Systematic molecular profiling of non-native N6-substitution effects on m6A binding to the YTH domains of human RNA m6A readers in diabetes

The RNA N⁶-adenosine methylation, resulting in N⁶-methyl adenosine (m6A), is one of the most important post-transcriptional modification events in the eukaryotic transcriptome, which is dynamically regulated by methyltransferases (writers), recognition proteins (readers) and demethylases (erasers). Human has five m6A readers namely YTHDC1, YTHDC2, YTHDF1, YTHDF2 and YTHDF3 that specifically recognize and bind to the methylated m6A residue of RNA through their YT521-B homology (YTH) domains, which have been involved in the pathogenesis of diabetes mellitus and its diverse complications such as diabetic nephropathy. Instead of the native N⁶-methylation, we herein attempted to explore the molecular effect of various non-native N⁶-substitutions on adenosine (A) binding behavior to YTH domains. A systematic interaction profile of 40 reported N⁶-substituted adenosine (x6A) mononucleotides with 5 human reader YTH domains was created computationally. Heuristic clustering of the profile divided these YTH domains and these x6A mononucleotides into two subfamilies and three classes, respectively; they represent distinct intrinsic interaction modes between the domains and mononucleotides. Statistical survey unraveled that the volume (V_g) and hydrophobicity (H_g) of N⁶-substituted chemical groups exhibit linear and nonlinear correlations with the binding energy (ΔG_ttl) of x6A mononucleotides to YTH domains, respectively; N⁶-substitutions with moderate size and weak polarity are favorable for the x6A binding. From the profile the N⁶-bromomethyl adenosine (brm6A) was identified as a potent binder of YTHDF2 YTH domain; its affinity was improved significantly by 77.2-fold from A and considerably by 19.5-fold from m6A. Structural modeling observed that the N⁶-bromomethyl group of brm6A is tightly packed against an aromatic cage defined by the Trp432-Trp486-Trp491 triad of YTHDF2 YTH domain. Electron-correlation analysis revealed that the bromine atom can form geometrically and energetically satisfactory halogen-π interactions with the aromatic cage, thus conferring considerable affinity and specificity to the domain–brm6A interaction.