Abstract
We describe a general antisense strategy to inhibit target gene expression. The substitution of a cis-acting ribozyme for a polyadenylylation signal in an antisense expression vector results in the nuclear retention of RNAs and the efficient degradation of their targets. We demonstrate the utility of this system in polyoma virus, where early-strand RNA levels are downregulated in the nucleus by antisense late-strand counterparts. We show that mutations destabilizing these naturally occurring antisense transcripts lead to increased levels of early-strand RNAs. Furthermore, expression in trans of nuclear antisense transcripts lowers early-strand RNA levels and quantitatively mimics the natural regulation.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Acheson N. H. Efficiency of processing of viral RNA during the early and late phases of productive infection by polyoma virus. J Virol. 1981 Feb;37(2):628–635. doi: 10.1128/jvi.37.2.628-635.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Acheson N. H. Kinetics and efficiency of polyadenylation of late polyomavirus nuclear RNA: generation of oligomeric polyadenylated RNAs and their processing into mRNA. Mol Cell Biol. 1984 Apr;4(4):722–729. doi: 10.1128/mcb.4.4.722. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Acheson N. H. Polyoma virus giant RNAs contain tandem repeats of the nucleotide sequence of the entire viral genome. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4754–4758. doi: 10.1073/pnas.75.10.4754. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Acheson N. H. Transcription during productive infection with polyoma virus and Simian virus 40. Cell. 1976 May;8(1):1–12. doi: 10.1016/0092-8674(76)90179-3. [DOI] [PubMed] [Google Scholar]
- Adami G. R., Carmichael G. G. Polyomavirus late leader region serves an essential spacer function necessary for viability and late gene expression. J Virol. 1986 May;58(2):417–425. doi: 10.1128/jvi.58.2.417-425.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Adami G. R., Carmichael G. G. The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability. Nucleic Acids Res. 1987 Mar 25;15(6):2593–2610. doi: 10.1093/nar/15.6.2593. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Adami G. R., Marlor C. W., Barrett N. L., Carmichael G. G. Leader-to-leader splicing is required for efficient production and accumulation of polyomavirus late mRNAs. J Virol. 1989 Jan;63(1):85–93. doi: 10.1128/jvi.63.1.85-93.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barrett N. L., Carmichael G. G., Luo Y. Splice site requirement for the efficient accumulation of polyoma virus late mRNAs. Nucleic Acids Res. 1991 Jun 11;19(11):3011–3017. doi: 10.1093/nar/19.11.3011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bass B. L., Weintraub H. An unwinding activity that covalently modifies its double-stranded RNA substrate. Cell. 1988 Dec 23;55(6):1089–1098. doi: 10.1016/0092-8674(88)90253-x. [DOI] [PubMed] [Google Scholar]
- Beard P., Acheson N. H., Maxwell I. H. Strand-specific transcription of polyoma virus DNA-early in productive infection and in transformed cells. J Virol. 1975 Jan;17(1):20–26. doi: 10.1128/jvi.17.1.20-26.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Birg F., Favaloro J., Kamen R. Analysis of polyoma virus nuclear RNA by mini-blot hybridization. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3138–3142. doi: 10.1073/pnas.74.8.3138. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cahill K. B., Roome A. J., Carmichael G. G. Replication-dependent transactivation of the polyomavirus late promoter. J Virol. 1990 Mar;64(3):992–1001. doi: 10.1128/jvi.64.3.992-1001.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cogen B. Virus-specific early RNA in 3T6 cells infected by a tsA mutant of polyoma virus. Virology. 1978 Mar;85(1):222–230. doi: 10.1016/0042-6822(78)90426-9. [DOI] [PubMed] [Google Scholar]
- Colman A. Antisense strategies in cell and developmental biology. J Cell Sci. 1990 Nov;97(Pt 3):399–409. doi: 10.1242/jcs.97.3.399. [DOI] [PubMed] [Google Scholar]
- Cornelissen M. Nuclear and cytoplasmic sites for anti-sense control. Nucleic Acids Res. 1989 Sep 25;17(18):7203–7209. doi: 10.1093/nar/17.18.7203. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Denhardt D. T. Antisense strategies come of age. Antisense strategies sponsored by the New York Academy of Sciences, Philadelphia, PA, USA, January 12-15, 1992. New Biol. 1992 May;4(5):473–481. [PubMed] [Google Scholar]
