Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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 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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

  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).

    CAS  PubMed  Google Scholar 

  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).

    Article  PubMed  Google Scholar 

  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).

    CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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).

    Article  PubMed  Google Scholar 

  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).

    CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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).

    Chapter  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

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

Authors and Affiliations

Authors

Contributions

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.

Corresponding author

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). https://doi.org/10.1038/nchembio.1070

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1070

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing