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.

Exceptional structured noncoding RNAs revealed by bacterial metagenome analysis

Nature volume 462, pages 656–659 (2009)Cite this article

Abstract

Estimates of the total number of bacterial species1,2,3 indicate that existing DNA sequence databases carry only a tiny fraction of the total amount of DNA sequence space represented by this division of life. Indeed, environmental DNA samples have been shown to encode many previously unknown classes of proteins4 and RNAs5. Bioinformatics searches6,7,8,9,10 of genomic DNA from bacteria commonly identify new noncoding RNAs (ncRNAs)10,11,12 such as riboswitches13,14. In rare instances, RNAs that exhibit more extensive sequence and structural conservation across a wide range of bacteria are encountered15,16. Given that large structured RNAs are known to carry out complex biochemical functions such as protein synthesis and RNA processing reactions, identifying more RNAs of great size and intricate structure is likely to reveal additional biochemical functions that can be achieved by RNA. We applied an updated computational pipeline17 to discover ncRNAs that rival the known large ribozymes in size and structural complexity or that are among the most abundant RNAs in bacteria that encode them. These RNAs would have been difficult or impossible to detect without examining environmental DNA sequences, indicating that numerous RNAs with extraordinary size, structural complexity, or other exceptional characteristics remain to be discovered in unexplored sequence space.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Size and structural complexity of new-found RNAs compared to the ten largest known bacterial ncRNAs with complex structures.
Figure 2: GOLLD RNAs.
Figure 3: HEARO RNAs.

References

  1. Whitman, W. B., Coleman, D. C. & Wiebe, W. J. Prokaryotes: the unseen majority. Proc. Natl Acad. Sci. USA 95, 6578–6583 (1998)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  2. Curtis, T. P., Sloan, W. T. & Scannell, J. W. Estimating prokaryotic diversity and its limits. Proc. Natl Acad. Sci. USA 99, 10494–10499 (2002)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bent, S. J. & Forney, L. J. The tragedy of the uncommon: understanding limitations in the analysis of microbial diversity. ISME J. 2, 689–695 (2008)

    Article  CAS  PubMed  Google Scholar 

  4. Yooseph, S. et al. The Sorcerer II Global Ocean Sampling expedition: expanding the universe of protein families. PLoS Biol. 5, e16 (2007)

    Article  PubMed  PubMed Central  Google Scholar 

  5. Shi, Y., Tyson, G. W. & DeLong, E. F. Metatranscriptomics reveals unique microbial small RNAs in the ocean's water column. Nature 459, 266–269 (2009)

    Article  ADS  CAS  PubMed  Google Scholar 

  6. Gelfand, M. S., Mironov, A. A., Jomantas, J., Kozlov, Y. I. & Perumov, D. A. A conserved RNA structure element involved in the regulation of bacterial riboflavin synthesis genes. Trends Genet. 15, 439–442 (1999)

    Article  CAS  PubMed  Google Scholar 

  7. Rivas, E. & Eddy, S. R. Noncoding RNA gene detection using comparative sequence analysis. BMC Bioinformatics 2, 8 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wassarman, K. M., Repoila, F., Rosenow, C., Storz, G. & Gottesman, S. Identification of novel small RNAs using comparative genomics and microarrays. Genes Dev. 15, 1637–1651 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Barrick, J. E. et al. New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control. Proc. Natl Acad. Sci. USA 101, 6421–6426 (2004)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Yao, Z. et al. A computational pipeline for high-throughput discovery of cis-regulatory noncoding RNA in prokaryotes. PLOS Comput. Biol. 3, e126 (2007)

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  11. Weinberg, Z. et al. Identification of 22 candidate structured RNAs in bacteria using the CMfinder comparative genomics pipeline. Nucleic Acids Res. 35, 4809–4819 (2007)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. Meyer, M. M. et al. Identification of candidate structured RNAs in the marine organism ‘Candidatus Pelagibacter ubique’. BMC Genomics 10, 268 (2009)

    Article  PubMed  PubMed Central  Google Scholar 

  13. Montange, R. K. & Batey, R. T. Riboswitches: emerging themes in RNA structure and function. Annu. Rev. Biophys. 37, 117–133 (2008)

