Abstract
RNA modifications serve as dynamic regulators of neural plasticity through their ability to fine-tune transcript stability and splicing. Pseudouridine (Ψ), an evolutionarily conserved RNA modification catalyzed by pseudouridine synthases, plays established roles in neurodevelopment, yet its functional significance in activity-dependent behavioral adaptation remains poorly defined. Here, we investigate Ψ-mediated epitranscriptomic regulation within the infralimbic prefrontal cortex (ILPFC), a brain region requiring precise synaptic remodeling for the clinically relevant form of fear extinction memory. Combining transcriptome-wide pseudouridylation profiling with behavioral analysis in mice, we identified selective Ψ enrichment at exons of synaptic regulatory genes within ILPFC during fear extinction learning. Fear extinction in the ILPFC drives concomitant exonic Ψ deposition and upregulation of synaptogenic transcripts, processes that involve pseudouridine synthase PUS7. Crucially, PUS7 knockdown in the ILPFC selectively impaired fear extinction memory formation without altering baseline fear expression, establishing a causal link between Ψ-dependent RNA processing and activity-dependent synaptic structural remodeling in this microcircuit. Our findings demonstrate that PUS7-mediated Ψ modification spatiotemporally regulates activity-dependent RNA dynamics in the ILPFC, providing the evidence that epitranscriptomic mechanisms precisely coordinate synaptic gene expression within behaviorally defined brain sub-region. This work bridges molecular RNA biology with systems neuroscience, revealing a novel mechanism for activity-dependent regulation of fear extinction in ILPFC.