Rapid increase in poly(A) (+) mRNA content following inhibition of protein synthesis

When resting 3T6 cells undergo a serum-induced transition to the growing state, the cytoplasmic content of ribosomal, transfer and messenger RNA increase as the cells prepare for DNA synthesis. The normal linear increase in mRNA content occurs even when the production of ribosomes is blocked. In this paper we determine the effect of inhibiting protein synthesis on the increase in poly(A) (+) mRNA content. Resting cells were serum stimulated in the presence of cycloheximide or puromycin at levels which inhibit protein synthesis by greater than 95%. Cytoplasmic poly(A) (+) mRNA content was determined at various times thereafter. We found that mRNA content increased five to ten times more rapidly in drug treated cells than in control cells stimulated in the absence of inhibitors. mRNA content increased 50–70% by one hour, and 60–90% by two hours following stimulation in the presence of inhibitor, and remained more or less constant thereafter. In contrast, mRNA content increased linearly in control stimulated cultures and did not double until about 15 hours after stimulation. The rapid increase in mRNA content is most likely the result of inhibition of protein synthesis rather than a secondary effect of the drug since the same observations were made in growth stimulated cells if protein synthesis was blocked with either puromycin or cycloheximide. A similar effect was also observed with resting 3T6, exponentially growing 3T6 and growing HeLa cells following exposure to cycloheximide, although the magnitude of the increase was less than that observed with growth stimulated cells. Puromycin had negligible effect on mRNA content in resting or exponentially growing cells. The rapid increase in cytoplasmic poly(A) (+) mRNA content was not due to rapid unbalanced export of nuclear poly(A) (+) RNA into the cytoplasm since there was no decrease in nuclear poly(A) content following serum stimulation in the presence of cycloheximide.     

Mobilisation of NADPH-glutamate dehydrogenase mRNA in regreening cultures of Euglena gracilis

Exposure of dark-grown resting Euglena gracilis Klebs strain Z Pringsheim to light results in a transient increase in the specific activity of NADPH-glutamate dehydrogenase. NADPH-glutamate dehydrogenase antibody was used to detect NADPH glutamate dehydrogenase resulting from the translation of total polyadenylated RNA and polysomal RNA from Euglena in a cell-free rabbit reticulocyte lysate system. NADPH-glutamate dehydrogenase mRNA was present in cells at all stages of development and present on polysomes from dark-grown and regreening cells but not on polysomes from dark-grown resting cells. These results indicate that the light-induced increase in NADPH-glutamate dehydrogenase in dark-grown resting cells represent an increase in the rate enzyme synthesis resulting from the mobilisation of NADPH-glutamate dehydrogenase mRNA onto polysomes.     

Increased efficiency of translation of ornithine decarboxylase mRNA in mitogen-activated lymphocytes

Ornithine decarboxylase (ODC) mRNA was elevated ninefold by 6 h following concanavalin A (ConA) stimulation of bovine lymphocytes. Comparison of the increases in ODC mRNA and ODC activity revealed a fivefold discrepancy, which is consistent with a change in efficiency of translation of ODC mRNA. In resting cells, 45% of the total ODC mRNA was associated with particles sedimenting at about 40 S, and therefore was not translated. The untranslated ODC mRNA in resting cells could be completely shifted into polysomes by a 15-min treatment of the cells with appropriate concentrations of cycloheximide. In activated cells, the proportion of ODC mRNA in untranslated material was reduced to 18%. This shift in distribution of ODC mRNA occurred between 6 h and 12 h following mitogen stimulation with no increase in the cellular level of this message. The rate of synthesis of ODC protein was found in increase twofold between 6 h and 12 h, paralleling the increase in the amount of ODC mRNA associated with polysomes. Thus, in this time frame, a decrease in the amount of untranslated ODC mRNA with a corresponding increase in the amount associated with polysomes leads to an increase in the biosynthesis of ODC with no change in the cellular level of the message. These changes in translational efficiency were not observed with actin mRNA.     

Parallel changes in protein synthesis and messenger RNA content in growing and resting epithelial cells of Xenopus laevis

The regulation of cell growth can be achieved at many levels but ultimately the regulatory factors must alter protein synthesis since growing cells always exhibit an increased rate of protein synthesis compared to resting cells. Some studies using growing and nongrowing mammalian cells have shown that the rate of protein synthesis compared to resting cells. Some studies using growing and nongrowing mammalian cells have shown that the rate of protein synthesis is directly dependent on mRNA content. Other studies have shown that growing and resting cells have similar amounts of mRNA and that protein synthesis is regulated by the proportion of mRNA in polysomes. We have analyzed mRNA content in growing and resting epithelial cells of Xenopus laevis. Quantitation of poly(A)⁺ mRNA by uniform labeling with ³H-uridine and by ³H-poly(U)hybridization demonstrated a direct relationship between mRNA content and the relative rate of protein synthesis in growing and resting cells. Likewise, after serum stimulation of resting cells the increase in mRNA content closely paralleled the increase in protein synthesis. Our results suggest that control of protein synthesis in growing and nongrowing cells is exerted before the translational level.     

