Biogenesis and growth phase-dependent alteration of 5-methoxycarbonylmethoxyuridine in tRNA anticodons

Abstract

Post-transcriptional modifications at the anticodon first (wobble) position of tRNA play critical roles in precise decoding of genetic codes. 5-carboxymethoxyuridine (cmo(5)U) and its methyl ester derivative 5-methoxycarbonylmethoxyuridine (mcmo(5)U) are modified nucleosides found at the anticodon wobble position in several tRNAs from Gram-negative bacteria. cmo(5)U and mcmo(5)U facilitate non-Watson-Crick base pairing with guanosine and pyrimidines at the third positions of codons, thereby expanding decoding capabilities. By mass spectrometric analyses of individual tRNAs and a shotgun approach of total RNA from Escherichia coli, we identified mcmo(5)U as a major modification in tRNA(Ala1), tRNA(Ser1), tRNA(Pro3) and tRNA(Thr4); by contrast, cmo(5)U was present primarily in tRNA(Leu3) and tRNA(Val1). In addition, we discovered 5-methoxycarbonylmethoxy-2'-O-methyluridine (mcmo(5)Um) as a novel but minor modification in tRNA(Ser1). Terminal methylation frequency of mcmo(5)U in tRNA(Pro3) was low (≈30%) in the early log phase of cell growth, gradually increased as growth proceeded and reached nearly 100% in late log and stationary phases. We identified CmoM (previously known as SmtA), an AdoMet-dependent methyltransferase that methylates cmo(5)U to form mcmo(5)U. A luciferase reporter assay based on a +1 frameshift construct revealed that terminal methylation of mcmo(5)U contributes to the decoding ability of tRNA(Ala1).

Figure 1.

5-carboxymethoxyuridine (cmo⁵U) and E. coli tRNAs. (A) Chemical structures of 5-carboxymethoxyuridine (cmo⁵U, left), 5-methoxycarbonylmethoxyuridine (mcmo⁵U, center) and 5-methoxycarbonylmethoxy-2′-O-methyluridine (mcmo⁵Um, right). (B) Secondary structures of E. coli tRNA^Ala1 (left) and tRNA^Ser1 (right) with post-transcriptional modifications: 4-thiouridine (s⁴U), 2′-O-methylguanosine (Gm), dihydrouridine (D), 2′-O-methylcytidine (Cm), 2′-O-methyluridine (Um), 5-methoxycarbonylmethoxyuridine (mcmo⁵U), 5-methoxycarbonylmethoxy-2′-O-methyluridine (mcmo⁵Um), 2-methylthio-N⁶-isopentenyladenosine (ms²i⁶A), 7-methylguanosine (m⁷G), 5-methyluridine (m⁵U) and pseudouridine (Ψ). The position numbers of the residues (gray letters) are displayed according to the nucleotide numbering system (64). Pairs of gray triangles indicate the positions of cleavage by RNase T₁ that generate RNA fragments containing the wobble positions. The sequence of E. coli tRNA^Ala1 is the same as that of tRNA^Ala1B (65).

Figure 2.

