长链非编码RNA-AK131677表达的多肽对小鼠精原干细胞的调控作用及其机制研究

The self-renewal of spermatogonial stem cells (SSCs) is mainly depended on an important microenvironmental factor: GDNF. Although there are some basic researches on the intrinsic signaling pathways stimulated by GDNF, less is known about the functions of lncRNA during this process. Recently, more and more evidences showed that there may be short open reading frame hidden in the sequence regions of noncoding genes, which could express functional polypeptide. Based on previously identified differentially expressed lncRNA between GDNF starvation and refreshment, combining the results of proteomic analysis, we obtained a list of candidate polypeptides encoded by lncRNA. We found that AK131677 was down-regulated after GDNF starvation and up-regulated after GDNF refreshment. Proteomic results indicate that it expressed a polypeptide with 42 amino acids and two phosphorylated serine sites. The present study will focus on the polypeptide encoded by AK131677 and aim to determine its function on the self-renewal of SSC, as well as to explore the signaling pathway. Our work will provide evidence for the non-canonical fucntion of polypeptide encoded by lncRNA in the field of stem cell, and will also provide new researching direction for identifying key regulators involving in SSC self-renewal.

精原干细胞（SSC）的自我更新依赖于一种重要的微环境因子：GDNF，虽然GDNF激活胞内信号通路已有一定的研究基础，但是长链非编码RNA在此过程中的作用研究较少。近年来越来越多的证据表明非编码基因区域暗藏着短开放读码框架，并可表达具有活性功能的多肽。基于前期鉴定的在GDNF饥饿与再补充条件下差异表达的lncRNA，结合蛋白质谱结果，我们筛选到了一批备选的由lncRNA表达的功能性多肽。其中，我们发现AK131677转录本在GDNF饥饿状态明显下调，而在补充GDNF后表达回升，质谱数据显示该基因表达42个氨基酸的多肽，并且有两个丝氨酸位点发生磷酸化修饰。本课题将围绕由AK131677编码的多肽，明确其对小鼠SSC自我更新的调控作用，并深入分析其发挥功能的信号通路。本研究将在干细胞领域为lncRNA通过翻译多肽发挥作用这一非经典途径提供证据，并为鉴定干细胞自我更新调控的关键分子提供新的思路。

精原干细胞（SSC）的自我更新依赖于一种重要的微环境因子：GDNF，虽然GDNF激活胞内信号通路已有一定的研究基础，但是长链非编码RNA在此过程中的作用研究较少。近年来越来越多的证据表明非编码基因区域暗藏着短开放读码框架，并可表达具有活性功能的多肽。本研究首先基于优化的算法系统分析了GDNF介导SSC自我更新相关的关键基因、调控网络及其信号通路。其次，在GDNF饥饿与加回的转录组学谱系中鉴定了差异表达的lncRNAs，并在这些lncRNA中预测了暗藏的sORF及其编码多肽，随后利用蛋白质组学和磷酸化蛋白组学方法进一步筛选和验证了这些多肽。最终，我们发现一种关键lncRNA：AK131677，其转录本在GDNF饥饿状态明显下调，而在补充GDNF后表达回升，质谱数据显示该基因表达42个氨基酸的多肽，并且有两个丝氨酸位点发生磷酸化修饰。通过激酶-底物位点分析发现其上游调控激酶可能为Erk1/2和ATM，因此Gsp42可能通过磷酸化相关信号通路参与GDNF介导SSC自我更新相关功能的调控。本研究为干细胞领域为lncRNA通过翻译多肽发挥作用这一非经典途径提供了直接证据，同时也为鉴定干细胞自我更新调控的关键分子提供了新的思路。