Synthesis of RNA-cofactor conjugates and structural exploration of RNA recognition by an m6A RNA methyltransferase

Abstract

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.

Synthesis of Bisubstrate Analogues for RNA Methylation Studies using two Transition‐Metal‐Catalyzed Reactions

“…We have recently demonstrated the effectiveness of bisubstrate analogues containing a di or a tri-nucleotide to mimic the RNA part to probe MTases active site. [13] To synthesize SAM-RNA conjugates, we investigated the opportunity to prepare a 5iodo-triazole compound from a dinucleotide as an alkynesubstrate. The previously described method was optimized to apply the iCuAAC reaction conditions to the dinucleotides 10 and 11 with 5'-azido-adenosine 12 (Scheme 3).…”

“…We have recently demonstrated the effectiveness of bisubstrate analogues containing a di or a tri‐nucleotide to mimic the RNA part to probe MTases active site [13] . To synthesize SAM‐RNA conjugates, we investigated the opportunity to prepare a 5‐iodo‐triazole compound from a dinucleotide as an alkyne‐substrate.…”

“…In our study we used the enzyme RlmJ as a m 6 A RNA MTases model. [13] The docking was based on the RlmJ complex structure solved in the presence of a bisubstrate…”

“…Activation of the N6 position of an adenosine with isopropenyl carbamate allowed us to obtain SAM-adenosine conjugates containing an urea linker, whereas the convertible nucleoside strategy, starting from N6-hydroxybenzotriazole-inosine led to bisubstrates with alkyl linkers (with two and three carbons). [11][12][13] (Figure 1D) Interestingly, the former strategy was successfully employed to access to oligonucleotides linked to SAM cofactor analogues (SAM-RNA conjugates). [13] These compounds were then used in crystallization assays with RlmJ, a bacterial m 6 A rRNA MTase.…”

“…[11][12][13] (Figure 1D) Interestingly, the former strategy was successfully employed to access to oligonucleotides linked to SAM cofactor analogues (SAM-RNA conjugates). [13] These compounds were then used in crystallization assays with RlmJ, a bacterial m 6 A rRNA MTase. Three crystal structures of RlmJ in complex with the bisubstrate conjugates were solved and revealed the RNAspecific recognition elements used by RlmJ to bind the RNA substrate in its active site.…”