Ribosomal RNA Turnover in Contact Inhibited Cells

CONTACT inhibition of animal cell growth is accompanied by a decreased rate of incorporation of nucleosides into RNA^1–3. Contact inhibited cells, however, transport exogenously-supplied nucleosides more slowly than do rapidly growing cells^4,5, suggesting that the rate of incorporation of isotopically labelled precursors into total cellular RNA may be a poor measure of the absolute rate of RNA synthesis by these cells. Recently, Emerson⁶ determined the actual rates of synthesis of ribosomal RNA (rRNA) and of the rapidly labelled heterogeneous species (HnRNA) by labelling with ³H-adenosine and measuring both the specific activity of the ATP pool and the rate of incorporation of isotope into the various RNA species. He concluded that contact inhibited cells synthesize ribosomal precursor RNA two to four times more slowly than do rapidly growing cells, but that there is little if any reduction in the instantaneous rate of synthesis of HnRNA by the non-growing cells. We have independently reached the same conclusion from simultaneous measurements on the specific radioactivity of the UTP pool and the rate of ³H-uridine incorporation into RNAs (unpublished work of Edlin and myself). However, although synthesis of the 45S precursor to ribosomal RNA is reduced two to four times in contact inhibited cells, the rate of cell multiplication and the rate of rRNA accumulation are reduced ten times. This suggests either “wastage”⁷ of newly synthesized 45S rRNA precursor, or turnover of ribosomes in contact inhibited cells Two lines of evidence suggest that “wastage” of 45S RNA does not play a significant role in this system. (1) The rate of synthesis of 45S RNA in both growing and contact inhibited cells agrees well with that expected from the observed rates of synthesis of 28S and 18S RNAs (unpublished work of Edlin and myself). Emerson has made similar calculations⁶. (2) 45S RNA labelled with a 20 min pulse of ³H-uridine is converted in the presence of actinomycin D to 28S and 18S RNAs with the same efficiency (approximately 50%) in both growing and contact inhibited cells. These results indicate that, in order to maintain a balanced complement of ribosomal RNAs, contact inhibited cells must turn over their ribosomes. We present evidence here that rRNA is stable in rapidly growing chick cells, but begins to turn over with a half-life of approximately 35–45 h as cells approach confluence and become contact inhibited.     

Changes in rate of RNA synthesis and ribosomal gene number during oogenesis of Drosophila melanogaster.

A new method for separating Drosophila egg chambers into different developmental classes (Jacobs-Lorena and Crippa, 1977) made it possible to study changes in the rate of ribosomal RNA (rRNA), 5S RNA, and tRNA synthesis and the changes in ribosomal gene number during oogenesis. Synthesis of RNA was measured by [³H]uridine incorporation in vivo and subsequent analysis on sucrose gradients or gel electrophoresis. Specific radioactivity of nucleotide pools has also been determined. Ribosomal gene number has been measured by hybridization of egg chamber DNA to rRNA of high specific radioactivity. Our findings led us to conclude that in Drosophila melanogaster: (i) rRNA, 5S RNA, and tRNA are synthesized in all stages of oogenesis. (ii) In every stage, rRNA is the main RNA species synthesized. (iii) The rate of rRNA, 5S RNA, and tRNA synthesis increases greatly during oogenesis and is paralleled by a similar increase in ribosomal gene number resulting from the polyploidization of the nurse cell nuclei.     

The pattern of protein and nucleic acid synthesis in germinating spores of Phycomyces blakesleeanus

Summary Synthesis of proteins, RNA and DNA is measured by incorporation of labelled precursors at different times during germination of Phycomyces spores.RNA and protein synthesis increases immediately after activation. DNa synthesis begins at a later stage (± 8 h) of germination when germ tubes are already present. Nuclear division occurs earlier in germination (±4–5 h) and is accompanied by a decrease in RNA synthesis. It can be concluded that at least most of the dormant spores are in the G2 phase of the cell cycle.Analysis of ribosomal RNA after pulse-chase labelling shows only three labelled compounds: a precursor molecule (2.25×10⁶ daltons) and the two mature ribosomal RNA compounds (1.4×10⁶ and 0.7×10⁶ daltons). This suggests that the two rRNAs are formed directly from the precursor molecule. Cycloheximide totally blocks the transformation of the ribosomal precursor molecule into mature rRNA.     

