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: Proximity labeling reveals a distinct transcriptome associated with RAB7A-positive endosomes in motor neurons. (A) Schematic representation of the APEX2-based proximity labeling strategy used to biotinylate RNAs in the vicinity of RAB7A-positive endosomes in iPSCs-derived MNs. (B) Western blot analysis of the APEX2-pulldown on iPSCs-derived MNs expressing flagged APEX2-RAB7A protein. Early endosomal markers (EEA1, RAB5), Late endosomal marker (RAB7A), Late endosomal/lysosomal marker (LAMP1), APEX2-RAB7A construct (FLAG), Nuclear marker (H3), and mitochondrial marker (TOM20) were used to assess compartment purification. INP = 2,5% input, PD = pull down, H2O2= hydrogen peroxide treatment. (C) Volcano plot showing the log2FC pull-down/input and the −log10Pvalue for each RNA detected. Enriched and Depleted RNAs (Padj < 0.05, |log2FC| > 0.59) or Unspecific RNAs are indicated by red, blue and gray dots, respectively. (D) Dotplot depicting the cellular component GO over-represented categories of the Enriched RNAs. Only significant categories (FDR < 0.05) were depicted. X-axis represents category enrichment score, while Y-axis reports the GO category description. Dot size represents the amount of RNAs in the analyzed group that overlap the category. (E) Representative RNA FISH and immunofluorescence images showing localization of an endosome- enriched (DNAJC5) or endosome- depleted (ARGLU1) mRNA (magenta) relative to RAB7A-positive endosomes (cyan) in soma and neurites. Insets show 3D renderings of boxed regions. Scale bar = 5 μm. Colocalization is indicated by yellow arrows. (F) Quantification of the percentage of endosome- enriched (red), unspecific (gray), or depleted (blue) RNAs associated with RAB7A-positive particles. Data are shown as boxplots; p-values are indicated with asterisks (**p<0.01, ***p<0.001, ****p<0.0001). Statistical significance was assessed with One-way Brown-Forsythe and Welch ANOVA tests. N = 3 biological replicates (total cells: NORAD n = 315, DNAJC5 n = 270, TMOD2 n = 278, CACNG4 n = 272, LRRC4B n = 295, AGPAT4 n = 269, ARGLU1 n = 274).

Article Snippet: Plasmid encoding N-terminal FLAG-APEX2–tagged human RAB7A was obtained from Addgene (plasmid #135652).

Techniques: Labeling, Derivative Assay, Western Blot, Expressing, Marker, Construct, Purification, Immunofluorescence

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: Conserved 5′UTR elements are sufficient to target RNAs to RAB7A-positive endosomes. (A) Distribution of PhyloP100 conservation scores for untranslated regions (UTRs). Boxplots show PhyloP100 scores for enriched (red) and depleted (blue) elements in the 5′ UTR (left pair) and 3′ UTR (right pair). (B) Quantification of the fraction of RLUC reporter mRNAs associated with RAB7A-positive endosomes when fused to 5′UTRs derived from endosome- enriched (red), unspecific (gray), or depleted (blue) transcripts. Data are shown as boxplots; p-values are indicated with asterisks (**p<0.01, ***p<0.001, ****p<0.0001). Statistical significance was assessed with One-way Brown-Forsythe and Welch ANOVA tests. N = 3 biological replicates (total cells: DNAJC5 n = 338, TMOD2 n = 354, CHKA n = 275, AGPAT4 n = 300, ARGLU1 n = 301). (C) Representative RNA FISH and immunofluorescence images showing localization of RLUC reporter mRNAs (magenta) fused to an endosome- enriched (DNAJC5) or unspecific (AGPAT4) 5′UTR relative to RAB7A-positive endosomes (cyan) in soma and neurites. 3D rendering of selected white boxes showing RAB7A-particles (cyan) and reporter mRNA (magenta). Scale bar = 5 μm. Colocalization is indicated by yellow arrows.

Article Snippet: Plasmid encoding N-terminal FLAG-APEX2–tagged human RAB7A was obtained from Addgene (plasmid #135652).

Techniques: Derivative Assay, Immunofluorescence

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: Plasmid encoding N-terminal FLAG-APEX2–tagged human RAB7A was obtained from Addgene (plasmid #135652).

