Discovery and biological characterization of geranylated RNA in bacteria


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 tRNAGluUUC, tRNALysUUU and tRNAGlnUUG 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 tRNAGluUUC, tRNALysUUU and tRNAGlnUUG affects codon bias and frameshifting during translation. These RNAs represent the first reported examples of oligoisoprenylated cellular nucleic acids.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Discovery of two hydrophobic small molecule–RNA conjugates with [M-H] m/z = 824.200 and 868.189.
Figure 2: MS characterization of two new hydrophobic small molecule–RNA conjugates.
Figure 3: Structural elucidation of two geranylated nucleosides.
Figure 4: Characterization of geranylated cellular RNAs.
Figure 5: Biological abundance and properties of geranylated RNA.


  1. 1

    He, L. & Hannon, G.J. MicroRNAs: small RNAs with a big role in gene regulation. Nat. Rev. Genet. 5, 522–531 (2004).

  2. 2

    Serganov, A. & Patel, D.J. Ribozymes, riboswitches and beyond: regulation of gene expression without proteins. Nat. Rev. Genet. 8, 776–790 (2007).

  3. 3

    Fedor, M.J. & Williamson, J.R. The catalytic diversity of RNAs. Nat. Rev. Mol. Cell Biol. 6, 399–412 (2005).

  4. 4

    Ding, S.W. RNA-based antiviral immunity. Nat. Rev. Immunol. 10, 632–644 (2010).

  5. 5

    Cantara, W.A. et al. The RNA Modification Database, RNAMDB: 2011 update. Nucleic Acids Res. 39, D195–D201 (2011).

  6. 6

    Ikeuchi, Y. et al. Agmatine-conjugated cytidine in a tRNA anticodon is essential for AUA decoding in archaea. Nat. Chem. Biol. 6, 277–282 (2010).

  7. 7

    Mandal, D. et al. Agmatidine, a modified cytidine in the anticodon of archaeal tRNA(Ile), base pairs with adenosine but not with guanosine. Proc. Natl. Acad. Sci. USA 107, 2872–2877 (2010).

  8. 8

    Ikeuchi, Y., Shigi, N., Kato, J., Nishimura, A. & Suzuki, T. Mechanistic insights into sulfur relay by multiple sulfur mediators involved in thiouridine biosynthesis at tRNA wobble positions. Mol. Cell 21, 97–108 (2006).

  9. 9

    Miles, Z.D., McCarty, R.M., Molnar, G. & Bandarian, V. Discovery of epoxyqueuosine (oQ) reductase reveals parallels between halorespiration and tRNA modification. Proc. Natl. Acad. Sci. USA 108, 7368–7372 (2011).

  10. 10

    Noma, A., Kirino, Y., Ikeuchi, Y. & Suzuki, T. Biosynthesis of wybutosine, a hyper-modified nucleoside in eukaryotic phenylalanine tRNA. EMBO J. 25, 2142–2154 (2006).

  11. 11

    Kowtoniuk, W.E., Shen, Y., Heemstra, J.M., Agarwal, I. & Liu, D.R. A Chemical screen for biological small molecule-RNA conjugates reveals CoA-linked RNA. Proc. Natl. Acad. Sci. USA 106, 7768–7773 (2009).

  12. 12

    Chen, Y.G., Kowtoniuk, W.E., Agarwal, I., Shen, Y. & Liu, D.R. LC/MS analysis of cellular RNA reveals NAD-linked RNA. Nat. Chem. Biol. 5, 879–881 (2009).

  13. 13

    Scott, A.I. How were porphyrins and lipids synthesized in the RNA world? Tetrahedr. Lett. 38, 4961–4964 (1997).

  14. 14

    Hagervall, T.G., Edmonds, C.G., McCloskey, J.A. & Bjork, G.R. Transfer RNA(5-methylaminomethyl-2-thiouridine)-methyltransferase from Escherichia coli K-12 has two enzymatic activities. J. Biol. Chem. 262, 8488–8495 (1987).

