A CCHC metal-binding domain in Nanos is essential for translational regulation.
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.
Curtis
D
D
Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge 02142, USA.
Treiber
D K
DK
Tao
F
F
Zamore
P D
PD
Williamson
J R
JR
Lehmann
R
R
D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D030801:Animals, Genetically Modified; D017124:Conserved Sequence; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics; D005260:Female; D018507:Gene Expression Regulation, Developmental; D007301:Insect Hormones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
D029721:Drosophila Proteins; D007301:Insect Hormones; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C070574:nos protein, Drosophila; D015032:Zinc