Drosophila germ granules are assembled from protein components through different modes of competing interactions with the multi-domain Tudor protein.

Membraneless organelles are RNA-protein assemblies which have been implicated in post-transcriptional control. Germ cells form membraneless organelles referred to as germ granules, which contain conserved proteins including Tudor domain-containing scaffold polypeptides and their partner proteins that interact with Tudor domains. Here, we show that in Drosophila, different germ granule proteins associate with the multi-domain Tudor protein using different numbers of Tudor domains. Furthermore, these proteins compete for interaction with Tudor in vitro and, surprisingly, partition to distinct and poorly overlapping clusters in germ granules in vivo. This partition results in minimization of the competition. Our data suggest that Tudor forms structurally different configurations with different partner proteins which dictate different biophysical properties and phase separation parameters within the same granule.

Glial granules contain germline proteins in the Drosophila brain, which regulate brain transcriptome.

Membraneless RNA-protein granules play important roles in many different cell types and organisms. In particular, granules found in germ cells have been used as a paradigm to study large and dynamic granules. These germ granules contain RNA and proteins required for germline development. Here, we unexpectedly identify large granules in specific subtypes of glial cells ("glial granules") of the adult Drosophila brain which contain polypeptides with previously characterized roles in germ cells including scaffold Tudor, Vasa, Polar granule component and Piwi family proteins. Interestingly, our super-resolution microscopy analysis shows that in the glial granules, these proteins form distinct partially overlapping clusters. Furthermore, we show that glial granule scaffold protein Tudor functions in silencing of transposable elements and in small regulatory piRNA biogenesis. Remarkably, our data indicate that the adult brain contains a small population of cells, which express both neuroblast and germ cell proteins. These distinct cells are evolutionarily conserved and expand during aging suggesting the existence of age-dependent signaling. Our work uncovers previously unknown glial granules and indicates the involvement of their components in the regulation of brain transcriptome.

Protein components of ribonucleoprotein granules from Drosophila germ cells oligomerize and show distinct spatial organization during germline development.

The assembly of large RNA-protein granules occurs in germ cells of many animals and these germ granules have provided a paradigm to study structure-functional aspects of similar structures in different cells. Germ granules in Drosophila oocyte's posterior pole (polar granules) are composed of RNA, in the form of homotypic clusters, and proteins required for germline development. In the granules, Piwi protein Aubergine binds to a scaffold protein Tudor, which contains 11 Tudor domains. Using a super-resolution microscopy, we show that surprisingly, Aubergine and Tudor form distinct clusters within the same polar granules in early Drosophila embryos. These clusters partially overlap and, after germ cells form, they transition into spherical granules with the structural organization unexpected from these interacting proteins: Aubergine shell around the Tudor core. Consistent with the formation of distinct clusters, we show that Aubergine forms homo-oligomers and using all purified Tudor domains, we demonstrate that multiple domains, distributed along the entire Tudor structure, interact with Aubergine. Our data suggest that in polar granules, Aubergine and Tudor are assembled into distinct phases, partially mixed at their "interaction hubs", and that association of distinct protein clusters may be an evolutionarily conserved mechanism for the assembly of germ granules.

In vivo mapping of a dynamic ribonucleoprotein granule interactome in early Drosophila embryos.

Macromolecular complexes and organelles play crucial roles within cells, but their native architectures are often unknown. Here, we use an evolutionarily conserved germline organelle, the germ granule, as a paradigm. In Drosophila embryos, we map one of its interactomes using a novel in vivo crosslinking approach that employs two interacting granule proteins and determines their common neighbor molecules. We identified an in vivo granule assembly of Tudor, Aubergine, motor and metabolic proteins, and RNA helicases, and provide evidence for direct interactions within this assembly using purified components. Our study indicates that germ granules contain efficient biochemical reactors involved in post-transcriptional gene regulation.