Partial purification of a ribosomal ribonucleic acid methylase from rat liver nuclei and methylation of undermethylated nuclear ribonucleic acid from regenerating liver of ethionine-treated rat

S-Adenosylmethionine-dependent ribosomal RNA (rRNA) methylase has been purified approx. 90-fold from rat liver nuclei. The partially purified methylase catalyzes the methylation of base and ribose in hypomethylated nuclear rRNA prepared from the regenerating rat liver after treatment with ethionine and adenine. The enzyme has an apparent molecular weight of about 3 x 10(4) and a sedimentation coefficient of 3.0 S. The enzyme is optimally active at pH 9.5 and sensitive to p-chloromercuribenzoate. Thiol-protecting reagents, such as dithiothreitol, are necessary for its activity, and the enzyme requires no divalent cations for its full activity. This enzyme did not efficiently transfer the methyl group to nuclear rRNA from normal rat liver, compared with hypomethylated nuclear rRNA. Methyl groups were mainly incorporated into pre-rRNA larger than 28 S, and the extent of 2'-O-methylation of ribose by this enzyme was greater than that of base methylation in the hypomethylated rRNA. No other nucleic acids, including transfer RNA (tRNA) and microsomal RNA from normal as well as ethionine-treated rat livers, tRNA from Escherichia coli, yeast RNA, and DNA from rat liver and calf thymus, were significantly methylated by this methylase. These results suggest that partially purified rRNA methylase from rat liver nuclei incorporates methyl groups into hypomethylated pre-rRNA from S-adenosylmethionine.     

The effect of ethionine on ribonucleic acid synthesis in rat liver.

1. By 1h after administration of ethionine to the female rat the appearance of newly synthesized 18SrRNA in the cytoplasm is completely inhibited. This is not caused by inhibition of RNA synthesis, for the synthesis of the large ribosomal precursor RNA (45S) and of tRNA continues. Cleavage of 45S RNA to 32S RNA also occurs, but there was no evidence for the accumulation of mature or immature rRNA in the nucleus. 2. The effect of ethionine on the maturation of rRNA was not mimicked by an inhibitor of protein synthesis (cycloheximide) or an inhibitor of polyamine synthesis [methylglyoxal bis(guanylhydrazone)]. 3. Unlike the ethionine-induced inhibition of protein synthesis, this effect was not prevented by concurrent administration of inosine. A similar effect could be induced in HeLa cells by incubation for 1h in a medium lacking methionine. The ATP concentration in these cells was normal. From these two observations it was concluded that the effect of etionine on rRNA maturation is not caused by an ethionine-induced lack of ATP. It is suggested that ethionine, by lowering the hepatic concentration of S-adenosylmethionine, prevents methylation of the ribosomal precursor. The methylation is essential for the correct maturation of the molecule; without methylation complete degradation occurs.     

Quantitative alterations in the nucleolar and nucleoplasmic ribosomal ribonucleic acids in regenerating rat liver

A quantitative analysis of the nuclear pre-rRNA (precursor to rRNA) and rRNA in normal and 12h-regenerating rat liver was carried out, and the absolute amounts of the identified pre-rRNA and rRNA species in the nucleolus and nucleoplasm were determined. Characteristic changes in the pre-rRNA and rRNA pool sizes in regenerating liver are found which reveal alternations in both pre-rRNA processing and nucleocytoplasmic transition of ribosomes.     

Differences in the patterns of methylation in rat liver ribosomal ribonucleic acid after reaction in vivo with methyl methanesulphonate and NN-dimethylnitrosamine

1. rRNA was isolated from rat liver at short intervals after the intraperitoneal injection of [(14)C]methyl methanesulphonate (50mg/kg) or NN-di[(14)C]methylnitrosamine (2mg/kg). These doses were chosen to minimize the effects of toxicity. 2. The following methods of hydrolysis of [(14)C]methylated rRNA were employed: enzymic digestion to nucleosides at pH8; alkaline hydrolysis and conversion into nucleosides; acid hydrolysis to bases. 3. The methylation products were analysed by chromatography on columns of Dowex-50 (H(+) form) and Dowex-50 (NH(4) (+) form). 4. With both methylating agents the principal product of methylation was 7-methylguanine. Differences were obtained, however, in the molar proportions of the minor bases 3-methylcytosine, 1-methyladenine and 7-methyladenine. Methylation at the O-6 position of guanine was a significant feature of rRNA obtained from the NN-di[(14)C]methylnitrosamine-treated animals but was not detected in rRNA after treatment with [(14)C]methyl methanesulphonate.     

Effect of colchicine and vincristine on DNA synthesis in regenerating rat liver

The increases in the activities of hepatic thymidylate synthetase and thymidine kinase were significantly suppressed at 24 h after 70% partial hepatectomy in rats which had been administered a microtubule disrupter, colchicine or vincristine. The decrease of these enzymic activities was accompanied by a reduction of DNA content in 24 h regenerating liver. The immunoblotting assay showed that the depression of the thymidylate synthetase activity by the injection of colchicine or vincristine was due to the decrease of the enzyme protein. These results indicate that colchicine and vincristine inhibit the DNA synthesis during liver regeneration by inhibiting the induction of the key enzyme in DNA synthesis.