Specific inhibition of transfer RNA methylation and modification in tissues of mice treated with 5-fluorouracil.
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Animals↗
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Results for “RNA methylation”
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Regulation of ribosomal RNA methylation in a temperature-sensitive mutant of BHK cells.
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Cell Line↗
Drug effects on nucleic acid modification. I. A specific effect of 5-azacytidine on mammalian transfer RNA methylation in vivo.
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Animals↗
High-resolution liquid chromatographic analysis of methylated purine and pyrimidine bases in transfer RNA.
Methylated and major purine and pyrimidine bases were separated and quantified by high-resolution liquid chromatography after hydrolyzing transfer ribonucleic acids (tRNAs). Separation was accomplished by eluting the hydrolyzed samples from an anion-exchange column with a concentration gradient of ammonium acetate at pH 9.2. Isolated sample of tRNA were hydrolyzed to the free bases with a trifluoroacetic acid-formic acid mixture of 200 degrees. Detection limits of 100-200 ng/ml were measured for the methylated bases; analytical data are reported for ten methylated bases plus the four major bases of calf liver and rat liver tRNA.
Adenine↗
5'-Terminal m-7G(5')ppp(5')G-m-p in vivo: identification in reovirus genome RNA.
Methylated reovirus mRNA was synthesized in vitro in the presence of S-adenosyl-L-[methyl-3H]-methionine. Viral genome double-stranded RNA that was uniformly labeled with 32-P was isolated from purified virions. The RNAs were mixed and their 5'-terminal structures compared by electrophoretic and chromatographic analyses after enzymatic digestion. Both the mRNA and the corresponding strand in the genome RNA contain m-7G(5')ppp(5')G-m-pCp, indicating that infected cells synthesize viral RNA with blocked, methylated 5' termini.
Base Sequence↗
Biosynthesis of ribothymidine in the transfer RNA of Streptococcus faecalis and Bacillus subtilis. A methylation of RNA involving 5,10-methylenetetrahydrofolate.
The methyl moiety of ribothymidine in the tRNA of Streptococcus faecalis, Bacillus subtilis, and probably Bacillus cereus is dervied from a 1-carbon folate derivative and not S-adenosylmethionine. Micrococcus luteus (M. lysodeikticus) tRNA appears to be almost devoid of ribothymidine. S. faecalis tRNA lacking ribothymidine can be methylated in vitro with formation of ribothymidine. 5,10-Methylenetetrahydrofolate has been identified as the 1-carbon donor involved in ribothymidine formation in S. faecalis and implicated in the analogous reaction in B. subtilis. The reaction requires FADH2 and/or another reducing agent present in cell extracts for the reduction of the methylene moiety.
Bacillus subtilis↗
RNA and protein metabolism in the oviduct and endometrium of the ewe at pro-oestrus: Regulation by oestradiol and progesterone.
The effects on RNA and protein metabolism in the oviduct and endometrium at pro-oestrus of oestradiol and progesterone secreted during the oestrous cycle were examined, using the ovariectomized, hormone-treated ewe as a model system. Thirty ewes received hormone injections during a period of 13 days, according to schedules designed to simulate endogenous ovarian secretion of oestradiol and progesterone during the oestrous cycle. Hormone effects on RNA:DNA ratios and on rates of synthesis of protein and methylated RNA in vitro, as well as effects on oviducal and uterine weight, were examined. The results obtained suggest that endogenous ovarian hormones have the following effects in the intact ewe. The secretion of oestradiol at pro-oestrus rapidly increases rates of synthesis of protein and methylated RNA, and mean cell content of RNA in both the endometrium and oviduct. Oestradiol secreted during the previous luteal phase of the oestrous cycle markedly increases mean cell content of RNA and amounts of protein and methylated RNA synthesis occurring in both tissues at pro-oestrus. In the endometrium, progesterone secreted during the luteal phase increases the RNA:DNA ratio, and probably also the amounts of protein and methylated RNA synthesized at pro-oestrus, but there are no significant interactions between the effects of oestradiol and progesterone. Progesterone had no effect on either the amounts or rates of synthesis of protein or methylated RNA in the oviduct. The results are discussed in relation to the hormonal regulation of physiological functions of the oviduct and endometrium during the first few days after the onset of oestrus.
