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.     

Isolation and characterization of a nucleolar 2'-O-methyltransferase from Ehrlich ascites tumor cells

A 2'-O-methyltransferase that transfers the methyl group from S-adenosylmethionine to the 2'-hydroxyl group of ribose moieties of RNA has been purified from Ehrlich ascites tumor cell nucleoli. The partially purified enzyme is devoid of other RNA methylase activities and is free of ribonucleases. The enzyme has optimal activity in tris(hydroxymethyl)aminomethane buffer, pH 8.0, in the presence of 0.4 mM ethylenediaminetetraacetic acid, 2 mM dithiothreitol, and 50 mM KCl, and has an apparent Km for S-adenosylmethionine of 0.44 microM. Gel filtration studies of this enzyme gave a Stokes radius of 43 A. Sedimentation velocity measurements in glycerol gradients yield an S20,w of 8.0 S. From these values, a native molecular weight of 145,000 was calculated. The enzyme catalyzes the methylation of synthetic homoribopolymers as well as 18S and 28S rRNA; however, poly(C) is the preferred synthetic substrate, and preference for unmethylated sequences of rRNA was observed. For each RNA substrate examined, only methylation of the 2'-hydroxyl group of the ribose moieties was detected.     

Quantitative analysis of rat liver nucleolar and nucleoplasmic ribosomal ribonucleic acids.

rRNA from detergent-purified nuclei was fractionated quantitatively, by two independent methods, into nucleolar and nucleoplasmic RNA fractions. The two RNA fractions were analysed by urea/agar-gel electrophoresis and the amount of pre-rRNA (precursor of rRNA) and rRNA components was determined. The rRNA constitutes 35% of total nuclear RNA, of which two-thirds are in nucleolar RNA and one-third in nucleoplasmic RNA. The identified pre-rRNA components (45 S, 41 S, 39 S, 36 S, 32 S and 21 S) are confined to the nucleolus and constitute about 70% of its rRNA. The remaining 30% are represented by 28 S and 18 S rRNA, in a molar ratio of 1.4. The bulk of rRNA in nucleoplasmic RNA is represented by 28 S and 18 S rRNA in a molar ratio close to 1.0. Part of the mature rRNA species in nucleoplasmic RNA originate from ribosomes attached to the outer nuclear membrane, which resist detergent treatment. The absolute amount of nuclear pre-rRNA and rRNA components was evaluated. The amount of 32 S and 21 S pre-rRNA (2.9 x 10(4) and 2.5 x 10(4) molecules per nucleus respectively) is 2-3-fold higher than that of 45 S, 41 S and 36 S pre-rRNA.     

Intranuclear maturation pathways of rat liver ribosomal ribonucleic acids.

The maturation of pre-rRNA (precursor to rRNA)in liver nuclei is studied by agar/ureagel electrophoresis, kinetics of labelling in vivo with [14C] orotate and electron-microscopic observation of secondary structure of RNA molecules. (1) Processing starts from primary pre-rRNA molecules with average mol. wt. 4.6X10(6)(45S) containing the segments of both 28S and 18S rRNA. These molecules form a heterogeneous peak on electrophoresis. The 28S rRNA segment is homogeneous in its secondary structure. However, the large transcribed spacer segment (presumably at the 5'-end) is heterogeneous in size and secondary structure. A minor early labelled RNA component with mol.wt. about 5.8X10(6) is reproducibly found, but its role as a pre-rRNA species remains to be determined. (2) The following intermediate pre-rRNA species are identified: 3.25X10(6) mol.wt.(41S), a precursor common to both mature rRNA species ; 2.60X10(6)(36S) and 2.15X10(6)(32S) precursors to 28S rRNA; 1.05X10(6) (21S) precursor to 18S rRNA. The pre-rRNA molecules in rat liver are identical in size and secondary structure with those observed in other mammalian cells. These results suggest that the endonuclease-cleavage sites along the pre-rRNA chain are identical in all mammalian cells. (3) Labelling kinetics and the simultaneous existence of both 36S and 21S pre-rRNA reveal that processing of primary pre-rRNA in adult rat liver occurs simultaneously by at least two major pathways: (i) 45S leads to 41S leads to 32S+21S leads to 28S+18S rRNA and (ii) 45S leads to 41S leads to 36S+18S leads to 32S leads to 28S rRNA. The two pathways differ by the temporal sequence of endonuclease attack along the 41 S pre-rRNA chain. A minor fraction (mol.wt.2.9X10(6), 39S) is identified as most likely originating by a direct split of 28S rRNA from 45S pre-rRNA. These results show that in liver considerable flexibility exists in the order of cleavage of pre-rRNA molecules during processing.     

The methylation of transfer ribonucleic acid during regeneration of the liver

Transfer ribonucleic acid¹ is methylated after the molecule is synthesized; at least eight enzymes are involved in the transfer of methyl groups (derived from methionine). The time courses of methylation and synthesis of tRNA during rat liver regeneration have been compared in an in vivo radioisotopic study, using 6-orotic acid-¹⁴C and ³H-methyl-L-methionine as precursors in double label pulses. Liver regeneration is a synchronized system in which biochemical events of the cell cycle are separable. Transfer RNA methylation increase precedes by several hours tRNA synthesis during regeneration, although the curves overlap. A ratio of the relative rate of methylation to the relative rate of synthesis has been made; that curve positively correlates with the rise and fall of protein synthesis during regeneration. It is clear that methylation and synthesis of tRNA are only weakly coupled; changing methyl content of the tRNA "pool" resulting from differential tRNA methylase and polymerase activities may regulate the rate of protein synthesis in the cell cycle at the translational level. The "pool sizes" of uridine monophosphate (UMP) and S-adenosylmethionine (SAM) were measured indirectly; UMP and SAM were isolated from perchloric acid supernatants and their specific activities were computed. Differential changes in radioactivity available to tRNA methylases and polymerases are not a source of artifact. That is, the control of both the synthesis and methylation of tRNA is at the enzyme level in vivo, rather than at some enzymatic step prior to those enzymatic reactions.     

Transfer ribonucleic acid methylase activity in adult and foetal rat liver.

Foetal rat liver extracts were found to have higher tRNA methylene activities than corresponding extracts of adult liver. When the specific activities were expressed per mg of liver or per mg of protein, the foetal tRNA methylating enzymes were respectively 2.5 and 6 times higher than those of adult livers. The presence of an inhibitor in adult liver can be excluded, since the same recoveries of total tRNA methylase activity were obtained after partial purification of both adult and foetal liver extracts: yields were close to 100%. The apparent Km's for the substrates in the methylating reactions were the same when tRNA methylases from either adult or foetal liver were used: values were 0.2 muM for Escherichia coli tRNA and 2.1 muM for S-adenosyl-L-methionine. After T1-T2 ribonuclease digestion of an in vitro methylated tRNA, similar methyl nucleotide patterns were observed in foetal and adult enzymatic extracts. It is concluded that the same tRNA methylase pool is present in adult and foetal liver. In addition, it is hypothesized that the different reaction rates exhibited by these enzymes might be due to the tRNA functional requirements rather than to the presence of a tRNA methylase inhibitor.     

The stability of rat liver ribonucleic acid in vivo after methylation with methyl methanesulphonate or dimethylnitrosamine

1. RNA was isolated from rat liver at selected times after the intraperitoneal injection of either [¹⁴C]methyl methanesulphonate (50mg/kg) or [¹⁴C]dimethylnitrosamine (2mg/kg). These doses were chosen to minimize effects due to toxicity. 2. Two methods of extraction and purification of RNA were used and an analysis of the radioactivity present was made by column chromatography of acid hydrolysates of the purified RNA. 3. The extent of methylation of guanine, the principal site of alkylation in rat liver RNA, was determined at times up to 14 days after injection. Although dimethylnitrosamine is a potent liver carcinogen and methyl methanesulphonate is not carcinogenic to rat liver, the rate of disappearance of 7-methylguanine from RNA was similar for both compounds, with a half-life of about 3.5 days. 4. An estimate of the biological half-life of rRNA was made by using [³H]orotic acid. A half-life of 5 days was obtained and this was not affected by injecting animals with unlabelled methyl methanesulphonate at the same dosage of 50mg/kg used in the studies of RNA methylation. 5. After administration of labelled orotic acid, reutilization of labelled RNA degradation products probably results in an overestimation of the biological half-life for rRNA. It is suggested that non-toxic doses of methylating agents such as methyl methanesulphonate and dimethylnitrosamine may prove to be a more effective way of accurately estimating the biological turnover of RNA species.     

