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Nuclear antisense RNA

An efficient new method to inhibit gene expression

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Abstract

We describe an efficient new antisense RNA method to inhibit gene expression. Antisense RNAs that are retained in the nucleus bind to target transcripts and appear to lead to the degradation of their targets. Antisense RNAs can be expressed and accumulated specifically in the nucleus if they are not polyadenylated at their 3′ ends. In antisense expression vectors we use a cis-acting ribozyme to generate 3′-ends independently of the polyadenylation machinery and thereby inhibit transport of RNA molecules from the nucleus to the cytoplasm. We have evaluated this method in the mouse polyoma virus model system, where nuclear antisense transcripts to the viral early transcription region efficiently reduced the level of viral early-strand RNAs. Nonspecific antisense RNA had no effects on viral gene expression. In comparative studies, nuclear antisense RNAs were significantly more effective in downregulating polyoma virus early RNAs than were conventional antisense molecules, which were processed by polyadenylation.

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References

  1. Inouye, M. (1988) Antisense RNA: Its functions and applications in gene regulation.Gene 72, 25–34.

    Article  PubMed  CAS  Google Scholar 

  2. Takayama, K. M. and Inouye, M. (1990) Antisense RNA.Crit. Rev. Biochem. Mol. Biol. 25, 155–184.

    Article  PubMed  CAS  Google Scholar 

  3. Tosic, M., Roach, A., Rivaz, J.-C. D., Dolivo, M., and Matthieu, J.-M. (1990) Posttranscriptional events are responsible for low expression of myelin basic protein in myelin deficient mice: role of natural antisense RNA.EMBO J. 2, 401–406.

    Google Scholar 

  4. Volk, R., Köster, M., Pöting, A., Hartmann, L., and Knöchel, W. (1989) An antisense transcript from the Xenopus laevis bFGF gene coding for an evolutionarily conserved 24 kd protein.EMBO J. 8, 2983–2988.

    PubMed  CAS  Google Scholar 

  5. Williams, T. and Fried, M. (1986) A mouse locus at which transcription from both DNA strands produces mRNAs complementary at their 3′ ends.Nature 322, 275–279.

    Article  PubMed  CAS  Google Scholar 

  6. Adelman, J. P., Bond, C. T., Douglass, J., and Herbert, E. (1987) Two mammalian genes transcribed from opposite strands of the same DNA locus.Science 235, 1514–1517.

    Article  PubMed  CAS  Google Scholar 

  7. Spencer, C. A., Gietz, R. D., and Hodgetts, R. B. (1986) Overlapping transcription units in the Dopa decarboxylase region of Drosophila.Nature 322, 279–281.

    Article  PubMed  CAS  Google Scholar 

  8. Cornelissen, M. (1989) Nuclear and cytoplasmic sites for anti-sense control.Nucleic Acids Res. 17, 7203–7209.

    Article  PubMed  CAS  Google Scholar 

  9. Höfgen, R., Axelsen, K. B., Kannangara, C. G., Schüttke, I., Pohlenz, H.-D., Willmitzer, L., et al. (1994) A visible marker for antisense mRNA expression in plants: Inhibition of chlorophyll synthesis with a glutamate-1-semialdehyde aminotransferase antisense gene.Proc. Natl. Acad. Sci. USA 91, 1726–1730.

    Article  PubMed  Google Scholar 

  10. Murray, J. A. H. and Crockett, N. (1992) Antisense techniques: an overview, inModern Cell Biology, Wiley-Liss, New York, pp. 1–49.

    Google Scholar 

  11. Liu, Z., Batt, D. B., and Carmichael, G. G. (1994) Targeted nuclear antisense RNA mimics natural antisense-induced degradation of polyoma virus early RNA.Proc. Natl. Acad. Sci. USA 91, 4258–4262.

    Article  PubMed  CAS  Google Scholar 

  12. Cogen, B. (1978) Virus-specific early RNA in 3T6 cells infected by a tsA mutant of polyoma virus.Virology 85, 222–230.

    Article  CAS  Google Scholar 

  13. Piper, P. (1979) Polyoma virus transcription early during productive infection of mouse 3T6 cells.J. Mol. Biol. 131, 399–407.

    Article  PubMed  CAS  Google Scholar 

  14. Fenton, R. G. and Basilico, C. (1982) Changes in the topography of early region transcription during polyomavirus lytic infection.Proc. Natl. Acad. Sci. USA 79, 7142–7146.

    Article  PubMed  CAS  Google Scholar 

  15. Farmerie, W. G. and Folk, W. R. (1984) Regulation of polyoma-virus transcription by large tumor antigen.Proc. Natl. Acad. Sci. USA 81, 6919–6923.

    Article  PubMed  CAS  Google Scholar 

  16. Hyde-DeRuyscher, R. P., and Carmichael, G. G. (1990) Polyomavirus late pre-mRNA processing: DNA-replication-associated changes in leader exon multiplicity suggest a role for leader-to-leader splicing in the early-late switch.J. Virol. 64, 5823–5832.

    PubMed  CAS  Google Scholar 

  17. Liu, Z. and Carmichael, G. G. (1993) Polyoma virus early-late switch: Regulation of late RNA accumulation by DNA replication.Proc. Natl. Acad. Sci. USA 90, 8494–8498.

