Exogenous control of mammalian gene expression through modulation of RNA self-cleavage

Article metrics


Recent studies on the control of specific metabolic pathways in bacteria have documented the existence of entirely RNA-based mechanisms for controlling gene expression. These mechanisms involve the modulation of translation, transcription termination or RNA self-cleavage through the direct interaction of specific intracellular metabolites and RNA sequences1,2,3,4. Here we show that an analogous RNA-based gene regulation system can effectively be designed for mammalian cells via the incorporation of sequences encoding self-cleaving RNA motifs5 into the transcriptional unit of a gene or vector. When correctly positioned, the sequences lead to potent inhibition of gene or vector expression, owing to the spontaneous cleavage of the RNA transcript. Administration of either oligonucleotides complementary to regions of the self-cleaving motif or a specific small molecule results in the efficient induction of gene expression, owing to inhibition of self-cleavage of the messenger RNA. Efficient regulation of transgene expression is shown in a variety of mammalian cell lines and live animals. In conjunction with other emerging technologies6, this methodology may be particularly applicable to the development of gene regulation systems tailored to any small inducer molecule, and provide a novel means of biological sensing in vivo that may have an important application in the regulated delivery of protein therapeutics.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Strategy for controlling gene expression through the modulation of RNA self-cleavage and optimization of Schistosome Sm1 ribozyme self-cleavage activity.
Figure 2: Efficient self-cleavage can occur in different cells, with different vectors and with ribozyme sequences positioned in different locations.
Figure 3: Induction of gene expression in cultured cells through inhibition of ribozyme self-cleavage.
Figure 4: Effective control of gene expression in vivo using ribozyme-based gene regulation system.


  1. 1

    Winkler, W., Nahvi, A. & Breaker, R. R. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419, 952–956 (2002)

  2. 2

    Winkler, W. C., Nahvi, A., Roth, A., Collins, J. A. & Breaker, R. R. Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428, 281–286 (2004)

  3. 3

    Mandal, M. & Breaker, R. R. Adenine riboswitches and gene activation by disruption of a transcription terminator. Nature Struct. Mol. Biol. 11, 29–35 (2004)

  4. 4

    Cech, T. R. RNA finds a simpler way. Nature 428, 263–264 (2004)

  5. 5

    Cech, T. R. Nobel lecture. Self-splicing and enzymatic activity of an intervening sequence RNA from Tetrahymena. Biosci. Rep. 10, 239–261 (1990)

  6. 6

    Silverman, S. K. Rube Goldberg goes (ribo)nuclear? Molecular switches and sensors made from RNA. RNA 9, 377–383 (2003)

  7. 7

    Ory, D. S., Neugeboren, B. A. & Mulligan, R. C. A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc. Natl Acad. Sci. USA 93, 11400–11406 (1996)

  8. 8

    Rojas, A. A. et al. Hammerhead-mediated processing of satellite pDo500 family transcripts from Dolichopoda cave crickets. Nucleic Acids Res. 28, 4037–4043 (2000)

  9. 9

    Ferbeyre, G., Smith, J. M. & Cedergren, R. Schistosome satellite DNA encodes active hammerhead ribozymes. Mol. Cell. Biol. 18, 3880–3888 (1998)

  10. 10

    Hertel, K. J. et al. Numbering system for the hammerhead. Nucleic Acids Res. 20, 3252 (1992)

  11. 11

    Ruffner, D. E., Stormo, G. D. & Uhlenbeck, O. C. Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry 29, 10695–10702 (1990)

  12. 12

    Chowrira, B. M., Pavco, P. A. & McSwiggen, J. A. In vitro and in vivo comparison of hammerhead, hairpin and hepatitis delta virus self-processing ribozyme cassettes. J. Biol. Chem. 269, 25856–25864 (1994)

  13. 13

    Zillmann, M., Limauro, S. E. & Goodchild, J. In vitro optimization of truncated stem-loop II variants of the hammerhead ribozyme for cleavage in low concentrations of magnesium under non-turnover conditions. RNA 3, 734–747 (1997)

  14. 14

    Conaty, J., Hendry, P. & Lockett, T. Selected classes of minimised hammerhead ribozyme have very high cleavage rates at low Mg2+ concentration. Nucleic Acids Res. 27, 2400–2407 (1999)

