m6A 修饰调控小鼠视网膜神经发育的机制研究

REGULATION OF MOUSE RETINALNEURDEVELOPMENT BY m6A MODIFICATION

牛富贵

11649085

硕士

生物学

姬生健

2018-05-26

2018-07-06

哈尔滨工业大学

深圳

m6A 是一种广泛存在于 mRNA 和非编码 RNAs 的一种动态且可逆的甲基化修饰，在干细胞增殖与分化、癌症发生、神经元发育、神经环路形成等过程中有 着重要调控作用。目前为止 m6A 修饰调控神经系统的研究主要集中于其甲基化酶即‘writers’和去甲基化酶即‘erasers’，而对其阅读器蛋白即‘readers’调控所知甚少。本课题选取小鼠视网膜神经系统，主要通过免疫荧光、体外视网膜细胞培养、慢病毒转染、条件性敲除小鼠等手段研究 m6A 及其相关蛋白在视网膜发育中的表达与功能。我们得到了 m6A 及其相关蛋白在小鼠视网膜内的表达谱；建立了体外视网膜细胞培养体系；针对 YTHDF2 蛋白功能，我们在体外与体内环境下进行研究发现，在体外培养过程中 YTHDF2 参与调控视网膜神经节细胞（RGC） 的树突形成与双极细胞（bipolar cell）分化形成的过程，并在 Ythdf2 cKO 小鼠视网膜中进行了初步分析。为进一步研究 YTHDF2 介导的 m6A 修饰在小鼠视网膜神经发育中的功能与具体调控机制提供了合理、可行的实验方案。实验结果表明m6A 修饰在小鼠视网膜神经发育中的调控作用，其中 YTHDF2 参与调控 RGC 的树突形成过程。

N6-methyladenosine (m6A) is a dynamic and reversible modification in mRNA and non-coding RNAs and has been shown to regulate the proliferation and differentiation in stem cells, tumorigenesis, neurodevelopment and nerve circuit formation. However, current studies on m6A modification mediating nerve system mainly focus on its ‘writers’ and ‘erasers’. How m6A ‘readers’ regulate neurodevelopment by targeting specific neural target mRNAs is still unclear. In this project, using mouse retinal system, we studied expression patterns of m6A-related enzymes and proteins and functions in retinal neurodevelopment in vivo and in vitrowith immunofluorescence, primary neuron culture, lenti-virus-based knock-down and overexpression, conditonal knockout mice. Firstly, we checked expression patterns of m6A and its-related enzymes and proteins in mouse retinas in different stages. Then we knocked down YTHDF2 expression in retinal neuron culture, and we found that YTHDF2 could control dendrite development of retina ganglion cell (RGC) and differentiation of bipolar cell. We also did priliminary analysis of Ythdf2 condition knockout mice. Moreover, we make reasonable and feasible future plans to further explore the functions and mechanisms of YTHDF2 in mouse retina neurodevelopment. These findings collectively suggest that m6A modification can regulate mouse retinal neurodevelopment.

m6A YTHDF2 视网膜 神经发育 细胞分化 树突形成

m6A YTHDF2 Retina Neurodevelopment Dendrites Growth Neuron Differentiation

中文

联合培养

南方科技大学

牛富贵. m6A 修饰调控小鼠视网膜神经发育的机制研究[D]. 深圳. 哈尔滨工业大学,2018.