EPILEPTOGENIC INSULTS IMPACT THE EPITRANSCRIPTOMIC (N6-METHYLADENOSINE/M6A) SIGNATURE OF MATURE MICRORNAS AND REGULATES THEIR ROLE IN EPILEPSY DEVELOPMENT

Epilepsy is a chronic brain disease characterised by spontaneous recurrent seizures, affecting 50–65 million people worldwide. Evidence suggests that epileptogenic insults drive pathogenic gene expression and gene expression regulation which in turn promotes aberrant cellular and network function. MicroRNAs (miRNAs) regulate these processes and are emerging therapeutic targets. The regulation of miRNAs in epilepsy, remains obscure. We identified significant disruption of the epitranscriptomic m6A signature in epilepsy. Here we extend this analysis to non-coding RNAs, specifically miRNAs to test whether mature miRNAs harbour m6A tags in the brain, and if so, what effect this has on the function and activity of the miRNA in the context of epilepsy.
Epileptogenesis is induced in male C57 mice using the intra-amygdala kainic acid model of temporal lobe epilepsy. M6A-tagged small RNAs were enriched by immunoprecipitation, followed by small RNA-seq. To explore the function of m6A-tagged miRNAs we generated m6A-tagged miRNA mimics for 5 of the differentially methylated miRNAs identified in our initial screen and transfecting them (as well as non-targeting and non-methylated targeting mimics) into N2A cells and performing RNA sequencing analyses to uncover the effects of each.
Epileptogenic insults provoke widespread differential tagging of mature miRNAs in the hippocampus. Methylated miRNAs are detected in the RISC complex and therefore may be functional. Methylated miRNAs however display less effective silencing of target mRNAs than their non-methylated counterparts.
These results suggest that m6A-tagging of miRNAs may represent an additional layer of regulation of miRNA activity which is rapid and responsive to cellular needs/environment.