Jiang Bing, Wang Kehan, Hu Haoyue, Gao Wenxin, Shen Cong, Chen Xia, Huang Xiaoyan, Yu Jun, Wu Yibo, Zheng Bo
Human Reproductive and Genetic Center, Affiliated Hospital of Jiangnan University, Wuxi, 214062, China.
State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproduction and Genetics, Gusu School, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Nanjing Medical University, Suzhou, 215002, China.
BMC Biol. 2025 Aug 7;23(1):247. doi: 10.1186/s12915-025-02363-z.
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, and its role in SSC function.
We utilized molecular biology techniques, including gene knockdown and functional assays, to assess the effects of CRL2 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. We specifically focused on ATP-dependent RNA helicase DDX5, a known regulator of spermatogenesis, to explore its interaction with CRL2 and the downstream molecular mechanisms involved.
Our findings revealed that the disruption or dysfunction of CRL2 led to reduced proliferative and migratory abilities in human SSCLCs. Through our investigation, we identified DDX5 as a ubiquitination substrate of CRL2. 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.
This study provides the first identification of the CRL2 complex in human SSCLCs and elucidates the molecular mechanisms by which CRL2 facilitates SSCLC function via ubiquitination-mediated protein interactions. These findings offer novel insights into the molecular underpinnings of male infertility.
人类精原干细胞（SSCs）具有显著的增殖能力，这对于维持一生的精子发生至关重要。虽然已知Cullin-RING E3泛素连接酶2（CRL2）复合物可调节多种细胞功能，但其在人类SSCs中的精确作用尚未完全阐明。本研究旨在研究CRL2复合物的一种新型变体，称为CRL2，及其在SSC功能中的作用。
我们利用分子生物学技术，包括基因敲低和功能测定，来评估CRL2对人类精原干细胞样细胞（SSCLC）系增殖和迁移能力的影响。此外，我们采用蛋白质组学和生化方法来鉴定CRL2的潜在底物。我们特别关注ATP依赖性RNA解旋酶DDX5，一种已知的精子发生调节因子，以探索其与CRL2的相互作用以及涉及的下游分子机制。
我们的研究结果表明，CRL2的破坏或功能障碍导致人类SSCLCs的增殖和迁移能力降低。通过我们的研究，我们鉴定出DDX5是CRL2的泛素化底物。值得注意的是，DDX5的泛素化促进了其与RNA结合蛋白ELAVL1的相互作用，而不是通过泛素-蛋白酶体系统（UPS）将DDX5导向降解。这种相互作用增强了下游转录本Noggin（NOG）的稳定性，从而支持人类SSCLC的增殖和迁移。
本研究首次在人类SSCLCs中鉴定出CRL2复合物，并阐明了CRL2通过泛素化介导的蛋白质相互作用促进SSCLC功能的分子机制。这些发现为男性不育的分子基础提供了新的见解。
