The Role of m6A-RNA Methylation in Memory Formation and Recall and Its Modulation and Influence on Long-Term Outcomes as a Consequence of Early Life Lead Exposure.
Regulation of transcription is central to proper nervous system development and functioning. Dysregulation of transcriptional and post-transcriptional regulatory pathways are associated with various neurodevelopmental diseases and disorders including developmental lead (Pb) exposure, which is associated with significant and persistent cognitive and behavioral deficits. Recent data from our lab have shown Pb-induced methylation changes at gene promoter regions and effects of Pb on various post-translational histone modifications (PTHMs), however, gene expression changes have not always corresponded to promoter methylation status and mismatches between PTHM levels and transcriptional regulation were also observed, suggesting that other factors may be influencing transcriptional changes in response to Pb exposures. Thus, mechanisms un- derlying altered transcriptional regulation in specific brain regions following Pb exposure cannot be completely explained by alterations in DNA methylation and PTHM levels. Preliminary data from our lab support the hy- pothesis that another mechanism, direct RNA methylation via N6-methyladenosine (m6A), may be an important post-transcriptional mechanism contributing to the functionally altered transcriptome after developmental Pb exposure. Like DNA, RNA is susceptible to dynamic, reversible chemical modification and this epitranscrip- tomic mechanism may play an important role in gene transcription and behavioral plasticity. This has not been investigated as a potential molecular mechanism involved in environmental neurotoxicology. The goal of this proposal is to test the novel hypothesis that the most abundant epitranscriptomic mark, m6A, plays an im- portant role in modulating RNA and influencing memory processes in male and female rats and, based on preliminary data, in regulating transcriptional and behavioral responses to developmental Pb exposure. The following aims will examine the contribution of m6A to memory processing in normal and Pb-exposed animals: Aim1: Examine transcriptome-wide profile of the m6A modification (using methylated RNA immunoprecipitation sequencing (MeRIP-seq)) in mPFC and hippocampus (CA1) in behaviorally na?ve and behaviorally trained ani- mals of both sexes to assess its potential role as a dynamic epitranscriptomic mechanism involved in encoding and maintenance of memory, and, examine the extent to which the transcriptome-wide m6A enrichment profile is altered in animals with developmental Pb exposure; Aim2: Assess the functional significance of m6A in PFC and CA1 in memory processes in Pb-exposed animals using site-directed knockdown of the RNA demethylase gene, Fto, which will increase m6A levels in mPFC and CA1 and allow us to evaluate the effects of brain region -dependent reversal of decreased m6A levels on Pb-induced impaired memory processes. This work, the first to examine the dynamic modulation of RNA by m6A in PFC and CA1 in male and female normal and Pb- exposed rats, will result in new information regarding mRNA methylation as an effector mechanism for gene expression and behavioral plasticity in response to an environmental neurotoxicant.