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. 1994 Sep 11;22(18):3722–3727. doi: 10.1093/nar/22.18.3722

Identification of phosphate oxygens that are important for self-cleavage activity of the HDV ribozyme by phosphorothioate substitution interference analysis.

Y H Jeoung 1, P K Kumar 1, Y A Suh 1, K Taira 1, S Nishikawa 1
PMCID: PMC308353  PMID: 7937083

Abstract

A phosphorothioate substitution interference assay was used to investigate the role of the pro-Rp oxygens of phosphate groups in the self-cleavage reaction of the genomic human hepatitis delta virus (HDV) ribozyme. Incorporation of several different phosphorothioates (NTP alpha S) into the HDV ribozyme inhibited the self-cleavage activity. Incorporation of uridine 5' phosphorothioate or adenosine 5' phosphorothioate maintained 72% of the original self-cleavage activity whereas incorporation of guanosine 5' phosphorothioate or cytosine 5' phosphorothioate into the precursor reduced self-cleavage activity to about 20% in each case. Using partially substituted phosphorothioate-modified transcripts, we identified the pro-Rp oxygens that are important for the ribozyme activity, and they are located at positions 0, 1, 4, 5, 21, 24, 25, 27, 28, 30-34, 40, 43 and 75. In particular, the pro-Rp oxygens at positions 0, 1 and 21 are appear to be critical for the self-cleavage activity of the HDV ribozyme.

