Abstract
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.
Highlights
In eukaryotes, N6-methyladenosine (m6A) and the cap-adjacent N6,2′‐O‐dimethyladenosine (m6Am), are highly abundant and reversible mRNA modifications [1, 2]. m6A/m6Am modification is regulated by effector proteins which orchestrate the transfer of methyl groups to and from adenosines located internally or at the first transcribed position
Dcp1a was commonly adjacent to two m6A modification signals suggesting that there is clustering of Dcp1a with modified transcripts. These results indicated that m6A-mediated regulation of protein expression is important at pre- and postsynaptic sites, and that in differentiated but quiescent, i.e. non-synaptically activated cells, m6A modified RNA is bound to the reader proteins, YTHDF1 and YTHDF3 more often than to the eraser ALKBH5
We presented new insight into m6A methylation processes as a fine-tuning mechanism of translation at pre- and post- synaptic sites and which we propose is an intrinsic component of synaptic tagging and capture
Summary
N6-methyladenosine (m6A) and the cap-adjacent N6,2′‐O‐dimethyladenosine (m6Am), are highly abundant and reversible mRNA modifications [1, 2]. m6A/m6Am modification is regulated by effector proteins (writers, erasers and readers) which orchestrate the transfer of methyl groups to and from adenosines located internally or at the first transcribed position. M6A/m6Am modification is regulated by effector proteins (writers, erasers and readers) which orchestrate the transfer of methyl groups to and from adenosines located internally or at the first transcribed position. 1234567890();,: what constitutes the ‘synaptic tag’ remains unclear, and response to NMDA or KCl, calcium imaging was performed on live it may include processes that regulate RNA stability, RNA differentiated cultures using a fluorescent calcium indicator Fluo4-AM degradation and protein translation. We examined changes induced by NMDA-mediated early and late phase plasticity stages, and at actively translating synaptic ribosomes, as well as during the periods of synaptogenesis and synaptic maturation in co-cultures of differentiating neurons, astrocytes and oligodendrocytes. By m6A mapping of grey and white matter from the human parahippocampus and late stage foetal brain, we characterised m6A regulated processes and m6A methylome characteristics in neuronal and glial cell-rich.
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