Methylated Nucleosides Research Articles

Inhibition of methylation of nuclear ribonucleic acid in L1210 cells by tubercidin, 8-azaadenosine and formycin

The adenosine analogs tubercidin (7-deazaadenosine), formycin (7-amino-3-[β- d-ribofuranosyl] pyrazolo[4,3-d]pyrimidine) and 8-azaadenosine were examined for their effects on the synthesis and methylation of nuclear RNA in L1210 cells in vitro. Total RNA and DNA synthesis was affected to the greatest extent by tubercidin (IC 50 = 7 × 10 −6M) and to an insignificant degree by 8-azaadenosine and formycin; however, the effects of the latter two drugs, but not of tubercidin, were potentiated by 2'-deoxycoformycin, an inhibitor of adenosine deaminase. In the presence of 2'-deoxycoformycin, RNA synthesis was inhibited by 40 per cent at 1 × 10 −4 M 8-azaadenosine and by 50 per cent at 2 × 10 −4 M formycin, while DNA synthesis was inhibited less extensively. Alkaline hydrolysis of nuclear RNA labeled with [ 14C]uridine and l-[ methyl- 3H]methionine showed preferential inhibition of base methylation in mononucleotides, but not of 2′- O-methylation in dinucleotides, for all three drugs. This differential effect persisted to varying degrees in ⪢18S and 4S nuclear RNA separated by electrophoresis. The reduction in base methylation in 4S RNA was associated with seven of the eight methylated nucleosides in 4S RNA separated by two-dimensional thin-layer chromatography. These results indicate that tubercidin, 8-azaadenosine and formycin can preferentially inhibit the base methylation of nuclear RNA relative to its synthesis.

The nucleus as the site of tRNA methylation.

Mouse L-cells were enucleated by exposure to cytochalasin B followed by centrifugation. The resulting karyoplasts, nuclei surrounded by a thin shell of cytoplasm and an outer cell membrane, and cytoplasts, the enucleated cell cytoplasm, were assayed for tRNA methyltransferase activity. The bulk of the enzyme activity was found to be localized in the nuclei. Analysis of the methylated nucleosides produced by the enzyme from the two sources showed that all the base-specific enzyme activities which are found in whole cell extracts were present in the nuclear extracts. The cytoplast extracts retained a low but detectable enzyme activity, which was composed predominantly of only two base-specific activities. This may represent tRNA methyltransferases of the mitochondria or may be cytoplasmic enzymes for late modification reactions.

Improved separation of modified nucleosides from tRNA hydrolysates: The patterns of tRNA methylation in rat tissues

A sensitive and reproducible method for the isolation of minor nucleosides derived from tRNA is described. The nucleosides obtained from enzymatic digestion of tRNA are separated into several groups using a QAE Sephadex column and increasing concentration of boric acid in a step-wise manner. The nucleosides in each group are separated by isocratic elution from a preparative Partisil 10-SCX column and high-performance liquid chromatography at ambient temperature. With this method we have determined the patterns of tRNA methylation in vitro with extracts from rat bone, liver, kidney and adrenal glands. Although different tissues appear to contain the same tRNA methyltransferases, the patterns of methylated nucleosides are different.

Restriction and Modification in Bacillus subtilis. Localization of the Methylated Nucleotide in the BsuRI Recognition Sequence

Calf thymus DNA was methylated in vitro with cell extracts of Bacillus subtilis OG3R (r+m+) and S-adenosyl[Me-3H]methionine. After depurination of the [3H]methylated DNA, the analysis of the pyrimidine dinucleotides revealed the following positions of the methylated nucleosides (indicated by an asterisk) within the BsuRI recognition sequence: 5' dG--dG--dC--dC dC--dC--dG--dG 5'.