    Article  CAS  PubMed  Google Scholar 

  14. Roth, A. & Breaker, R. R. The structural and functional diversity of metabolite-binding riboswitches. Annu. Rev. Biochem. 78, 305–334 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Barrick, J. E., Sudarsan, N., Weinberg, Z., Ruzzo, W. L. & Breaker, R. R. 6S RNA is a widespread regulator of eubacterial RNA polymerase that resembles an open promoter. RNA 11, 774–784 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Puerta-Fernandez, E., Barrick, J. E., Roth, A. & Breaker, R. R. Identification of a large noncoding RNA in extremophilic eubacteria. Proc. Natl Acad. Sci. USA 103, 19490–19495 (2006)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. Tseng, H. H., Weinberg, Z., Gore, J., Breaker, R. R. & Ruzzo, W. L. Finding non-coding RNAs through genome-scale clustering. J. Bioinform. Comput. Biol. 7, 373–388 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Michel, F. & Westhof, E. Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J. Mol. Biol. 216, 585–610 (1990)

    Article  CAS  PubMed  Google Scholar 

  19. Pace, N. R., Thomas, B. C. & Woese, C. R. in The RNA World 2nd edn (eds Gesteland, R. F., Cech, T. R. & Atkins, J. F.) Ch. 4 113–141 (Cold Spring Harbor Laboratory Press, 1999)

    Google Scholar 

  20. Toor, N., Keating, K. S. & Pyle, A. M. Structural insights into RNA splicing. Curr. Opin. Struct. Biol. 19, 260–266 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Rusch, D. B. et al. The Sorcerer II Global Ocean Sampling expedition: northwest Atlantic through eastern tropical Pacific. PLoS Biol. 5, e77 (2007)

    Article  PubMed  PubMed Central  Google Scholar 

  22. Raya, R. R. & Hébert, E. M. in Bacteriophages: Methods and Protocols Vol. 1 (ed. Clokie, M. R. J.) (Humana, 2009)

    Google Scholar 

  23. Stoddard, B. L. Homing endonuclease structure and function. Q. Rev. Biophys. 38, 49–95 (2005)

    Article  CAS  PubMed  Google Scholar 

  24. Lambowitz, A. M. & Zimmerly, S. Mobile group II introns. Annu. Rev. Genet. 38, 1–35 (2004)

    Article  CAS  PubMed  Google Scholar 

  25. Wassarman, K. M., Zhang, A. & Storz, G. Small RNAs in Escherichia coli . Trends Microbiol. 7, 37–45 (1999)

    Article  CAS  PubMed  Google Scholar 

  26. Frias-Lopez, J. et al. Microbial community gene expression in ocean surface waters. Proc. Natl Acad. Sci. USA 105, 3805–3810 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wassarman, K. M. 6S RNA: a regulator of transcription. Mol. Microbiol. 65, 1425–1431 (2007)

    Article  CAS  PubMed  Google Scholar 

  28. Pichon, C. & Felden, B. Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains. Proc. Natl Acad. Sci. USA 102, 14249–14254 (2005)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Altuvia, S., Weinstein-Fischer, D., Zhang, A., Postow, L. & Storz, G. A. Small, Stable RNA induced by oxidative stress: role as a pleiotropic regulator and antimutator. Cell 90, 43–53 (1997)

    Article  CAS  PubMed  Google Scholar 

  30. Yao, Z., Weinberg, Z. & Ruzzo, W. L. CMfinder—a covariance model based RNA motif finding algorithm. Bioinformatics 22, 445–452 (2006)

    Article  CAS  PubMed  Google Scholar 

  31. Weinberg, Z. & Ruzzo, W. L. Sequence-based heuristics for faster annotation of non-coding RNA families. Bioinformatics 22, 35–39 (2006)

    Article  CAS  PubMed  Google Scholar 

  32. Eddy, S. R. & Durbin, R. RNA Sequence Analysis Using Covariance Models. Nucleic Acids Res. 22, 2079–2088 (1994)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Klein, R. J. & Eddy, S. R. RSEARCH: finding homologs of single structured RNA sequences. BMC Bioinformatics 4, 44 (2003)

    Article  PubMed  PubMed Central  Google Scholar 

  34. Knudsen, B. & Hein, J. Pfold: RNA secondary structure prediction using stochastic context-free grammars. Nucleic Acids Res. 31, 3423–3428 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Yao, Z. Genome scale search of noncoding RNAs: bacteria to vertebrates. Dissertation, Univ. of Washington (2008)

