Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1998 Mar 15;26(6):1522–1527. doi: 10.1093/nar/26.6.1522

RNase L dimerization in a mammalian two-hybrid system in response to 2',5'-oligoadenylates.

S Naik 1, J M Paranjape 1, R H Silverman 1
PMCID: PMC147421  PMID: 9490801

Abstract

RNase L, a key enzyme in the anti-viral activity of interferons, requires activation by 2',5'-linked oligoadenylates (2-5A) to cleave viral and cellular single-stranded RNA. Here we demonstrate that 2-5A causes formation of stable dimers of RNase L in intact human cells as measured with a mammalian two-hybrid system. Hybrid proteins consisting of the GAL4 DNA binding domain fused to RNase L and the VP16 transactivation domain fused to RNase L were able to associate and drive transcription of a reporter gene, but only after cells were transfected with 2-5A. Several functional forms of 2-5A, such as p3A2'p5'A2'p5'A, were capable of activating transcription in human HeLa cells. In contrast, p3A2'p5'A, which can neither activate nor dimerize RNase L, did not induce gene expression. Evidence for the involvement of the C-terminal region of RNase L in dimerization was obtained by expressing truncated forms of RNase L. These findings describe a convenient, high-throughput screening method for RNase L activators which could lead to the discovery of novel anti-viral and anti-cancer agents.

Full Text

The Full Text of this article is available as a PDF (114.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allen J. B., Walberg M. W., Edwards M. C., Elledge S. J. Finding prospective partners in the library: the two-hybrid system and phage display find a match. Trends Biochem Sci. 1995 Dec;20(12):511–516. doi: 10.1016/s0968-0004(00)89119-7. [DOI] [PubMed] [Google Scholar]
  2. Cole J. L., Carroll S. S., Kuo L. C. Stoichiometry of 2',5'-oligoadenylate-induced dimerization of ribonuclease L. A sedimentation equilibrium study. J Biol Chem. 1996 Feb 23;271(8):3979–3981. doi: 10.1074/jbc.271.8.3979. [DOI] [PubMed] [Google Scholar]
  3. Dong B., Silverman R. H. 2-5A-dependent RNase molecules dimerize during activation by 2-5A. J Biol Chem. 1995 Feb 24;270(8):4133–4137. doi: 10.1074/jbc.270.8.4133. [DOI] [PubMed] [Google Scholar]
  4. Dong B., Silverman R. H. A bipartite model of 2-5A-dependent RNase L. J Biol Chem. 1997 Aug 29;272(35):22236–22242. doi: 10.1074/jbc.272.35.22236. [DOI] [PubMed] [Google Scholar]
  5. Dong B., Xu L., Zhou A., Hassel B. A., Lee X., Torrence P. F., Silverman R. H. Intrinsic molecular activities of the interferon-induced 2-5A-dependent RNase. J Biol Chem. 1994 May 13;269(19):14153–14158. [PubMed] [Google Scholar]
  6. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  7. Hassel B. A., Zhou A., Sotomayor C., Maran A., Silverman R. H. A dominant negative mutant of 2-5A-dependent RNase suppresses antiproliferative and antiviral effects of interferon. EMBO J. 1993 Aug;12(8):3297–3304. doi: 10.1002/j.1460-2075.1993.tb05999.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hovanessian A. G., Wood J., Meurs E., Montagnier L. Increased nuclease activity in cells treated with pppA2'p5'A2'p5' A. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3261–3265. doi: 10.1073/pnas.76.7.3261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kerr I. M., Brown R. E. pppA2'p5'A2'p5'A: an inhibitor of protein synthesis synthesized with an enzyme fraction from interferon-treated cells. Proc Natl Acad Sci U S A. 1978 Jan;75(1):256–260. doi: 10.1073/pnas.75.1.256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ma J., Ptashne M. Deletion analysis of GAL4 defines two transcriptional activating segments. Cell. 1987 Mar 13;48(5):847–853. doi: 10.1016/0092-8674(87)90081-x. [DOI] [PubMed] [Google Scholar]
  11. Maitra R. K., Li G., Xiao W., Dong B., Torrence P. F., Silverman R. H. Catalytic cleavage of an RNA target by 2-5A antisense and RNase L. J Biol Chem. 1995 Jun 23;270(25):15071–15075. doi: 10.1074/jbc.270.25.15071. [DOI] [PubMed] [Google Scholar]
  12. Maran A., Maitra R. K., Kumar A., Dong B., Xiao W., Li G., Williams B. R., Torrence P. F., Silverman R. H. Blockage of NF-kappa B signaling by selective ablation of an mRNA target by 2-5A antisense chimeras. Science. 1994 Aug 5;265(5173):789–792. doi: 10.1126/science.7914032. [DOI] [PubMed] [Google Scholar]
  13. Patel R. C., Stanton P., McMillan N. M., Williams B. R., Sen G. C. The interferon-inducible double-stranded RNA-activated protein kinase self-associates in vitro and in vivo. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8283–8287. doi: 10.1073/pnas.92.18.8283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rivera V. M., Clackson T., Natesan S., Pollock R., Amara J. F., Keenan T., Magari S. R., Phillips T., Courage N. L., Cerasoli F., Jr A humanized system for pharmacologic control of gene expression. Nat Med. 1996 Sep;2(9):1028–1032. doi: 10.1038/nm0996-1028. [DOI] [PubMed] [Google Scholar]
  15. Sadowski I., Ptashne M. A vector for expressing GAL4(1-147) fusions in mammalian cells. Nucleic Acids Res. 1989 Sep 25;17(18):7539–7539. doi: 10.1093/nar/17.18.7539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sobol R. W., Henderson E. E., Kon N., Shao J., Hitzges P., Mordechai E., Reichenbach N. L., Charubala R., Schirmeister H., Pfleiderer W. Inhibition of HIV-1 replication and activation of RNase L by phosphorothioate/phosphodiester 2',5'-oligoadenylate derivatives. J Biol Chem. 1995 Mar 17;270(11):5963–5978. doi: 10.1074/jbc.270.11.5963. [DOI] [PubMed] [Google Scholar]
  17. Takacs A. M., Das T., Banerjee A. K. Mapping of interacting domains between the nucleocapsid protein and the phosphoprotein of vesicular stomatitis virus by using a two-hybrid system. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10375–10379. doi: 10.1073/pnas.90.21.10375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Vasavada H. A., Ganguly S., Germino F. J., Wang Z. X., Weissman S. M. A contingent replication assay for the detection of protein-protein interactions in animal cells. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10686–10690. doi: 10.1073/pnas.88.23.10686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Zhou A., Hassel B. A., Silverman R. H. Expression cloning of 2-5A-dependent RNAase: a uniquely regulated mediator of interferon action. Cell. 1993 Mar 12;72(5):753–765. doi: 10.1016/0092-8674(93)90403-d. [DOI] [PubMed] [Google Scholar]
  20. Zhou A., Paranjape J., Brown T. L., Nie H., Naik S., Dong B., Chang A., Trapp B., Fairchild R., Colmenares C. Interferon action and apoptosis are defective in mice devoid of 2',5'-oligoadenylate-dependent RNase L. EMBO J. 1997 Nov 3;16(21):6355–6363. doi: 10.1093/emboj/16.21.6355. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES