Synthesis Of Ribosomal Precursor RNA Research Articles

Regulation of ribosome synthesis during compensatory renal hypertrophy in mice.

Ribosomal synthesis was studied at the transcriptional and translational levels to investigate the mechanisms of ribosome accretion during compensatory renal hypertrophy. As measured by in vitro transcriptional runoff comparisons 6-48 h after surgery, nuclei from the kidney remaining after contralateral nephrectomy show an increase of up to 150% in the rate of synthesis of ribosomal precursor RNA. The rate of rDNA transcription is 40-50% greater than control values as early as 6 h after nephrectomy; by 48 h, the rate returns to normal. In contrast to the stimulated transcription of rDNA and accretion of rRNA, the steady-state levels and the cytoplasmic distribution of ribosomal protein mRNAs S16 and L10 remain unchanged during induced renal growth. Thus coordinate production of adequate protein for increased assembly of ribosomes during induced renal growth appears to be accomplished by increasingly efficient translation of existing ribosomal protein mRNAs or by post-translational stabilization of ribosomal proteins. The rate of rDNA transcription may be regulated by accelerating the transcription of already functioning genes or, more likely, by recruiting transcription units that are transcriptionally inactive in the normal kidney.

The effect of herpesvirus infection on ribosomal RNA synthesis and on nucleolar size and number in HeLa cells.

The effects of infection by the herpesvirus pseudorabies virus on the nucleolus and on ribosomal RNA synthesis in HeLa cells were examined. The size of individual nucleoli and the total nucleolar content per cell are reduced to approximately 35 per cent of normal by 6 hours of infection. The proportion of cells containing one nucleolus rises from 10 to 50 per cent in the same time. The synthesis of ribosomal precursor RNA declines to approximately 40 per cent in this period.

The regulation of RNA metabolism in suspended and reattached anchorage-dependent 3T6 fibroblasts.

AbstractSuspending anchorage‐depent fibroblasts in methocel results in marked reduction in a number of macromolecular metabolic processes. These are restored when cells make contact with a solid substrate and are allowed to spread. The response of 3T6 cells to suspension culture and their recovery upon reattachment has been extensively studied. (Benecke et al., '78; Farmer et al., '78). In these cells, message production and its turnover rate both drop abruptly when cells are suspended, while protein synthesis declines very slowly but extensively. Recovery of protein synthesis in 3T6 cells only requires surface contact and not cell spreading and proceeds rapidly, reaching control values within a few hours. The recovery of messenger RNA production in spread cells is much slower, and commences about 18 hours after cells are replated.The present report extends the studies on RNA regulation in suspended and reattached 3T6 fibroblasts. All RNA synthesis systems respond to cell suspension. Production of small nuclear RNA species is shown to behave very similarly to the production of messenger RNA, suggesting these may be coordinately regulated. Ribosomal precursor RNA synthesis ceases promptly upon cell suspension and recovers only slowly after reattachment. The control of ribosome formation appears independent of the regulation of protein synthesis in this system. The products of polymerase III including transfer RNA, 5S RNA, and species K and L are all regulated together and show a distinct pattern of behavior unlike that of any other RNA species. Their formation is rapidly inhibited at the beginning of suspension, but then recovers to the level of control cells, even during continued suspension culture. This unusual property of polymerase III RNA species is quite different from the behavior of the small nuclear (snRNA) RNA species, which strengthens the previous suggestion that these two classes of RNA molecules are formed by different mechanisms and regulated independently.

