Immunospecific retention of oligonucleotides possessing N6-methyladenosine and 7-methylguanosine.

Antibodies specific for N6-methyladenosine (m6A) and for 7-methylguanosine (m7G) were immobilized on Sepharose and the resulting immunoadsorbents tested for their ability to retain specific oligonucleotides possessing the corresponding antigenic haptens (i.e. m6A and m7G). Results obtained with oligonucleotides derived from ribonuclease T1 digests of Escherichia coli tRNA (previously labeled with [methyl-3H]methionine) indicated that each immunoadsorbent quantitatively and exclusively retained those methyl-3H-labeled oligonucleotides possessing [methyl-3H]m6A and [methyl-3H]m7G. Elution and subsequent characterization of the retained methyl-3H-labeled oligonucleotides via DEAE-cellulose chromatography revealed the presence of several small oligonucleotides containing m7G and a single, larger oligonucleotide containing m6A. These findings are in accord with previously sequenced structures which indicate that numerous bacterial tRNA species possess m7G while only tRNAVal contains m6A.     

Identification of the catalytic nucleophile of tRNA (m5U54)methyltransferase

A covalent complex between tRNA (m5U54)methyltransferase, 5-fluorouridine tRNA(Phe), and S-adenosyl-L-[methyl-3H]methionine was formed in vitro and purified. Previously, it was shown that in this complex the 6-position of fluorouridine-54 is covalently linked to a catalytic nucleophile and the 5-position is bound to the transferred methyl group of AdoMet [Santi, D. V., & Hardy, L. W. (1987) Biochemistry 26, 8599-8606]. Proteolysis of the complex generated a [3H]methyl-FUtRNA-bound peptide, which was purified by 7 M urea-15% polyacrylamide gel electrophoresis. The peptide component of the complex was sequenced by gas-phase Edman degradation and found to contain two cysteines. The tritium was shown to be associated with Cys 324 of the methyltransferase, which unequivocally identifies this residue as the catalytic nucleophile.     

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.     

The Biosynthesis of Transfer Ribonucleic Acid in the Developing Rat Brain and in Cultured Glial Cells

Abstract: The biosynthesis of tRNA was investigated in cultured astroglial cells and the 3-day-old rat brain in vivo. In the culture system astrocytes were grown for 19 days and were then exposed to [³H]guanosine for 1.5–7.5 h; 3-day-old rats were injected with [³H]guanosine and were killed 5–45 min later. [³H]tRNA was extracted, partially purified, and hydrolyzed to yield [³H]-guanine and [³H]methyl guanines. The latter were separated from the former by high performance liquid chromatography and their radioactivity determined as a function of the time of exposure to [³H]guanosine. The findings indicate that labeling of astrocyte tRNA continued for 7.5 h and was maximal, relative to total RNA labeling, at 3 h, while in the immature brain tRNAs were maximally labeled at 20 min after [³H]guanosine administration. The labeling pattern of the individual methyl guanines differed considerably between astrocyte and brain tRNAs. Thus, [³H]1-methylguanine represented up to 35% of the total [³H]methyl guanine radioactivity in astrocyte [³H]tRNA, while it became only negligibly labeled in brain [³H]tRNA. Conversely, brain [³H]tRNA contained more [³H]N²-methylguanine than did astrocyte [³H]tRNA. Approximately equal proportions of [³H]7-methylguanine were found in the [³H]tRNAs of both neural systems. The [³H]methylguanine composition of brain [³H]tRNA was followed through several stages of tRNA purification, including benzoylated DEAE-cellulose and reverse phase chromatography (RPC-5), and differences were found between the [³H]methylguanine composition of RPC-5 fractions containing, respectively, tRNA^lys and tRNA^phe. The overall results of this study suggest that developing brain cells biosynthesize their particular complement of tRNAs actively and in a cell-specific manner, as attested by the significant differences in the labeling rates of their methylated guanines. The notion is advanced that cell-specific tRNA modifications may be a prerequisite for the successful synthesis of cell-specific neural proteins.     

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.     

Arangement of transfer-RNA -genes in yeast.

The redundancy and the arrangement of the genes for specific transfer ribonucleic acids in yeast were studied by the hybridization techniques developed by Birnstiel et al., e.g.[1]. The redundancy was found to be in the order of 10 genes for tRNA1Met, tRNA3Met, tRNA2Ser, and tRNA-Pro. High molecular weight yeast DNA was fractionated by density gradient centrifugation in cesium chloride and the [32p]tRNAs were hybridized to the single fractions. The results together with earlier findings [2] suggest that the cistrons for these tRNAs are arranged in tandem interspersed by 6 to 10 times longer segments of spacer DNA which varies in (G+C) content for the different tRNA species.     

