Half-lives and relative amounts of stored and polysomal ribosomes and poly(A)+ RNA in mouse oocytes

Growing mouse oocytes were labeled in vitro with [³H]uridine and chased for 2 or for 7 days to estimate the relative amounts of RNA appearing in different fractions and to follow their turnover. Oocytes were lysed and thoroughly dispersed in the presence of 1% DOC, and centrifuged on sucrose gradients to separate polysomes from smaller components not engaged in translation. After the short chase, one-third of the labeled ribosomes appeared in EDTA-sensitive polysomes. The proportion of ribosomes in both fractions remained stable during the long chase, demonstrating no net flow from one fraction to the other. When gradient fractions were analyzed by poly(U) Sepharose chromatography, it was found that about 20% of the labeled poly(A)+ RNA appeared in polysomes after the short chase. The half-lives of stored and translated mRNA were followed relative to stable rRNA during the long chase. Stored mRNA was completely stable, but translated mRNA turned over with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of about 6 days. Other methods for separating stored from translated components were not successful, including sedimentation of putative large complexes (fibrillar lattices) containing stored components, or chromatography of lysates on oligo(dT)-cellulose. Results presented here combined with our previous results demonstrate that, during meiotic maturation, the percent of labeled stable RNA which is polyadenylated declines from 19 to 10%, suggesting deadenylation or degradation of half of the accumulated maternal mRNA.     

Crystallography of liver alcohol dehydrogenase complexed with substrates.

The cytoplasmic metabolism of two adenovirus (Ad2)-specific messenger RNAs, encoded within the left-hand 11% of the Ad2 genome, has been examined in a transformed rat embryo line (strain 8617). Both the accumulation of labeled mRNA and the decay of pulse-labeled mRNA after an actinomycin D chase indicate a stochastic turnover of these Ad2-specific mRNA with half lives of approximately 35 and 100 minutes, considerably shorter than the half-life of the bulk of mRNA.The separation of mRNA molecules according to the length of poly(A) showed that the poly(A) of the Ad2-specific mRNAs was shortened both during continuous and pulse-chase labeling. Moreover, consistent with the observed rates of mRNA turnover, the rate of poly(A) shortening in the Ad2 mRNAs was found to be faster than that of the bulk mRNA population. In addition, the results suggested that both mRNA turnover and poly(A) shortening proceeded by random endonucleolytic cleavage.     

Size and turnover of polyadenylic acid-containing ribonucleic acids in a fragile mutant of Saccharomyces cerevisiae.

Ribonucleic acid-containing polyadenylic acid [poly(A)+-RNA] was studied in lysates from an osmotic-sensitive mutant of Saccharomyces cerevisiae characterized by low nuclease activity. The poly(A)+-RNA fraction, analyzed by electrophoresis in polyacrylamide-formamide gels, constitutes a heterogeneous population of molecules, with molecular weights ranging from 0.2 X 10(6) to 3 X 10(6) and having an average of 1.2 X 10(6). The turnover rate of poly(A)+-RNA was determined by the decay of radioactivity after a cold uracil chase, and the observed half-life of 21 min corresponds to about 10% of the cell doubling time. Poly(A)+-RNA was analyzed by gel electrophoresis under denaturing and non-denaturing conditions. A correlation was established between the apparent secondary structure and the turnover rate of poly(A)+-RNA species.     

Turnover of the cystic fibrosis transmembrane conductance regulator (CFTR): Slow degradation of wild-type and ΔF508 CFTR in surface membrane preparations of immortalized airway epithelial cells

The protein product of the cystic fibrosis (CF) gene, termed the cystic fibrosis transmembrane conductance regulator (CFTR), is known to function as an apical chloride channel at the surface of airway epithelial cells. It has been proposed that CFTR has additional intracellular functions and that there is altered processing of mutant forms. In examining these functions we found a stable form of CFTR with slow turnover in surface membrane preparations from CF and non-CF immortalized airway epithelial cell lines. The methods used to study the turnover of CFTR were pulse/chase experiments utilizing saturation labeling of [³⁵S]Met with chase periods of 5–24 h in the presence of 8 mM Met and cell fractionation techniques. Preparations of morphologically identifiable surface membranes were compared to total cell membrane preparations containing intracellular membranes. Surface membrane CFTR had lower turnover defined by pulse/chase ratios than that of the total cell membrane preparations. Moreover, mutant CFTR was stable in the surface membrane fraction with little degradation even after a 24 h chase, whereas wild-type CFTR had a higher pulse/chase ratio at 24 h. In the presence of 50 μM castanospermine, which is an inhibitor of processing α-glucosidases, a more rapid turnover of mutant CFTR was found in the total cell membrane preparation, whereas wild-type CFTR had a lower response. The results are compatible with a pool of CFTR in or near the surface membranes which has an altered turnover in CF and a glycosylation-dependent alteration in the processing of mutant CFTR. © 1996 Wiley-Liss, Inc.     

