RNA methyltransferases (MTases) catalyse the transfer of a methyl group to their RNA substrates using most-often S-adenosyl-L-methionine (SAM) as cofactor. Only few RNA-bound MTases structures are currently available due to the difficulties in crystallising RNA:protein complexes. The lack of complex structures results in poorly understood RNA recognition patterns and methylation reaction mechanisms. On the contrary, many cofactor-bound MTase structures are available, resulting in well-understood protein:cofactor recognition, that can guide the design of bisubstrate analogues that mimic the state at which both the substrate and the cofactor is bound. Such bisubstrate analogues were recently synthesized for proteins monomethylating the N6-atom of adenine (m 6 A). These proteins include, amongst others, RlmJ in E. coli and METLL3:METT14 and METTL16 in human. As a proof-of-concept, we here test the ability of the bisubstrate analogues to mimic the substrate:cofactor bound state during catalysis by studying their binding to RlmJ using differential scanning fluorimetry, isothermal titration calorimetry and X-ray crystallography. We find that the methylated adenine base binds in the correct pocket, and thus these analogues could potentially be used broadly to study the RNA recognition and catalytic mechanism of m 6 A MTases. Two bisubstrate analogues bind RlmJ with micro-molar affinity, and could serve as starting scaffolds for inhibitor design against m 6 A RNA MTases. The same analogues cause changes in the melting temperature of the m 1 A RNA MTase, TrmK, indicating non-selective protein:compound complex formation. Thus, optimization of these molecular scaffolds for m 6 A RNA MTase inhibition should aim to increase selectivity, as well as affinity.

RNA methyltransferases (RNMTs) catalyze the methylation of RNA using S‐adenosyl‐l‐methionine (SAM) as the methyl donor. Methylation at the N‐6 position of adenosine is the most abundant modification found in nearly all classes of RNAs and contributes to the regulation of many biological processes in the three domains of life. However, this family of enzymes remains relatively unexplored by the medicinal chemistry community and new molecules are needed for their studies. Since RNMTs are suitable for bisubstrate binding, we report here the synthesis of SAM‐adenosine conjugates as bisubstrate analogues for RNMTs responsible for methylation of the N6‐position of adenosine. Six compounds were synthesized by connecting an analogue of SAM to an adenosine unit chosen to mimic the RNA substrate, via alkyl and urea linkers.

Chemical synthesis of RNA conjugates has opened new strategies to study enzymatic mechanisms in RNA biology. To gain insights into poorly understood RNA nucleotide methylation processes, we developed a new method to synthesize RNA-conjugates for the study of RNA recognition and methyl-transfer mechanisms of SAM-dependent m6A RNA methyltransferases. These RNA conjugates contain a SAM cofactor analogue connected at the N6-atom of an adenosine within dinucleotides, a trinucleotide or a 13mer RNA. Our chemical route is chemo- and regio-selective and allows flexible modification of the RNA length and sequence. These compounds were used in crystallization assays with RlmJ, a bacterial m6A rRNA methyltransferase. Two crystal structures of RlmJ in complex with RNA–SAM conjugates were solved and revealed the RNA-specific recognition elements used by RlmJ to clamp the RNA substrate in its active site. From these structures, a model of a trinucleotide bound in the RlmJ active site could be built and validated by methyltransferase assays on RlmJ mutants. The methyl transfer by RlmJ could also be deduced. This study therefore shows that RNA-cofactor conjugates are potent molecular tools to explore the active site of RNA modification enzymes.