Journal: bioRxiv

Article Title: FUS post-transcriptional splicing is autoregulated via RNA condensation with therapeutic potential for ALS-FUS

doi: 10.1101/2025.02.01.633781

Figure Lengend Snippet: a Experimental pipeline for HyPro-MS analysis of FUSint6&7-RNA condensates. b Efficient labelling of the endogenous FUSint6&7-RNA condensates using HyPro probes. Stellaris FUSint6-specific probe was used for co-staining. Arrows indicate the foci labelled by both Stellaris and HyPro probes. Scale bar, 10 µm. c Principal component analysis (PCA) demonstrating clustering of triplicated HyPro-MS samples for both probes and the no-probe control. d Volcano plot for FUSint6&7-RNA condensates versus no-probe control (Ctrl). Proteins with padj<0.05 are labelled in black. Proteins with padj<0.05 and involved in RNA metabolism-related pathways and/or implicated in neurodegeneration, are labelled in red. e Volcano plot for FUSint6&7-RNA condensates versus ACTB probe. Proteins with padj<0.1 are labelled in black and the top 15 hits are labelled in red. f Dot plot of GO Biological Process term enrichment analysis for nuclear proteins identified in FUSint6&7-RNA condensates and significantly enriched as compared to no-probe control. g Dot plot of GO Biological Process term enrichment analysis for nuclear proteins identified in FUSint6&7-RNA condensates and significantly enriched as compared to ACTB probe control. h Validation of HyPro-MS proteins enriched in FUSint6&7-RNA condensates, as well as FUS and other proteins previously shown to bind FUSint6&7-RNA. Cells expressing exoFUSint7 were analysed by RNA-FISH and immunofluorescence with appropriate antibodies. Representative images are shown. Top graph, 28-35 transfected cells with condensates were analysed per protein; bottom graph, 41 and 27 individual condensates were analysed for YTHDC1 and FUS enrichment, respectively, ****p<0.0001, two-tailed unpaired t test.

Article Snippet: The following commercial primary antibodies were used: FUS (mouse monoclonal, Santa Cruz, sc-47711 and rabbit polyclonal, Proteintech, 11570-1-AP); SMN (mouse monoclonal, BD Biosciences, 610646); coilin p80 (mouse monoclonal, BD Biosciences, 612074); TDP-43 (rabbit polyclonal, C-terminal, Sigma); YTHDC1 (rabbit polyclonal, Proteintech, 14392-1-AP); HDGFL2 (rabbit polyclonal, Proteintech, 15134-1-AP); hnRNPC (rabbit polyclonal, Proteintech, 11760-1-AP); PNN/pinin (rabbit polyclonal, Proteintech, 18266-1-AP); ANP32B (rabbit polyclonal, Proteintech, 10843-1-AP); FUBP1 (rabbit polyclonal, Proteintech, 24864-1-AP).

Techniques: Staining, Control, Expressing, Immunofluorescence, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: FUS post-transcriptional splicing is autoregulated via RNA condensation with therapeutic potential for ALS-FUS

doi: 10.1101/2025.02.01.633781

Figure Lengend Snippet: a,b YTHDC1 depletion or m6A downregulation leads to a loss of FUSint6&7-RNA condensate integrity. Representative images (general plane) and quantification of condensates (a) as well as high-resolution images (b) are shown. 107 and 65 cells (4 or 5 FoV) were analysed for scrambled and YTHDC1 siRNA, respectively, from a representative experiment. **p<0.01, Mann-Whitney U test. HeLa cells were used in a and SH-SY5Y cells were used in b . Representative images are shown. STM2457-treated cells are also shown. In b , PNN was used as a speckle marker. Scale bar, 20 μm in a and 5 μm in b . c YTHDC1 depletion does not affect FUSint6&7-RNA level but downregulates FUS mRNA. qRT-PCR analysis with FUS intron 6- and 7-specific primers was performed in HeLa cells. N=5-7, *p<0.05, Mann-Whitney U test. d High-confidence DRACH motif in FUS intron 7 shown in a structural context, as predicted by SRAMP. e,f FUS introns 6 and 7 are extensively methylated. m6A-Atlas 2.0 database was used for mapping methylation sites (e) and calculating the m6A mark density (f). Also see Supplementary Table S3. g,h Pharmacological inhibition of a m6A writer METTL3 affects FUSint6&7-RNA condensate integrity without changing levels of this RNA. Representative images and quantification of the condensate number in STM2457-treated cells (g) and qRT-PCR analysis of RNA level (h) are shown. In g , cells were treated for 16 h, and >200 cells (5 FoV) were analysed per condition from a representative experiment, **p<0.01, Mann-Whitney U test. Scale bar, 20 μm. In h , qRT-PCR analysis was done using FUS intron 6- and -7 specific primers. N=4. i Pharmacological inhibition of a m6A eraser FTO promotes FUSint6&7-RNA condensate assembly. Representative images and quantification of the condensate number in FB23-2 treated cells are shown. Cells were treated with the compound for either 4 or 16 h. 79, 106 and 117 cells (5 FoV) were analysed for DMSO, 4-h FB23-2 and 16-h FB23-2 treatments, respectively, from a representative experiment. *p<0.05, Kruskal-Wallis with Dunn’s test. Scale bar, 20 μm. j FUS intron 6 RNAscope-ISH reveals partial cytoplasmic redistribution of FUSint6&7-RNA in STM2457-treated cells. Arrows indicate cytoplasmic accumulations of this RNA; nucleus is circled in the insets. Scale bar, 20 μm. k FUSΔNLS lines have preserved or enhanced ability to form FUSint6&7-RNA condensates. Representative images and quantification are shown. 234, 239, 234, 141 and 62 cells were analysed for WT, ΔNLS4, ΔNLS7, ΔNLS10 and ΔNLS11 lines, respectively (from 4-9 FoV). *p<0.05, **p<0.01, Kruskal-Wallis with Dunn’s test. l MeRIP demonstrates increased FUSint6&7-RNA methylation in FUSΔNLS lines. Total RNA purified from ΔNLS7 (homozygous) and ΔNLS10 (heterozygous) lines was subjected to pulldown using m6A antibody-coated beads and used for qRT-PCR analysis with FUSint6-specific primers. Methylated FUSint6&7-RNA level was normalised to GAPDH mRNA (extensively methylated transcript), then to total FUSint6&7-RNA level in the respective cell line, and finally, to no-antibody beads control. N=4, *p<0.05, Kruskal-Wallis with Dunn’s test. m Enhanced association of FUSint6&7-RNA with YTHDC1 in FUSΔNLS lines. RIP was performed using Flag-Trap agarose in YTHDC1-Flag expressing WT and FUSΔNLS cells, followed by qRT-PCR analysis with FUSint6-specific primers. Venus-Flag was used as a control (“vector”). FUSint6&7-RNA level was normalised to GAPDH and then to total FUSint6&7-RNA level in the respective cell line. Results for ΔNLS4, ΔNLS7 and ΔNLS10 lines were combined. N=2. *p<0.05, Mann-Whitney U test (WT vs . ΔNLS).

Article Snippet: The following commercial primary antibodies were used: FUS (mouse monoclonal, Santa Cruz, sc-47711 and rabbit polyclonal, Proteintech, 11570-1-AP); SMN (mouse monoclonal, BD Biosciences, 610646); coilin p80 (mouse monoclonal, BD Biosciences, 612074); TDP-43 (rabbit polyclonal, C-terminal, Sigma); YTHDC1 (rabbit polyclonal, Proteintech, 14392-1-AP); HDGFL2 (rabbit polyclonal, Proteintech, 15134-1-AP); hnRNPC (rabbit polyclonal, Proteintech, 11760-1-AP); PNN/pinin (rabbit polyclonal, Proteintech, 18266-1-AP); ANP32B (rabbit polyclonal, Proteintech, 10843-1-AP); FUBP1 (rabbit polyclonal, Proteintech, 24864-1-AP).

Techniques: MANN-WHITNEY, Marker, Quantitative RT-PCR, Methylation, Inhibition, RNAscope, Purification, Control, Expressing, Plasmid Preparation