Curtis D, Treiber D K, Tao F, Zamore P D, Williamson J R, Lehmann R
Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge 02142, USA.
EMBO J. 1997 Feb 17;16(4):834-43. doi: 10.1093/emboj/16.4.834.
The Drosophila Nanos protein is a localized repressor of hunchback mRNA translation in the early embryo, and is required for the establishment of the anterior-posterior body axis. Analysis of nanos mutants reveals that a small, evolutionarily conserved, C-terminal region is essential for Nanos function in vivo, while no other single portion of the Nanos protein is absolutely required. Within the C-terminal region are two unusual Cys-Cys-His-Cys (CCHC) motifs that are potential zinc-binding sites. Using absorption spectroscopy and NMR we demonstrate that the CCHC motifs each bind one equivalent of zinc with high affinity. nanos mutations disrupting metal binding at either of these two sites in vitro abolish Nanos translational repression activity in vivo. We show that full-length and C-terminal Nanos proteins bind to RNA in vitro with high affinity, but with little sequence specificity. Mutations affecting the hunchback mRNA target sites for Nanos-dependent translational repression were found to disrupt translational repression in vivo, but had little effect on Nanos RNA binding in vitro. Thus, the Nanos zinc domain does not specifically recognize target hunchback RNA sequences, but might interact with RNA in the context of a larger ribonucleoprotein complex.
果蝇的Nanos蛋白是早期胚胎中驼背（hunchback）mRNA翻译的局部阻遏物，是建立前后体轴所必需的。对nanos突变体的分析表明，一个小的、进化上保守的C末端区域对于Nanos在体内的功能至关重要，而Nanos蛋白的其他单一部分并非绝对必需。在C末端区域内有两个不寻常的Cys-Cys-His-Cys（CCHC）基序，它们是潜在的锌结合位点。利用吸收光谱和核磁共振，我们证明每个CCHC基序都以高亲和力结合一当量的锌。在体外破坏这两个位点中任何一个的金属结合的nanos突变会消除Nanos在体内的翻译阻遏活性。我们表明全长和C末端Nanos蛋白在体外以高亲和力结合RNA，但序列特异性很小。发现影响Nanos依赖性翻译阻遏的驼背mRNA靶位点的突变会破坏体内的翻译阻遏，但对体外Nanos RNA结合影响很小。因此，Nanos锌结构域不会特异性识别靶驼背RNA序列，而是可能在更大的核糖核蛋白复合物的背景下与RNA相互作用。
