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. 1994 Jun 15;13(12):2904–2912. doi: 10.1002/j.1460-2075.1994.tb06585.x

Facilitation of hammerhead ribozyme catalysis by the nucleocapsid protein of HIV-1 and the heterogeneous nuclear ribonucleoprotein A1.

E L Bertrand 1, J J Rossi 1
PMCID: PMC395172  PMID: 8026475

Abstract

In order to improve the activity of hammerhead ribozymes in vivo, we have analyzed the effect of several prototypical RNA binding proteins on the ribozyme cleavage reaction: bacteriophage T4 gene 32 protein (gp32), hnRNP A1 (A1) and the nucleocapsid protein of HIV-1 (NCp7). We show that, while gp32 has no effect on the cleavage reaction, A1 and NCp7 affect different steps of the reaction. Moreover, some of these effects depend upon the ribozyme-substrate hybrid length. A1 and NCp7 inhibit the reaction of the least stable ribozyme-substrate complexes, which have 12 bp of duplex. NCp7, but not A1, inhibits the cleavage of substrates that have long ribozyme-substrate duplexes (17 or 20 bp), while cleavage of complexes having shorter duplexes (13 or 14 bp) is not affected. NCp7 and A1 enhance the turnover of ribozymes by increasing the rate of product dissociation, but only when both cleavage products are bound with < or = 7 bp. A1 and NCp7 enhance ribozyme binding to long substrates, such as mRNAs, the structure of which otherwise limits ribozyme binding. Therefore, the effects of A1 or NCp7 on the different steps of the cleavage reaction define a length of the ribozyme-substrate duplex which allows enhancement of the rate of binding and product release without inhibiting the cleavage step. Interestingly, this duplex length (14 bases, or 7 on each side of the cleavage site) is identical for A1 and NCp7. Since A1 is thought to interact with most, if not all mRNAs in vivo, it may enhance the intracellular activity of ribozymes targeted against any mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

