Department of Andrology, The Center for Men's Health, Urologic Medical Center, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
State Key Lab of Reproductive Medicine, Nanjing Medical University, Nanjing, China.
Cell Prolif. 2021 Sep;54(9):e13098. doi: 10.1111/cpr.13098. Epub 2021 Jul 23.
RNA-binding proteins (RBPs) play essential post-transcriptional roles in regulating spermatogonial stem cells (SSCs) maintenance and differentiation. We identified a conserved and SSCs-enriched RBP ELAVL2 from our single-cell sequencing data, but its function and mechanism in SSCs were unclear.
Expressions of ELAVL2 during human and mouse testis development were validated. Stable C18-4 and TCam-2 cell lines with overexpression and knockdown of ELAVL2 were established, which were applied to proliferation and apoptosis analysis. RNA immunoprecipitation and sequencing were used to identify ELAVL2 targets, and regulatory functions of ELAVL2 on target mRNAs were studied. Proteins interacting with ELAVL2 in human and mouse testes were identified using immunoprecipitation and mass spectrometric, which were validated by in vivo and in vitro experiments.
ELAVL2 was testis-enriched and preferentially expressed in human and mouse SSCs. ELAVL2 was down-regulated in NOA patients. ELAVL2 promoted proliferation and inhibited apoptosis of C18-4 and TCam-2 cell lines via activating ERK and AKT pathways. ELAVL2 associated with mRNAs encoding essential regulators of SSCs proliferation and survival, and promoted their protein expression at post-transcriptional level. ELAVL2 interacted with DAZL in vivo and in vitro in both human and mouse testes.
Taken together, these results indicate that ELAVL2 is a conserved SSCs-enriched RBP that down-regulated in NOA, which regulates spermatogonia proliferation and apoptosis by promoting protein expression of targets.
RNA 结合蛋白（RBPs）在调控精原干细胞（SSCs）的维持和分化方面发挥着重要的转录后作用。我们从单细胞测序数据中鉴定了一种保守且在 SSCs 中丰富表达的 RBP——ELAVL2，但它在 SSCs 中的功能和机制尚不清楚。
验证了 ELAVL2 在人及小鼠睾丸发育过程中的表达情况。构建了 ELAVL2 过表达和敲低的稳定 C18-4 和 TCam-2 细胞系，用于增殖和凋亡分析。利用 RNA 免疫沉淀和测序鉴定 ELAVL2 的靶标，并研究 ELAVL2 对靶 mRNA 的调控作用。采用免疫沉淀和质谱技术鉴定人及小鼠睾丸中与 ELAVL2 相互作用的蛋白，通过体内和体外实验进行验证。
ELAVL2 在睾丸中高表达且具有组织特异性，在人及小鼠 SSCs 中特异性表达。NOA 患者中 ELAVL2 的表达下调。ELAVL2 通过激活 ERK 和 AKT 通路促进 C18-4 和 TCam-2 细胞系的增殖并抑制其凋亡。ELAVL2 与编码 SSCs 增殖和存活的关键调控因子的 mRNA 结合，并在转录后水平促进其蛋白表达。ELAVL2 在体内和体外均与 DAZL 相互作用，在人及小鼠睾丸中均如此。
综上所述，这些结果表明 ELAVL2 是一种保守的 SSCs 丰富表达的 RBP，在 NOA 中下调，通过促进靶标蛋白的表达来调节精原细胞的增殖和凋亡。
