Figure 4

CREST aggregation disrupts paraspeckles. (A) CREST aggregates might originate from the sites of paraspeckle formation. Paraspeckles (anti-NONO/p54nrb staining, arrowheads) and CREST nuclear aggregates exist as distinct structures in COS7 cells (top panel). In a fraction of cells CREST aggregates are found in close apposition to/partially overlapping with paraspeckles (bottom panel). (B) In response to transcriptional inhibition CREST redistributes to the same nucleolar caps as a typical paraspeckle protein FUS but not to the caps formed by coilin p80. (C) CREST lacking autoregulatory domain is efficiently recruited in paraspeckles (top panel) and redistributes to nucleolar caps (bottom panel). (D) Endogenous FUS is not essential for nuclear aggregation of CREST. Cells were co-transfected with FUS siRNA and a plasmid to express CREST-Flag and were analysed 48 hours post-transfection. (E-G) CREST efficiently sequesters endogenous FUS into dot-like nuclear aggregates in COS7 cells. In contrast, two other paraspeckle components, p54nrb and PSPC1, are not recruited to small and medium-sized CREST aggregates (F and G, top panels), and are detected in aggregates only in nuclei with extensive CREST aggregation (F and G, bottom panels). (H, I) Presence of CREST aggregates in the nucleus negatively affects paraspeckles. The fraction of cells with paraspeckles among COS7 cells expressing CREST-Flag (anti-p54nrb staining) or CREST-GFP (FISH with NEAT1 probe) was quantified separately for cells with diffuse CREST distribution and with nuclear CREST aggregates (mean ± s.e.m, *p < 0.05, **p < 0.01; 150-250 cells counted from each of the four or three independent experiments). (J) NEAT1 levels are decreased in CREST-expressing cells. Untagged CREST or GFP (vector) were expressed in SH-SY5Y cells for 24 hours; NEAT1 levels were measured by qPCR (**p < 0.01; results from four independent experiments run in duplicates). Scale bars, A – 5 μm; B-G – 10 μm.