Gene expression is regulated at many levels including co- or post-transcriptionally, where chemical modifications are added to RNA on riboses and bases. Expression control via RNA modifications has been termed ‘epitranscriptomics’ to keep with the related ‘epigenomics’ for DNA modification. One such RNA modification is the N6-methylation found on adenosine (m6A) and 2′-O-methyladenosine (m6Am) in most types of RNA. The N6-methylation can affect the fold, stability, degradation and cellular interaction(s) of the modified RNA, implicating it in processes such as splicing, translation, export and decay. The multiple roles played by this modification explains why m6A misregulation is connected to multiple human cancers. The m6A/m6Am writer enzymes are RNA methyltransferases (MTases). Structures are available for functionally characterized m6A RNA MTases from human (m6A mRNA, m6A snRNA, m6A rRNA and m6Am mRNA MTases), zebrafish (m6Am mRNA MTase) and bacteria (m6A rRNA MTase). For each of these MTases, we describe their overall domain organization, the active site architecture and the substrate binding. We identify areas that remain to be investigated, propose yet unexplored routes for structural characterization of MTase:substrate complexes, and highlight common structural elements that should be described for future m6A/m6Am RNA MTase structures.

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.