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Comparative tRNA sequencing and RNA mass spectrometry for surveying tRNA modifications

Abstract

Chemical modifications of the nucleosides that comprise transfer RNAs are diverse. However, the structure, location and extent of modifications have been systematically charted in very few organisms. Here, we describe an approach in which rapid prediction of modified sites through reverse transcription-derived signatures in high-throughput transfer RNA-sequencing (tRNA-seq) data is coupled with identification of tRNA modifications through RNA mass spectrometry. Comparative tRNA-seq enabled prediction of several Vibrio cholerae modifications that are absent from Escherichia coli and also revealed the effects of various environmental conditions on V. cholerae tRNA modification. Through RNA mass spectrometric analyses, we showed that two of the V. cholerae-specific reverse transcription signatures reflected the presence of a new modification (acetylated acp3U (acacp3U)), while the other results from C-to-Ψ RNA editing, a process not described before. These findings demonstrate the utility of this approach for rapid surveillance of tRNA modification profiles and environmental control of tRNA modification.

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Fig. 1: Profiling tRNA modifications in V. cholerae through tRNA-seq.
Fig. 2: Frequency of acp3U is dependent on growth phase.
Fig. 3: Structure of acacp3U.
Fig. 4: Biogenesis and function of acacp3U.
Fig. 5: Cytidine at position 32 in tRNA-Tyr undergoes C-to-Ψ RNA editing.

Data availability

All data is available from the corresponding authors upon request. The data reported in this paper have been deposited in the NCBI Gene expression omnibus https://www.ncbi.nlm.nih.gov/geo/ (accession code, GSE147614). Source data for Figs. 1, 2 and 4 and Extended Data Figs. 15 are presented with the paper.

Code availability

All codes are available from the corresponding authors upon request.

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Acknowledgements

We thank V. Srisuknimit, B. Davis, T. Hubbard and Waldor laboratory members for helpful comments on the project, the manuscript, the Harvard Medical School East Quad NMR Facility for assistance with nuclear magnetic resonance, the Harvard Medical School Analytical Chemistry Core for use of the QTOF mass spectrometer and the Harvard FAS Science Core Facility for use of the MALDI equipment. This work was supported by NIH R01-AI-042347 (M.K.W.), HHMI (M.K.W.), NIH R01-ES026856 (P.C.D.), R01-ES024615 (P.C.D.) and National Research Foundation of Singapore through the Singapore-MI Alliance for Research and Technology Antimicrobial Resistance Interdisciplinary Research Group (P.C.D.).

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S.K. and M.K.W. designed the research. S.K. performed all experiments and analyzed data. M.K.W. and P.C.D. discussed the results. S.K. and M.K.W. wrote the paper.

Corresponding authors

Correspondence to Satoshi Kimura or Matthew K. Waldor.

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Extended data

Extended Data Fig. 1 tRNA-seq profiling of tRNA modifications in E. coli.

a, Example of the analysis of reverse transcription derived signatures in tRNA-seq data. The bars in the left panel represent mapped read depth with the left side and right side corresponding to the 5′ and 3′ end of tRNAs, respectively. Bars in which the misincorporation frequency is less than 1% are colored in grey; additional colors are shown at sites where there are higher levels of misincorporation (red corresponds to U, blue, C, orange, G and yellow, A). The importance of the misincorporation signal at position 10 is not known. Several drops in depth around sites of known modifications are also apparent (for example DD at positions 16, 17); however, the correspondence between the decrease in read depth and sites of modification is less precise than with nucleotide misincorporation. The right panel shows the secondary structure of tRNA-Lys. The structures of some of the modifications that lead to reverse transcription derived signatures are shown in lower panels. b, Heatmaps of the frequency of misincorporation (left) and stop (termination) of reverse transcription (right) signals in tRNA from stationary phase E. coli. Each row represents an individual tRNA and each column represents a position within tRNAs. The modifications are assigned based on the reference tRNA sequences (Supplementary Data 1 and Supplementary Table 1). The color keys indicated above upper right corners of the heatmaps correspond to the frequencies of misincorporation and termination. Representative data from one replicate is shown.

Source data

Extended Data Fig. 2 Validation of modifications inferred from RT-derived signals by nucleoside analysis.

Nucleosides from purified tRNAs were analyzed by mass spectrometry. The area values of a nucleoside was normalized using the signal of T, which is present in all tRNA species as an internal control. The values relative to the maximum number across the nine tRNA species are shown in the heatmap. Glu-Q was not observed in any tRNAs. The presence of most of the modifications were also confirmed in the fragment analyses except for Q in tRNA-Tyr, s2C and I in tRNA-Arg2A, and Ψ (Supplementary Data 3). This experiment was performed once.