Mass spectrometric analysis of individual tRNAs isolated from stationary-phase E. coli. (A) Mass chromatograms of RNase T₁-digested fragments containing cmo⁵U and its derivatives from tRNA^Ala1 (left panels) and tRNA^Ser1 (right panels) isolated from stationary-phase E. coli. Top and middle panels: extracted-ion chromatograms (XIC) for doubly-charged negative ions of cmo⁵U34-containing fragments (CUUcmo⁵UGp of tRNA^Ala1, MW 1660.18, m/z 829.08; UCmUcmo⁵UGp of tRNA^Ser1, MW 1674.19, m/z 836.09) and mcmo⁵U34-containing fragments (CUUmcmo⁵UGp of tRNA^Ala1, MW 1674.19, m/z 836.09; UCmUmcmo⁵UGp of tRNA^Ser1, MW 1688.21, m/z 843.10), respectively. Bottom left and bottom right panels: XICs for doubly-charged negative ions of Um32/mcmo⁵U34-containing fragment of tRNA^Ala1 (CUmUmcmo⁵UGp, MW 1688.21, m/z 843.10) and mcmo⁵Um34-containing fragment of tRNA^Ser1 (UCmUmcmo⁵UmGp, MW 1702.22, m/z 850.10), respectively. The peaks marked with asterisks represent Um32/cmo⁵U-containing fragment (CUmUcmo⁵UGp, MW 1674.19, m/z 836.09) in tRNA^Ala1 and cmo⁵Um-containing fragment (UCmUcmo⁵UmGp, MW 1688.21, m/z 843.10) in tRNA^Ser1. The RNA fragments containing unmodified C32 are also present in tRNA^Ser1, but they are not described here due to high frequency of Cm32 in tRNA^Ser1 isolated from stationary-phase E. coli. (B) Modification frequencies of cmo⁵U and its derivatives at position 34 in six species of tRNAs isolated from stationary-phase E. coli. Relative composition of each modification was calculated from the peak area ratio of mass chromatograms of RNase T₁-digested fragments containing mcmo⁵Um34 (red), mcmo⁵U34 (green), cmo⁵U34 (blue) or U (gray). (C) Collision-induced dissociation (CID) spectrum of a fragment of E. coli tRNA^Ser1 containing mcmo⁵Um34. The doubly-charged negative ion of the RNase T₁-digested fragment containing mcmo⁵Um34 (m/z 850.10) was used as the precursor ion for CID. The product ions were assigned as described previously (66). Sequences of parent ion and assigned product ions are described on the upper left side of this panel. (D) Nucleoside analysis of E. coli tRNA^Ser1. Top panel: UV chromatogram at 254 nm of the four major nucleosides (C, U, G and A). Second panel: mass chromatograms of the protonated nucleosides (MH⁺) on the base peak chromatogram (BPC). Third to bottom panels: XICs for cmo⁵U (m/z 319.08), mcmo⁵U (m/z 333.09) and mcmo⁵Um (m/z 347.11), respectively. The peak marked with an asterisk represents unspecific peak. (E) CID spectrum of mcmo⁵Um nucleoside. The protonated mcmo⁵Um (MH⁺, m/z 347.11) was used as the precursor ion for CID. The N-glycoside bond cleaved to generate the base-related ion (BH₂⁺) and other product ions are assigned on the chemical structure.

Figure 3.

Shotgun analysis of tRNA fragments and growth phase-dependent alteration of mcmo⁵U. (A) Shotgun analysis of total tRNA digested by RNase T₁. Top panel: base peak chromatogram (BPC) of RNase T₁-digested total tRNA isolated from early log phase (2 h) E. coli. Second panel: XICs for doubly-charged negative ions of the cmo⁵U34-containing fragment (UmUcmo⁵UGp, m/z 683.57) and the mcmo⁵U34-containing fragment (UmUmcmo⁵UGp, m/z 690.57) from tRNA^Pro3. Bottom panel: XICs for doubly-charged negative ions of the cmo⁵U34-containing fragment (ACmUcmo⁵UGp, m/z 847.60) and the mcmo⁵U34-containing fragment (ACmUmcmo⁵UGp, m/z 854.61) from tRNA^Thr4. (B) mcmo⁵U level is altered by growth phase. tRNA^Pro3 (filled circle and rectangle) or tRNA^Thr4 (open circle and rectangle) were calculated from the peak area ratio of XICs for the fragments containing mcmo⁵U34 and cmo⁵U34 at different cultivation times. Cell growth (green line) was monitored by absorbance at 610 nm.

Figure 4.

Reverse genetic approach identified a gene responsible for terminal methylation of mcmo⁵U. (A) Nucleoside analyzes by LC/MS using reverse phase chromatography of total RNA from wild-type (left panels), ΔsmtA (middle panels) and ΔsmtA rescued with psmtA (right panels). Top panels: UV trace at 254 nm. Second and bottom panels: XICs for cmo⁵U (m/z 319) and mcmo⁵U (m/z 333), respectively. Intensity of each peak was normalized to that of cyclic t⁶A (m/z 395). (B) Mass chromatograms of RNase T₁-digested fragments containing cmo⁵U and its derivatives from tRNA^Ala1 isolated from wild-type (left panels) and ΔsmtA (right panels) strains. Top, middle and bottom panels: XICs for doubly-charged negative ions of the cmo⁵U34-containing fragments (CUUcmo⁵UGp, m/z 829.08), the mcmo⁵U34-containing fragments (CUUmcmo⁵UGp, m/z 836.09) and the Um32/mcmo⁵U34-containing fragment (CUmUmcmo⁵UGp, m/z 843.10), respectively. The peak marked with an asterisk represent the Um32/cmo⁵U-containing fragment (CUmUcmo⁵UGp, m/z 836.09). (C) Mass chromatograms of RNase T₁-digested fragments containing cmo⁵U and its derivatives from tRNA^Ser1 isolated from wild-type (left panels), ΔsmtA (middle panels) and ΔtrmL (right panels) strains. Top, middle and bottom panels: XICs for doubly-charged negative ions of the cmo⁵U34-containing fragments (UCmUcmo⁵UGp, m/z 836.09), the mcmo⁵U34-containing fragments (UCmUmcmo⁵UGp, m/z 843.10) and the mcmo⁵Um34-containing fragment (UCmUmcmo⁵UmGp, m/z 850.10), respectively.