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Selected References

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

  1. Berkowitz R. D., Luban J., Goff S. P. Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays. J Virol. 1993 Dec;67(12):7190–7200. doi: 10.1128/jvi.67.12.7190-7200.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bertrand E., Pictet R., Grange T. Can hammerhead ribozymes be efficient tools to inactivate gene function? Nucleic Acids Res. 1994 Feb 11;22(3):293–300. doi: 10.1093/nar/22.3.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bodner M., Castrillo J. L., Theill L. E., Deerinck T., Ellisman M., Karin M. The pituitary-specific transcription factor GHF-1 is a homeobox-containing protein. Cell. 1988 Nov 4;55(3):505–518. doi: 10.1016/0092-8674(88)90037-2. [DOI] [PubMed] [Google Scholar]
  4. Buvoli M., Cobianchi F., Riva S. Interaction of hnRNP A1 with snRNPs and pre-mRNAs: evidence for a possible role of A1 RNA annealing activity in the first steps of spliceosome assembly. Nucleic Acids Res. 1992 Oct 11;20(19):5017–5025. doi: 10.1093/nar/20.19.5017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cameron F. H., Jennings P. A. Specific gene suppression by engineered ribozymes in monkey cells. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9139–9143. doi: 10.1073/pnas.86.23.9139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Casas-Finet J. R., Karpel R. L. Bacteriophage T4 gene 32 protein: modulation of protein-nucleic acid and protein-protein association by structural domains. Biochemistry. 1993 Sep 21;32(37):9735–9744. doi: 10.1021/bi00088a028. [DOI] [PubMed] [Google Scholar]
  7. Casas-Finet J. R., Smith J. D., Jr, Kumar A., Kim J. G., Wilson S. H., Karpel R. L. Mammalian heterogeneous ribonucleoprotein A1 and its constituent domains. Nucleic acid interaction, structural stability and self-association. J Mol Biol. 1993 Feb 20;229(4):873–889. doi: 10.1006/jmbi.1993.1093. [DOI] [PubMed] [Google Scholar]
  8. Castanotto D., Rossi J. J., Deshler J. O. Biological and functional aspects of catalytic RNAs. Crit Rev Eukaryot Gene Expr. 1992;2(4):331–357. [PubMed] [Google Scholar]
  9. Cobianchi F., Karpel R. L., Williams K. R., Notario V., Wilson S. H. Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. Large-scale overproduction in Escherichia coli and cooperative binding to single-stranded nucleic acids. J Biol Chem. 1988 Jan 15;263(2):1063–1071. [PubMed] [Google Scholar]
  10. Cobianchi F., SenGupta D. N., Zmudzka B. Z., Wilson S. H. Structure of rodent helix-destabilizing protein revealed by cDNA cloning. J Biol Chem. 1986 Mar 15;261(8):3536–3543. [PubMed] [Google Scholar]
  11. Cotten M., Birnstiel M. L. Ribozyme mediated destruction of RNA in vivo. EMBO J. 1989 Dec 1;8(12):3861–3866. doi: 10.1002/j.1460-2075.1989.tb08564.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cotten M., Schaffner G., Birnstiel M. L. Ribozyme, antisense RNA, and antisense DNA inhibition of U7 small nuclear ribonucleoprotein-mediated histone pre-mRNA processing in vitro. Mol Cell Biol. 1989 Oct;9(10):4479–4487. doi: 10.1128/mcb.9.10.4479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Covey S. N. Amino acid sequence homology in gag region of reverse transcribing elements and the coat protein gene of cauliflower mosaic virus. Nucleic Acids Res. 1986 Jan 24;14(2):623–633. doi: 10.1093/nar/14.2.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Darlix J. L., Gabus C., Nugeyre M. T., Clavel F., Barré-Sinoussi F. Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1. J Mol Biol. 1990 Dec 5;216(3):689–699. doi: 10.1016/0022-2836(90)90392-Y. [DOI] [PubMed] [Google Scholar]
  15. De Rocquigny H., Ficheux D., Gabus C., Allain B., Fournie-Zaluski M. C., Darlix J. L., Roques B. P. Two short basic sequences surrounding the zinc finger of nucleocapsid protein NCp10 of Moloney murine leukemia virus are critical for RNA annealing activity. Nucleic Acids Res. 1993 Feb 25;21(4):823–829. doi: 10.1093/nar/21.4.823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. De Rocquigny H., Gabus C., Vincent A., Fournié-Zaluski M. C., Roques B., Darlix J. L. Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6472–6476. doi: 10.1073/pnas.89.14.6472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dorfman T., Luban J., Goff S. P., Haseltine W. A., Göttlinger H. G. Mapping of functionally important residues of a cysteine-histidine box in the human immunodeficiency virus type 1 nucleocapsid protein. J Virol. 1993 Oct;67(10):6159–6169. doi: 10.1128/jvi.67.10.6159-6169.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dreyfuss G. Structure and function of nuclear and cytoplasmic ribonucleoprotein particles. Annu Rev Cell Biol. 1986;2:459–498. doi: 10.1146/annurev.cb.02.110186.002331. [DOI] [PubMed] [Google Scholar]
