Global Co-regulatory Cross Talk Between m6A and m5C RNA Methylation Systems Coordinate Cellular Responses and Brain Disease Pathways

N6 adenosine and C5 cytosine modification of mRNAs, tRNAs and rRNAs are regulated by the behaviour of distinct sets of writer, reader and eraser effector proteins which are conventionally considered to function independently. Here, we provide evidence of global cross-regulatory and functional interaction between the m⁶A and m⁵C RNA methylation systems. We first show that m⁶A and m⁵C effector protein transcripts are subject to reciprocal base modification supporting the existence of co-regulatory post-transcriptional feedback loops. Using global mass spectrometry proteomic data generated after biological perturbation to identify proteins which change in abundance with effector proteins, we found novel co-regulatory cellular response relationships between m⁶A and m⁵C proteins such as between the m⁶A eraser, ALKBH5, and the m⁵C writer, NSUN4. Gene ontology analysis of co-regulated proteins indicated that m⁶A and m⁵C RNA cross-system control varies across cellular processes, e.g. proteasome and mitochondrial mechanisms, and post-translational modification processes such as SUMOylation and phosphorylation. We also uncovered novel relationships between effector protein networks including contributing to intellectual disability pathways. Finally, we provided in vitro confirmation of colocalisation between m⁶A-RNAs and the m⁵C reader protein, ALYREF, after synaptic NMDA activation. These findings have important implications for understanding control of RNA metabolism, cellular proteomic responses, and brain disease mechanisms.