Nanos recruits the CCR4-NOT complex to induce degradation of mRNA targets

The Nanos protein is a non-specific RNA-binding protein, which together with Pumilio represses translation of the maternally deposited hunchback (hb) mRNA and plays a major role in the establishment of the anterior-posterior body axis in Drosophila melanogaster. Previous studies have indicated that the hb 3’ UTR is sufficient for translational repression and that reporter mRNAs containing this 3’ UTR are translationally repressed and deadenylated in a Nanos- and Pumilio-dependent manner. However, the exact molecular mechanism of deadenylase complex recruitment to the mRNA reporter has so far remained elusive. Here we investigated the role of Nanos in the regulation of the hb 3’ UTR. The Nanos protein contains an N-terminal region predicted to be unstructured and two conserved C-terminal CCHC type zinc finger motifs. Using coimmunoprecipitation assays in Drosophila S2 cells, we show that Nanos interacts with both the CCR4-NOT and PAN2-PAN3 deadenylase complexes. Furthermore, we mapped the deadenylase- binding region to a fragment within the unstructured part of the protein. An in vitro pull-down experiment revealed that Nanos interacts directly with the NOT module of the CCR4-NOT complex. We also show that the deadenylase- binding region of Nanos is required to elicit mRNA degradation via the 5’-to-3’ decay pathway. In addition, our results show that tethered Nanos can promote both translational repression and mRNA degradation independently of Pumilio. Interestingly, the three human Nanos proteins, Nanos 1-3, also interact with the CCR4-NOT deadenylase complexes in human cells. Similar to Dm Nanos, human Nanos 1-3 promote the degradation of bound mRNAs. We conclude that Nanos proteins can recruit the CCR4-NOT complex to mRNA targets independently of Pumilio and that this activity is conserved amongst Nanos orthologs