  15. 15

    Kambampati, R. & Lauhon, C.T. MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli. Biochemistry 42, 1109–1117 (2003).

  16. 16

    Nicolas, E.C. & Scholz, T.H. Active drug substance impurity profiling part II. LC/MS/MS fingerprinting. J. Pharm. Biomed. Anal. 16, 825–836 (1998).

  17. 17

    Chen, P., Crain, P.F., Nasvall, S.J., Pomerantz, S.C. & Bjork, G.R.A. “Gain of function” mutation in a protein mediates production of novel modified nucleosides. EMBO J. 24, 1842–1851 (2005).

  18. 18

    Jühling, F. et al. tRNAdb 2009: compilation of tRNA sequences and tRNA genes. Nucleic Acids Res. 37, D159–D162 (2009).

  19. 19

    Yaniv, M. & Folk, W.R. The nucleotide sequences of the two glutamine transfer ribonucleic acids from Escherichia coli. J. Biol. Chem. 250, 3243–3253 (1975).

  20. 20

    Yokogawa, T., Kitamura, Y., Nakamura, D., Ohno, S. & Nishikawa, K. Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts. Nucleic Acids Res. 38, e89 (2010).

  21. 21

    Volkin, E. & Cohn, W.E. On the structure of ribonucleic acids. 2. The products of ribonuclease action. J. Biol. Chem. 205, 767–782 (1953).

  22. 22

    Mcluckey, S.A., Vanberkel, G.J. & Glish, G.L. Tandem mass spectrometry of small, multiply charged oligonucleotides. J. Am. Soc. Mass Spectrom. 3, 60–70 (1992).

  23. 23

    Dong, H., Nilsson, L. & Kurland, C.G. Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates. J. Mol. Biol. 260, 649–663 (1996).

  24. 24

    Jakubowski, H. & Goldman, E. Quantities of individual aminoacyl-tRNA families and their turnover in Escherichia coli. J. Bacteriol. 158, 769–776 (1984).

  25. 25

    Wolfe, M.D. et al. Functional diversity of the rhodanese homology domain: the Escherichia coli ybbB gene encodes a selenophosphate-dependent tRNA 2-selenouridine synthase. J. Biol. Chem. 279, 1801–1809 (2004).

  26. 26

    Looman, A.C. et al. Influence of the codon following the AUG initiation codon on the expression of a modified lacZ gene in Escherichia coli. EMBO J. 6, 2489–2492 (1987).

  27. 27

    Sørensen, M.A. & Pedersen, S. Absolute in vivo translation rates of individual codons in Escherichia coli. The two glutamic acid codons GAA and GAG are translated with a threefold difference in rate. J. Mol. Biol. 222, 265–280 (1991).

  28. 28

    Wittwer, A.J. & Ching, W.M. Selenium-containing tRNA(Glu) and tRNA(Lys) from Escherichia coli: purification, codon specificity and translational activity. Biofactors 2, 27–34 (1989).

  29. 29

    Gurvich, O.L. et al. Sequences that direct significant levels of frameshifting are frequent in coding regions of Escherichia coli. EMBO J. 22, 5941–5950 (2003).

  30. 30

    Lindsley, D. & Gallant, J. On the directional specificity of ribosome frameshifting at a “hungry” codon. Proc. Natl. Acad. Sci. USA 90, 5469–5473 (1993).

  31. 31

    Suzuki, T. Biosynthesis and function of tRNA wobble modifications. in Fine-Tuning of RNA Functions by Modification and Editing, Vol. 12 (ed. Grosjean, H.) 23–69 (Springer, Berlin/Heidelberg, 2005).

  32. 32

    Agris, P.F., Vendeix, F.A. & Graham, W.D. tRNA's wobble decoding of the genome: 40 years of modification. J. Mol. Biol. 366, 1–13 (2007).

