Metabolism of tRNA in rats with aflatoxin B1-induced hepatomas

This study describes effects of aflatoxin B1-induced hepatomas on RNA metabolism in rats. At 4 and 24 hours after the administration of L-(14CH3)-methionine, tRNA was isolated from the livers and hydrolyzed enzymatically to nucleosides which were quantitatively measured by HPLC. Radioactivity of the nucleosides was also determined. The data indicate that although tRNA methylation may be more rapid in livers with hepatomas, catabolism of tRNA in tumorous tissue is slower than in control livers. The large increase in some radioactive methylated nucleosides and bases by the tumor-bearing rats during the 24-hour period following the administration of labeled methionine indicates increased turnover of mRNA and rRNA as well as tRNA. Since degradation of tumor tRNA appears to be delayed, the excessive amounts of the urinary methylated nucleosides must be derived from RNA in nonneoplastic tissue.     

Inhibition of tRNA methylation in vitro and in whole cells by an oncostatic S-adenosyl-homocysteine (SAH) analogue: 5''-deoxy 5''-S-isobutyl adenosine (SIBA).

A high increase in the amount of methylated tRNA bases was found in vivo in Rous sarcoma virus infected and transformed chick embryo fibroblasts in comparison with normal cells, tRNA methylases extracted from transformed cells showed also higher activity in vitro with a heterologous substrate. 5'-deoxy-5'-S-isobutyl adenosine, (a structural analogue of S-adenosyl-L homocysteine), which inhibits virus-induced cell transformation, inhibits also the increase of incorporation of labelled methyl groups into tRNA in infected and transformed cells. When normal cells are grown in the presence of this inhibitor, undermethylated tRNAs are obtained. The effect of the drug is different in normal, infected and transformed cells. The methylation of the different bases is inhibited in vitro and in vivo to various extent. The effect of this substance on tRNA methylation may be the cause of its inhibitory effect on cell transformation.     

Effects of DL-ethionine on mouse liver tRNA base composition.

Treatment of mice with DL-ethionine and adenine causes a reduction of all methylated bases of liver tRNA. This effect is dose-dependent and specific for the methylated bases. Individual methylated components are affected to different extents, m22G being most sensitive to inhibition.     

Sites of methylation of purified transfer ribonucleic acid preparations by enzymes from normal tissues and from tumours induced by dimethylnitrosamine and 1,2-dimethylhydrazine

1. The sites within the tRNA sequence of nucleosides methylated by the action of enzymes from mouse colon, rat kidney and tumours of these tissues acting on tRNA(Asp) from yeast and on tRNA(Glu) (2), tRNA(fMet) and tRNA(Val) (1) from Escherichia coli were determined. 2. The same sites in a particular tRNA were methylated by all of these extracts. Thus tRNA(Glu) (2) was methylated at the cytidine residue at position 48 and the adenosine residue at position 58 from the 5'-end of the molecule; tRNA(Asp) was methylated at the guanosine residue at position 26 from the 5'-end of the molecule; tRNA(fMet) was methylated at the guanosine residues 9 and 27, the cytidine residue 49 and the adenosine residue 59 from the 5'-end; tRNA(Val) (1) was methylated at the guanosine residue 10, the cytidine residue 48 and the adenosine residue 58 from the 5'-end. 3. All of these sites within the clover leaf structure of the tRNA sequence are occupied by a methylated nucleoside in some tRNA species of known sequence. It is concluded that methylation of tRNA from micro-organisms by enzymes from mammalian tissues in vitro probably does accurately represent the specificity of these enzymes in vivo. However, there was no evidence that the tumour extracts, which had considerably greater tRNA methylase activity than the normal tissues, had methylases with altered specificity capable of methylating sites not methylated in the normal tissues.     

