Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Aug 1;105(2):705–711. doi: 10.1083/jcb.105.2.705

Early hypomethylation of 2'-O-ribose moieties in hepatocyte cytoplasmic ribosomal RNA underlies the protein synthetic defect produced by CCl4

PMCID: PMC2114784  PMID: 3114267

Abstract

Carbon tetrachloride (CCl4) treatment of rats produces an early defect in methylation of hepatocyte ribosomal RNA, which occurs concurrently with a defect in the protein synthetic capacity of isolated ribosomes. The CCl4-induced methylation defect is specific for the 2'-O-ribose position, and a corresponding proportional increase in m7G base methylation occurs in vivo. Undermethylated ribosomal subunits (rRNA) from CCl4-treated preparations can be methylated in vitro to a much greater extent than those from control preparations, and in vitro methylation restores their functional capacity. In vitro methylation of treated ribosomal subunits (which restores functional capacity) occurs at 2'-O-ribose positions (largely G residues). In contrast, in vitro methylation of control ribosomal subunits (which does not affect functional activity) represents base methylation as m7G, sites which are apparently methylated in treated preparations in vivo. Methylation/demethylation of 2'-O-ribose sites in rRNA exposed on the surface of cytoplasmic ribosomal subunits may represent an important cellular mechanism for controlling protein synthesis in quiescent hepatocytes, and it appears that CCl4 disrupts protein synthesis by inhibiting this 2'-O-ribose methylation.

Full Text

The Full Text of this article is available as a PDF (790.5 KB).

