Modification of yeast ribosomal proteins. Methylation.

Two-dimensional polyacrylamide-gel electrophoretic analysis of yeast ribosomal proteins uniformly labelled in vivo with [methyl-3H]methionine and [1-14C]methionine revealed that four ribosomal proteins are methylated, i.e. proteins S31, S32, L15 and L41. Lysine and arginine appear to be the predominant acceptors of the methyl groups. The degree of methylation ranges from 0.09 to 0.20 methyl group per modified ribosomal protein species.     

Methylation of ribosomal proteins in Bacillus subtilis

We measured the methylation of ribosomal proteins from the 30S and 50S subunits of Bacillus subtilis after growing the cells in the presence of [1-14C]methionine and [methyl-3H]methionine. Two-dimensional polyacrylamide gel electrophoretic analysis revealed a preferential methylation of the 50S ribosomal proteins. Proteins L11 and L16, and possibly L9, L10, L18, and L20, were methylated. On the other hand, only two possibly methylated proteins were found on the 30S subunit. A comparison of these results with those for Escherichia coli suggests a common methylation pattern for the bacterial ribosomal proteins.     

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.     

Studies on the poky mutant of eurospora crassa. Fingerprint analysis of mitochondrial ribosomal RNA.

Base sequence and methylation of mitochondrial ribosomal RNAs from wild type and poky strains of Neurospora crassa were compared to determine whether a mutational lesion exists in poky 19 S RNA. At the outset, new procedures were developed for the isolation of intact nucleic acids from Neurospora mitochondria based on the substitution of Ca2+ for Mg2+ in the isolation media to inhibit mitochondrial nuclease activity. Using these procedures, intact and highly purified 32P-labeled ribosomal RNAs were extracted from purified mitochondrial ribosomal subunits of wild type and poky and compared using three complementary fingerprinting systems: two-dimensional electrophoresis of T1 plus phosphatase digests and homochromatography of T1 and pancreatic RNase digests. In supplementary experiments, 32P-labeled wild type RNA was co-fingerprinted with 32P-labeled poky and ratios of 32P/33P radioactivity were determined in each fragment to detect possible differences in stoichiometry. In addition, levels and patterns of methylated nucleotides were compared using procedures based on in vivo labeling with [methyl-3H]methionine and [32P]orthophosphate. In all these experiments, no difference was detected between wild type and poky in base sequence or methylation of either 19 S or 25 S RNA. Levels of methylation of Neurospora mitochondrial ribosomal RNAs were extremely low (less than 0.1% of the nucleotides), and results based on fingerprint analysis and DEAE-cellulose chromatography of alkaline hydrolysates of the [3H]methyl-labeled RNA suggested that 25 S RNA contains two ribose methylations, while 19 S RNA contains no methylated nucleotides.     

Ribonucleic acid labelling and nucleotide pools during compensatory renal hypertrophy

During the first 48h of compensatory renal hypertrophy induced by unilateral nephrectomy, RNA content per cell increased by 20-40%. During this period, rates of RNA synthesis derived from the rates of labelling of UTP and RNA after a single injection of [5-(3)H]uridine showed no change in the rate of RNA synthesis (3.1nmol of UTP incorporated into RNA/min per mg of RNA). ATP and ADP pools were not changed. The rate of RNA synthesis was considerably in excess of the increment of total RNA appearing in the kidneys. With [5-(3)H]uridine as label, only continuous infusion for 24h could produce an increase (60%) in the specific radioactivity of renal rRNA in mice with contralateral nephrectomies. With a single injection of [methyl-(3)H]methionine used to identify methyl groups inserted into newly synthesized rRNA, the specific radioactivity of this rRNA was unchanged 5h after contralateral nephrectomy, increased by 60% at 9-48h, and returned to normal values at 120h. Most RNA synthesized in both nephrectomized and sham-nephrectomized mice has a short half-life. Since total cellular RNA content increases in compensatory hypertrophy despite unchanged rates of rRNA synthesis, the accretion of RNA might involve conservation of ribosomal precursor RNA or a change in rate of degradation of mature rRNA.     

STUDIES ON RAPIDLY LABELLED NUCLEAR RNA OF RAT BRAIN

—Methyl albumin kieselguhr chromatography (MAK) has been employed to separate rat brain nuclear RNA, labelled in vivo with [³H]uridine, into three major fractions. The first fraction (QI RNA) is ribosomal in nature for it has a high G + C/U ratio and is methylated by [methyl-³H] methionine. The other two fractions (Q2 RNA and TD RNA) are DNA-like for they exhibit a low G + C/U ratio and are labelled minimally by methionine. Pure ribosomal RNA chromatographs almost entirely in the Q1 RNA fraction. Labelling studies indicate that ribosomal RNA and DNA-like RNA behave differently. Initially, the label in the DNA-like RNA fractions increases rapidly and in a linear fashion for the first 30 min, but thereafter decreases rapidly and reaches a steady state level by 1 h and remains so up to at least the 2 h period. In contrast, the labelling of ribosomal RNA is much slower than that of DNA-like RNA during the first 30 min; however, unlike DNA-RNA, the labelling of ribosomal RNA still continues to increase linearly thereafter. Thus, during longer labelling periods, ribosomal RNA is labelled more rapidly than DNA-like RNA. It appears that the labelling of ribosomal RNA relative to DNA-like RNA is more rapid in liver than in brain.     

Labelling kinetics of ribosomal RNA's in rat brain

Slices of cerebral cortex and cerebellum from two-week-old rats were incubated in the presence of [14C] uridine and [methyl-3H] methionine. Incorporation of 14C- and 3H-radioactivity into 18S and 28S RNA's of the two tissues was analysed by sucrose-density-gradient centrifugation. The results show that the rates of synthesis of ribosomal RNA's as well as the pattern of methylation in the two tissues were different. Some of these differences may be ascribed to factors such as pool sizes, intracellular rate of transport of the precursors by other pathways, etc. Examination of the results indicates that some differences may consist in the actual biosynthesis and maturation of ribosomal RNA's.     

Characterization of preribosomal ribonucleoprotein particles from Tetrahymena pyriformis.

Ribonucleoprotein particles present in extracts of nuclei prepared from Tetrahymena pyriformis labelled for 1, 2.5, 5 and 10 min with [3H]uridine during exponential growth were analysed by sedimentation through linear 10--30% sucrose gradients. After 1 min of labelling, the early ribosomal RNA precursor (36-S) is found to be associated with slowly sedimenting particles which form a broad peak centred at approximately 50 S. Other kinds of particles sedimenting at 80 S, 66 S, 60 S and 44 S are observed when labelling is carried out for longer periods (2.5, 5 and 10 min). The 80-S particle contains 29-S and 18-S RNA species together with traces of 36-S RNA; the 60-S and 44-S particles contain 26-S and 17-S RNAs respectively. Similar results were obtained when [Me-3H]methionine was used for labelling in place of [3H]uridine. Methylation of the RNA present in slowly sedimenting nuclear components (30-70-S) is rapid, reaching a plateau at 5 min while that of the faster sedimenting (70--90-S) components is still increasing after 10 min. Only three types of ribonucleoprotein particles (80-S, 66-S, and 44-S) were observed when the cells were labelled after prolonged starvation. A scheme of ribosome biogenesis based on these results is presented.     

Enzymatic methylation of band 3 anion transporter in intact human erythrocytes

Band 3, the anion transport protein of erythrocyte membranes, is a major methyl-accepting substrate of the intracellular erythrocyte protein carboxyl methyltransferase (S-adenosyl-L-methionine: protein-D-aspartate O-methyltransferase; EC 2.1.1.77) [Freitag, C., & Clarke, S. (1981) J. Biol. Chem. 256, 6102-6108]. The localization of methylation sites in intact cells by analysis of proteolytic fragments indicated that sites were present in the cytoplasmic N-terminal domain as well as the membranous C-terminal portion of the polypeptide. The amino acid residues that serve as carboxyl methylation sites of the erythrocyte anion transporter were also investigated. 3H-Methylated band 3 was purified from intact erythrocytes incubated with L-[methyl-3H]methionine and from trypsinized and lysed erythrocytes incubated with S-adenosyl-L-[methyl-3H]methionine. After proteolytic digestion with carboxypeptidase Y, D-aspartic acid beta-[3H]methyl ester was isolated in low yields (9% and 1%, respectively) from each preparation. The bulk of the radioactivity was recovered as [3H]methanol, and the amino acid residue(s) originally associated with these methyl groups could not be determined. No L-aspartic acid beta-[3H]methyl ester or glutamyl gamma-[3H]methyl ester was detected. The formation of D-aspartic acid beta-[3H]methyl esters in this protein in intact cells resulted from protein carboxyl methyltransferase activity since it was inhibited by adenosine and homocysteine thiolactone, which increases the intracellular concentration of the potent product inhibitor S-adenosylhomocysteine, and cycloleucine, which prevents the formation of the substrate S-adenosyl-L-[methyl-3H]methionine.     

Inhibitors of cyclic nucleotide phosphodiesterases inhibit protein carboxyl methylation in intact blood platelets

The cycle of protein-carboxyl methylation and demethylation was studied in intact blood platelets. Platelets rapidly incorporated L-[methyl-3H]methionine and after a delay of about 20 min, they evolved [3H]methanol. This evolution, and the amount of [3H] methanol liberated by treatment with base, was inhibited in a dose-dependent fashion by the cyclic nucleotide phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine, papaverine, dipyridamole, and RA233 (2,6-bis(diethanolamino)-4-piperidinopyrimido[5,4-d] pyrimidine). Each of these compounds increased the incorporation of [3H]methionine into platelets. The effects of RA233 were studied in more detail. Inhibition of [3H]methanol production was not potentiated by stimulators of the adenylate cyclase or the guanylate cyclase. The majority of the base-labile radioactivity was trichloroacetic acid precipitable. Thin layer chromatography of extracts of platelets incubated with L-[35S]methionine showed that RA233 did not induce a cellular accumulation of [35S]S-adenosylhomocysteine, and that it actually increased the amount of cellular [35S]S-adenosylmethionine. Discontinuous polyacrylamide gel electrophoresis at acid pH using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride of platelets incubated with [3H]methionine showed incorporation of radioactivity into more than 30 protein bands, including one which co-migrates with calmodulin. The incorporation into the majority of these bands was inhibited by RA233 in a dose-dependent fashion. It is suggested that caution should be used in ascribing the pharmacological effects of known phosphodiesterase inhibitors to increases in cyclic nucleotides, because some of these effects could be due to inhibition of protein carboxyl methylation.     

Effects of sinefungin on rRNA production and methylation in the yeast Saccharomyces cerevisiae

The antifungal agent, Sinefungin (SF), has been shown to be an inhibitor of transmethylation reactions. We report here the effects of SF on the production and methylation of rRNA in the yeast, Saccharomyces cerevisiae. Under conditions of SF treatment which have been shown to affect the regulation of cell proliferation in this yeast, pulse-chase labeling experiments using [methyl-3H]methionine and [3H]uracil indicated that methyl incorporation into rRNA during a short labeling period was inhibited, and stable 18 S rRNA production was differentially decreased. Other experiments quantitating modified nucleotides in newly produced rRNA showed that stable molecules were methylated. Taken together, these results suggest that SF slows methylation of rRNA, and is associated with differential loss of undermethylated 18 S rRNA species.     

Studies on myelin-basic-protein methylation during mouse brain development.

The synthesis and methylation in vivo of myelin basic protein (MBP) during the mouse brain development has been investigated. When mice ranging in age from 13 to 60 days were injected intracerebrally with L-[methyl-3H]methionine, the incorporation of radioactivity into MBP isolated from youngest brain was found to be the highest and declined progressively in mature brains. This pattern of radioactivity incorporation was inversely correlated with the total amount of MBP in the brains, suggesting a higher ratio of MBP methylation to synthesis in younger brain. To differentiate the relative rate of protein synthesis and methylation, animals were given intracerebral injections of a L-[methyl-3H]methionine and L-[35S]methionine mixture and the ratio of 3H/35S (methylation index) was determined. The ratios in the isolated MBP fractions were higher than those of 'acid extracts' and 'breakthrough' fractions, with a maximal ratio in the youngest brain. This high ratio was well correlated with the higher protein methylase I (PMI) activity in younger brains. The MBP fractions were further separated on SDS/polyacrylamide-gel electrophoresis into several species with apparent Mr ranging from 32,400 to 14,500. The results indicated that each protein species accumulated at a characteristic rate as a function of age. The high-Mr (32,400) species was predominant in younger brain, whereas the smaller MBP was the major species in older brain tissue. The importance of this developmental pattern of MBP synthesis and methylation is discussed in relation to PMI activity.     

Evidence of methylation of B77 avian sarcoma virus genome RNA subunits.

B77 avian sarcoma virus RNA was labeled with (methyl-3H) methionine under conditions that prevent non-methyl incorporation of 3H radioactivity into purine rings. From the determined values for the extent of methylation of 4S RNA isolated from infected chicken embryo cells, it was estimated that 30 to 40S RNA subunits that results from heat denaturation of the 60 to 70S RNA contain approximately 21 methyl groups, of which 14 to 16 are present at internal positions as N6 -methyladenosine residues. In addition, each of the virion RNA subunits appears to contain about two methyl groups in the "capped" 5' -terminal structure m7G(5')ppp(5') gm. These properties are consistent with the hypothesis that the 30 to 40S genome RNA os oncornaviruses also serves an mRNA function in infected cells.     

Further investigation of methylation on sickle erythrocyte membranes

The methylation of erythrocyte membrane proteins has been investigated with fractionated reversible and irreversible sickle erythrocytes to better understand conflicting results obtained from two laboratories (Green and Kalra (6), Ro et al. (1). When subpopulations of intact erythrocytes obtained by two different separation methods (33% bovine serum albumin and Stractan II gradient centrifugations) were incubated with L-[methyl-3H] methionine at pH 7.2 and 37 degrees C, membranes from both reversible and irreversible sickle erythrocyte populations showed about half the [3H]methyl group incorporation than that observed in normal erythrocytes. In addition, this difference in the level of methylation between normal and sickle cells was maintained during the entire course of a 2-hr incubation utilizing S-adenosyl-L-[methyl-3H]methionine, the immediate in vivo methyl donor.     

"In vivo" (35S)methionine interaction with rat liver tRNA

As part of a study to characterize the methionine role in tumorigenesis, we report that methionine sulfur interacts with rat liver tRNA "in vivo" (35S) radioactivity remained associated to the nucleic acid after a number of treatments, including tRNA deacylation. Similar data were obtained after administration of (methyl-3H) methionine, while no comparable tRNA labelling was detected when the aminoacid labelled in the aliphatic chain was given. Hplc analysis of (35S) tRNA enzymic hydrolysate showed two unidentified UV-absorbing radioactive peaks. NMR spectra of these two peaks did not reveal any thiomethyl group.     

Sequential methylation of globin mRNA in nucleated erythroid cells and reticulocytes of mice

The order of methylation of the 5'-terminus of globin mRNA of mice was studied by incubation of staged nucleated erythroid cells and peripheral reticulocytes with [methyl-3H] methionine. Methylation of the 5'-termini of alpha and beta- globin mRNAs in enucleated reticulocytes was demonstrated as follows: (a) [methyl-3H] incorporation into poly(A)+ RNA of reticulocytes co-migrated with the alpha- and beta- globin mRNAs on gel electrophoresis, and (b) following digestion of this RNA, radioactivity was localized to the four methyl sites at the 5'-capped structure of mouse globin mRNAs. However, this methylation is only 5 to 8% as efficient as in nucleated erythroid precursor cells, suggesting that most globin mRNA molecules are fully methylated prior to the reticulocyte stage. Incubations of early and late nucleated erythroid precursor cells and pulse-chase experiments with reticulocytes demonstrate that addition of the four 5'-terminal methyl groups follows an orderly sequence. In addition, the pulse-chase experiments suggest the turnover of the N7-methyl group on the 5'-terminal guanosine, but not of the other methyl groups in the 5'-terminus of globin mRNA. Thus, 5'-terminal methylation of globin mRNA is a nonrandom, dynamic process.