Turnover of Labelled DNA in Differentiated Collenchyma

The incorporation of ³H-thymidine into the nuclear DNA of differentiated, non-dividing collenchyma tissue in cut shoots of Lycopersicon esculentum was studied autoradiographically. When the fed shoots were transferred for up to 168 h to a non-radioactive solution, a significant loss of label was observed following a chase for 48 h. Thereafter, the only further change in the labelling pattern was demonstrated by a 168 h chase. When fed shoots were alternatively re-rooted and sampled over a longer chase period, there was a consistent decline in the mean grain count per nucleus until 14 weeks after re-rooting. A final sampling at 32 weeks revealed that almost all of the labelled DNA had been lost or turned over from this tissue.     

Degradation and replenishment of the labile protein fraction in non-growing cells of the asporogenic mutant ofBacillus megaterium

Kinetics of degradation of labelled proteins was followed in two asporogenic mutants ofBacillus megaterium during incubation in a sporulation medium. Both the mutant producing exocellular protease (KM 1prn ⁺) and the mutant not producing the enzyme (KM 12prn) were found to contain a labile protein fraction, whose proportion decreases with prolonged time of labelling and whose half-life is about 1 h. Most proteins were relatively stable and were degraded at a rate of 1 %/h and 2 %/h in strains KM 1 and KM 12, respectively (half life 70–80 h and 35–40 h in strains KM 1 and KM 12, respectively). The intracellular proteolytic activity of the KM 12 mutant remains practically the same during incubation in the sporulation medium or slowly increases. The labile protein fraction practically disappears from the cells after a 3.5-h incubation. When such a culture is then subjected to a shift-up and transferred again to the sporulation medium, the rate of protein turnover temporarily increases. The temporary increase of the turnover rate is caused by a partial replenishment of the labile protein fraction rather than by an accelerated degradation of the relatively stable fraction. The intracellular proteolytic activity does not increase under these conditions. The wild sporogenic strain ofB. megaterium also contains the labile protein fraction. Its half protein life is 1 h or less. However, the second protein fraction is degraded much more rapidly than in the asporogenic mutants and its half life is 6–7 h.     

Interactions of CCCH zinc finger proteins with mRNA: tristetraprolin-mediated AU-rich element-dependent mRNA degradation can occur in the absence of a poly(A) tail

The CCCH family of tandem zinc finger proteins has recently been shown to promote the turnover of certain mRNAs containing class II AU-rich elements (AREs). In the case of one member of this family, tristetraprolin (TTP), absence of the protein in knockout mice leads to stabilization of two mRNAs containing AREs of this type, those encoding tumor necrosis factor alpha (TNFalpha) and granulocyte-macrophage colony-stimulating factor. To begin to decipher the mechanism by which these zinc finger proteins stimulate the breakdown of this class of mRNAs, we co-transfected TTP and its related CCCH proteins into 293 cells with vectors encoding full-length TNFalpha, granulocyte-macrophage colony-stimulating factor, and interleukin-3 mRNAs. Co-expression of the CCCH proteins caused the rapid turnover of these ARE-containing mRNAs and also promoted the accumulation of stable breakdown intermediates that were truncated at the 3'-end of the mRNA, even further 5' than the 5'-end of the poly(A) tail. To determine whether an intact poly(A) tail was necessary for TTP to promote this type of mRNA degradation, we inserted the TNFalpha ARE into a nonpolyadenylated histone mRNA and also attached a histone 3'-end-processing sequence to the 3'-end of nonpolyadenylated interleukin-3 and TNFalpha mRNAs. In all three cases, TTP stimulated the turnover of the ARE-containing mRNAs, despite the demonstrated absence of a poly(A) tail. These studies indicate that members of this class of CCCH proteins can promote class II ARE-containing mRNA turnover even in the absence of a poly(A) tail, suggesting that the processive removal of the poly(A) tail may not be required for this type of CCCH protein-stimulated mRNA turnover.     

