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

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.