Influence of mycoplasma infection on the incorporation of different precursors into RNA components of tissue culture cells

Seven different tissue culture cells have been cultured with and without mycoplasma (M. hyorhinis) in the presence of various precursors of RNA. Total cellular RNA was isolated and analysed by electrophoresis on polyacrylamide gels. The results obtained with mycoplasma-infected cells can be summarized as follows:

1. 1. When cells are labelled with [8-³H]guanosine or [5-³H]uridine there is some incorporation into host cell 28S and 18S rRNA, but it is less than into mycoplasma 23S and 16S rRNA. [8-³H]guanosine or [5-³H]uridine are also incorporated into host cell and mycoplasma tRNA and mycoplasma 4.7S RNA, but the incorporation into host cell 5S rRNA and low molecular weight RNA components (LMW RNA) is reduced.

2. 2. [5-³H]uracil is not incorporated into host cell RNA but into mycoplasma tRNA, 4.7S RNA, a mycoplasma low molecular weight RNA component M₁ and 23S and 16S rRNA.

3. 3. [³H]methyl groups are incorporated into mycoplasma tRNA, 23S and 16S rRNA, but not into host cell 28S, 18S, 5S rRNA nor into mycoplasma 4.7S RNA.

4. 4. With [³²P]orthophosphate or [³H]adenosine as precursors, the labelling is primarily in the host RNA.

Mycoplasma infection influences the labelling of RNA primarily by an effect on the utilization of the exogenously added radioactive RNA precursors, since the generation time of mycoplasma infected cells is about the same as that of uninfected cells. Mycoplasma infection may completely prevent the identification of LMW RNA components.     

Frequency of N-benzyladenine incorporation into major RNA fractions of tobacco cell suspensions grown at stimulatory or cytostatic cytokinin concentration

The frequency of incorporation of the cytokinin N⁶-[p-³H]benzyladenine into major RNA species of tobacco (Nicotiana tabacum cv W 38) cells steadily increased as a function of its concentration in the culture medium, up to a 10 micromolar cytostatic overdose. During a 55-hour incubation of cells with 0.4 micromolar benzyladenine (BA), which is the optimal concentration for cell division, the incorporation frequency increased to one BA per 1.5 to 2.0 × 10⁴ conventional bases in total RNA. Frequencies of BA incorporation into 18S and 25S rRNA and into RNA precursors were very similar, 2- to 3-fold higher than the frequency of BA incorporation into the 4S + 5S RNA fraction. In cells incubated with 10 micromolar BA, the rate of RNA synthesis between 24 and 55 hours was lower than at optimal growth conditions; 18S and 25S rRNA synthesis was depressed more than the synthesis of 4S + 5S RNA. At 55 hours, BA was incorporated into total RNA at the steady state frequency of one per 1,300 conventional bases. All major RNA species were BA-labeled to approximately the same level, except that the labeling of the RNA precursors was 2-fold higher than the labeling of mature RNA species. These results may reflect an alteration in the processing of the RNA precursors at supra-optimal cytokinin concentration.     

A cell-free assay system for the analysis of changes in RNA synthesis during the development of Xenopus laevis.