    Google Scholar 

  36. Pruitt, K., Tatusova, T. & Maglott, D. NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 33, D501–D504 (2005)

    Article  CAS  PubMed  Google Scholar 

  37. Tyson, G. W. et al. Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428, 37–43 (2004)

    Article  ADS  CAS  PubMed  Google Scholar 

  38. Tringe, S. G. et al. Comparative metagenomics of microbial communities. Science 308, 554–557 (2005)

    Article  ADS  CAS  PubMed  Google Scholar 

  39. Gill, S. R. et al. Metagenomic analysis of the human distal gut microbiome. Science 312, 1355–1359 (2006)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kurokawa, K. et al. Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes. DNA Res. 14, 169–181 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Turnbaugh, P. J. et al. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027–1031 (2006)

    Article  ADS  PubMed  Google Scholar 

  42. Woyke, T. et al. Symbiosis insights through metagenomic analysis of a microbial consortium. Nature 443, 950–955 (2006)

    Article  ADS  CAS  PubMed  Google Scholar 

  43. Martín, H. G. et al. Metagenomic analysis of two enhanced biological phosphorus removal (EBPR) sludge communities. Nature Biotechnol. 24, 1263–1269 (2006)

    Article  Google Scholar 

  44. Warnecke, F. et al. Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. Nature 450, 560–565 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  45. Konstantidinis, K. T. et al. Comparative metagenomic analysis of a microbial community residing at a depth of 4,000 meters at station ALOHA in the North Pacific subtropical gyre. Appl. Environ. Microbiol. 75, 5345–5355 (2009)

    Article  Google Scholar 

  46. Venter, J. C. et al. Environmental genome shotgun sequencing of the Sargasso Sea. Science 304, 66–74 (2004)

    Article  ADS  CAS  PubMed  Google Scholar 

  47. Noguchi, H., Park, J. & Takagi, T. MetaGene: prokaryotic gene finding from environmental genome shotgun sequences. Nucleic Acids Res. 34, 5623–5630 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Markowitz, V. M. et al. IMG/M: a data management and analysis system for metagenomes. Nucleic Acids Res. 36, D534–D538 (2008)

    Article  CAS  PubMed  Google Scholar 

  49. Marchler-Bauer, A. et al. CDD: a Conserved Domain Database for protein classification. Nucleic Acids Res. 33, D192–D196 (2005)

    Article  CAS  PubMed  Google Scholar 

  50. Gardner, P. P. et al. Rfam: updates to the RNA families database. Nucleic Acids Res. 37, D136–D140 (2009)

    Article  CAS  PubMed  Google Scholar 

  51. Liu, C. et al. NONCODE: an integrated knowledge database of non-coding RNAs. Nucleic Acids Res. 33, D112–D115 (2005)

    Article  CAS  PubMed  Google Scholar 

  52. Gutell, R. R., Larsen, N. & Woese, C. R. Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective. Microbiol. Rev. 58, 10–26 (1994)

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Dai, L. & Zimmerly, S. Compilation and analysis of group II intron insertions in bacterial genomes: evidence for retroelement behavior. Nucleic Acids Res. 30, 1091–1102 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Boudvillain, M. & Pyle, A. M. Defining functional groups, core structural features and inter-domain tertiary contacts essential for group II intron self-splicing: a NAIM analysis. EMBO J. 17, 7091–7104 (1998)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Toor, N., Hausner, G. & Zimmerly, S. Coevolution of group II intron RNA structures with their intron-encoded reverse transcriptases. RNA 7, 1142–1152 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Haas, E. S., Brown, J. W., Pitulle, C. & Pace, N. R. Further perspective on the catalytic core and secondary structure of ribonuclease P RNA. Proc. Natl Acad. Sci. USA 91, 2527–2531 (1994)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  57. Zwieb, C., Wower, I. & Wower, J. Comparative sequence analysis of tmRNA. Nucleic Acids Res. 27, 2063–2071 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Barrick, J. E. & Breaker, R. R. The distributions, mechanisms, and structures of metabolite-binding riboswitches. Genome Biol. 8, R239 (2007)