Animals↗
Interrelationships between synthesis and methylation of ribosomal RNA in isolated Novikoff Tumor nucleoli.
Nucleoli isolated from Novikoff hepatoma cells of the rat were previously shown to carry out synthesis of predominantly ribosomal precursor RNA and methylation of this RNA in vitro. In order to develop in vitro systems for further detailed study of these processes and their interrelationships, isolated nucleoli were incubated in a complete RNA-synthesizing medium using (5-3H)cytidine 5'-triphosphate or S-adenoxyl(methyl-3H)methionine to measure the activities of RNA synthesis and methylation, respectively, under the same reaction conditions. Methylation of the ribose of the nascent ribosomal precursor RNA predominated. It occurred in close coordination with the transcriptional step by RNA polymerase as shown by the kinetic data, the analysis of labeled RNA in sucrose gradients, the inhibition by increased ionic strength or actinomycin D, and the release of labeled nucleotides by a 3'-exonuclease, venom phosphodiesterase. Methylation of the RNA bases occurred more slowly, continued longer after transcription ceased, and appeared to follow later in the processing of the RNA. Certain divalent cations (Mg2+, Mn2+, and Ca2+ at higher concentrations, and Zn2+ and Cu2+) inhibited both RNA synthesis and methylation to similar extents. RNase inhibitors (bentonite and dextran sulfate) at low concentration inhibited methylation while stimulating RNA synthesis, and pyrophosphate greatly decreased RNA synthesis with relatively little effect on methylation. These results indicated that RNA polymerase and ribosomal RNA methylases can function independently despite their close relationship. An exogenous substrate for the nucleolar rRNA methylases was found: nuclear RNA prepared from Novikoff hepatoma cells, cultured in the absence of methionine, served as a good substrate for methylation of both ribose and bases. Other exogenous RNAs, including cytoplasmic ribosomal RNA from these methionine-starved cells, nucleolar RNA from normal cells, and wheat germ ribosomal RNA were almost devoid of methyl-acceptor activity. A description of these parameters helps establish isolated nucleoli as a suitable system for further study of interaction of RNA polymerase, methylases, and nucleases in control of synthesis of ribosomal RNA.
Animals↗
[Methylation of prokaryotic RNA by S-methyl methionine in in vivo experiments].
While cultivating the E. coli 113-3 strain on the mineral medium containing S-methyl-(methyl-3H)-methionine, the incorporation of methyl groups into 4S, 16S and 23S RNA proved to be over 5 times more effective as compared with the control, when L-(methyl-3H)-methionine acted as a donor of methyl groups. The ratio of methylated components has much in common and significant differences. All the types of RNA of E. coli grown in the mineral medium containing S-methyl methionine showed an increased ratio of m2G as compared with the corresponding types of RNA of E. coli after its cultivation in the methionine containing mineral medium.
Escherichia coli↗
Cycloleucine blocks 5'-terminal and internal methylations of avian sarcoma virus genome RNA.
Cycloleucine, a competitive inhibitor of ATP: L-methionine S-adenosyltransferase in vitro, has been used to reduce intracellular concentrations of S-adenosylmethionine and by this means to inhibit virion RNA methylation in chicken embryo cells that are infected with B77 avian sarcoma virus. Under conditions of cycloleucine treatment, where virus production as measured by incorporation of radioactive precursors or by number of infectious particles is not significantly affected, the internal m6A methylations of the avian sarcoma virus genome RNA are inhibited greater than 90%. The predominant 5'-terminal structure in viral RNA produced by treated cells in m7G(5')pppG (cap zero) rather than m7G-(5')pppGm (cap 1). It appears from these results that internal m6A and penultimate ribose methylations are not required for avian sarcoma RNA synthesis and function. Furthermore, these methylations are apparently not required for transport of genome RNA to virus assembly sites. The insensitivity of the 5'-terminal m7G methylation to inhibition by cycloleucine suggests that the affinity of S-adenosylmethionine for 7-methylguanosine methyltransferase is significantly greater than for the 2'-0-methyltransferases or the N6-methyltransferases.
Amino Acids↗
1H nuclear magnetic resonance studies of transfer RNA: the methyl and methylene resonances of baker's yeast phenylalanine transfer RNA and its fragments.
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Magnetic Resonance Spectroscopy↗
Biosynthesis of S-N6-methyladenosylhomocysteine, an inhibitor of RNA methyltransferases.
