Figure S4.
Staufen and Egl association with oskar RNPs. (A)
oskar mRNA content distribution in wild-type nurse cells and oocytes at stages 5–7 (yellow) and 9 (blue) of oogenesis. In the nurse cells, most oskar RNPs contain 1–2 copies of the RNA, consistent with previous reports (Little et al., 2015). oskar RNP content increases in the oocyte: at stages 5–7 most RNPs contain 4+ copies of the RNA, which decreases to >2 copies during oskar posterior localization (stage 9; Little et al., 2015). (B) Normalized Staufen-GFP signal intensity as a function of oskar mRNA content at stages 5–7 (yellow) and stage 9 (blue and red). Staufen-GFP signal intensity was measured in the complete absence (yellow and blue) or presence (red) of endogenous, unlabeled Staufen. Fitted linear models showing the correlation between Staufen-GFP signal intensity and oskar mRNA copy number as solid lines and equations. Underscored parameters of the models are significantly different from zero (P < 0.05). The slopes of the two fitted models are significantly different (P < 0.0001, ANOVA). (C) Mean signal intensity of Staufen-GFP measured at multiple locations throughout developing oocytes. Size of the oocytes (x-axis) is used as a proxy of developmental time and, along with morphological features, for staging of the oocytes (shaded areas as indicated in the panel). (D) Western blot showing Egl protein detected by anti-Egl antibody in the indicated genotypes. Tubulin was used as a loading control. (E) Normalized Egl-GFP signal intensity as a function of oskar mRNA content at stages 5–7 (yellow) and stage 9 (blue). Fitted linear models showing the correlation between Egl-GFP signal intensity and oskar mRNA copy number as solid lines and equations (top—stages 5–7, bottom—stage 9). Underscored parameters of the models are significantly different from zero (P < 0.05). The slopes of the two fitted models are significantly different (P < 0.0001, ANOVA). (F–G′) Association of Egl-GFP with oskar RNPs in oocytes with BicD1 (purple) or BicD2 (pink) alleles (F and F′) or expressing stau (red) or control (brown) RNAi (G and G′). Note that knock-down of Staufen results in similar retention of Egl on oskar RNPs as in the complete absence of Staufen protein (Fig. 4, E and E′). In E, G, and G′, egg chambers expressed a single copy of Egl-GFP in the presence of two endogenous wild-type egl alleles, except in the case of the rescued egl mutants (G, egl1/egl2, green). Although we observed a slightly elevated fraction of Egl positive RNPs when unlabeled Egl was absent (egl1/egl2, green), larger RNPs containing 16+ copies of oskar mRNA displayed no significant association with Egl (G) and the relative amounts of Egl on oskar RNPs were identical to what was observed in the presence of endogenous, unlabeled Egl (G′, blue). 14,321–43,299 oskar RNPs per genotype were analyzed. Triangles indicate that the fraction of GFP-positive oskar RNPs is not significantly different from zero (P > 0.01, one sample t test). In F, datapoints are slightly offset in the x-axis to facilitate comparison. (H) Western blot of input lysates and eluates after RNA immunoprecipitation in the presence (lane 3) or the absence (lane 4) of Staufen. Bait proteins—monomeric EGFP (lane 1), GFP-Staufen (lane 2) and Egl-GFP (lanes 3,4)—are detected by anti-GFP antibody. Anti-tubulin staining was used to monitor potential contamination of the eluates. In D and H, blue and yellow indicate low and high intensity of signal, respectively. Source data are available for this figure: SourceData S4.