mRNA methylation: A novel mechanism that blocks signaling by drugs of abuse

描述(申请人提供)：可卡因的生理作用是通过抑制多巴胺的再摄取，以及随后增强多巴胺能神经传递来实现的。我们最近有了一个令人惊讶和意想不到的发现，可卡因对动物的生理效应需要mRNA中腺苷残基的甲基化。腺苷甲基化是去年发现的，是第一个，也可能是唯一一个可逆的mRNA修饰。我们的小组发现，mRNA中腺苷残基的甲基化形成N6-甲基腺苷(M6A)是非常普遍的，并影响到大脑中超过8,000个转录本。此外，腺苷甲基化和去甲基化受信号通路的调节，这表明腺苷甲基化和蛋白质磷酸化一样，可能是细胞内调节信号通路效应的基本机制。这项应用的目标是大大提高我们对这种新发现的、基本上是神秘的mRNA修饰的理解，这种修饰似乎在突触传递中起着核心作用。作为我们了解m6A的细胞功能和确定它如何调节多巴胺能神经传递的总体目标的一部分，这一提议的具体目标是：(1)在单核苷酸分辨率下定位中脑转录组中的m6A位点。我们将开发下一代测序方法，该方法结合了一种新的化学生物学策略来标记活细胞中的M6A。这些实验将为m6A的潜在生物学功能提供新的见解，并将导致确定中脑转录组中的m6A位点，以便在AIMS 2和3中进行分析和突变；(2)确定FTO如何影响其目标mRNAs的翻译。M6A通过翻译抑制mRNAs的机制尚不清楚。在这一目标中，我们将探讨m6A影响细胞内mRNA命运的潜在分子途径。(3)确定多巴胺能神经元中可卡因信号传导所需的FTO靶点。在这里，我们将测试影响神经传递和突触信号的FTO靶向mRNAs，看看它们是否有能力挽救FTO-/-脑片中受损的可卡因效应。这些实验将阐明第一条信号通路，将特定mRNAs的甲基化与神经元的生理变化联系起来。这里提出的实验将开始破译一种新的、高度流行的、具有重要医学意义的mRNA修饰的作用，这种修饰有望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 have recently made a surprising and unexpected finding that physiological effects of cocaine in animals require methylation of adenosine residues in mRNA. Adenosine methylation was discovered last year, and constitutes the first, and potentially only, reversible mRNA modification. 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. 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 incorporates a novel chemical biology strategy to label 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 and 3; (2) To determine how FTO affects the translation of its target mRNAs. The mechanism by which mRNAs are translationally suppressed by m6A is not known. In this aim, we will address the potential molecular pathways by which m6A influence mRNA fate in cells. (3) To identify the FTO targets that is required for cocaine signaling in dopaminergic neurons. Here we will test FTO-target mRNAs that influence neurotransmission and synaptic signaling for their ability to rescue the impaired cocaine effects in FTO-/- slices. These experiments will elucidate the first signaling pathway linking methylation of specific mRNAs to physiological changes in neurons. The experiments proposed here will begin to decipher the role of a novel, highly prevalent, and medically important mRNA modification that is poised to have important roles in dopamine neurotransmission as well as other signaling pathways in neurons and other tissues.