m6A修饰与mRNA在神经元轴突中转运与定位关系的研究探索

EXPLORATION OF THE POTENTIAL ROLES OF M6A MODIFICATION IN MRNA TRANSPORT AND LOCALIZATION TO NEURONAL AXONS

刘建辉

11849125

硕士

生物工程

姬生健

2020-06-03

2020-07-13

哈尔滨工业大学

深圳

m6A修饰是mRNA中最为普遍的化学修饰，在它的调控蛋白writers、erasers、readers的作用下发挥重要的功能，m6A修饰因其广泛的存在mRNAs中而正在被研究。它的动态调节过程能够影响到细胞的增殖与分化、神经系统的发育、一些人类疾病的发生、mRNAs的局部翻译，以及神经系统行使正常的生理功能等。本课题之前的研究表明，神经元轴突中去甲基化反应被抑制，会导致mRNAs上的m6A水平的维持或者升高，进而导致轴突中mRNAs的翻译被抑制。mRNAs在轴突中进行局部翻译之前，必须首先要转运与定位到轴突中， m6A修饰是否能够通过其阅读蛋白参与调控mRNAs的转运与定位目前还不清楚。神经元作为一种高度极化的亚细胞结构，是在核外研究m6A修饰的功能、以及m6A修饰对于mRNA在亚细胞结构特别是轴突中的转运与定位的理想模型系统。围绕着这个目的，本课题探索了有效收集大量轴突RNAs的方法，建立体外敲低m6A修饰相关基因的系统。本课题将使用到神经元分离与培养技术、RNA测序技术、原位杂交技术（FISH）等。本文结合神经元原代培养技术和微流控技术来达到分离神经元胞体与轴突的目的，并通过改进微流控芯片的参数以及设计创新的微流控芯片以提高轴突RNAs收集的效率。本课题后续将利用敲低m6A相关基因的慢病毒来体外转染神经元，收集到对照组与实验组的胞体与轴突RNA，进行RNA测序，鉴定那些受m6A修饰调控其向轴突转运和定位的mRNAs。我们将进一步检测和验证这些mRNAs的转运与定位是否受m6A修饰及其阅读器蛋白的调控。本文改进了微流控装置的参数，优化了微流控装置在神经科学上的应用，接下来研究m6A修饰对轴突中mRNA的转运和定位的调控机制对于了解以及阐释m6A修饰在神经系统中的功能和作用机制具有重要意义，有利于更深入地研究神经元轴突这一亚细胞结构的发育过程等。

m6A modification is the most popular chemical modification in mRNAs. It plays an important role with its regulatory proteins: writers, erasers, and readers. Its dynamic regulation process can affect cells proliferation and differentiation, nervous system development, human diseases, local translation of mRNAs. Recently, m6A modification is being studied in various fields because of its widespread presence in mRNAs. Previous studies have shown that loss of function of FTO in axons can lead to the higher level of m6A on mRNAs, which leads to the translation of mRNAs in axons suppressed. Before local translation of mRNAs in axons, mRNAs should be transported into axons firstly. It is unclear whether m6A modifications can participate in regulating the transport and localization of mRNAs through their readers. Neuron, as a highly polarized structure, is an ideal model for studying the transport and localization of mRNAs in axons. Our study establishes an assay for collecting axonal RNAs, a system for knocking down m6A modification-related genes in vitro. We will use neuron isolation and culture technology, RNA collection and sequencing technology, fluorescence in situ hybridization (FISH), etc. We combine microfluidic technology and neuron culture technology to achieve the purpose of separating neuron cell bodies and axons. By improving the parameters of the microfluidic chips or using innovative microfluidic chip, we can improve the collection efficiency of axonal RNAs. We will knock down the expression of m6A-related genes by transfecting neurons with lenti-virus in vitro, the somal and axonal RNAs of the control group and experimental group will be collected. Next, we will analyze those mRNA-seq data, and find some mRNAs localization regulated by m6A modification. Studying the regulation mechanism of mRNA m6A modification on mRNA transport and localization in axons plays an important role in understanding the function of m6A modification in nervous system development, which is also conducive to further research on the maturation and polarization process of neurons, especially in axonal subcellular structure.

m6A修饰 mRNA转运与定位 神经元培养 微流控芯片 轴突RNA测序 轴突RNA的分离

m6A microfluidics neuron culture mRNAs transport and localization axonal RNAs sequencing isolate axonal RNAs

中文

联合培养

南方科技大学

刘建辉. m6A修饰与mRNA在神经元轴突中转运与定位关系的研究探索[D]. 深圳. 哈尔滨工业大学,2020.