The process of sex determination in Drosophila is regulated by the RNA-binding protein Sex lethal (Sxl) in both the germline and the soma. While the mechanism by which SXL brings about sexual dimorphism in the soma is well studied, its function in the germline is less well understood. To identify genes that are regulated by SXL in the germline, RNA sequencing was done in a bam mutant background to compare Sxl-RNAi ovaries to control ovaries. Analysis of this expression data uncovered a previously uncharacterized gene CG15930 with a 16-fold increase in expression in Sxl-RNAi females. Comparison of bam mutant testes and ovaries also showed a 16-fold higher expression level in males suggesting that SXL functions in the female germline to repress CG15930. CG15930 contains a single tudor domain and is most closely related to the mouse TDRD5 and the Drosophila TDRD5 homolog tejas, so we have named it tdrd5-prime (tdrd5p).
To elucidate the function of TDRD5P in the male germline, we generated mutant alleles using CRISPR/CAS9 along with an HA tagged genomic BAC construct for analysis of the TDRD5P protein. The tdrd5p mutant testes show several phenotypes such as hub displacement, germline loss, and a 50% reduction in fecundity indicative of an important role for TDRD5P in germline differentiation. Confocal microscopy of HA-TDRD5P testes shows an interesting localization of the protein to discrete cytoplasmic punctae near the nuclear periphery. This suggests that TDRD5P may localize to an RNA body such as the P-body or nuage similar to other tudor domain containing proteins. Since tudor domain proteins are known to function in the piRNA pathway, RT-PCR was conducted for numerous transposons in tdrd5p mutants and found no change in expression. We are currently conducting RNA-seq to compare tdrd5p mutants to wildtype to investigate whether TDR5P regulates the levels of other cellular RNAs. To determine what type of RNA body TDR5P functions in, we are conducting co-immunoprecipitation followed by mass spectrometry. Additionally, to understand the mechanism by which TDR5P functions we plan to do a series of truncations to identify potential important regions of the protein that have yet to be annotated. Based on this data we propose that the previously uncharacterized TDR5P functions to ensure the proper development of the male germline and must be repressed by SXL to allow for female germline development.