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: GEMIN5 is required for 5′UTR-dependent RNA recruitment to RAB7A-positive endosomes. (A) Quantification of endogenous RNA association with RAB7A-positive endosomes in control (Scramble) and GEMIN5 knockdown (siGEMIN5) SK-N-BE cells. Endosome- enriched (red), unspecific (gray), and depleted (blue) transcripts are shown. Data are shown as boxplots; p-values are indicated with asterisks (*p<0.05, **p<0.01). Statistical significance was assessed with two-tailed, unpaired T-test. (B) Representative RNA FISH and immunofluorescence images showing localization of an endosome- enriched (DNAJC5) or endosome- depleted (ARGLU1) mRNA (magenta) relative to RAB7A-positive endosomes (cyan) in control (Scramble) and GEMIN5 knockdown (siGEMIN5) SK-N-BE cells. Scale bar = 10 μm. Colocalization is indicated by yellow arrows. (C) Quantification of RAB7A association for RLUC reporter mRNAs fused to 5′UTRs from endosome- enriched (red), unspecific (gray), or depleted (blue) transcripts in control and GEMIN5 KD conditions. Data are shown as boxplots; p-values are indicated with asterisks (**p<0.01, ***p<0.001). Statistical significance was assessed with two-tailed, unpaired T-test.

Article Snippet: Plasmid encoding N-terminal FLAG-APEX2–tagged human RAB7A was obtained from Addgene (plasmid #135652).

Techniques: Control, Knockdown, Two Tailed Test, Immunofluorescence

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: Plasmid encoding N-terminal FLAG-APEX2–tagged human RAB7A was obtained from Addgene (plasmid #135652).

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

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Fluorescence images of RPE-1 cells expressing exogenous BLTP3A-mRFP (shown in inverted grays) and (not shown) GFP-tagged wild-type Rab7 (left) or dominant negative (DN) Rab7 (T22N) (right). Scale bar, 5 μm. ( B ) CLEM of a BLTP3A-mRFP positive cluster in an RPE-1 cell expressing GFP-tagged dominant negative Rab7. Left: fluorescence image of BLTP3A-mRFP (magenta). Scale bar, 1 μm. Right: EM micrograph of the field shown at left revealing that the BLTP3A-mRFP fluorescence reflects clusters of small vesicles. Scale bar, 500 nm. ( C ) BLTP3 chimeras design. Left: Surface representation of the predicted RBG core of BLTP3A. Red and blue indicate positive and negative charges, respectively, and gray indicates hydrophobic surfaces. Right: Surface representation (top) and ribbon representation (bottom) of the “untwisted” protein showing individual RBG motifs. Bottom: Cartoon of chimeras consisting of BLTP3A (dark orange) and BLTP3B (light orange) RBG motifs. ( D ) High-magnification live fluorescence images of RPE-1 cells expressing the indicated BLTP3-mRFP constructs (magenta) and LAMP1-GFP (green). Individual channels are shown as inverted grays. Scale bar, 1 μm. ( E ) Ribbon representation of the AlphaFold prediction of a.a. 1–336 of BLTP3A. Blue indicates loops connecting adjoining RBG motifs, and gray indicates the first beta-strand of the third RBG motif. ( F ) Fluorescence images (inverted grays) of RPE-1 cells expressing BLTP3A-1-336-mRFP and either (not shown) GFP-Rab7 (left), or GFP-Rab7 T22N (right). Scale bar, 5 μm. A zoom of an area of the cell at left (dotted square) expressing BLTP3A-1-336-mRFP (magenta) is also shown, along with the Rab7 fluorescence (green), demonstrating the localization of BLTP3A-1-336-mRFP around the entire profile of lysosomes. Individual channels are shown as inverted grays. Scale bar, 2 μm. ( G ) Cartoon depicting the proposed association of BLTP3A vesicle clusters with the surface of lysosomes and the dependence of this association on Rab7.

Article Snippet: GFP-Rab7 T22N , Addgene , RRID:Addgene_28048.

Techniques: Fluorescence, Expressing, Dominant Negative Mutation, Construct