Antibodies to N6-(delta2-isopentenyl) adenosine and its nucleotide: interaction with purified tRNAs and with bases, nucleosides and nucleotides of the isopentenyladenosine family.

The interaction of antibodies directed toward N6-(delta2-isopentenyl)adenosine, i6Ado, or its nucleotide with related bases, nucleosides, nucleotides and purified tRNAs is described. The selectivity of the antibody preparation was tested in inhibition experiments utilizing a sensitive radioimmunoassay to quantitate the binding of [3H]i6Ado to the antibody. Purified tRNAs containing various modified nucleosides adjacent to the 3'-end of the anticodon were tested to provide information about the selectivity of the antibody preparation toward nucleotides in this position of the tRNA chain. Antibodies directed against the nucleotide hapten were used to purify tRNAs which contain i6Ado and to quantitate the amount of that nucleotide. The same order of selectivity was expressed whether the nucleotides were free or in a tRNA molecule. Interaction of the antibody with compounds from the i6Ado family demonstrated dominance of the hydrophobic isopentenyl group and the importance of positional differences of modifications.     

Sequence of methylated nucleotides at the 5''-terminus of adenovirus-specific RNA.

RNA labeled with [methyl-3H] methionine and [14C]uridine was isolated from the cytoplasm of adenovirus-infected cells and purified by poly(U)-Sepharose chromatography and hybridization to filters containing immobilized adeovirus DNA. Analysis by dimethyl sulfoxide-sucrose gradient sedimentation suggested that the major mRNA species were methylated. 7-Methylguanosine was identified at the 5'-terminus of the advenovirus-specific RNA and could be removed by periodate oxidation and beta-elimination. Structures of the type m7G(5')ppp(5')Nm containing the unusual nucleoside N6, O2'-dimethyladenosine, and smaller amounts of 2'-O-methyladenosine were isolated by DEAE-cellulose chromatography after P1 nuclease digestion of the RNA. Evidence for some 5'-terminal sequences, m7G(5')ppp(5')m6AmpNm, with additional 2'-O-methylribonucleosides was also obtained. A base-methylated nucleoside, N6-methyladenosine, is located within the RNA chain and is released as a mononucleotide by alkali hydrolysis.     

The postnatal methylation of transfer ribonucleic acid in brain. Evidence for the methylation of precursor transfer ribonucleic acid.

Incubation of 3-day-old rat brain with L-[methyl-3H]methionine resulted in the rapid labeling of low-molecular-weight cytoplasmic RNA. Electrophoresis in 15% polyacrylamide gels provided evidence for the methylation of precursor tRNA molecules, and high-performance liquid chromatography demonstrated N2-methylguanine to be the predominant methylated base formed during the first 2 min of labelling.     

Interaction of chick oviduct progesterone receptor with immobilized aurintricarboxylic acid

Aurintricarboxylic acid (ATA) was immobilized on Sepharose 4B via a carbodiimide coupling mechanism. A majority of the chick oviduct progesterone receptor was retained on the affinity resin and could be recovered upon washing the column with buffer containing free ligand or 3 M guanidine-HCl. The [3H]progesterone-receptor complex retained its integrity following the chromatography on ATA-Sepharose as judged by sedimentation analysis. The procedure allowed significant purification of progesterone receptor: SDS-polyacrylamide gel electrophoresis of the purified preparation revealed elimination of many peptide bands present in the cytosol prior to ATA-Sepharose chromatography. The technique thus has a clear potential in characterization and purification of progesterone receptor.     

Synthesis of elastin and procallagen by cells from embryonic aorta. Differences in the role of hydroxyproline and the effects of proline analogs on the secretion of the two proteins.

Transfer ribonucleic acid (tRNA) profiles in human lymphocytes stimulated by various mitogens have been compared with profiles from nonstimulated cells and from leukemic cells using reversed-phase chromatography. Comparisons of [³H]- or [¹⁴C]uridine- or [³²P]phosphate-labeled tRNAs showed that the greatest changes in tRNA composition upon phytohemagglutinin (PHA) stimulation occurred in the first 8 h after mitogen addition. Stimulation of lymphocytes by pokeweed mitogen, anti-human immunoglobulin, or bacterial lipopolysaccharide resulted in tRNA species which showed distinct differences from each other and also from the tRNAs produced by phytohemagglutinin stimulation. Leukemic lymphocyte tRNAs showed the most extensive differences in profile when compared with chromatograms from non-neoplastic cells stimulated by a variety of mitogens. Specific isoaccepting species of tyrosyl-, aspartyl-, and phenylalanyl-tRNAs were also compared in PHA-stimulated and resting lymphocytes and no differences were found. When these same species were studied in leukemic cells, tyrosyl-tRNA profiles were shifted to elute at a lower salt concentration, while the aspartyl-tRNA profile showed a new peak not present in noncancerous cells.     