Ribosomal proteins synthesis and exchange in the absence of 28-S and 18-S ribosomal RNA synthesis in L5178Y cells

The effect of the adenosine analogue toyocamycin on ribosomal proteins synthesis and assembly within ribosomal particles was investigated in the murine cells, L5178Y. The analogue was used for periods not exceeding 5 h, at a concentration which permits the synthesis of ribosomal precursor RNA but inhibits the maturation process. The following observations were made: 1. 1. Ribosomal proteins, synthesized de novo in the presence of the drug, were associated with toyocamycin-containing 45-S pre-rRNA in preribosomal-like 80-S ribonucleoproteins which accumulated in the nucleolus. Two-dimensional electrophoresis revealed a full protein complement of these particles, although minor discrepancies were observed in the relative proportions of a limited number of polypeptides. 2. 2. In the absence of 28-S and 18-S rRNA formation, a surprisingly high proportion of newly synthesized ribosomal proteins were incorporated into high-salt washed ribosomal subunits. The extent of individual protein exchange as well as their apparent turnover rates were markedly heterogeneous. Most of these exchangeable proteins were shown to be labeled rapidly in ribosomal subunits of normal cells. Some alternative interpretations of these results are discussed.

In the absence of ribosomal RNA synthesis, the ribosomal proteins of HeLa Cells are synthesized normally and degraded rapidly

It is possible to solubilize essentially the total protein complement from a lysate of HeLa cells. When such an extract is displayed on a two-dimensional polyacrylamide gel (pH 5·0 × sodium dodecyl sulfate) it is possible to isolate and identify 45 ribosomal proteins, five histones, and numerous other proteins. Using this method we have measured directly the synthesis of ribosomal proteins in the presence of actinomycin D. The results show that the synthesis of ribosomal proteins does not depend on concurrent transcription of ribosomal precursor RNA. Furthermore, if ribosomal precursor RNA synthesis is blocked for 25 hours by 10 ng of actinomycin/ml, ribosomal protein synthesis as well as histone synthesis continues normally. This suggests either that messenger RNA for ribosomal proteins is exceedingly long-lived or that the synthesis of mRNA for ribosomal proteins can be uncoupled from the synthesis of ribosomal precursor RNA. Ribosomal proteins synthesized in the presence of either high or low concentrations of actinomycin are unstable. They disappear, presumably through degradation, with half-lives of 30 to 100 minutes, depending on the individual proteins. Under these conditions, histones and several other cell proteins are stable.

Differential effects of phenobarbital and 3-methylcholanthrene on rat hepatic ribosomal precursor RNA

Synthesis of hepatic ribosomal precursor RNA (45S rRNA) was investigated in rats treated with single intraperitoneal injections of methylcholanthrene (MC) (30 mg/kg) or phenobarbital (PB) (100 mg/kg). Labelling in vivo of total nuclear RNA and of nuclear 45S rRNA with [ 3H]orotic acid was unchanged in rats sacrificed 3 or 20 hr after reveiving a single dose of MC. These results agreed with measurements in vitro of nucleolar RNA polymerase activity, which was unaffected by either MC or PB pretreatment. In rats sacrificed 6 hr after receiving an injection of PB or 3 or 20 hr after receiving an injection of MC, incorporation of [ 3H]UTP into isolated nucleoli was not enhanced. Labeling in vivo of microsomal 28S and 18S rRNA with [ 3H]orotic acid increased 50 per cent 6 hr after a single dose of PB but was unchanged 3 or 20 hr after a single dose of MC. The stability of 45S rRNA in rats treated with PB or MC was tested in a nuclear incubation system. After incubation of nuclei at 37° for 2 min in 0.25 M sucrose containing 5 mM MgCl 2, RNA was extracted and layered on 10–40% sucrose gradients. Planimetric analysis of these sucrose gradients demonstrated that, 16 hr after PB treatment, the stability of 45S rRNA was increased over that of controls, whereas 20 hr after MC, 45S rRNA stability was unchanged. These results suggest that PB, but not MC, enhances post-transcriptional stability of 45S rRNA and labeling in vivo of microsomal RNA. Neither MC nor PB increases synthesis of ribosomal precursor RNA.

Action of dichlorobenzimidazole riboside on RNA synthesis in L-929 and HeLa cells.

5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibits RNA synthesis in L-929 cells (mouse fibroblast line) and HeLa cells (human epitheloid carcinoma line) within 2 min of addition of the compound to the medium. By removing DRB from the medium, the inhibition is promptly and completely reversed after treatment of cells for as long as 1 h or even longer. The inhibitory effect of DRB on the overall rate of RNA synthesis is similar in L and HeLa cells and is markedly concentration-dependent in the low dose range (5-20 muM or 1.6-6.4 mug/ml), but not as higher concentrations of DRB. At a concentration of 12 muM, DRB has a highly selective inhibitory effect on the synthesis of nuclear heterogenous RNA in L cells. At higher concentrations, there is also inhibition of 45 S ribosomal precursor RNA synthesis, but at all concentrations the effect on heterogeneous RNA synthesis in L cells in considerably greater than that on preribosomal RNA synthesis. In HeLa cells, too, DRB has a selective effect on heterogeneous RNA synthesis, but quantitatively the selectivity of action is somewhat less pronounced. In both L and HeLa cells, the inhibition of synthesis of nuclear heterogeneous RNA is incomplete even at very high concentrations of DRB (150 muM). Thus, while DRB is a selective inhibitor of nuclear heterogeneous RNA synthesis, not all such RNA synthesis is sensitive to inhibition. It is proposed that messenger precursor RNA synthesis may largely be sensitive to inhibition by DRB. In short-term experiments, DRB has no effect on protein synthesis in L or HeLa cells. DRB has a slight to moderate inhibitory effect on uridine uptake into L cells and a moderate to marked effect on uptake of uridine into HeLa cells.

Amino acid starvation affects the initiation frequency of nucleolar RNA polymerase

The synthesis of ribosomal precursor RNA in mouse ascites nucleoli derived from cells starved for amino acids is compared with the activity of nucleoli from control cells cultivated in the presence of all amino acids. It is shown that deprivation of a single essential amino acid from the culture medium results in a drastic decrease of the RNA-forming capacity of the isolated nucleoli by a factor of 2–3. This switchoff in rRNA synthesis is a very fast process. Half-maximal inactivation occurs after only 30 min. Addition of amino acids to starved cells leads to a rapid recovery, which is reflected by a sharp increase in the RNA polymerase activity of the isolated nucleoli. Studies on the molecular mechanism of this amino acid-mediated control of rRNA synthesis indicate that this effect is not caused by different growth rates of the RNA chains, but rather by an altered initiation frequency of the RNA polymerase in vivo. Whereas in nucleoli derived from cells grown in full medium almost all the polymerase is tightly bound in a transcriptional complex, a high amount of “free” polymerase which becomes active after addition of exogenous template is present in nucleoli from starved cells.

The sensitivity of RNA polymerases I and II from Novikoff hepatoma (N1S1) cells to 3'-deoxyadenosine 5'-triphosphate.

The synthesis of ribosomal precursor RNA in Novikoff hepatoma (N1S1) cells is very sensitive to cordycepin (3'-dA). The synthesis of hnRNA, however, is resistant to inhibition concentrations of 3'-dA that completely block the synthesis of 45S ribosomal RNA precursor. We have examined the RNA polymerases present in these cultured cells with regard to their sensitivity to cordycepin 5'-triphosphate (3'-dATP) in an effort to explain the differential inhibition of RNA synthesis observed in vivo. RNA polymerases I and II were characterized on the basis of their chromatographic behavior on DEAE-Sephadex, as well as the response of their enzymatic activities to ionic strength, the divalent metal ions Mn2+ and Mg2+, and the toxin alpha-amanitin. For both enzymes the inhibition of in vitro RNA synthesis by 3'-dATP was competitive for ATP. The km values for ATP and the K1 values for 3'-dATP for the two enzymes were quite similar. RNA polymerase II, the enzyme presumed responsible for hnRNA synthesis, was actually slightly more sensitive to 3'-dATP than RNA polymerase I, the enzyme presumed responsible for ribosomal precursor RNA synthesis. Similar data were obtained when the RNA polymerases were assayed in isolated nuclei. These results indicate that the differential inhibition of RNA synthesis caused by 3'-dA in vivo cannot be simply explained by differential sensitivity of RNA polymerases I and II to 3'-dATP.

