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. 1997 Nov 15;25(22):4545–4550. doi: 10.1093/nar/25.22.4545

The RNA moiety of chick embryo 5-methylcytosine- DNA glycosylase targets DNA demethylation.

J P Jost 1, M Frémont 1, M Siegmann 1, J Hofsteenge 1
PMCID: PMC147099  PMID: 9358164

Abstract

We have previously shown that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. RNA from enzyme purified by SDS-PAGE was isolated and cloned. The clones have an insert ranging from 240 to 670 bp and contained on average one CpG per 14 bases. All six clones tested had different sequences and did not have any sequence homology with any other known RNA. RNase-inactivated 5-MeC-DNA glycosylase regained enzyme activity when incubated with recombinant RNA. However, when recombinant RNA was incubated with the DNA substrate alone there was no demethylation activity. Short sequences complementary to the labeled DNA substrate are present in the recombinant RNA. Small synthetic oligoribonucleotides (11 bases long) complementary to the region of methylated CpGs of the hemimethylated double-stranded DNA substrate restore the activity of the RNase-inactivated 5-MeC-DNA glycosylase. The corresponding oligodeoxyribonucleotide or the oligoribonucleotide complementary to the non-methylated strand of the same DNA substrate are inactive when incubated in the complementation test. A minimum of 4 bases complementary to the CpG target sequence are necessary for reactivation of RNase-treated 5-MeC-DNA glycosylase. Complementation with double-stranded oligoribonucleotides does not restore 5-MeC-DNA glycosylase activity. An excess of targeting oligoribonucleotides cannot change the preferential substrate specificity of the enzyme for hemimethylated double-stranded DNA.

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Selected References

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  1. Angell S. M., Baulcombe D. C. Consistent gene silencing in transgenic plants expressing a replicating potato virus X RNA. EMBO J. 1997 Jun 16;16(12):3675–3684. doi: 10.1093/emboj/16.12.3675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Antequera F., Bird A. CpG islands. EXS. 1993;64:169–185. doi: 10.1007/978-3-0348-9118-9_8. [DOI] [PubMed] [Google Scholar]
  3. Brandeis M., Frank D., Keshet I., Siegfried Z., Mendelsohn M., Nemes A., Temper V., Razin A., Cedar H. Sp1 elements protect a CpG island from de novo methylation. Nature. 1994 Sep 29;371(6496):435–438. doi: 10.1038/371435a0. [DOI] [PubMed] [Google Scholar]
  4. Cavaillé J., Nicoloso M., Bachellerie J. P. Targeted ribose methylation of RNA in vivo directed by tailored antisense RNA guides. Nature. 1996 Oct 24;383(6602):732–735. doi: 10.1038/383732a0. [DOI] [PubMed] [Google Scholar]
  5. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Frémont M., Siegmann M., Gaulis S., Matthies R., Hess D., Jost J. P. Demethylation of DNA by purified chick embryo 5-methylcytosine-DNA glycosylase requires both protein and RNA. Nucleic Acids Res. 1997 Jun 15;25(12):2375–2380. doi: 10.1093/nar/25.12.2375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Glisin V., Crkvenjakov R., Byus C. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry. 1974 Jun 4;13(12):2633–2637. doi: 10.1021/bi00709a025. [DOI] [PubMed] [Google Scholar]
  9. Greider C. W., Blackburn E. H. The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell. 1987 Dec 24;51(6):887–898. doi: 10.1016/0092-8674(87)90576-9. [DOI] [PubMed] [Google Scholar]
  10. Guerrier-Takada C., Gardiner K., Marsh T., Pace N., Altman S. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell. 1983 Dec;35(3 Pt 2):849–857. doi: 10.1016/0092-8674(83)90117-4. [DOI] [PubMed] [Google Scholar]
  11. Jost J. P. Nuclear extracts of chicken embryos promote an active demethylation of DNA by excision repair of 5-methyldeoxycytidine. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4684–4688. doi: 10.1073/pnas.90.10.4684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jost J. P., Siegmann M., Sun L., Leung R. Mechanisms of DNA demethylation in chicken embryos. Purification and properties of a 5-methylcytosine-DNA glycosylase. J Biol Chem. 1995 Apr 28;270(17):9734–9739. doi: 10.1074/jbc.270.17.9734. [DOI] [PubMed] [Google Scholar]
  13. Kirillov A., Kistler B., Mostoslavsky R., Cedar H., Wirth T., Bergman Y. A role for nuclear NF-kappaB in B-cell-specific demethylation of the Igkappa locus. Nat Genet. 1996 Aug;13(4):435–441. doi: 10.1038/ng0895-435. [DOI] [PubMed] [Google Scholar]
  14. Kiss-László Z., Henry Y., Bachellerie J. P., Caizergues-Ferrer M., Kiss T. Site-specific ribose methylation of preribosomal RNA: a novel function for small nucleolar RNAs. Cell. 1996 Jun 28;85(7):1077–1088. doi: 10.1016/s0092-8674(00)81308-2. [DOI] [PubMed] [Google Scholar]
  15. Lundblad V., Wright W. E. Telomeres and telomerase: a simple picture becomes complex. Cell. 1996 Nov 1;87(3):369–375. doi: 10.1016/s0092-8674(00)81358-6. [DOI] [PubMed] [Google Scholar]
  16. Macleod D., Charlton J., Mullins J., Bird A. P. Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. Genes Dev. 1994 Oct 1;8(19):2282–2292. doi: 10.1101/gad.8.19.2282. [DOI] [PubMed] [Google Scholar]
  17. Neumann U., Hofsteenge J., Arkema A. H., Dijkstra B. W. Crystallization of porcine liver ribonuclease inhibitor a member of the family of proteins containing leucine-rich repeats. J Mol Biol. 1993 May 20;231(2):505–508. doi: 10.1006/jmbi.1993.1299. [DOI] [PubMed] [Google Scholar]
  18. Tollervey D. Small nucleolar RNAs guide ribosomal RNA methylation. Science. 1996 Aug 23;273(5278):1056–1057. doi: 10.1126/science.273.5278.1056. [DOI] [PubMed] [Google Scholar]
  19. Wassenegger M., Heimes S., Riedel L., Sänger H. L. RNA-directed de novo methylation of genomic sequences in plants. Cell. 1994 Feb 11;76(3):567–576. doi: 10.1016/0092-8674(94)90119-8. [DOI] [PubMed] [Google Scholar]
  20. Weiss A., Keshet I., Razin A., Cedar H. DNA demethylation in vitro: involvement of RNA. Cell. 1996 Sep 6;86(5):709–718. doi: 10.1016/s0092-8674(00)80146-4. [DOI] [PubMed] [Google Scholar]
  21. Zimmerly S., Guo H., Eskes R., Yang J., Perlman P. S., Lambowitz A. M. A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility. Cell. 1995 Nov 17;83(4):529–538. doi: 10.1016/0092-8674(95)90092-6. [DOI] [PubMed] [Google Scholar]

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