primary antibodies include rabbit anti rrp40  (Proteintech)

Journal: Cell reports

Article Title: The RNA exosome maintains cellular RNA homeostasis by controlling transcript abundance in the brain

doi: 10.1016/j.celrep.2025.116729

Figure Lengend Snippet: (A) Illustration of the RNA exosome, an evolutionarily conserved ribonuclease complex composed of structural subunits (EXOSC1–9; EXOSC4 not shown) and a catalytic subunit (Dis3). (B) Pathogenic variants in EXOSC3 (orange, labeled with “3,” termed Rrp40 in Drosophila ) cause pontocerebellar hypoplasia type 1b (PCH1b). Domain structures of human EXOSC3 and fly Rrp40 proteins highlight the conservation and disease-associated amino acid changes (in red). Sequence alignments from human, mouse, Drosophila , and yeast show conserved regions surrounding the disease-linked residues. Variants modeled in this study map to the N-terminal (N) and S1 RNA-binding domains of EXOSC3. (C) Schematic of CRISPR-Cas9 genome-editing strategy used to generate Rrp40 mutant fly models of EXOSC3-linked PCH1b amino acid substitutions. (D) Chart of patient genotypes located in EXOSC3 , the Drosophila equivalent genotype in Rrp40 , and the viability of homozygous Rrp40 wild-type (WT) and mutant flies modeling PCH1b-linked recessive genotypes (WT/WT, G11A/G11A, and G146C/G146C) shown as %eclosion relative to expected Mendelian ratios. n = number of individual flies analyzed. (E) Experimental design for snRNA-seq study. For each genotype (WT/WT, G11A/G11A, and G146C/G146C), two biological replicates of 20 newly eclosed (day 1) adult female fly heads were pooled. Tissues were dissociated to isolate nuclei, followed by flow cytometry for quality. Libraries were prepared using the 10× Genomics Chromium Next GEM Single Cell 3′ kit, targeting 20,000 nuclei per sample. (F) 2D uniform manifold approximation and projection (UMAP) for dimension reduction of integrated snRNA-seq data from ~116,619 nuclei derived from brain-enriched tissue of age-matched Rrp40 WT (WT/WT; yellow) and mutant (G11A/G11A, blue; G146C/G146C, teal) flies. (G) 2D UMAPs colored by broad cell classes (neurons, blue; glia, pink; head specific, tan; unannotated, gray), separated by genotype. Based on the integrated UMAP in (F). (H) 2D UMAPs colored by 25 cell types (right), separated by genotype. Based on the integrated UMAP in (F). (I) UMAPs of subclustered neuron, glial, and head-specific cell classes, each colored by genotype (WT/WT, yellow; G11A/G11A, blue; G146C/G146C, tan), highlighting distinct transcriptomic profiles in Rrp40 mutant flies.

Article Snippet: Primary antibodies include rabbit anti-Rrp40 (custom made with Pacific Immunology), rabbit anti-Arc1 (gift from Dr. Travis Thomson) and mouse anti-GAPDH (1:1000, Proteintech).

Techniques: Labeling, Sequencing, RNA Binding Assay, CRISPR, Mutagenesis, Flow Cytometry, Single Cell, Derivative Assay

Journal: Cell reports

Article Title: The RNA exosome maintains cellular RNA homeostasis by controlling transcript abundance in the brain

