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Site-Specific RNA Methylation by a Methyltransferase Ribozyme

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Site-Specific RNA Methylation by a Methyltransferase Ribozyme Article Site-specific RNA methylation by a methyltransferase ribozyme https://doi.org/10.1038/s41586-020-2854-z Carolin P. M. Scheitl1, Mohammad Ghaem Maghami1, Ann-Kathrin Lenz1 & Claudia Höbartner1 ✉ Received: 29 March 2020 Accepted: 27 July 2020 Nearly all classes of coding and non-coding RNA undergo post-transcriptional Published online: 28 October 2020 modifcation, including RNA methylation. Methylated nucleotides are among the Check for updates evolutionarily most-conserved features of transfer (t)RNA and ribosomal (r)RNA1,2. Many contemporary methyltransferases use the universal cofactor S-adenosylmethionine (SAM) as a methyl-group donor. SAM and other nucleotide-derived cofactors are considered to be evolutionary leftovers from an RNA world, in which ribozymes may have catalysed essential metabolic reactions beyond self-replication3. Chemically diverse ribozymes seem to have been lost in nature, but may be reconstructed in the laboratory by in vitro selection. Here we report a methyltransferase ribozyme that catalyses the site-specifc installation of 1-methyladenosine in a substrate RNA, using O6-methylguanine as a small-molecule cofactor. The ribozyme shows a broad RNA-sequence scope, as exemplifed by site-specifc adenosine methylation in various RNAs. This fnding provides fundamental insights into the catalytic abilities of RNA, serves a synthetic tool to install 1-methyladenosine in RNA and may pave the way to in vitro evolution of other methyltransferase and demethylase ribozymes. More than 70 methylated nucleotides have important functional roles riboswitches that regulate the expression of associated genes, which in present-day RNA4,5. Nucleotide modifications are mostly known are often involved in the biosynthesis of the respective metabolite or for shaping the structures and tuning the functions of non-coding its transport across membranes24. Six classes of SAM-binding ribos- rRNA, tRNA and small nuclear (sn)RNA, but some also influence witches accommodate the ligand with its reactive methyl group in gene-expression programs by regulating the fate and function of mes- various different conformations25,26; however, these RNAs apparently senger (m)RNA6–8. The majority of methylated nucleotides currently avoid self-methylation. known in RNA are installed through post-synthetic (that is, post- or Therefore, it has remained an open question whether RNA can cata- co-transcriptional) methylation by protein enzymes that use SAM as lyse site-specific methylation reactions to produce defined methylated the universal methyl-group donor. Methyltransferases are considered RNA products. Previously, in vitro selection efforts have identified ancient enzymes, and methylated nucleotides have also been discussed SAM-binding aptamers, but methyl transfer reactions were not as molecular fossils of the early Earth produced by prebiotic methyl- observed—probably because the aptamer established a binding site ating agents9,10. For the era preceding modern life based on DNA and for the adenine moiety of the cofactor, but did not specifically interact proteins, RNA has been proposed to have functioned both as primary with its 5′ substituent27. genetic material and as a catalyst or ribozyme11. Ribozymes have been We speculated that methyl-group donors other than SAM or discovered in nature, where they catalyse RNA cleavage and ligation methylene-THF could be substrates for RNA-catalysed RNA methyla- reactions—mostly in the context of RNA splicing and retrotransposi- tion, and took inspiration from an enzyme class that is responsible tion12–14. In vitro-selected ribozymes have been evolved to act as RNA for the repair of alkylated DNA (that is, catalyses the demethylation of ligases and replicases that are able to reproduce themselves or their DNA). The O6-methylguanine (m6G) DNA methyltransferase releases ancestors, and that are able to produce functional RNAs (including unmodified DNA, accompanied by the irreversible methylation of the ribozymes and aptamers)15–17. Self-alkylating ribozymes have been protein28. By analogy, we hypothesized that RNA-catalysed methyl described using reactive iodo- or chloro-acetyl derivatives18–20, or elec- transfer would result in methylated RNA upon release of guanine. trophilic epoxides21, but the design of earlier in vitro selection strategies Using in vitro selection, we identified a ribozyme that uses m6G as a prevented the emergence of catalysts that are capable of transferring small-molecule