The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80–90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein’s central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.

The RNA binding protein TDP-43 forms intranuclear or cytoplasmic aggregates in age-related neurodegenerative diseases. In this study, we found that RNA binding–deficient TDP-43 (produced by neurodegeneration-causing mutations or posttranslational acetylation in its RNA recognition motifs) drove TDP-43 demixing into intranuclear liquid spherical shells with liquid cores. These droplets, which we named “anisosomes”, have shells that exhibit birefringence, thus indicating liquid crystal formation. Guided by mathematical modeling, we identified the primary components of the liquid core to be HSP70 family chaperones, whose adenosine triphosphate (ATP)–dependent activity maintained the liquidity of shells and cores. In vivo proteasome inhibition within neurons, to mimic aging-related reduction of proteasome activity, induced TDP-43–containing anisosomes. These structures converted to aggregates when ATP levels were reduced. Thus, acetylation, HSP70, and proteasome activities regulate TDP-43 phase separation and conversion into a gel or solid phase.

Binding specificity of Cas9–guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9–RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from <0.006 s−1 to >2 s−1 upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate <0.006 s−1), suggesting that extremely slow rejection could sequester Cas9–RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.

We measure the stability and folding relaxation rate of phosphoglycerate kinase (PGK) Förster resonance energy transfer (FRET) constructs localized in the nucleus or in the endoplasmic reticulum (ER) of eukaryotic cells. PGK has a more compact native state in the cellular compartments than in aqueous solution. Its native FRET signature is similar to that previously observed in a carbohydrate-crowding matrix, consistent with crowding being responsible for the compact native state of PGK in the cell. PGK folds through multiple states in vitro, but its folding kinetics is more two-state-like in the ER, so the folding mechanism can be modified by intracellular compartments. The nucleus increases PGK stability and folding rate over the cytoplasm and ER, even though the density of crowders in the nucleus is no greater than in the ER or cytoplasm. Nuclear folding kinetics (and to a lesser extent, thermodynamics) vary less from cell to cell than in the cytoplasm or ER, indicating a more homogeneous crowding and chemical environment in the nucleus.

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80-90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that a centrally located 40 amino acid intrinsically disordered domain drives phase separation of N protein when bound to RNA, with the morphology of the resulting condensates affected by inclusion in the RNA of the putative SARS-CoV-2 packaging signal. The SARS-CoV-2 M protein, normally embedded in the virion membrane with its C-terminus extending into the virion core, independently induces N protein phase separation that is dependent on the N protein's C-terminal dimerization domain and disordered region. Three-component mixtures of N+M+RNA form condensates with mutually exclusive compartments containing N+M or N+RNA, including spherical annular structures in which the M protein coats the outside of an N+RNA condensate. These findings support a model in which phase separation of the N protein with both the viral genomic RNA and the SARS-CoV-2 M protein facilitates RNA packaging and virion assembly.