Journal: bioRxiv

Article Title: Neuronal late endosomes serve as selective RNA hubs disrupted by ALS-linked FUS mutation

doi: 10.64898/2026.01.19.700337

Figure Lengend Snippet: GEMIN5 binds the 5’UTR of endosome- enriched RNAs and localizes on LEs. (A) Pie chart showing the fraction of enriched, depleted or unspecific RBPs among 49 cytoplasmic RBPs from the ENCODE eCLIP compendium, as determined by RBP binding enrichment analysis on the 5′UTRs of RNAs enriched or depleted in endosomes (B) Meta-transcript heatmap showing the z-scored fraction of bound endosomal RNAs across transcript regions (5′UTR, CDS and 3′UTR) for the indicated RBPs (GRSF1, FTO, DDX3X and GEMIN5). Colour indicates relative enrichment (red) or depletion (blue) along the 5′–3′ axis. (C) qPCR quantification of GEMIN5 binding to endogenous endosome- enriched (red), unspecific (gray), and depleted (blue) RNAs in SK-N-BE cells, expressed as percentage of input. GEMIN5 mRNA serves as a positive control (yellow). p-values are indicated with asterisks (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Statistical significance was assessed with One-way ANOVA with Tukey multiple comparisons. (D) qPCR analysis of GEMIN5 binding to RLUC reporter mRNAs fused to 5′UTRs from endosome- enriched (red), unspecific (gray), or depleted (blue) transcripts in SK-N-BE cells, expressed as percentage of input. p-values are indicated with asterisks (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Statistical significance was assessed with One-way ANOVA with Tukey multiple comparisons. (E) Co-immunoprecipitation showing interaction between V5–GEMIN5 and GFP–RAB7A in neuroblastoma cells; GAPDH serves as a negative control. (F) Left, immunofluorescence analysis of GEMIN5 or the P-body marker DCP1A (magenta) relative to RAB7A-positive endosomes (cyan). Insets show 3D renderings of boxed regions. Scale bar = 10 μm. Right, quantification of colocalization by Pearson’s correlation coefficient. Data are shown as boxplots; p-value is indicated with asterisks (****p<0.0001). Statistical significance was assessed with two-tailed, unpaired T-test. N = 3 biological replicates (total cells: GEMIN5 n = 323, DCP1A = 259)

Article Snippet: After BCA quantification, 500 μg of lysate were combined with 2 μg of anti-V5 antibody (Novus Biologicals, NB600-381) or control IgGs (Thermo Fisher, 02-6102), and incubated for 2:30 hours at 4°C in a final volume of 500 μL.

Techniques: Binding Assay, Positive Control, Immunoprecipitation, Negative Control, Immunofluorescence, Marker, Two Tailed Test

Journal: bioRxiv

Article Title: Neuronal late endosomes serve as selective RNA hubs disrupted by ALS-linked FUS mutation

doi: 10.64898/2026.01.19.700337

Figure Lengend Snippet: ALS-related FUS P 525 L disrupts endosomal RNA recruitment. (A) Venn diagram showing the overlap between endosome- enriched RNAs identified in FUS WT and FUS P 525 L iPSCs-derived MNs. (B) Quantification of RAB7A association for representative RNAs that are commonly enriched (NORAD), selectively lost in FUS P 525 L neurons (CACNG4, TMOD2), or commonly excluded (AGPAT4). Data are shown as boxplots; p-values are indicated with asterisks (*p<0.05, **p<0.01, ***p<0.001). Statistical significance was assessed with a two-way ANOVA test. N = 3 biological replicates (total cells: FUS WT NORAD n = 315, CACNG4 n = 272, TMOD2 n = 278, AGPAT4 n = 269; FUS P 525 L NORAD n = 349, CACNG4 n = 257, TMOD2 n = 282, AGPAT4 n = 260). (C) Representative RNA FISH and immunofluorescence images showing CACNG4 mRNA (magenta) relative to RAB7A-positive endosomes (cyan) in soma and neurites, in FUS WT and FUS P 525 L MNs. Scale bar = 5 μm. Colocalization is indicated by yellow arrows. 3D rendering of selected white boxes showing RAB7A-particles (cyan) and CACNG4 mRNA (magenta). (D) Pearson’s correlation coefficient analysis of GEMIN5 and RAB7A colocalization, in FUS WT and FUS P 525 L MNs. Data are shown as boxplots; p-values is indicated with asterisks (**p<0.01). Statistical significance was assessed with two-tailed, unpaired T-test. N = 3 biological replicates (total cells: FUS WT n = 323, FUS P 525 L = 298) (E) Co-IP for V5-GEMIN5 showing its interaction with FLAG-FUS P 525 L and GFP-RAB7A in SK-N-BE cells. GAPDH serves as a negative control. (F) Co-IP for FLAG-FUS P 525 L demonstrating its interaction with V5-GEMIN5 and GFP-RAB7A. GAPDH serves as a negative control.

Article Snippet: After BCA quantification, 500 μg of lysate were combined with 2 μg of anti-V5 antibody (Novus Biologicals, NB600-381) or control IgGs (Thermo Fisher, 02-6102), and incubated for 2:30 hours at 4°C in a final volume of 500 μL.

Techniques: Derivative Assay, Immunofluorescence, Two Tailed Test, Co-Immunoprecipitation Assay, Negative Control