Genome-wide RNA structure changes during human neurogenesis modulate gene regulatory networks. Genome-wide RNA structure changes during human neurogenesis modulate gene regulatory networks

RNAs structures play central roles in various cellular processes, and can be remodeled upon binding to cellular factors such as RNA binding proteins (RBP). However, how widespread this remodeling is and whether structural changes can serve as additional regulatory steps for other regulators remains under-explored. Here, we assayed RNA structural dynamics, gene expression, translation and decay during different stages of human neuronal differentiation to understand the impact of RNA structures in stem cell regulation. RBP binding resulted in wide-spread RNA structure changes during early neuronal differentiation. Structure changes act as a common mechanism for RBPs to modulate the binding of additional RBPs and miRNAs to coordinate gene expression temporally. In particular, we showed that PUM2 binding on LIN28A resulted in RNA structure changes that enabled miR-30 binding, leading to downregulation of LIN28A. Compensatory mutagenesis experiments that lock specific structural states confirmed the importance of PUM2-induced structure changes in miR-30 regulation. This systematic study broadens our understanding of RNA structural dynamics during human neuronal differentiation and illustrates the wide-spread and complex role of RBPs in regulating RNA structures for gene regulation during human development. Overall design: High throughput RNA structure probing experiments in vivo using icSHAPE in hESCs (Day 0, D0), neuronal precursor cells (Day 7, D7), early neurons (Day 8, D8) and neurons (Day 14, D14).

RNA结构在诸多细胞过程中发挥核心作用，并可在结合RNA结合蛋白（RNA binding protein, RBP）等细胞因子后发生重塑。然而，这种结构重塑的普及程度如何，以及结构变化能否作为其他调控因子的额外调控步骤，目前尚未得到充分探索。本研究针对人类神经元分化的不同阶段，检测了RNA结构动态、基因表达、翻译及降解水平，以解析RNA结构对干细胞调控的影响。在神经元分化早期，RNA结合蛋白的结合可引发广泛的RNA结构变化。结构变化是RNA结合蛋白调控其他RNA结合蛋白及微小RNA（microRNA, miRNA）结合，从而时序性协调基因表达的常见机制。具体而言，本研究证实，PUM2结合LIN28A后会引发RNA结构变化，使miR-30得以结合，最终导致LIN28A的表达下调。通过锁定特定结构状态的补偿性诱变实验，本研究证实了PUM2诱导的结构变化在miR-30调控中的重要作用。这项系统性研究加深了我们对人类神经元分化过程中RNA结构动态的认识，并阐明了RNA结合蛋白在人类发育过程中通过调控RNA结构参与基因调控的广泛且复杂的作用。实验设计：在人类胚胎干细胞（hESCs，第0天，D0）、神经前体细胞（第7天，D7）、早期神经元（第8天，D8）及神经元（第14天，D14）中，采用icSHAPE技术开展体内高通量RNA结构探测实验。