Fig. 5: dZnf598 and Fmr1 interact with 40S ribosomal subunit recycling complex.

a Immunostaining of mitochondrial markers showing the regulation of Fmr1 and dZnf598 in muscle tissue of Usp10 and rin OE flies. Control (w-) serves as a negative control. Mitochondrial morphology is visualized by mitoGFP (Usp10 group) and ATP5α (rin group). b Quantification of mitochondrial size shown in (a). Mitochondria counts were obtained from three samples (n = 3, 7 areas counted per sample). c Immunostaining showing the effect of Fmr1 and dZnf598 on mitochondrial morphology in muscle tissue of Pink1 RNAi flies. Mitochondrial morphology is visualized using mitoGFP. d Violin plots showing the quantification of mitochondrial aggregations in (c). Significance was calculated using a two-proportion Z test with a threshold set as 3 µm². Three samples (n = 3) were analyzed. Statistical parameters are introduced in the Methods section and source code. e Mito-lysosome analysis showing increased mitophagy levels promoted by Fmr1 OE and dZnf598 OE. Control (w-) serves as a negative control. White arrowheads indicate the mito-lysosomes. f Quantification of positive mito-lysosomal signals shown in (e). An increased mito-lysosome number indicates elevated mitophagy. Twenty replicates were analyzed (n = 20). g Immunoblotting showing the effect of Usp10 OE on the Fmr1 protein level. Control (w-) serves as a negative control; Actin is used as a loading control in blots. h Tandem co-IP analysis using Flag tag (Fmr1) and rin antibodies in sequence, showing the interacting proteins with the Usp10-rin-Fmr1 complex. Three experiments (n = 3) were conducted. i MitoGCaMP imaging in fly muscle tissue showing the regulation of mitochondrial Ca²⁺ levels by Usp10, rin, and Fmr1. j Quantification of images shown in (i). Data were obtained from ten samples (n = 10). One-way ANOVA test (two-sided) followed by post hoc Dunnett’s multiple comparisons test 95% confidence interval (CI) was used in (b, f, j). k A working model illustrating the interactions between the 40S ribosomal subunit recycling complex and Fmr1 and dZnf598. The figure was created in BioRender¹³⁵. All scale bars, 5 µm. Data are means ± SEM. The p values are included in the figure. Source data are provided as a Source Data file.