Evidence that Axonal tRNAs Are Resistant to RNase and ATPase and Can Be Aminoacylated in the Absence of Exogenous ATP

A high molecular weight (HMW) fraction of the 150,000 g supernatant of rat brain homogenates contains protein-tRNA complexes which are able to incorporate [3H]Arg and [3H]Lys into tRNA. The aminoacylation of tRNA(Arg) was found to be dependent on ATP and inhibited by RNase. Conversely, the aminoacylation of tRNA(Lys) did not require exogenous ATP and was resistant to RNase and ATPase. In HMW fractions of regenerating rat sciatic nerves, the charging of both tRNA(Arg) and tRNA(Lys) was resistant to RNase and ATPase and did not require exogenous ATP. Because sciatic nerves are rich in axoplasm and tRNAs are known to be present in axons, we tested the hypothesis that degradative enzyme-resistant, ATP-tRNA complexes were of axonal origin. In HMW fractions from rat liver (containing no axons), both tRNA(Arg) and tRNA(Lys) were sensitive to RNase and required exogenous ATP for charging. But, in similar fractions of axoplasm obtained from the giant axon of squid, both tRNAs were insensitive to RNase and ATPase and did not require exogenous ATP for charging. These results suggest that tRNAs in axons are present in protected HMW complexes and contain endogenous stores of ATP. The presence of ATP in the HMW complexes was demonstrated by the luciferase-luciferin assay for ATP. The nature of the protection of tRNAs from RNases was examined by dissociating proteins from HMW complexes by boiling, treating with proteinase K, or overhomogenizing the tissue. These procedures failed to render brain tRNA(Lys) susceptible to RNase. But phenol-extracted, ethanol-precipitated brain tRNA(Lys) was sensitive to RNase, suggesting that the protection of tRNA(Lys) may be by a protease- and heat-resistant polypeptide or by a nonproteinaceous mechanism.     

Interaction of RNA with transformed glucocorticoid receptor. II. Identification of the RNA as transfer RNA

An endogenous RNA (designated as PIVB RNA), which is capable of associating with the 4 S glucocorticoid receptor (GR) to generate the 6 S form, has been purified from AtT-20 cells (Ali, M., and Vedeckis, W. V. (1987) J. Biol. Chem., 262, 6771-6777). We describe here the physiochemical properties, GR-RNA interaction characteristics, and the chemical identification of PIVB RNA. 32P-Labeled PIVB RNA was similar to transfer RNA (tRNA) in its sedimentation coefficient (4 S) on sucrose gradients, electrophoretic mobility on formaldehyde-agarose gels, and receptor binding characteristics. The amino acid acceptor activity of PIVB RNA displayed a typical tRNA-dependent saturation curve and was 2-3-fold higher than that of homologous rabbit liver tRNA when tested using rabbit liver aminoacyl-tRNA synthetase. The purified [3H] aminoacyl-PIVB complex was also capable of binding to the 4 S GR to generate the 6 S form. The analysis of PIVB RNA on an acrylamide-urea sequencing gel revealed that it contained a major tRNA of 76 nucleotides and other minor tRNA species of 74 and 78 nucleotides. The identity of the tRNA present in the PIVB RNA was indirectly deduced by analyzing the 3H-amino acids, liberated from the [3H]aminoacyl-PIVB RNA (tRNA) complex, and subsequent analysis on an amino acid analyzer. PIVB RNA mainly contained tRNAArg (51.8%), tRNALys (17.1%), and tRNAHis (9.2%) which together accounted for 78% of the total PIVB tRNA. The remaining 22% of tRNA was contributed by threonine, valine, aspartic acid, alanine, and phenylalanine tRNAs. The GR displayed no species specificity, and tRNA samples from mouse, cow, rabbit, yeast, and Escherichia coli can bind to the mouse 4 S GR to generate the 6 S form. However, PIVB RNA did not affect the sedimentation profiles of albumin, chymotrypsinogen, and histone, indicating that PIVB RNA does not bind to all proteins. Thus, there may exist some specificity both at the level of protein (GR) and the selection of RNA (tRNA). The GR binding to PIVB RNA occurred at low (nM) receptor concentration, and PIVB RNA showed limited capacity to shift 4 S GR to the 6 S form. 22.4 X 10(-11) mol of PIVB RNA can completely shift 4.8 X 10(-13) mol of 4 S GR to 6 S. That is, PIVB RNA has to be in a 500-600-fold excess over the amounts of GR to observe a stable 6 S GR X RNA complex on sucrose gradients. These results conclusively demonstrate that the transformed GR specifically binds to endogenous tRNA.     

