A single methyltransferase YefA (RlmCD) catalyses both m5U747 and m5U1939 modifications in Bacillus subtilis 23S rRNA

Abstract

Methyltransferases that use S-adenosylmethionine (AdoMet) as a cofactor to catalyse 5-methyl uridine (m(5)U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, and are also found in certain Archaea. These enzymes belong to the COG2265 cluster, and the Gram-negative bacterium Escherichia coli possesses three paralogues. These comprise the methyltransferases TrmA that targets U54 in tRNAs, RlmC that modifies U747 in 23S rRNA and RlmD that is specific for U1939 in 23S rRNA. The tRNAs and rRNAs of the Gram-positive bacterium Bacillus subtilis have the same three m(5)U modifications. However, as previously shown, the m(5)U54 modification in B. subtilis tRNAs is catalysed in a fundamentally different manner by the folate-dependent enzyme TrmFO, which is unrelated to the E. coli TrmA. Here, we show that methylation of U747 and U1939 in B. subtilis rRNA is catalysed by a single enzyme, YefA that is a COG2265 member. A recombinant version of YefA functions in an E. coli m(5)U-null mutant adding the same two rRNA methylations. The findings suggest that during evolution, COG2265 enzymes have undergone a series of changes in target specificity and that YefA is closer to an archetypical m(5)U methyltransferase. To reflect its dual specificity, YefA is renamed RlmCD.

Figure 1.

The three characterized sites of m⁵U modification in B. subtilis and E. coli RNAs. The 23S rRNA secondary structures are based on the B. subtilis sequence and nucleotide differences in E. coli are indicated by the arrows pointing to the italicized nucleotides. (A) Portion of domain II of 23S rRNA, containing hairpins 34 and 35 and part of helix 33, showing the location of m⁵U747 modified by YefA in B. subtilis rRNA and RlmC in E. coli. The m¹G745 modification is seen in E. coli rRNA but is absent in B. subtilis; the presence of pseudouridine Ψ746 could not be tested by mass spectrometry. (B) Region from domain IV of 23S rRNA containing m⁵U 1939 that is modified by YefA in B. subtilis rRNA, and by RlmD in E. coli. The B. subtilis rRNA is methylated on the ribose of nucleotide C1920 presumably by an orthologue of the enzyme TlyA (42) and this modification is missing in E. coli; the m⁵C1962 modification is present in the E. coli rRNA (47) but was not detected in B. subtilis; the pseudouridines Ψ1911, Ψ1915 and Ψ1917 could not be verified by mass spectrometry. (C) Schematic consensus of tRNA structures indicating the U54 target of the E. coli TrmA (48) and the structurally unrelated enzyme TrmFO that modifies the same nucleotide in B. subtilis using a tetrahydrofolate cofactor as the methyl donor (13,25).

Figure 2.

MALDI-MS analyses of m⁵U methylation sites in 23S rRNA. The upper row of spectra were generated from 23S rRNA of the B. subtilis wild-type strain 168 and show sequences containing m⁵U nucleotides (shaded in grey) at (A) U747 (in an RNase T1 oligo), (B) U1939 (RNase T1 oligo) and (C) U1939 (RNase A oligo). The corresponding spectral regions from the B. subtilis ΔyefA::Cm strain are shown in panels (D–F). The rRNA spectra from the B. subtilis ΔyfjO strain (data not shown) were identical to the wild-type rRNA. The rRNA analysed in spectra (G and H) was from B. subtilis ΔyefA::Cm strain complemented with an active copy of the yefA gene. (I and J) are spectra from the same rRNA regions of the E. coli ΔrlmC/ΔrlmD/ΔtrmA triple mutant expressing an active copy of yefA (note the E. coli m¹G745 is resistant to RNase T1 digestion, producing a longer fragment). Before complementation with yefA, the rRNA from the E. coli ΔrlmC/ΔrlmD/ΔtrmA mutant produced none of the peaks containing m⁵U (data not shown). The table shows relevant fragments (sequences 5′–3′) generated from digestion of B. subtilis (Bs) and/or E. coli (Ec) rRNAs with RNase A (leaving a 3′-U or -C) and RNase T1 (3′-G). RNase T1 produced a mixture of fragments with a 2′–3′-cyclic phosphate (>p) and a linear 3′-phosphate (p), the latter having an 18 Da larger mass; the theoretical monoisotopic mass/charge values (m/z) are given for the predominant peaks.

Figure 3.

Amino acid sequence alignment of m⁵U methyltransferases. The E. coli RlmC and RlmD enzymes, which are respectively specific for 23S rRNA nucleotides U747 and U1939, are aligned with the B. subtilis YefA sequence. Identical amino acid residues are highlighted in black, and grey indicates highly similar residues. Full-length sequences are shown; RlmC lacks the N-terminal extension seen in RlmD and YefA. The common core sequences were used for calculating percentages of amino acid identity and similarity.