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.