转录因子Mesp1招募RNA结合蛋白Quaking调控人类多能干细胞心血管定向分化的分子机制

Mesp1 is a master regulator for cardiovascular differentiation of pluripotent stem cells and mammalian heart development. It is at the top of the hierarchy of the gene network responsible for cardiovascular cell-fate determination. Owing to its transient expression nature during cardiac development and differentiation, however, the detailed mechanisms of Mesp1 regulating cardiovascular differentiation as well as its interacting proteins are unclear. Recently, using Mesp1 as the bait in a yeast two hybrid study, we have found that the RNA-binding protein Quaking directly interacts with Mesp1. In this proposed study, we will further confirm the roles of Quaking in directed cardiovsacular differentiation of human pluripotent stem cells. We will also investigate how Mesp1 regulates expressions of mRNAs related to cardiovascular differentiation through interacting with Quaking and clarify the detailed mechanisms of Mesp1 and Quaking in regulating cardiovascular differentiation. This will provide important information for improving the efficiency of cardiovascular differentiation for pluripotent stem cells as well as for early diagnosis and prevention of human congenital heart diseases.

转录因子Mesp1是调控人类多能干细胞心血管定向分化与心脏发育的关键分子，位于调控心血管细胞命运决定的转录层级之最高层。但由于其在早期心脏发育与分化过程中非常短暂的表达特性，其调控心血管细胞分化的具体机制尚未明确，与其相互结合的蛋白也未见报道。我们近期通过酵母双杂交实验，首次发现在人类多能干细胞心血管定向分化过程中，Mesp1可以与RNA结合蛋白Quaking直接结合。本项研究中，我们将进一步明确Quaking在人类多能干细胞心血管定向分化中的作用，探索Mesp1如何通过Quaking调控心血管定向分化相关mRNA的表达，阐明Mesp1结合Quaking调控人类多能干细胞心血管定向分化的分子机制，为提高人类多能干细胞分化为心血管细胞的效率，以及先天性心脏病的早期诊断防治提供理论依据。

RNA结合蛋白QKI属于KH结构域蛋白家族成员之一，其作为RNA可变剪切的新型调控因子，可能在神经发育异常及心血管疾病中起到关键性作用。我们发现在小鼠胚胎发育早期E7.5天QKI即特异性高表达于原始心管中，E9.5天在神经管中开始表达。本课题利用人胚胎干细胞建立QKI基因敲除细胞系，通过体外心血管定向分化体系探究QKI对心肌细胞分化成熟的调控机制。我们发现QKI敲除并不影响胚胎干细胞的多能性及心血管祖细胞早期标记物的表达，但能引起分化的心肌细胞肌丝结构紊乱，显著减低成熟心肌细胞的收缩力。进一步单细胞及bulk RNA-seq测序结果表明，QKI通过调控Z线形成相关基因（如ACTN2、NEBL、ABLIM1、PDLIM5等）的转录后可变剪切，参与调控心肌细胞肌小节的形成及功能的发挥。同时在QKI敲除小鼠模型中，我们亦证实QKI基因的缺失会引起小鼠心脏发育结构的异常。由此，本课题阐明了RNA结合蛋白QKI可通过调控肌丝收缩相关基因ACTN2等的可变剪切参与了心脏的发育过程，这将为先天性心脏病的产前诊断防治提供新的理论依据。