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How a ribozyme catalyses site- specific RNA methylation

Methylated nucleotides are important for the function of natural RNAs. A recently discovered methyltransferase ribozyme specifically installs a methyl group in another RNA. X-ray crystallography revealed the ribozyme s crystal structure, showing how the RNA folds to form the binding site for the methyl donor and suggesting a plausible and novel reaction mechanism.

In addition to proteins, ribonucleic acids (RNAs) are also able to specifically catalyse chemical reactions. Such catalytically active RNAs are called ribozymes. Recently, a ribozyme was discovered that attaches a small chemical modification, more precisely a single methyl group, to a defined site within another RNA with high specificity [1]. This RNA-catalysed reaction generates 1-methyladenosine (m1A), a naturally occurring modified nucleotide that is found at conserved positions in tRNAs and rRNAs in all domains of life. This first-known methyltransferase ribozyme was named MTR1 and was identified by in vitro evolution from synthetic RNA. Using X-ray crystallography at beamlines ID23-1, ID30A-1 and ID30A-3, the crystal structure of MTR1 and the underlying mechanism of this RNA-catalysed reaction has been elucidated.

The ribozyme was crystallised along with its target RNA and O6-methylguanine, which acts as the cofactor and methyl group donor. Analysis of the crystal structure revealed that the methyl group was completely transferred to the RNA during the crystallisation process, thus resulting in the capture of the post-catalytic state in the crystal (Figure 26). The arrangement of the nucleotides and the binding mode of the reacted cofactor directly suggested the catalytic mechanism, which was

subsequently confirmed by numerous biochemical experiments. It became apparent that a protonated nucleobase adjacent to the cofactor plays a crucial role in the methyl transfer reaction. This protonated cytidine serves as a general acid that facilitates the methyl transfer to the substrate RNA by donating its proton to the guanine leaving group (Figure 27). In addition, the binding pocket shows surprising similarities to the substrate-binding site in so-called purine riboswitches [2]. These are naturally occurring RNA segments that bind small molecules and act as on/off switches for selected genes.

Besides the protonated cytidine, two additional nucleotides in the active pocket of the MTR1 ribozyme are of special importance for its catalytic activity. Attaching a methyl group to the ribose at each of these two positions enhanced the ribozyme s activity by another order of magnitude. This observation demonstrates that even small modifications on certain nucleotides can be crucial for ribozyme activity as they can affect the conformational dynamics of the RNA.

Such a strong acceleration, achieved by a protonated nucleobase in concert with two strategically positioned methyl groups, has not yet been observed for any other catalytically active DNA or RNA. Together with the surprising similarity between laboratory-evolved MTR1 and naturally occurring RNA motifs, these results support the so-called RNA world hypothesis. It postulates that RNA was among the first biopolymers to act as both a storage unit for information and as a catalyst. At that evolutionary stage, the activity of the first RNA enzymes could have also been greatly enhanced by installing simple modifications within the RNA. Moreover, this research suggests that present-day riboswitches could potentially represent remnants of earlier ribozymes that have lost their catalytic activity over time.

Fig 26: MTR1 transfers the methyl group from m6G to adenosine in the substrate

RNA, generating m1A and demethylated G in the post-catalytic active site.