Methylation of newly synthesized ribonucleic acid by isolated rat liver nuclei. Characterization of the ribonucleic acid synthesized by nuclei from starved animals

1. Nuclei from rat liver incubated with S-adenosyl[methyl-(14)C]methionine incorporated radioactivity into RNA and into lipid and protein. 2. All of the labelled RNA was extracted from the nuclei with trichloroacetic acid at 90 degrees C. 3. The [(14)C]methyl-group incorporation into the hot-trichloroacetic acid extract was 30% inhibited by the addition of actinomycin D (100mug/mg of DNA) or by the omission of CTP, GTP and UTP. 4. Assuming that the main substrate for this triphosphate-dependent methylation was newly synthesized precursor rRNA containing one methyl group/30 uridylate residues, it was calculated that approx. 60% of the [(14)C]UMP incorporated under similar conditions represented precursor rRNA synthesis. 5. In agreement with this, low concentrations of actinomycin D (approx. 1mug/mg of DNA) sufficient to abolish the triphosphate-dependent incorporation of [(14)C]methyl group inhibited 68% of the [(14)C]UMP incorporation. 6. The incorporation of [(14)C]UMP by nuclei from starved animals decreased progressively with increasing periods of starvation, whereas the triphosphate-dependent [(14)C]methyl-group incorporation was not further decreased after 1 day of starvation. 7. This suggests that precursor rRNA synthesis decreased within 1 day whereas other species of RNA were affected only after longer periods of starvation.     

Isolation of nucleolar methylase producing only 5-methylcytidine in ribosomal RNA

Methylation of rRNA mostly occurs in the nucleolus of mammalian cells. We have isolated nucleoli from Ehrlich ascites tumor cells of mice and purified RNA methylase taken from them. This highly purified nucleolar methylase produces only 5-methylcytidine in hypomethylated 18S and 28S rRNAs prepared from the mouse tumor cells after treatment with cycloleucine. This enzyme, however, did not transfer the methyl-group to normally methylated rRNA from the same mouse tumor cells. This high substrate specificity and enrichment of this enzyme in the nucleoli strongly suggest that we have isolated one of the enzymes which physiologically methylate rRNA precursor in the nucleoli.     

Processing and migration of ribosomal ribonculeic acids in the nucleolus and nucleoplasm of rat liver nuclei.

Kinetic studies on the labelling in vivo with [14C]orotate of rat liver nucleolar and nucleoplasmic pre-rRNA (precursor of rRNA) and rRNA, isolated from detergent-purified nuclei, were carried out. The mathematical methods used for the computer analysis of specific-radioactivity curves are described. Evaluation of the experimental data permitted the selection of the most probable models for the processing of pre-rRNA and the nucleo-cytoplasmic transfer of rRNA. It was shown that considerable flexibility exists in the sequence of endonuclease attacks at critical sites of 45 and 41 S pre-rRNA chains, resulting in the simultaneous occurrence of several processing pathways. However, the phosphodiester bonds involved in the formation of mature 28 and 18 S rRNA appear to be protected until the generation of their immediate pre-rRNA. The turnover rates and half-lives of all pre-rRNA and rRNA pools were determined. The turnover rate of 45 S pre-rRNA corresponds to the formation of 1100 ribosomes/min per nucleus. The model for the nucleolus-nucleoplasm-cytoplasm migration of rRNA includes a 'nucleoplasm' compartment in which the small ribosomal subparticle is in rapid equilibrium with the respective cytoplasmic pool. At equimolar amounts of nuclear 28 and 18 S rRNA this model explains the faster appearance of labelled small ribosomal subparticles in the cytoplasm simultaneous with a lower labelling of nuclear 18 S rRNA as compared with 28 S rRNA.     

