View Program - 63rd Annual Drosophila Research Conference

Ubiquitination is a post-translational modification in which a small protein called Ubiquitin is covalently attached to Lysine residues of target proteins to specify their degradation, alter their activity or localization. In a multistep process, an E1 ubiquitin ligase activates Ubiquitin and transfers it to an E2 ligase. The activated E2 then interacts with an E3 ligase which specifies the target. In D. melanogaster there is a single E1 ligase (Uba1), 29 predicted E2 ligases and 156 predicted E3 ligases, allowing for a multitude of potential ubiquitin ligase complexes. This suggests that this complexity is needed for temporal or tissue-specific ubiquitination, however relatively few complexes and their targets have been defined. Here we sought to identify which E2 ligases are required in the male germ line. We used four GAL4 drivers (bam, C135, nanos, T110) in combination with UAS-RNAi constructs to knock down expression of all 29 E2 ligases and Uba1 in the male germline. Test males were assayed for fertility, were examined cytologically for visible defects in spermatogenesis and monitored genetically for fourth chromosome missegregation. Knockdowns of both taf1 and ubc6 produced the most severe phenotype of male sterility resulting from failure to enter meiosis. This phenotype was also observed for the knockdown of uba1. Meiotic chromosome segregation defects were observed in the knockdowns of six E2 ligases (bruce, CG7656, CG8188, ubc2, ubc10 and ubcE2H). Fourth chromosome missegregation (which may not be detectable cytologically) was observed via genetic crosses for knockdowns of four additional E2 ligases (CG4443, CG9602, CG10862 and CG17030). Our results reveal that multiple ubiquitin ligase complexes are necessary for entry into meiosis and meiotic chromosome segregation in males. Several of these have not formerly been implicated in meiosis and future work towards understanding their roles will be focused on identification of their E3 ligase partners and their protein targets.

Identification of E2 ubiquitin-conjugating enzymes required in Drosophila male meiosis