Figure 7. An RNA matrix prevents full fusion of spherical condensates, thus promoting arrangement into filamentous structures in vitro.

(A) Confocal 3D time-lapse imaging of phase separation experiments using purified mGFP-FUS-TIS (10 µM) in the presence of the TLR8 3′UTR (200 nM) after 30 min of incubation. Scale bar, 2 µm. Snapshots show a fusion event of two FUS-TIS condensates. (B) Same as (A), but in the presence of the CD47 3′UTR (30 nM). Snapshots show the contact of two FUS-TIS condensates. As they do not fully mix, they grow into condensates with irregular shapes. The contact site is indicated by the white arrow. (C) Same as (B). Snapshots show two fusion events between FUS-TIS condensates 1 and 2 and between 2 and 3 and demonstrate how irregularly shaped condensates grow into large filamentous networks. (D) Representative high-resolution confocal images of phase separation experiments using purified mGFP-FUS-TIS (10 µM) in the presence of Cy5-labeled CD47 3ʹUTR RNA after 16 hr of incubation. Scale bar, 1 µm. (E) Model showing how structured RNAs induce spherical condensates and how RNAs with large unstructured regions induce formation of filamentous and mesh-like condensates. Purple indicates the condensate. For details, see text. (F) Fluorescence recovery after photobleaching of mGFP-FUS-TIS and the indicated Cy5-labeled RNAs performed at 2 hr after setting up the phase separation experiments.

Figure 7—figure supplement 1. Extensive intermolecular RNA–RNA interactions are responsible for formation of mesh-like condensates in vitro.

(A) Representative high-resolution confocal images of phase separation experiments using purified mGFP-FUS-TIS (10 µM) in the presence of Cy5-labeled TLR8 3′UTR RNA after 16 hr of incubation. Scale bar, 1 µm. (B) Fluorescence recovery after photobleaching of mGFP-FUS-TIS and the indicated Cy5-labeled RNAs at 16 hr after setting up the phase separation experiments.