Characterization of Neuronal RNA Modifications during Non-associative Learning in Aplysia Reveals Key Roles for tRNAs in Behavioral Sensitization

[Image: see text] Subtle changes in the landscape of post-transcriptional modifications have emerged as putative regulators of central nervous system plasticity and activity-induced protein synthesis. However, simultaneous characterization of multiple RNA modifications and their covariation during learning and memory paradigms has been impeded by the complexity of animal models and lack of untargeted approaches for identifying pathway-relevant RNA modifications in small-volume samples. Here, we used mass spectrometry to profile spatiotemporal changes in dozens of neuronal RNA modifications in Aplysia californica during behavioral sensitization of a simple defensive reflex. Unique RNA modification patterns were observed in the major ganglia of trained and naı̇ve animals, with two tRNA modifications, namely, 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U) and 1-methyladenosine (m(1)A), at significantly higher levels in trained subjects. We report that tRNAs, and their modifications, correlate with increased polyglutamine synthesis and excitability in neurons, characterizing the first link between noncoding RNA modifications and non-associative learning.