Abstract
BACKGROUND: Human spermatogonial stem cells (SSCs) exhibit a remarkable capacity for proliferation, crucial for sustaining spermatogenesis throughout life. While the Cullin-RING E3 ubiquitin ligase 2 (CRL2) complex is known to regulate various cellular functions, its precise role in human SSCs has not been fully elucidated. This study aimed to investigate a novel variant of the CRL2 complex, termed CRL2(LRRC41), and its role in SSC function. METHODS: We utilized molecular biology techniques, including gene knockdown and functional assays, to assess the effects of CRL2(LRRC41) on the proliferative and migratory abilities of human spermatogonial stem cell-like cell (SSCLC) line. Additionally, we employed proteomics and biochemical approaches to identify potential substrates of CRL2(LRRC41). We specifically focused on ATP-dependent RNA helicase DDX5, a known regulator of spermatogenesis, to explore its interaction with CRL2(LRRC41) and the downstream molecular mechanisms involved. RESULTS: Our findings revealed that the disruption or dysfunction of CRL2(LRRC41) led to reduced proliferative and migratory abilities in human SSCLCs. Through our investigation, we identified DDX5 as a ubiquitination substrate of CRL2(LRRC41). Notably, the ubiquitination of DDX5 fosters its interaction with the RNA-binding protein ELAVL1, without directing DDX5 towards degradation via the ubiquitin-proteasome system (UPS). This interaction enhances the stability of the downstream transcript, Noggin (NOG), thereby supporting human SSCLC proliferation and migration. CONCLUSIONS: This study provides the first identification of the CRL2(LRRC41) complex in human SSCLCs and elucidates the molecular mechanisms by which CRL2(LRRC41) facilitates SSCLC function via ubiquitination-mediated protein interactions. These findings offer novel insights into the molecular underpinnings of male infertility.