Conditions were established for the maximal synthesis of RNA by Xenopus cultured cell nuclei. These differed from those for mammalian nuclei in having a lower K⁺ optimum. The Xenopus nuclei showed all three RNA polymerase activities and processed rRNA to 28 S and 18 S species. Extracts of full-grown oocytes stimulated the rate of RNA synthesis 2.5-fold and caused it to continue linearly for at least 6 hr. This full effect could be produced by preincubation of the nuclei with oocyte extract, followed by their reisolation and assay under standard conditions, provided that the four ribonucleotide triphosphates were present during the preincubation. The stimulatory factor(s) were mainly present in the cytoplasm of the oocyte. They produced quantitatively identical stimulations of RNA synthesis in hamster nuclei. The overall stimulatory effect of cell extracts disappears in the egg, remains absent through cleavage, but reappears at the late blastula stage. This corresponds to the changes in RNA synthesis believed to occur in early development. The extracts affect polymerases I and III, but not II to a significant extent. They also stimulate the incorporation of [γ-³²P]ATP and GTP into RNA, though to a lesser extent than the incorporation of [³H]UTP. The egg extract inhibits γ-³²P incorporation. There therefore seems to be some effect on the initiation of new chain synthesis, but its magnitude is uncertain, and the effect could be indirect.     

The Kinetics of the Synthesis of Ribosomal RNA in E. coli

The kinetics of the synthesis of ribosomal RNA in E. coli has been studied using C¹⁴-uracil as tracer. Two fractions of RNA having sedimentation constants between 4 and 8S have kinetic behavior consistent with roles of precursors. The first consists of a very small proportion of the RNA found in the 100,000 g supernatant after ribosomes have been removed. It has been separated from the soluble RNA present in much larger quantities by chromatography on DEAE-cellulose columns. The size and magnitude of flow through this fraction are consistent with it being precursor to a large part of the ribosomal RNA.

A fraction of ribosomal RNA of similar size is also found in the ribosomes. This fraction is 5 to 10 per cent of the total ribosomal RNA and a much higher proportion of the RNA of the 20S and 30S ribosomes present in the cell extract. The rate of incorporation of label into this fraction and into the main fractions of ribosomal RNA of 18S and 28S suggests that the small molecules are the precursors of the large molecules. Measurements of the rate of labeling of the 20, 30, and 50S ribosomes made at corresponding times indicate that ribosome synthesis occurs by concurrent conversion of small to large molecules of RNA and small to large ribosomes.

     

The differential regulation of the synthesis of ribosomal RNA, 5 S RNA, and 4 S RNA in the polytenic salivary gland cells of the blowfly, Calliphora erythrocephala

Third-instar larvae of the blowfly Calliphora erythrocephala were injected with [2-³H]adenosine, and its flow into the salivary gland ATP pool and each of several electrophoretically resolved salivary gland RNA species were quantitated. From these data, the individual in vivo rates of synthesis, accumulation, and processing of salivary gland ribosomal RNA (rRNA), 4 S RNA, and 5 S RNA have been measured at several different developmental stages. These results indicate that the synthesis of 5 S RNA and rRNA are coordinate, developmentally regulated, and independent of the synthesis of 4 S RNA. A nonribosomal, heterodisperse RNA component (hdRNA) was also identified. This species contributes to both the rapidly turning over pulse-labeled RNA and the accumulating pulse-labeled RNA populations. Indirect measurements suggest that the developmental pattern of regulation of this RNA species is also independent of 5 S RNA and rRNA synthesis. The rate of synthesis and accumulation of each of these RNA species either remained constant or declined during the first three-fourths of the instar, despite a six- to sevenfold increase in the content of cellular DNA.     

Rates of synthesis of ribosomal protein and total ribonucleic acid through the cell cycle of the fission yeast Schizosaccharomyces pombe

The rates of synthesis of ribosomal proteins through the cell cycle of the fission yeast Schizosaccharomyces pombe have been examined by spec. act. estimations of isolated 80S ribosomes pulse-labelled with ³⁵S-sulphate. The spec. act. have minimum values at the beginning (0.0) and maxima between 0.6 and 0.9 of the cell cycle. This pattern in spec. act. is also shown by isolated 80S ribosomes pulse-labelled with ³H-uridine during synchronous cultures and is in marked contrast to the small, random variations in the spec. act. of isolated 80S ribosomes from control, asynchronous cultures pulse-labelled with ³⁵S-sulphate or ³H-uridine.A detailed examination of the rates of synthesis of total RNA through the cell cycle measured by the rates of incorporation of ³H-uridine and ³H-adenine shows a step in the rates of incorporation at the time of DNA synthesis. This step has further been shown to be independent both of the uridine concentration, over a range from 0.03 μM to 820 μM, and of pre-filling the adenine pool. This step thus appears to be independent of variations in rates of uptake of both purines and pyrimidines, or fluctuations in the pool size of the precursors and may be explained as a gene-dosage effect.The step in the pattern of synthesis of total RNA has been shown to yield a cyclic pattern in the spec. act. of the total RNA through the cell cycle. This pattern is similar to that of the spec, act. of RNA and of protein recovered from ribosomes. The variation exhibited by the ribosomal proteins is believed to be a consequence of the step in the pattern of RNA synthesis, with a concomitant fluctuation in the pool of ribosomal proteins synthesised continuously through the cell cycle.     

Ribosomal ribonucleic acid maturation in synchronous strain l cells

Summary The incorporation of [³H]-5-uridine into cytoplasmic 18S and 28S ribosomal ribonucleic acid (rRNA) was examined in Colcemid-synchronized strain L cells during G1 and S phases of the cell cycle in the presence of 5×10⁻⁵ m uridine, which was determined to be the saturating concentration for this system. The data show that in S phase a significant increase occurs in the level of [³H]-5-uridine incorporation into each rRNA species. During a 90-min exposure period, S phase cells incorporate 3.4 times as much [³H]-5-uridine into 18S rRNA and 1.9 times as much into 28S rRNA as do G1 cells. The time required for maturation of the ribosomal RNA species during G1 and during S phase is the same, with 18S rRNA appearing in the cytoplasm in 20 min and 28S rRNA in 40 min.     

The 32 S RNA of Neurospora crassa mitochondria is not a precursor of the mitochondrial ribosomal RNAs.

Studies on the synthesis of Neurospora mitochondrial ribosomal RNAs by Kuriyama &; Luck 1973 have led to the currently accepted idea that the mature 19 S and 25 S rRNA species are synthesized via a common 32 S precursor RNA. The present results provide evidence that the 32 S RNA band analyzed by Kuriyama &; Luck was in fact a mixture of low concentrations of rapidly labeled RNA species, probably including separate precursors of 19 S and 25 S RNA, along with higher concentrations of aggregates of mature 19 S and 25 S RNA. The former account for the pulse-labeling characteristics of the 32 S band, whereas the latter contribute most of the mass-label, resulting in misleading hybridization data.     

RNA transcription and translation in sea urchin oocytes and eggs

The steady-state concentrations and absolute rates of synthesis of ribosomal RNA (rRNA) molecules were measured in oocytes, eggs, embryos, and larvae of the Hawaiian sea urchin Tripneustes gratilla. The steady-state concentration per genome of the RNA precursor sequences measured by hybridization to a cloned rDNA fragment was approximately 100- to 300-fold greater in the RNA obtained from oocytes and eggs than in the RNA extracted from embryos and larvae. Since the rate of processing of the rRNA precursor at different stages is not greatly different, the rates of rRNA synthesis must be considerably greater in oocytes than in embryo cells. The absolute rate of RNA synthesis in oocytes and embryos was determined from the incorporation of [³H]guanosine into cellular GTP pools and into both precursor and mature rRNA species. The data indicate an approximately 40-fold higher rate of rRNA synthesis in oocytes than that measured in embryos or previously in larvae (J. Griffith and T. Humphreys, 1979, Biochemistry18, 2178–2185). Together these results indicate that the ribosomal genes are transcribed much more rapidly during sea urchin oogenesis than during embryogenesis or larval stages.     