Source data

Extended Data Fig. 3 Validation of modifications inferred from RT-derived signals using mutant V. cholerae strains.

a, Heatmap of misincorporation frequency at position 8 in V. cholerae tRNAs. Most of the misincorporation signals, except for tRNA-Ser1 and tRNA-Gln1A, are eliminated in the ΔthiI strain, consistent with the idea that misincorporation results from the associated modification, s4U. The data for tRNA-Ile2 is not shown (black) because of insufficient read depth (<100 reads). b, Heatmap of misincorporation frequency at position 32 in V. cholerae tRNAs. The signals in tRNAs that are expected to have s2C (tRNA-Arg2A, tRNA-Arg2C, tRNA-Arg3, tRNA-Ser3A, tRNA-Ser3B, and tRNA-Arg4) are eliminated in the ΔttcA strain, whereas the signal in tRNA-Tyr remains due to C to Ψ RNA editing (see Fig 5). The data for tRNA-Ile2 is not shown (black) because of insufficient read depth. c, Heatmap of misincorporation frequency at position 37 in V. cholerae tRNAs. The signals in tRNAs that are expected to have ms2io6A (tRNA-Leu5, tRNA-Phe1, tRNA-Phe2, tRNA-Leu4, tRNA-Trp, tRNA-Cys1, tRNA-Cys2, tRNA-Ser1, and tRNA-Tyr) are eliminated in the ΔmiaA strain, whereas the signals in tRNA species that are predicted to have m1G at position 37 remain. d, Heatmap of misincorporation frequency at position 22 in V. cholerae tRNAs. The signal in tRNA-Tyr was absent in the ΔtrmK strain, suggesting that this signal is derived from m1A. The data for tRNA-Ile2 is not shown (black) because of insufficient read depth. In all panels, representative data from three replicates with similar results for WT and one replicate for knockout strains is shown.

Source data

Extended Data Fig. 4 tRNA-seq profiles of log phase (a) and cecal fluid-derived (b)V. cholerae.

Heatmaps of frequency of misincorporation (left) and termination of reverse transcription (right) signals in indicated samples. Types and positions of modifications that are presumed shared with E. coli are shown in black. Positions of V. cholerae-specific signals are indicated in white letters. Representative data from three replicates with similar results is shown.

Source data

Extended Data Fig. 5 VC0317 is a candidate acetyltransferase required for acacp3U synthesis.

Nucleoside analysis of total tRNAs derived from strains containing transposon insertions in putative acetyltransferases. Relative abundances of acacp3U (a) and acp3U (b), normalized to that of T, are shown. This experiment was performed once.

Source data

Extended Data Fig. 6 Nucleoside analysis of tRNA-Tyr from the Δvc1231 strain RNA cultured with stable isotope labeled cytidine (15N3-C) or unlabeled cytidine (non-SI).

The detecting bases are shown on the left of panels. Representative data from two independent experiments with similar results for G, A, and T is shown. Experiments for other nucleosides were performed once.

Extended Data Fig. 7 RNA mass spectrometric analyses of tRNA-Tyr.

a, Nucleoside analysis detecting D, Ψ, m1A, T, oQ, Q, s4U, ms2io6A. The peak heights between different nucleosides are not comparable. Representative data from two independent experiments with similar results is shown. b, Fragment analyses of RNase T1 (left) and RNase A (right) digests. The fragments with or without modifications are shown in red and black, respectively. Measurement was conducted in positive polarity mode. Representative data from two independent experiments with similar results is shown.

Supplementary information

Supplementary information

Supplementary Figs. 1–9, Tables 1 and 2, Synthetic procedures and references

Reporting Summary

Supplementary Data

Supplementary Datasets. Supplementary Data 1. Reference E. coli tRNA sequences with modifications. tRNA sequences were retrieved from tRNAdb and partial or full sequences were changed or added based on the literature. Bold letters indicate the changes and addition of sequences based on references. Supplementary Data 2. Conservation of tRNA modification enzymes between V. cholerae and E. coli. Supplementary Data 3. Primary sequences of V. cholerae tRNAs with modifications The nucleosides that were detected in the RNase T1 (top rows), RNase A (middle rows) and either RNase (bottom rows) fragment analyses of digests are colored in black. The abbreviation of nucleosides is shown on the right. Supplementary Data 4. Parameters of mass spectrometry for dynamic MRM analyses. Supplementary Data 5. Comparative genomics for narrowing down candidate acetyltransferases required for acacp3U biogenesis. Putative acetyltransferases in V. cholerae are listed with E values calculated by BLAST among homologs between V. cholerae and indicated organisms. n.d. means the E value is higher than 1E-10 or no detectable homologs were found. Supplementary Data 6. Primer list Supplementary Data 7. Reference DNA sequences of tRNAs for mapping

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Kimura, S., Dedon, P.C. & Waldor, M.K. Comparative tRNA sequencing and RNA mass spectrometry for surveying tRNA modifications. Nat Chem Biol 16, 964–972 (2020). https://doi.org/10.1038/s41589-020-0558-1

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