Pathways controlling mRNA translation pathways in dopaminergic neurons

描述（由申请人提供）：可卡因的生理作用是通过抑制多巴胺再摄取和随后增强多巴胺能神经传递来介导的。我们最近有一个令人惊讶和意想不到的发现，可卡因在动物体内的生理作用需要mRNA中腺苷残基的甲基化。腺苷甲基化是第一个，可能也是唯一一个可逆的mRNA修饰。去年，我们的研究小组发现，mRNA中腺苷残基甲基化形成n6 -甲基腺苷（m6A）是非常普遍的，影响了大脑中8000多个转录本。此外，腺苷甲基化和去甲基化受信号通路的调控，提示腺苷甲基化可能与蛋白磷酸化一样，是细胞中介导信号通路作用的基本机制。这项应用的目的是大大提高我们对这种新发现的、很大程度上神秘的mRNA修饰的理解，这种修饰似乎在突触传递和神经元功能中起着核心作用。作为我们了解m6A的细胞功能并确定其如何调节多巴胺能神经传递的总体目标的一部分，本提案的具体目标是：(1)以单核苷酸分辨率绘制中脑转录组中的m6A位点。我们将开发下一代测序方法，使用一种新的方法来检测活细胞中的m6A。这些实验将为m6A潜在的生物学功能提供新的见解，并将导致在Aims 2中鉴定中脑转录组中的m6A位点进行分析和诱变；(2)了解m6A调控神经元蛋白翻译的机制。我们将探索m6A与蛋白质翻译之间的关系，并确定使m6A能够影响体外和神经元中mRNA翻译的结构和序列背景。我们还将确定介导m6A对mRNA翻译影响的特定m6A结合蛋白；(3)我们将确定FTO如何通过其去甲基化m6A的能力来控制翻译。我们发现FTO在体内选择性地去甲基化m6A残基。我们将测试FTO通过影响与多巴胺能神经传递相关的特定转录物的翻译来重编程神经元中蛋白质翻译途径的假设。这里提出的实验将从根本上为神经元蛋白翻译是如何被调节提供新的见解。这里描述的途径可能影响神经元功能的各个方面。这些实验将开始破译一种新颖的、高度普遍的、富含大脑的mRNA修饰的作用，这种修饰在多巴胺神经传递以及神经元和其他组织中的其他信号通路中发挥着重要作用。

DESCRIPTION (provided by applicant): The physiological effects of cocaine are mediated by inhibition of dopamine reuptake, and subsequent enhancement of dopaminergic neurotransmission. We recently made a surprising and unexpected finding that physiological effects of cocaine in animals requires methylation of adenosine residues in mRNA. Adenosine methylation constitutes the first, and potentially only, reversible mRNA modification. Last year, our group showed that methylation of adenosine residues in mRNA to form N6-methyladenosine (m6A) is highly prevalent and affects over 8,000 transcripts in the brain. Furthermore, adenosine methylation and demethylation is regulated by signaling pathways, suggesting that adenosine methylation, like protein phosphorylation, may be a fundamental mechanism mediating effects of signaling pathways in cells. The goal of this application is to substantially advance our understanding of this newly discovered and largely mysterious mRNA modification that appears to have a central role in synaptic transmission and neuronal function. As part of our overall goal to understand the cellular functions of m6A and to determine how it regulates dopaminergic neurotransmission, the specific aims of this proposal are: (1) To map m6A sites in the midbrain transcriptome at single-nucleotide resolution. We will develop a next-generation sequencing approach that uses a novel to detect m6A in living cells. These experiments will provide new insights into the potential biological functions of m6A and will result in the identification of m6A sites in the midbrain transcriptome for analysis and mutagenesis in Aims 2; (2) To determine how m6A regulates protein translation in neurons. We will explore the relationship between m6A and protein translation and determine the structural and sequence contexts that enable m6A to influence mRNA translation in vitro and in neurons. We will also determine the specific m6A-binding proteins that mediate the effects of m6A on mRNA translation; (3) We will determine how FTO controls translation via its ability to demethylate m6A. We find that FTO selectively demethylates m6A residues in vivo. We will test the hypothesis that FTO reprograms protein translation pathways in neurons by influencing the translation of specific transcripts relevant to dopaminergic neurotransmission. The experiments proposed here will provide fundamentally novel insights into how neuronal protein translation is regulated. The pathways described here are likely to influence diverse aspects of neuronal function. These experiments will begin to decipher the role of a novel, highly prevalent, and brain-enriched mRNA modification that is poised to have important roles in dopamine neurotransmission as well as other signaling pathways in neurons and other tissues.