A CCHC METAL-BINDING DOMAIN IN NANOS IS ESSENTIAL FOR TRANSLATIONAL REGULATION

The Drosophila Nanos protein is a localized repressor of hunchback mRN A translation in the early embryo, and is required for the establishme nt of the anterior-posterior body axis. Analysis of nanos mutants reve als that a small, evolutionarily conserved, C-terminal region is essen tial 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-bin ding sites. Using absorption spectroscopy and NMR we demonstrate that the CCHC motifs each bind one equivalent of zinc with high affinity, n anos mutations disrupting metal binding at either of these two sites i n vitro abolish Nanos translational repression activity in vivo. We sh ow that full-length and C-terminal Nanos proteins bind to RNA in vitro with high affinity, but with little sequence specificity. Mutations a ffecting the hunchback mRNA target sites for Nanos-dependent translati onal 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 sequ ences, but might interact with RNA in the context of a larger ribonucl eoprotein complex.