SUMMARY Germ cells belong to a unique class of stem cells which give rise to eggs and sperm and ultimately to an entire organism after the fusion of the gametes. In many organisms, germ cells contain electron-dense structures also known as nuage or germ granules. Although the germ granules were discovered more than 100 years ago, their composition, structure, assembly and function are not fully understood. Germ granules contain noncoding RNAs, mRNAs and proteins required for germline development. Here we review recent studies which highlighted the importance of several protein families in germ granule assembly and function, including germ granule inducers, which initiate the granule formation, and downstream components such as RNA helicases and Tudor domain - Piwi protein - piRNA complexes. Assembly of these RNAs and proteins in one granule is likely to result in a highly efficient molecular machine which ensures translational control and protects germline DNA from mutations caused by mobile genetic elements. Furthermore, recent studies have shown that different somatic cells, including stem cells and neurons, produce germ granule components which play a crucial role in stem cell maintenance and memory formation, indicating a much more diverse functional potential of the granules than previously thought.

Germ cells give rise to all cell lineages in the next-generation and are responsible for the continuity of life. In a variety of organisms, germ cells and stem cells contain large ribonucleoprotein granules. Although these particles were discovered more than 100 years ago, their assembly and functions are not well understood. Here we report that glycolytic enzymes are components of these granules in Drosophila germ cells and both their mRNAs and the enzymes themselves are enriched in germ cells. We show that these enzymes are specifically required for germ cell development and that they protect their genomes from transposable elements, providing the first link between metabolism and transposon silencing. We further demonstrate that in the granules, glycolytic enzymes associate with the evolutionarily conserved Tudor protein. Our biochemical and single-particle EM structural analyses of purified Tudor show a flexible molecule and suggest a mechanism for the recruitment of glycolytic enzymes to the granules. Our data indicate that germ cells, similarly to stem cells and tumor cells, might prefer to produce energy through the glycolytic pathway, thus linking a particular metabolism to pluripotency.

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.

Drosophila Me31B is a conserved protein of germ granules, ribonucleoprotein complexes essential for germ cell development. Me31B post-transcriptionally regulates mRNAs by interacting with other germ granule proteins. However, a Me31B interactome is lacking. Here, we use an in vivo proteomics approach to show that the Me31B interactome contains polypeptides from four functional groups: RNA regulatory proteins, glycolytic enzymes, cytoskeleton/motor proteins, and germ plasm components. We further show that Me31B likely colocalizes with the germ plasm components Tudor (Tud), Vasa, and Aubergine in the nuage and germ plasm and provide evidence that Me31B may directly bind to Tud in a symmetrically dimethylated arginine-dependent manner. Our study supports the role of Me31B in RNA regulation and suggests its novel roles in germ granule assembly and function.

Me31B is a protein component of Drosophila germ granules and plays an important role in germline development by interacting with other proteins and RNAs. To understand the dynamic changes that the Me31B interactome undergoes from oogenesis to early embryogenesis, we characterized the early embryo Me31B interactome and compared it to the known ovary interactome. The two interactomes shared RNA regulation proteins, glycolytic enzymes, and cytoskeleton/motor proteins, but the core germ plasm proteins Vas, Tud, and Aub were significantly decreased in the embryo interactome. Our follow-up on two RNA regulations proteins present in both interactomes, Tral and Cup, revealed that they colocalize with Me31B in nuage granules, P-bodies/sponge bodies, and possibly in germ plasm granules. We further show that Tral and Cup are both needed for maintaining Me31B protein level and mRNA stability, with Tral's effect being more specific. In addition, we provide evidence that Me31B likely colocalizes and interacts with germ plasm marker Vas in the ovaries and early embryo germ granules. Finally, we show that Me31B's localization in germ plasm is likely independent of the Osk-Vas-Tud-Aub germ plasm assembly pathway although its proper enrichment in the germ plasm may still rely on certain conserved germ plasm proteins. Germ cells are essential for sexual reproduction and the survival of many species, and species-specific strategies exist to form germ cells 1-5. Drosophila melanogaster uses maternally inherited germ granules to determine germ cell fate. Germ granules are heterogeneous aggregates of ribonucleoprotein (RNP) complexes 6 that undergo dynamic positional, morphological, and compositional changes during germline development, a process that spans oogenesis and early embryogenesis 7-11. Me31B, a conserved germ granule component 9,12 , is expressed in nurse cells, oocytes, and early embryos 13. In these cells, Me31B exists in different types of RNP granules, including nuage granules, P-bodies, sponge bodies, and germ plasm granules 12-15. In these granules, Me31B has been suggested to function as a putative ATP-dependent RNA helicase that interacts with other germline proteins and RNAs to exert post-transcriptional regulation on those RNAs 10,11,13,16,17. As an important example, Me31B associates with osk mRNA to ensure its proper translation into Osk protein only at the posterior pole of developing oocytes. Then, the Osk protein initiates a step-wise assembly pathway that recruits downstream proteins including Vas, Tud, and Aub to form the germ plasm and eventually dictates germ cell formation 13,18-21. Me31B exhibits changes in its localization pattern, aggregation status, and even function as germline cells develop during the ovary-to-embryo transition 13,17. It is believed that these changes are correlated with the different biological contexts in which Me31B exists 17. Therefore, to understand the role of Me31B during germ