A CCHC metal-binding domain in Nanos is essential for translational regulation
A CCHC metal-binding domain in Nanos is essential for translational regulation
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DOI：
10.1093/emboj/16.4.834
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发表时间：
1997-02-17
期刊：
影响因子：
11.4
通讯作者：
Lehmann, R
中科院分区：
文献类型：
--
作者：
Curtis, D;Treiber, DK;Lehmann, R
果蝇Nanos蛋白是一种在早期胚胎中局部抑制驼背mRNA翻译的蛋白，是建立前后体轴所必需的。对Nanos突变体的分析表明，一个进化上保守的小C末端区域是Nanos在体内发挥作用所必需的，而Nanos蛋白的其他单一部分并不是绝对必需的。在C-末端区域有两个不寻常的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技术，我们证明了CCHC基序每个都与一个相当于锌的高亲和力结合，在体外这两个位点上的Nanos突变破坏了这两个位点上的金属结合，在体内取消了Nanos的翻译抑制活性。我们发现全长和C末端的Nanos蛋白在体外与RNA结合的亲和力很高，但序列特异性很小。影响Nanos依赖的翻译抑制的驼背mRNA靶点的突变被发现在体内扰乱了翻译抑制，但在体外对Nanos RNA的结合几乎没有影响。因此，Nanos锌结构域不能特异性识别目标驼背RNA序列，但可能在更大的核糖核蛋白复合体的背景下与RNA相互作用。
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.