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 nonpolysomal messenger ribonucleoprotein containing actin messenger RNA in chicken embryonic muscles.

Cytoplasmic nonpolysomal mRNAs have been isolated in the form of 16-40S ribonucleoprotein particles from the postribosomal supernatant of 14-day-old chick embryonic muscles. An 8-20S RNA fraction isolated from these particles directs the synthesis of actin in a wheat germ embryo S-30 system, as judged by copurification of the products with chicken muscle actin by repeated cycles of G- to F-actin transformation; mobilities of the purified product on sodium dodecyl sulfate-polyacrylamide gels and urea gels; and analysis of the CNBr-cleaved peptides. The 16-40S particles have a buoyant density of 1.4 g/cm3 which corresponds to an RNA/protein ratio of 1:3. They do not contain detectable levels of ribosomal subunits, as judged by the absence of typical ribosomal proteins in the range of 15,000-30,000. They contain at least eight distinct polypeptide species in the molecular weight range of 44,000-100,000, including a prominent 44,000 species. The presence of these particles suggests that they may have a role in the regulation of translation in developing muscles.     

In vivo inhibition of Novikoff cytoplasmic messenger RNA methylation by S-tubercidinylhomocysteine.

The analogue S-tubercidinylhomocysteine (STH) has been used to study the methylation of mRNA in vivo. Partial inhibition of cytoplasmic poly(A)-RNA methylation was observed using a level of inhibitor which still permitted cell growth. Characterization of the partially methylated mRNA indicated the presence of cap structures lacking 2'-O-methylnucleosides, m7GpppN', which are normally not found in mammalian mRNA. Inhibition of additional methylated sites in mRNA at the second 2'-O-methynucleoside, and at internal N6-methyladenosine was also observed Methylation of 7-methylguanosine was not affected under the conditions used in these experiments. The methylnucleoside composition of cap structures differed in STH-inhibited and uninhibited cells. These results indicate that a completely methylated cap is not required for transport of mRNA into the cytoplasm. Furthermore, it may now be possible to assess in vivo the sequential nature of mRNA methylation and its potential role in mRNA processing.     

Aminoacylation of undermethylated mammalian transfer RNA.

To study the role of 5-methylcytidine in the aminoacylation of mammalian tRNA, bulk tRNA specifically deficient in 5-methylcytidine was isolated from the livers of mice treated with 5-azacytidine (18 mg/kg) for 4 days. For comparison, more extensively altered tRNA was isolated from the livers of mice treated with DL-ethionine (100 mg/kg) plus adenine (48 mg/kg) for 3 days. The amino acid acceptor capacity of these tRNAs was determined by measuring the incorporation of one of eight different 14C-labeled amino acids or a mixture of 14C-labeled amino acids in homologous assays using a crude synthetase preparation isolated from untreated mice. The 5-methylcytidine-deficient tRNA incorporated each amino acid to the same extent as fully methylated tRNA. The tRNA from DL-ethionine-treated livers showed an overall decreased amino-acylation capacity for all amino acids tested. The 5-methylcytidine-deficient tRNA from DL-ethionine-treated mice were further characterized as substrates in homologous rate assays designed to determine the Km and V of the aminoacylation reaction using four individual 14C-labeled amino acids and a mixture of 14C-labeled amino acids. The Km and V of the reactions for all amino acids tested using 5-methylcytidine-deficient tRNA as substrate were essentially the same as for fully methylated tRNA. However, the Km and V were increased when liver tRNA from mice treated with DL-ethionine plus adenine was used as substrate in the rate reaction with [14C]lysine as label. Our results suggest that although extensively altered tRNA is a poorer substrate than control tRNA in both extent and rate of aminoacylation, 5-methylcytidine in mammalian tRNA is not involved in the recognition of the tRNA by the synthetase as measured by aminoacylation activity.     

Polysomal and non-polysomal messenger RNA in neuroblastoma cells: Lack of correlation between polyadenylation or initiation efficiency and messenger RNA location

Neuroblastoma cytoplasm was fractionated on sucrose gradients into polysomes (>90 S) and non-polysomal particles (<90 S). Purified RNA from these fractions was translated using a wheat germ lysate and translation products were compared by two-dimensional gel electrophoresis. Non-polysomal messenger RNA directed the synthesis of a specific subset of polysomal mRNA translation products. Careful comparison of individual translation products demonstrated that specific mRNAs were not randomly distributed between polysomes and the non-polysomal fraction.Fractionation of both RNA populations into polyadenylated (poly(A)⁺) and non-adenylated (poly(A)^?) species indicated that specific, abundant non-polysomal mRNAs were not less adenylated than their polysomal counterparts. Furthermore, comparison of translation products from assays of subsaturating and supersaturating RNA concentrations demonstrated that no simple correlation could be made between the relative initiation efficiency of a specific mRNA and its distribution between polysomes and non-polysomal particles.     

Intracellular location of human fibroblast interferon messenger RNA

Human diploid fibroblast (FS-4) cells were induced to produce interferon mRNA by exposure to poly(rI)·poly(rC) plus cycloheximide. The intracellular location of interferon mRNA was investigated by differential centrifugation of the cytoplasm into a membrane (pellet) and a free (supernatant) fraction, followed by injection of mRNA isolated from either fraction into $\text{X}\text{.}\text{laevis}$ oocytes. When translation in FS-4 cells was prevented, most (85–90%) of the interferon mRNA activity was found in the free fraction. However, when translation was permitted, most (80–95%) of the interferon mRNA activity was found in the membrane fraction. These results are consistent with the predictions of the “signal hypothesis” (Blobel and Dobberstein, J. Cell Biol. 1975, $\text{67}$:835) for secretory proteins.     

Cytoplasmic processing events in the polyadenylate region of Physarum messenger RNA

Cytoplasmic processing events in the poly(A) region of mRNA fromPhysarum polycephalum are reviewed. Two classes of poly-containing RNA [poly(A)⁺ RNA] exist in the cytoplasm. One contains very short poly(A) sequences, averaging about 15 adenylate residues, while the other contains relatively long poly(A) sequences, averaging about 60 residues. Molecules with short poly(A) sequences are found exclusively in the polysomes while those with long poly(A) sequences are restricted to the free cytoplasmic mRNP. Since proteins are associated with only the long poly(A) sequences the poly(A) · protein complex is also restricted to the free mRNP. The long poly(A) sequences are relatively short-lived. They are degraded by two distinct processes, a shortening process in which 15–20 residues are gradually removed and a turnover process in which long poly(A) tracts are rapidly converted to the short sequences. This process, along with the dissociation of the poly(A) · protein complex, occurs when poly(A)⁺ RNA molecules located in free mRNP are transferred to the polysomes. Poly(A) · protein complex dissociation appears to preceed poly(A) turnover during translational selection. The significance of these processing events in relation to mRNA maturation is discussed.     

RNA methylation and control of eukaryotic RNA biosynthesis: processing and utilization of undermethylated tRNAs in CHO cells.

The role of RNA methylations in the control of tRNA production and utilization for protein biosynthesis has been investigated through a study of the effects in vivo of cycloleucine a specific and potent inhibitor of S adenosyl-methionine mediated methylation. During the cycloleucine treatment, the rate of appearance of newly synthetized tRNAs into the cytoplasm is markedly reduced (about 50%). These molecules are extensively (more than 90%) undermethylated and are integrated into polysomes, but at a slower rate than normally methylated tRNAs.     

Cytoplasmic Ribonucleoprotein Components of the Novikoff Hepatoma

Prominent nucleoprotein sedimentation boundaries were demonstrable in cytoplasmic extracts of Novikoff hepatoma. Fractionation of the homogenates by differential centrifugation or a density gradient method revealed that 65 to 75 per cent of the cytoplasmic ribonucleic acid was present in the form of free ribonucleoprotein particles. After purification by differential centrifugation in dilute buffer, the particles contained 37 per cent RNA, very little lipid, and no demonstrable membrane material. Ultracentrifugal boundaries corresponding to those seen in the original extracts were present, the main component having an s20, _w of 81 S. Upon exposure to chelating agents, the particles dissociated through an intermediate component with sedimentation rate of 56 S to a final stage in which 46 and 28 S subunits were present in a weight ratio of 2:1. ATP and pyrophosphate were equally effective in causing dissociation. ADP was considerably less effective. Treatment of the purified particles with deoxycholate removed one-third of the protein and significantly altered the ultracentrifugal pattern. The particles now dissociated directly to the 46 and 28 S subunit when exposed to chelating agents. Upon electron microscopy, the 81 S particle appeared as an oblate spheroid 24 mµ in diameter. The 46 and 28 S subunits also appeared spheroidal.     

Regulation of messenger RNA stability in eukaryotic cells

Regulation of the cytoplasmic stability of mRNAs has recetly been identified as a major control mechanism which governs mRNA levels in a variety of eukaryotic systems. In this review we discuss what is known about several experimental systems that exhibit regulated mRNA stability, describe the mechanisms that cells may use to achieve control of mRNA degradation, and suggest areas of future investigation likely to provide new insights into this process.     

Comparison of proteins bound to heterogeneous nuclear RNA and messenger RNA in HeLa cells.

Ribonucleoprotein particles containing either heterogeneous nuclear RNA or polyribosomal messenger RNA were isolated from growing HeLa cells in order to compare their respective protein components. The major obstacle to analysing the proteins bound to HeLa cell mRNA proved to be the cosedimentation of a large fraction of the mRNP² particles with ribosomal subunits following puromycin or EDTA disassembly of polyribosomes. This was circumvented by oligo(dT)-cellulose chromatography, in which essentially all of the ribosomal subunits passed through the column without retention, while approximately 80% of the pulse-labeled, poly(A)-containing mRNP became bound and could be eluted with formamide. Polyacrylamide gel electrophoresis of the non-bound fraction (ribosomal subunits) revealed polypeptides between 15,000 and 55,000 molecular weight, with no detectable components greater than 55,000. The oligo-(dT)-bound mRNP contained a much simpler protein complement, consisting of three major components having molecular weights of 120,000, 76,000 and 52,000.In the case of the nuclear ribonucleoprotein particles that contain heterogeneous nuclear RNA, oligo(dT)-cellulose chromatography revealed two classes of particles. The first contained 10 to 20% of the hnRNA, did not bind to oligo(dT)-cellulose in 0.25 m-NaCl, 10 mm-sodium phosphate buffer, pH 7.0 (4 °C), and contained primarily a single polypeptide component having an estimated molecular weight of 40,000 (“informofers”). A second population of hnRNP particles comprised approximately 80% of the hnRNA, displayed strong binding to oligo(dT)-cellulose at 0.25 m-NaCl, and contained a very complex population of proteins, having molecular weights between 40,000 and 180,000, the same as unfractionated hnRNP. The results indicate that, at the resolution of gel electrophoresis and at the sensitivity of Coomassie blue dye, the proteins bound to HeLa cell hnRNA are qualitatively distinct from those bound to polyribosomal mRNA and, in addition, that the hnRNP proteins are the more complex of the two. These results are discussed in relation to the possible nucleotide sequence elements in hnRNA and mRNA to which these specific proteins are bound.     

Effects of 5-azacytidine on nucleolar RNA and the preribosomal particles in Novikoff hepatoma cells.

Examination of nucleolar RNA from cultured Novikoff hepatoma cells treated for 3 hr with 5 x 10-4 M 5-azacytidine shows that significant amounts of analog-substituted 45S RNA are processed to the 32S RNA species, but 28S RNA formation is completely inhibited. Under these conditions of analog treatment 37% of the cytidine residues in the 45S RNA is replaced by 5-azacytidine. During coelectrophoresis of nucleolar RNA from 5-azacytidine-treated and control cells, the analog-substituted 45S RNA and 32S RNA display reduced mobilities compared to the control 45S RNA and 32S RNA. Coelectrophoresis of analog-substituted and control RNA after formaldehyde denaturation shows no differences in electrophoretic mobility between the two RNA samples, suggesting that 5-azacytidine incorporation may alter the secondary structure of the 45S RNA and the 32S RNA. 5-Azacytidine at 5 x 10-4 M severely inhibits protein synthesis in Novikoff cells by 3 hr. After this length of treatment, however, CsCl buoyant density analysis reveals no difference in density of either the 80S or 55S preribosomal ribonucleoprotein particles when compared to normal particles. Also 5-azacytidine treatment does not appear to cause major changes in the polyacrylamide gel electrophoresis patterns of the proteins in the 80S and 55S preribosomal particles. These results together with previous findings suggest that 5-azacytidine's inhibition of rRNA processing is possibly related to its alteration of the structure of the ribosomal precursor RNAs and is not a consequence of a general inhibition of ribosomal protein formation.