Editing and methylation at a single site by functionally interdependent activities

Abstract

Nucleic acids undergo naturally occurring chemical modifications. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified1. Despite recent progress, the mechanism for the biosynthesis of most modifications is not fully understood, owing, in part, to the difficulty associated with reconstituting enzyme activity in vitro. Whereas some modifications can be efficiently formed with purified components, others may require more intricate pathways2. A model for modification interdependence, in which one modification is a prerequisite for another, potentially explains a major hindrance in reconstituting enzymatic activity in vitro3. This model was prompted by the earlier discovery of tRNA cytosine-to-uridine editing in eukaryotes, a reaction that has not been recapitulated in vitro and the mechanism of which remains unknown. Here we show that cytosine 32 in the anticodon loop of Trypanosoma brucei tRNAThr is methylated to 3-methylcytosine (m3C) as a pre-requisite for C-to-U deamination. Formation of m3C in vitro requires the presence of both the T. brucei m3C methyltransferase TRM140 and the deaminase ADAT2/3. Once formed, m3C is deaminated to 3-methyluridine (m3U) by the same set of enzymes. ADAT2/3 is a highly mutagenic enzyme4, but we also show that when co-expressed with the methyltransferase its mutagenicity is kept in check. This helps to explain how T. brucei escapes ‘wholesale deamination’5 of its genome while harbouring both enzymes in the nucleus. This observation has implications for the control of another mutagenic deaminase, human AID, and provides a rationale for its regulation.

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Figure 1: TRM140a modifies position 32 of tRNAThr.
Figure 2: TRM140a and ADAT2/3 are required for methylation and editing of tRNAThrAGU.
Figure 3: LC-MS/MS analysis confirms the in vitro formation of m3U.
Figure 4: Complex formation with TRM140a dampens the mutagenicity of ADAT2/3.

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Acknowledgements

The research was funded by NIH grants GM084065 to J.D.A. and GM058843 to P.A.L. and a Czech Science Foundation (15-21450Y) grant to Z.P. We thank V. Gopalan, J. Rinehart and D. Schoenberg for comments and suggestions on the manuscript.

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Authors

Contributions

All authors contributed to the design of the experiments and data interpretation; experiments were performed by M.A.T. (Figs 1a, b, e, 2a–c), Z.P. (Fig. 1c–d), K.W.G (Fig. 3a–i), K.M.M. (Fig. 4a–d), I.M.C.F. (Fig. 4e). The manuscript was written by J.D.A. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Juan D. Alfonzo.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks J. Chaudhuri and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Anticodon sequences for tRNAThr from various organisms.

This limited set represents the few tRNAThr molecules for which modifications have been mapped to specific nucleotide positions as deposited in tRNAdb8. Highlighted in grey is position 32 of the anticodon, showing that this position is often modified to 3-methylcytosine (m3C).

Extended Data Figure 2 TRM140a methyltransferase is a nuclear enzyme.

a, Western blot analysis of total (T), nuclear (N) and cytoplasmic (C) fractions isolated from T. brucei as previously described24 probed with polyclonal antibodies raised against recombinant TRM140a. The same membrane was probed with antibodies against enolase, serving as a cytosolic marker and also as a control fraction to show the degree of nuclear contamination in our cytoplasmic fraction. b, Immunofluorescence localization of TRM140a using the same antibodies as in top panel of a. The cells were also stained with DAPI to establish the position of the nucleus (N) and mitochondrial (K) genomes for reference. ‘Merge’ refers to the superposition of the two panels, which confirms the nuclear localization of this protein. c, Primer extension analysis of total RNA isolated from the same fractions as above, primer +8 marks the positions of the strong stop one nucleotide short of position 32 (the methylated position), indicating that m3C is already present in the nuclear tRNA before cytoplasmic export. G, A, T and C refer to an unrelated sequencing ladder used as a size marker. T, N and C are as in a and O represents a mock primer extension with oligonucleotide primer alone but in the absence of template.

Extended Data Figure 3 Position 32 in the other tRNAThr isoacceptors is also methylated in the nucleus of T. brucei.

a, b, Similar reactions as in Extended Data Fig. 3c, but this time with oligonucleotide primers specific for tRNAThrCGU (a) and tRNAThrUGU (b), confirming that the strong stop in these tRNAs already appears in the nuclear fractions. This time, however, the expected product is at primer +7, as indicated.

Extended Data Figure 4 Concentration curves for the formation of m3C and m3U.

a, Concentration curve with increasing amounts of TRM140, ADAT2/3 was kept at the same molar ratio as the methyltransferase. The specific activities of TRM140 are as indicated and expressed in pmol per min per mg. b, Similar experiment as in a but this time showing the m3U activity of ADAT2/3 in the presence of stoichiometric amounts of the methyltransferase.

Supplementary information

Supplementary Figure 1

The file shows the uncropped western blots results from Figure 4. The relative electrophoretic migration of the size markers are shown by black lines on the left margin of each gel. The panels are representative of at least 5 independent experiments. TbADAT2, TbADAT3 (tagged version) and TbTRM140 have predicted molecular masses of 27, 39 and 47 kDa respectively. (PDF 7183 kb)

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Rubio, M., Gaston, K., McKenney, K. et al. Editing and methylation at a single site by functionally interdependent activities. Nature 542, 494–497 (2017). https://doi.org/10.1038/nature21396

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