  19. Dropulić B., Lin N. H., Martin M. A., Jeang K. T. Functional characterization of a U5 ribozyme: intracellular suppression of human immunodeficiency virus type 1 expression. J Virol. 1992 Mar;66(3):1432–1441. doi: 10.1128/jvi.66.3.1432-1441.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Dupraz P., Spahr P. F. Specificity of Rous sarcoma virus nucleocapsid protein in genomic RNA packaging. J Virol. 1992 Aug;66(8):4662–4670. doi: 10.1128/jvi.66.8.4662-4670.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Forster A. C., Symons R. H. Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell. 1987 Apr 24;49(2):211–220. doi: 10.1016/0092-8674(87)90562-9. [DOI] [PubMed] [Google Scholar]
  22. Fu X. D., Maniatis T. The 35-kDa mammalian splicing factor SC35 mediates specific interactions between U1 and U2 small nuclear ribonucleoprotein particles at the 3' splice site. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1725–1729. doi: 10.1073/pnas.89.5.1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gaillard C., Strauss F. Ethanol precipitation of DNA with linear polyacrylamide as carrier. Nucleic Acids Res. 1990 Jan 25;18(2):378–378. doi: 10.1093/nar/18.2.378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Heidenreich O., Eckstein F. Hammerhead ribozyme-mediated cleavage of the long terminal repeat RNA of human immunodeficiency virus type 1. J Biol Chem. 1992 Jan 25;267(3):1904–1909. [PubMed] [Google Scholar]
  26. Ingraham H. A., Chen R. P., Mangalam H. J., Elsholtz H. P., Flynn S. E., Lin C. R., Simmons D. M., Swanson L., Rosenfeld M. G. A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell. 1988 Nov 4;55(3):519–529. doi: 10.1016/0092-8674(88)90038-4. [DOI] [PubMed] [Google Scholar]
  27. Jeffries A. C., Symons R. H. A catalytic 13-mer ribozyme. Nucleic Acids Res. 1989 Feb 25;17(4):1371–1377. doi: 10.1093/nar/17.4.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Karpel R. L., Miller N. S., Fresco J. R. Mechanistic studies of ribonucleic acid renaturation by a helix-destabilizing protein. Biochemistry. 1982 Apr 27;21(9):2102–2108. doi: 10.1021/bi00538a019. [DOI] [PubMed] [Google Scholar]
  29. Kenan D. J., Query C. C., Keene J. D. RNA recognition: towards identifying determinants of specificity. Trends Biochem Sci. 1991 Jun;16(6):214–220. doi: 10.1016/0968-0004(91)90088-d. [DOI] [PubMed] [Google Scholar]
  30. Khan R., Giedroc D. P. Recombinant human immunodeficiency virus type 1 nucleocapsid (NCp7) protein unwinds tRNA. J Biol Chem. 1992 Apr 5;267(10):6689–6695. [PubMed] [Google Scholar]
  31. Kiledjian M., Dreyfuss G. Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box. EMBO J. 1992 Jul;11(7):2655–2664. doi: 10.1002/j.1460-2075.1992.tb05331.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kumar A., Wilson S. H. Studies of the strand-annealing activity of mammalian hnRNP complex protein A1. Biochemistry. 1990 Dec 4;29(48):10717–10722. doi: 10.1021/bi00500a001. [DOI] [PubMed] [Google Scholar]
  33. L'Huillier P. J., Davis S. R., Bellamy A. R. Cytoplasmic delivery of ribozymes leads to efficient reduction in alpha-lactalbumin mRNA levels in C127I mouse cells. EMBO J. 1992 Dec;11(12):4411–4418. doi: 10.1002/j.1460-2075.1992.tb05541.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lee C. G., Zamore P. D., Green M. R., Hurwitz J. RNA annealing activity is intrinsically associated with U2AF. J Biol Chem. 1993 Jun 25;268(18):13472–13478. [PubMed] [Google Scholar]
  35. Mayeda A., Helfman D. M., Krainer A. R. Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF. Mol Cell Biol. 1993 May;13(5):2993–3001. doi: 10.1128/mcb.13.5.2993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mayeda A., Krainer A. R. Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2. Cell. 1992 Jan 24;68(2):365–375. doi: 10.1016/0092-8674(92)90477-t. [DOI] [PubMed] [Google Scholar]
  37. Mayrand S. H., Pederson T. Crosslinking of hnRNP proteins to pre-mRNA requires U1 and U2 snRNPs. Nucleic Acids Res. 1990 Jun 11;18(11):3307–3318. doi: 10.1093/nar/18.11.3307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Munroe S. H., Dong X. F. Heterogeneous nuclear ribonucleoprotein A1 catalyzes RNA.RNA annealing. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):895–899. doi: 10.1073/pnas.89.3.895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nadler S. G., Merrill B. M., Roberts W. J., Keating K. M., Lisbin M. J., Barnett S. F., Wilson S. H., Williams K. R. Interactions of the A1 heterogeneous nuclear ribonucleoprotein and its proteolytic derivative, UP1, with RNA and DNA: evidence for multiple RNA binding domains and salt-dependent binding mode transitions. Biochemistry. 1991 Mar 19;30(11):2968–2976. doi: 10.1021/bi00225a034. [DOI] [PubMed] [Google Scholar]
  40. Piñol-Roma S., Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature. 1992 Feb 20;355(6362):730–732. doi: 10.1038/355730a0. [DOI] [PubMed] [Google Scholar]
  41. Pontius B. W., Berg P. Rapid assembly and disassembly of complementary DNA strands through an equilibrium intermediate state mediated by A1 hnRNP protein. J Biol Chem. 1992 Jul 15;267(20):13815–13818. [PubMed] [Google Scholar]