Further investigation of the increased transfer ribonucleic acid methylase activity in tumours of the mouse colon

1. Extracts prepared from tumours of the mouse colon induced by 1,2-dimethylhydrazine were considerably more active in catalysing the methylation of tRNA than were extracts from normal colon. The enhanced activity was observed when both unfractionated ;methyl-deficient' tRNA and purified tRNA preparations from yeast and bacteria were used as substrates for methylation. 2. The methylated bases produced in these reactions were identified. There were no differences between the products of the reaction catalysed by extracts of tumour and normal colon. 3. The increased activity of tRNA methylases was not due to the presence in the extracts of stimulatory or inhibitory molecules of low molecular weight such as polyamines or S-adenosylhomocysteine. 4. Other enzymes concerned with tRNA metabolism (RNA polymerase, ATP-tRNA adenylyltransferase, aminoacyl-tRNA ligases) were also increased in activity in the tumour tissue. 5. The extent of methylation of a limiting amount of tRNA was greater when tumour extracts were compared with controls, but in no case was it possible to achieve a stoicheiometric methylation of the purified tRNA preparations used as substrates, and the tumour extracts were not able to methylate tRNA obtained from normal mouse colon. We conclude that the tumours contained greater activities of tRNA methylases but that there was no evidence for changes in the specificity of these enzymes during neoplastic growth.     

Hypermethylation and higher stability of a plant (Eleusin coracana) tRNA

Total tRNA isolated from four-day-old ragi (Eleusin coracana) seedlings has been shown to be highly methylated. Each tRNA molecule on average contains two 2′-O-ribose methylated nucleosides. The high molar yields of 1-methyladenosine (1.6%) indicate that nearly a third of all the tRNA molecules contains more than one residue of 1-methyladenosine. Thermal denaturation studies with total tRNA show that the hypermethylated ragi tRNA melts slower that the yeast tRNA which is less methylated but otherwise has similar base composition. Ragi tRNA is also less susceptible to ribonucleases A, T₂ and T₂.     

Transfer RNA methyltransferase activity in paramecium aurelia.

The tRNA methyltransferases from Paramecium aurelia were investigated. The effects of varying the Mg2+ and NH4+ concentrations, pH, and temperature on the methylation of Escherichia coli B tRNA using extracts from P. aurelia were determined. Optimum tRNA methyltransferase activity was observed at pH 7.8 and 37 degrees C. The Mg2+ optimum occurred at 0.66 mM in the absence of NH4+ while the NH4+ optimum occurred at 100 mM in the absence of Mg2+. Analysis of the bases methylated in (E. coli B) tRNA by extracts of P. aurelia showed the presence of 1-methyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine and methylated pyrimidine nucleotides. In comparison, an analysis of the in vivo methylation of tRNA from P. aurelia showed the presence of 1-methyladenine, 6-methyladenine, 6,6-dimethyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine, 7-methylguanine, and methylated pyrimidine nucleotides. The pattern of methylation of tRNA in P. aurelia is similar to that observed in other eukaryotes.     

Composition and Characterization of tRNA from Methanococcus vannielii.

Purified bulk tRNA from Methanococcus vanielii (carbon source, formate) showed variation in the modified nucleoside pattern reported for Escherichia coli as analyzed by both ion-exchange and thin-layer chromatography. Ribothymidine and 7-methylguanosine were absent; 1-methyladenosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, thiolated nucleosides, pseudouridine, dihydrouridine, and O2'-methylcytidine were quantitated. In vitro methylation by M. Vannielii extracts with S-adenosylmethionine and undermethylated E. coli tRNA revealed active tRNA methyltransferases for formation of methylated residues found in native M. vannielii tRNA, but none for the formation of 7-methylguanosine or ribothymidine. The native M. vannielii tRNA became methylated in the 7-methylguanosine position by E. Coli extracts, but ribothymidine was not formed. Both M. vannielii and E. coli tRNA methyltransferases produced unidentified methylated residues in tRNA's lacking or deficient in ribothymidine.     

In vitro methylation of rat liver tRNA-synthesis of 3-methylcytosine and 1-methylhypoxanthine

Rat liver methylating enzymes could methylate tRNA extracted from the livers of rats treated with 35 mg/100 g L-ethionine 19 h prior to sacrifice. 1-methylhypoxanthine and 3-methylcytosine were among the methylated bases synthesized $\text{in vitro}$. The synthesis of 3-methylcytosine was dependent on the presence of Mg⁺⁺ although this ion inhibited the overall methylation of the tRNA.     

