A tRNA modification in Mycobacterium tuberculosis facilitates optimal intracellular growth
- Department of Immunology and Infectious Diseases Harvard T. H. Chan School of Public Health, United States
- Division of Infectious Diseases, Brigham and Women's Hospital, United States
- Department of Microbiology, Harvard Medical School, United States
- Howard Hughes Medical Institute, United States
Figures
Figure 1
Phylogenetic distribution of Mtb tRNA modifying enzyme homologs.
Heatmap of log10 E-values from BLAST search results. BLAST searches were conducted against 120 manually picked organisms using Mtb tRNA modifying enzymes as queries. When one organism has multiple …
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Figure 1—source data 1
log10 E-values from BLAST searching for the homologs of Mtb tRNA modifying enzymes in 120 organisms.
- https://cdn.elifesciences.org/articles/87146/elife-87146-fig1-data1-v1.xlsx
Figure 2 with 1 supplement
Heatmap of misincorporation and early termination frequency in sequencing of tRNAs from wild-ype Mtb.
Heatmaps show misincorporation (A) and termination (B) frequencies at all positions across tRNAs (read 5′ to 3′). Predicted modifications are labeled based on similarity to known modifications in …
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Figure 2—source data 1
Misincorporation and early termination frequencies in sequencing of tRNAs from wild-type Mtb.
- https://cdn.elifesciences.org/articles/87146/elife-87146-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
E. coli tRNA modifications detected by tRNA sequencing (tRNA-seq).
Schematic tRNA secondary structure with sites of modifications. Modifications and positions that are detected by RT-derived signature in yellow (without chemical treatment in Zhang et al., 2022) and …
Figure 3 with 1 supplement
Iodoacetamide (IAA) treatment promotes detection of sulfur modifications on tRNAs by enhancing termination signals.
Heatmaps of the termination signals of E. coli tRNAs treated with (A) or without (B) IAA. Known modification sites, including sulfur modifications (s4U, s2C, s2U in white) are shown. (C) Termination …
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Figure 3—source data 1
Termination frequencies of E. coli tRNAs treated with or without iodoacetamide (IAA).
- https://cdn.elifesciences.org/articles/87146/elife-87146-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Iodoacetamide (IAA) treatment promotes detecting sulfur modifications by enhancing misincorporation signals.
Heatmaps of the misincorporation signals of E. coli tRNAs treated with (A) or without (B) IAA. Known modification sites, including sulfur modifications (s4U, s2C, s2U in white) are shown. (C) …
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Figure 3—figure supplement 1—source data 1
Misincorporation frequencies of E. coli tRNAs treated with or without iodoacetamide (IAA).
- https://cdn.elifesciences.org/articles/87146/elife-87146-fig3-figsupp1-data1-v1.xlsx
Figure 4
Heatmap and plot of early termination frequency from sequencing of tRNAs from wild-type and MtbΔmnmA with and without RNA alkylation.
Heatmap of early termination frequencies across tRNA molecules and positions for wild-type (WT) (A) and MtbΔmnmA (B). Sulfur modification is shown in white. (C) Plot of termination frequencies at …
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Figure 4—source data 1
Termination frequencies from sequencing of tRNAs from wild-type and MtbΔmnmA with and without RNA alkylation.
- https://cdn.elifesciences.org/articles/87146/elife-87146-fig4-data1-v1.xlsx
Figure 5 with 4 supplements
1-Cyclohexyl-(2-morpholinoethyl) carbodiimide (CMC) and alkali treatment facilitate detection of ψ, D, and m7G modifications in E. coli.
Heatmaps of the misincorporation signals of E. coli tRNAs treated with (A) or without (B) CMC. In both conditions, tRNAs are incubated in alkali condition. Known Ψ, D, and m7G sites are shown. Ψ is …
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Figure 5—source data 1
Misincorporation frequencies from sequencing of E. coli tRNAs treated with or without 1-cyclohexyl-(2-morpholinoethyl) carbodiimide (CMC).
- https://cdn.elifesciences.org/articles/87146/elife-87146-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
1-Cyclohexyl-(2-morpholinoethyl) carbodiimide (CMC) and the following alkali treatment facilitate detecting additional modifications.
Heatmaps of the termination signals of E. coli tRNAs treated with (A) or without (B) CMC. In both conditions, tRNAs are incubated at an alkali condition. Known Ψ (in white), D, and m7G sites are …
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Figure 5—figure supplement 1—source data 1
Termination frequencies from sequencing of E. coli tRNAs treated with or without 1-cyclohexyl-(2-morpholinoethyl) carbodiimide (CMC).
- https://cdn.elifesciences.org/articles/87146/elife-87146-fig5-figsupp1-data1-v1.xlsx
Figure 5—figure supplement 2
1-Cyclohexyl-(2-morpholinoethyl) carbodiimide (CMC) treatment facilitate detecting ψ.
Heatmaps of the termination frequencies at the uridine in E. coli tRNAs at positions 40 (A), 41 (B), and 57 (ψ55) (C). tRNA species where U at the indicated position is known to be ψ are shown in …
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Figure 5—figure supplement 2—source data 1
Termination frequencies at the uridine in E. coli tRNAs at positions 40, 41, and 57.
- https://cdn.elifesciences.org/articles/87146/elife-87146-fig5-figsupp2-data1-v1.xlsx
Figure 5—figure supplement 3
Misincorporation and termination signals derived from D in E. coli tRNAs.