Role of RNA and protein synthesis and turnover in the heat-induced increase in nuclear protein

The proteins which become associated with nuclei during hyperthermic exposure were characterized by labeled amino acid incorporation. Actinomycin-D (Act-D) or cycloheximide (CHM) pretreatment was used to determine whether concurrent RNA or protein synthesis is required for hyperthermia to induce the increase in nuclear protein content. Prior to heat exposure exponentially growing HeLa cells were (i) pulse labeled for 1 h, (ii) labeled for 36 h, or (iii) labeled for 24 h followed by 17 h chase. The nuclear specific activity (CPM/microgram protein) of [3H]lysine-labeled proteins did not change under any of the labeling conditions, whereas that of [3H]leucine-containing proteins increased significantly with (i) but not with (ii) or (iii), while that of [3H]tryptophan-labeled protein increased significantly with (i) and (ii) but not with (iii). Act-D treatment 1 h prior to and during heating did not affect nuclear protein increase, while CHM-treated cells showed generally less nuclear protein content (70% of control at 60 min) but nevertheless significant nuclear protein increase upon heating (60% increase at 60 min from 0 min). These results suggest that those proteins associated with nuclei following heat exposure are nonhistones with a high turnover rate, and the process dose not require the synthesis of RNA or proteins.     

Studies on the turnovers in vivo of adenosine di- and triphosphates in a coupling factor of Escherichia coli.

The metabolic stabilities of bound adenine nucleotides in a membrane-bound ATPase (EF1) [EC 3.6.1.3] of Escherichia coli were studied by estimating their rates of turnover in vivo. Two-thirds of the bound ATP prelabelled with 32Pi in EF1 molecules was retained after 3 h in a chase medium. The bound ADP was chased rapidly with a half time of decrease of less than 1 h, the rate similar to that of cytoplasmic free nucleotides. These results suggest that bound ATP in the EF1 is not a direct intermediate in oxidative phosphorylation.     

Synthesis and Turnover of Cell-Wall Polysaccharides and Starch in Photosynthetic Soybean Suspension Cultures

Soybean (Glycine max [L.] Merr.) suspension cultures grown under photoautotrophic and photomixotrophic (1% sucrose) culture conditions were used in 14CO2 pulse-chase experiments to follow cell-wall polysaccharide and starch biosynthesis and turnover. Following a 30-min pulse with 14CO2, about one-fourth of the 14C of the photoautotrophic cells was incorporated into the cell wall; this increased to about 80% during a 96-h chase in unlabeled CO2. Cells early in the cell culture cycle (3 d) incorporated more 14C per sample and also exhibited greater turnover of the pectin and hemicellulose fractions as shown by loss of 14C during the 96-h chase than did 10- and 16-d cells. When the chase occurred in the dark, less 14C was incorporated into the cell wall because of the cessation of growth and higher respiratory loss. The dark effect was much less pronounced with the photomixotrophic cells. Even though the cell starch levels were much lower than in leaves, high 14C incorporation was found during the pulse, especially in older cells. The label was largely lost during the chase, indicating that starch is involved in the short-term storage of photosynthate. Thus, these easily labeled and manipulated photosynthetic cells demonstrated extensive turnover of the cell-wall pectin and hemicellulose fractions and starch during the normal growth process.     

Stability of polysome-associated polyadenylated RNA from soybean suspension culture cells.

The half-life of polysome-associated, poly(A)-RNA in exponentially growing soybean (Glycine max) suspension culture cells was determined with pulse-chase experiments. Based on a best fit from a computer analysis of the data, two decay components for poly(A)-RNA were found. One component had a half-life of approximately 0.6 h, while the other had a half-life of about 30 h, similar to the doubling time of the cultures. At the beginning of the chase period, the short-lived component represented approximately 90% of the total poly(A)-RNA in the polysomes. This percentage decreased with time so that, under steady-state conditions, the long-lived component probably represented the majority of poly(A)-RNA.     

Selective Degradation of Newly Synthesized Nonmessenger Simian Virus 40 Transcripts

