Functional redundancy in tRNA dihydrouridylation

Abstract

Dihydrouridine (D) is a common modified base found predominantly in transfer RNA (tRNA). Despite its prevalence, the mechanisms underlying dihydrouridine biosynthesis, particularly in prokaryotes, have remained elusive. Here, we conducted a comprehensive investigation into D biosynthesis in Bacillus subtilis through a combination of genetic, biochemical, and epitranscriptomic approaches. Our findings reveal that B. subtilis relies on two FMN-dependent Dus-like flavoprotein homologs, namely DusB1 and DusB2, to introduce all D residues into its tRNAs. Notably, DusB1 exhibits multisite enzyme activity, enabling D formation at positions 17, 20, 20a and 47, while DusB2 specifically catalyzes D biosynthesis at positions 20 and 20a, showcasing a functional redundancy among modification enzymes. Extensive tRNA-wide D-mapping demonstrates that this functional redundancy impacts the majority of tRNAs, with DusB2 displaying a higher dihydrouridylation efficiency compared to DusB1. Interestingly, we found that BsDusB2 can function like a BsDusB1 when overexpressed in vivo and under increasing enzyme concentration in vitro. Furthermore, we establish the importance of the D modification for B. subtilis growth at suboptimal temperatures. Our study expands the understanding of D modifications in prokaryotes, highlighting the significance of functional redundancy in this process and its impact on bacterial growth and adaptation.

Graphical Abstract

Figure 1.

Location of D-sites in tRNA and the corresponding enzyme involved in site dihydrouridylation determined experimentally. Schematic representation of the secondary structure of tRNA, showing the location of D residues and the corresponding Dus enzyme responsible for their synthesis in E. coli, T. thermophilus and M. capricolum for eubacteria and S. cerevisiae for eukaryotes. In the lower panel is shown the sequence of B. subtilis tRNAs used to analyze the D-sites in the MALDI-MS experiments.

Figure 2.

Quantification of D-level in tRNA from B. subtilis. (A) Extracted ion chromatograms of dihydrouridine in tRNAs isolated from B. subtilis WT strain (W168 in light green), ΔdusB1::kan, ΔdusB2::erm double deletion strain (orange) and ΔdusB1::kan (blue) and ΔdusB2::erm single mutant strains (cyan). The signals were normalized to the respective UV signal of Adenosine. (B) D levels determined in bulk tRNAs of B. subtilis WT strains (W168 in light green), ΔdusB1 (blue) and ΔdusB2 (cyan) single mutants, ΔdusB1ΔdusB2 (orange) or double deletion complemented with either DusB1 of M. capricolum (magenta) or DusB2 from S. aureus (green). The strains were grown in LB media at 37°C. Results are shown as average of three biological replicates in relation to the wild type strain W168.

Figure 3.

MALDI-TOF analysis of position 17, 20, 20a and 47 in tRNAs from B. subtilis WT and Dus deletion mutants. (A) D17-containing MS relative isotope patterns of derived oligonucleotides after RNAse T1 treatment of isolated from wild type, ΔdusB1ΔdusB2, ΔdusB1 and ΔdusB2, respectively. (B) D20-containing MS relative isotope patterns of derived oligonucleotides after RNAse A treatment of isolated from wild type, ΔdusB1, ΔdusB2 and ΔdusB1ΔdusB2, respectively. (C) D20a-containing MS relative isotope patterns of derived oligonucleotides after RNAse T1 treatment of isolated from wild type, ΔdusB1, ΔdusB2 and ΔdusB1ΔdusB2, respectively. (D) D47-containing MS relative isotope patterns of derived oligonucleotides after RNAse T1 treatment of isolated from wild type, ΔdusB1, ΔdusB2 and ΔdusB1ΔdusB2, respectively. Further details of the tRNA-derived oligonucleotide fragments and their sizes (m/z) used for the identification of DusB specificities are shown in supplementary figures.

Figure 4.

Heatmaps for the assessment of dihydrouridylation changes in individual modified sites in tRNAs from B. subtilis and its DusB mutants. The heatmap displays one specific D-modification's stoichiometry across the different samples (in X-axis) and the different D-sites retained for analysis (in Y-axis). The stoichiometry is blue-coded and relies on through stop ratio of the AlkAnilineSeq detection method, which detects m⁷G, m³C and D. R1, R2 and R3 represent the results for the three different replicas.

Figure 5.

Structural and functional characterization of B. subtilis DusB. (A) UV-visible absorption spectra of BsDusB1 (blue) and BsDusB2 (teal) holoproteins. (B) Comparative structural models of the active sites of BsDusB1, BsDusB2 and DusB of E. coli (EcDusB). The active site view is centered on the overlay of a section encompassing the FMN isoalloxazine (yellow) of the respective active site of the three DusB (BsDusB1 in pink, BsDusB2 in blue and EcDusB in deep olive). Residues around the FMN are shown in stick in the respective color codes of the Dus. (C) In vitro dihydrouridylation activity test of recombinant BsDusB at 1μM of enzyme after 1 hour incubation at 37°C. Dihydrouridine levels were determined by LC-MS/MS and normalized to the UV signal of adenosine. To compare the activity of BsDusB, the activity of BsDusB1 was set to 100%. Results are shown as average of biological duplicates. (D) Structural models of the DusB holoenzymes from B. subtilis, E. coli and M. capricolum. Except for EcDusB, which is a crystallographic structure (PDB, 6EI9), the other three models are from Alphafold. TBD = TIM Barrel Domain, HD = Helical domain. The FMN is shown in yellow stick. (E) Electrostatic surface of the Dus model. The dashed line represents the line of demarcation (LOD) mentioned in the text. (F) Isotherm of tRNA binding to BsDusB. ΔF529nm is the change in FMN fluorescence at 529 nm resulting from tRNA titration to BsDusB1 (blue) and BsDusB2 (teal).

Figure 6.

In vitro biosynthesis of D in B. subtilis tRNAs catalyzed by the recombinant BsDusB1 and BsDusB2 proteins. (A) Recombinant enzymes expressed in E. coli and purified were incubated with D-unmodified B. subtilis total RNA fraction extracted from ΔdusB1::kan,ΔdusB2::erm strain. Quantification of D17 level was done using NormCount score of AlkAnilineSeq (the signal normalized to median of background cleavages in the surrounding 10 nucleotides). NormCount score (as well as other AlkAnilineSeq Scores) does not show linear dependence from D content, but provides good compromise between sensitivity and specificity of detection for low D levels in tRNA. Only 8 best modified tRNA sites are shown (out of 18 altogether). Concentration of the recombinant BsDusB1 and BsDusB2 is expressed in μM. Identity of tRNA substrates analyzed is shown at the right. (B) Modification efficiency of the D-sites measured at 25 μM of enzymes. Quantification of D level was done using NormCount score of AlkAnilineSeq.

Figure 7.

Phylogenetic analysis of DusB1, DusB2, DusC and DusA proteins in 120 reference and complete Bacteria. DusA proteins are in blue. DusC proteins are in green. DusB/DusB1 proteins are in black. DusB2 proteins are in red. The BV-BRC annotations seem to correctly group the proteins with one exception, the Caur_0210 protein annotated as DusB but clustering with the DusB2 proteins. This section of the tree has however very low bootstrap values as the thickness of the tree branches are reflective of the bootstrap percentage values. E. coli proteins are highlighted in yellow and B. subtilis proteins in purple.