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Targeting mRNAs by Engineered Sequence-Specific RNase P Ribozymes

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Ribozymes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 848))

Abstract

The methods of using engineered RNase P catalytic RNA (termed as M1GS RNA) for in vitro and in vivo in trans-cleavage of target viral mRNA are described in this chapter. Detailed information is focused on (1) mapping accessible regions of target viral mRNA in infected cells, (2) generation and in vitro cleavage assay of the customized M1GS ribozyme, (3) stable expression of M1GS RNAs and evaluation of its antiviral activity in cultured cells. Using these methods, we have constructed functional M1GS ribozyme that can cleave an overlapping region of the mRNAs coding for the human cytomegalovirus (HCMV) capsid scaffolding protein (CSP) and assemblin in vitro. Further study has demonstrated that, in cultured human cells expressing the functional M1GS ribozyme and infected with HCMV, more than 85% reduction in the expression of CSP and assemblin and a 4,000-fold reduction in viral growth were achieved. Our study provided the direct evidence that the customized M1GS ribozyme can be used as an effective gene-targeting agent for in trans-cleavage of viral genes and inhibition of viral growth in cultured cells.

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References

  1. Evans D, Marquez SM, & Pace NR (2006) RNase P: interface of the RNA and protein worlds. (Translated from eng) Trends Biochem Sci 31(6):333–341.

    Article  PubMed  CAS  Google Scholar 

  2. Gopalan V & Altman S eds (2007) Ribonuclease P: structure and catalysis. The RNA World. (Cold Spring Harbor Laboratory Press, New York).

    Google Scholar 

  3. Xiao S, Scott F, Fierke CA, & Engelke DR (2002) Eukaryotic ribonuclease P: a plurality of ribonucleoprotein enzymes. (Translated from eng) Annu Rev Biochem 71:165–189.

    Article  PubMed  CAS  Google Scholar 

  4. Frank DN & Pace NR (1998) Ribonuclease P: unity and diversity in a tRNA processing ribozyme. Annu Rev Biochem 67:153–180.

    Article  PubMed  CAS  Google Scholar 

  5. Altman S & Kirsebom LA (1999) Ribonuclease P. In The RNA world. 2nd Ed (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) 2nd Ed pp 351–380.

    Google Scholar 

  6. Altman S (2010) History of RNase P and overview of its catalytic activity. Ribonuclease P, eds Liu F & Altman S (Springer, New York).

    Google Scholar 

  7. Forster AC & Altman S (1990) External Guide Sequences for an RNA Enzyme. Science 249(4970):783–786.

    Article  PubMed  CAS  Google Scholar 

  8. Yuan Y, Hwang ES, & Altman S (1992) Targeted cleavage of mRNA by human RNase P. Proc Natl Acad Sci USA 89(17):8006–8010.

    Article  PubMed  CAS  Google Scholar 

  9. Liu F (2010) Ribonuclease P as a tool. Ribonuclease P, eds Liu F & Altman S (Springer, New York).

    Google Scholar 

  10. Guerriertakada C, Li Y, & Altman S (1995) Artificial Regulation of Gene-Expression in Escherichia coli by Rnase-P. Proceedings of the National Academy of Sciences of the United States of America 92(24):11115–11119.

    Article  CAS  Google Scholar 

  11. Raj SML & Liu F (2003) Engineering of RNase P ribozyme for gene-targeting applications. Gene 313:59–69.

    Article  PubMed  CAS  Google Scholar 

  12. Liu FY & Altman S (1995) Inhibition of Viral Gene-Expression by the Catalytic RNA Subunit of Rnase-P from Escherichia coli. Genes & Development 9(4):471–480.

    Article  CAS  Google Scholar 

  13. Plehn-Dujowich D & Altman S (1998) Effective inhibition of influenza virus production in cultured cells by external guide sequences and ribonuclease P. Proceedings of the National Academy of Sciences of the United States of America 95(13):7327–7332.

    Article  PubMed  CAS  Google Scholar 

  14. Cobaleda C & Sanchez-Garcia I (2000) In vivo inhibition by a site-specific catalytic RNA subunit of RNase P designed against the BCR-ABL oncogenic products: a novel approach for cancer treatment. Blood 95(3):731–737.

