Glucocorticoids modify the rate of ribosomal RNA synthesis in rat thymus cells by regulating the polymerase elongation rate

0022-4731/84 $3.00 + 0.00 Copyright 0 1984 Pergamon Press Ltd

J. steroid Bioehem. Vol. 21, No. 5, pp. 497-504, 1984 Printed in Great Britain. All rights reserved

GLUCOCORTICOIDS MODIFY THE RATE OF RIBOSOMAL RNA SYNTHESIS IN RAT THYMUS CELLS BY REGULATING THE POLYMERASE ELONGATION RATE THOMAS C. DEMBINSKI*

and PHILIP A. BELL?

Tenovus Institute for Cancer Research, Welsh National School of Medicine, Heath Park, Cardiff CF4 4Xx, U.K. (Received 19 December

1983)

Summary-The mechanism by which glucocorticoids inhibit RNA polymerase A activity, and hence rRNA synthesis, in rat thymus cells has been investigated. Studies of the intranuclear distribution of RNA polymerase A between chromatin bound (“engaged”) and unbound (“free”) forms revealed that the steroid-mediated inhibition of the activity of the “engaged” form of the enzyme was not accompanied by significant changes in “free” pool activity. In the presence of rifamycin AF/O-13, an inhibitor of re-initiation of RNA polymerase A, the rate of [‘H]UMP incorporation into RNA was slower in nuclei from steroid-treated cells than in those from control cells, although in both conditions similar plateau levels of UMP incorporation were attained. Direct measurements of the numbers of transcribing RNA polymerase A molecules and of elongation rates showed that the inhibition of pre-rRNA synthesis was the result of a decrease in enzyme elongation rate; no significant change was observed in the number of transcribing enzymes. The steroid-induced inhibition of pre-rRNA synthesis was selectively abolished by mild proteolysis of nuclei, suggesting the involvement of a labile, regulatory glucocorticoid-induced protein. It is concluded that glucocorticoid treatment of rat thymus cells decreases 4% rRNA synthesis primarily by decreasing the polyribonucleotide elongation rate of RNA polymerase A, possibly by modification of the enzyme.

INTRODUCTION

Treatment of rat thymocytes and other steroidsensitive lymphoid cells with glucocorticoids results in the inhibition of a variety of metabolic processes and, ultimately, in growth inhibition and cell death. The expression of most, if not all, glucocorticoid effects in these cells can he blocked by inhibitors of RNA or protein synthesis [l-4]. Furthermore, for each effect, the period of sensitivity to cycloheximide corresponds with the course of emergence of the effect at the cellular level [2, 51. Such circumstantial

evidence has led to the hypothesis that glucocorticoids act in lymphoid cells at the transcriptional level, to initiate or stimulate the synthesis of species of RNA coding for regulatory proteins that are responsible for producing the observed inhibitory effects. Thus, one approach to the evaluation of the mechanism of action of glucocorticoids may be through an analysis of their effects at the transcriptional level. In early studies, glucocorticoid-induced changes in the DNA-dependent RNA polymerase (nucleoside triphosphate-RNA nucleotidyltransferase : E.C.

*Present address: Department of Physiology, Faculty of Medicine, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada. tTo whom correspondence should be addressed.

2.7.7.6) activity of rat thymus cell nuclei were observed 3 h after steroid injection in viva [5,6]. In more detailed investigations of the actions of glucocorticoids on the individual RNA polymerases [7], inhibitory effects of the synthetic glucocorticoid, dexamethasone, were observed on the activities of RNA polymerases A and B in rat thymus cell nuclei, both in vivo and in vitro. These effects were first apparent about 1 h after steroid administration and were preceded by the selective, but transient, stimulation of RNA polymerase B activity within 10 min of steroid addition. The inhibitory effects were concentrationdependent, glucocorticoid-specific, and could be blocked by glucocorticoid antagonists such as progesterone or cortexolone [l]. Studies with inhibitors of mRNA and protein synthesis have indicated that the suppressive effects of glucocorticoids on RNA polymerase A and B activities in rat thymus cells are mediated by different mechanisms [I, 81. Pretreatment of cell suspensions with a-amanitin, cordycepin (3’-deoxyadenosine) or cycloheximide all resulted in the abolition of the steroid effect on RNA polymerase A activity, but cycloheximide was unable to block the effect of the steroid on the activity of the form B polymerase. The steroid effect on RNA polymerase A activity could only be blocked if the inhibitors were added within 20 min of the steroid, the period during which form B activity was stimulated, suggesting that the inhibitory 497