5'-N-ethylcarboxamido [3H] adenosine binding sites of mouse P815 mastocytoma cell membranes: solubilization and partial purification by affinity chromatography

A 5'-N-ethylcarboxamido[3H]adenosine ([3H]NECA) binding site of mouse mastocytoma P815 cell membranes has been purified approximately 100-fold by affinity chromatography. This adenosine binding site, which has a similar specificity to that of the A2 adenosine receptor, was absorbed on NECA-linked Sepharose 6B and eluted with NECA. The adsorption of the [3H]NECA binding site to the affinity matrix was specifically blocked by NECA. The [3H]NECA binding site bound on the affinity matrix was also specifically eluted by NECA. This affinity matrix adsorbed approximately 90% of the digitonin-solubilized [3H]NECA binding activity applied, and after the gel was washed, 30-50% of the adsorbed binding activity could be eluted with 500 microM NECA with specific binding activity of 50-70 pmol/mg of protein. The affinity-purified [3H]NECA binding site retained the same ligand binding specificities as the original membrane preparation. The results indicate that the NECA-Sepharose Sepharose 6B should provide a powerful tool for the eventual purification of [3H]NECA binding sites of P815 cell membranes.     

The ''polysemous'' codon--a codon with multiple amino acid assignment caused by dual specificity of tRNA identity.

In some Candida species, the universal CUG leucine codon is translated as serine. However, in most cases, the serine tRNAs responsible for this non-universal decoding (tRNA(Ser)CAG) accept in vitro not only serine, but also, to some extent, leucine. Nucleotide replacement experiments indicated that m1G37 is critical for leucylation activity. This finding was supported by the fact that the tRNA(Ser)CAGs possessing the leucylation activity always have m1G37, whereas that of Candida cylindracea, which possesses no leucylation activity, has A37. Quantification of defined aminoacetylated tRNAs in cells demonstrated that 3% of the tRNA(Ser)CAGs possessing m1G37 were, in fact, charged with leucine in vivo. A genetic approach using an auxotroph mutant of C.maltosa possessing this type of tRNA(Ser)CAG also suggested that the URA3 gene inactivated due to the translation of CUG as serine was rescued by a slight incorporation of leucine into the polypeptide, which demonstrated that the tRNA charged with multiple amino acids could participate in the translation. These findings provide the first evidence that two distinct amino acids are assigned by a single codon, which occurs naturally in the translation process of certain Candida species. We term this novel type of codon a 'polysemous codon'.     

Covalent cross-linking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid.

