Distinct Recruitment Mechanisms for Aub and Ago3 to Nuage Lead to Functional Compartmentalization of the pIRNA Pathway in Drosophila

In Drosophila, two Piwi proteins, Aubergine (Aub) and Argonaute-3 (Ago3) localize to perinuclear ‘nuage’ granules and use guide piRNAs to target and destroy transposable element transcripts. Using imaging of fluorophore-tagged proteins in the Drosophila ovary we show that Aub and Ago3 have distinct localization and dynamics in nuage. Furthermore, we find that the mechanism of nuage localization is different for each of the two proteins. Aub requires a piRNA guide for recruitment to nuage indicating that its localization depends on recognition of RNA targets. Although wild-type Aub is highly mobile between nuage and a diffused cytoplasmic fraction, inactivation of its nuclease activity results in immobilization of Aub in nuage. Contrary to Aub, Ago3 localization in nuage is independent of a piRNA cargo and relies on interactions with Krimper. Our study reveals unexpected dynamics of nuage granule components and a multi-step process responsible for the assembly of nuage complexes.