498

THOMASC. DEMBINSKIand F%ILIP A. BELL

effect of the steroid on 4% rRNA synthesis, unlike that on hnRNA synthesis, depends on the synthesis of a species of mRNA and on its translation. Differences in the behaviour of the steroid-mediated effects on the form A and B enzymes have also been observed in response to energy deprivation [8]. The inhibitory effect of dexamethasone on RNA polymerase A activity in vitro, unlike the effects on RNA polymerase B, was dependent on the presence in the incubation medium of energy-yielding substrates such as glucose or amino acids. We now report the results of further experiments concerned with elucidating the mechanism by which glucocorticoids inhibit RNA polymerase A activity in rat thymus cells. These studies have involved: (a) determination of steroid effects on “free” and “engaged’ pools of form A activity, (b) “engaged” activity measurements in the presence of rifamycin AF/O- 13, and (c) direct measurements of the numbers of transcribing RNA polymerase molecules and of elongation rates. The results indicate that glucocorticoids suppress 45s rRNA synthesis primarily by decreasing the polyribonucleotide elongation rate of RNA polymerase A, possibly by modification of the enzyme. MATERIALS AND METHODS

Reagents

[5-3H]UTP (2 Ci/mmol), [5-3H]UMP (11.2 Ci/ mmol), [5-3H]uridine (5 Ci/mmol) and [5-‘H]GTP (2 Ci/mmol) were obtained from Amersham International, Amersham, Bucks, U.K. The sodium salts of ATP, CTP, GTP, UTP, UDP and UMP, uridine, Poly[d(A-T)] and a-amanitin were purchased from the Boehringer Corp., Lewes, Sussex, U.K. Trypsin, grade TRTPCK, was the product of Worthington Biochemical Corp., Freehold, NJ, U.S.A. Dexamethasone (9a-fluoro-16a-methyl-11/?,17,21trihydroxypregna-l+diene-3,20-dione), cycloheximide, actinomycin D, 2-mercaptoethanol, dithiothreitol and calf thymus DNA were supplied by Sigma Chemical Co., Poole, Dorset, U.K. Medium 199 with Earle’s salts and L-glutamine was obtained from Gibco Biocult, Paisley, Renfrewshire, U.K. Rifamycin AF/O-13 was a generous gift from Dr R. Cricchio, Gruppo Lepetit, Milan, Italy. Polyethylenecoated TLC sheets were imine(PEI)-cellulose purchased from Camlab Ltd, Cambridge, U.K. PPO(2,5-diphenyl- 1,3&oxadiazole), hyamine hydroxide (1 M solution in methanol), Triton X-100 and toluene were of scintillation grade and were supplied by Koch-Light Laboratories, Colnbrook, Bucks, U.K. All other reagents were of Analar grade and were obtained from BDH Chemicals, Poole, Dorset, U.K. Preparation and incubation of thymus cell suspensions

Thymus tissue was obtained from male Sprague-Dawley rats (8-12 weeks old) of the Insti-

tute colony, adrenalectoldized 1-2 weeks before use. Animals were killed by decapitation under light ether anaesthesia. Excised thymus glands were minced and hand-homogenized in a loose-fitting Teflon-glass homogenizer (1 mm clearance) in approx 20 ml of medium 199 at 4°C. The homogenate was filtered through 4 layers of nylon gauze and centrifuged for 2 min at 800 g. After discarding the supematant, the cell pellet was resuspended in medium and recentrifuged. The pellet was then suspended in medium to a final cell concentration of 5% (w/v). Cell suspensions in medium 199 were incubated at 37°C in a shaking water bath under an atmosphere of O2 + CO2 (95: 5). The cells were allowed to equilibrate for 15 min before dexamethasone was added, in medium, to a final concentration of 1 PM; control incubations received medium alone. Preparation of nuclei

Nuclei were isolated from thymus cell suspensions by the hypo-osmotic shock technique [9]. Thymocyte suspensions (5 ml) were diluted with 2mM MgClz (1: 10) and left for 15 min at 0°C. Nuclei were then collected by centrifugation at 8OOg for 2 min and the pellet was suspended in TKMS buffer (50mM Tris-HCl, pH 7.4 at 20°C containing 25mM KCl, 5 mM MgCl, and 0.25 M sucrose). The nuclei were recentrifuged and resuspended in TKMS buffer prior to the determination of nuclear RNA polymerase activities. Determination of RNA polymerase activity

RNA polymerase activities in isolated nuclei were assayed by a method based on published procedures [7, lo]. The reaction mixture contained 0.94mM ATP, CTP and GTP, 62.5pM [3H]UTP (O.SpCi), 10 mM Tris-HCl pH 8.0,2.5 mM 2-mercaptoethanol and 5% (v/v) glycerol in a final volume of 0.08m1, including the nuclear suspension in TKMS buffer. The assay mixture also contained 4mM MgC12, 50 mM KC1 and a-amanitin (1.25 pg/ml) for the determination of RNA polymerase A activity. Assays were initiated by the addition of nuclear suspension (0.05 ml) to the reaction mixture, which was then incubated at 37°C for 15 min. Incubations were terminated by cooling to 4°C and the incorporation of radioactivity into acid-insoluble material was determined by methods described previously [lo]. The DNA content of nuclear suspensions was estimated by the method of Burton [l l] with the modifications introduced by Giles and Myers [12], using calf thymus DNA as standard. The assays were unaffected by nucleotide pool size, since variations in the UTP concentration of the assay between 0.0625 and 6.25 mM had no effect on the steroid-induced changes in enzyme activity. Furthermore, the enzyme activity measured under low salt conditions was not affected by an increase in the concentration of a-amanitin from 1.25 to 125 pg/ml, indicating that the contribution of RNA