Transfer ribonucleic acid methylase activity in adult and fœtal rat liver

Foetal rat liver extracts were found to have higher tRNA methylase activities than corresponding extracts of adult liver. When the specific activities were expressed per mg of liver or per mg of protein, the foetal tRNA methylating enzymes were respectively 2.5 and 6 times higher than those of adult livers.The presence of an inhibitor in adult liver can be excluded, since the same recoveries of total tRNA methylase activity were obtained after partial purification of both adult and foetal liver extracts: yields were close to 100 per cent.The apparent Km's for the substrates in the methylating reactions were the same when tRNA methylases from either adult or foetal liver were used: values were 0.2 μM for Escherichia coli tRNA and 2.1 μM for S-adenosyl-l-methionine.After T₁-T₂ ribonuclease digestion of an in vitro methylated tRNA, similar methyl nucleotide patterns were observed in foetal and adult enzymatic extracts.It is concluded that the same tRNA methylase pool is present in adult and foetal liver. In addition, it is hypothesized that the different reaction rates exhibited by these enzymes might be due to the tRNA functional requirements rather than to the presence of a tRNA methylase inhibitor.     

A comparison of methods for extracting ribonucleic acid polymerases from rat liver nuclei.

Nuclei were prepared from rat liver after homogenization of the tissue in hyperosmotic sucrose and RNA polymerases (EC 2.7.7.6) extracted by two methods applied sequentially. Optimal conditions for washing loosely bound enzymes out of nuclei were determined first, and involved short (10 min) incubations at 0 degrees C in the presence of 5 mM-Mg2+ and 60 mM-(NH4)2SO4. Subsequent sonication of the residual nuclear pellet after resuspension and lysis at high ionic strength resulted in further release of RNA polymerases. The primary wash yielded about 2 x 10(4) molecules of RNA polymerases I and III (altogether) and 1 x 10(4) molecules of form-II enzymes per original nucleus, whereas subsequent sonication released 2 x 10(4)-2.5 x 10(4) form-I and -III enzyme molecules (altogether) and a further 7 x 10(3)-8 x 10(3) form-II enzyme molecules, as measured by end-labelling of nascent RNA. RNA polymerase II was partially purified from both types of extracts and shown to initiate very poorly on high-molecular-weight homologous DNA irrespective of the source of the enzyme.     

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.     

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.     

Studies of the ethylation of rat liver transfer ribonucleic acid after administration of l-ethionine

1. The ethylated nucleosides present in tRNA isolated from the livers of rats treated with 0.5g of l-ethionine/kg body wt. were investigated. Evidence that this tRNA contained N(2)-ethylguanine, N(2)N(2)-diethylguanine, N(2)-ethyl-N(2)-methylguanine, 7-ethylguanine, two ethylated pyrimidines and ethylated ribose groups was obtained. 2. Ethylation of bacterial tRNA was catalysed by extracts containing tRNA methylases prepared from rat liver by using S-adenosyl-l-ethionine as an ethyl donor, but the rate of ethylation was 20 times less than the rate of methylation with S-adenosyl-l-methionine as a methyl donor. 3. The principal product of such ethylation in vitro was N(2)-ethylguanine and traces of the other ethylated guanines and pyrimidines found in tRNA isolated from rats treated with ethionine in vivo were also found. 1-Ethyladenine was not formed, although 1-methyl-adenine is a major product of methylation of bacterial tRNA by these extracts, and 1-ethyladenine was not present in the rat liver tRNA isolated from ethionine-treated animals. 4. After injection of actinomycin D (15mg/kg body wt.) or l-methionine (1.0g/kg body wt.) before the ethionine, ethylation of tRNA was diminished by about 80% but not completely abolished. Administration of 1-aminocyclopentanecarboxylic acid (2.5g/kg body wt.) to inhibit the formation of S-adenosyl-l-ethionine inhibited ethylation of tRNA by 44%. 5. These results suggest that not all of the ethylation of tRNA that occurs in the livers of rats treated with ethionine is mediated by the action of tRNA methylases acting with S-adenosyl-l-ethionine as a substrate, but that this pathway does occur and accounts for a major part of the observed ethylation. 6. The results are discussed with reference to ethionine-induced hepatocarcinogenesis.     

