Fig. 3
Structural and functional deficiency of paraspeckles in FUS ΔNLS lines. a Interaction of FUS with SFPQ and NONO is reduced in FUS ΔNLS lines as revealed by proximity ligation assay (PLA). PLA was performed in a heterozygous (ΔNLS2_het) and a homozygous (ΔNLS1_ho) lines; FUS KO cells were used as a negative control. Representative images and quantification (number of single interactions (dots) per cell (foci per cell)) are shown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Holm-Sidak test). b Extractability of NEAT1_2 is increased in FUS ΔNLS lines. NEAT1_2 extractability was analysed by determining its levels in QIAzol-lysed heated versus non-heated samples (“fold extraction”) by qRT-PCR. Note near-complete NEAT1_2 extractability in FUS KO cells (fold extraction ~ 1). See also Additional file 1: Figure S4B. N = 3 per line. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA). c NEAT1_1 accumulates in soluble nuclear extract (SNE) in FUS ΔNLS lines. Left, representative PCR (non-saturated conditions, 26 cycles); right, qRT-PCR analysis. A primer pair located immediately upstream NEAT1_1 polyA-tail (NEAT1 pA) was used to quantify NEAT1_1 in cDNA of polyadenylated RNA. Note that NEAT1_2 which is not polyadenylated is undetectable under these conditions. *p < 0.05, **p < 0.01 (one-way ANOVA with Holm-Sidak test). d NEAT1 displays diffuse distribution in poly(I:C)-stimulated ΔNLS_het lines. Cells were analysed 8 h after poly(I:C) transfection by NEAT1 RNA-FISH. Representative images and quantification of the fraction of cells with diffuse NEAT1 distribution are shown. e Paraspeckle-regulated miRNAs are decreased in FUS ΔNLS lines. Levels of six mature miRNAs produced from pri-miR17~92 were measured by qRT-PCR separately for heterozygous and homozygous FUS ΔNLS lines, and combined average values were plotted. *p < 0.05 (Mann-Whitney U-test). Combined data for three heterozygous and three homozygous lines are referred as “het pooled” and “ho pooled”, respectively. In a and d, numbers of cells analysed are indicated within each bar. Scale bars, 10 μm