Abstract
N[6]-methyladenosine (m[6]A) 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 m[6]A-dependent regulatory networks across coding and non-coding RNA species remains poorly understood. We investigated the impact of volatilized cocaine (vCOC) exposure on m[6]A RNA methylation, m[6]A pathway components, transcriptome, and cocaine-induced locomotor sensitization in Drosophila melanogaster. Acute vCOC administration significantly increased global m[6]A levels in total and poly(A)-enriched RNA, with a stronger effect in polyadenylated transcripts. This increase occurred without changes in the m[6]A methyltransferases Mettl3 and Mettl14 transcripts, but was accompanied by robust upregulation of the levels of m[6]A reader YTHDC and YTHDF transcripts. Genetic and cell-type-specific analyses revealed distinct and context-dependent roles for m[6]A writers and readers in neurons and glia, with m[6]A readers being essential for vCOC-induced locomotor sensitization. Integration of RNA-seq and MeRIP-seq demonstrated that vCOC selectively amplifies m[6]A 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, m[6]A-modified RNAs shared in CTRL and vCOC were enriched for core metabolic and mitochondrial pathways, such as oxidative phosphorylation. Notably, vCOC increased m[6]A 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 m[6]A-defined regulatory RNA network, spanning coding and non-coding transcripts that is mechanistically linked to m[6]A reader-dependent behavioral plasticity.
