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Structure and mechanism of the methyltransferase ribozyme MTR1

Nature Chemical Biology volume 18, pages 547–555 (2022)Cite this article

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Abstract

RNA-catalyzed RNA methylation was recently shown to be part of the catalytic repertoire of ribozymes. The methyltransferase ribozyme MTR1 catalyzes the site-specific synthesis of 1-methyladenosine (m1A) in RNA, using O6-methylguanine (m6G) as a methyl group donor. Here, we report the crystal structure of MTR1 at a resolution of 2.8 Å, which reveals a guanine-binding site reminiscent of natural guanine riboswitches. The structure represents the postcatalytic state of a split ribozyme in complex with the m1A-containing RNA product and the demethylated cofactor guanine. The structural data suggest the mechanistic involvement of a protonated cytidine in the methyl transfer reaction. A synergistic effect of two 2′-O-methylated ribose residues in the active site results in accelerated methyl group transfer. Supported by these results, it seems plausible that modified nucleotides may have enhanced early RNA catalysis and that metabolite-binding riboswitches may resemble inactivated ribozymes that have lost their catalytic activity during evolution.

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Fig. 1: MTR1 ribozyme-catalyzed methyl transfer reaction and overall structure.
Fig. 2: The catalytic core domain of MTR1.
Fig. 3: The methyltransferase ribozyme uses general acid catalysis.
Fig. 4: Acceleration of methyl transfer by synergistic effects of Cm12 and Um42.
Fig. 5: Comparison of MTR1 guanine-binding site to purine riboswitches.

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Data availability

Structural data obtained by X-ray crystallography were deposited in the PDB and are available with the following accession codes: 7Q7X, 7Q7Y, 7Q7Z, 7Q80, 7Q81 and 7Q82. All relevant data are provided in the figures, Extended Data Figs. 1–6, Supplementary Tables 1 and 2 and Supplementary Fig. 1. Publicly available datasets from rcsb.org used in this study include 7OAX, 3SKI, 1Y27, 3FO6, 3MUM, 3Q50 and 7ELR. Source data are provided with this paper.

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Acknowledgements

This work was supported by the European Research Council (ERC; 682586), the Deutsche Forschungsgemeinschaft (DFG; HO4436/3-1) and the University of Würzburg. We thank C. Pfeuffer and A. Lenz for technical assistance and the beamline staff at DESY (PETRA III, P11) and ESRF (ID23) for assistance with data collection.

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Author notes

  1. These authors contributed equally. Carolin P.M. Scheitl, Mateusz Mieczkowski.

Authors and Affiliations

  1. Institute of Organic Chemistry, Julius-Maximilians-Universität Würzburg, Am Hubland, Würzburg, Germany

    Carolin P. M. Scheitl, Mateusz Mieczkowski & Claudia Höbartner

  2. Rudolf Virchow Center for Integrative and Translational Bioimaging, Julius-Maximilians-Universität Würzburg, Würzburg, Germany

    Hermann Schindelin

  3. Center for Nanosystems Chemistry (CNC), Julius-Maximilians-Universität Würzburg, Theodor-Boveri-Weg, Würzburg, Germany

    Claudia Höbartner

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Contributions

C.P.M.S. and C.H. designed the study. H.S. and C.P.M.S. collected diffraction data. M.M. solved the structure. C.P.M.S. performed biochemical experiments. C.H. wrote the paper with input from all authors.

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Correspondence to Claudia Höbartner.

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Extended data

Extended Data Fig. 1 Heavy atom derivatives of MTR1 crystal structure.

(a) Overall structure of MTR1 co-crystallized with Tl+. (b) MTR1 containing 2′-Selenomethyl-uridine modified residue in the RNA substrate. Yellow mesh indicates anomalous difference Fourier map contoured at (a) 3σ and (b) 5σ. The difference maps were computed from data collected at the Thallium L-III edge and Selenium K-edge, respectively.

Extended Data Fig. 2 Overall structures of MTR1 and crystal contacts.

(a) Secondary structure scheme of trimolecular MTR1. (b) Packing of overhangs of P1 and P2 to form a semi-continuous double helix. Arrangement of two copies in the asymmetric unit for crystals grown with (c) ab6G (ab1A in the product) and (d) with m6G (m1A in the crystal) but no added Mg2+. (e) Crystal contact via stacking of P3 in the structure shown in (d). (f,g) Anion exchange HPLC analyses of dissolved crystals corresponding to (c) and (d).