  42. Pontius B. W., Berg P. Renaturation of complementary DNA strands mediated by purified mammalian heterogeneous nuclear ribonucleoprotein A1 protein: implications for a mechanism for rapid molecular assembly. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8403–8407. doi: 10.1073/pnas.87.21.8403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pontius B. W. Close encounters: why unstructured, polymeric domains can increase rates of specific macromolecular association. Trends Biochem Sci. 1993 May;18(5):181–186. doi: 10.1016/0968-0004(93)90111-y. [DOI] [PubMed] [Google Scholar]
  44. Portman D. S., Dreyfuss G. RNA annealing activities in HeLa nuclei. EMBO J. 1994 Jan 1;13(1):213–221. doi: 10.1002/j.1460-2075.1994.tb06251.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Prats A. C., Sarih L., Gabus C., Litvak S., Keith G., Darlix J. L. Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA. EMBO J. 1988 Jun;7(6):1777–1783. doi: 10.1002/j.1460-2075.1988.tb03008.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rozen F., Edery I., Meerovitch K., Dever T. E., Merrick W. C., Sonenberg N. Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F. Mol Cell Biol. 1990 Mar;10(3):1134–1144. doi: 10.1128/mcb.10.3.1134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sakaguchi K., Zambrano N., Baldwin E. T., Shapiro B. A., Erickson J. W., Omichinski J. G., Clore G. M., Gronenborn A. M., Appella E. Identification of a binding site for the human immunodeficiency virus type 1 nucleocapsid protein. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5219–5223. doi: 10.1073/pnas.90.11.5219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sarver N., Cantin E. M., Chang P. S., Zaia J. A., Ladne P. A., Stephens D. A., Rossi J. J. Ribozymes as potential anti-HIV-1 therapeutic agents. Science. 1990 Mar 9;247(4947):1222–1225. doi: 10.1126/science.2107573. [DOI] [PubMed] [Google Scholar]
  49. Saxena S. K., Ackerman E. J. Ribozymes correctly cleave a model substrate and endogenous RNA in vivo. J Biol Chem. 1990 Oct 5;265(28):17106–17109. [PubMed] [Google Scholar]
  50. Scanlon K. J., Jiao L., Funato T., Wang W., Tone T., Rossi J. J., Kashani-Sabet M. Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10591–10595. doi: 10.1073/pnas.88.23.10591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sioud M., Drlica K. Prevention of human immunodeficiency virus type 1 integrase expression in Escherichia coli by a ribozyme. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7303–7307. doi: 10.1073/pnas.88.16.7303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sioud M., Natvig J. B., Førre O. Preformed ribozyme destroys tumour necrosis factor mRNA in human cells. J Mol Biol. 1992 Feb 20;223(4):831–835. doi: 10.1016/0022-2836(92)90244-e. [DOI] [PubMed] [Google Scholar]
  53. Steinecke P., Herget T., Schreier P. H. Expression of a chimeric ribozyme gene results in endonucleolytic cleavage of target mRNA and a concomitant reduction of gene expression in vivo. EMBO J. 1992 Apr;11(4):1525–1530. doi: 10.1002/j.1460-2075.1992.tb05197.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sullenger B. A., Cech T. R. Tethering ribozymes to a retroviral packaging signal for destruction of viral RNA. Science. 1993 Dec 3;262(5139):1566–1569. doi: 10.1126/science.8248806. [DOI] [PubMed] [Google Scholar]
  55. Surovoy A., Dannull J., Moelling K., Jung G. Conformational and nucleic acid binding studies on the synthetic nucleocapsid protein of HIV-1. J Mol Biol. 1993 Jan 5;229(1):94–104. doi: 10.1006/jmbi.1993.1011. [DOI] [PubMed] [Google Scholar]
  56. Symons R. H. Small catalytic RNAs. Annu Rev Biochem. 1992;61:641–671. doi: 10.1146/annurev.bi.61.070192.003233. [DOI] [PubMed] [Google Scholar]
  57. Tsuchihashi Z., Khosla M., Herschlag D. Protein enhancement of hammerhead ribozyme catalysis. Science. 1993 Oct 1;262(5130):99–102. doi: 10.1126/science.7692597. [DOI] [PubMed] [Google Scholar]
  58. Uhlenbeck O. C. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. doi: 10.1038/328596a0. [DOI] [PubMed] [Google Scholar]
  59. Wassarman D. A., Steitz J. A. RNA splicing. Alive with DEAD proteins. Nature. 1991 Feb 7;349(6309):463–464. doi: 10.1038/349463a0. [DOI] [PubMed] [Google Scholar]
  60. Weerasinghe M., Liem S. E., Asad S., Read S. E., Joshi S. Resistance to human immunodeficiency virus type 1 (HIV-1) infection in human CD4+ lymphocyte-derived cell lines conferred by using retroviral vectors expressing an HIV-1 RNA-specific ribozyme. J Virol. 1991 Oct;65(10):5531–5534. doi: 10.1128/jvi.65.10.5531-5534.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wu J. Y., Maniatis T. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell. 1993 Dec 17;75(6):1061–1070. doi: 10.1016/0092-8674(93)90316-i. [DOI] [PubMed] [Google Scholar]
  62. Zhao J. J., Pick L. Generating loss-of-function phenotypes of the fushi tarazu gene with a targeted ribozyme in Drosophila. Nature. 1993 Sep 30;365(6445):448–451. doi: 10.1038/365448a0. [DOI] [PubMed] [Google Scholar]

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