Effects of ethionine on tRNA methylation in male and female rats

Administration of hepatocarcinogens to rats results in an increase in tRNA methyltransferase activity in the target tissues. Ethionine is active as a carcinogen only in female rats and only in females is this increase in enzyme activity seen. However, ethionine also causes the formation of methyl-deficient tRNA in the liver. Other hepatocarcinogens do not do this. Ethionine is equally effective in this action in males and females. Thus, the two actions of ethionine are completely separable, and the methyl-deficiency of tRNA is caused by an activity not identical with the carcinogenic one.     

Drug effects on nucleic acid modification. I. A specific effect of 5-azacytidine on mammalian transfer RNA methylation in vivo.

Administration of the potent antineoplastic agent, 5-azacytidine (1-β-D-ribofuranosyl-4-amino-$\text{s}\text{-triazine-2}$(1$\text{H}$)-one), to mice causes a marked reduction of the 5-methylcytidine content of liver transfer RNA. This effect is dose-dependent and specific for the methylation of the 5-position of cytidine; the drug has no effect on 3-methylcytidine, 4-acetylcytidine, 5-methyluridine, dihydrouridine, pseudouridine, and modified purines. The mechanism of this effect has not yet been elucidated but probably involves the selective inhibition of 5-methylcytosine methyltransferase(s) by 5-azacytidine itself or its metabolites. The results presented show that the modified constituents of nucleic acids provide potential targets for chemotherapeutic agents.     

Alterations of tRNA modification in mammalian systems: the effect of ethionine.

The relationship between the modification of tRNA and its ability to act as a substrate for homologous tRNA modification enzymes in vitro was studied. The tRNA extracted from the livers of rats was active as a substrate for in vitro methylation with extracts from normal rat liver 19 h after treatment with L-ethionine (35 mg/100 g/24 h). After 4 weeks of feeding a diet containing o.25% DL-ethionine, the tRNA was a poor substrate for methylation in vitro, even though it was deficient in methylated nucleosides. Only 18% and 7% of the available sites could be methylated after 67 h and 4 weeks, respectively, of ethionine treatment. 3-(3-amino-3-carboxypropyl)uridine, a nucleoside that is also synthesized from S-adenosylmethionine, was assayed in individual tRNAs by their reactivity with the N-hydroxysuccinimide ester of phenoxyacetic acid. The reactivity of tRNAIle, tRNAAsn, and tRNAThr was decreased by treatment with ethionine at 67 h as well as at 2 and 4 weeks, although no difference could be detected at 19 h.     

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.     

Role of modifications in tyrosine transfer RNA. II. Ribothymidylate-deficient tRNA

The ability of suppressor tyrosine transfer RNA to participate in protein synthesis was studied in an E. coli mutant unable to synthesize ribothymidylate. Sequence analysis has shown that uracil replaces thymine in the common TψCG sequence. This transfer RNA is able to support protein synthesis at a rate equivalent to that observed with normally methylated transfer RNA.     

Effect of ethionine on synthesis and methylation of ribosomal ribonucleic acid in regenerating rat liver

Ethionine, a hepatocarcinogen, was administered into rats 24 h before partial hepatectomy and immediately thereafter. Hepatic precursor ribosomal RNA (pre-rRNA) obtained 20 h after the operation of rats injected with ethionine and adenine resulted in methyl deficiency as judged by the incorporation of [3H]methyl group of S-adenosylmethionine into nuclear rRNA by partially purified rRNA methylase. The ethionine and adenine treatment causes methyl deficiency of nuclear rRNA at 2'-hydroxyribose sites of cytidine and uridine, but not at base sites. Although the ethionine and adenine treatment produced no significant change in total hepatic RNA synthesis in vivo assayed by the incorporation of labeled orotate, a one-third increase in nuclear rRNA synthesis as well as a one-third decrease in microsomal rRNA synthesis was found under the treatment. These results suggest that the undermethylation at 2'-hydroxyribose of pre-rRNA in liver nucleus, which is caused by ethionine and adenine administration into rats, causes an inhibition of the processing of nuclear pre-rRNA to cytoplasmic rRNA.     

Methyldeficient mammalian 4S RNA: evidence for L-ethionine-induced inhibition of N6-dimethyladenosine synthesis in rat liver tRNA