Inhibition of macromolecular synthesis in L1210 mouse leukemia ascites cells by hycanthone

Hycanthone inhibits macromolecular synthesis in L1210 cells. At the same concentrations of hycanthone, DNA synthesis is inhibited to a greater extent than the synthesis of RNA. There is an inhibition by the drug of the synthesis of ribosomal precursor RNA; the synthesis of other types of RNA including heterogeneous and small-molecular-weight, also appears to be inhibited. Maturation of 45-S RNA does not seem to be affected, and there is a partial reversal of inhibition of both DNA and RNA synthesis by washing the cells free of hycanthone.

A comparison of the effects of alkylating agents 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and nitrogen mustard on nuclear RNA synthesis/and processing

The effect of three alkylating agents on the RNA metabolism of HeLa cells were compared. The agents, nitrogen mustard (HN 2), 1,3-bis(2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, are quite different in their modes of inhibition and relatively specific in their effects on RNA synthesis and processing. Nitrogen mustard severely inhibits the formation of 45 S ribosomal RNA precursor in the nucleolus by producing shortened RNA chains. The remnant 45 S RNA that is produced appears to process normally at least as far as 32 S. The drug produces shortened nucleoplasmic RNA (HnRNA) molecules. Cytoplasmic messenger RNA is completely inhibited, presumably as a result of the aberrant nucleoplasmic synthesis. 1,3-bis(2-chloroethyl)-1-nitrosourea has little immediate effect on ribosomal precursor RNA synthesis in the nucleolus. However, subsequent processing of the 45 S precursor RNA is severely inhibited. Prolonged exposure to the drug leads to an inhibition of 45 S RNA synthesis, possibly because of the block in the processing step. The drug has little effect on HnRNA synthesis. The addition of poly(A) to HnRNA is normal. However, the subsequent appearance of poly(A)-containing cytoplasmic mRNA is severely inhibited. 1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea directly inhibits the synthesis of 45 S precursor RNA in the nucleolus. The processing of such RNA as is formed is also inhibited. In contrast, it has little or no effect on HnRNA synthesis and does not affect the appearance of poly(A)-containing cytoplasmic message.

Comparison of regulation of synthesis and utilization of 45S ribosomal precursor RNA in diploid and heteroploid human cells in response to valine deprivation

Diploid human fibroblasts respond similarly to heteroploid, malignant cells (HeLa) in decreasing both the synthesis and the utilization of 45S ribosomal precursor RNA when cells are blocked in growth by valine deprivation. During valine deprivation the diploid cells, like HeLa cells, decrease 45S r-pre-RNA synthesis to approximately half the rate in active growth, but utilize only about one-fourth of this RNA for ribosome manufacture. The diploid cells also continue synthesis, and partial utilization, of 45S r-pre-RNA for some time after protein synthesis is completely blocked with cycloheximide, as has previously been observed with HeLa cells. Thus the synthesis of ribosomal precursor RNA appears to be under non-stringent control in diploid, normal, as well as heteroploid, malignant human cells. A significant element in the regulation of ribosome synthesis in both types of cells is turnover of unused 45S r-pre-RNA.

Further studies on the stimulation of protein synthesis in androgen-dependent tissues by testosterone.

1. By using centrifugation through a discontinuous sucrose gradient, four microsomal fractions are obtained from the prostate gland. 2. Administration of androgens to castrated rats stimulates protein synthesis in all fractions, particularly in the heavy rough fraction. 3. Androgens also increase the content of protein, RNA and phospholipid in the heavy rough fraction. 4. Time-course experiments in vivo show that androgens induce a rapid increase in the synthesis of ribosomal precursor RNA preceding the synthesis of new microsomal fraction and the increase in protein synthesis.