Heatmaps showing termination frequencies at positions 18 (A), 21 (D, E), and 22 (F), and misincorporation frequencies at positions 16 (B), 17 (C), 20 (G), 20A (H), and 21 (I) to predict the presence …
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Figure 5—figure supplement 3—source data 1
Termination frequencies at positions 18, 21, and 22, and misincorporation frequencies at positions 16, 17, 20, 20A, and 21 to predict the presence of D in E. coli tRNAs.
- https://cdn.elifesciences.org/articles/87146/elife-87146-fig5-figsupp3-data1-v1.xlsx
Figure 5—figure supplement 4
RT-derived signatures originated from tandem Ds or singlet Ds.
Figure 6 with 3 supplements
Heatmap of early termination frequency from sequencing of tRNAs isolated from wild-type (WT) and MtbΔtruB following 1-cyclohexyl-(2-morpholinoethyl) carbodiimide (CMC) treatment.
Heatmap of early termination frequencies across tRNA molecules and positions for WT (left) and MtbΔtruB (right). Termination signals derived from position 55 are shown in white. The experiment was …
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Figure 6—source data 1
Termination frequencies across tRNA molecules and positions for wild-type (WT) and MtbΔtruB.
- https://cdn.elifesciences.org/articles/87146/elife-87146-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Misincorporation and termination signals derived from D at positions 20, 20A, and 21 in Mtb tRNAs.
Heatmaps showing termination frequencies at positions 18 (A) and 21 (D), and misincorporation frequencies at positions 16 (B), 17 (C), 20 (E), and 20A (F) to predict the presence of D. tRNAs bearing …
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Figure 6—figure supplement 1—source data 1
Termination frequencies at positions 18 and 21, and misincorporation frequencies at positions 16 (B), 17, 20, and 20A to predict the presence of D in Mtb tRNAs.
- https://cdn.elifesciences.org/articles/87146/elife-87146-fig6-figsupp1-data1-v1.xlsx
Figure 6—figure supplement 2
Misincorporation signals derived from m7G at position 46.
Heatmaps showing termination frequencies at position 46 to predict the presence of m7G. tRNAs bearing G at position 46 are shown. tRNAs that have consistently high signals when tRNAs are treated …
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Figure 6—figure supplement 2—source data 1
Termination frequencies at position 46 to predict the presence of m7G in Mtb tRNAs.
- https://cdn.elifesciences.org/articles/87146/elife-87146-fig6-figsupp2-data1-v1.xlsx
Figure 6—figure supplement 3
Termination signals derived from pseudouridine at positions 55, 38, and 39.
Heatmaps showing termination frequencies at positions 57 (A), 40 (B), and 41 (C) to assess the pseudouridylation states at positions 55 (A), 38 (B), and 39 (C). tRNAs bearing U at the indicated …
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Figure 6—figure supplement 3—source data 1
Termination frequencies at positions 57, 40, and 41 to assess the pseudouridylation states at positions 55, 38, and 39 in Mtb tRNAs.
- https://cdn.elifesciences.org/articles/87146/elife-87146-fig6-figsupp3-data1-v1.xlsx
Figure 7
MtbΔmnmA is attenuated in a macrophage infection model.
(A) Wild-type and MtbΔmnmA do not display growth differences in 7H9 medium. (B) Auto-luminescent wild-type and ΔmnmA Mtb strains were diluted to a multiplicity of infection of 2 bacteria per mouse …
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Figure 7—source data 1
Mtb growth curve.
- https://cdn.elifesciences.org/articles/87146/elife-87146-fig7-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mycobacterium tuberculosis) | mnmA | NA | Uniprot: Rv3024c; Refseq: NP_217540.1 | |
| Gene (Mycobacterium tuberculosis) | truB | NA | Uniprot: Rv2793c; Refseq: NP_217309.1 | |
| Strain, strain background (Mycobacterium tuberculosis) | H37Rv | PMID:9634230 | ||
| Genetic reagent (Mycobacterium tuberculosis) | MtbΔmnmA::zeo | This paper | Mtb H37Rv strain lacking mnmA | |
| Genetic reagent (Mycobacterium tuberculosis) | MtbΔtruB::zeo | This paper | Mtb H37Rv strain lacking truB | |
| Recombinant DNA reagent | pNit-RecET SacBR | NA | For homologous recombination | |
| Chemical compound | Iodoacetamide (IAA) | Sigma | ||
| Chemical compound | 1-Cyclohexyl-(2-morpholinoethyl) carbodiimide (CMC) | Sigma |
Additional files
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Supplementary file 1
tRNA modifying enzymes used as queries for BLAST.
- https://cdn.elifesciences.org/articles/87146/elife-87146-supp1-v1.xlsx
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Supplementary file 2
Exploration of tRNA modifying enzymes in Mtb by BLAST.
- https://cdn.elifesciences.org/articles/87146/elife-87146-supp2-v1.xlsx
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Supplementary file 3
Predicted tRNA modifying enzymes in Mtb.
- https://cdn.elifesciences.org/articles/87146/elife-87146-supp3-v1.xlsx
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Supplementary file 4
List of primers, plasmids, and strains.
- https://cdn.elifesciences.org/articles/87146/elife-87146-supp4-v1.xlsx
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Supplementary file 5
Mtb tRNA sequences.
- https://cdn.elifesciences.org/articles/87146/elife-87146-supp5-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/87146/elife-87146-mdarchecklist1-v1.pdf
