Fig. 2

Accumulation of NEAT1 and augmented paraspeckle assembly in heterozygous FUS ΔNLS lines. a, b Cells heterozygous for the FUS NLS deletion (ΔNLS_het) have increased number of paraspeckles, whereas homozygous (ΔNLS_ho) and FUS knockout (KO) lines are almost devoid of paraspeckles. Arrows indicate clusters of paraspeckles in ΔNLS_het lines and arrowheads – residual paraspeckles in ΔNLS_ho lines (a). The number of NEAT1-positive foci and their area were quantified for ΔNLS_het lines (b). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Holm-Sidak test). c Paraspeckles in ΔNLS_het cells contain both NEAT1_2 and a core paraspeckle protein NONO. d NEAT1 isoforms are upregulated in FUS ΔNLS lines. Representative tracks for poly(A) capture RNA-Seq analysis of NEAT1 gene in a heterozygous (ΔNLS8_het) and a homozygous (ΔNLS4_ho) lines are shown. NEAT1_1 levels were measured by RNA-Seq and NEAT1_2 levels – by qRT-PCR. N = 4 per line. *p < 0.05, ***p < 0.001, ****p < 0.0001 (one-way ANOVA). e A NEAT1-repressed transcript ADARB2 is downregulated in FUS ΔNLS lines. ADARB2 mRNA levels were measured by RNA-Seq (left) and qRT-PCR (right). N = 3 per line. ****p < 0.0001 (one-way ANOVA with Dunnett’s test). f-h Overexpression of FUS or its mutants restores paraspeckles in FUS KO and ΔNLS_ho cells. Arrowheads indicate mature paraspeckles or their clusters (f, g). Inset in g shows paraspeckle primary units in a non-transfected FUS KO cell. Bar chart shows the fraction of transfected ΔNLS1_ho and FUS KO cells with one or more paraspeckle (large NEAT1-positive dot) (h). **p < 0.01, ***p < 0.001, ****p < 0.0001 as compared to non-transfected (NT) cells (one-way ANOVA with Holm-Sidak test). All FUS variants were expressed as N-terminal GFP-fusions. Paraspeckles were visualised by NEAT1 RNA-FISH. Combined data for three heterozygous and three homozygous lines are referred as “het pooled” and “ho pooled”, respectively. In b and h, numbers of cells analysed are indicated within each bar. Scale bars, 10 μm