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. 2021 Mar 2;10:e64252. doi: 10.7554/eLife.64252

Figure 5. A multivalent RNA matrix is responsible for mesh-like condensate formation in vitro.

(A) Schematic of RNAs with strong local secondary structures that are predicted to induce spherical condensates. (B) Schematic of RNAs with large disordered regions that form extensive intermolecular RNA–RNA interactions that are predicted to form network-like condensates. (C) Native and denaturing agarose gel electrophoresis of sphere-forming (lanes 1–3) and network-forming (lanes 4–6) RNAs (5 µM, each [1.7, 1.6, 1.9, 1.7, 1.8, 1.7 µg/µl]). (D) Schematic of a complex RNA network characterized by extensive intermolecular RNA–RNA interactions mediated by two dimerization elements (D1 and D2) that were added to structured RNAs. (E) Native and denaturing agarose gel electrophoresis of the indicated RNAs (1 µM, each [326, 154, 345, 193 ng/µl]). (F) Representative images of phase separation experiments using purified mGFP-FUS-TIS (10 µM) in the presence of the indicated Cy3- or Cy5-labeled RNAs generated by in vitro transcription after 16 hr of incubation. Scale bar, 2 µm.

Figure 5.

Figure 5—figure supplement 1. Extensive intermolecular RNA–RNA interactions are responsible for formation of mesh-like condensates in vitro.

Figure 5—figure supplement 1.

(A) The TLR8 3′UTR (gray) and the MYC 3′UTR (green) are predicted by RNAfold to dimerize. The secondary structure prediction shows extensive base-pairing between the two RNAs, indicated by the black marks. The nucleotide positions that demarcate the interaction sites are indicated by arrows, and the numbers show their positions in their respective 3′UTRs. The MYC 3′UTR was added directly downstream of the TLR8 3′UTR for structure prediction. Although both RNAs are able to base-pair, they are unable to form a complex RNA network. (B) Schematic of the D1 RNA dimerization motif. Shown is the sequence and base-pairing of the directional RNA dimerization motif D1 that was obtained from the CRISPR/Cas9 system and represents tracrRNA/crRNA. The two components form a heterologous RNA–RNA interaction. (C) Schematic of the D2 RNA dimerization motif. Shown is the sequence and base-pairing of the RNA dimerization motif D2, which is the HIV dimerization motif. It forms a homologous RNA–RNA interaction. (D) Centroid RNA secondary structure of the TLR8 3′UTR with the RNA dimerization elements D1 and D2 as predicted by RNAfold is shown. The color code represents base-pairing probability. NED: normalized ensemble diversity. (E) As in (D), but centroid RNA secondary structure of the MYC 3′UTR with the RNA dimerization elements D1 and D2 as predicted by RNAfold is shown.