    Article  PubMed  PubMed Central  Google Scholar 

  59. Guindon, S. & Gascuel, O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704 (2003)

    Article  PubMed  Google Scholar 

  60. Cazenave, C. & Uhlenbeck, O. C. RNA-template-directed RNA synthesis by T7 polymerase. Proc. Natl Acad. Sci. USA 91, 6972–6976 (1994)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  61. Wu, T., Ogilvie, T. T. & Pon, R. T. Prevention of chain cleavage in the chemical synthesis of 2′ silylated oligoribonucleotides. Nucleic Acids Res. 17, 3501–3517 (1989)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Regulski, E. E. & Breaker, R. R. in Methods in Molecular Biology Vol. 419 Post-Transcriptional Gene Regulation (ed. Wilusz, J.) (Humana, 2008)

    Google Scholar 

Download references

Acknowledgements

We thank N. Carriero and R. Bjornson for assisting our use of the Yale Life Sciences High Performance Computing Center (NIH grant RR19895-02), T. Gruczka for advice and assistance in ocean water collection, J. Yang for assistance with the analysis of the dct-1 motif, D. Rodrigues for E. sibiricum, D. Bryant for A. maxima genomic DNA and P. O’Donoghue, M. Hammond, N. Sudarsan, S. Li, J. Barrick, Z. Yao, W. L. Ruzzo and E. Tseng for advice. J.P. and M.M.M. were supported by postdoctoral fellowships from the Canadian Institutes of Health Research and National Institutes of Health, respectively. R.R.B. is a Howard Hughes Medical Institute Investigator.

Author Contributions Z.W. and R.R.B. conceived the study and R.R.B. supervised the research. Z.W. created bioinformatics scripts and prepared RNA sequence alignments. J.P. conducted GOLLD and IMES RNA experiments. M.M.M. conducted GOLLD RACE and HEARO RNA experiments. Z.W. and R.R.B. wrote the manuscript, and all authors participated in editing.

Author information

Authors and Affiliations

  1. Howard Hughes Medical Institute,,

    Zasha Weinberg & Ronald R. Breaker

  2. Department of Molecular, Cellular and Developmental Biology,

    Zasha Weinberg, Jonathan Perreault, Michelle M. Meyer & Ronald R. Breaker

  3. Department of Molecular Biophysics and Biochemistry, Yale University, Box 208103, New Haven, Connecticut 06520-8103, USA,

    Ronald R. Breaker

Authors
  1. Zasha Weinberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jonathan Perreault
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michelle M. Meyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ronald R. Breaker
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ronald R. Breaker.

Supplementary information

Supplementary Information

This file contains Supplementary Notes, Supplementary Tables 1-3, Supplementary Figures 1-11 with Legends and Supplementary References. (PDF 1207 kb)

Supplementary Data 1

This file presents detailed data on GOLLD, HEARO and IMES RNAs in printable format. For each RNA class, the organisms containing representatives are listed, and the nucleotide coordinates and genes surrounding each representative are depicted. The file also contains a full multiple-sequence alignment with consensus secondary structure for each RNA class. Also included are proposed alignments of regions of the 5' half of GOLLD RNA in Streptococus species, and a smaller structure that is more broadly detected. (PDF 3544 kb)

Supplementary Data 2

This compressed archive file houses multiple-sequence alignments in machine-readable format. The alignments were presented in printable form in Supplementary Data 1. The alignments include additional annotation used to generate drawings and printable data, as well as sequences that flank the RNAs. Each alignment is stored in "Stockholm" text format (http://en.wikipedia.org/wiki/Stockholm_format). The Stockholm files can be extracted from the .tar.gz format archive using programs such as WinZip (Windows), StuffIt Expander (Mac) or the tar/gzip commands (UNIX). (TAR 2050 kb)

Supplementary Data 3

This compressed archive file houses multiple-sequence alignments in Stockholm text format (http://en.wikipedia.org/wiki/Stockholm_format). However, annotation beyond the consensus secondary structure and flanking sequence is not included. The Stockholm files can be extracted from the .tar.gz format archive using programs such as WinZip (Windows), StuffIt Expander (Mac) or the tar/gzip commands (UNIX). (TAR 580 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Weinberg, Z., Perreault, J., Meyer, M. et al. Exceptional structured noncoding RNAs revealed by bacterial metagenome analysis . Nature 462, 656–659 (2009). https://doi.org/10.1038/nature08586

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08586

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced 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