doi: 10.1016/j.celrep.2025.116729

Figure Lengend Snippet: (A) Multidimensional scaling (MDS) plot of snRNA-seq data, with each point colored by cell class (neuronal, blue; glial, pink; head specific, tan; unannotated, gray) and separated by genotype (WT/WT, square; G11A/G11A, circle; and G146C/G146C, triangle). (B) Bar graphs showing the number of differentially expressed transcripts increased (gray; log2 fold change [log2FC] > 2, FDR < 0.05) or decreased (white, log2FC < −2, FDR < 0.05) in each genotype compared to WT/WT. Left, G11A/G11A vs. WT/WT; right, G146C/G146C vs. WT/WT. Bars are colored by cell class (neuron, blue; glia, pink; and head specific, tan). (C) UpSet plot showing the overall increased differentially expressed transcripts (log2FC > 2, FDR < 0.05) in neurons (blue) and glia (pink) in G11A/G11A and G146C/G146C mutants compared to WT/WT. A red box highlights 60 transcripts shared between genotypes and cell classes. (D) GO biological process enrichment (FlyEnrichr) for the 60 overlapping transcripts in (C), integrated across neuronal and glial subpopulations in both mutants relative to WT/WT. The bars in blue represent significant enrichment (adjusted p value < 0.05), ordered by significance. (E) Heatmap of the differentially expressed transcripts in G11A/G11A (top) and G146C/G146C (bottom) relative to WT/WT. Functionally important neuronal transcripts are highlighted in red. Normalized log2FC is shown on a gradient from +10 (red) to −10 (blue). (F) Quantification of the percentage of cells that express Arc1 in each Rrp40 mutant (G11A/G11A or G146C/G146C) and WT control (WT/WT) from the FeaturePlot (G). Results are presented as mean ± standard error of the mean (SEM) for n = 2 biological replicates (ns, not statistically significant and * p < 0.05). (G) 2D UMAP FeaturePlots of Arc1 -expressing cells (blue) in WT/WT (left), G11/G11A (middle), and G146C/G146C (right) brains. The color gradient from blue to teal indicates high to low expression. Data are from two pooled biological replicates per genotype. (H) Hybridization chain reaction RNA-FISH (HCR RNA-FISH) of Arc1 mRNA (teal) combined with immunohistochemistry (magenta; neuropil marker CadN/cadherin) in day 1 whole-mount fly brains. Rows: Arc1 mRNA (top), CadN (middle), and merged images (bottom). Scale bars, 50 μm. Cadherin staining is shown only to provide spatial context within the brain. (I) Quantification of mean maximum Arc1 mRNA fluorescence intensity from Rrp40 mutant and control brains ( n = 6) in (H). Values represent the mean ± SEM (* p < 0.05, statistically significant; ns, not significant).

Article Snippet: Primary antibodies include rabbit anti-Rrp40 (custom made with Pacific Immunology), rabbit anti-Arc1 (gift from Dr. Travis Thomson) and mouse anti-GAPDH (1:1000, Proteintech).

Techniques: Mutagenesis, Control, Expressing, Hybridization, Immunohistochemistry, Marker, Staining, Fluorescence

Journal: Cell reports

Article Title: The RNA exosome maintains cellular RNA homeostasis by controlling transcript abundance in the brain

doi: 10.1016/j.celrep.2025.116729

Figure Lengend Snippet: (A) Schematic of the Drosophila central complex (left) and the mushroom body (right). The central complex structures highlighted include the fan-shaped body (FB; light purple), protocerebral bridge (PB; dark purple; dorsal to FB), ellipsoid body (EB; orange), noduli (NOs; yellow), and antennae lobes (ALs; green). (B) Mushroom-body structures include vertical α- (orange) and α′ - (light green) lobes, medially projecting β- (orange) and β′ - (light green) lobes, γ-lobes (light purple), the calyx (dark purple), and Kenyon cell bodies (red). (C) Confocal max intensity projection images of central complex structures labeled with anti-DLG antibody in WT (WT/WT) and Rrp40 mutant (G11A/G11A or G146C/G146C) brains from newly eclosed (day 1) or aged (day 14) flies. Single coronal sections show (from top to bottom) ALs, EB, FB, and PB. The columns represent WT/WT (left), G11A/G11A (middle), and G146C/G146C (right) for each age group ( n = 10 per genotype and age). The region of interest is outlined in green. Scale bar, 100 μm. (D) Quantification of central complex structures region of interest area from DLG labeling in day 1 and 14 brains from WT/WT and Rrp40 mutants, normalized to WT/WT values. The data correspond to images in (C). Values represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns, not significant. (E) Confocal images of mushroom bodies in WT/WT (top), G11A/G11A (middle), and G146C/G146C (bottom) flies expressing GFP under UAS control in combination with the R13F02 -Gal4 driver ( R13F02 -Gal4> UAS -GFP) on days 1 (left) and 14 (right) from WT/WT (top row), G11A/G11A (middle row), and G146C/G146C (bottom row) flies on days 1 (left) and 14 (right). The brains were co-labeled with anti-GFP (mushroom-body γ-lobe neurons; green) and anti-DLG (central complex; magenta). Columns: merged GFP/DLG (left), GFP alone (middle), and DLF alone (right). The region of interest is outlined in white. Scale bars, 50 μm. (F and G) Quantification of γ-lobe region of interest size from GFP labeling on days 1 (F) and 14 (G) in WT/WT and Rrp40 mutants, based on images in (E). Thinned γ-lobes are indicated by white arrows. The areas of the gamma lobes were normalized to WT/WT controls. Values represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns, not significant. (H and I) Quantification of normalized distances between beta lobes on days 1 (H) and 14 (I) in WT/WT and Rrp40 mutants, based on images in (E). The beta lobe midline is indicated by white arrows. The distance between beta lobes were normalized to WT/WT controls. Values represent the mean ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001, and ns, not significant. (J) Human EXOSC3 (fly Rrp40) rescue experiment. Confocal images of day 14 brains from WT, G11A/G11A, w 1118 flies expressing UAS-EXOSC3-myc in mushroom bodies ( +/+; R13F02 -Gal4> UAS-EXOSC3-myc ), and G11A/G11A mutants expressing UAS-EXOSC3-myc in the mushroom bodies (G11A/G11A ;R13F02-Gal4 > UAS-EXOSC3-myc ) are included. The brains were labeled with anti-DLG (central complex, magenta). Anti-DLG labeling highlights β-lobe midline-crossing defects in G11A/G11A mutants that are rescued by EXOSC3 expression (white arrows). Scale bar, 50 μm. (K and L) Quantification of γ-lobe size (K) and β-lobe midline distance (L) in day 14 WT/WT, G11A/G11A, w 1118 with R13F02-Gal4 > UAS-EXOSC3-myc , and G11A/G11A mutants with EXOSC3 rescue in mushroom bodies ( G11A/G11A;R13F02 -Gal4> UAS-EXOSC3-myc ). Values represent the mean ± SD. **** p < 0.0001 and ns, not significant.

