ELAVL2调控精原干细胞自我更新和分化的作用及机制

RNA-binding protein, proteins bind to the double or single stranded RNA, could regulate gene expression via post-transcriptional level. It is essential in spermatogenesis. Previous study revealed that RNA-binding protein ELAVL2 was specifically expressed in the neuronal and reproductive system. However, the expression, function and mechanism of ELAVL2 in spermatogenesis still remained unclear. Through comparing the mRNA profiles among spermatogonia, spermatocytes and spermatids, we predicted and verified that ELAVL2 was specifically expressed in speramtogonia. Notably, it is located in the nuclei of SSCs, whereas in differentiated spermatogonia, it is located in the plasma, suggesting ELAVL2 maybe plays essential roles in self-renew and differentiation of SSCs. In this project, the following studies will be conducted: 1) Identification of ELAVL2 in adult testis and its expression in developmental process from PGC to spermatogonia. 2) The function of ELAVL2 in self-renew and differentiation regulation of SSCs. 3) the mechanism of ELAVL2 in self-renew and differentiation regulation of SSCs. This study will provide insightful information in the regulation of spermatogenesis and offer a new theory for the establishment of in-vitro SSCs culture system and in-vitro induction from SSCs to haploid spermatids.

RNA结合蛋白与RNA分子相互作用，在转录后水平调控精子发生基因表达，为揭示精子发生机制的一类关键分子。前期研究表明RNA结合蛋白ELAVL2在哺乳类动物生殖和神经系统特异表达，但在睾丸中的表达、功能以及机制至今不得而知。本团队前期研究证实了ELAVL2在人精原细胞特异表达，其在精原干细胞中定位于胞核，在分化型精原细胞定位于胞浆，而在精母细胞和精子细胞中均不表达，提示其对精原干细胞自我更新和分化发挥关键作用。本项目拟开展以下研究：1）明确ELAVL2在睾丸组织中的时空表达特征以及发育过程中表达模式；2）应用模式动物及体外实验探讨ELAVL2对精原干细胞自我更新和分化的影响；3）利用RIP和irCLIP技术揭示ELAVL2参与精原干细胞自我更新和分化的机制。本研究将有助于阐明精原干细胞自我更新和分化新机制，为非梗阻性无精子症的治疗提供新靶标，为精原干细胞长期培养及诱导分化提供理论基础。

RNA结合蛋白（RNA-binding proteins, RBPs）在转录后水平调控基因表达，是调控精原干细胞（Spermatogonial stem cells, SSCs）自我更新和分化的关键分子，但具体的机制目前仍知之甚少。基于生精正常和非梗阻性无精子症（Non-obstructive azoospermia, NOA）患者睾丸单细胞转录组差异，课题组筛选出在人睾丸特异表达、精原细胞特异表达和NOA患者低表达的RBP ELAVL2。前期研究证实ELAVL2在人精原细胞特异表达，其在SSCs中定位于胞核，在分化型精原细胞定位于胞浆，提示其对SSCs自我更新和分化发挥关键作用，但相关研究从未见报道。在本项目的资助下，本课题组进一步开展了下述工作：1）利用免疫荧光等技术明确了ELAVL2在睾丸组织中的时空表达特征，发现在人和小鼠睾丸发育各阶段，ELAVL2均特异表达于SSCs；2）在体外研究中证明，ELAVL2显著抑制SSCs凋亡并通过激活ERK和AKT通路促进SSCs增殖；3）RNA免疫共沉淀联合高通量测序发现，ELAVL2在SSCs靶向结合一系列自我更新关键基因，包括Id4、Taf4b、Plzf、Lin28a、Bcl6b、Sall4、Foxo1和Etv5；4）体外敲低细胞系证明ELAVL2通过转录后调控促进靶基因蛋白表达；5）免疫共沉淀-质谱分析发现，在人和小鼠SSCs中，ELAVL2与生精关键调控蛋白DAZL互相作用。综上所述，本项目揭示了ELAVL2通过和DAZL互作，促进靶mRNA的蛋白表达，从而维持SSCs自我更新和增殖的分子机制，进一步证明了RBPs介导的转录后调控在SSCs命运决定以及NOA发病中的重要作用，为研究生精障碍的病因及诊治提供了新思路和新靶点。