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Tudor domain-containing proteins are conserved across the animal kingdom for their important role in germline development and fertility. Previously, we demonstrated that Tudor domain-containing protein 5-like (Tdrd5l) plays an important role in the germline where it promotes male identity. However, Tdrd5l is also expressed in both the ovary and testis during later stages of germline development, suggesting that it may play a role in germline differentiation in both sexes. Through immunohistochemistry and genetic interaction assays we found that Tdrd5l localizes to a potentially novel germline RNA granule and plays a role in post-transcriptional gene regulation. RNA sequencing of Tdrd5l mutant ovaries compared to wild-type showed an enrichment for maternally deposited RNAs in differentially expressed genes. Additionally, embryos laid by Tdrd5l-mutant females exhibited reduced viability and displayed dorsal appendage defects suggesting a failure of proper dorsal-ventral patterning. We also observed defects in posterior localization of the oskar mRNA and protein, as well as Kinesin-LacZ, which indicate defects on anterior-posterior polarization of the oocyte. As both A/P and D/V patterning are dependent on gurken, we examined Grk expression during oogenesis. We observed premature accumulation of Gurken (Grk) protein, as well as a translational regulator of Grk, Oo18 RNA-Binding Protein (Orb or CPEB), in nurse cells, indicating that translation of these proteins is no longer properly repressed during mRNA transport to the oocyte. We also found that decreased orb function suppressed the Tdrd5l-mutant phenotype, and so defects in Orb are likely a primary cause of the defects in Tdrd5l mutants. Our data indicate that Tdrd5l is important for translational repression of maternal mRNAs such as orb, and possibly others, following their synthesis in the nurse cells and during their transport and subsequent activation in the oocyte.Author SummaryAcross the animal kingdom, Tudor-domain containing proteins are important for germline development, gametogenesis and fertility. One important aspect of oogenesis is the post-transcriptional regulation of maternal mRNAs that must be translationally repressed prior to their activation at the correct time and place in the oocyte or early embryo. In this study we show that Drosophila Tudor-domain containing protein 5-like (Tdrd5l) is important for post-transcriptional regulation of maternal mRNAs. Tdrd5l localizes to an apparently new type of RNA granule in the germline and interacts with post-transcriptional regulation pathways. Embryos from Tdrd5l-mutant mothers exhibit patterning defects and lethality. We observe premature accumulation of the important embryonic patterning factor Gurken (Grk/EGF) in nurse cells during oogenesis. Grk is known to be regulated by a cytoplasmic polyadenylation element binding protein (CPEB) called Orb (Oo18 RNA-Binding Protein), and we also observe premature accumulation of Orb in nurse cells in Tdrd5l mutants. Decreased orb function suppresses the Tdrd5l-mutant phenotype, indicating that this is a primary defect in these mutants. Our data indicate that the “Tdrd5l Granule” is important for post-transcriptional regulation of maternal mRNAs such as orb, leading to their repression until they are required in the oocyte or early embryo.

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