This paper demonstrates that N6-methyladenosine (6-methylaminopurine ribonucleoside) will condense in vitro with homocysteine to form S-N6-methyladenosylhomocysteine in a reaction catalyzed by mouse liver S-adenosylhomocysteine hydrolase. Injection of mice with N6-methyladenosine is followed by accumulation of S-N6-methyladenosylhomocysteine in the liver. Studies from other laboratories have shown that S-N6-methyladenosylhomocysteine is nearly as potent an RNA methyltransferase inhibitor as S-adenosylhomocysteine. This indicates that administration of N6-methyladenosine may be a general method for blocking in vivo RNA methylation in studies to determine the role of methylation in RNA processing and translational function.
Animals↗
Marked for Success: How RNA m6A Methylation Fine-Tunes Gut Epithelial Function.
Post-transcriptional gene regulation-particularly through RNA modifications-plays an essential but understudied role in development, homeostasis, and regeneration of rapidly changing tissues like the mammalian intestinal epithelium. RNA modifications such as N6-methyladenosine (m⁶A) represent a burgeoning area of research in posttranscriptional regulation, with m⁶A being the most abundant modification found in approximately 25% of all mRNA transcripts. Multiple groups have begun to report m⁶A and associated regulation of mRNA fate as critical to key process in the intestinal epithelium. In this review, we synthesize key findings to date into the following 3 categories: m⁶A changes in response to the homeostatic luminal environment, m⁶A as a mediator of stemness in the crypt, and m⁶A as a tool for reacting to inflammation and injury. Over the course of this review, we will demonstrate how m⁶A is uniquely positioned to regulate homeostasis and disease states in the challenging and dynamic environment of the intestinal epithelium.
Humans↗
Methylated messenger RNA in mouse kidney.
Polyadenylated messenger RNA from mouse kidney labeled in vivo exhibited a pattern of methylation distinct from that of rRNA and tRNA. After mice were given L-[methyl-3H]methionine, 4% of the polyribosomal RNA label was bound to oligo (dT)-cellulose; 20-24% of orotate- or adenine-labeled polyribosomal RNA eluted in the poly(A)+ RNA fraction under similar conditions. [3H]Methyl radioactivity was not incorporated into low molecular weight (5-5.8 S) rRNA, indicating the extent of nonmethylpurine ring labeling was negligible. [3H]Methyl-labeled poly(A)+ RNA sedimented heterogeneously in sodium dodecyl sulfate containing gradients similarly to poly(A)+ mRNA labeled with [3H]orotic acid. Based on an average molecular length of 2970 nucleotides, renal mRNA was estimated to contain 8.6 methyl moieties per molecule. Analysis of alkaline-hydrolyzed RNA sampled by DEAE-Sephadex-urea chromatography provided estimates of the relative amounts of base and ribose methylation. Although 83% of the [3H]methyl radioactivity in rRNA was in the 2'-0-methylnucleotide fraction, no methylated dinucleotides were found in mRNA. In poly(A)+ mRNA 60% of the [3H]methyl label was in the mononucleotide fraction; the remainder eluted between the trinucleotide and tetranucleotide markers and had a net negative charge between -4 and -5. The larger structure, not yet charcterized, could result from two or three consecutive 2'-0-ribose methylations and is estimated to contain 2.6 methyl residues. Alternatively, the oligonucleotide could be a 5'-terminal methylated nucleotide species containing 5'-phosphate(s) in addition to the 3'-phosphate moiety resulting from alkaline hydrolysis. Either structure could have a role in the processing or translation of mRNA in mammalian cells.
Animals↗
Inhibition of methylated nucleoside synthesis in vivo: accumulation of incompletely methylated transfer RNA in ethionine-treated cells of Escherichia coli B.
tRNA prepared from cells of E. coli B that had been incubated with 0.5% DL-ethionine (Ethio sRNA) was found to accept methyl groups from 14CH3-S-adenosyl-methionine in the enzymatic reaction catalyzed in vitro by tRNA methyl transferases from untreated cells of the same organism. tRNA from cells that were not exposed to ethionine did not accept a significant level of methyl groups when incubated with the same enzyme system. Base ratio analysis of the product obtained after in vitro addition of methyl groups to Ethio sRNA by enzymes from normal E. coli B indicated that a high proportion of uracil sites in this tRNA were available for enzymatic methylation. These results indicated that tRNA from ethionine-treated organisms was recognized by the homologous enzymes to be incompletely methylated, while, as previously shown, all methyl-acceptor sites on tRNA from normal cells were already filled, and that Ethio sRNA was preferentially deficient in methyl groups on uracil moieties in the RNA molecules. Ethionine thus appears to interfere with normal tRNA modification in vivo.