Quantitative determination of transfer RNA base composition using high-pressure liquid chromatography

An analytical method is presented for the quantitative determination of certain major and modified bases in unfractionated rat liver transfer RNA (tRNA), tRNA was hydrolyzed with perchloric acid, and the liberated bases were separated by high-pressure liquid chromatography. Bases were selectively detected in tRNA hydrolysates at wavelengths near their uv-absorption maxima. Recovery values for individual bases generally were in the 80–100% range. The composition of rat liver tRNA with respect to 10 bases was determined, and the levels of these bases were in agreement with published values determined by other methods.     

A yeast mutant which accumulates precursor tRNAs.

It has been proposed that the conditional yeast mutant ts136 is defective in the transport of mRNA from the nucleus to the cytoplasm (Hutchinson, Hartwell and McLaughlin, 1969). We have examined ts136 to determine whether it is defective in tRNA biosynthesis. At the restrictive temperature, the mutant accumulates twelve new species of RNA. These species co-migrate on polyacrylamide gels with some of the pulse-labeled precursor tRNAs. Three of the new RNAs (species 1a, 1b and 1c are large enough to contain two tandom tRNAs. Although RNAs 1a, 1b, and 1c do not contain detectable levels of modified and methylated bases, at least one of them hybridizes to DNA from an E. coli plasmid containing a yeast tRNA gene. All the remaining RNAs (2--8) contain modified and methylated bases typical of tRNA. Three of these species were tested and were found to hybridize to tRNA genes. Ribosomal RNA synthesis is also defective in ts136. It is suggested that ts136 may be defective in a nucleolytic activity, which is a prerequisite to RNA transport.     

Selective methylation of newly synthesized transfer RNA from an rel mutant of Escherichia coli during methionine starvation

The observation that the change in concentration of several methylated nucelosides from normal to methyl-deficient tRNA populations is not the same has been explained on the basis of continued methylation of newly synthesized tRNA by several but not all tRNA methyltransferases.     

Occurrence of 1-methyladenosine and absence of ribothymidine in transfer ribonucleic acid of Mycobacterium smegmatis.

The minor base composition of Mycobacterium smegmatis tRNA has been studied. Thin-layer chromatographic patterns of a ribonuclease T2 digest of mycobacterial tRNA indicated the presence of appreciable amounts of 1-methyladenosine (which is commonly present only in eucaryotic tRNA), dihydrouridine, and 7-methylguanosine. Ribothymidine was absent. The S-adenosylmethionine-dependent tRNA methylases of M. smegmatis catalyzed the formation of 1-methyladenosine when Escherichia coli tRNA was used as acceptor. Similarly, E. coli extracts methylated the tRNA of M. smegmatis, forming ribothymidine.     

Transfer of the methyl group of methionine to choline and to tRNA in the honeybee Apis mellifica L.

Contrary to some previous reports on the absence of biological transmethylation reactions in some insect species, the transfer of the methyl group of methionine-methyl 14C leading to choline and to methylated bases in tRNA was shown in the honeybee Apis mellifica. The addition of antibiotics to the food of the insect does not diminish the incorporation of radioactivity, proving that intestinal bacteria are not responsible for the methylation reactions observed.     

Bases methylated in vitro by cell-free extracts of adenovirus-18-induced tumours

The tRNA methylase activity in vitro of adenovirus-18-induced tumour was studied. The activity expressed per mg of protein was the same for extracts of tumours induced by either adenovirus-12 or adenovirus-18. The methylated bases isolated after incubation with extract from adenovirus-18-induced tumour were N(1)-methylguanine, 1-methyladenine, 3-methyladenine and N(6)-methyladenine.     

Undermethylated transfer ribonucleic acid from a relaxed strain of Bacillus subtilis: construction of the strain and analysis of the transfer ribonucleic acid.

A strain of Bacillus subtilis is described from which undermethylated transfer ribonucleic acid (tRNA) can be obtained. The tRNA's from a methionine-limited culture were compared with those from a control culture with respect to general nucleoside composition, methylated components, and amino acid acceptor activity. The undermethylated tRNA's had the normal amounts of the four major nucleosides, pseudouridine, and 5-methyluridine (ribothymidine), but were deficient in methylated nucleosides other than 5-methyluridine. These methyl-deficient nucleosides can be fully remethylated in the presence of the appropriate methylases. Since the majority of the work characterizing undermethylated tRNA's has been done using Escherichia coli, the work with B. subtilis presents some interesting comparisons and offers an alternative substrate for methylase studies.     

