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Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity

Nature Structural & Molecular Biology volume 10, pages 708–712 (2003)Cite this article

A Corrigendum to this article was published on 01 October 2003

Abstract

The hammerhead ribozyme (HHRz) is a small, naturally occurring ribozyme that site-specifically cleaves RNA and has long been considered a potentially useful tool for gene silencing. The minimal conserved HHRz motif derived from natural sequences consists of three helices that intersect at a highly conserved catalytic core of 11 nucleotides. The presence of this motif is sufficient to support cleavage at high Mg2+ concentrations, but not at the low Mg2+ concentrations characteristic of intracellular environments. Here we demonstrate that natural HHRzs require the presence of additional nonconserved sequence elements outside of the conserved catalytic core to enable intracellular activity. These elements may stabilize the HHRz in a catalytically active conformation via tertiary interactions. HHRzs stabilized by these interactions cleave efficiently at physiological Mg2+ concentrations and are functional in vivo. The proposed role of these tertiary interacting motifs is supported by mutational, functional, structural and molecular modeling analysis of natural HHRzs.

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Figure 1: In vitro, in vivo, and mutational analysis of natural HHRzs.
Figure 2: Sequence alignment of natural HHRzs integrated through stem III.
Figure 3: Structural modeling of the sTRSV HHRz.
Figure 4: In vitro, in vivo, and structural analysis of chimeric HHRzs.

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References

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

    Article  CAS  Google Scholar 

  2. Darnell, J., Lodish, H.F. & Baltimore, D. Molecular Cell Biology (W.H. Freeman, New York, 1986).

    Google Scholar 

  3. Deshler, J.O., Li, H., Rossi, J.J. & Castanotto, D. Ribozymes expressed within the loop of a natural antisense RNA form functional transcription terminators. Gene 155, 35–43 (1995).

    Article  CAS  Google Scholar 

  4. Buzayan, J.M., Hampel, A. & Bruening, G. Nucleotide sequence and newly formed phosphodiester bond of spontaneously ligated satellite tobacco ringspot virus RNA. Nucleic Acids Res. 14, 9729–9743 (1986).

    Article  CAS  Google Scholar 

  5. Forster, A.C. & Symons, R.H. Self-cleavage of virusoid RNA is performed by the proposed 55-nucleotide active site. Cell 50, 9–16 (1987).

    Article  CAS  Google Scholar 

  6. Hernandez, C. & Flores, R. Plus and minus RNAs of peach latent mosaic viroid self-cleave in vitro via hammerhead structures. Proc. Natl. Acad. Sci. USA 89, 3711–3715 (1992).

    Article  CAS  Google Scholar 

  7. Long, D.M. & Uhlenbeck, O.C. Kinetic characterization of intramolecular and intermolecular hammerhead RNAs with stem II deletions. Proc. Natl. Acad. Sci. USA 91, 6977–6981 (1994).

    Article  CAS  Google Scholar 

  8. Long, D.M., LaRiviere, F.J. & Uhlenbeck, O.C. Divalent metal ions and the internal equilibrium of the hammerhead ribozyme. Biochemistry 34, 14435–14440 (1995).

    Article  CAS  Google Scholar 

  9. Pley, H.W., Flaherty, K.M. & McKay, D.B. Three-dimensional structure of a hammerhead ribozyme. Nature 372, 68–74 (1994).

    Article  CAS  Google Scholar 

  10. Scott, W.G., Finch, J.T. & Klug, A. The crystal structure of an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. Cell 81, 991–1002 (1995).

    Article  CAS  Google Scholar 

  11. Hainzl, T., Huang, S. & Sauer-Eriksson, A.E. Structure of the SRP19 RNA complex and implications for signal recognition particle assembly. Nature 417, 767–771 (2002).

    Article  CAS  Google Scholar 

  12. Oubridge, C., Kuglstatter, A., Jovine, L. & Nagai, K. Crystal structure of SRP19 in complex with the S domain of SRP RNA and its implication for the assembly of the signal recognition particle. Mol. Cell 9, 1251–1261 (2002).

    Article  CAS  Google Scholar 

  13. Berman, H.M. et al. The nucleic acid database. A comprehensive relational database of three-dimensional structures of nucleic acids. Biophys. J. 63, 751–759 (1992).

    Article  CAS  Google Scholar 

  14. Massire, C. & Westhof, E. MANIP: an interactive tool for modelling RNA. J. Mol. Graph. Mod. 16, 197–205 (1999).

    Article  CAS  Google Scholar 

  15. McKay, D.B. Structure and function of the hammerhead ribozyme: an unfinished story. RNA 2, 395–403 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Hammann, C., Norman, D.G. & Lilley, D.M. Dissection of the ion-induced folding of the hammerhead ribozyme using 19F NMR. Proc. Natl. Acad. Sci. USA 98, 5503–5508 (2001).

