Cap analog substrates reveal three clades of cap guanine-N2 methyltransferases with distinct methyl acceptor specificities

Abstract

The Tgs proteins are structurally homologous AdoMet-dependent eukaryal enzymes that methylate the N2 atom of 7-methyl guanosine nucleotides. They have an imputed role in the synthesis of the 2,2,7-trimethylguanosine (TMG) RNA cap. Here we exploit a collection of cap-like substrates to probe the repertoire of three exemplary Tgs enzymes, from mammalian, protozoan, and viral sources, respectively. We find that human Tgs (hTgs1) is a bona fide TMG synthase adept at two separable transmethylation steps: (1) conversion of m 7 G to m 2,7 G, and (2) G nucleotides by hTgs1 and GlaTgs2 underscored the importance of guanine N7 alkylation in providing a key p-cation interaction in the methyl acceptor site. Mimivirus Tgs (MimiTgs) shares with the Giardia homolog the ability to catalyze only a single round of methyl addition at guanine-N2, but is distinguished by its capacity for guanine-N2 methylation in the absence of prior N7 methylation. The relaxed cap specificity of MimiTgs is revealed at alkaline pH. Our findings highlight both stark and subtle differences in acceptor specificity and reaction outcomes among Tgs family members.

An enzyme-coupled high-throughput assay for screening RNA methyltransferase activity inE. Colicell lysate

“…18 In vitro experiments have shown that hTGS1 specifically recognizes the cap 0-structure also if truncated to dimeric m 7 GpppX (X = A, G and C) or monomeric m 7 GTP. 19 To date several assays have been developed to monitor the activity of SAM-dependent methyltransferases. Incorporation of radioactively labeled methyl groups from the respective SAM substrates has been used since the early 1960s and is still very popular because of its high sensitivity.…”

“…20 Today, measuring methyltransferase activity is mainly based on using [methyl-3 H] SAM as cosubstrate. 15,19,21 Although this approach can be used for most enzymes, it requires access to an accordingly equipped isotope laboratory, which is not always available. Downstream processing requires gel electrophoresis and analysis by autoradiography which makes the overall process long and tedious and severely limits throughput.…”

“…This observation was in agreement with kinetic data on hTGS1. 19 Homocysteine is prone to oxidation by air which renders it unavailable for detection by thiol-reactive probes after a certain time. This may explain the decrease in signal after 60 min at 50 μM SAM.…”

“…23 The structure of the C-terminal part was solved and this part was shown to be sufficient for cap 0-methylation of monomeric m 7 GTP and the minimum cap structure m 7 GpppX (X = A, C or G) in vitro. 19,23 Since these compounds are commercially available we chose them as substrates for our high-throughput assay. The dinucleotide m 7 GpppA contains the 5'-5'-triphosphate bridge characteristic of the RNA cap structure (Fig.…”

“…19,24,28 Therefore, production of SAH is the rate-limiting step of these consecutive reactions producing homocysteine from SAM. Thus, every molecule of SAH 2 produced by hTGS1 will be quickly converted to homocysteine 5 and thus be available for detection.…”