Selected References

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

  1. Blobel G., Potter V. R. Nuclei from rat liver: isolation method that combines purity with high yield. Science. 1966 Dec 30;154(3757):1662–1665. doi: 10.1126/science.154.3757.1662. [DOI] [PubMed] [Google Scholar]
  2. Brand R. C., Klootwijk J., Van Steenbergen T. J., De Kok A. J., Planta R. J. Secondary methylation of yeast ribosomal precursor RNA. Eur J Biochem. 1977 May 2;75(1):311–318. doi: 10.1111/j.1432-1033.1977.tb11531.x. [DOI] [PubMed] [Google Scholar]
  3. Brattin W. J., Pencil S. D., Waller R. L., Glende E. A., Jr, Recknagel R. O. Assessment of the role of calcium ion in halocarbon hepatotoxicity. Environ Health Perspect. 1984 Aug;57:321–323. doi: 10.1289/ehp.8457321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown G. E., Kolb A. J., Stanley W. M., Jr A general procedure for the preparation of highly active eukaryotic ribosomes and ribosomal subunits. Methods Enzymol. 1974;30:368–387. doi: 10.1016/0076-6879(74)30039-0. [DOI] [PubMed] [Google Scholar]
  5. CHRISTIE G. S., JUDAH J. D. Mechanism of action of carbon tetrachloride on liver cells. Proc R Soc Lond B Biol Sci. 1954 Mar 25;142(907):241–257. doi: 10.1098/rspb.1954.0024. [DOI] [PubMed] [Google Scholar]
  6. Caboche M., Bachellerie J. P. RNA methylation and control of eukaryotic RNA biosynthesis. Effects of cycloleucine, a specific inhibitor of methylation, on ribosomal RNA maturation. Eur J Biochem. 1977 Mar 15;74(1):19–29. doi: 10.1111/j.1432-1033.1977.tb11362.x. [DOI] [PubMed] [Google Scholar]
  7. Camper S. A., Albers R. J., Coward J. K., Rottman F. M. Effect of undermethylation on mRNA cytoplasmic appearance and half-life. Mol Cell Biol. 1984 Mar;4(3):538–543. doi: 10.1128/mcb.4.3.538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Erwin B. G., Stoscheck C. M., Florini J. R. A rapid fluorometric method for the estimation of DNA in cultured cells. Anal Biochem. 1981 Jan 15;110(2):291–294. doi: 10.1016/0003-2697(81)90194-9. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Goddard J. P., Maden B. E. Reaction of HeLa cell methyl-labelled 28S ribosomal RNA with sodium bisulphite: a conformational probe for methylated sequences. Nucleic Acids Res. 1976 Feb;3(2):431–440. doi: 10.1093/nar/3.2.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Helser T. L., Davies J. E., Dahlberg J. E. Mechanism of kasugamycin resistance in Escherichia coli. Nat New Biol. 1972 Jan 5;235(53):6–9. doi: 10.1038/newbio235006a0. [DOI] [PubMed] [Google Scholar]
  13. Kane S. E., Beemon K. Precise localization of m6A in Rous sarcoma virus RNA reveals clustering of methylation sites: implications for RNA processing. Mol Cell Biol. 1985 Sep;5(9):2298–2306. doi: 10.1128/mcb.5.9.2298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kelker H. C., Pogo A. O. The stringent and relaxed phenomena in Saccharomyces cerevisiae. J Biol Chem. 1980 Feb 25;255(4):1526–1535. [PubMed] [Google Scholar]
  15. Khan M. S., Maden B. E. Conformation of methylated sequences in HeLa cell 18-S ribosomal RNA: nuclease S1 as a probe. Eur J Biochem. 1978 Mar;84(1):241–250. doi: 10.1111/j.1432-1033.1978.tb12162.x. [DOI] [PubMed] [Google Scholar]
  16. Klootwijk J., Planta R. J. Analysis of the methylation sites in yeast ribosomal RNA. Eur J Biochem. 1973 Nov 15;39(2):325–333. doi: 10.1111/j.1432-1033.1973.tb03130.x. [DOI] [PubMed] [Google Scholar]
  17. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  18. Lai E. K., McCay P. B., Noguchi T., Fong K. L. In vivo spin-trapping of trichloromethyl radicals formed from CCl4. Biochem Pharmacol. 1979 Jul 15;28(14):2231–2235. doi: 10.1016/0006-2952(79)90212-0. [DOI] [PubMed] [Google Scholar]
  19. Li A. W., Singer R. A., Johnston G. C. Effects of sinefungin on rRNA production and methylation in the yeast Saccharomyces cerevisiae. Arch Biochem Biophys. 1985 Aug 1;240(2):613–620. doi: 10.1016/0003-9861(85)90068-2. [DOI] [PubMed] [Google Scholar]