Level and turnover of polyadenylate-containing ribonucleic acid in Neurospora crassa in different steady states of growth.

Mycelia of Neurospora crassa in a steady state of growth in different media have a ribosomal content proportional to the rate of growth. Moreover, both the percentage of polysomes and the average ribosomal activity are about the same at all different growth rates. The content of polyadenylated RNA was determined in three different conditions of exponential growth, which allowed growth rates that ranged from 0.26 to 0.51 duplications/h, and was found to constitute about the same fraction of total RNA (4.5--5.2%). Using a kinetic approach, an equation was derived which allowed determination of the average half-lives of polyadenylated RNA: in each medium the cultures were labeled from the moment of the inoculation with [32P]orthophosphate and were then given a 10-min pulse with [5-3H]uridine when they were in the exponential phase. It was found that the determined half-lives of polyadenylated RNA vary, depending on the growth medium, between 30 and 60 min, but with no direct correlation with the growth rate. Moreover, the rate of synthesis of polyadenylated RNA relative to that of stable RNA decreased with the growth rate. On the basis of previous data on the rates of synthesis of stable RNA, it was possible to make an evaluation of the absolute rate of synthesis of polyadenylated RNA. Whereas the rate of synthesis of stable ribosomal RNA increases as a function of the square of the number of duplications per hour, the increase in the rate of synthesis of polyadenylated RNA with the growth rate is much less consistent. It is concluded that in Neurospora the growth rate does not depend on the rate of synthesis of mRNA but rather on the rate of synthesis of rRNA, which sets both the ribosomal level and the steady-state level of mRNA.     

Direct measurement of tubulin and bulk message distributions on polysomes of growing, starved and deciliated Tetrahymena using RNA gel blots of sucrose gradients containing acrylamide.

A method was developed using sucrose gradients containing acrylamide which greatly simplifies the measurement of the polysomal distribution of messages. After centrifugation, the acrylamide was polymerized, forming a "polysome gel". RNA gel blots of polysome gels were used to determine the polysomal distributions of alpha-tubulin and total polyadenylated mRNA in growing, starved (nongrowing) and starved-deciliated Tetrahymena and the number of messages loaded onto polysomes was calculated. These measurements indicated that the translational efficiencies of alpha-tubulin mRNA and total polyadenylated mRNA are largely unaffected when the rates of tubulin and total protein synthesis vary dramatically. Thus, differential regulation of alpha-tubulin mRNA translation initiation does not contribute to the greater than 100-fold induction of tubulin synthesis observed during cilia regeneration and in growing cells. The major translation-level process regulating tubulin synthesis in Tetrahymena appears to be a change in message loading mediated by a non-specific message recruitment or unmasking factor.     

Insulin-stimulated ribosomal protein synthesis in maize embryonic axes during germination

Addition of insulin to maize seed ( Zea mays L. cv. Chalqueño) was found to accelerate germination and seedling growth. Insulin-stimulated maize axes showed enhancement of 35 S-methionine incorporation into ribosomal proteins (rp) and mobilization of S₆ rp mRNA into polysomes. Increase in S₆ rp phosphorylation of the small ribosomal subunit (40S) was observed in 32 P-orthophosphate pulse-labeled experiments when maize axes were stimulated by insulin. Application of either wortmannin or rapamycin, inhibitors of protein kinases of the insulin transduction pathway, abolished the insulin stimulatory effect on S₆ rp phosphorylation and on ribosomal protein synthesis. The above data are interpreted as an indication of the existence of an insulin-stimulated signal transduction pathway in maize tissues that is involved in the regulation of translation.     