The nucleotide composition of 4s RNA from livers of rats fed with a diet containing 0.3% D-ethionine was found to be identical with that from untreated animals. In contrast, one single modified nucleotide was absent in 4s RNA from livers of rats fed with a 0.3% L-ethionine diet. The minor nucleo=tide was also absent in liver 4s RNA from rats fed with a 0.3% L-ethionine diet followed by ten days of normal food. It was identified after dephosphorylation by ultraviolet absorption spectra, cochromatography with authentic material and mass spectra as N(6)-dimethyladenosine. It is concluded that S-adenosylethionine, the primary product of L-ethionine in the liver, causes strong and selective inhibition of the specific RNA-methylase responsible for adenosine to N(6)-dimethyl=adenosine methylation in rat liver 4s RNA. Compared to the strong inhibition of N(6)-dimethyladenosine formation described here, L-ethionine-dependent ethylation of liver 4s RNA is far less efficient. The quantitation of l-methyladenosine, ribothymidine and 3'-terminal adenosine in this 4s RNA as well as its aminoacid acceptor activity is typical for tRNA; hence it may be concluded that N(6)-dimethyladenosine is a component of rat liver tRNA. This may demonstrate the first evidence for the existence of specifically methyl-deficient mammalian tRNA. A possible correlation between the activity of L-ethionine as a liver carcinogen and its ability to induce the formation of methyl-deficient tRNA by selectively inhibiting the synthesis of N(6)-dimethyladenosine on the tRNA level in the same organ is discussed.     

The effect of growth temperatures on the in vivo ribose methylation of Bacillus stearothermophilus transfer RNA

When Bacillus stearothermophilus was cultured at 70 and at 50 °C, 1.4 times as many methyl groups were incorporated into tRNA produced at the higher temperature compared to that produced at the lower. This was due predominantly to a threefold increase in the 2′-O-methylribose moieties of the tRNA. The type and quantity of the base methylated nucleotides in the tRNAs produced in cultures grown at 70 and 50 °C were almost identical. The base methylated nucleotides found were: m²Ap, ms²Ap, ms²i⁶Ap, an unidentified i⁶Ap derivative, m⁶Ap, m₂⁶Ap, m¹Gp, m⁷Gp, m⁵Up-(Tp), and an unidentified methylated Up or Cp.³ The nucleotide m⁷Ap, never before reported to be a constituent of tRNA, has been tentatively identified as a component of B. stearothermophilus tRNA.     

Effect of the hepatocarcinogen ethionine on hepatic messenger RNA synthesis.

An experiment was conducted to determine if any changes resulted in the proportion of hepatic messenger RNA following treatment with ethionine, a hepatocarcinogen. The relative specific activity of the total RNA isolated from nontumor-like tissues was increased in Sprague-Dawley rats fed ethionine. However, the percentage of total RNA that was message was found to be decreased in the ethionine-treated rats.     

Functional differences in protein synthesis between rat liver tRNA and tRNA from Novikoff hepatoma.

Synthesis of ovalbumin in fragmented oviduct magnum explants of immature, estrogen-stimulated chicks has been studied in the presence of exogenous tRNA. tRAN from Novikoff hepatoma specifically inhibited ovalbumin synthesis, determined by precipitation with antisera. In addition, the major protein(s) synthesized in the presence of hepatoma tRNA had higher electrophoretic mobility than ovalbumin, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. tRNAs from rat liver, rooster liver, and hen oviduct did not affect ovalbumin synthesis, although oviduct tRNA is stimulatory during the earlier stages of estrogen stimulation.     

Isolation of the transfer RNA genes of bacteriophage T4 and transfer RNA synthesis in vitro.

Non-glucosylated T4 DNA was restricted with the endonuclease EcoRI and the mixture of DNA fragments separated by gel electrophoresis and transcribed with purified Escherichia coli RNA polymerase. Three purified fragments were shown to act as templates for tRNA synthesis. A smaller fragment, shown to be hybridizable to 32P-labeled T4 tRNA was not transcribable. It was concluded that the promoter for T4 tRNA synthesis had been separated from the structural genes in the smaller fragment by EcoRI and that the distal portion of the tRNA gene cluster lacks internal promoters which display in vitro activity. Preparations of non-glucosylated T4 DNA were never fully restricted with EcoRI and when the larger purified fragments carrying the tRNA were restricted with excess enzyme only a slight cleavage to yield the smaller fragments was obtained. The property of the DNA-limiting complete restriction is not know.     

Mechanism of action of silibinin. V. Effect of silibinin on the synthesis of ribosomal RNA, mRNA and tRNA in rat liver in vivo

The influence of the flavonolignane Silibinin on the rate of RNA synthesis in rat livers was studied in detail and the time course of the stimulatory effect was determined: 8 h after i.p. application a maximal increase of about 60% in nuclear RNA synthesis can be observed. The analysis of the RNA by electrophoresis on agarose and by sucrose gradient centrifugation demonstrated that in particular the ribosomal RNA (28S, 18S, 5.8S) synthesis is accelerated followed by enhanced incorporation of rRNA into mature ribosomes. During stimulation also changes in the pattern of 45S RNA can be observed. The synthesis of mRNAs, 5S RNA and tRNAs is not influenced by Silibinin, which was shown after separation of these moieties on oligo(dT)-cellulose, and by polyacrylamid electrophoresis, respectively. The clinically observed enhancement of liver cell regeneration during Silibinin treatment thus can be explained by an increase of the protein synthetic apparatus.