Fig 2: Assessment of neuronal activity in Fus?NLS/+ mice in vivo.a Neuronal activity was monitored in frontal cortex of anesthetized mice. Scheme of coronal section, indicating the expression of GCaMP6s in cortex assessed through a cranial window. Magnified view of imaged cortical area demonstrates neuronal expression of GCaMP (green) across all cortical layers. b Timeline of experiments. Male and female mice were injected with AAV9-syn-jGCaMP7s (at 3 months of age) or AAV2/1-hsyn-GCaMP6m (at 9 months of age) into frontal cortex and implanted with a cranial window. In vivo imaging began 4 weeks after implantation. c Representative examples (average projections) of field of views (FOV) imaged in Fus+/+ and Fus?NLS/+ mice at 4 months (N = 8 Fus+/+ mice and N = 3 Fus?NLS/+ mice, left) and at 10 months (N = 5 Fus+/+ mice and N = 6 Fus?NLS/+ mice, right) are shown together with fluorescence calcium traces of selected regions of interest (ROIs). d The fraction of active cells per FOV was not affected in 4-month-old Fus?NLS/+ mice. N = 13 FOVs in 3 Fus?NLS/+ and N = 25 FOVs in 8 Fus+/+ mice. Data are presented as mean ± SEM p = 0.1627, Two-sided Unpaired Student’s t-test. e, f The calcium transient frequencies (e) were increased while the average transient amplitudes (f) were decreased in Fus?NLS/+ mice. N = 1107 ROIs in 3 Fus?NLS/+ and N = 2264 ROIs in 8 Fus+/+, superimposed by the median (e). Kolmogorov–Smirnov test, ***p < 0.0001 for both panel e and f. g–i The fraction of active cells per FOV (g) as well as (h) the frequencies and (i) the average amplitudes of calcium transients of each ROI were increased in 10-month-old Fus?NLS/+ mice. Data are individual FOVs (g; N = 14 FOVs in 6 Fus?NLS/+ and N = 10 FOVs in 5 Fus+/+ mice) or individual ROIs (h, i; N = 855 ROIs in 6 Fus?NLS/+ and N = 631 ROIs in 5 Fus+/+ mice) superimposed by the mean ± SEM (g) or the median (h, i). panel g: Two-tailed Unpaired Student’s t-test, *p = 0.0126; panel h and i: Kolmogorov–Smirnov test, ***p < 0.0001 for both panels. Source data are provided as a Source Data file.

Fig 3: Accumulation of paraspeckles in spinal neurons and glial cells in ALS-FUS. a Examples of paraspeckles in spinal neurons and glial cells of ALS-FUS and sALS patients visualised using RNA-FISH with fluorescently-labelled (Quasar 570) 5' segment NEAT1 probe. Images were taken both in the orange and green channels to distinguish between specific NEAT1 signal and autofluorescence from lipofuscin. See also Additional file 1: Table S2. Arrowheads point to paraspeckles in a glial cell. Scale bars, 10 µm. b Examples of paraspeckles in spinal neurons (left panels) and glial cells (right panels) in an ALS-FUS patient visualised with RNAscope® ISH using NEAT1_2 probe. Neuronal nuclei are circled. Scale bars, 10 µm (left panels) and 50 µm (right panels)

Fig 4: Structural and functional deficiency of paraspeckles in FUS ?NLS lines. a Interaction of FUS with SFPQ and NONO is reduced in FUS ?NLS lines as revealed by proximity ligation assay (PLA). PLA was performed in a heterozygous (?NLS2_het) and a homozygous (?NLS1_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 ?NLS 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 ?NLS 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 ?NLS_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 ?NLS lines. Levels of six mature miRNAs produced from pri-miR17~92 were measured by qRT-PCR separately for heterozygous and homozygous FUS ?NLS 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

Fig 5: RGG Domains Are Required for Efficient Binding of the FUS C-Terminal Domain to Phase-Separated QGSY Hydrogels and Liquid Droplets(A) Scheme illustrating the C-terminal deletion constructs tested for QGSY droplet and hydrogel binding.(B) The RGG domains are the main domains affecting binding of GFP fusion constructs to mCherry-QGSY hydrogels or QGSY droplets. Asterisk indicates higher exposure setting to show that there is still some residual binding of single ?RGG mutants to the hydrogel.(C) Partitioning of the C-terminal deletion constructs in the QGSY droplets and hydrogels in vitro correlates with the toxicity of the corresponding full-length deletion construct in fly. The red fit takes into account all values; the blue fit considers ?RGG1 as an outlier.