The effect of cycloheximide upon polyribosome stability in two yeast mutants defective respectively in the initiation of polypeptide chains and in messenger RNA synthesis

Summary The effect of cycloheximide upon protein synthesis, RNA metabolism, and polyribosome stability was investigated in the parent and in two temperature-sensitive mutant yeast strains defective respectively in the initiation of polypeptide chains and in messenger RNA synthesis. Cycloheximide at high concentrations (100 g/ml) severely inhibits but does not completely stop protein synthesis (Fig. 1); the incorporation of ¹⁴C-amino acids into polyribosome-associated nascent polypeptide chains continues at a slow but measurable rate (Figs. 2 and 3). Polyribosome structures are stable in the parent strain at 36° whether or not cycloheximide is present (Fig. 5). However, in Mutant ts^- 136, a mutant defective in messenger as well as in stable RNA production, polyribosomes decay at the restrictive temperature (36° C) at the same rate whether or not cycloheximide is present (Fig. 5). Thus the maintenance of polyribosome structures is dependent upon the continued synthesis of messenger RNA even under conditions of extremely slow polypeptide chain elongation. In mutant ts^- 187, a mutant defective in the initiation of polypeptide chains, all of the polyribosomes decay to monoribosomes within 2 minutes after a shift to the restrictive temperature; cycloheximide completely prevents this decay demonstrating that this mutant is capable of continued messenger RNA synthesis at 36° C. Consistent with these observations is the fact that a newly synthesized heterogeneously sedimenting RNA fraction continues to enter polyribosomes in the presence of cycloheximide whereas the entrance of newly synthesized ribosomal RNA is severely inhibited (Figs. 7, 8, 9). The decay or lack of decay of polyribosomes at the restrictive temperature is, therefore, a rapid and discriminating test for the analysis of mutants defective in macromolecule synthesis. Mutants which exhibit a decay of polyribosomes in the presence of cycloheximide are likely to be defective directly or indirectly in the synthesis of messenger RNA whereas mutants in which decay is prevented or slowed by cycloheximide are likely to be defective in some factor required for the association of ribosomes and messenger RNA.     

Evidence for differential regulation of two classes of poly(A) RNA inBlastocladiella emersonii zoospores

The poly(A) RNA in zoospores ofBlastocladiella emersonii contains RNA synthesized during the growth phase (GP poly(A) RNA) and late sporulation (LS poly(A) RNA). LS poly(A) RNA synthesized during the final 30 minutes of sporulation is bound exclusively to polyribosomes which comprise approximately 50% of the total zoospore ribosome population. In contrast, GP poly(A) RNA is bound to zoospore monoribosomes. During the final 30 minutes of sporulation, GP poly(A) RNA which is bound to polyribosomes makes a transition to monoribosomes. Zoospore monoribosomes and RNA extracted from zoospore monoribosomes are inactivein vitro while both zoospore polyribosomes and RNA extracted from zoospore polyribosomes stimulate protein synthesis in the wheat germin vitro system. Sedimentation of poly(A) RNA from zoospore monoribosomes on dimethyl sulfoxide gradients revealed that the GP poly(A) RNA was of sufficiently high molecular weight to code for average-sized proteins. These denaturing gradients failed to activate the zoospore monoribosome RNA. The results suggest that the inability to translate zoospore monoribosomesin vitro is due to some property or modification of the zoospore monoribosome poly(A) RNA. Zoospore monoribosomes bound to poly(A) RNA contain an average of two tRNA molecules while zoospore polyribosomes have an average of less than one tRNA bound. This suggests the two classes of ribosomes are blocked at different steps in the elongation process.     

Trichodermin,a possible inhibitor of the termination process of protein synthesis

The mode of action of the antibiotic, trichodermin, on yeast cells has been investigated. Trichodermin specifically inhibits protein synthesis and, during the in vivo inhibition of protein synthesis, ribosomes remain in polyribosomes rather than shifting to monoribosomes. This observation suggests that trichodermin inhibits either an elongation step or a termination step of protein biosynthesis. These two possibilities were distinguished by comparing the action of trichodermin with that of cycloheximide, a known elongation inhibitor, upon the reformation of polyribosomes during recovery from a block in polypeptide chain initiation. Cycloheximide slows the recovery of polyribosomes from monoribosomes following a block in polypeptide chain initiation whereas trichodermin enhances the recovery of polyribosomes. This observation is interpreted to mean that trichodermin primarily inhibits the termination step of protein biosynthesis.     

Differences in the cytoplasmic distribution of newly synthesized poly (A) in serum-stimulated and resting cultures of BALB/c 3T3 cells.

Density-inhibited, serum-stimulated, and SV40 virus-transformed BALB/c 3T3 cultures were compared with respect to the rates of accumulation of cytoplasmic RNA molecules and with respect to the distribution of newly synthesized messenger RNA (mRNA) between polyribosomes and the post-ribosomal cell fraction. mRNA was isolated and quantitated by virtue of its association with radioactive polyadenylate (poly(A))-synthesized during a 90 min exposure of the cultures to ³H-adenosine. The rate of accumulation of cytoplasmic poly(A) rose slowly after serum stimulation and reached a value of 1.8 times that of resting cultures at 12 h after serum stimulation, which was also the time of onset of DNA synthesis. A change in the cytoplasmic distribution of newly synthesized poly(A) occurred more rapidly than the change in the rate of its synthesis, however. Resting cultures contained 37% of newly synthesized cytoplasmic poly(A)-containing RNA large enough to be mRNA in the post-ribosomal cell fraction, whereas virtually all of this material was found in polyribosomes at 3, 6 and 12 h after stimulation and in transformed cultures. The relatively infrequently translated mRNA of resting cultures was shown to be functional by cycloheximide treatment. (All BALB/c 3T3 cultures, resting or stimulated, contained about 20% of newly synthesized cytoplasmic poly(A) as nearly pure poly(A) in molecules of 4–6 Svedbergs in size, presumably too small to be mRNA.) We conclude that serum stimulation of density-inhibited cultures resulted in a more efficient use of the protein-synthesizing ability of the cell, and that the change in efficiency preceded increases in numbers of ribosomes and mRNA molecules.     

Kinetics of synthesis of cytoplasmic messenger-like RNA not associated with ribosomes in HeLa cells

The turn-over of cytoplasmic messenger-like RNA not associated with polyribosomes as well as that of polyribosomal mRNA was investigated by labelling with [3H]uridine in conditions of arrested ribosomal RNA and mitochondrial RNA synthesis. The synthesis of ribosomal RNA was inhibited with toyokamycin and that of mitochondrial RNA with ethidium bromide. In both accumulation kinetics and actinomycin-D-chase experiments, cytoplasmic messenger-like ribonucleoprotein particles and polyribosomes were fractionated by buoyant density centrifugation in CsCl gradients. The half-life of free m1RNA was found to be of 1--2 h whereas the bulk of polyribosomal mRNA was stable over the time period considered (up to 8 h) but with a minor short-lived component. Purification of RNA from polyribosomes labelled under the same conditions and fractionation of it into polyadenylated and non-polyadenylated fractions showed that this short-lived minor component of half-life less than 1 h is non-polyadenylated.     

The cytoskeletal framework of sea urchin eggs and embryos: developmental changes in the association of messenger RNA

Extraction of sea urchin eggs and embryos with Triton X-100 generated a cytoskeletal framework (CSK) composed of a cortical filamentous network and an internal system of filaments associated with ribosomes. The CSK contained only 10-20% of the cellular protein, RNA, and lipid. A specific subset of proteins was enriched in the CSK. Several lines of evidence suggest that mRNA is a component of the CSK of both eggs and embryos. First, the CSK contained poly(A) sequences which hybridized with [3H]poly(U). Second, the CSK contained polyribosomes. Finally, RNA extracted from the CSK showed translational activity in an in vitro system. The nonhistone messages present in the CSK were qualitatively similar to those solubilized by detergent, as determined by separation on polyacrylamide gels of the products of in vitro translation. In the unfertilized egg, most mRNA was present as nonpolyribosomal messenger ribonucleoprotein complexes which, along with monoribosomes, were efficiently extracted by Triton X-100. The converse was found in blastulae, as most of the mRNA was present as polyribosomes associated with the CSK, although monoribosomes were still efficiently extracted by detergent. These results indicate a correlation between the activation of protein synthesis in eggs and the association of polyribosomes with the CSK.     

Requirement of protein synthesis for the degradation of host mRNA in Friend erythroleukemia cells infected wtih herpes simplex virus type 1.

We describe experiments which demonstrate that shortly after infection of Friend erythroleukemia cells with herpes simplex virus (HSV), polyribosomes dissociate and cellular mRNA degrades. Analysis of infected cell extracts on sucrose density gradients demonstrates that the majority of the polyribosomes have dissociated to monoribosomes at 2 h postinfection. Physical measurements of infected-cell RNAs support this conclusion and demonstrate that the polyadenylated RNAs decrease in size. The degradation of mRNA is apparently a stochastic process as judged by the failure to detect a shift in the Crt1/2 when polyadenylated RNA extracted from infected cells at different times is hybridized to globin complementary DNA. In experiments designed to determine whether dissociation of polyribosomes is sufficient to cause degradation of globin mRNA, the amount of globin mRNA in uninfected cells did not change when cells were treated with NaF or pactamycin at concentrations sufficient to dissociate all polyribosomes. In cells infected with UV-irradiated virus polyribosomes dissociate but globin mRNA does not degrade, suggesting that it is possible to separate dissociation from degradation.     

Gene affecting longevity of messenger RNA: a mutant of Escherichia coli with altered mRNA stability.

Summary we have screened 897 temperature sensitive growth mutants ofE. coli for mutant strains showing longer mRNA half-life. The fate of pulse-labelled RNA was examined at 42° C after cessation of RNA synthesis and with prior exposure to nonpermissive temperature (42° C). Eight stains showed altered turn-over of RNA (presumably mRNA), and further analysis on mutant strain JE15144 indicated that the stability of pulse-labeled RNA as well as of tryptophan (trp) mRNA increased four to seven fold over its parental strain at 42° C. At 4 min or 10 min after addition of rifampicin, some 70 to 80% of polyribosome in the growing cells could still be conserved in JE15144 cultured at the nonpermissive temperature while little, if any, polyribosomes remained in its parental strain (PA3092) under the same condition. Two generation times were required for complete stoppage of growth of this mutant strain after shifting to 42° C, and protein synthesis continued at a significant, but slightly reduced, rate at 42° C. However, functional decay of mRNA in the mutant strain, with respect to the capacity for producing peptides, appeared to be similar to the parent strain, with half-lives of 3.5 min in PA3092 and 4.7 min in JE15144.     

The half-life of mRNA in Saccharomyces cerevisiae.

Summary The decay kinetics of mRNA was studied in a yeast temperature-sensitive mutant, ts136, which is defective in cytoplasmic RNA production at 37° C. The disappearance of the synthetic capacity of mRNA was determined by withdrawing equal volumes of ts136 cell culture and pulse-labelling with [³⁵S]methionine at various time intervals after the shift to 37° C from 23° C. The synthesized proteins were separated on a two-dimensional gel electrophoretic system and then quantitatively analyzed for their incorporated radioactivities by scintillation counting. Our results show that yeast mRNAs have divergent functional half-lives ranging from 4.5 to 41 min, with an average value of 22 min. Each mRNA exhibits a simple exponential decay with its own characteristic decay pattern. Of the approximately 500 major polypeptides made by yeast cells, which are detectable on autoradiograms of the gels, 80 were arbitrarily selected and the mRNAs coding for those polypeptides were examined for their decay kinetics.     

Inhibition of host protein synthesis by vaccinia virus: fate of cell mRNA and synthesis of small poly (A)-rich polyribonucleotides in the presence of actinomycin D.

Purified vaccinia virus rapidly inhibited HeLa cell protein synthesis in the presence of actinomycin D. Under these conditions host polyribosomes were extensively degraded but the mRNA was stable as indicated by a greater than 90% recovery of prelabeled polyadenylylated RNA. Although actinomycin D prevented the synthesis of host mRNA and poly(A) in uninfected cells, incorporation of adenosine into poly(A) was inhibited by less than 50% in infected cells. Further analysis indicated that there was little or no normal size viral mRNA but that a unique class of small poly(A)-rich RNA was made in the presence of actinomycin D. From measurements of the RNase resistance and base composition of the RNA, approximately 40% of the nucleotide sequence was estimated to be poly(A). The poly(A)-rich RNA was found associated with small polyribosomes and monoribosomes that were inactive in protein synthesis. It was suggested that the poly(A) segment of the RNA is formed by the poly(A) polymerase previously found in vaccinia virus cores and that the inactive RNA, by competing with host mRNA, may contribute to the virus-mediated inhibition of host protein synthesis observed in the presence of actinomycin D.     

Cytokinin-induced activation of polyribosomes in the protonema of Ceratodon purpureus

A ribosomal preparation from N⁶-isopentenyladenine-treated protonema of Ceratodon purpureus (Hedw.) Brid. exhibited an increased activity of protein synthesis in a cell-free system as compared to a control preparation. The ratio of polyribosomes to monoribosomes was the same in both preparations, and it is assumed that an activation of pre-existing polyribosomes was responsible for the increased efficiency in protein synthesis. An electrophoretic fractionation of the in vitro translation product showed an enhanced synthesis of some polypeptide fractions in the cytokinin variant.     

The interaction between newly-formed mRNA and ribosomal particles during retarded translation]

The formation of polyribosomes in mouse liver cells at the reduced-rate translation was studied by treatment with cycloheximide (CHI) and aurintricarboxylic (ATA) acid. An increase of polypeptide synthesis time by 1.7-2.7 times (0.5 mg CHI per 25 g of weight or 15 mg ATA per 25 g) leads to a delay of the entrance of newly formed cytoplasmic D-RNA into polyribosomes. These results are in agreement with the model of polyribosome formation from ribonucleoprotein precursors containing cytoplasmic D-RNA. On the other hand, in the presence of a CHI dose (5 mg/25 g) causing a dramatic (240-fold) increase of polypeptide synthesis time, the kinetics of entrance of newly formed D-RNA into polyribosomes does not differ from the normal one, and amount of the incorporated mRNA is even somewhat higher than under normal conditions. It is suggested that in this situation ribosomes are moving along the newly formed mRNA, and their movement is not accompanied by the synthesis of completed polypeptide chain.     

Analysis of cytoplasmic RNA and polyribosmomes from feline leukemia virus-infected cells.

Cytoplasmic virus-specific RNA and polyribosomes from a chronically infected feline thymus tumor cell line, F-422, were analyzed by using in vitro-synthesized feline leukemia virus (Rickard strain) (R-FeLV) complementary DNA (cDNA) probe. By hybridization kinetics analysis, cytoplasmic, polyribosomat, and nuclear RNAs were found to be 2.1, 2.6, and 0.7% virus specific, respectively. Size classes within subcellular fractions were determined by sucrose gradient centrifugation in the presence of dimethyl sulfoxide followed by hybridization. The cytoplasmic fraction contained a 28S size class, which corresponds to the size of virion subunit RNA, and 36S, 23S, and 15 to 18S RNA species. The virus-specific 36S, 23S, and 15 to 18S species but not the 28S RNA were present in both the total and polyadenylic acid-containing polyribosomal RNA. Anti-FeLV gamma globulin bound to rapidly sedimenting polyribosomes, with the peak binding at 400S. The specificity of the binding for nascent virus-specific protein was determined in control experiments that involved mixing polyribosomes with soluble virion proteins, absorption of specific gamma globulin with soluble virion proteins, and puromycin-induced nascent protein release. The R-FeLV cDNA probe hybridized to RNA in two polyribosomal regions (approximately 400 to 450S and 250S) within the polyribosomal gradients before but not after EDTA treatment. The 400 to 450S polyribosomes contained three major peaks of virus-specific RNA at 36S, 23S, and 15 to 18S, whereas the 250S polyribosomes contained predominantly 36S and 15 to 18S RNA. Further experiments suggest that an approximately 36S minor subunit is present in virion RNA.