  20. MURAMATSU M., BUSCH H. STUDIES ON NUCLEOLAR RNA OF THE WALKER 256 CARCINOSARCOMA AND THE LIVER OF THE RAT. Cancer Res. 1964 Jul;24:1028–1034. [PubMed] [Google Scholar]
  21. Maden B. E.H., Lees C. D., Salim M. Some methylated sequences and the numbers of methyl groups in HeLa cell rRNA. FEBS Lett. 1972 Dec 15;28(3):293–296. doi: 10.1016/0014-5793(72)80734-8. [DOI] [PubMed] [Google Scholar]
  22. Maden B. E., Salim M., Summers D. F. Maturation pathway for ribosomal RNA in the Hela cell nucleolus. Nat New Biol. 1972 May 3;237(70):5–9. doi: 10.1038/newbio237005a0. [DOI] [PubMed] [Google Scholar]
  23. Monlux G., Smuckler E. A. An autoradiographic study of protein synthesis in mouse liver parenchymal cells during CC14 intoxication. Am J Pathol. 1969 Jan;54(1):73–82. [PMC free article] [PubMed] [Google Scholar]
  24. Moody D. E., James J. L., Clawson G. A., Smuckler E. A. Correlations among the changes in hepatic microsomal components after intoxication with alkyl halides and other hepatotoxins. Mol Pharmacol. 1981 Nov;20(3):685–693. [PubMed] [Google Scholar]
  25. Muthukrishnan S., Moss B., Cooper J. A., Maxwell E. S. Influence of 5'-terminal cap structure on the initiation of translation of vaccinia virus mRNA. J Biol Chem. 1978 Mar 10;253(5):1710–1715. [PubMed] [Google Scholar]
  26. Ouellette A. J., Bandman E., Kumar A. Regulation of ribosomal RNA methylation in a temperature-sensitive mutant of BHK cells. Nature. 1976 Aug 12;262(5569):619–621. doi: 10.1038/262619a0. [DOI] [PubMed] [Google Scholar]
  27. Perry R. P., Kelley D. E. Kinetics of formation of 5' terminal caps in mRNA. Cell. 1976 Jul;8(3):433–442. doi: 10.1016/0092-8674(76)90156-2. [DOI] [PubMed] [Google Scholar]
  28. Perry R. P. Processing of RNA. Annu Rev Biochem. 1976;45:605–629. doi: 10.1146/annurev.bi.45.070176.003133. [DOI] [PubMed] [Google Scholar]
  29. Poldermans B., Bakker H., Van Knippenberg P. H. Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3' end of 16S ribosomal RNA of Escherichia coli. IV. The effect of the methylgroups on ribosomal subunit interaction. Nucleic Acids Res. 1980 Jan 11;8(1):143–151. doi: 10.1093/nar/8.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Poldermans B., Goosen N., Van Knippenberg P. H. Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3' end of 16 S ribosomal RNA of Escherichia coli. I. The effect of kasugamycin on initiation of protein synthesis. J Biol Chem. 1979 Sep 25;254(18):9085–9089. [PubMed] [Google Scholar]
  31. Poldermans B., Van Buul C. P., Van Knippenberg P. H. Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3' end of 16 S ribosomal RNA of Escherichia coli. II. The effect of the absence of the methyl groups on initiation of protein biosynthesis. J Biol Chem. 1979 Sep 25;254(18):9090–9093. [PubMed] [Google Scholar]
  32. Prince D. L., Kotin R. M., Dubin D. T. Evidence that the methylation inhibitor cycloleucine causes accumulation of a discrete ribosomal RNA precursor in hamster mitochondria. Mol Biol Rep. 1986;11(1):51–55. doi: 10.1007/BF00417596. [DOI] [PubMed] [Google Scholar]
  33. RECKNAGEL R. O., LOMBARDI B. Studies of biochemical changes in subcellular particles of rat liver and their relationship to a new hypothesis regarding the pathogenesis of carbon tetrachloride fat accumulation. J Biol Chem. 1961 Feb;236:564–569. [PubMed] [Google Scholar]
  34. REYNOLDS E. S. LIVER PARENCHYMAL CELL INJURY. I. INITIAL ALTERATIONS OF THE CELL FOLLOWING POISONING WITH CARBON TETRACHLORIDE. J Cell Biol. 1963 Oct;19:139–157. doi: 10.1083/jcb.19.1.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rechnagel R. O., Glende E. A., Jr Carbon tetrachloride hepatotoxicity: an example of lethal cleavage. CRC Crit Rev Toxicol. 1973 Nov;2(3):263–297. doi: 10.3109/10408447309082019. [DOI] [PubMed] [Google Scholar]
  36. Recknagel R. O. A new direction in the study of carbon tetrachloride hepatotoxicity. Life Sci. 1983 Aug 1;33(5):401–408. doi: 10.1016/0024-3205(83)90787-7. [DOI] [PubMed] [Google Scholar]
  37. Reynolds E. S. Liver parenchymal cell injury. IV. Pattern of incorporation of carbon and chlorine from carbon tetrachloride into chemical constituents of liver in vivo. J Pharmacol Exp Ther. 1967 Jan;155(1):117–126. [PubMed] [Google Scholar]
