Fig. 3

Figure 3. hub translation is regulated by miR430. A: The level of the endogenous hub mRNA is increased in 10hpf MZdicer embryos compared to the level observed in wildtype embryos. As a comparison, qPCR analysis was performed to quantify nanos mRNA level in the same embryos. Error bars show SEM. ***P < 0.001 in t-test. B: Expression of GFP from mRNA containing the hub 32UTR is increased in the soma of MZdicer embryos (top right) as compared with the wild type control embryo (top left). mCherry fluorescent protein expressed from a co-injected mRNA that is not regulated by miRNA (mCherry-f-globin) was used as an internal control (bottom panels). Inset highlights the expression of GFP in the PGCs. C: The miR430 seed sequences within the hub 32UTR are labeled in blue and the mutations inactivating them in red. D–F: The predicted miR430 seed sequences in the hub 32UTR are functional in mediating translational repression. D: Expression of GFP from mRNA containing the wild type hub 32UTR (top left), compared to the expression from mRNA carrying mutations in the four predicted miR430 seeds in the 32 UTR (top right). Fluorescence intensity was measured in the soma (outline) at 16hpf and co-injected mCherry-F-globin served as internal control for the injection volume (bottom panels). E: A graph depicting the intensity of GFP expression measured for different mutated reporters as compared to that of wild type. n is the number of embryos analyzed and the error bars represent the SEM. ***P < 0.001 in t-test. F: Western blot of total protein extracted from 16-hpf embryos injected with RNA encoding GFP including the wild type or mutated hub 32UTR.