Antibody nucleic acid complexes. Immunospecific retention of N6-methyladenosine-containing transfer ribonucleic acid

Antibodies specific for N6-methyladenosine (m6A) were immobilized on Sepharose and the resulting immunoadsorbent was tested for its ability to retain those Escherichia coli tRNAs containing the antigenic hapten, i.e., m6A. Results obtained with [32P]PO4- and [methyl-3H]-methionine-labeled tRNAs indicated that approximately 3 to 5% of the radioactive RNA was retained by the immunoadsorbent. Under identical conditions, but in the presence of m6A (1 mg/mL), less than 0.2% of the radioactivity was retained. Subsequent characterization of the retained tRNA via (a) analysis of methyl-3H-labeled, methylated nucleosides, (b) two-dimensional gel electrophoresis, and (c) analysis of the retention of [3H]aminoacyl-tRNA species led to the conclusion that the anti-m6A/Sepharose adsorbent quantitatively and exclusively retained a single tRNA species containing m6A, namely, tRNAVal.

Chemical and physical properties of mammalian mitochondrial aminoacyl-transfer RNAs II. Analysis of 7-methylguanosine in mitochondrial and cytosolic aminoacyl-transfer RNAs

The 7-methylguanosine (m 7G) content of two individual mitochondrial tRNAs, labelled in the aminoacyl moiety was assayed by the specific cleavage of the tRNA at this nucleotide followed by electrophoretic analysis to identify the 3′-terminal fragment of the tRNA. Neither Syrian hamster mitochondrial tRNA Leu nor tRNA Met were found to contain m 7G. In contrast, cytosolic tRNA Mets were cleaved indicating the presence of m 7G, apparently 27–28 and 29 nucleotides from their 3′ terminus. Cytosolic tRNA Leu was not cleaved. These results are discussed in relationship to the reported low content of methylated nucleosides in mitochondrial 4 S RNA.

General screening procedure for RNA modificationless mutants: isolation of Escherichia coli strains with specific defects in RNA methylation

A general method for the isolation of mutants of Escherichia coli that are defective in RNA modification is described. The method is based on the fact that RNA with specific undermodifications accumulates under nonpermissive growth conditions and that such a defect can be detected by remodification either in vivo at permissive conditions or in vitro. The method provides a means by which to study mutations affecting essential modification reactions. The usefulness of the method was demonstrated by the isolation of two rRNA and two tRNA methylation defective mutants. Both rRNA mutants accept methyl groups into their 23S rRNA in vitro. Analyses of in vitro methylated 23S rRNA from one of the mutants revealed the presence of several methylated nucleosides, of which 6-methyladenosine was the most abundant (40% of recovered radioactivity). In 23S rRNA from the other mutant, the only product formed in vitro was 5-methylcytidine. The tRNA mutants are characterized in the accompanying paper.

5'-Terminal and internal methylated nucleosides in herpes simplex virus type 1 mRNA

RNA labeled with [methyl-3H]methionine and/or [32P]orthophosphate was isolated from the polyribosomes of herpes simplex virus (HSV) types 1-infected cells and separated into polyadenylylated [poly(A+)]and non-polyadenylylated [poly(A-)] fractions. Virus-specific RNA was obtained by hybridization in liquid to either excess HSV DNA or filters containing immobilized HSV DNA. Analysis in denaturing sucrose gradients indicated that HSV-specific poly(A+) RNA sedimented in a broad peak, with a modal S value of 20. The ratio of [3H]methyl to 32P decreased with increasing size of RNA, suggesting that each RNA chain contains a similar sumber of methyl groups. Further analysis indicated an average of one RNase-resistant structure of the type m7G(5')pppNmpNp or m7G(5')pppNmpNmpNp per 2,780 nucleotides. The following components were identified in the 5'-terminal oligonucleotides of polyribosome-associated HSV-specific poly(A+) and poly(A-) RNA: 7-methylguanosine, N6,2'-O-dimethyladenosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and denosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and cytidine. The most common 5'-terminal sequences were m7G(5')pppm6Am and m7G(5')pppGm. An additional modified nucleoside, N6-methyladenosine, was present in an internal position of HSV-specific RNA.

