Discovery and biological characterization of geranylated RNA in bacteria

A general mass spectrometry-based screen for unusually hydrophobic cellular small-molecule RNA conjugates revealed geranylated RNA in E. coli, Enterobacter aerogenes, Pseudomonas aeruginosa, and Salmonella thyphimurium. 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 tRNAGluUUC, tRNALysUUU, tRNAGlnUUG at a frequency of up to 6.7% (~400 geranylated nucleotides per cell). RNA geranylation levels 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 tRNAGluUUC, tRNALysUUU, and tRNAGlnUUG affects codon bias and frameshifting during translation. These RNAs represent the first reported examples of oligoisoprenylated cellular nucleic acids.

For the comparison of chemically synthesized and biologically generated ges2U samples, we used a linear LC gradient from 0.1% aqueous formic acid (A2) to acetonitrile (B2) on an Aquity UPLC BEH C18 column (1.7 µm, 2.1 mm x 100 mm, Waters) at a constant flow rate of 0.3 mL/min. The mobile phase composition was as follows: 100% A2 for 1 min; linear increase over 21 min to 100% B2; maintain at 100% B2 for 8 min; return to 100 % A2 over 5 min. Electrospray ionization was used with a capillary voltage of 3 kV, a sampling cone voltage of 20 V, collision energies of 2, 10, 15, 20, and 25 eV, a LM resolution of 13, and the detector was operating in positive ion mode.
For analysis of oligonucleotides resulting from digestion of RNA with RNase T1, samples were dissolved in 50 µL of deinionized water and run using a linear gradient from 6 mM triethylammonium bicarbonate (A3) to methanol (B3) on an Aquity UPLC BEH C18 column (1.7 µm, 2.1 mm x 100 mm, Waters) at a constant flow rate of 0.3 mL/min. The mobile phase composition was a linear gradient from 100% A3 to 100% B3 over 60 min. Electrospray ionization was used with a capillary voltage of 3 kV, a sampling cone voltage of 40 V, collision energies of 2, 10, 20, and 30 eV and the detector was operating in negative ion mode.
Data analysis of small molecule-RNA conjugate screening results. Replicates of three nuclease P1-digested RNA samples and three control samples were used to identify candidate hits. Ions with average intensities below 50 counts were not considered for further analysis.
The XCMS program 1 was used to quantify ion abundance as it stepped through each ion chromatogram; the step size was set to 0.050 Da. Integrated ion abundances were averaged among replicates, and the enrichment values reported represent the ratios of average ion abundances in the nuclease-treated samples to average ion abundances in the heat-inactivated nuclease (control) samples.
The product was crystallized in a mixture of methanol and ethyl acetate. A single crystal was mounted on a diffractometer and data was collected at 100 K. The intensities of the reflections were collected using a Bruker APEX II DUO CCD diffractometer (Cu Kα radiation, λ=1.54178 Å) equipped with an Oxford Cryosystems nitrogen flow apparatus. The collection method involved 1.0° scans in ω at 30°, 55°, 80° and 105° in 2θ. Data integration to 0.84 Å resolution was carried out using SAINT V7.46 A (Bruker diffractometer, 2009) with reflection spot size optimization. Absorption corrections were made with the SADABS software package (Bruker diffractometer, 2009). The structure was solved by the direct methods procedure and refined by least-squares methods again F 2 using SHELXS-97 and SHELXL-97 (Sheldrick, 2008). Nonhydrogen atoms were refined anisotropically, and hydrogen atoms were allowed to ride on the respective atoms. Crystal data as well as details of data collection and refinement are summarized in Supplementary Quantitation of ges2U levels per cell. To establish a standard curve relating mass spectrometry ion counts with ges2U levels, 0.78, 1.56, 3.13, 6.25, 12.5, and 25 pmols of synthetic ges2U were analyzed by LC/MS. The ges2U signal from 50 µg of total E. coli ΔmnmE RNA was compared to the standard curves to determine the number of pmols of cellular ges2U per µg RNA. Based on a yield of 2.5 to 3 mg of total RNA per L and 4.6x10 11 E. coli cells per litre of culture at OD 600 = 0.6 (determined by plating serial dilutions of such a culture), the number of ges2U molecules per E. coli cell was estimated to be 398±81. The percentage of geranylated nucleotides was calculated by dividing this number by the sum of the number of tRNA Glu UUC , tRNA Lys UUU , and tRNA Gln UUG per E. coli per cell previously determined using two different methods (13993 at 2.5 doublings per hour 2 , 2.8%; 5910 3 , 6.7%).
Size fractionation of total RNA. Size fractionation was performed using high-pressure liquid chromatography (Agilent 1100) with an anion-exchange Dionex DNAPac PA200 4x250mm analytical column. Mobile phase A was 300 mM sodium perchlorate in water and mobile phase B was 20% acetonitrile in water. The flow rate was a constant 1.2 mL/min, temperature was 80 °C and the mobile phase composition was as follows: linear increase from 30% A to 70% A over over 2 min; return to 30% A over 3 min. Detection was carried out at 260 nm and 230 nm. 50 µg of total RNA from E. coli were injected for each size fractionation run. Fractions were collected every 5 min and the corresponding fractions from four to ten runs were combined.
The first two fractions (0-5 min and 5-10 min elution) were concentrated using Millipore's Centriprep Centrifugal Filter Unit with Ultracel-3 membrane (YM-3) and the rest of the fractions were concentrated using the Centriprep Centrifugal Filter Unit with Ultracel-10 membrane (YM-10) by centrifugation (3,000 x g) until the total volume was less than 1.5 mL. The retentate was lyophilized and precipitated with ethanol prior to nuclease P1 digestion. Digestion by nuclease P1, purification, and mass spectrometric analysis was performed as described above.
Pull-down of individual tRNAs. Individual tRNAs were isolated using a slightly modified version of a previously described protocol. 4  Biosystems Expedite 8909 DNA synthesizer using commercially available standard base monomers and 3'-BiotinTEG-CPG from Glen Research. 7.5 mg of E. coli tRNA (Roche) were dissolved in 1 mL of 10 mM Tris-HCl, 0.9 M tetramethylammonium chloride, 0.1 mM EDTA, pH 7.8. To this solution sepharose beads bound to the pull-down oligonucleotides were added and incubated for 10 minutes at 65 ºC. The beads were cooled to 25 ºC over 10 min and transferred to Ultrafree-MC spin columns PVDF 5 µM (Millipore) to remove unbound RNA. The resin was washed six times with 10 mM Tris-HCl, pH 7.8. Bound tRNAs were eluted twice by successive additions of 200 µL deionized water and incubation for 10 minutes at 65 ºC. The eluted RNA was digested either with nuclease P1 or RNase T1 as described above.
Expression and partial purification of SelU. E.coli selU containing a C-terminal hexahistidine tag was cloned into a pET vector. The protein was expressed in BL21(DE3) cells in 1 L of LB / 6 After 3 hours at 24 °C cells were harvested by centrifugation. Cell pellets were resuspended in 30 mL of buffer A (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM 2-mercaptoethanol, 5% glycerol) containing 20 units TURBO DNase and EDTA-free Protease Inhibitor Cocktail (Roche). Cells were disrupted by sonication. After centrifugation for 20 min at 17,000 G, the clarified extract was loaded onto 1 mL of Ni-NTA resin (Qiagen) pre-equilibrated in buffer A.
After washing with 10 mL of buffer A and 10 mL of buffer A containing 10 mM imidazole, the bound protein was eluted with 6 mL buffer A containing 200 mM imidazole. The eluted protein was dialyzed twice against 1 L of 50 mM Tricine-NaOH, pH 8.0, 1 mM dithiothreitol, 10% glycerol. Purification of the dialyzed protein was then repeated as described above except the protein was elution in a stepwise manner using 2 mL buffer A containing, successively, 20, 40, 60, 80, 100, or 200 mM imidazole. All fractions were analyzed by SDS-PAGE. The elution fractions with 100 mM imidazole contained the most pure protein and were dialyzed twice against 1 L of 50 mM Tricine-NaOH, pH 8.0, 1 mM dithiothreitol, 10% glycerol. After dialysis the protein was either used immediately or stored at -20 °C. Characterization of the protein by UV spectroscopy showed similar absorption values at 260 and 280 nm, suggesting that a significant portion of the protein may be associated with oligonucleotides.
In vitro geranylation with SelU. 140 nM partially purified selU-His 6 was incubated with 500 µg of E. coli tRNA (Roche) in the presence or absence of 135 µM ammonium geranyl pyrophosphate (Sigma) in 100 µL of 10 mM Tricine-NaOH, pH 8.0, 0.2 mM dithiothreitol, 2% glycerol at 25 °C. After 24 h the RNA was digested with Nuclease P1 / alkaline phosphatase and analyzed by LC/MS as described above.
Complementation with selU. The USER cloning method 5 was used to assemble a plasmid containing selU, bla, and repA and the origin of replication of pSC101. The selU genes including 200 nucleotides upstream of the translation initiation ATG as well 140 nucleotides downstream of the stop codon were PCR amplified from E. coli BW25113 and S. typhimurium LT2. The ampicillin resistance gene bla was taken from pUC19, and repA and the origin of replication originate from pSC101. The point mutations G67E and G67R were introduced in a separate USER cloning step.
Codon usage reporter assay. USER cloning was used to assemble a plasmid containing luxAB under control of the tet promotor, aadA, tetR, rop and the origin of replication from ColE1.
The luciferase fusion luxAB was constructed as previously described. 6 In addition, six codons were inserted between the starting ATG and the second codon of luxAB. The resulting first eight codon sequences were as follows: Glu ( Transcript level measurement in the codon usage assay. RNA was isolated from 1 mL of E. coli culture grown to OD = 0.4 as described for the luciferase assay using RNeasy kit (Qiagen).
Reverse transcription and quantitative PCR were carried out using the following primer pairs for the transcript of the bacterial luciferase gene fusion luxAB and the α-subunit of bacterial RNA polymerase rpoA as a reference. 7  for 1 h. The membrane was washed three times with PBS, 0.05% Tween 20 and visualized using an Odyssey infrared imaging system (Li-COR Biosciences). Images were analyzed using Odyssey imaging software version 2.0.