Translational regulation in growing clonal human astrocytoma cells in culture

The in vivo and in vitro protein synthesis by polysomes prepared from Cox astrocytoma cells grown in the presence of 100 mM ethanol were examined during transition from exponential to stationary growth phase. A sharp decline of translational activities of Cox poly (A)+messenger RNAs (mRNAs) occurred during this transition. This decline was accentuated when cells were grown in the presence of ethanol. The observed decline in mRNA translational activity was investigated in vitro in a micrococcal nuclease treated, mRNA depleted postmitochondrial supernatant (PMS) fraction containing [³⁵S]methionine. The formation of the³⁵S-labeled 40S ternary complex in the absence of mRNA and of the³⁵S-labeled 80S initiation complex in the presence of Cox or brain poly (A)+mRNAs were reduced substantially when the source of PMS was from stationary phase or ethanol exposed cells. The sedimentation of peaks containing 40S ternary and 80S initiation complexes following sucrose density gradient analysis showed marked reductions in [³⁵S] methionine labeling during the transition to stationary phase and also following ethanol exposure. The reduced formation of initiation complexes suggests possible functional modifications of eukaryotic initiation factor-2 (eIF-2) present in the PMS fraction and of mRNAs under these conditions. Data suggest that cells initiate adaptive or protective mechanisms by reducing the rate of the initiation reaction following environmental alterations produced by ethanol.     

Changes in state of adenylation and time course of degradation of maternal mRNAs during oocyte maturation and early embryonic development in the mouse

Previous work has shown that more than 50% or about 50 pg of polyadenylated RNA found in the full-grown mouse oocyte is deadenylated or degraded during meiotic maturation. Here we show that rRNA declines by 60 pg during this period, accounting for most of the 80-pg decline in total RNA and indicating that a significant amount of mRNA is deadenylated but not degraded during maturation. Actin mRNA is deadenylated at about 7 hr of in vitro maturation, following the decline in its translation. The poly(A) tail on hypoxanthine phosphoribosyltransferase (HPRT) mRNA is elongated at 7 hr of maturation, preceding an increase in HPRT activity. Actin mRNA is partially degraded in the one-cell embryo and falls to near the limit of detection in the late two-cell stage, while HPRT mRNA shows no change in early two-cell embryos, but is deadenylated and declines greatly during the two-cell stage. In aging unfertilized eggs, most of these changes occur on a delayed schedule. The various species of alpha-tubulin mRNA are largely deadenylated and more than half are degraded during maturation. Taken together with other published results, we conclude that each mRNA has its own pattern of changes in the length of the poly(A) tail (correlated with translation) and degradation during the period of maternal control of protein synthesis, and, for those examined, the maternal mRNAs remaining in the early two-cell embryo are degraded to low levels by the late two-cell stage.     

RNA synthesis in the early stage of aerobic incubation of potato tuber discs

RNA synthesis of potato tuber discs during the early periodof their aerobic incubation was investigated by feeding thediscs with ³H-uridine. The rate of total RNA synthesis increasedin two steps during the incubation. The increase during thefirst 2 to 3 hr was small, but that after 3 hr was large. Thelabeled RNAs were separated into poly(A) containing RNA [poly(A)(+) RNA] and poly(A) lacking RNA [poly(A) (–) RNA] bythe use of a poly(U)-Sepharose column. Poly(A) (+) RNA was synthesizedeven in the freshly prepared discs which incorporated little¹⁴C-leucine into a protein fraction, and the synthetic rateof poly(A) (+) RNA increased by about 50% during the first 3hr incubation period, then gradually decreased thereafter. Synthesisof poly(A) (–) RNA continued to increase up to 7 hr afterslicing. When the discs were pulse labeled, the proportion ofradioactivity in poly(A) (+) RNA to that in the total RNA wasmaintained at about 50% until about 3 hr after slicing, butit abruptly decreased between 3 and 5 hr to about 35% whichwas maintained up to 9 hr after slicing. (Received October 12, 1977; )     

The correlation between the synthesis of skeletal muscle actin,myosin heavy chain,and myosin light chain and the accumulation of corresponding mRNA sequences during myogenesis

Cloned cDNA probes were used to measure the accumulation of myosin heavy chain, myosin light chain 2, and actin mRNA during differentiation of rat skeletal muscle cell cultures. This was compared with the changes in the rate of synthesis of the corresponding proteins. Accumulation of those mRNA sequences was detectable a few hours before the onset of the phase of cell fusion; however, the main increase in hybridizable RNA occurred during the phase of rapid cell fusion. A close correlation was found between the amounts of mRNAs coding for these proteins and the rate of synthesis of the proteins. The results suggest that the activation of stored mRNA is not a major mechanism for controlling the time at which these proteins are synthesized.     