  15. 15

    Hermann, T. & Westhof, E. Aminoglycoside binding to the hammerhead ribozyme: a general model for the interaction of cationic antibiotics with RNA. J. Mol. Biol. 276, 903–912 (1998)

  16. 16

    Jenne, A. et al. Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology. Nature Biotechnol. 19, 56–61 (2001)

  17. 17

    Murray, J. B. & Arnold, J. R. Antibiotic interactions with the hammerhead ribozyme:tetracyclines as a new class of hammerhead inhibitor. Biochem. J. 317, 855–860 (1996)

  18. 18

    Stage, T. K., Hertel, K. J. & Uhlenbeck, O. C. Inhibition of the hammerhead ribozyme by neomycin. RNA 1, 95–101 (1995)

  19. 19

    Tor, Y., Hermann, T. & Westhof, E. Deciphering RNA recognition: aminoglycoside binding to the hammerhead ribozyme. Chem. Biol. 5, R277–R283 (1998)

  20. 20

    von Ahsen, U., Davies, J. & Schroeder, R. Antibiotic inhibition of group I ribozyme function. Nature 353, 368–370 (1991)

  21. 21

    Braasch, D. A. & Corey, D. R. Novel antisense and peptide nucleic acid strategies for controlling gene expression. Biochemistry 41, 4503–4510 (2002)

  22. 22

    Morcos, P. A. Achieving efficient delivery of morpholino oligos in cultured cells. Genesis 30, 94–102 (2001)

  23. 23

    Aszalos, A., Lemanski, P., Robison, R., Davis, S. & Berk, B. Identification of antibiotic 1037 as toyocamycin. J. Antibiot. (Tokyo) 19, 285 (1966)

  24. 24

    Contag, P. R., Olomu, I. N., Stevenson, D. K. & Contag, C. H. Bioluminescent indicators in living mammals. Nature Med. 4, 245–247 (1998)

  25. 25

    Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl Acad. Sci. USA 89, 5547–5551 (1992)

  26. 26

    Rivera, V. M. et al. A humanized system for pharmacologic control of gene expression. Nature Med. 2, 1028–1032 (1996)

  27. 27

    Suhr, S. T., Gil, E. B., Senut, M. C. & Gage, F. H. High level transactivation by a modified Bombyx ecdysone receptor in mammalian cells without exogenous retinoid X receptor. Proc. Natl Acad. Sci. USA 95, 7999–8004 (1998)

  28. 28

    Wang, Y., O'Malley, B. W. Jr, Tsai, S. Y. & O'Malley, B. W. A regulatory system for use in gene transfer. Proc. Natl Acad. Sci. USA 91, 8180–8184 (1994)

  29. 29

    Breaker, R. R. Engineered allosteric ribozymes as biosensor components. Curr. Opin. Biotechnol. 13, 31–39 (2002)

  30. 30

    Wilson, D. S. & Szostak, J. W. In vitro selection of functional nucleic acids. Annu. Rev. Biochem. 68, 611–647 (1999)

Download references


We thank K. Salehi-Ashtlani and J. Szostak for helpful discussions, Y. Tang and R. Weissleder for help with imaging experiments performed during the early course of the work, and M. Chung for her technical assistance. This work was supported by grants from AMGEN and L'Association Francaise contre les Myopathies (AFM). R.C.M. is an AMGEN consultant and equity holder.

Author information

Correspondence to Richard C. Mulligan.

Ethics declarations

Competing interests

Funding for this work was provided by AMGEN corporation and L'Association Francaise contre les Myopathies (AFM). R.C.M. holds a non-paying consultant position and AMGEN equity.

Supplementary information

Supplementary Table

Survey of ability of different ‘self-cleaving’ ribozymes to function in mammalian cells. Including a list of references. (PDF 106 kb)

Supplementary Methods (DOC 24 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Yen, L., Svendsen, J., Lee, J. et al. Exogenous control of mammalian gene expression through modulation of RNA self-cleavage. Nature 431, 471–476 (2004) doi:10.1038/nature02844

Download citation

Further reading


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.