<b>Temporal Dynamics of tsRNA Regulation Mark an Abrupt Transition After Epileptogenesis</b>

Recent studies increasingly recognize transfer RNA-derived small RNAs or tsRNAs, especially those bound to Argonaute 2 (Ago2), as functional regulators of gene expression. Here, we analyzed Ago2-immunoprecipitated small RNA-Seq data along with matching transcriptomic and proteomic data across seven defined timepoints in a rat model of epilepsy that was induced using perforant pathway stimulation (PPS).The analysis showed dynamic shifts in Ago-2 bound tsRNA expression, with early and intermediate stages showing upregulation of shorter tsRNA fragments, whereas Day of First Seizure (DOFS) and chronic timepoints showing a shift toward 5’tiRNAs, including highly upregulated GlyGCC-derived fragments. Cluster analysis using Weighted Gene Co-Expression Network Analysis (WGCNA) identified modules specific to DOFS timepoint where tsRNAs clustered together with genes enriched in pathways including neuronal metabolism, mitochondrial function, and synaptic stability. Target prediction analysis using RNAhybrid at DOFS predicted targets in 3’UTR, 5’UTR, and CDS regions showing association with glycolysis, protein localization, and vesicle trafficking. Subsequent gene-disease association analysis further associated the predicted targets with neurodegenerative conditions including but not limited to Alzheimer's disease, intellectual disability, and epilepsy.This study highlights that tsRNAs potentially play a temporal dynamic regulatory role in epileptogenesis with an evident shift in tsRNA accumulation at DOFS suggesting rewiring of post-transciptional control at the completion of epileptogenesis. This work also highlights a first integrative approach of tsRNA downstream effects on the transcriptome and proteome in epilepsy and suggests innovative tsRNA-driven mechanisms relevant to disease progression.