The regulation of m6A related proteins during whole-body freezing of the wood frog, Rana sylvatica.

Rana sylvatica is one of the few vertebrates that spends extreme winters showing no physiological signs of life. Up to 70% total body water of the wood frog freezes as extracellular ice. Survival in extreme conditions requires regulation at transcriptional and translational levels to activate pro-survival pathways. N6-methyladenosine (m6A) methylation is one of the most common RNA modifications, regulating transcript processing and translation by executing important functions that affect regulatory pathways in stress conditions. In the study, regulation of m6A-related proteins in the liver of R. sylvatica was analysed during 24H frozen and 8H thaw conditions. Decreases in activity of demethylases of 28.44 ± 0.4% and 24.1 ± 0.9% of control values in frozen and thaw tissue respectively were observed. Total protein levels of m6A methyltransferase complex components METTL14 and WTAP were increased by 1.28-fold and 1.42-fold respectively during freezing. Demethylase FTO however, showed a decreasing trend, with a significant decrease during recovery from frozen conditions. Levels of mRNA degraders YTHDF2 and YTHDC2, also decreased under stress. Overall, increased levels of m6a methylation complex components, and suppressed levels of readers/erasers, provide evidence for the potential role of RNA methylation in freezing survival and its regulation in a hypometabolic state.