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

In the Drosophila germline, Me31B is a putative ATP-dependent, RNA helicase that plays an important role in post-transcriptional RNA regulation to ensure mRNA′s correct spatial and temporal expression, a process crucial for proper germline development and fertility. However, Me31B′s in vivo working mechanism remains unclear. In this study, we aim to analyze the functions of Me31B′s key domains/motifs to understand how these domains/motifs operate to fulfill the protein′s overall activities. We generated mutant Drosophila strains for six important motifs including three ATPase/helicase motifs (DEAD-box, DVLARAK, and HRIGR), the N-terminal domain (N-ter), the C-terminal domain (C-ter), and a protein-binding motif (FDF motif-binding motif). In characterizing these mutants, we observed that the three ATPase/helicase motif mutations cause dominant female sterility, which is associated with developmental defects in oogenesis and embryogenesis. Follow-up examinations of the DVLARAK motif mutant revealed abnormalities in germline mRNA localization and transcript level. The Me31B N-ter domain (deletion of C-ter), C-ter domain (deletion of N-ter), and mutation of the FDF motif-binding motif led to a decrease in female fertility and abnormal subcellular Me31B localizations within the egg chambers. Moreover, deletion of Me31B′s N-ter or C-ter motif decreases Me31B protein levels in the ovaries. This study indicates that these six motifs of Me31B play distinct roles in contributing to Me31B′s whole-protein functions such as ATPase, RNA helicase, protein stability, protein localization, and partner protein binding, crucial for germline development and fertility. Considering the Me31B protein family′s conserved presence in both Drosophila germline and soma and in other eukaryotes such as yeast, worms, mice, and humans, the results from this study could expand our understanding of Me31B family helicases′ general working mechanisms in different cell types and species.

Me31B/DDX6 is a DEAD-box family RNA helicase playing roles in posttranscriptional RNA regulation in different cell types and species. Despite the known motifs/domains of Me31B, the in vivo functions of the motifs remain unclear. Here, we used the Drosophila germline as a model and used CRISPR to mutate the key Me31B motifs/domains: helicase domain, N-terminal domain, C-terminal domain and FDF-binding motif. Then, we performed screening characterization on the mutants and report the effects of the mutations on the Drosophila germline, on processes such as fertility, oogenesis, embryo patterning, germline mRNA regulation and Me31B protein expression. The study indicates that the Me31B motifs contribute different functions to the protein and are needed for proper germline development, providing insights into the in vivo working mechanism of the helicase.

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