Rapid analysis of the base-methylated and 2′- O-methylated ribonucleosides in messenger RNA

A simple two-dimensional chromatographic procedure which readily resolves a mixture of ribonucleosides, 2′- O-methylated ribonucleosides, and a variety of base-methylated ribonucleosides is described. The method involves enzymatic conversion of tritiated methyl-labeled RNA to nucleosides, followed by two-dimensional thin-layer chromatography and detection of the tritium-labeled ribonucleosides by fluorography. The method is illustrated using in vitro synthesized methylated reovirus mRNA. Using this method, a complete fingerprint of the methylated nucleosides in a sample of mRNA may be obtained in a single experiment. About 90% of the radioactivity applied to the chromatogram can be recovered for further analysis.

Secondary Methylation of Yeast Ribosomal Precursor RNA

The timing of methylation of the ribosomal sequences of ribosomal precursor RNA (pre-rRNA) from the yeast Saccharomyces carlsbergensis was investigated by fingerprint analysis of the methylated oligonucleotides derived from the various precursors. From the total of 37 ribose and 6 base-methyl groups found in 26-S rRNA, the two copies of the base-methylated nucleoside m3U as well as the doubly methylated sequence Um-Gm psi are not yet present in 37-S RNA, the predominant common precursor of 26-S and 17-S rRNA. Introduction of these methyl groups into the ribosomal sequences appears to take place at the level of 29-S pre-rRNA, the immediate precursor to 26-S rRNA. From the total of 18 ribose-methylated and 6 base-methylated nucleosides found in 17-S rRNA, the latter group (one copy of m7G, the m62A-m62A- sequence and the hypermodified methylated nucleoside "mX") is completely missing in 37-S pre-rRNA. The methyl group of m7G is introduced into 18-S pre-rRNA, the direct precursor of 17-S rRNA, in the nucleus. The -m62A-m62A- sequence is methylated after transport of the 18-S pre-rRNA to the cytoplasm prior to the final maturation into 17-S rRNA.

Methylated nucleosides in globin mRNA from mouse nucleated erythroid cells.

Poly(A)-containing mRNAs labeled with [methyl-3H]methionine were isolated from nucleated erythroid cells obtained from the spleens of anemic mice. The RNAs were further separated into non-globin poly(A)-containing RNAs and highly purified globin mRNA by globin cDNA-cellulose affinity chromatography. DEAE-Sephadex column chromatography of the T2 ribonuclease digestion products of the cDNA-purified globin mRNA fraction yielded methylated resistant fragments with charges of -4.7 (Cap 1) and -5.3 (Cap 2). Digestion of the non-globin RNA fraction revealed a similar pattern with the addition of a methylated mononucleotide identified as 6-methyladenosine at -2 charges. Alkaline phosphatase treatment of the T2 resistant fragments reduced their charges by approximately 2, which is consistent with the removal of one terminal phosphate. Treatment of the globin T2 and alkaline phosphatase-resistant fragments withpenicillium P1 nuclease and alkaline phosphatase yielded a P1-resistant core structure in both fragments. In addition to the core, 2'-O-methylcytidine (Cm) was released from the more negatively charged globin fragment. The P1-resistant cores of the cap structures eluted from DEAE-Sephadex with the known standard m2G5'ppp5'Am and were found to be pyrophosphatase-sensitive establishing a 5'-5'-triphosphate linkage. The pyrophosphatase and alkaline phosphatase digestion products of the globin Cap 1 and Cap 2 core structures were analyzed by high voltage electrophoresis and paper chromatography and found to be 7-methyiguanosine (m7G) and the dimethylated nucleoside 6-methyl-2'-O-methyladenosine (N6mAm). A small amount of the singularly methylated adenosine, 2'-O-methyladenosine (Am) was also observed. The predominant sequences of the methylated nucleosides in the globin cap structures are therefore m7G5'ppp5'N6mAm and m7G5'ppp5'N6mAmpCm.

