Neuronal tRNA modifications in Aplysia californica are repatterned during behavioral habituation

Transfer RNA (tRNA) modifications are increasingly implicated as posttranscriptional regulators of basic neuronal functions. Characterizing tRNA modification profiles in specific neural circuits that control well-defined behaviors is challenging because of the complexity of conventional neurobiological models. Here, we leveraged the numerically simple central nervous system (CNS) of the marine mollusk Aplysia californica to investigate tRNA modification dynamics during habituation of a defensive reflex. We identified and quantified dozens of tRNA modifications and characterized the distribution of select small RNA modifications across different neuronal and nonneuronal tissues. Upon behavioral habituation of the siphon-elicited siphon withdrawal reflex (SSWR), we found that tRNA modification profiles in the major ganglion that controls the SSWR were repatterned with predictable, learning-related changes. We found a family of anticodon loop modifications that changed with habituation learning, including N⁶-isopentenyladenosine (i⁶A), which increased in abundance, and additional modifications N¹-methyladenosine (m¹A) and N⁷-methylguanosine (m⁷G), which decreased. These tRNA modification dynamics occurred independently of changes in the expression of their parent tRNAs, illustrating that behavioral habituation altered the activity of tRNA-modifying enzymes. Overall, our work reveals a link between tRNA modifications and behavioral habituation and provides insights toward understanding the posttranscriptional mechanisms of learning and memory.