Modifying the m⁶A brain methylome by ALKBH5-mediated demethylation: a new contender for synaptic tagging

Synaptic plasticity processes, which underlie learning and memory formation, require RNA to be translated local to synapses. The synaptic tagging hypothesis has previously been proposed to explain how mRNAs are available at specific activated synapses. However how RNA is regulated, and which transcripts are silenced or processed as part of the tagging process is still unknown. Modification of RNA by N6-methyladenosine (m⁶A/m) influences the cellular fate of mRNA. Here, by advanced microscopy, we showed that m⁶A demethylation by the eraser protein ALKBH5 occurs at active synaptic ribosomes and at synapses during short term plasticity. We demonstrated that at activated glutamatergic post-synaptic sites, both the YTHDF1 and YTHDF3 reader and the ALKBH5 eraser proteins increase in co-localisation to m⁶A-modified RNAs; but only the readers showed high co-localisation to modified RNAs during late-stage plasticity. The YTHDF1 and YTHFDF3 readers also exhibited differential roles during synaptic maturation suggesting that temporal and subcellular abundance may determine specific function. m⁶A-sequencing of human parahippocampus brain tissue revealed distinct white and grey matter m⁶A methylome profiles indicating that cellular context is a fundamental factor dictating regulated pathways. However, in both neuronal and glial cell-rich tissue, m⁶A effector proteins are themselves modified and m⁶A epitranscriptional and posttranslational modification processes coregulate protein cascades. We hypothesise that the availability m⁶A effector protein machinery in conjunction with RNA modification, may be important in the formation of condensed synaptic nanodomain assemblies through liquid-liquid phase separation. Our findings support that m⁶A demethylation by ALKBH5 is an intrinsic component of the synaptic tagging hypothesis and a molecular switch which leads to alterations in the RNA methylome, synaptic dysfunction and potentially reversible disease states.