Sequence specificity of internal methylation in B77 avian sarcoma virus RNA subunits

Following ribonuclease digestion of methyl-3H-labeled B77 avian sarcoma virus RNA subunits, methylated oligonucleotides were isolated by diethylaminoethylcellulose chromotogrpahy. Partial nucleotide sequences were deduced from the known enzymatic specificities of the ribonucleases. In addition to methylated nucleosides in the 5'-terminal cap structure, m7G(5')GmpCp, N6-methyladenosine(m6A) was found to be present in only two internal sequences of the RNA molecule, Gpm6ApC and Apm6ApC. The average numbers of methylated nucleosides per RNA subunit are about 12-13 in Gpm6ApC, 1-2 in Apm6ApC, and 2 in m7GpppGmpCp. The sequences containing m6A in B77 sarcoma virus RNA are identical to m6A-containing sequences previously reported for the bulk mRNA from HeLa cells (Wei, C.M., Gershowitz, A., and Moss, B. (1976), Biochemistry 15, 397-401). Analysis of the oligonucleotides produced by RNase A digestion indicated that the sequence of bases on the 5' side of these trinucleotides is not specific. The oligonucleotide profile, however, was highly reproducible in different virus preparations. This suggests that the methylations occur at specific positions on the RNA molecule. Some of the methylated oligonucleotides produced by RNase A digestion appear to be present in less than molar amounts. Several hypotheses are proposed to explain this result.

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.

Analysis of purines and their nucleosides by the reverse phase partition mode of high pressure liquid chromatography.

Although the analysis of nucleotides in blood is now being carried out routinely by high pressure liquid chromatography (HPLC) (1–4), and work is in progress to determine alterations casued by various disease states of free nucleotide concentrations in physiological fluids and cell extracts (5,6), less attention has been paid to free nucleoside levels in cells. Recent investigations, however, have focused attention on methylated nucleosides in cells as biological markers in cancer (7,8). Moreover, nucleoside concentrations are thought to be important in cardiac disease and birth defects. The intracellular level of adenosine, one of the physiological regulators of coronary blood flow, has been found to increase in cardiac hypertrophy and after brief periods of myocardial ischemia and hypoxia (9,10). In abnormalities in purine metabolic pathways caused by disease, the concentrations of purine bases and/or their nucleosides often are involved. For example in patients with adenosine deaminase deficiency, it has been observed that excess adenosine in cultured mammalian cells is toxic (11) and it has been postulated that accumulated adenosine is associated with severe immunological defects in children (12).KeywordsXanthine OxidaseHigh Pressure Liquid ChromatographyPurine BasePotassium Dihydrogen PhosphatePartition ModeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Histone mRNAs contain blocked and methylated 5′ terminal sequences but lack methylated nucleosides at internal positions

Histone mRNA, labeled with 32P or 3H-methionine during the S phase of partially synchronized HeLa cells, was isolated from the polyribosomes and purified as a “9S” component by sucrose gradient sedimentation. We identified two types of 5′ terminals, m 7G(5′)pppN mpN and m 7G(5′)pppN m-pN mpN, in which the first methylated nucleoside is 7-methylguanosine, the second is either N 6,2′-O-dimethyladenosine, 2′-O-methyladenosine, or 2′-O-methylguanosine, and the third is 2′-O-methyluridine, 2′-O-methylcytidine, or 2′-O-methyladenosine. Approximately 1.7% of the 32P label was present in the 5′ terminal structures. Assuming a similar specific radioactivity for all phosphates, this percentage corresponds to an average of one terminal per 335 nucleotides. Histone mRNA differed from bulk polyadenylylated mRNA of HeLa cells in lacking significant amounts of 2′-O-methyluridine or 2′-O-methylcytidine in the second position of the 5′ terminal oligonucleotide and in lacking N 6-methyladenosine residues at internal positions.

Isolation and characterization of N6-succinyladenosine from human urine.

From the urines of colon carcinoma patients and normal subjects we have isolated a nucleoside in which an amino group of aspartic acid is attached to the six position of purine ribonucleoside. The structure, N6-succinyladenosine, N-(9-B-D-ribofuranosylpurin-6-yl)aspartic acid was assigned on the basis of spectral data, chemical degradation, and by synthesis. The ultraviolet and mass spectra, chromatographic and electrophoretic mobilities, and the chemical properties of the naturally occurring nucleoside were identical to those of the synthetic N6-succinyladenosine. In contrast to the methylated and hypermodified nucleosides which are products of RNA catabolism, this urinary nucleoside appears to be derived from adenylosuccinic acid, a key intermediate required in the biosynthesis of ubiquitous, natural purine nucleotide adenosine-5'-monophosphate (AMP).

