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bioRxiv · 10.1101/2025.11.13.688303

Neuronal tRNA Modifications in Aplysia californica are Repatterned during Behavioral Habituation

Huang, H. X.·Floro, G. M.·Asare, E.·Huang, W.·Clark, K. D.
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Abstract

Transfer RNA (tRNA) modifications have been increasingly implicated as post-transcriptional regulators of basic neuronal functions. However, characterizing tRNA modification profiles in specific neural circuits that control well-defined behaviors is notoriously difficult due to 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 in functionally identified neurons during habituation of a defensive reflex. We identified and quantified dozens of neuronal tRNA modifications using liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing characteristic distributions of select small RNA modifications across different neuronal and non-neuronal 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 discovered a family of anticodon loop modifications including N6-isopentenyladenosine (i6A) and its downstream product, 2-methylthio-N6-isopentenyladenosine (ms2i6A), that displayed a significant increase and trend toward lower levels, respectively. These tRNA modification dynamics occurred independently of changes in expression of their parent tRNAs, illustrating that behavioral habituation alters the activity of tRNA-modifying enzymes. Overall, our work reveals an underexplored link between tRNA modifications and behavioral habituation and provides new insights toward understanding the post-transcriptional mechanisms of learning and memory. One-Sentence SummaryCharacterization of neuronal tRNA modifications during behavioral habituation in Aplysia californica revealed a post-transcriptional mechanism of learning and memory. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/688303v1_ufig1.gif" ALT="Figure 1000"> View larger version (25K): org.highwire.dtl.DTLVardef@48d01forg.highwire.dtl.DTLVardef@57d28eorg.highwire.dtl.DTLVardef@1c0816dorg.highwire.dtl.DTLVardef@1714716_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Huang, H. X., Floro, G. M., Asare, E., Huang, W., Clark, K. D.. 2025-11-13. Neuronal tRNA Modifications in Aplysia californica are Repatterned during Behavioral Habituation. https://doi.org/10.1101/2025.11.13.688303

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