Extended data Fig. 4 |. Heat shock limits asincRNAs and triggers sincRNA-dependent nucleolar phase transitions.

(a) Heat shock (43°C) rapidly induces the formation of intranucleolar liquid droplets harbouring the amyloid-converting protein motif (ACM)-containing VHL protein. (b) Gradual amyloid body (A-body) formation. The stress-induced, mobile, and spherical liquid-like foci (yellow arrowhead) gradually transition into irregularly shaped solid-like amyloid bodies (cyan arrowhead) in cells subjected to heat shock (43°C)¹⁹. (c) The appearance of early stage ACM-marked liquid-like foci^18,19 in cells subjected to a 15 min heat shock treatment is abrogated upon siRNA-mediated knockdown of either sincRNA16 or sincRNA22. (d) In a cell-free in vitro system, the low-complexity sincRNA forms liquid droplets when mixed with the ACM of human VHL or β-amyloid proteins. Droplets were detected using fluorescently labelled RNA (5’FAM) and differential interference contrast (DIC). (e) Strand-specific RNA-seq (ss-RNA-seq) reveals that sincRNA levels increase while asincRNA levels decrease across the IGS following a 30 min heat shock. Heat shock increases sincRNA levels by 607% and decreases asincRNA levels by 38%. (a-e) Nucleolar stress hyper-responsive MCF7 cells were used where applicable; images are representative of two independent experiments; scale bars, 5 μm.