Inhibition of dihydrofolate reductase gene expression following serum withdrawal or db-cAMP addition in methotrexate-resistant mouse fibroblasts

We have used a methotrexate (MTX)-resistant mouse 3T6 cell line (M50L3), that overproduces dihydrofolate reductase (DHFR) and its mRNA by a factor of 300, to study the mechanism for turning off DHFR gene expression following withdrawal of serum factors or elevation of the intracellular level of cAMP. When resting (G0) M50L3 cells are serum-stimulated to reenter the cell cycle, the level of DHFR activity begins to increase at about the same time the cells begin synthesizing DNA. The increase in enzyme activity is preceded by increases in the synthesis rate of the enzyme, and the content and production rate of DHFR mRNA. These increases, as well as entry into S phase, are blocked when the cells are serum-stimulated in the presence of dibutyryl cyclic AMP (db-cAMP) and theophylline. In this study, we found that when these drugs were added, or the serum stimulus was withdrawn during S phase (20 h following stimulation), the subsequent increase in DHFR level was blocked. Immunoprecipitation of DHFR from pulse-labelled cells showed that both treatments led to a rapid decrease in synthesis rate of the enzyme. The effect on total protein synthesis was much less than on DHFR synthesis. In DNA-excess filter hybridization experiments, we found that the content of cytoplasmic DHFR mRNA decreased in parallel with the synthesis rate of the enzyme. This was due in part to a decrease in the production rate of DHFR mRNA relative to total mRNA. In addition, drug addition or serum withdrawal led to a significant destabilization of DHFR (as well as total) mRNA. About 85% of poly(A)(+) DHFR mRNA was associated with polysomes in resting, growing, or cAMP-treated cells, suggesting that DHFR gene expression was not controlled at the translational level under these conditions.     

Changes in RNA in relation to growth of the fibroblast: II. The lifetime of mRNA,rRNA, and tRNA in resting and growing cells

The stabilities of the principal classes of RNA have been studied in resting and exponentially growing mouse fibroblast lines 3T6 and 3T3. Cytoplasmic mRNA, labeled with tritiated uridine and isolated by virtue of its poly A content, is equally stable in resting and growing cells, displaying a half-life of about 9 hr. We conclude that the accumulation of poly A(+) mRNA during transition from resting to growing state is due not to an increase in its stability, but to an increase in its rate of formation.The stability of cytoplasmic rRNA was measured after labeling with ³H-methyl-methionine. In agreement with the results of previous studies, we found that rRNA is stable in growing cells and unstable in resting cells. Quite unexpectedly, the 18S and 28S rRNA of resting cultures were found to differ appreciably in turnover rate. In both 3T6 and 3T3, the half-life of 28S RNA is about 50 hr, and that of 18S RNA about 72 hr. For this reason, though growing cells should synthesize the two ribosomal subunits in equal numbers, resting cells should synthesize more of the larger subunits than of the smaller. tRNA is unstable under all conditions. Its half-life is 36 hr in resting cells and about 60 hr in growing cells.     

Utilization of stored messenger RNA during early aging of Jerusalem artichoke tuber slices

Using dissociation in 0.8 M KCl, it was established that in freshly excised Jerusalem artichoke (Helianthus tuberosus L.) tuber slices less than 8% of the ribosomes were in polysomes. The first hour of aging in water was the period of most rapid polysome accumulation; over 32% of the ribosomes carried nascent polypeptide chains at the end of this time. Thereafter polysome accumulation continued to increase, but more gradually. While synthesis of high-molecular-weight RNA (presumed mRNA) was inhibited more than 95% by -amanitin during the first hour of aging, the inhibitor had no effect on polysome formation. As determined by [³H]polyuridylic acid hybridization, unaged cells contained polyadenylated RNA with a size range of 6–30S. The amount of polyadenylated RNA did not change during the first hour of aging. In control cells in water the in-vivo rate of protein synthesis increased exponentially during the first 4 h of aging without a comparable increase in polysomes. In -amanitintreated tissues a similar increase in protein synthesis was not observed despite the presence of near control levels of polysomes. It is suggested that early polysome formation depends on stored mRNA. Inhibition of mRNA synthesis by -amanitin prevents the normal development of an enhanced rate of protein synthesis which is not directly related to numbers of ribosomes in polysomes.Abbreviations Poly(A) polyadenylic acid - Poly(A)+RNA polyadenylated RNA - Poly(U) polyuridylic acid - TCA trichloroacetic acid     

The mTOR signaling pathway mediates control of ribosomal protein mRNA translation in rat liver

