Rbm24磷酸化修饰在心肌分化中调控可变剪切的机制

Rbm24 (RNA binding motify protein 24, Rbm24) is a tissue-specific RNA binding protein, which is closely related to cardiac function. The loss-of-function of Rbm24 suggests that Rbm24 plays an important role in regulation of cardiac differentiation by modulating splicing. Unfortunately, the regulation of Rbm24 or its context signaling remains unknown. Recently, we identified Rbm24 as a phosphoprotein. We found that a kinase Stk38 could directly phosphorylate Rbm24 and affected its biological function. Therefore we hypothesize that Rbm24 phosphorylation play an important role on its biological function. In this proposal, we will combine mass spectrometry analysis and mutagenesis-based experiments to identify the phosphorylation site of Rbm24 protein. We will then demonstrate the role and the mechanism of Rbm24 phosphorylation on splicing activity and cardiac differentiation. Finally, we will detect the expression of phosphorylated Rbm24 in the hearts of mice at different developmental stages. This study will further our understanding of the regulatory mechanism of cardiac differentiation in both physiologic and pathologic contexts, and uncovers a potential novel pathway to cardiac differentiation through modulating the Rbm24 protein activity.

Rbm24是组织特异性的RNA结合蛋白，我们发现Rbm24通过调节可变剪切，精准调控了影响心脏发育和功能的mRNA，进而调控了多能干细胞向心肌的定向分化。但Rbm24本身调控的信号网络尚不清楚。我们通过筛选与Rbm24相互作用的蛋白，发现Stk38激酶可以直接磷酸化Rbm24并影响其生物学功能。磷酸化是重要的蛋白修饰方式。我们猜测磷酸化是调节Rbm24生物学功能的重要环节。本项目将在此发现的基础上，着眼于研究Rbm24磷酸化在心肌分化中的作用和机制。首先利用质谱分析与点突变确定Rbm24的磷酸化位点；进而建立Rbm24磷酸化位点点突变胚胎干细胞系，结合RNA-seq探索Rbm24磷酸化在干细胞向心肌分化中的作用，及与相关基因可变剪切的联系和作用机制；最后在不同发育阶段的小鼠心脏中，检测Rbm24磷酸化形式的表达情况。预期研究结果可为探讨干细胞向心肌分化提供新的理论依据，具有重要的临床意义。

Rbm24是组织特异性的RNA结合蛋白，我们发现Rbm24调控了多能干细胞向心肌细胞的定向分化。通过蛋白互作，我们首次发现其是一个磷酸化的蛋白。本项目在此基础上，利用质谱分析与点突变确定了Rbm24的磷酸化位点，并且发现Rbm24的磷酸化是其入核发挥生物学功能的重要方式，其磷酸化状态影响ESC的心肌分化。为了进一步研究Rbm24的作用机制，我们构建了Rbm24的条件敲除小鼠，进而利用RNA-seq测序分析，我们发现Rbm24调控的可变剪切，是调控心脏发育的重要转录后调控机制，Rbm24通过调控一系列心肌骨架相关基因如Ttn等的可变剪接，调控心脏发育。相关结果已整理发表，德国研究小组Anna Gaertneret al发表了同期评论性文章，认为我们的研究成果是心脏领域的重大发现。