Extended Data Fig. 3 In-line probing of MTR1.

(a-c) Full gel images for the excerpts shown in Fig. 2. (a) In-line probing of MTR1 hybridized to unmethylated RNA R1, with increasing concentrations of m6G. (b) In-line probing of guanine binding to the MTR1 product complex containing m1A. (c) In-line probing of guanine binding to the MTR1 starting complex hybridized to unmethylated RNA R1. (a, b, c) Incubation at pH 8.0, 20 °C, 36 h. (d) Normalized band intensities seen in (a), shown for U36 (red), A37 (black), G38 (blue). The [m6G]1/2 value is ca 800 µM, however, this value cannot be interpreted as a Kd or Km because of multiple overlapping equilibria (with m6G and G since partial/slow methylation occurred during incubation). (e) Normalized in-line probing band intensities seen in (b) for U36 (red), A37 (black), G38 (blue). Kd,app = 2.0 ± 0.3 µM. Data in d) and e) are fitted to a one-site binding model. Error bars denote ± s.d. of the mean for n = 3 (d) or 4 (e) independent replicates. (f) Secondary structure scheme of MTR1 used in in-line probing experiments (with connecting loop at P3; numbers of nucleotides correspond to the split version). (g) Excerpt of the catalytic core showing solvent exposed location of U36 and stacking of A37 on m1A. (h) Excerpt of the metal ion binding site in the transition of the catalytic core to P3.

Source data

Extended Data Fig. 4 MTR1 mutagenesis of the core nucleotides and pH dependence.

(a) Scheme of MTR1 with mutated nucleotides and base-pairs indicated. (b) Summary of rate constants determined at pH 6.0 and 7.5. Individual data points of two independent replicates are shown as black dots. Reaction of MTR1-wt at pH 7.5 war repeated three times. (c) Representative gel images of kinetic experiments of two independent replicates. Reaction conditions: 5′-32P-labeled R1, 10 µM MTR1 or mutant, 100 µM m6G, 40 mM MgCl2, 25 °C. Timepoints: 0, 0.2, 0.5, 1, 2, 4, 7, 23 h (ON).

Source data

Extended Data Fig. 5 MTR1 as alkyltransferase ribozyme and pH dependence.

(a, b) Chemical structures of substrates (adenosine and benzyl guanine cofactor) before the RNA-catalyzed reaction, and after transfer to the target adenosine in R1 and release of guanine. (c) Overlay of active sites containing m1A, bn1A or ab1A in stick representation. (d) Kinetics of benzyl group transfer at pH 6.0 and pH 7.5, with exemplary gel images as inset. (e) Same as (d), but reaction with ab6G. The fastest reaction was observed with ab6G at pH 6.0, yielding ca 90% ab1A-RNA within 2 min. All reactions were performed in duplicate, individual data points and representative gel images are shown (timepoints shown in gels up to 60 min).

Source data

Extended Data Fig. 6 Comparison to binding sites of purine riboswitches also involving protonated nucleobases or metal ion binding.

(a, b) G20A mutant of Vc2 riboswitch with c-di-GMP (pdb 3mum), showing (a) Gβ and (b) Gα bound via stably protonated A20. (c) T. tengcongensis preQ1 riboswitch with bound preQ1 (pdb 3q50). (d) NMT1 riboswitch in complex with xanthine (pdb 7elr).

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2 and Fig. 1.

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Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Individual data points.

Source Data Fig. 3

Unprocessed gels.

Source Data Fig. 4

Individual data points.

Source Data Fig. 4

Unprocessed gels.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Individual data points.

Source Data Extended Data Fig. 4

Unprocessed gels.

Source Data Extended Data Fig. 5

Individual data points.

Source Data Extended Data Fig. 5

Unprocessed gels.

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Scheitl, C.P.M., Mieczkowski, M., Schindelin, H. et al. Structure and mechanism of the methyltransferase ribozyme MTR1. Nat Chem Biol 18, 547–555 (2022). https://doi.org/10.1038/s41589-022-00976-x

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