探究RNA结合蛋白的可变剪接作用对胚胎干细胞的分化调控

Pluripotency maintenance as well as the exit and re-acquisition of pluripotency by de-differentiation and somatic cell reprogramming, require the interplay of transcriptional regulators, epigenetic modifiers, and extracellular signaling pathways. Tremendous efforts have been directed toward studying chromatin binding proteins such as DNA binding transcription factors and chromatin modifying proteins However, much less is known about the roles of RNA-binding proteins (RBPs) in pluripotency, differentiation, and reprogramming. RBPs participate in every step of RNA biology, from transcription, splicing, and polyadenylation to RNA modification, transport, translation, and turnover. RNAs in cells are associated with RNA-binding proteins (RBPs) to form ribonucleoprotein (RNP)..Here, by functional screening some pre-selected RBPs in ES cells, we identified a few critical RBPs required for exit from self-renewal and further explored their molecular mechanisms. Firstly, 13 candidate RBPs were picked by bioinformatics analysis based on the mouse ES cell RBPs database and published literatures. And then we disrupted these candidates by using CRISPR-Cas9 system in mouse haploid ES cells and got the mutant cell lines respectively. The differentiation ability of these mutant cell lines were evaluated through embryoid body formation and withdraw LIF from the medium. RBP-X RBP-null cell clones showed significant differentiation defect compared with wide-type ES cells..RBP-X is a tissue-specific hnRNP, which was identified as a regulator of CD45RA to CD45RO switching during memory T-cell development. Since then, RBP-X has emerged as a critical regulator of lymphocyte homeostasis and terminal differentiation, controlling alternative splicing or expression of critical genes for the lymphocytes development. There was no evidence of functional roles about RBP-X in mouse ES cells. .In this study we will disrupt RBP-X in diploid mESC, We will perform RNA-seq and CLIP-seq by using the EB samples of RBP-X-null ESCs and wide-type ESCs, and compare the change of the whole transcriptome and alternative splicing events. We will focus on the influence of RBP-X on alternative splicing events. And finally reveal the functional role RBP-X in ESC Exit from Pluripotency.

多能性的维持与退出需要转录调控因子、表观遗传修饰因子和细胞外信号通路的相互作用。目前已经有大量研究是针对于染色质结合蛋白的，然而对于RNA 结合蛋白（RBPs）所发挥的作用了解的不多。我们在前期工作中通过生物信息学分析获得了13个可能参与ESC退出多能性的RBPs。随后我们利用CRISPR-Cas9技术在单倍体mESC中对这些RBPs进行了敲除，并利用分化实验对RBPs缺失后mESC的分化能力进行了检测，其中RBP-X缺失后分化缺陷表型最为显著，已有研究表明RBP-X可以通过调控可变剪接影响B细胞分化，但在mESC中的作用机制尚未明确。在本研究中，我们将RBP-X作为目标，为排除细胞背景影响，在二倍体mESC中对其进行敲除，利用RNA-seq及CLIP-seq等实验手段找到RBP-X调控的靶RNA以及影响的RNA加工过程，揭示其在ESC分化过程中的作用机制。

目前，胚胎干细胞（embryonic stem cells，ESCs）的多能性调控网络已经被很好地揭示，但是这一调控网络是如何被打破，使ESCs进入特异性分化的还有待进一步探究。RNA结合蛋白（RNA-binding protein，RBP）在RNA介导的基因调控中扮演了重要的角色，已有研究表明RBPs可以调控ESCs的命运选择。在我们的研究中我们将生物信息学分析与现有已发表的退出多能性相关的遗传筛选工作数据相结合，共筛选获得了7个候选RBPs，它们可能在ESCs多能性退出中发挥了调控作用。我们的进一步研究揭示hnRNPLL可以调控ESCs的可变剪接（alternative splicing，AS），当ESCs起始分化时hnRNPLL可以促进ESCs特异的外显子跳跃事件。当hnRNPLL缺失时，ESCs持续表达多能性维持相关的转录本，使ESCs表现出分化缺陷，在hnRNPLL敲除的小鼠也表现出生长发育异常。CLIP-seq、RIP和质谱等实验证明hnRNPLL是通过调控两个转录因子Bptf和Tbx3的可变剪接来影响ESCs的多能性退出。我们的研究结果进一步揭示了RBPs在ESCs多能性退出中扮演了重要角色，可以通过调控RBPs来控制ESCs的命运选择。