  38. SMUCKLER E. A., BENDITT E. P. STUDIES ON CARBON TETRACHLORIDE INTOXICATION. 3. A SUBCELLULAR DEFECT IN PROTEIN SYNTHESIS. Biochemistry. 1965 Apr;4:671–679. doi: 10.1021/bi00880a009. [DOI] [PubMed] [Google Scholar]
  39. SMUCKLER E. A., ISERI O. A., BENDITT E. P. An intracellular defect in protein synthesis induced by carbon tetrachloride. J Exp Med. 1962 Jul 1;116:55–72. doi: 10.1084/jem.116.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Salim M., Maden B. E. Early and late methylations in HeLa cell ribosome maturation. Nature. 1973 Aug 10;244(5415):334–336. doi: 10.1038/244334a0. [DOI] [PubMed] [Google Scholar]
  41. Shatkin A. J. Capping of eucaryotic mRNAs. Cell. 1976 Dec;9(4 Pt 2):645–653. doi: 10.1016/0092-8674(76)90128-8. [DOI] [PubMed] [Google Scholar]
  42. Skeggs P. A., Thompson J., Cundliffe E. Methylation of 16S ribosomal RNA and resistance to aminoglycoside antibiotics in clones of Streptomyces lividans carrying DNA from Streptomyces tenjimariensis. Mol Gen Genet. 1985;200(3):415–421. doi: 10.1007/BF00425725. [DOI] [PubMed] [Google Scholar]
  43. Slater T. F. Necrogenic action of carbon tetrachloride in the rat: a speculative mechanism based on activation. Nature. 1966 Jan 1;209(5018):36–40. doi: 10.1038/209036a0. [DOI] [PubMed] [Google Scholar]
  44. Smuckler E. A. Alterations produced in the endoplasmic reticulum by carbon tetrachloride. Panminerva Med. 1976 Sep-Oct;18(9-10):292–309. [PubMed] [Google Scholar]
  45. Thompson J., Skeggs P. A., Cundliffe E. Methylation of 16S ribosomal RNA and resistance to the aminoglycoside antibiotics gentamicin and kanamycin determined by DNA from the gentamicin-producer, Micromonospora purpurea. Mol Gen Genet. 1985;201(2):168–173. doi: 10.1007/BF00425655. [DOI] [PubMed] [Google Scholar]
  46. Tomasi A., Albano E., Lott K. A., Slater T. F. Spin trapping of free radical products of CC14 activation using pulse radiolysis and high energy radiation procedures. FEBS Lett. 1980 Dec 29;122(2):303–306. doi: 10.1016/0014-5793(80)80461-3. [DOI] [PubMed] [Google Scholar]
  47. Trudell J. R., Bösterling B., Trevor A. J. Reductive metabolism of carbon tetrachloride by human cytochromes P-450 reconstituted in phospholipid vesicles: mass spectral identification of trichloromethyl radical bound to dioleoyl phosphatidylcholine. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2678–2682. doi: 10.1073/pnas.79.8.2678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Van Buul C. P., Damm J. B., Van Knippenberg P. H. Kasugamycin resistant mutants of Bacillus stearothermophilus lacking the enzyme for the methylation of two adjacent adenosines in 16S ribosomal RNA. Mol Gen Genet. 1983;189(3):475–478. doi: 10.1007/BF00325912. [DOI] [PubMed] [Google Scholar]
  49. Van Charldorp R., Van Knippenberg P. H. Sequence, modified nucleotides and secondary structure at the 3'-end of small ribosomal subunit RNA. Nucleic Acids Res. 1982 Feb 25;10(4):1149–1158. doi: 10.1093/nar/10.4.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Waller R. L., Glende E. A., Jr, Recknagel R. O. Carbon tetrachloride and bromotrichloromethane toxicity. Dual role of covalent binding of metabolic cleavage products and lipid peroxidation in depression of microsomal calcium sequestration. Biochem Pharmacol. 1983 May 15;32(10):1613–1617. doi: 10.1016/0006-2952(83)90336-2. [DOI] [PubMed] [Google Scholar]
  51. Winicov I., Perry R. P. Enzymological aspects of processing of mammalian rRNA. Brookhaven Symp Biol. 1975 Jul;(26):201–213. [PubMed] [Google Scholar]
  52. Zimmerman E. F., Holler B. W. Methylation of 45 s ribosomal RNA precursor in HeLa cells. J Mol Biol. 1967 Jan 28;23(2):149–161. doi: 10.1016/s0022-2836(67)80023-8. [DOI] [PubMed] [Google Scholar]
  53. Zimmerman E. F. Secondary methylation of ribosomal ribonucleic acid in HeLa cells. Biochemistry. 1968 Sep;7(9):3156–3164. doi: 10.1021/bi00849a019. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES