Kinetics of Novikoff cytoplasmic messenger RNA methylation.

Methylation patterns of Novikoff cytoplasmic mRNA were determined as a function of labeling time with L-[methyl-3H]methionine. The 5'-terminal m7G could be released from whole mRNA by treatment with nucleotide pyrophosphatase. Subsequent alkaline phosphatase treatment of this mRNA, followed by KOH digestion, yielded N'mpNp and N'mpNp from cap 1 (m7GpppN'mpN) and cap 2 (m7GpppN'mpN'mpN), respectively. Our results indicate that the relative amounts of labeled cap structures do change with time and that the amount of internal N6-methyladenosine decreases, relative to 5'-cap structures, as the cytoplasmic mRNAs age and the average size decreases. The formation of cap-2 structures by the addition of second 2'-O-methyl group at position N'm appears to be cytoplasmic event. Thus, after very short labeling times, greater than 80% of the labeled methyl groups in cap 2 are found in this position. These results, along with earlier data obtained on L-cell heterogeneous nuclear RNA methylation, are consistent with a model in which the nucleus is the cellular site of three mRNA methylation events producing 5'-terminal m7G, the first 2'-O-methylnucleoside (N'm) found in cap-1 structures and internal N6-methyladenosine. Subsequently, these nuclear methylations are followed by the cytoplasmic methylation at N'm. Analysis of the methynucleoside composition of cap-1 structures, along with comparable "core" structures (m7GpppN'm) generated from cap-2 by removal of N'm, indicates that at any single labeling time the methylnucleoside composition of a given cap-1 and the cap-2 "core" structure is remarkably similar. On the other hand, comparisons of the methylnucleoside composition of the cap structures at different labeling times indicate an increase in Cm in the first 2'-O-methylnucleoside (N'm) with time.     

Cytoplasmic location of undermethylated messenger RNA in Novikoff cells.

Novikoff cells in culture were labeled with L-[methyl-3H]methionine and [U-14C]uridine in the presence of (a) TubHcy2, (b) AdoHcy, (c) homocysteine, (d) tubercidin, or (e) without any additions. Only in cultures labeled in the presence of TubHcy were undermethylated cap structures observed to represent a significant portion of [3H]methyl radioactivity. Novikoff cells in culture were then simultaneously labeled with L-[methyl-3H]methionine and [32P]orthophosphate in the presence or absence of TubHcy. Total cytoplasmic, polysomal and monosomal poly(A)-containing RNAs were analyzed. Both monosomal and polysomal mRNA fractions from TubHcy-treated cells contain partially methylated cap structures, suggesting that 2'-O-methylation of the nucleoside adjacent to the pyrophosphate linkage in caps is not required for transport, ribosomal binding or translation. Comparison of nuclear and cytoplasmic cap structures from normal and inhibited cultures indicate that an altered mRNA population is generated in the presence of TubHcy.     

In vitro translation studies of the cytoplasmic nonpolysomal particles containing messenger RNA.

We analyzed the translational capacity of different kinds of free cytoplasmic messenger ribonucleoprotein complexes (free mRNP) in a Hela cell cell free system. Native free mRNP are not translated although free mRNP washed with 0.5 M KC1 can direct polypeptide synthesis. Furthermore, the 0.5 M KC1 wash possesses a factor which inhibits the translation of 0.5 M KC1 washed free mRNP as well as globin mRNA naked mRNA from plasmocytoma, or Hela cells. We also demonstrated that native free mRNP are able to form a complex with ribosomal subunits in the presence of initiation factors. This indicates that inhibition of translation by the 0.5 M KC1 wash occurs either at some point after initiation complex formation or at the elongation step.     

Minimal methylation of yeast messenger RNA

Most, if not all, yeast mRNAs are capped at their 5-terminus by m⁷G. Apart from m⁷G no other methylated nucleotides could be detected in poly (A)⁺ mRNA isolated from yeast polysomes.Abbreviations used poly (A)⁺ mRNA messenger RNA containing poly (A) - poly (A)^- RNA RNA lacking poly (A) - m⁷G N₇-methyl guanosine - N_m any 2-0 methylated nucleoside - mN any basemethylated nucleoside     

In vivo degradation of rat globin messenger RNA during maturation of reticulocytes.

The degradation of globin mRNA in rat reticulocytes maturing in the peripheral blood was investigated. Poly(A) and non poly(A) portions of mRNA molecules were determined quantitatively by hybridization with radioactive poly(U) and complementary DNA, respectively. During the degradation of mRNA in vivo, it was shown that (1) globin mRNA and the bulk of RNA decrease in parallel, (2) the average chain length of poly(A) segments in the mRNA does not change, (3) the percentage of poly(A) (-) globin mRNA in total globin mRNA does not change, and (4) fragments of large molecular weight do not accumulate. Possible mechanisms of degradation of globin mRNA in the reticulocytes are discussed on the basis of these observations.     

The methylation of adenovirus-specific nuclear and cytoplasmic RNA.

Each poly(A) containing cytoplasmic AD-2 MRNA contains at its 5' terminus the general structure m7 GpppN1 pN2p or m7 GpppN1mpN2mpNp as well as an average of 4 m6A and 0.5-1 m5C residues per molecule. Almost all of the N1m residues are adenine derivatives including Am, m6Am and probably m26,6Am. The N2m is mostly Cm but small amounts of the other three methylated bases are also present. All the methylated constitutents of mRNA are distant from the 3' terminal poly(A). The amount of m6A appears to be greater in larger mRNA than in smaller mRNA. Nuclear Ad-2 specific RNA also contains caps, m6A, and m5C with about twice as much m6A relative to caps as cytoplasmic mRNA. The similarity of Ad-2 nuclear and mRNA to HeLa hnRNA and mRNA suggests that adenovirus mRNA production is a good model for eukaryotic mRNA production.     

In vivo synthesis and turnover of cytoplasmic ribosomal RNA by stage 6 oocytes of Xenopus laevis.

Cytoplasmic ribosomal RNA (rRNA) synthesis was detected in white-banded stage 6 oocytes taken from female Xenopus laevis which were injected with [³H]guanosine 7 days previously. The specific radioactivity of the rRNA in oocytes collected from injected females by weekly laparotomies displays first-order exponential decay. Calculated values for the half-life of rRNA ranged from 9.1–30.9 days in experiments on four animals. The concept of ribosomes in large ovarian oocytes of amphibians as an absolutely stable, long-term storage product appears incorrect.     

In vivo expression of beta-galactosidase by rat oviduct exposed to naked DNA or messenger RNA

Intra-oviductal administration of RNA obtained from oviducts of estradiol-treated rats resulted in accelerated egg transport (Ríos et al., 1997). It is probable that estradiol-induced messenger RNA (mRNA) entered oviductal cells and was translated into the proteins involved in accelerated egg transport. In order to test this interpretation we deposited in vivo 50 micrograms of pure beta-galactosidase (beta-gal) mRNA, 50 micrograms of pure DNA from the reporter gene beta-gal under SV40 promoter or the vehicle (control oviducts) into the oviductal lumen of rats. Twenty four hours later the beta-gal activity was assayed in oviductal tissue homogenates using o-nitrophenyl-beta-D-galactopyranoside as a substrate. The administration of beta-gal mRNA and pSVBgal plasmid increased beta-gal activity by 71% and 142%, respectively, over the control oviducts. These results indicate that naked DNA and mRNA coding for beta-gal can enter oviductal cells and be translated into an active enzyme. They are consistent with the interpretation that embryo transport acceleration caused by the injection of estradiol-induced RNA in the oviduct involves translation of the injected mRNA.     

Physical aspects and cytoplasmic distribution of messenger RNA in mouse kidney.

As a prerequisite to examining mRNA metabolism in compensatory renal hypertrophy, polyadenylated RNA has been purified from normal mouse kidney polysomal RNA by selection on oligo(dT)-cellulose. Poly(A)-containing RNA dissociated from polysomes by treatment with 10 mM EDTA and sedimented heterogeneously in dodecyl sulfate-containing sucrose density gradients with a mean sedimentation coefficient of 20 S. Poly(A) derived from this RNA migrated at the rate of 6-7 S RNA in dodecyl sulfate-containing 10% polyacrylamide gels. Coelectrophoresis of poly(A) labeled for 90 min with poly(A) labeled for 24 h indicated the long-term labeled poly(A) migrated faster than pulse-labeled material. Twenty percent of the cytoplasmic poly(A)-containing mRNA was not associated with the polysomes, but sedimented in the 40-80 S region (post-polysomal). Messenger RNA from the post-polysomal region had sedimentation properties similar to those of mRNA prepared from polysomes indicating post-polysomal mRNA was not degraded polysomal mRNA. Preliminary labeling experiments indicated a rapid equilibration of radioactivity between the polysomal and post-polysomal mRNA populations, suggesting the post-polysomal mRNA may consist of mRNA in transit to the polysomes.     

The methylation state of poly A-containing messenger RNA from cultured hamster cells.

The poly A-containing mRNA of cultured hamster (BHK-21) cells has been examined with regard to methylation status. Steady state-labeled mRNA was obtained by incubating cells for 20-22h in the presence of [methyl-3H]-methionine and 32Pi. The degree of methylation of this RNA was 1.8 methyl groups per 1000 nucleotides, or 4-5 methyl groups on the average per molecule. The nature of the methylated residues was determined by paper chromatography and electrophoresis of acid and alkaline hydrolysates, by DEAE cellulose chromatography of alkaline hydrolysates and of T2 RNase digests, and by examining the effect of subjecting samples to "beta-elimination." Approx. half of the methyl groups occurred in standard ("internal") linkage, 10% as m5Cp and 40% as m6Ap residues. The remainder occurred at least for the most part in "blocked" 5'-termini with the presumptive structure m7G(5')ppp(Nm)p.., where Nm was Gm, m6Am, Um, or Cm.     

In vivo methylation of prokaryotic elongation factor Tu.

In Salmonella typhimurium and Escherichia coli, elongation factor Tu (EF-Tu) is methylated as shown by its incorporation of labeled methyl residues from [methyl-3H]methionine. Analysis of the nature of the methyl-containing residues by protein hydrolysis, followed by paper chromatography and high voltage electrophoresis showed that both mono- and dimethyllysine are present. Eighty per cent of the EF-Tu molecules are methylated if methylation occurs at a unique lysine residue. The EF-Tu fraction which is not methylated is still able to accept methyl groups, as shown by methylation of approximately 10% of the EF-Tu after addition of chloramphenicol (D-(-)-threo-2,2-dichloro-N-[beta-hydroxy-alpha-(hydroxymethyl)-o-nitrophenethyl] acetamide) to inhibit further protein synthesis. There is no evidence of turnover of the methyl residues. We attempted to separate the methylated from the nonmethylated form of EF-Tu by isoelectric focusing on polyacrylamide gel, but were unable to do so.     

In vivo inhibition of superoxide dismutase in mice by diethyldithiocarbamate.

Superoxide dismutase was assayed by a method which takes advantage of the inhibitory action of superoxide dismutase (or tissues which contain superoxide dismutase) on the rate of autooxidation of 6-hydroxydopamine. Incubation of pure superoxide dismutase of homogenates of brain or liver with 10(-3) M diethyldithiocarbamate for 1.5 hours resulted in total loss of superoxide dismutase activity. Inhibition of superoxide dismutase was not reversed by dialysis, but after dialysis, enzymatic activity was restored with CuSO4. When 1.5 g of diethyldithiocarbamate/kg were injected into mice, the superoxide dismutase activity at 3 hours was decreased by 86%, 71%, and 48%, respectively, in whole blood, liver, and brain. A dose of 0.5 g of diethyldithiocarbamate/kg lowered the superoxide dismutase activity by 42% in liver at 3 hours. A study of the time course for inhibiton of superoxide dismutase in liver after 1.5 g of diethyldithiocarbamate/kg, showed a maximum decrease (81%) within 1 hour, with a slow return to 64% of normal by 24 hours. Inhibition of superoxide dismutase in vivo and in vitro was confirmed with other assay systems based on the autooxidation of pyrogallol or epinephrine or on reduction of cytochrome c or intro blue tetrazolium. Treatment of animals with diethyldithiocarbamate may provide a useful experimental model to study the role of superoxide dismutase in various tissues.