elavl1a在斑马鱼心脏发育中的调控机制研究

Elavl1, an RNA binding protein, is essential for embryonic development. However, its role in zebrafishcardiac development has not been studied. Our preliminary data has shown cardiac looping defect and decreased nkx2.5 expression in the elavl1a knock-downmorphant, which is similiar to gata5 mutant phenotype. Interestingly, zebrafish gata5 3’UTR region contains 3 potential elavl1a binding sites through bioinformatic analysis. Therefore, we hypothesize that elavl1a is essential for cardiac development; mechanistically, elavl1a directly interacts with gata5 3’UTR region, protectgata5 mRNA stability and enhance gata5 expression. In the proposed research project, we will validate cardiac phenotype and marker genes in CRISPR-Cas9 mediated mutants. We will elucidate the association between RNA binding protein elavl1a and gata5 3’UTR in vitro and in vivo by RNA-IP. Furthermore, we will investigate ELAVL1 regutes mRNA stability in lentivirus-mediated ELAVL1knock-down HEK293 cell lines. Thereby, our aim is to uncover a novel role of elavl1a in regulation of cardiac development by protecting gata5 mRNA stability, which provide a signaling pathway for understanding congenial heart disease.

Elavl1是一种RNA结合蛋白，对于胚胎的早期发育至关重要。然而，elavl1在早期心脏发育的研究甚少。我们前期工作表明敲降elavl1a斑马鱼胚胎中心脏发育不正常，同时nkx2.5表达下降。这个表型跟gata5突变体非常相似。生物信息学分析发现gata53’UTR存在三个elavl1a的可能结合位点。因此，我们假设elavl1a对心脏的发育具有至关重要的作用；其可能通过直接结合gata5 3’ UTR，保护mRNA稳定性，从而调控gata5的表达。本课题拟在elavl1a敲除的斑马鱼和细胞系中验证elavl1a可以直接结合gata5非编码区，增加 gata5的表达，保证心脏正常发育。本课题是第一次证明elavl1a在心脏发育中的作用，为先天性心脏病的治疗提供一定的理论基础。

RNA结合蛋白elavl1从属于Elavl家族，通过与3'-非编码区（UTR）的AU富集元件（AREs）结合以稳定靶mRNA并促进翻译，链接区经过磷酸化控制着ELAVL1蛋白在细胞核细胞质中的穿梭，在个体发育和器官形成起着重要作用。PKC介导ELAVL1的磷酸化对于其核质穿梭是必不可少的。为了验证elavl1a在斑马鱼心脏胚胎发育中的影响，我们发现elavl1a在斑马鱼心脏胚胎发育中起重要作用，PKC介导Elavl1a的Serv19和Ser316位点磷酸化将促进细胞核-质穿梭，这是胚胎心脏分化所需的一个过程。同时PKC介导的elavl1a磷酸化在斑马鱼心脏胚胎发育中起着关键作用。无论是敲除elavl1a或抑制PKC都将对胚胎心脏发育产生干扰。总之，本课题主要阐明PKC介导了elavl1a的Ser219和Ser316潜在位点磷酸化，导致elavl1a从细胞核向细胞质的输出，从而促进胚胎心脏发育。