  33. 33

    Krüger, M.K., Pedersen, S., Hagervall, T.G. & Sorensen, M.A. The modification of the wobble base of tRNAGlu modulates the translation rate of glutamic acid codons in vivo. J. Mol. Biol. 284, 621–631 (1998).

  34. 34

    Yarian, C. et al. Accurate translation of the genetic code depends on tRNA modified nucleosides. J. Biol. Chem. 277, 16391–16395 (2002).

  35. 35

    Sylvers, L.A., Rogers, K.C., Shimizu, M., Ohtsuka, E. & Soll, D. A 2-thiouridine derivative in tRNAGlu is a positive determinant for aminoacylation by Escherichia coli glutamyl-tRNA synthetase. Biochemistry 32, 3836–3841 (1993).

  36. 36

    Ashraf, S.S. et al. Single atom modification (O→S) of tRNA confers ribosome binding. RNA 5, 188–194 (1999).

  37. 37

    Yarian, C. et al. Modified nucleoside dependent Watson-Crick and wobble codon binding by tRNALysUUU species. Biochemistry 39, 13390–13395 (2000).

  38. 38

    Lane, B.G. Historical Perspectives on RNA Nucleoside Modifications. in Modification and Editing of RNA (eds. Grosjean, H. & Benne, R.) 1–20 (ASM Press, 1998).

  39. 39

    Tsuchihashi, Z. & Brown, P.O. Sequence requirements for efficient translational frameshifting in the Escherichia coli dnaX gene and the role of an unstable interaction between tRNA(Lys) and an AAG lysine codon. Genes Dev. 6, 511–519 (1992).

  40. 40

    Licznar, P. et al. Programmed translational −1 frameshifting on hexanucleotide motifs and the wobble properties of tRNAs. EMBO J. 22, 4770–4778 (2003).

  41. 41

    Begley, U. et al. Trm9-catalyzed tRNA modifications link translation to the DNA damage response. Mol. Cell 28, 860–870 (2007).

  42. 42

    Chan, C.T. et al. A quantitative systems approach reveals dynamic control of tRNA modifications during cellular stress. PLoS Genet. 6, e1001247 (2010).

  43. 43

    Keiler, K.C. RNA localization in bacteria. Curr. Opin. Microbiol. 14, 155–159 (2011).

  44. 44

    Nevo-Dinur, K., Nussbaum-Shochat, A., Ben-Yehuda, S. & Amster-Choder, O. Translation-independent localization of mRNA in E. coli. Science 331, 1081–1084 (2011).

  45. 45

    Chan, Y.H., van Lengerich, B. & Boxer, S.G. Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides. Proc. Natl. Acad. Sci. USA 106, 979–984 (2009).

Download references


This work was supported by the Howard Hughes Medical Institute and the US National Institutes of Health (NIH)-National Institute of General Medical Sciences (NIGMS) (R01GM065865). C.E.D. acknowledges fellowship from the Novartis Foundation. A.M.L. was supported by a NIH National Research Service Award Postdoctoral Fellowship (F32GM095028). We thank S.-L. Zheng for his help with X-ray diffraction and structural determination of synthetic geranyl-2-thiouracil. We thank A. Saghatelian (Harvard University) for providing P. aeruginosa and I. Chen (Harvard University) for providing S. Typhimurium and BL2 facilities. We are also grateful to J. Carlson for his assistance and M. Ibba for helpful discussions.

Author information

C.E.D., Y.C., A.M.L. and Y.G.C. designed and performed the experiments. All authors analyzed the results and wrote the manuscript.

Correspondence to David R Liu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Results (PDF 3375 kb)

Supplementary Data Set 1

geranyl-2-thiouracil (CIF 14 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Dumelin, C., Chen, Y., Leconte, A. et al. Discovery and biological characterization of geranylated RNA in bacteria. Nat Chem Biol 8, 913–919 (2012).

Download citation

Further reading