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Selected References

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  1. Belinsky M. G., Britton E., Dinter-Gottlieb G. Modification interference analysis of a self-cleaving RNA from hepatitis delta virus. FASEB J. 1993 Jan;7(1):130–136. doi: 10.1096/fasebj.7.1.8422959. [DOI] [PubMed] [Google Scholar]
  2. Buzayan J. M., Feldstein P. A., Bruening G., Eckstein F. RNA mediated formation of a phosphorothioate diester bond. Biochem Biophys Res Commun. 1988 Oct 14;156(1):340–347. doi: 10.1016/s0006-291x(88)80846-5. [DOI] [PubMed] [Google Scholar]
  3. Buzayan J. M., Feldstein P. A., Segrelles C., Bruening G. Autolytic processing of a phosphorothioate diester bond. Nucleic Acids Res. 1988 May 11;16(9):4009–4023. doi: 10.1093/nar/16.9.4009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chowrira B. M., Burke J. M. Extensive phosphorothioate substitution yields highly active and nuclease-resistant hairpin ribozymes. Nucleic Acids Res. 1992 Jun 11;20(11):2835–2840. doi: 10.1093/nar/20.11.2835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Christian E. L., Yarus M. Analysis of the role of phosphate oxygens in the group I intron from Tetrahymena. J Mol Biol. 1992 Dec 5;228(3):743–758. doi: 10.1016/0022-2836(92)90861-d. [DOI] [PubMed] [Google Scholar]
  6. Christian E. L., Yarus M. Metal coordination sites that contribute to structure and catalysis in the group I intron from Tetrahymena. Biochemistry. 1993 May 4;32(17):4475–4480. doi: 10.1021/bi00068a001. [DOI] [PubMed] [Google Scholar]
  7. Griffiths A. D., Potter B. V., Eperon I. C. Stereospecificity of nucleases towards phosphorothioate-substituted RNA: stereochemistry of transcription by T7 RNA polymerase. Nucleic Acids Res. 1987 May 26;15(10):4145–4162. doi: 10.1093/nar/15.10.4145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Griffiths A. D., Potter B. V., Eperon I. C. Substitution of pre-mRNA with phosphorothioate linkages reveals a new splicing-related reaction. J Biol Chem. 1988 Sep 5;263(25):12295–12304. [PubMed] [Google Scholar]
  9. Kawakami J., Kumar P. K., Suh Y. A., Nishikawa F., Kawakami K., Taira K., Ohtsuka E., Nishikawa S. Identification of important bases in a single-stranded region (SSrC) of the hepatitis delta (delta) virus ribozyme. Eur J Biochem. 1993 Oct 1;217(1):29–36. doi: 10.1111/j.1432-1033.1993.tb18214.x. [DOI] [PubMed] [Google Scholar]
  10. Kierzek R. Hydrolysis of oligoribonucleotides: influence of sequence and length. Nucleic Acids Res. 1992 Oct 11;20(19):5073–5077. doi: 10.1093/nar/20.19.5073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kumar P. K., Suh Y. A., Miyashiro H., Nishikawa F., Kawakami J., Taira K., Nishikawa S. Random mutations to evaluate the role of bases at two important single-stranded regions of genomic HDV ribozyme. Nucleic Acids Res. 1992 Aug 11;20(15):3919–3924. doi: 10.1093/nar/20.15.3919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kumar P. K., Suh Y. A., Taira K., Nishikawa S. Point and compensation mutations to evaluate essential stem structures of genomic HDV ribozyme. FASEB J. 1993 Jan;7(1):124–129. doi: 10.1096/fasebj.7.1.8422958. [DOI] [PubMed] [Google Scholar]
  13. Kumar P. K., Taira K., Nishikawa S. Chemical probing studies of variants of the genomic hepatitis delta virus ribozyme by primer extension analysis. Biochemistry. 1994 Jan 18;33(2):583–592. doi: 10.1021/bi00168a025. [DOI] [PubMed] [Google Scholar]
  14. Kuo M. Y., Sharmeen L., Dinter-Gottlieb G., Taylor J. Characterization of self-cleaving RNA sequences on the genome and antigenome of human hepatitis delta virus. J Virol. 1988 Dec;62(12):4439–4444. doi: 10.1128/jvi.62.12.4439-4444.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McSwiggen J. A., Cech T. R. Stereochemistry of RNA cleavage by the Tetrahymena ribozyme and evidence that the chemical step is not rate-limiting. Science. 1989 May 12;244(4905):679–683. doi: 10.1126/science.2470150. [DOI] [PubMed] [Google Scholar]
  16. Moore M. J., Sharp P. A. Evidence for two active sites in the spliceosome provided by stereochemistry of pre-mRNA splicing. Nature. 1993 Sep 23;365(6444):364–368. doi: 10.1038/365364a0. [DOI] [PubMed] [Google Scholar]
  17. Perrotta A. T., Been M. D. A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature. 1991 Apr 4;350(6317):434–436. doi: 10.1038/350434a0. [DOI] [PubMed] [Google Scholar]
  18. Piccirilli J. A., Vyle J. S., Caruthers M. H., Cech T. R. Metal ion catalysis in the Tetrahymena ribozyme reaction. Nature. 1993 Jan 7;361(6407):85–88. doi: 10.1038/361085a0. [DOI] [PubMed] [Google Scholar]
  19. Pyle A. M. Ribozymes: a distinct class of metalloenzymes. Science. 1993 Aug 6;261(5122):709–714. doi: 10.1126/science.7688142. [DOI] [PubMed] [Google Scholar]
  20. Ruffner D. E., Uhlenbeck O. C. Thiophosphate interference experiments locate phosphates important for the hammerhead RNA self-cleavage reaction. Nucleic Acids Res. 1990 Oct 25;18(20):6025–6029. doi: 10.1093/nar/18.20.6025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schatz D., Leberman R., Eckstein F. Interaction of Escherichia coli tRNA(Ser) with its cognate aminoacyl-tRNA synthetase as determined by footprinting with phosphorothioate-containing tRNA transcripts. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6132–6136. doi: 10.1073/pnas.88.14.6132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sharmeen L., Kuo M. Y., Dinter-Gottlieb G., Taylor J. Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage. J Virol. 1988 Aug;62(8):2674–2679. doi: 10.1128/jvi.62.8.2674-2679.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Steitz T. A., Steitz J. A. A general two-metal-ion mechanism for catalytic RNA. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6498–6502. doi: 10.1073/pnas.90.14.6498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Suh E., Waring R. B. A phosphorothioate at the 3' splice-site inhibits the second splicing step in a group I intron. Nucleic Acids Res. 1992 Dec 11;20(23):6303–6309. doi: 10.1093/nar/20.23.6303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Suh Y. A., Kumar P. K., Kawakami J., Nishikawa F., Taira K., Nishikawa S. Systematic substitution of individual bases in two important single-stranded regions of the HDV ribozyme for evaluation of the role of specific bases. FEBS Lett. 1993 Jul 12;326(1-3):158–162. doi: 10.1016/0014-5793(93)81782-u. [DOI] [PubMed] [Google Scholar]
  26. Suh Y. A., Kumar P. K., Nishikawa F., Kayano E., Nakai S., Odai O., Uesugi S., Taira K., Nishikawa S. Deletion of internal sequence on the HDV-ribozyme: elucidation of functionally important single-stranded loop regions. Nucleic Acids Res. 1992 Feb 25;20(4):747–753. doi: 10.1093/nar/20.4.747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tanner N. K., Schaff S., Thill G., Petit-Koskas E., Crain-Denoyelle A. M., Westhof E. A three-dimensional model of hepatitis delta virus ribozyme based on biochemical and mutational analyses. Curr Biol. 1994 Jun 1;4(6):488–498. doi: 10.1016/s0960-9822(00)00109-3. [DOI] [PubMed] [Google Scholar]
  28. Thill G., Vasseur M., Tanner N. K. Structural and sequence elements required for the self-cleaving activity of the hepatitis delta virus ribozyme. Biochemistry. 1993 Apr 27;32(16):4254–4262. doi: 10.1021/bi00067a013. [DOI] [PubMed] [Google Scholar]
  29. Uchimaru T., Uebayasi M., Tanabe K., Taira K. Theoretical analyses on the role of Mg2+ ions in ribozyme reactions. FASEB J. 1993 Jan;7(1):137–142. doi: 10.1096/fasebj.7.1.8422960. [DOI] [PubMed] [Google Scholar]
  30. Waring R. B. Identification of phosphate groups important to self-splicing of the Tetrahymena rRNA intron as determined by phosphorothioate substitution. Nucleic Acids Res. 1989 Dec 25;17(24):10281–10293. doi: 10.1093/nar/17.24.10281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wu H. N., Lin Y. J., Lin F. P., Makino S., Chang M. F., Lai M. M. Human hepatitis delta virus RNA subfragments contain an autocleavage activity. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1831–1835. doi: 10.1073/pnas.86.6.1831. [DOI] [PMC free article] [PubMed] [Google Scholar]

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