Significant roles in RNA-binding for the amino-terminal domains of Drosophila Pumilio and Nanos.

The Drosophila Pumilio (Pum) and Nanos (Nos) RNA-binding proteins govern abdominal segmentation in the early embryo, as well as a variety of other events during development. They bind together to a compound Nanos Response Element (NRE) present in thousands of maternal mRNAs in the ovary and embryo, including hunchback ( hb ) mRNA, thereby regulating poly-adenylation, translation, and stability. Many studies support a model in which mRNA recognition and effector recruitment are achieved by distinct regions of each protein. The well-ordered Pum and Nos RNA-binding domains (RBDs) are sufficient to specifically recognize NREs; the relatively larger low-complexity N-terminal domains (NTDs) of each protein have been thought to act by recruiting mRNA regulators. Here we use yeast interaction assays to show that the NTDs also play a significant role in recognition of the NRE, acting via two mechanisms. First, the Pum and Nos NTDs interact in trans to promote assembly of the Pum/Nos/NRE ternary complex. Second, the Pum NTD acts via an unknown mechanism in cis, modifying base recognition by its RBD. These activities of the Pum NTD are important for its regulation of maternal hb mRNA in vivo.

Widespread regulation of the maternal transcriptome by Nanos in Drosophila.

The translational repressor Nanos (Nos) regulates a single target, maternal hunchback (hb) mRNA, to govern abdominal segmentation in the early Drosophila embryo. Nos is recruited specifically to sites in the 3'-UTR of hb mRNA in collaboration with the sequence-specific RNA-binding protein Pumilio (Pum); on its own, Nos has no binding specificity. Nos is expressed at other stages of development, but very few mRNA targets that might mediate its action at these stages have been described. Nor has it been clear whether Nos is targeted to other mRNAs in concert with Pum or via other mechanisms. In this report, we identify mRNAs targeted by Nos via two approaches. In the first method, we identify mRNAs depleted upon expression of a chimera bearing Nos fused to the nonsense mediated decay (NMD) factor Upf1. We find that, in addition to hb, Upf1-Nos depletes ~2600 mRNAs from the maternal transcriptome in early embryos. Virtually all of these appear to be targeted in a canonical, hb-like manner in concert with Pum. In a second, more conventional approach, we identify mRNAs that are stabilized during the maternal zygotic transition (MZT) in embryos from nos- females. Most (86%) of the 1185 mRNAs regulated by Nos are also targeted by Upf1-Nos, validating use of the chimera. Approximately 60% of mRNAs targeted by Upf1-Nos are not stabilized in the absence of Nos. However, Upf1-Nos mRNA targets are hypo-adenylated and inefficiently translated at the ovary-embryo transition, whether or not they suffer Nos-dependent degradation in the embryo. We suggest that the late ovarian burst of Nos represses a large fraction of the maternal transcriptome, priming it for later degradation by other factors during the MZT in the embryo.

No significant regulation of bicoid mRNA by Pumilio or Nanos in the early Drosophila embryo.

Drosophila Pumilio (Pum) is a founding member of the conserved Puf domain class of RNA-binding translational regulators. Pum binds with high specificity, contacting eight nucleotides, one with each of the repeats in its RNA-binding domain. In general, Pum is thought to block translation in collaboration with Nanos (Nos), which exhibits no binding specificity in isolation but is recruited jointly to regulatory sequences containing a Pum binding site in the 3'-UTRs of target mRNAs. Unlike Pum, which is ubiquitous in the early embryo, Nos is tightly restricted to the posterior, ensuring that repression of its best-characterized target, maternal hunchback (hb) mRNA, takes place exclusively in the posterior. An exceptional case of Nos-independent regulation by Pum has been described-repression of maternal bicoid (bcd) mRNA at the anterior pole of the early embryo, dependent on both Pum and conserved Pum binding sites in the 3'-UTR of the mRNA. We have re-investigated regulation of bcd in the early embryo; our experiments reveal no evidence of a role for Pum or its conserved binding sites in regulation of the perdurance of bcd mRNA or protein. Instead, we find that Pum and Nos control the accumulation of bcd mRNA in testes.