CRL2LRRC41-Mediated DDX5 Ubiquitination Enhances Interaction with ELAVL1 Preventing NOG mRNA Degradation and Sustaining Proliferation and Migration of Human Spermatogonial Stem Cell-Like Cell Line.

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.