Fig. 3: The central intrinsically disordered region is important for RNA-dependent phase separation of N protein.
a–l DIC and fluorescence images of the mixtures of 2 µM 17-mer ssRNA and 10 µM N protein variants: a full-length N1–419; b N1–364; c N49–419; d N49–364; e N1–246; f N247–419; g N49–246; h N49–209; i N1–174; j N49–174; k N175–419; l N175–364. m–p DIC and fluorescence images of mixtures of 20 ng/µL Cy3-UTR265 RNA and 10 µM of N variants: m N∆SR (note that this construct shows significant aggregation on its own; see Supplementary Fig. 3e); n N∆210–246; o N11SD; p N14SA. Images were taken 30 min after mixing N protein variants with RNA. Scale bar, 5 µm (a–p). Percentage values indicate the percentage of total RNA fluorescence within condensates, calculated from two (f–m) or three (a–e, n–p) fields. See Supplementary Fig. 3b for SDS–PAGE analysis of purified proteins, Supplementary Fig. 3d for phase separation of proteins without RNA, and Supplementary Fig. 3f for phase separation of N1–364, N1–246, and N49–246 with UTR265 RNA. q Summary of phase separation behaviors of N protein variants shown in a–p. Domain schematic of the SARS-CoV-2 N protein, with known domains marked. Gray shading indicates the region implicated in N + RNA phase separation. + indicates 1–15% of RNA fluorescence within condensates; ++ indicates 16% or higher. r Fusion events of phospho-resistant (N14SA) and phospho-mimetic (N11SD) N protein droplets (representative of four fusion events observed). Imaging was performed 30 min after initial mixing. Scale bar, 1 µm.