methyl-group donor and catalyses site-specific meth- a one-carbon unit. Thus, ribozymes that catalyse RNA methylation ylation of adenosine at position N1, resulting in position-specific instal- have so far remained unknown. This lack of ribozymes that act as lation of 1-methyladenosine (m1A) in the target RNA (Fig. 1a). methyltransferases seems surprising, as numerous natural aptamers are known to specifically bind nucleotide-derived metabolites associ- ated with methyl-group transfer or one-carbon metabolism, including Search for methyltransferase ribozymes SAM, methylene tetrahydrofolate (THF) and adenosylcobalamin In vitro selection is a powerful method for enriching functional RNAs (vitamin B12)22,23. These aptamers are found as components of by repeated cycles of selection and amplification from a random RNA 1Institut für Organische Chemie, Julius-Maximilians-Universität Würzburg, Würzburg, Germany. ✉e-mail: [email protected] Nature | Vol 587 | 26 November 2020 | 663 Article CH a O 3 b NH NH 2 N 2 SL=5′-GACAUACUGAGCCUUCAAAUA(22 nt) CA13 +BG-NH2 N CH N N N N 3 Time Substrate N Product N NH2 N H N U A U A _ N 6 N 1 G UA G UA SL m G m A A CA A AA C G C G C A C CA21 A G CA13 G CA21 +BG-NH U U 2 Ribozyme Guanine MethylatedRNA A C A C C A C A Time C CGACA C CGACA CG GC CG CG _ c CUUUCCG A G CAUA -5′ SL 0.20 C U U A 5′-GGGAGGC CAAUU ) GC A C U A –2 A U C A AG UAC 0 CA13 +BG C A 0.15 C C A A Time C G C U A C MTR1 A , ×1 CU UA G U –1 _ A C 0.10 5 23 h SL CC C GACAA 0 2 (min Time C G O O _ C G obs 0.05 P CA21 +BG k _ N NH2 Time C G S N N N G C _ A U 0 N N NH N N NH SL G C 050 100 150 200 H 2 H 2 U G 6 U C m G (μM) BG-NH2 BG CH d O 3 CH CH CH S 3 O 3 HN 3 N G N G 6 H A dG – 6 s 6 6 6 N N N m m m m m G DMSOSAM N N N HO N N NH2 _ P O _ N N NH N N N N S H 2 H H ms6Gm6Hm6A HO m6dG Fig. 1 | Methyltransferase ribozyme-catalysed synthesis of m1A in RNA using methyl-group transfer. The insert shows a gel image of a 3′-fluorescein-labelled m6G as methyl-group donor. a, Reaction scheme with intermolecular 13-mer RNA substrate reacted with MTR1 and m6G (10 μM). The observed rate hybridization of ribozyme to target RNA. b, Sequences and predicted constant kobs was determined with a 3′-fluorescein-labelled 17-mer RNA at 5 secondary structure of CA13 and CA21 ribozymes identified by in vitro concentrations of m6G, ranging from 5 to 200 μM. The red line represents a 6 6 selection, and their trans-activity for modification of a 22-nt RNA (designated curve fit to kobs = kmax[m G]/(Km,app + [m G]). Individual data points (white) (n = 3), 6 SL) with BG-NH2 or BG, analysed by 20% denaturing PAGE (100 μM guanine mean ± s.e.m. (black). P, product; S, substrate. d, Structures of m G analogues 6 derivative, 40 mM MgCl2, pH 7.5, 37 °C, and time points 0, 0.5, 1, 2 and 5 h). tested. Gel image shows that product formation occurs with m G, and to a minor Representative images of three independent experiments with similar results. extent with m6dG (24-h reaction time, 25 °C, with 100 μM m6G or analogue). c, Methyltransferase ribozyme MTR1 with stabilized stem-loop shows efficient Representative image from two independent experiments. G, guanine. library. We used a structured RNA pool containing 40 random nucleo- (which we named MTR1) (Fig. 1c) used a 13-nt or a 17-nt RNA as target and tides that was designed according to a previously used strategy to direct m6G as cofactor to generate methylated RNA products in 80–90% yield the RNA-catalysed labelling of a specific adenosine in a target RNA29,30. after 23 h incubation at 37 °C, pH 7.5. The reaction rate was dependent 6 RNA methylation would most probably occur at an O or N nucleobase on m G concentration, with an apparent Michaelis constant Km of about heteroatom, on the 2′-OH group or on the phosphate backbone. In any 100 μM. The presence of the stem loop in the core of MTR1 was con- of these cases, the attachment of a single methyl group would not be firmed by compensatory mutations of individual base pairs, resulting likely to enable the physical separation of the active sequences on the in mutants that retained catalytic activity. Shortening the stem led to basis of size or charge. only slightly reduced activity, whereas deletion of the stem resulted in We therefore searched for alkylating ribozymes that catalysed the inactive ribozymes (Extended Data Fig. 2). Chemical structure probing transfer of a biotin group attached via a benzyl linker to the target experiments (using dimethylsulfate (DMS) and selective 2′-hydroxyl RNA, and speculated that the resulting ribozymes could later be engi- acylation analysed by primer extension (SHAPE)) also confirmed the neered to enable RNA methylation.
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