

My work also addressed how interactions between the nucleocapsid (N) protein and viral RNA in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) drive LLPS. SARS-CoV-2 infection causes COVID-19, a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. Packaging of the viral genome into the nascent virion is mediated by the N protein, but the underlying mechanism is not fully understood. I found that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base-pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry identifies a region that mediates interactions between N proteins in condensates, and truncation of this region disrupts phase separation. I also identified small molecules that alter the formation of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may promote biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.