Article Snippet: Primary antibodies include rabbit anti-Rrp40 (custom made with Pacific Immunology), rabbit anti-Arc1 (gift from Dr. Travis Thomson) and mouse anti-GAPDH (1:1000, Proteintech).

Techniques: Labeling, Mutagenesis, Expressing, Control

Journal: Cell reports

Article Title: The RNA exosome maintains cellular RNA homeostasis by controlling transcript abundance in the brain

doi: 10.1016/j.celrep.2025.116729

Figure Lengend Snippet: (A) Overview of the Drosophila melanogaster ribosomal RNA (rRNA) biogenesis pathway. , The sizes of rRNA intermediates are indicated in dark gray. Red arrows denote external or internal spacer regions, which are cleaved and excised to produce mature 18S, 5.8S, 2S, 28Sa, and 28Sb rRNA. A 5.8S probe (dark blue arrow) was used in near-infrared northern (irNorthern) blots in this study. (B) irNorthern blot analysis of 5.8S rRNAs in brain-enriched tissue from Rrp40 mutants and WT controls. (C) “Nanoblot” generated by targeting mature 5.8S rRNA with a 5.8S nanoprobe using Nanopore-based RNA sequencing data from Rrp40 mutants and WT controls. Note the similarity to the irNorthern blot in (B). (D) IGV browser screenshots showing single-track read coverage mapping to 5.8S rRNA, 2S rRNA, and ITS2 in Rrp40 mutants and WT controls. The blue asterisk (*) labels 5.8S species extending into ITS2, while the red asterisk (*) denotes 2S-ITS2 transcripts resulting from impaired degradation. (E) Quantification of the ratio of extended 5.8S rRNA and 2S rRNA species to mature 5.8S in Rrp40 mutants and WT controls, shown as a bar graph. Values represent the mean ± SD for 3 biological replicates. (F) Nanoblot generated by targeting mature 2S rRNA with a 2S nanoprobe using the Nanopore-based RNA sequencing data from Rrp40 mutants and WT controls.

Article Snippet: Primary antibodies include rabbit anti-Rrp40 (custom made with Pacific Immunology), rabbit anti-Arc1 (gift from Dr. Travis Thomson) and mouse anti-GAPDH (1:1000, Proteintech).