Origin and formation of 1,7-dimethylguanosine in tRNA chemical and enzymatic methylation

tRNA chemical methylation: 1. 1,7-Dimethylguanosine was found in in vivo methylated tRNA from liver and kidney of rat after exposure to a low dose of dimethylnitrosamine (4 mg/kg body weight). 2. At 4 h after dimethylnitrosamine administration, the 1,7-dimethylguanosine:7-methylguanine ratio (product ratio) for liver and kidney tRNA was 0.017 and 0.091, respectively. At 24 h after dimethylnitrosamine administration, the product ratio was lower in both hepatic and renal tRNA. 3. When dimethylnitrosamine was given in four separate daily injections, the product ratio in hepatic tRNA 4 h after the last dose was the same as for the same total dose given by a single injection, but in renal tRNA it was lower. No dialkyl compound was found in liver and kidney tRNA 24 h after the last multiple injection. tRNA enzymatic methylation: 1. Base analyses of Escherichia coli B tRNA methylated in vitro, by using S-adenosylmethionine as physiological methyl donor and enzyme preparations from liver and kidney of normal rat, indicated that 1,7-dimethylguanosine was also a product of enzymatic methylation. 2. The amount of 1,7-dimethylguanosine formed by kidney enzyme preparation was 3-times that produced by the liver extract. 3. A second type of enzymatic methylation assay where chemically methylated tRNA was used as substrate indicated that the 7-methylguanosine residues in the nucleic acid are not the substrate of the methylase activity forming the 1,7-dimethylguanosine moieties. Analogous data were obtained for the origin of 1,7-dimethylguanosine residues in tRNA chemical methylation by dimethyl sulphate.     

N2-guanine specific transfer RNA methyltransferase I from rat liver and leukemic rat spleen

An enzyme was purified from rat liver and leukemic rat spleen which methylates guanosine residues in tRNA to N(2)-methylguanosine. By sequence analysis of bulk E. coli tRNA methylated with crude extracts it was shown that the enzyme is responsible for about 50% of total m(2)G formed invitro. The extent of methylation of a number of homogenous tRNA species was measured using the purified enzyme from both sources. Among tested E. coli tRNAs only tRNA(Arg), tRNA(Phe), and tRNA(Val) yielded significantly more m(2)G than the bulk tRNA. The K(m) for tRNA(Arg) in the methylation reaction with enzymes from either tissue was 7.8 x 10(-7) M as compared to the value 1 x 10(-5) M obtained for the bulk tRNA. In a pancreatic RNase digest of bulk tRNA as well as of pure tRNA(Arg), tRNA(Phe), and tRNA(Val), A-m(2)G-Cp was found to be the only sequence methylated. Thus, the mammalian methyltransferase specifically recognizes the guanylate residue at position 10 from the 5'-end contained in a sequence (s(4))U-A-G-Cp. Furthermore, there is no change between the enzyme from normal liver and leukemic spleen in the affinity for tRNA, the methylating capacity, and tRNA site and sequence recognition specificity.     

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.     

Characterization of messenger-like ribonucleic acid from Saccharomyces cerevisiae by the use of chromatography on methylated albumin–kieselguhr

Chromatography on methylated albumin–kieselguhr of RNA from Saccharomyces cerevisiae was used to separate stable RNA from a tenaciously bound DNA-like RNA fraction. The tenaciously bound RNA, which was eluted with a dilute solution of sodium dodecyl sulphate, was characterized as messenger-like RNA by its sedimentation behaviour, nucleotide composition, lack of methylated bases and labelling kinetics. Chromatography of purified ribosomal RNA indicated a minor contamination of the tenaciously bound fraction with ribosomal RNA. On the other hand, a large portion of pulse-labelled polyribosomal RNA from protoplasts of Saccharomyces cerevisiae was tenaciously bound to the columns. The `chase' of isotopic label from the messenger-like RNA was found to be retarded during inhibition of protein synthesis both by cycloheximide and by starvation for a carbon source.