- Eckner R., Ellmeier W., Birnstiel M. L. Mature mRNA 3' end formation stimulates RNA export from the nucleus. EMBO J. 1991 Nov;10(11):3513–3522. doi: 10.1002/j.1460-2075.1991.tb04915.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Farmerie W. G., Folk W. R. Regulation of polyomavirus transcription by large tumor antigen. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6919–6923. doi: 10.1073/pnas.81.22.6919. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fenton R. G., Basilico C. Regulation of polyoma virus early transcription in transformed cells by large T-antigen. Virology. 1982 Sep;121(2):384–392. doi: 10.1016/0042-6822(82)90176-3. [DOI] [PubMed] [Google Scholar]
- Haseloff J., Gerlach W. L. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature. 1988 Aug 18;334(6183):585–591. doi: 10.1038/334585a0. [DOI] [PubMed] [Google Scholar]
- Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
- Hyde-DeRuyscher R. P., Carmichael G. G. 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. 1990 Dec;64(12):5823–5832. doi: 10.1128/jvi.64.12.5823-5832.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hyde-DeRuyscher R., Carmichael G. G. 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 U S A. 1988 Dec;85(23):8993–8997. doi: 10.1073/pnas.85.23.8993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Inouye M. Antisense RNA: its functions and applications in gene regulation--a review. Gene. 1988 Dec 10;72(1-2):25–34. doi: 10.1016/0378-1119(88)90124-2. [DOI] [PubMed] [Google Scholar]
- Izant J. G., Weintraub H. Constitutive and conditional suppression of exogenous and endogenous genes by anti-sense RNA. Science. 1985 Jul 26;229(4711):345–352. doi: 10.1126/science.2990048. [DOI] [PubMed] [Google Scholar]
- Kamen R., Lindstrom D. M., Shure H., Old R. W. Virus-specific RNA in cells productively infected or transformed by polyoma virus. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):187–198. doi: 10.1101/sqb.1974.039.01.025. [DOI] [PubMed] [Google Scholar]
- Kim S. K., Wold B. J. Stable reduction of thymidine kinase activity in cells expressing high levels of anti-sense RNA. Cell. 1985 Aug;42(1):129–138. doi: 10.1016/s0092-8674(85)80108-2. [DOI] [PubMed] [Google Scholar]
- Krystal G. W., Armstrong B. C., Battey J. F. N-myc mRNA forms an RNA-RNA duplex with endogenous antisense transcripts. Mol Cell Biol. 1990 Aug;10(8):4180–4191. doi: 10.1128/mcb.10.8.4180. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lichtler A., Barrett N. L., Carmichael G. G. Simple, inexpensive preparation of T1/T2 ribonuclease suitable for use in RNase protection experiments. Biotechniques. 1992 Feb;12(2):231–232. [PubMed] [Google Scholar]
- Liu Z., Carmichael G. G. Polyoma virus early-late switch: regulation of late RNA accumulation by DNA replication. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8494–8498. doi: 10.1073/pnas.90.18.8494. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Munholland J. M., Kelly J. J., Hassell J. A., Wildeman A. G. Cell specificity of transcription regulation by papovavirus T antigens and DNA replication. EMBO J. 1992 Jan;11(1):177–184. doi: 10.1002/j.1460-2075.1992.tb05040.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Munroe S. H., Lazar M. A. Inhibition of c-erbA mRNA splicing by a naturally occurring antisense RNA. J Biol Chem. 1991 Nov 25;266(33):22083–22086. [PubMed] [Google Scholar]
- Piper P. W. Polyoma virus transcription early during productive infection of mouse 3T6 cells. J Mol Biol. 1979 Jun 25;131(2):399–407. doi: 10.1016/0022-2836(79)90083-4. [DOI] [PubMed] [Google Scholar]
- Takayama K. M., Inouye M. Antisense RNA. Crit Rev Biochem Mol Biol. 1990;25(3):155–184. doi: 10.3109/10409239009090608. [DOI] [PubMed] [Google Scholar]
- Treisman R., Kamen R. Structure of polyoma virus late nuclear RNA. J Mol Biol. 1981 May 25;148(3):273–301. doi: 10.1016/0022-2836(81)90539-8. [DOI] [PubMed] [Google Scholar]
- Volloch V., Schweitzer B., Rits S. Inhibition of pre-mRNA splicing by antisense RNA in vitro: effect of RNA containing sequences complementary to exons. Biochem Biophys Res Commun. 1991 Sep 30;179(3):1593–1599. doi: 10.1016/0006-291x(91)91756-3. [DOI] [PubMed] [Google Scholar]