The effect of vitamin A deficiency on testicular transfer RNA methyltransferase activity

Testicular transfer RNA methyltransferase activity was examined in normal and vitamin A-deficient rats. The specific activity was reduced by 50% in the vitamin A-deficient rats. In addition, a 5-fold decrease in the extent of tRNA methylation was observed with enzyme preparations from deficient testes. Both the rate and extent of tRNA methylation returned to control levels in vitamin A-repleted rats. In contrast, retinoic acid repletion did not reverse the effect of vitamin A deficiency on testicular tRNA methyltransferase activity. The methylated nucleoside composition of tRNA methylated by extracts of vitamin A-deficient testes was altered dramatically compared to that of tRNA methylated by control testicular enzymes. Decreased testicular tRNA methyltransferase activity was noted in mildly deficient rats before the onset of testicular degeneration suggesting that the decreased tRNA methyltransferase activity in the testes is primarily the result of vitamin A deficiency.

Separation of methylated nucleosides by high-pressure liquid chromatography: The pattern of tRNA methylation in stimulated rat lymphocytes

An analytical method has been developed for the quantitative analysis of radiolabeled methyl ribonucleosides derived from transfer ribonucleic acids. The technique utilizes QAE-Sephadex in the borate form to separate 17 ribosides into three groups: quaternary base-methylated, sugar- O-methylated, and nonquaternary base-methylated nucleosides. The components of each group are subsequently separated on a strong cation-exchange resin of the polystyrene-divinyl-benzene type with baseline resolution for 14 species. Quantitative analysis is effected by counting column effluent fractions in a scintillation spectrometer.

An automated procedure for the analysis of methylated nucleosides in nuclear RNA and mRNA

A commercial high pressure liquid chromatographic system is used for the rapid analysis of 2′-O-methylnucleosides and pyrophosphate-linked methylated termini of nuclear RNAs and mRNAs. Oligonucleotides or RNA samples were enzymatically hydrolyzed to nucleosides and analyzed automatically by cationexchange chromatography in less than 1 hr using a 0.4 m ammonium formate buffer system at 55° c. This method is sufficiently sensitive to detect the N7-methylguanosine residue in 0.1 mg of Novikoff ascites hepatoma mRNA and may be used for qualitative or quantitative studies on RNA methylation in vivo or in vitro.

Modified nucleosides of Bacillus subtilis transfer ribonucleic acids

An analysis of the kinds and amounts of minor nucleosides of transfer ribonucleic acids (tRNA's) from Bacillus subtilis 168 trpC2 is presented. Identification and quantitation were accomplished using ion exclusion chromatography, thin-layer and paper chromatography, and ultraviolet absorption properties. Nucleosides and their amount in moles per 80 residues are as follows: guanosine (25.7), cytidine (22.0), adenosine (15.2), uridine (13.1), 5-methyluridine (0.98), pseudouridine (1.54), 1-methyladenosine (0.15), N6-methyladenosine (0.01), 7-methyladenosine (0.10), 2-methyladenosine (0.03), 7-methylguanosine (0.20), N2-methylguanosine (0.14), 1-methylguanosine (0.14), a methylated pyrimidine (0.17), a methylated derivative of N6-(delta 2-isopentenyl)adenosine (0.02), ribose methylated nucleosides (0.02), 4-thiouridine (0.12), 2-thio-5-(N-methylaminomethyl) (0.09), and an unknown thionucleoside (0.12). Although the composition is similar to that of Escherichia coli in the proportion of major nucleosides, the content of pseudouridine and 5-methyluridine, and the degree of base and ribose methylation, the composition is more similar to that of the tRNA's of yeast and higher organisms in its lower degree of thiolation, the presence of significant amounts of 1-methyladenosine, and the low levels of 2-methyladenosine and 6-methyladenosine. Therefore, the nucleoside composition of B. subtilis presents some different aspects from those usually given as characteristic for bacterial tRNA's. It is not known whether these differences are due to variation between bacterial species in general or related to the process of differentiation.