Regulation of messenger RNA stability in mouse erythroleukemia cells

The decay rates of several messenger RNA species were determined in mouse erythroleukemia cells. The t1/2 values for the actin and tubulin mRNAs were 16 to 26 hours and about seven hours, respectively. The globin mRNA, and two mRNA species subject to translation repression, the P40 and P21 mRNAs, were about as stable as the ribosomal RNA. A stable tubulin mRNA component also appeared to be present in the cells. Exposure of the cells to dimethylsulfoxide for 48 hours led to considerable increases in the rates of decay of all but the globin mRNA. The induction of erythroid differentiation caused by the drug appears to lead to activation of a mRNA-degradation process that affects individual species to different degrees. The newly synthesized actin and tubulin mRNAs lost their poly(A) rather rapidly. This was accompanied by accumulation of poly(A)-deficient mRNA chains, particularly in the case of actin mRNA. The steady-state distribution of mRNA components, determined by Northern blot analysis, also showed that the actin mRNA and one tubulin mRNA species have a high proportion of poly(A)-deficient molecules. The globin, P40 and P21 mRNAs showed little tendency to lose their poly(A) sequence. The steady-state globin and P40 mRNAs also had a low proportion of chains depleted of poly(A). For all five species, the proportions of poly(A)-deficient chains in newly synthesized mRNA were about the same in uninduced and induced cells, in spite of the large decreases in mRNA stability in the induced cells. The lack of correlation between tendency to lose poly(A) and rate of mRNA decay, and the large accumulation of poly(A)-deficient molecules in the cases of the actin and tubulin mRNAs suggest that the stability of mRNA is not determined solely by the presence of poly(A) on the RNA chains. The behavior of the untranslated species in induced and uninduced cells also fails to support the notion of a relationship between translation and mRNA decay.     

Change in message sequences during erythrodifferentiation.

The change in the poly A(+) mRNA population during erythrodifferentiation was analyzed by cDNA-RNA hybridization. Poly A(+) RNA was isolated from spleen erythroblasts. When mice became anemic, the amount of globin mRNA increased to 50% of the total poly A(+) mRNA. cDNA from anemic spleen erythroblasts that did not contain globin mRNA sequences was cross-hybridized with mRNAs from mouse reticulocytes and cultured Friend leukemia (FL) cells. Only half the spleen cDNA hybridized with reticulocyte mRNA, whereas most of it hybridized with mRNA from FL cells. The results suggest that decrease in the complexity of the message population and increase in the concentration of globin mRNA are important in erythrodifferentiation.     

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.     

The metabolism of hyaluronan in cultured rabbit growth plate chondrocytes during differentiation

Hyaluronan (HA) is one of the major extracellular matrix components in cartilage. In addition to the biomechanical functions, HA has various important roles in the differentiation of chondrocytes. The purpose of this study was to clarify the nature of HA synthesis during chondrocyte differentiation. Growth plate chondrocytes were isolated from rabbit ribs and cultured in chondrocyte differentiation medium. The amount of HA and HA synthase (HAS) mRNA levels were analyzed for each stage of chondrocyte differentiation by means of high-performance liquid chromatography (HPLC) and real-time PCR, respectively. The distribution of HA in cultured chondrocytes was observed by histochemical staining. The amount of HA, ranging widely in size, was increased substantially during the hypertrophic stage. The expression levels of HAS2 and HAS3 mRNAs were low during the matrix-forming stage. HAS2 mRNA level was substantially enhanced at the pre-hypertrophic stage, whereas HAS3 mRNA level exhibited a slight increase. HAS1 mRNA was not detected. The intensity of HA staining was high around the hypertrophic chondrocytes. These results suggest that HA metabolism in chondrocyte differentiation is regulated by the selective expression of HASs, and HAS2 and the related large size-HA may have a certain association with the hypertrophic changes of chondrocytes.     

Model for the positional differentiation of the cap in Acetabularia.

A late stage during the biological cycle of the unicellular alga Acetabularia is the differentiation of a cap at the apical end of the stalk. A minimal model of the spatio-temporal regulation of this event is proposed on the basis of biological data available and current hypotheses. This involves the interaction between a diffusing inhibitor specific to the translation of cap mRNAs and a graded distribution of these messengers. The model accounts for delayed protein synthesis which occurs preferably at the apex and is likely to initiate the formation of the cap. The biological and theoretical implications are discussed.     

Accelerated poly(A) loss and mRNA stabilization are independent effects of protein synthesis inhibition on alpha-tubulin mRNA in Chlamydomonas.

In Chlamydomonas, the usual rapid degradation of tubulin mRNAs induced by flagellar amputation is prevented by inhibition of protein synthesis with cycloheximide. Evidence is presented that the ability of cycloheximide to stabilize alpha-tubulin mRNA depends on the time of addition. Addition of cycloheximide to cells before induction strongly stabilizes the induced mRNAs, while addition after their synthesis stabilizes them only transiently. Moreover, cycloheximide inhibition does not stabilize the same alpha-tubulin mRNA species in uninduced cells. These results suggest that cycloheximide is not acting to stabilize the induced alpha-tubulin mRNAs simply by preventing ribosome translocation. The stabilized state of tubulin mRNA was found to correlate with its occurrence on smaller polysomes but larger EDTA-released mRNP particles than the unstable state. A second effect of cycloheximide on the metabolism of induced tubulin mRNAs is to accelerate complete poly(A) removal. This effect of cycloheximide inhibition, unlike stabilization, occurs whenever cycloheximide is added to cells, and appears unrelated to stabilization. The effect is shown to be mRNA-specific; poly(A)-shortening on the rbcS2 mRNA is not altered in the presence of cycloheximide, nor do completely deadenylated molecules accumulate. Experiments in which cells were released from cycloheximide inhibition suggest that deadenylated alpha-tubulin mRNAs may be less stable than their polyadenylated counterparts during active translation.