Alteration in the processing of ribosomal ribonucleic acid inSaccharomyces cerevisiae caused by ethidium bromide

Ethidium bromide in a concentration of 200 μg/ml causes a full inhibition of RNA synthesis in aSaccharomyces cerevisiae ρ° strain, while protein synthesis continues at a reduced rate. Under these conditions, processing of rRNA is slowed down and part of the 37S rRNA precursor molecules are cleaved to a 32S RNA fraction (molecular weight 2.15×10⁶). The 32S RNA accumulates in cells treated with ethidium bromide but cannot be processed to mature 25S and 18S rRNA and is degraded. The 32S RNA fraction also appears when processing of rRNA occurs in cells starved for required amino acids. The degradation of 37S precursor molecules through 32S RNA may be a regulatory mechanism of rRNA biosynthesis in yeast, which operates when excess rRNA must be wasted.     

DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1

RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.     

Effect of 5-fluoroorotic acid administration of the 32 P base composition, DNA-RNA hybridization properties, and labeling of polyadenylate-rich RNA in the cytoplasm of rat liver cells

5-Fluoroorotic acid treatment lowered the (Guanine + Cytosine)/(Adenine + Uracil) base ratio of ³²P-labeled microsomal RNA from a control value of 1.36 to 1.00. Low doses of actinomycin D, which are effective in inhibiting ribosomal RNA synthesis without significantly affecting messenger RNA synthesis, caused a similar decrease in the base ratio. Microsomal RNA labeled by [³H]orotate in the presence of 5-fluoroorotic acid had approximately $\text{1}\text{2}$ the specific radioactivity but twice the hybridization efficiency of RNA labeled in its absence. Evidence is presented that this RNA (1) has a different structure from that of ribosomal RNA, (2) hybridizes to DNA with an efficiency consistent with that of other published studies of polysome-associated messenger RNA, and (3) possesses sequences which are present in other samples of liver microsomal RNA but not in kidney microsomal RNA. These properties differ from those known to be exhibited by 18 S and 28 S ribosomal RNA. Electrophoretic analysis of this [³H]orotate-labeled microsomal RNA indicated that the analogue greatly inhibited precursor incorporation into ribosomal RNA but had little or no effect on incorporation into messenger RNA. Ribosomal RNA and polyadenylate-rich nonribosomal RNA were prepared from total polyribosomes by phenol extraction at pH 7.6 and pH 9.0, respectively. 5-Fluoroorotic acid inhibited [³H]orotate or ³²P_i incorporation into the pH 7.6 fraction much more effectively than incorporation into the pH 9.0 fraction. A subfraction of the pH 9.0 RNA which was retained by a polythymidylate-cellulose column had a greatly increased adenylate content.     

Control of the yeast cell cycle by protein synthesis

The increased synthesis of ribosomal RNA (rRNA) is correlated with enhanced cell proliferation, and it has been suggested that rRNA metabolism may have a regulatory role in the progression of the cell cycle. Alternatively, it might be the ensuing more active protein synthesis that drives the cell cycle progression. We have found that treatment with low doses of cycloheximide dissociates rRNA and protein synthesis. In fact, after the addition of cycloheximide the protein synthesis rate is strongly inhibited, whereas the rate of rRNA synthesis is unaffected for some time. The progression of the cell cycle, monitored as analysis of DNA distribution by flow cytometry and as bud emergence, is quickly and largely inhibited, thus indicating that a sustained rRNA metabolism is not sufficient to allow continuous cycle progression. The effects of cycloheximide on the daughter and mother duplication times, on the mean cell volume, and on the volume at budding were also analyzed. The results suggest that protein synthesis, rather than rRNA synthesis, may have a key role in the control of cell cycle progression in Saccharomyces cerevisiae.