Extended Data Fig. 3: Imbalanced DGCR8:DROSHA stoichiometry drives irreversible Microprocessor aggregation in vitro.

a, Top, prediction of disordered regions in DGCR8 protein by PONDR (http://pondr.com/). Bottom, schematic diagram showing the domains of DGCR8. b, Coomassie blue staining of purified rDGCR8 and rDROSHA proteins at different concentrations, as well as two mutant versions of rDGCR8 proteins with deletion of ΔCTT and ΔRhed domains. c, Representative images of phase separation of rDGCR8, rDGCR8-ΔCTT, rDGCR8-ΔRhed and rDROSHA at different concentrations in physiological buffer. d, Representative images of the aggregates of labelled rDGCR8 (30 μM) before and after treatment with 10% 1,6-hexanediol for 5 min. e, Representative images of FRAP analysis of rDGCR8 puncta. Targeted region is highlighted in a white box. Normalized fluorescence intensity of rDGCR8 in FRAP analysis is represented as mean ± s.d. (n = 6, independent observations in six separated aggerates). f, g, Representative confocal images of pre-formed Microprocessor aggregates (32 μM rDGCR8:8 μM rDROSHA) under conditions of dilution and high salt (1 M NaCl). rDGCR8 (red), rDROSHA (green) and merged (yellow) signals are shown. h, i, Images of pre-formed Microprocessor aggregates (32 μM rDGCR8:8 μM rDROSHA) followed by the addition of extra rDROSHA to achieve a 2:1 ratio or treatment with 10% 1,6-hexanediol for 10 min. j, Representative time-lapse images of two proximate Microprocessor aggregates for the times indicated. rDGCR8 (red), rDROSHA (green) and merged (yellow) signals are shown. k, FRAP analysis of Microprocessor aggregates in vitro. Targeted droplet region is highlighted in a white box, and rDGCR8 (red), rDROSHA (green) and merged (yellow) signals are shown. Right, normalized fluorescence intensities. Data are represented as mean ± s.d. (n = 7, independent observations in seven separated aggregates). All experiments were repeated at least three times with similar results (Methods).