Figure 2. Multiple Cen domains support mRNA localization to the centrosome.

(A) Schematic of the full-length and truncated Cen protein products with positions of predicted domains (Paysan-Lafosse et al., 2023), antibody epitopes (Kao and Megraw, 2009), and the transposon f04787 within null mutants indicated. (B) Immunoblots from 0.5–2.5 hr embryo extracts from the indicated genotypes showing truncated Cen protein products in the CenΔC (~35 kDa) and CenΔN (~70 kDa) samples relative to the Asl loading control. The N-terminal anti-Cen antibody was used for the top two blots (α-Cen N), while the C-terminal anti-Cen antibody was used below (α-Cen C; see also (Kao and Megraw, 2009)). (C–F) Maximum-intensity projections of Cen smFISH (magenta) in NC 13 interphase embryos expressing GFP-Cnn (green) with DAPI-stained nuclei (blue). (C) Control embryo with Cen mRNA localized at centrosomes (arrow). In contrast, (D) Cen mutants and (E) CenΔC embryos fail to localize Cen mRNA to centrosomes. (F) Although CenΔN is partially sufficient to form small RNA granules (arrow) near centrosomes, neither fragment recapitulates WT localization. In all experiments, CenΔC and CenΔN are expressed in the Cen null background. Percentage of Cen mRNA (G) overlapping with centrosomes or (H) in granules 0 μm from the Cnn surface. Each dot represents a measurement from N= 15 control, 11 Cen, 13 CenΔC, and 17 CenΔN embryos. Mean ± SD is displayed (red). Significance was determined by (G) one-way ANOVA followed by Dunnett’s T3 multiple comparison test or (H) Kruskal-Wallis test followed by Dunn’s multiple comparison test with n.s., not significant; *, P<0.05; **, P<0.01; and ****, P<0.0001. Scale bars: 5μm; 1μm (insets).