The influence of N6-methyladenosine (m6A) RNA methylation on synaptic function and local protein synthesis

Abstract

Leading theories on learning and memory postulate the importance of strengthening specific synaptic pathways, communication of post- and pre-synaptic terminals, and dendritic local protein synthesis. RNA-binding proteins which recognise epigenomic N6-methyladenosine (m6A) messenger RNA modifications can regulate a number of molecular functions including polyribosome loading, RNA decay, splicing, and translation. To investigate whether and how synaptic plasticity may be regulated by m6A RNA processing, three approaches were undertaken. First, I analysed m6A-sequencing data of human grey matter, white matter, and fetal tissue to identify transcripts that were m6A-methylated in human brain, as well as the biological processes and diseases enriched in this data. Several transcripts whose protein product is important in synaptic structure and function, particularly neurotransmitter receptors, were found to be methylated. The most highly enriched gene ontology terms included “synapses” and “nervous system development,” suggesting a function for m6A methylation in plasticity. Second, I performed immunofluorescent assays on quiescent and activated neuronal cell lines to examine changes in colocalisation of m6A modifications with m6A-binding proteins at synapses. Colocalisation of m6A and YTHDF1, YTHDF3, and ALKBH5 at synapses all increased significantly after 15 minutes of glutamate receptor activation. Changes were also observed 24 hours after activation. These results suggest the binding of these proteins to m6A-mRNAs responds to plasticity processes and is time-dependent. Third, abundance of m6A-modified RNA and expression of YTHDF1/YTHDF3 in the cerebellum, frontal and cingulate gyrus cortex, and hippocampus of normal and neurological disease-affected human brain was examined. Using machine learning quantitative analysis, I found m6A abundance and YTH protein expression to be differently dysregulated in each region and neuronal population of brain affected by Parkinson’s disease, Lewy Body Dementia, and cognitive defects. Overall, these findings provide evidence highlighting the importance of m6A-mediated regulation of local protein synthesis at synapses, providing a clearer understanding of the processes involved in normal synaptic and neuronal function and how m6A regulation may contribute to the pathophysiology of brain disease