January 1, 2011;
Nanos1 functions as a translational repressor in the Xenopus germline.
Nanos family members have been shown to act as translational repressors in the Drosophila and Caenorhabditis elegans germline, but direct evidence is missing for a similar function in vertebrates. Using a tethered function assay, we show that Xenopus Nanos1
is a translational repressor and that association with the RNA is required for this repression. We identified a 14 amino acid region within the N-terminal domain of Nanos1
that is conserved in organisms as diverse as sponge and Human. The region is found in all vertebrates but notably lacking in Drosophila and C. elegans. Deletion and substitution analysis revealed that this conserved region was required for Nanos1
repressive activity. Consistent with this observation, deletion of this region was sufficient to prevent abnormal development that results from ectopic expression of Nanos1
in oocytes. Although Nanos1
can repress capped and polyadenylated RNAs, Nanos1
mediated repression did not require the targeted RNA to have a cap or to be polyadenylated. These results suggest that Nanos1
is capable of repressing translation by several different mechanisms. We found that Nanos1
, like Drosophila Nanos, associates with cyclin B1
RNA in vivo indicating that some Nanos targets may be evolutionarily conserved. Nanos1
protein was detected and thus available to repress mRNAs while PGCs were in the endoderm
, but was not observed in PGCs after this stage.
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Fig.7. Nanos1 protein expression during X. laevis development. Endogenous Nanos1 was detected by confocal immunofluorescence with anti-Nanos1 antibody (red in all images). (A) Nanos1 was not expressed in stage VI oocytes. (C) Nanos1 protein was concentrated in a sub-cellular cytoplasmic domain close to the cell periphery in stage 8 embryos. (B, E, G, I) Nanos1 protein co-localizes with the germ plasm at stages 4 (8 cell), 10.5, 12, and 13. Xiwi, a germ plasm specific protein, was detected by anti-Xiwi antibody (green). (D, F, H, J, K) Hyperacetylated histone H4 (Penta), a nuclear marker, was detected by immunostaining (Green). Nanos1 protein (red) was found only in perinuclear locations in stages 10.5, 12, 13, 14, and 28. (L and M) Stages 33/34 and 37/38. Xpat RNA was detected by whole mount in situ hybridization using fluorescein tyramide (green). L′ and M′, same sample as in L and M. Nanos1 protein could no longer be detected in PGCs after stage 34. C–K, images were from the endoderm mass. L and M, lateral view, with anterior to the left. Scale bars are as indicated.
Fig. 3. Nanos1 Nt-conserved region is essential for repressive function (A) Summary of results from tethered function assays testing deletion and point mutations in the Nanos1Nt-7-22 region. Identical residues between humans, mouse, and frogs are in red, conserved residues in blue with their respective numbers indicated. Lines below the sequence indicate region of deletions with loss (−) or retention (+) of repressive function shown. Arrows above sequence indicate those residues substituted for alanine and only the blue arrows indicate residues found to be required for translational repression. (B) Mutation analysis defining residues required for Nanos1 repressive ability using the tethered function assay (Fig. 1). (C) Host transfer with Myc-Nanos1 (green) or Myc-Nanos1 deletion mutant δ7–22 (red). Note that expression of Myc-Nanos1 causes abnormal development while the mutant δ7-22 does not. Blot shows that Myc-tagged proteins were expressed in donor oocytes used in host transfer.
Maternal Nanos regulates zygotic gene expression in germline progenitors of Drosophila melanogaster.