Discovery and biological characterization of geranylated RNA in bacteria

Abstract

A general MS-based screen for unusually hydrophobic cellular small molecule-RNA conjugates revealed geranylated RNA in Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa and Salmonella enterica var. Typhimurium. The geranyl group is conjugated to the sulfur atom in two 5-methylaminomethyl-2-thiouridine nucleotides. These geranylated nucleotides occur in the first anticodon position of tRNA(Glu)(UUC), tRNA(Lys)(UUU) and tRNA(Gln)(UUG) at a frequency of up to 6.7% (~400 geranylated nucleotides per cell). RNA geranylation can be increased or abolished by mutation or deletion of the selU (ybbB) gene in E. coli, and purified SelU protein in the presence of geranyl pyrophosphate and tRNA can produce geranylated tRNA. The presence or absence of the geranyl group in tRNA(Glu)(UUC), tRNA(Lys)(UUU) and tRNA(Gln)(UUG) affects codon bias and frameshifting during translation. These RNAs represent the first reported examples of oligoisoprenylated cellular nucleic acids.

Figure 1

Discovery of two hydrophobic small molecule-RNA conjugates with [M-H]⁻ m/z = 824.200 and 868.189. (a) Scheme of the general method for discovering biological small molecule-RNA conjugates applied in this work. Total cellular RNA samples are treated with active nuclease P1 or with heat-inactivated nuclease P1 under otherwise identical conditions, then subjected to comparative LC/MS analysis. Nucleotide ions more abundant in the active nuclease sample than in the heat-inactivated nuclease (control) sample represent candidate cellular small molecule-RNA conjugates. (b) Extracted ion chromatogram of AMP-tryptophan and the two unknown nucleotides elucidated in this work.

Figure 2

Mass spectrometric characterization of two novel hydrophobic small molecule-RNA conjugates. (a) Negative ion mode MS/MS of the two unknown nucleotides reveals a dinucleotide structure with uracil and two unknown nucleobases of 307.171 and 351.161 Da. (b and c) Positive mode MS/MS of the unknown nucleobases of 307.171 Da (b) and 351.161 Da (c) from unlabeled as well as ¹³C- and ¹⁵N-labeled RNA. The spectra of the unlabeled RNA are shown in red, the ¹³C-labeled samples are shown in black, and the ¹⁵N-labeled samples are shown in green. The +10 Da shifts, indicating 10 carbon atoms, of the geranyl fragment in the ¹³C-labeled RNA spectra are shown. (d) Proposed structures for the individual ion fragments observed in the MS/MS experiments.

Figure 3

Structural elucidation of two geranylated nucleosides. (a) Structures of geranylated 2-thiouridine (ges2U), geranylated 5-methylaminomethyl-2-thiouridine (mnm5ges2U), and geranylated 5-carboxymethylaminomethyl-2-thiouridine (cmnm5ges2U). (b) LC comparison of biologically generated and authentic synthetic ges2U. (c) Comparison of biologically generated and authentic ges2U by MS/MS fragmentation. Proposed structures of ion fragments are shown.

Figure 4

Characterization of geranylated cellular RNAs. (a) MS analysis of individual tRNAs isolated from cells reveals mnm5ges2U to be present on tRNA^Glu_UUC, tRNA^Lys_UUU, and (to a lesser extent) tRNA^Gln_UUG, and cmnm5ges2U to be present on tRNA^Gln_UUG. The same analysis is shown from tRNA^Asn_GUU as a negative control. (b) MS/MS fragmentation of the T1 digestion product of tRNA^Glu_UUC reveals that the geranylated nucleotide is U34.

Figure 5

Biological abundance and properties of geranylated RNA. (a) E. coli selU mutations affect geranylated nucleotide levels. No geranylated nucleotides were detected in E. coli cells lacking selU (ΔselU). Complementation with wild-type selU restores geranylation. Complementation with mutant selU(G67E) results in high levels of geranylation. Nucleoside levels are shown as relative ratios of MS counts of the individual nucleosides versus t6A. Error bars represent the standard deviation of three independent biological replicates. (b) SelU geranylates mnm5s2U and cmnm5s2U on tRNA in vitro in a geranyl pyrophosphate-dependant manner. Nucleoside levels are shown as relative ratios of MS counts of the individual nucleosides versus t6A. Error bars represent the standard deviation of three analytical replicates. (c) Luciferase reporter assay of glutamate codon translation efficiency for GAA and GAG reveals a strong bias favoring GAA translation under low geranylation conditions and a significantly lower preference under high geranylation conditions due to reduced efficiency of GAA decoding. Error bars represent the standard deviation of three independent biological replicates. (d) +1 and −1 frameshift efficiency at the sequences GCC AAGC and A AAA AAG inserted between GST and MBP reading frames. The Y-axis indicates the amount of GST-MBP fusion protein detected by western blot as a percentage of total GST-containing proteins produced. Error bars reflect the range of two technical replicates.