Figure 5.

In vitro reconstitution of cmo⁵U methylation by recombinant CmoM. (A) E. coli tRNA^Ser1 bearing cmo⁵U isolated from the ΔcmoM strain was incubated in the presence or absence of recombinant CmoM with or without AdoMet. Top and bottom panels: XICs for doubly-charged negative ions of the cmo⁵U34-containing fragments (UCmUcmo⁵UGp, m/z 836.09) and the mcmo⁵U34-containing fragments (UCmUmcmo⁵UGp, m/z 843.10), respectively. (B) A CID spectrum of RNase T₁-digested fragment of E. coli tRNA^Ser1 incubated in the presence of recombinant CmoM with AdoMet. The doubly-charged negative ion of the mcmo⁵U34-containing fragment (UCmUmcmo⁵UGp, m/z 843.10) was used as the precursor ion for CID. The product ions were assigned according to the literature (66). Sequences of parent ion and assigned product ions are described upper left side in this panel.

Figure 6.

Characterization of CmoM. (A) Sequence alignment of CmoM homologs from six γ-proteobacteria, Escherichia coli (NP_415441.1), Salmonella enterica (NP_455477.1), Aeromonas hydrophila (YP_856903.1), Vibrio cholerae (NP_231353.1), Pseudoalteromonas atlantica (ABG40695.1), Pseudomonas aeruginosa (NP_253478.1), and two Actinobacteria, Modestobacter marinus (WP_014741474.1), Streptomyces coelicolor (WP_011028138.1). Identical or similar residues are shaded in black or gray, respectively. Red triangles indicate residues that are essential (filled) or non-essential (open) for generic complementation. Motifs I to VI are conserved in Class I AdoMet-dependent methyltransferases. (B) XICs for doubly-charged negative ions of the cmo⁵U34-containing fragment (black lines, UmUcmo⁵UGp, m/z 683.57) and the mcmo⁵U34-containing fragment (red lines, UmUmcmo⁵UGp, m/z 690.57) from tRNA^Pro3 in the ΔcmoM strain rescued by plasmid-encoded wild-type cmoM or its mutant derivatives. The peak marked with an asterisk represents unspecific peak. (C) Close-up view of the AdoMet-binding site in the crystal structure of E. coli CmoM (PDB ID: 4HTF) containing ligands, AdoMet, acetate and sulfate. Predicted hydrogen bonds between ligands and CmoM are indicated by red dotted lines.

Figure 7.

Terminal methylation of mcmo⁵U contributes to GCG decoding. (A) Schematic depiction of the dual-luciferase reporter constructs based on the RF2 recoding system. SD, Shine-Dalgarno sequence. Renilla and firefly luciferases were fused with a linker containing the +1 frameshift signal of the RF2 recoding site. The frameshift target site was replaced with a GCG codon for tRNA^Ala1, a UCG codon for tRNA^Ser1or GG for zero frame (used as a control). (B) Relative pausing activity at the frameshift site with GCG (left), UCG (middle) or zero frame (right) was calculated based on relative Fluc activity normalized to Rluc activity in wild-type, ΔcmoM and ΔcmoB strains. Data are presented as means ± SD (n = 4). *, P < 0.01 versus control (Student's t-test).

Figure 8.

Biosynthesis of mcmo⁵U. In E. coli, U34 of six tRNAs responsible for decoding NCN codons is modified to ho⁵U34 by an unknown pathway, and subsequently modified to cmo⁵U in a reaction catalyzed by CmoM. CmoB uses SCM-SAH as a substrate and transfers its carboxymethyl group to ho⁵U34 on tRNAs. SCM-SAH is synthesized from prephenate and AdoMet catalyzed by CmoA. Four tRNAs (Ala1, Ser1, Pro3 and Thr4) are further modified to mcmo⁵U by CmoM, using AdoMet as a substrate. mcmo⁵U frequency is altered by growth phase only in tRNA^Pro3. In a minor pathway, mcmo⁵U34 in tRNA^Ser1 is further methylated by TrmL to yield mcmo⁵Um34. Alternatively, cmo⁵U34 could be first converted to cmo⁵Um34, then to mcmo⁵Um34.