m⁶A RNA Degradation Products Are Catabolized by an Evolutionarily Conserved N⁶-Methyl-AMP Deaminase in Plant and Mammalian Cells

Abstract

N⁶-methylated adenine (m⁶A) is the most frequent posttranscriptional modification in eukaryotic mRNA. Turnover of RNA generates N⁶-methylated AMP (N⁶-mAMP), which has an unclear metabolic fate. We show that <i>Arabidopsis thaliana</i> and human cells require an N⁶-mAMP deaminase (ADAL, renamed MAPDA) to catabolize N⁶-mAMP to inosine monophosphate in vivo by hydrolytically removing the aminomethyl group. A phylogenetic, structural, and biochemical analysis revealed that many fungi partially or fully lack MAPDA, which coincides with a minor role of N⁶A-RNA methylation in these organisms. MAPDA likely protects RNA from m⁶A misincorporation. This is required because eukaryotic RNA polymerase can use N⁶-mATP as a substrate. Upon abrogation of <i>MAPDA</i>, root growth is slightly reduced, and the N⁶-methyladenosine, N⁶-mAMP, and N⁶-mATP concentrations are increased in Arabidopsis. Although this will potentially lead to m⁶A misincorporation into RNA, we show that the frequency is too low to be reliably detected in vivo. Since N⁶-mAMP was severalfold more abundant than N⁶-mATP in <i>MAPDA</i> mutants, we speculate that additional molecular filters suppress the generation of N⁶-mATP. Enzyme kinetic data indicate that adenylate kinases represent such filters being highly selective for AMP versus N⁶-mAMP phosphorylation. We conclude that a multilayer molecular protection system is in place preventing N⁶-mAMP accumulation and salvage.