Temperature sensitive mutations affecting ribosome synthesis in Saccharomyces cerevisiae

Studies have been carried out on the effects of mutations in nine distinct genes which regulate ribosome biosynthesis in yeast. Although some mutants have more extensive effects than others, in each case there is an inhibition by 50 to 80% of the synthesis of ribosomal precursor RNA. In each case there is an inhibition of the processing of that RNA which is made, ranging from 80 to 95%. In each case there is degradation of 50 to 70% of that RNA which is processed. Much of the RNA which survives to become the mature 25 s and 18 s species is found in functioning polyribosomes. In three of the mutants, ribosomal protein synthesis has been examined. At the restrictive temperature there is a rapid decline in the synthesis of ribosomal proteins to about 25% of normal. The rate of decline is several-fold faster than the average rate of messenger RNA degradation, leading to the possibility that there is some translational control of this synthesis. The inhibition of ribosome synthesis has enabled us to demonstrate that three proteins become associated with the 60 s subunit only after it reaches the cytoplasm. In none of the mutants is there any accumulation of precursor RNA, of defective precursor particles, or of one of the mature ribosomal subunits. We conclude that there is a stringent regulation of all facets of ribosome biosynthesis in yeast. An interruption at any step rapidly causes a feedback inhibition of all other steps.

Ribosome formation is blocked by camptothecin, a reversible inhibitor of RNA synthesis.

A new drug, camptothecin, has been used to study the regulation of ribosome synthesis in HeLa cells. 5 muM camptothecin inhibits the synthesis of heterogeneously sedimenting nuclear RNA by about 70%. Camptothecin also blocks a specific step in the processing of ribosomal precursor RNA, allowing the conversion of 45S RNA to 32S RNA, but inhibiting the conversion of 32S RNA to 28S RNA. The action of camptothecin, unlike that of actinomycin D, is rapidly reversible. Within 5 min after the removal of the drug, ribosomal precursor RNA synthesis and maturation resume at the normal rate. Ribosomal proteins made in the presence of camptothecin do not accumulate in the nucleolus, and are not subsequently used to form ribosomes if RNA synthesis is allowed to resume.

Effects of camptothecin on RNA synthesis in leukemia L1210 cells

The plant alkaloid camptothecin rapidly inhibits synthesis of ribosomal precursor RNA in the L1210 murine leukemia. The drug also produces extensive inhibition of heterogeneous nuclear RNA synthesis, but does not initially affect incorporation of uridine into 4-S and 5-S RNA. Camptothecin does not interfere with maturation of 45-S RNA into cytoplasmic 18-S and 28-S RNA species. When camptothecintreated cells are washed free from the drug, RNA synthesis is quickly restored.

Ornithine decarboxylase and ribosomal RNA synthesis during the stimulation of lymphocytes by phytohaemagglutinin

Omithine decarboxylase (ODC) catalyses the first step in the biosynthesis of polyarnines, the conversion of omithine to putrescine [l] . Its activity rises dramatically at an early stage in the conversion of many types of cells from a non-growing to a growing state [2-41 and it has also been reported to have an unusually short half life [5] . The consequent rapid fluctuations in enzyme activity in response to growth stimulation suggest that this enzyme might be involved in cell growth regulation mechanisms. Prominent among the sequence of metabolic changes invariably associated with the change from the non-growing to the growing state is an early increase in the synthesis of ribosomal RNA (rRNA), and there are some suggestions that ODC activity may be associated with this [6,7] . The effect of growth stimulation on rRNA synthesis has been studied in most detail during the activation of cultured human lymphocytes by phytohaemagglutinin (PHA). Separate effects of the growth stimulant on the synthesis of ribosomal precursor RNA and on the rate and efficiency of the maturation of the precursor rRNA have been identified [8-lo] . We have found that ODC activity increases greatly when lymphocytes are cultured with PHA. This increase correlates with, and is dependent upon, the major increase in the net synthesis of rRNA.