    Article  CAS  Google Scholar 

  17. Esteban, J.A., Banerjee, A.R. & Burke, J.M. Kinetic mechanism of the hairpin ribozyme. Identification and characterization of two nonexchangeable conformations. J. Biol. Chem. 272, 13629–13639 (1997).

    Article  CAS  Google Scholar 

  18. Zhao, Z.-Y. et al. The folding of the hairpin ribozyme: dependence on the loops and the junction. RNA 6, 1833–1846 (2000).

    Article  CAS  Google Scholar 

  19. Walter, N.G., Burke, J.M. & Millar, D.P. Stability of hairpin ribozyme tertiary structure is governed by the interdomain junction. Nat. Struct. Biol. 6, 544–549 (1999).

    Article  CAS  Google Scholar 

  20. Stage-Zimmermann, T.K. & Uhlenbeck, O.C. A covalent crosslink converts the hammerhead ribozyme from a ribonuclease to an RNA ligase. Nat. Struct. Biol. 8, 863–867 (2001).

    Article  CAS  Google Scholar 

  21. Blount, K.F. & Uhlenbeck, O.C. Internal equilibrium of the hammerhead ribozyme is altered by the length of certain covalent cross-links. Biochemistry 41, 6834–6841 (2002).

    Article  CAS  Google Scholar 

  22. Fedor, M.J. Tertiary structure stabilization promotes hairpin ribozyme ligation. Biochemistry 38, 11040–11050 (1999).

    Article  CAS  Google Scholar 

  23. Pan, J., Thirumalai, D. & Woodson, S.A. Magnesium-dependent folding of self-splicing RNA: exploring the link between cooperativity, thermodynamics, and kinetics. Proc. Natl. Acad. Sci. USA 96, 6149–6154 (1999).

    Article  CAS  Google Scholar 

  24. Pan, J. & Woodson, S.A. The effect of long-range loop-loop interactions on folding of the Tetrahymena self-splicing RNA. J. Mol. Biol. 294, 955–965 (1999).

    Article  CAS  Google Scholar 

  25. Lehnert, V., Jaeger, L., Michel, F. & Westhof, E. New loop-loop tertiary interactions in self-splicing introns of subgroup IC and ID: a complete 3D model of the Tetrahymena thermophila ribozyme. Chem. Biol. 3, 993–1009 (1996).

    Article  CAS  Google Scholar 

  26. Collins, M.L. et al. A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml. Nucleic Acids Res. 25, 2979–2984 (1997).

    Article  CAS  Google Scholar 

  27. Milligan, J.F. & Uhlenbeck, O.C. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 180, 51–62 (1989).

    Article  CAS  Google Scholar 

  28. Stage-Zimmermann, T.K. & Uhlenbeck, O.C. Hammerhead ribozyme kinetics. RNA 4, 875–889 (1998).

    Article  CAS  Google Scholar 

  29. Massire, C., Gaspin, C. & Westhof, E. DRAWNA: a program for drawing schematic views of nucleic acids. J. Mol. Graph. 12, 201–206 (1994).

    Article  CAS  Google Scholar 

  30. Hertel, K.J. et al. Numbering system for the hammerhead. Nucleic Acids Res. 20, 12 (1992).

    Article  Google Scholar 

  31. Gorodkin, J., Knudsen, B., Zwieb, C. & Samuelsson, T. SRPDB (Signal Recognition Particle Database). Nucleic Acids Res. 29, 169–170 (2001).

    Article  CAS  Google Scholar 

  32. Leontis, N.B. & Westhof, E. Geometric nomenclature and classification of RNA base pairs. RNA 7, 499–512 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank O. Uhlenbeck, A. Wolfson, J. Rossi and D. Lilley for insightful discussions and critical reading of this manuscript, S. Suggs for comments and encouragement and A. Reynolds, M. Brewer and W. Marshall for their help and support.

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Author notes

  1. Aurélie Lescoute & Eric Westhof

    Present address: Institut de Biologie Moléculaire et Cellulaire, UPR9002 du CNRS, Université Louis Pasteur, 15 rue René Descartes, Strasbourg, F-67084, Cedex, France

  2. Anastasia Khvorova

    Present address: Dharmacon Research, Inc., 1376 Miners Drive, #101, Lafayette, Colorado, 80026, USA

Authors and Affiliations

  1. Amgen, Inc., One Amgen Center Drive, Thousand Oaks, 91320, California, USA

    Anastasia Khvorova & Sumedha D Jayasena

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Correspondence to Anastasia Khvorova or Sumedha D Jayasena.

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Khvorova, A., Lescoute, A., Westhof, E. et al. Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity. Nat Struct Mol Biol 10, 708–712 (2003). https://doi.org/10.1038/nsb959

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