    Article  PubMed  CAS  Google Scholar 

  18. Kamen, R., Lindstrom, D. M., Shure, H., and Old, R. W. (1974) Virus-specific RNA in cells productively infected or transformed by polyoma virus.Cold Spring Harb. Symp. Quant. Biol. 39, 187–198.

    Google Scholar 

  19. Beard, P., Acheson, N. H., and Maxwell, I. H. (1976) Strand-specific transcription of polyoma virus DNA early in productive infection and in transformed cells.J. Virol. 17, 20–26.

    CAS  Google Scholar 

  20. Hyde-DeRuyscher, R. and Carmichael, G. G. (1988) Polyomavirus early-late switch is not regulated at the level of transcription initiation and is associated with changes in RNA processing.Proc. Natl. Acad. Sci. USA 85, 8993–8997.

    Article  PubMed  CAS  Google Scholar 

  21. Acheson, N. (1976) Transcription during productive infection with polyoma virus and SV40.Cell 8, 1–12.

    Article  PubMed  CAS  Google Scholar 

  22. Acheson, N. H. (1978) Polyoma giant RNAs contain tandem repeats of the nucleotide sequence of the entire viral genome.Proc. Natl. Acad. Sci. USA 75, 4754–4758.

    Article  PubMed  CAS  Google Scholar 

  23. Acheson, N. (1981) Efficiency of processing of viral RNA during the early and late phases of productive infection by polyoma virus.J. Virol. 37, 628–635.

    PubMed  CAS  Google Scholar 

  24. Acheson, N. (1984) Kinetics and efficiency of polyacenylation of late polyomavirus nuclear RNA: Generation of oligomeric polyadenylated RNAs and their processing into mRNA.Mol. Cell. Biol. 4, 722–729.

    PubMed  CAS  Google Scholar 

  25. Birg, F., Favaloro, J., and Kamen, R. (1977) Analysis of polyoma viral nuclear RNA by miniblot hybridization.Proc. Natl. Acad. Sci. USA 74, 3138–3142.

    Article  PubMed  CAS  Google Scholar 

  26. Treisman, R. and Kamen, R. (1981) Structure of polyoma virus late nuclear RNA.J. Mol. Biol. 148, 273–301.

    Article  PubMed  CAS  Google Scholar 

  27. Adami, G. R., Marlor, C. W., Barrett, N. L., and Carmichael, G. G. (1989) Leader-to-leader splicing is required for the efficient production and accumulation of polyomavirus late mRNA's.J. Virol. 63, 85–93.

    PubMed  CAS  Google Scholar 

  28. Chen, C. and Okayama, H. (1987) High efficiency transformation of mammalian cells by plasmid DNA.Mol. Cell. Biol. 7, 2745–2752.

    PubMed  CAS  Google Scholar 

  29. Xie, W. and Rothblum, L. I. (1991) Rapid, small-scale RNA isolation from tissue culture cells.BioTechniques 11, 325–327.

    Google Scholar 

  30. Lichtler, A., Barrett, N. L., and Carmichael, G. G. (1992) Simple, inexpensive preparation of T1/T2 ribonuclease suitable for use in RNase protection experiments.Biotechniques 12, 231–232.

    PubMed  CAS  Google Scholar 

  31. Birchmeier, C., Folk, W., and Birnstiel, M. L. (1983) The terminal RNA stem-loop structure and 80 bp of spacer DNA are required for the formation of 3′ termini of sea urchin H2A mRNA.Cell 35, 433–440.

    Article  PubMed  CAS  Google Scholar 

  32. Eckner, R., Ellmeier, W., and Birnstiel, M. L. (1991) Mature messenger RNA 3′ end formation stimulates RNA export from the nucleus.EMBO J. 10, 3513–3522.

    PubMed  CAS  Google Scholar 

  33. Izant, J. G. and Weintraub, H. (1985) Constitutive and conditional suppression of exogenous and endogenous genes by anti-sense RNA. Science229, 345–352.

    Article  PubMed  CAS  Google Scholar 

  34. Colman, A. (1990) Antisense strategies in cell and developmental biology.J. Cell. Sci. 97, 399–409.

    PubMed  CAS  Google Scholar 

  35. Hélène, C. and Toulmé, J.-J. (1990) Specific regulation of gene expression by antisense and antigene nucleic acids.Biochim. Biophys. Acta. 1049, 99–125.

    PubMed  Google Scholar 

  36. Haseloff, J. and Gerlach, W. L. (1988) Simple RNA enzymes with new and highly specific endoribonuclease activities.Nature 334, 585–591.

    Article  PubMed  CAS  Google Scholar 

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Author notes

  1. Zhong Liu

    Present address: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 98104, Seattle, WA

Authors and Affiliations

  1. Department of Microbiology, University of Connecticut Health Center, 06030, Farmington, CT

    Gordon G. Carmichael

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  1. Zhong Liu
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  2. Gordon G. Carmichael
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Liu, Z., Carmichael, G.G. Nuclear antisense RNA. Mol Biotechnol 2, 107–118 (1994). https://doi.org/10.1007/BF02824803

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