Effects of 3′deoxyadenosine (cordycepin) and 2′deoxyadenosine on nucleoside transport, macromolecular synthesis, and replication of cultured Novikoff hepatoma cells

3′Deoxyadenosine (cordycepin) inhibits the incorporation of uridine and adenosine into RNA and, to a lesser extent, DNA by Novikoff rat hepatoma cells growing in suspension culture without affecting the intracellular phosphorylation of the nucleosides to the triphosphate level. Although 3′deoxyadenosine competitively inhibits adenosine and uridine transport into the cell, this effect plays a minor role in its inhibition of nucleoside incorporation into nucleic acids. In contrast, 2′deoxyadenosine inhibits uridine and adenosine incorporation into nucleic acids in direct proportion to its effect on nucleoside transport and it is three to four times more effective as competitive inhibitor of nucleoside transport than is 3′deoxyadenosine. The latter inhibits preferentially the synthesis of ribosomal precursor RNA and transfer RNA, whereas the synthesis of heterogeneous nucleoplasmic RNA is little affected. It also inhibits to a much greater extent uridine incorporation into CMP than UMP of RNA. In addition, 3′deoxyadenosine inhibits amino acid incorporation into protein to about the same extent as DNA synthesis and, at 0.4 m m, stops very rapidly and completely cell division. These and other results suggest that most of the multiple effects of 3′deoxyadenosine might be related to an interference by 3′deoxyadenosine nucleotides with various normal functions of ATP and 2′deATP.

A comparative study of RNA synthesis in rat hepatic nuclei and mitochondria under the influence of cortisone

1. A mixture of labeled [ 3H]uridine and [ 14C]guanine was employed in 10-minute pulse labeling experiments in vivo to study the nature of RNA synthesis in hepatic nuclei and mitochondria of adrenalectomized rats under the influence of cortisone. 2. Ten minutes after cortisone treatment there was a change in the nature of the newly synthesized total hepatic RNA. Specific activity measurements indicated that the U/G base labeling ratio of the total RNA of hormonally treated rats was elevated relative to controls as a consequence of a concurrent stimulation of U-rich RNA synthesis and a diminution of G-rich RNA synthesis. 3. Four hours after hormonal treatment, with a 10-minute pulse labeling period, the U/G base labeling ratio of the total hepatic RNA was shifted downward as a consequence of cortisone enhancement of G-rich ribosomal precursor RNA synthesis in the nucleus. 4. Mitochondria actively synthesized U-rich RNA which was stimulated by a 10-minute cortisone treatment. However, there was no reversal of the RNA labeling pattern after four hours of hormone treatment, as evidenced by an unchanged high U/G base labeling ratio. It is therefore concluded that the mitochondrial ribosomal RNA genome is under a different mechanism of control than its nuclear counterpart. 5. In view of the fact that after a 10-minute pulse incorporation the U/G base labeling ratio of the RNA species was low in nuclei and high in cytoplasm, it is inferred that U-rich RNA synthesized in nuclei was rapidly transported into cytoplasm leaving behind the immature G-rich ribosomal RNA, whereas mitochondria retained their U-rich RNA. 6. Separate acid-soluble RNA precursor pools in nuclei and mitochondria seem to be present in liver cells, the former being high in U/G base ratio, the latter low. The U/G ratios of the acid-soluble precursor pools were relatively insensitive to cortisone both in nuclei and mitochondria after either 10 min or 4 h of hormonal treatment.

Effects of valine deprivation on ribosome formation in HeLa cells

When HeLa cells are deprived of the essential amino acid, valine, the formation of new ribosomes continues at a reduced rate compared to growing cells. Examination of nucleolar RNA reveals that during valine starvation there is a considerable decrease in the rate of 45 s ribosomal precursor RNA synthesis and of its conversion into 32 s RNA. Restoration of valine reverses these effects, but cycloheximide prevents reversal. The total steady-state level of nucleolar RNA remains practically constant for many hours after the onset of valine deprivation, but small changes in the relative amounts of 45 s and 32 s RNA occur. Preliminary experiments on cells deprived of other amino acids give results similar to those observed during valine deprivation. The findings indicate that in HeLa cells the availability of some particular protein or proteins may regulate the rate of synthesis and cleavage of ribosomal precursor RNA.