By pretreating simian virus 40-infected BSC-1 cells with glucosamine, [(3)H]uridine labeling of both cellular and viral RNA can be halted instantaneously by addition of cold uridine. We have studied the fate of pulse-labeled viral RNA from cells at 45 h postinfection under these conditions. During a 5-min period of labeling, both the messenger and nonmessenger regions of the late strand were transcribed. After various chase periods, nuclear viral species which sediment at 19, 17.5, and 16S were observed. Nuclear viral RNA decays in a multiphasic manner. Of the material present at the beginning of the chase period, 50% was degraded rapidly with a half-life of 8 min (initial processing). This rapidly degraded material was that fraction of the late strand which did not give rise to stable late mRNA species. Forty percent was transported to the cytoplasm, and 10% remained in the nucleus as material which sedimented in the 2 to 4S region. These 2 to 4S viral RNAs had a half-life of 3 h, and hybridization studies suggest that they are in part coded for by the late-strand nonmessenger region and are derived from the initial nuclear processing step. Another part is coded for by the late-strand messenger region and may be generated by some subsequent nuclear cleavages of 19S RNA into 17.5 and 16S RNAs. Transport of nuclear viral RNA into the cytoplasm was detected after a 5-min pulse and a 7-min chase. The maximum amount of labeled viral RNA was accumulated in the cytoplasm after a 30-min to 1-h chase. At least two viral cytoplasmic species were observed. Kinetic data suggest that 19S RNA is transported directly from the nucleus. Whether cytoplasmic 16S is formed by cleavage of 19S RNA in the cytoplasm is not clear. The half-lives of cytoplasmic 19 and 16S RNAs can be approximated as 2 and 5 h, respectively.     

Regulation of species-specific ribosomal RNA in a somatic hybrid cell.

When 13B hamster-mouse hybrid cells are harvested either right after 4 h of incubation with [me-3H]methionine or following 26 h of "chase" with excess non-radioactive methionine, in both cases about half of the labeled cytoplasmic rRNA is of hamster type. It had been previously shown in this laboratory (Eliceiri, G.L. (1973) Biochim. Biophys. Acta 312, 737-741) that when [3H]uridine was the radioactive precursor about 80% of the labeled cytoplasmic rRNA was of hamster type after a short incubation, and about half after a long incubation. It is postulated that a temporary difference in the specific acitivity of [3H]UTP in possibly segregated mouse and hamster types of nucleoli might account for these results. The master/mouse ratio of cytoplasmic rRNA in hybrid 13B is similar in free and in membrane-bound ribosomes, and in ribosomes of sparse (rapidly growing) cell populations and of confluent (slowly growing) cells.     

Rapid inhibition by cycloheximide of rat hepatic nuclear free and engaged poly(A) polymerase activities

This paper reports the effect of cycloheximide on the activity of rat hepatic nuclear poly(A) polymerase. Three hours after cycloheximide treatment (3 mg/100 g body weight), total rat hepatic nuclear poly (A) polymerase activity was decreased to 50% of the normal level. This conclusion was reached when the enzyme activity was measured either in the whole nuclei $\text{in vitro}$ or with partly purified enzyme preparations. When examined at different times after a single injection of cycloheximide, it was observed that poly(A) polymerase activity decayed biphasically with an initial rapid decay phase reaching a minimum at one hour $\text{(}\text{t}\text{1}\text{2}\text{= 0.8}\text{hrs}\text{)}$, followed by a stable phase thereafter. These results have been interpreted to mean that poly(A) polymerase consists of either a mixture of two structurally distinct populations of enzymes with a different turnover rate, or of a single type of enzyme with a protein factor which is rapidly turning over and which is required for maximal enzyme activity.     

Biosynthesis and stability of globin mRNA in cultured erythroleukemic friend cells

Biosynthesis and stability of the mRNA population in DMSO-induced Friend erythroleukemic cells were studied after labeling the RNA with ³H-uridine and then chasing it with nonlabeled uridine. Globin RNA metabolism was studied by hybridization to excess complementary DNA covalently coupled to oligo(dT)-cellulose. After a labeling period of 120 min, 2–4% of the poly(A)-containing labeled RNA was in globin RNA; it decayed with a half-life of 16–17 hr. The rest of the poly(A)-containing RNA was composed of two kinetic populations: 85–90% decayed with a half-life of about 3 hr, while 10% decayed with a half-life of about 37 hr. The portion of globin RNA in labeled poly(A)-containing RNA behaved in an unexpected fashion during the chase period. During the initial chase period, the percentage of globin RNA increased rapidly, reaching a maximum of about 15% at 20 hr, but if subsequently declined gradually.Based on these findings, a model was built that describes the changes in the proportion of globin mRNA in poly(A)-containing RNA during continuous synthesis and after chase of the labeled RNA. It appears that if the parameters described remain constant during the maturation of erythroblasts, then this model would not account for the almost exclusive presence of globin RNA in the reticulocyte. By far the most effective way to achieve this high level of globin RNA is the destabilization of the mRNA population which is more stable than globin RNA, and not the stabilization of globin RNA itself.