The covalent cross-linking of unmodified Escherichia coli N-acetylvalyl-tRNA to the 16S RNA of Escherichia coli ribosomes upon near-UV irradiation previously reported by us [Schwartz, I., & Ofengand, J. (1978) Biochemistry 17, 2524--2530] has been studied further. Up to 70% of the unmodified tRNA, nonenzymatically bound to tight-couple ribosomes at 7 mM Mg2+, could be cross-linked by 310--335-nm light. Covalent attachment was solely to the 16S RNA. It was dependent upon both irradiation and the presence of mRNA but was unaffected by the presence or absence of 4-thiouridine in the tRNA. The kinetics of cross-linking showed single-hit behavior. Twofold more cross-linking was obtained w-th tight-couple ribosomes than with salt-washed particles. Puromycin treatment after irradiation released the bound N-acetyl[3H]valine, demonstrating that the tRNA was covalently bound at the P site and that irradiation and covalent linking did not affect the peptidyl transferase reaction. Cross-linking was unaffected by the presence of O2, argon, ascorbate (1 mM), or mercaptoethanol (10 mM). Prephotolysis of a mixture of tRNA and ribosomes in the absence of puly(U2,G) did not block subsequent cross-linking in its presence nor did it generate any long-lived chemically reactive species. There was a strong tRNA specificity. E. coli tRNA1Val and tRNA1Ser and Bacillus subtilis tRNAVal and tRNAThr could be cross-linked, but E. coli tRNA2Val, 5-fluorouracil-substituted tRNA1Val, tRNAPhe, or tRNAFMet could not. By sequence comparison of the reactive and nonreactive tRNAs, the site of attachment in the tRNA was deduced to be the 5'-anticodon base, cmo5U, or ,o5U in all of the reactive tRNAs. The attachment site in 16S RNA is described in the accompanying paper [Zimmerman, R. A., Gates, S. M., Schwartz, I., & Ofengand, J. (1979) Biochemistry (following paper in this issue)]. The link between tRNA and 16S RNA is either direct or involves mRNA bases at most two nucleotides apart since use of the trinucleotide GpUpU in place of poly(U2,G) to direct the binding and cross-linking of N-acetylvalyl-tRNA to the P site did not affect either the rate or yield of cross-linking. Both B. subtilis tRNAVal (mo5U) and E. coli tRNA1Val (cmo5U) gave the same rate and yield of cross-linking when directed by the trinucleotide GpUpU. Therefore, the presence of the charged carboxyl group in the cmo5U-containing tRNA apparently does not markedly perturb the orientation of this base with respect to its reaction partner in the 16S RNA. The cross-linking of AcVal-tRNA only takes place from the P site. At 75 mM KCl and 75 mM NH4Cl, less than 0.4% cross-linking was found at the A site, while 55.5% was obtained at the P site. However, when the salt concentration was lowered to 50 mM NH4Cl, 5% cross-linking to the A site was detected, compared to 49% at the P site. Thus, a simple change in the ionic strength of the incubation mixture was able to alter the affinity labeling pattern of the ribosome.     

Thiolation of transfer RNA in Escherichia coli varies with growth rate.

We have used an affinity electrophoresis assay which when combined with Northern hybridization techniques permits us to estimate the degree of thiolation of individual tRNA species in Escherichia coli. We observe that the levels of 4-thio 2'(3')-uridine (4-thioU) in many but not all tRNAs varies dramatically at different bacterial growth rates: Five tRNAs are completely thiolated at all growth rates, while another eight tRNAs are incompletely thiolated and the fraction of the unthiolated form of these tRNA species increases as the growth rates increase. Transfer RNA(2Glu) contains 4-thioU as well as (methylamino)methyl-2-thio uridine (mnm(5)2-thioU). The level of mnm(5)2-thioU of tRNA(2Glu) is invariant with growth rate. Surprisingly, none of the thirteen tRNA species that we have studied is completely unmodified in all growth media. In particular, at the slowest growth rates every tRNA class that we have studied contains a form that has 4-thioU residues.     

Stereochemistry of methyl transfer catalyzed by tRNA (m5U54)-methyltransferase--evidence for a single displacement mechanism.

tRNA (m5U54)-methyltransferase (RUMT) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the 5-carbon of uridine 54 of tRNA. We have determined the steric course of methyl transfer, using (methyl-R)- and (methyl-S)-[methyl-2H1,3H]-AdoMet as the chiral methyl donors, and tRNA lacking the 5-methyl group at position 54 as the acceptor. Following methyl transfer, ribothymidine was isolated and degraded to chiral acetic acid for configurational analysis. Transfer of the chiral methyl group to U54 proceeded with inversion of configuration of the chiral methyl group, suggesting that RUMT catalyzed methyl transfer occurs by a single SN2 displacement mechanism.     

Human placental protein methylase--I. Purification and characterization.

1. Protein methylase I (S-adenosylmethionine[:]protein-arginine N-methyltransferase; EC 2.1.1.23) which methylates protein-bound arginine residues has been purified from human term placenta 400-fold with an approximate yield of 6%. 2. When histone was used as in vitro substrate, the methylation products were found to be NG-mono-, NG, NG-di- and NG, N'G-dimethylarginine. The enzyme was found to be sensitive toward Cu2+ with Ki value of 8 x 10(-5) M. The Km value for S-adenosyl-L-methionine was 5 x 10(-6) M. 3. When this partially purified protein methylase I was incubated with isolated human placental nuclei and S-adenosyl-L-[methyl-3H]methionine, the major endogenous [methyl-3H]-labeled proteins were protein species of 23, 38, 45 and 68 kDa, the 23 kDa species being the most predominant. 4. The endogenous enzyme activity during the pregnancy increased significantly, reaching more than 4 times the initial activity at the end of term.