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Fig. 8.
Dnd1 relieves the inhibitory effect of eIF3f on nanos1 translation and PGC development. (A) nanos1 (4 ng) was translated alone or with FLAG-eIF3f (2 ng) in wheat germ extracts with enhanced translational efficiency (Promega WG+). Nanos1 protein was detected by western blot. eIF3f inhibited nanos1 translation. Addition of recombinant Dnd1 protein relieved the repressive activity of eIF3f in a dose-dependent manner. The experiment was repeated three times. (B) eIF3f represses nanos1 translation in vivo. nanos1 RNA was injected into fertilized eggs alone, or together with eIF3f or dnd1 RNA. At stage 11, Nanos1 protein was immunoprecipitated and analyzed by western blot. Experiments were performed twice. (C-E) In situ hybridization of stage 33 embryos showing PGCs by Xpat staining in uninjected control (C), eIF3f-injected (D) and eIF3f+dnd1-injected (E) embryos. Experiments were repeated three times. (F) Quantification of results shown in C-E. Two-tailed t-tests were performed. *P<0.05, **P<0.01. (G) Working model of Dnd1 function in regulating nanos1 translation. Before fertilization, very little Dnd1 protein is present. Translation of nanos1 RNA is blocked by TCE, a secondary structure within the ORF that prevents the preinitiation complex (PIC) from scanning and initiating translation. After fertilization, Dnd1 protein accumulates within the germ plasm and there binds to nanos1 RNA, altering the TCE structure. The PIC can now scan the nanos1 RNA. Meanwhile, Dnd1 binds with the eIF3 complex through the interaction with subunit eIF3f. This interaction blocks the repressive activity of eIF3f and promotes the translation of nanos1 RNA. The eIF3-Dnd1 complex is released from the 40S ribosomal subunit as translation proceeds. |
