Nanog RNA-binding proteins YBX1 and ILF3 affect pluripotency of embryonic stem cells
Nanog RNA-binding proteins YBX1 and ILF3 affect pluripotency of embryonic stem cells
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Nanog RNA结合蛋白YBX1和ILF3影响胚胎干细胞的多能性
DOI：
10.1002/cbin.10539
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发表时间：
2016-08-01
影响因子：
3.9
通讯作者：
Zeng, Fanyi
中科院分区：
文献类型：
--
作者：
Guo, Chuanliang;Xue, Yan;Zeng, Fanyi
Nanog是一种众所周知的转录因子，在干细胞自我更新和维持其多能细胞身份中起着重要作用。关于关键多能性转录因子的相互作用伴侣的谱，仍然存在很大的数据缺口。关于Nanog相关的RNA结合蛋白（RBP）以及Nanog调节活性的内在蛋白质-RNA相互作用特征的信息有限。本文中，我们使用改进的亲和方案从小鼠胚胎干细胞（ESC）中纯化Nanog相互作用的RBP，并且鉴定了49个Nanog的RBP。其中，YBX 1和ILF 3与Nanog mRNA的相互作用通过Western blot、RNA免疫沉淀（RIP）等体外实验和免疫荧光染色、荧光原位杂交（FISH）、MS 2体内生物素标记RNA亲和纯化（MS 2-BioTRAP）等离体方法进一步证实。有趣的是，RNAi研究显示YBX 1和ILF 3对Nanog和其他多能性相关基因的表达有积极影响。特别地，YBX 1或ILF 3的下调导致中胚层标志物的高表达。因此，YBX 1和ILF 3表达的减少控制了ESC中多能性相关基因的表达，表明它们在进一步调节ESC的多能性状态中的作用。
Nanog is a well-known transcription factor that plays a fundamental role in stem cell self-renewal and the maintenance of their pluripotent cell identity. There remains a large data gap with respect to the spectrum of the key pluripotency transcription factors' interaction partners. Limited information is available concerning Nanog-associated RNA-binding proteins (RBPs), and the intrinsic protein-RNA interactions characteristic of the regulatory activities of Nanog. Herein, we used an improved affinity protocol to purify Nanog-interacting RBPs from mouse embryonic stem cells (ESCs), and 49 RBPs of Nanog were identified. Among them, the interaction of YBX1 and ILF3 with Nanog mRNA was further confirmed by in vitro assays, such as Western blot, RNA immunoprecipitation (RIP), and ex vivo methods, such as immunofluorescence staining and fluorescent in situ hybridization (FISH), MS2 in vivo biotin-tagged RNA affinity purification (MS2-BioTRAP). Interestingly, RNAi studies revealed that YBX1 and ILF3 positively affected the expression of Nanog and other pluripotency-related genes. Particularly, downregulation of YBX1 or ILF3 resulted in high expression of mesoderm markers. Thus, a reduction in the expression of YBX1 and ILF3 controls the expression of pluripotency-related genes in ESCs, suggesting their roles in further regulation of the pluripotent state of ESCs.