Previous studies have shown that oral administration of leucine to fasted rats results in a preferential increase in liver in the translation of mRNAs containing an oligopyrimidine sequence at the 5'-end of the message (i.e. a TOP sequence). TOP mRNAs include those encoding the ribosomal proteins (rp) and translation elongation factors. In cells in culture, the preponderance of evidence suggests that translation of TOP mRNAs is regulated by the mammalian target of rapamycin (mTOR), a protein kinase that signals through ribosomal protein S6 kinase (S6K1) to rpS6. However, the results of previous studies were recently challenged by several reports suggesting that translation of TOP mRNAs is independent of mTOR, S6K1, and S6 phosphorylation. The purpose of the present study was to evaluate the role of mTOR in the stimulation of TOP mRNA translation by leucine in vivo. Fasted rats were treated with the mTOR inhibitor, rapamycin, prior to oral administration of leucine. It was found that rapamycin severely attenuated leucine-induced signaling through mTOR in liver. In addition, rapamycin prevented the enhanced translation of TOP mRNAs in rats administered leucine, as assessed by a decrease in the proportion of TOP mRNAs associated with polysomes (i.e. those mRNAs being actively translated). Instead, in rapamycin-treated rats, ribosomal protein mRNAs accumulated in the fraction containing monosomes (mRNA bound to one ribosome). The results suggest that in liver in vivo, mTOR-dependent signaling is critical for maximal stimulation of TOP mRNA translation.     

Regulation of protein synthesis in human diploid fibroblasts: reduced initiation efficiency in resting cultures

The level of poly A+ RNA in growing cultures of human diploid fibroblasts is 1.8-fold times greater than in resting cultures. The level of functional ribosomes in growing cultures is 2.8 times that in resting cultures. Since transit times are similar in both types of cells, it can be concluded that the rate of protein synthesis in growing cultures is 2.8 times that in resting cultures. a reduced efficiency of mRNA translation at the level of initiation in resting cultures is proposed as a probable explanation for the fact that the decrease in protein synthesis rates is greater than the decrease in mRNA levels. This hypothesis is supported by the observations that: (a) poly A+ RNA is associated with smaller polysomes in resting than in growing cells, and (b) cycloheximide treatment of resting cells results in recruitment of nonpolysomal poly A+ RNA into polysomes and a shift of polysomal poly A+ RNA into larger polysomes.     

Quantitative changes in protein synthesis during oogenesis in Xenopus laevis

Protein synthesis rates in Xenopus laevis oocytes from stage 1 through stage 6 were measured. In addition, the translational efficiencies, total RNA contents, and percentages of ribosomes in polysomes in growing oocytes at several stages were determined. Stage 1 oocytes synthesize protein at a mean rate of 0.18 ng hr⁻¹ while stage 6 oocytes make protein at a rate of 22.8 ng hr⁻¹. Polysomes from growing and full-grown oocytes sedimented in a sucrose gradient with a peak value of 300 S, corresponding to a weight-average packing density of 10 ribosomes per mRNA. Ribosome transit times of endogenous mRNAs were essentially unchanged at all stages examined. While the oocyte's total ribosomal RNA content was observed to increase about 115-fold during oogenesis, the percentage of ribosomes in polysomes remained constant at approximately 2%. Taken together, the data suggest that the 127-fold increase in protein synthesis which occurs during Xenopus oogenesis involves the progressive recruitment onto polysomes of mRNA from the maternal stockpile.     

Translational control of protein synthesis after infection by vesicular stomatitis virus.

Four hours after infection of BHK cells by vesicular stomatitis virus (VSV), the rate of total protein synthesis was about 65% that of uninfected cells and synthesis of the 12 to 15 predominant cellular polypeptides was reduced to a level about 25% that of control cells. As determined by in vitro translation of isolated RNA and both one- and two-dimensional gel analyses of the products, all predominant cellular mRNA's remained intact and translatable after infection. The total amount of translatable mRNA per cell increased about threefold after infection; this additional mRNA directed synthesis of the five VSV structural proteins. To determine the subcellular localization of cellular and viral mRNA before and after infection, RNA from various sizes of polysomes and nonpolysomal ribonucleoproteins (RNPs) was isolated from infected and noninfected cells and translated in vitro. Over 80% of most predominant species of cellular mRNA was bound to polysomes in control cells, and over 60% was bound in infected cells. Only 2 of the 12 predominant species of translatable cellular mRNA's were localized to the RNP fraction, both in infected and in uninfected cells. The average size of polysomes translating individual cellular mRNA's was reduced about two- to threefold after infection. For example, in uninfected cells, actin (molecular weight 42,000) mRNA was found predominantly on polysomes with 12 ribosomes; after infection it was found on polysomes with five ribosomes, the same size of polysomes that were translating VSV N (molecular weight 52,000) and M (molecular weight 35,000) mRNA. We conclude that the inhibition of cellular protein synthesis after VSV infection is due, in large measure, to competition for ribosomes by a large excess of viral mRNA. The efficiency of initiation of translation on cellular and viral mRNA's is about the same in infected cells; cellular ribosomes are simply distributed among more mRNA's than are present in growing cells. About 20 to 30% of each of the predominant cellular and viral mRNA's were present in RNP particles in infected cells and were presumably inactive in protein synthesis. There was no preferential sequestration of cellular or viral mRNA's in RNPs after infection.