    PubMed  CAS  Google Scholar 

  15. Kilani AF, et al. (2000) RNase P ribozymes selected in vitro to cleave a viral mRNA effectively inhibit its expression in cell culture. Journal of Biological Chemistry 275(14):10611–10622.

    Article  PubMed  CAS  Google Scholar 

  16. Trang P, et al. (2000) Effective inhibition of human cytomegalovirus gene expression and replication by a ribozyme derived from the catalytic RNA subunit of RNase P from Escherichia coli. (Translated from eng) Proc Natl Acad Sci USA 97(11):5812–5817.

    Article  PubMed  CAS  Google Scholar 

  17. Kim K, Trang P, Umamoto S, Hai R, & Liu F (2004) RNase P ribozyme inhibits cytomegalovirus replication by blocking the expression of viral capsid proteins. (Translated from eng) Nucleic Acids Res 32(11):3427–3434.

    Article  PubMed  CAS  Google Scholar 

  18. Mocarski ES, Shenk T, & Pass RF eds (2007) in Fields Virology, pp 2701–2772 (Lippincott, Philadelphia).

    Google Scholar 

  19. Gopalan V, Vioque A, & Altman S (2002) RNase P: variations and uses. J Biol Chem 277(9):6759–6762.

    Article  PubMed  CAS  Google Scholar 

  20. Liu F & Altman S (1995) Inhibition of viral gene expression by the catalytic RNA subunit of RNase P from Escherichia coli. (Translated from eng) Genes Dev 9(4):471–480.

    Article  PubMed  CAS  Google Scholar 

  21. Ares M, Jr. & Igel AH (1990) Lethal and temperature-sensitive mutations and their suppressors identify an essential structural element in U2 small nuclear RNA. (Translated from eng) Genes Dev 4(12A):2132–2145.

    Google Scholar 

  22. Zaug AJ & Cech TR (1995) Analysis of the structure of Tetrahymena nuclear RNAs in vivo: telomerase RNA, the self-splicing rRNA intron, and U2 snRNA. (Translated from eng) RNA 1(4):363–374.

    PubMed  CAS  Google Scholar 

  23. Bertrand E, et al. (1997) The expression cassette determines the functional activity of ribozymes in mammalian cells by controlling their intracellular localization. (Translated from eng) RNA 3(1):75–88.

    PubMed  CAS  Google Scholar 

  24. Das G, Henning D, Wright D, & Reddy R (1988) Upstream regulatory elements are necessary and sufficient for transcription of a U6 RNA gene by RNA polymerase III. (Translated from eng) EMBO J 7(2):503–512.

    PubMed  CAS  Google Scholar 

  25. Miller AD & Rosman GJ (1989) Improved Retroviral Vectors for Gene-Transfer and Expression. Biotechniques 7(9):980.

    PubMed  CAS  Google Scholar 

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Acknowledgments

Gratitude goes to Dr. Phong Trang and Dr. Kihoon Kim. This research has been supported by NIH (AI041927 and DE014842).

Author information

Authors and Affiliations

  1. Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, CA, USA

    Yong Bai

  2. Division of Infectious Diseases and Vaccinology, University of California, Berkeley, CA, USA

    Naresh Sunkara

  3. Division of Infectious Diseases and Vaccinology, School of Public Health, and Program in Comparative Biochemistry, University of California, Berkeley, CA, USA

    Fenyong Liu

Authors
  1. Yong Bai
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  2. Naresh Sunkara
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  3. Fenyong Liu
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Corresponding author

Correspondence to Fenyong Liu .

Editor information

Editors and Affiliations

  1. Department of Chemistry, Universität Konstanz, Universitätsstr. 10, Konstanz, 78457, Germany

    Jörg S. Hartig

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Bai, Y., Sunkara, N., Liu, F. (2012). Targeting mRNAs by Engineered Sequence-Specific RNase P Ribozymes. In: Hartig, J. (eds) Ribozymes. Methods in Molecular Biology, vol 848. Humana Press. https://doi.org/10.1007/978-1-61779-545-9_22

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  • DOI: https://doi.org/10.1007/978-1-61779-545-9_22

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-544-2

  • Online ISBN: 978-1-61779-545-9

  • eBook Packages: Springer Protocols

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