Cocaine remodels m6A RNA-dependent signaling to drive locomotor plasticity in Drosophila melanogaster

: N6-methyladenosine (m6A) is a dynamic RNA modification that regulates RNA stability, processing, and translation and is increasingly recognized as a key modulator of neuronal plasticity. However, how psychostimulant exposure reshapes m6A-dependent regulatory networks across coding and non-coding RNA species remains poorly understood. We investigated the impact of volatilized cocaine (vCOC) exposure on m6A RNA methylation, m6A pathway components, transcriptome, and cocaine-induced locomotor sensitization in Drosophila melanogaster. Acute vCOC administration significantly increased global m6A levels in total and poly(A)-enriched RNA, with a stronger effect in polyadenylated transcripts. This increase occurred without changes in the m6A methyltransferases Mettl3 and Mettl14 transcripts, but was accompanied by robust upregulation of the levels of m6A reader YTHDC and YTHDF transcripts. Genetic and cell-type-specific analyses revealed distinct and context-dependent roles for m6A writers and readers in neurons and glia, with m6A readers being essential for vCOC-induced locomotor sensitization. Integration of RNA-seq and MeRIP-seq demonstrated that vCOC selectively amplifies m6A modification of regulatory and plasticity-associated RNA classes, including mRNAs involved in RNA processing, antisense RNAs, long non-coding RNAs, and transposable element-derived transcripts. In contrast, m6A-modified RNAs shared in CTRL and vCOC were enriched for core metabolic and mitochondrial pathways, such as oxidative phosphorylation. Notably, vCOC increased m6A modification of non-coding RNAs and transposable elements with minimal overlap with control conditions, indicating cocaine-induced engagement of epitranscriptomic regulation at multiple layers of the transcriptome. Together, these findings reveal that cocaine exposure reinforces an m6A-defined regulatory RNA network, spanning coding and non-coding transcripts that is mechanistically linked to m6A reader-dependent behavioral plasticity.