Cell Division↗
Short-lived methylated messenger RNA in mouse kidney.
In experiments originally designed to examine selective turnover of methylated "caps" in renal mRNA, we observed that [3H]methyl label decayed from mRNA containing poly(A) with a half-life of 1-2 hr. (Caps are blocked, methylated mRNA sequences of the general structure m7GpppNm p(1 or 2)Np.). To distinguish between metabolism of short-lived mRNA and discriminate turnover of "caps", we compared residual [3H]methyl label in 5' and 3'mRNA fragments prepared from mRNA isolated during the decay period. Hydrolysis of mRNA at 0 degrees with dilute KOH before oligo(dT)-cellulose selection produced 5' mRNA fragments enriched with an alkali-resistant oligonucleotide with a -5 charge; the 3' mRNA fraction was correspondingly reduced in oligonucleotide content. Since methyl label disappeared at the same rate from both fractions, we conclude that mouse kidney contains short-lived mRNA and that the "caps" of these labile mRNAs turn over with the rest of the mRNA molecule.
Animals↗
Epigenetic alterations induced by ionizing radiation: pathways to cancer and prognostic strategies.
PURPOSE: Ionizing radiation (IR) is widely used not only in cancer diagnosis and therapy, but its biological effects also extend beyond radiation-induced lethal lesions, e.g., specifically DNA double-strand breaks (DNA-DSBs). This review aims to summarize current evidence on IR-induced epigenetic alterations and to integrate mechanistic insights from radiation chemistry and radiation biology that link DNA damage to long-term epigenetic dysregulation. RESULTS: Experimental and clinical studies collectively show that IR induces persistent epigenetic reprogramming, including global and gene-specific DNA methylation changes, radiation-responsive histone modifications, chromatin remodeling, and dysregulation of non-coding RNAs. Aberrant RNA methylation, including modifications like N6-methyladenosine (m6A), 5-methylcytosine (m5C), N1-methyladenosine (m1A), N7-methylguanine (m7G), and N3-methylcytosine (m3C), is closely linked to tumorigenesis and progression. Due to its tumor-specific properties, RNA methylation markers, specifically m6A, m5C, m1A, m7G, and m3C, emerge as valuable markers in liquid biopsy. Radiation chemistry studies indicate that epigenetically modified bases, for example, m5C, are preferential targets of radiation-induced oxidative damage, thereby promoting mutational hotspots and genomic instability. By altering DNA repair, apoptosis, immune responses, and cellular differentiation, these epigenetic changes promote carcinogenesis, radioresistance, and tissue toxicity. CONCLUSION: IR-induced epigenetic alterations represent a critical interface between initial DNA damage and long-term biological outcomes. Improved understanding of radiation-associated epigenetic signatures may enhance risk assessment, inform prognostic stratification, and support the development of epigenetic-targeted strategies to optimize radiotherapy and reduce adverse effects.
Ionizing radiation↗
Processing and function of undermethylated chicken embryo fibroblast mRNA.
Cycloleucine (1-aminocyclopentane-1-carboxylic acid) is a potent inhibitor of RNA methylation in B77 sarcoma virus-infected chicken embryo fibroblasts. Under conditions where 40 mM cycloleucine is present, internal N-6-methyladenosine and 5'-terminal cap 2'-O-ribose methylations of poly(A)+ RNA are inhibited greater than 90%. The methylation of the 5'-terminal 7-methylguanosine, however, does not appear to be significantly affected. The poly(A)+ RNA synthesized in cycloleucine-treated cells is transported from the nucleus to the cytoplasm and associates with polyribosomes at rates comparable to poly(A)+ RNA in untreated cells. On the other hand, the transport and utilization of newly synthesized ribosomal RNA in cycloleucine-treated cells is impaired, and the accumulation of mature 18 S and 28 S rRNA is reduced.
Animals↗