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The translational repressor Nanos is expressed in the germline and stem cell populations of jellyfish as well as humans. Surprisingly, we observed that unlike other mRNAs, synthetic nanos1 RNA translates very poorly if at all after injection into Xenopus oocytes. The current model of simple sequestration of nanos1 within germinal granules is insufficient to explain this observation and suggests that a second level of repression must be operating. We find that an RNA secondary structural element immediately downstream of the AUG start site is both necessary and sufficient to prevent ribosome scanning in the absence of a repressor. Accordingly, repression is relieved by small in-frame insertions before this secondary structure, or translational control element (TCE), that provide the 15 nucleotides required for ribosome entry. nanos1 is translated shortly after fertilization, pointing to the existence of a developmentally regulated activator. Oocyte extracts were rendered fully competent for nanos1 translation after the addition of a small amount of embryo extract, confirming the presence of an activator. Misexpression of Nanos1 in oocytes from unlocalized RNA results in abnormal development, highlighting the importance of TCE-mediated translational repression. Although found in prokaryotes, steric hindrance as a mechanism for negatively regulating translation is novel for a eukaryotic RNA. These observations unravel a new mode of nanos1 regulation at the post-transcriptional level that is essential for normal development.
Fig. 5. nanos1 repression is relieved only after fertilization. (A) nanos1 remains repressed during oocyte maturation events. Xenopus oocytes were injected with 10 ng of nanos1 transcript per oocyte and incubated at 18°C. To trigger maturation events, oocytes were incubated with progesterone (2 μM) and collected until at least 50% displayed germinal vesicle breakdown. Samples were analyzed by blotting with anti-Nanos1 antibody. nanos1-injected embryos served as a positive control. (B) Injected nanos1 transcripts are efficiently translated in embryos before first cleavage. One nanogram of capped transcript was injected into 1-cell stage embryos soon after fertilization. Embryos were collected at the indicated stages and protein extracts analyzed by blotting after immunoprecipitation (IP) with anti-Nanos1 antibody. (C) nanos1-Myc was translationally active in the presence of embryo extract in a dose-independent fashion. One microgram of capped transcript was translated in vitro with oocyte and/or embryo extract. Samples were analyzed by blotting with anti-Myc antibody. (D) Endogenous Nanos1 was easily detected in the germ plasm by the 8-cell stage. Confocal immunofluorescence with anti-Nanos1 antibody. (E) Confocal immunofluorescence of embryos previously injected with nanos1-Myc RNA shows Nanos1 accumulation in somatic cells. Scale bars: 200 μm.
Fig. 6. Misexpression of nanos1 in oocytes results in abnormal development. (A) Schematic of host transfer experiment. (B) Following the procedure in A, oocytes were injected with either Myc-nanos1 RNA or control Myc RNA and analyzed by blotting with anti-Myc antibody. RNAs were translated in a dose-dependent fashion. (C) Xenopus embryos from experiment 2 (see D) showing representative phenotypes that result from nanos1 expression in oocytes. Uninjected oocytes or Myc-injected oocytes served as controls and were normal. Embryos observed at stage 12.5 fail to close their blastopores. Surviving embryos display incomplete neural tube closure at stage 21. (D) The distribution of phenotypes from two independent experiments. Embryos with a gastrulation defect (GD) were counted at stage 12.5 and those with incomplete neural tube closure (INC) or that were dead were counted at stage 21. The key includes the total number of embryos in each category for each experiment.
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