Effects of estradiol on uterine ribonucleic acid metabolism. Assessment of transfer ribonucleic acid methylation.

Immature rats treated with estradiol for selected periods of time demonstrated both increased methylation of uterine transfer ribonucleic acid (tRNA) and methylase activities. Whereas the former parameter was assessed by incubating whole uteri with [methyl-14C]methionine and measuring the incorporation of isotope into the tRNA, methylase activity was obtained by measuring the rate of incorporation of methyl groups from S-adenosyl[methyl-14C]methionine into heterologous tRNA (Escherichia coli B) in the presence of uterine cytosol preparations (100,000g supernatants). Although increased methylation of tRNA during the estrogen response was demonstrated, additional studies indicated that these results were largely attributable to an increased rate of synthesis of tRNA rather than gross changes in either the type or amount of methylated constituents present. Evidence in this regard included the inability of estrogen treatment of alter significantly the (a) resulting patterns of methyl-14C-methylated constituents of uterine tRNA, (b) the extent ot which [2-14C]guanine residues, incorporated into tRNA, become methylated, (c) the extent of methylation of precursor tRNA in the absence of tRNA synthesis, and (d) the types of methylase activities expressed in vitro.     

Isolation of ribonucleic acid from the unactivated rat liver glucocorticoid receptor

We have reported that the 7-8S form of the rat liver glucocorticoid receptor is associated with RNA. Whether the unactivated 9-10S form of the glucorticoid receptor is also associated with RNA is less clear. Here we provide evidence that the unactivated 9-10S receptor is indeed associated with RNA. Unactivated 9-10S receptor was partially purified by diethylaminoethyl (DEAE)-cellulose chromatography in the presence of molybdate, an activation inhibitor. This preparation was then bound to BuGR-2, a mouse monoclonal antibody of the immunoglobulin G (IgG)-2 class to the rat liver glucocorticoid receptor, or to nonspecific mouse IgG-2. The antibody-antigen complex was then bound to protein A sepharose and washed to remove extraneous RNA. When the receptor was dissociated from the antibody and the RNA extracted and end-labeled, a distinct band of approximately 170 nucleotide (nt) was found that was specific for the BuGR-2 purified receptor. This band could also be found in DEAE-cellulose receptor that had been isolated from sucrose gradients. The DEAE-cellulose receptor was then cross-linked with formaldehyde before mixing with BuGR-2 in order to permit more vigorous washing of the antigen-antibody complex. In addition to the 170 nt RNA band, another distinct band at approximately 400 nt was seen that was specific to the BuGR-2 derived isolate. These results provide evidence that the 9-10S form of the glucocorticoid receptor from rat liver is associated with RNA.     

A 12 S precursor to 5.8 S rRNA associated with rat liver nucleolar 28 S rRNA

The pre-rRNA and rRNA components of rat and mouse liver nucleolar RNA were analysed. It was shown that upon denaturation, part of the 32 S pre-rRNA is converted into 28 S rRNA and 12 S RNA. The 12 S RNA from mouse (Mr, 0.36 X 10(6)) is larger than the one from rat (Mr, 0.32 X 10(6). The 12 S RNA chain is intact and resists denaturation treatment. The non-covalent binding of this RNA with nucleolar 28 S rRNA is stronger than that of 5.8 S rRNA with 28 S rRNA. Hybridization with a rat internal-transcribed spacer rDNA fragment identifies 12 S RNA as corresponding to the 5'-end non-conserved segment of 32 S pre-rRNA, including 5.8 S rRNA. The significance of the formation of a 12 S precursor to 5.8 S rRNA in the biogenesis of ribosomes in mammalian cells is discussed.