Abstract
Background: Spermatogenesis depends on precise cytoskeletal regulation, particularly the microtubule system; however, the mechanisms governing tubulin homeostasis during meiosis are poorly defined. While the E3 ubiquitin ligase Hyd (Hyperplastic discs), the Drosophila homolog of UBR5 (Ubiquitin Protein Ligase E3 Component N-Recognin 5), plays roles in diverse cellular processes, its precise role in male meiosis is unknown. This study aims to define the function and expression dynamics of Hyd during Drosophila spermatogenesis and elucidate whether it acts independently of its canonical ligase activity. Methods: Using Drosophila genetics, immunofluorescence, CRISPR/Cas9-mediated tagging, and mosaic clonal analysis, we characterized Hyd expression and function in the testis. Hyd knockdown and rescue experiments were performed with wild-type and catalytically inactive transgenes. β2-tubulin expression and microtubule organization were assessed in hyd mutant clones. Results: Hyd exhibits a dynamic, stage-specific expression pattern, localizing to nuclear and meiotic structures. Hyd loss led to meiotic arrest, disrupted spindle formation, aberrant centrosome behavior, and failure of spermatid elongation. Genetic rescue demonstrated that both wild-type and catalytically inactive Hyd partially restored spermatid elongation, indicating a catalysis-independent role. Furthermore, Hyd deficiency resulted in β2-tubulin overexpression, disrupted microtubule organization, and abnormal spermatocyte morphology. Conclusions: Hyd ensures meiotic fidelity in Drosophila by fine-tuning β2-tubulin expression independently of its E3 ubiquitin ligase activity. These findings reveal a non-proteolytic function for UBR5/Hyd in cytoskeletal regulation during male gametogenesis, providing new insights into tubulin homeostasis in meiosis.