Techniques: Northern Blot, Generated, RNA Sequencing

Journal: Cell reports

Article Title: The RNA exosome maintains cellular RNA homeostasis by controlling transcript abundance in the brain

doi: 10.1016/j.celrep.2025.116729

Figure Lengend Snippet: (A and B) Cellular proportion analysis from the snRNA-seq dataset showing broad cell types (neuron, glia, head specific, and unannotated) (A) and distinct mushroom-body Kenton cell neuron subtypes (αβ, α′ β′, and γ) (B) in Rrp40 mutants (G11A/G11A or G146C/G146C) compared to WT controls (WT/WT). In the plots, clusters more abundant in WT/WT are shown on the left, and clusters more abundant in Rrp40 mutants are shown on the right. The red dots indicate statistically significant differences (FDR < 0.05 and |log 2 fold difference| > 0.32), and the gray dots indicate non-significant differences. (C) Diagram of the mushroom-body Kenyon cells (red) and calyx (purple) from the posterior-medial and posterior views. (D) Representative confocal z-projections from posterior-medial and posterior views of WT/WT (top two rows), G11A/G11A (middle two rows), and G146C/G146C (bottom two rows) brains expressing GFP under UAS control in combination with mushroom-body driver ( R13F02 -Gal4) ( R13F02 -Gal4> UAS -GFP). The brains were labeled with anti-GFP (cell bodies and calyx; green), anti-DCP-1 (magenta), and DAPI (blue). Regions of interest for the cell body and calyx are outlined in white. Images are shown for newly eclosed (day 1, left) and aged (day 14, right) flies. Scale bar, 100 μm. (E–G) Quantification of percentage of DCP-1-positive puncta. Values represent the mean ± SD. (E), normalized Kenyon cell body numbers (posterior view) (F), and normalized calyx area (G) in WT/WT and Rrp40 mutant flies on days 1 and 14. * p < 0.05, ** p < 0.01, **** p < 0.0001, and ns, not significant.

Article Snippet: Primary antibodies include rabbit anti-Rrp40 (custom made with Pacific Immunology), rabbit anti-Arc1 (gift from Dr. Travis Thomson) and mouse anti-GAPDH (1:1000, Proteintech).

Techniques: Expressing, Control, Labeling, Mutagenesis

Journal: Cell reports

Article Title: The RNA exosome maintains cellular RNA homeostasis by controlling transcript abundance in the brain

doi: 10.1016/j.celrep.2025.116729

Figure Lengend Snippet: (A) A Kaplan-Meier survival analysis of WT/WT (tan), G11A/G11A (blue), and G146C/G146C (green) flies ( n = 100 per genotype). (B) Locomotor activity measured by negative geotaxis assay (right, schematic of climbing assay). Data are shown as the mean percentage of flies reaching the top of a cylinder within 60 s, averaged across all trials. Groups of age-matched (days 1, 8, and 14; n = 36, cohorts of 9–12 flies) were tested in at least three independent trials per genotype. The results are represented as the mean ± SEM (ns p > 0.05, * p < 0.05, and **** p < 0.0001; unpaired two-tailed t test vs. WT). (C) Pan-neuronal expression of human EXOSC3 ( nSyb -Gal4> UAS-EXOSC3 ) significantly rescues locomotor deficits in Rrp40 G11A/G11A mutants ( G11A/G11A;nSyb -Gal4> UAS-EXOSC3 ) on days 7 and 14 compared to Rrp40 controls ( WT/WT and nSyb -Gal4 /UAS-EXOSC3 ). Data are shown as the mean ± SEM (ns p > 0.05, * p < 0.05, and **** p < 0.0001; unpaired two-tailed t test vs. WT). (D) Short-term aversive taste memory assay in newly eclosed (day 1, left) and aged (day 14, right) flies. Rrp40 control ( WT/WT ) and mutant ( G11A/G11A or G146C/G146C ) flies ( n = 10 per genotype) were food deprived for 24 h prior to testing. A memory assay was performed as described in the . Proboscis extension rates (PERs) are presented as the mean ± SEM (two-way ANOVA; * p < 0.05, ** p < 0.01, and *** p < 0.001). (E) Pan-neuronal expression of human EXOSC3 ( nSyb -Gal4> UAS-EXOSC3 ) rescues memory deficits in G11A/G11A mutants ( G11A/G11A;nSyb -Gal4> UAS-EXOSC3 ) on day 14 compared to controls (WT/WT and nSyb-Gal4/UAS-EXOSC3 ). Flies ( n = 10 per genotype) were food deprived for 24 h prior to testing. A memory assay was performed as described in the . Proboscis extension rates (PERs) are presented as the mean ± SEM (two-way ANOVA; * p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: Primary antibodies include rabbit anti-Rrp40 (custom made with Pacific Immunology), rabbit anti-Arc1 (gift from Dr. Travis Thomson) and mouse anti-GAPDH (1:1000, Proteintech).

Techniques: Activity Assay, Climbing Assay, Two Tailed Test, Expressing, Control, Mutagenesis