Glucoeartieoids and RNA polymerase A polymerase C (or III) activity to that measured as RNA polymerase A was negligible. Q~a~t~tu~~~nof p~~yrjb~~~cleor~ elongation rates and numbers of RNA polymerase molecules Elongation rates and numbers of transcribing RNA poiymerase molecules were determined by a modification [13] of the methods of Palmiter and Haines[ 141 and Cox[lS] which rely on estimation of the proportions of labelled uridine and UMP in alkaline hydrolysis products of RNA chains. Incubations contained 13HJUTP (62.5 PM; 10 @i) of sp. act. 2 Ci/mmol instead of the material of lower specific activity used in the routine assessment of RNA polymerase A activity. Assays were initiated by the addition of nuclear suspension (0.05 ml), containing approx 1OOpg DNA, followed by incubation at 37°C for 7 min. The reaction was terminated by the addition of ice-cold 2 M perchloric acid-66mM Na4P207 (1 ml). The precipitated material was washed extensively with ice-cold 0.3 M perchloric acid-10 mM Na4P207 and then incubated in 0.4 M KQH at 37°C for 16 h to hydrolys RNA. Neutralwere chromatographed on ized hydrolysates polyethylene-imine cellulose TLC plates after the addition of carriers. Chromatog~~s were developed first with distilled water, to separate uridine from the phosphorylated derivatives, and then with 2 M sodium formate buffer, pH 3.45 to separate UMP, UDP and UTP. The areas corresponding to U, UMP, UDP and UTP were visualized under U.V.light, excised and counted for radioactivity. Elongation rates were calculated from the [yH]UMP/[3HJuridine ratios after correction for the

u

$0

loo

150

2ti

Actinomycin 0 cont. (pg/mll

Fig. I. Titration of RNA polymerase A activity of rat thymocyte nuclei with aetinomycin D. RNA polymerase A activity was determined for rat thymocyte nuclei incubated at 37°C for 15 min in the presence of increasing concentrations of actinomycin D, with (0) or without (M) poly[d(A-T)] (30pg/ml). Results are expressed as percentages of the control activity in the absence of both actinomycin D and poly[d(A-T)].

499

degradation of UMP to U during the hydrolysis procedure, The number of 3’-term&i, repr~enting the number of transcribing enzyme molecules per cell, was computed from the incorporation of label into uridine per mg DNA [f 51.Data were corrected for the proportion of uridine in the RNA product using published base compositions of rat liver ribosomal RNA precursor [16]. The mean cell DNA content determined experimentally, by diphenylamine DNA assay and electronic cell counting, was 6.43 pg DNA/cell, simifar to a previously quoted value of 6.6 pg DNA/cell for rat leucocytes [ 171.

Radioactivity was measured using a NuclearChicago Mk. II liquid scintillation spectrometer (counting efficiency 25-30x) or a Searfe Isocap liquid spectrometer (efficiency 30-35%). scintillation Corrections for quenching were made by automatic external standardization using calibration curves for tritium. RESULTS

Ilexamethasotre-induced changes in “free ” and “en gaged” ~001%ofRNA pofymerase A activity in r@t thymocyfes A “free”’ pool of RNA polymerase A activity in rat thymus cell nuclei was detected following the addition of an exogenous poly[d(A-T)J synthetic template and suppression of endogenous enzyme activity by titration with actinomycin D (Fig. 1). The functional autonomy of both “free” and “engaged” pools of polymerase A activity was established as described by Yu[18] using labelled UTP and GTP in the presence and absence of poly[d(A-T)). The stimulatory effect of the synthetic template on nuclear enzyme activity was confined to the UTP assay. When rH]GTP was used to monitor RNA synthesis, the addition of poly[$(A-T)J neither enhanced nor antagonized incorporation These results indicated that the endogenous chromatin template retained all of the functionally active RNA polymerase (“engaged” form) for [‘H]GTP incorporation in the presence of poly[d(A-T)]. Consequently, the RNA polymerase population that transcribed the synthetic template was derived from a pool of polymerase A enzyme which was functionally inactive towards the endogenous chromatin template (“free” enzyme). The activity of the “free” form of RNA polymerase A was determined sim~taneo~ly with that of the “engaged” form, in the presence of polyid(A-T)] (50 &$ml) and actinomycin D (160 ggfml). Table 1 shows that treatment of rat thymus cells wtih 1 PM dexamethasone for 3 h resulted in significant inhibition of the activity of the “engaged” form of RNA polymerase A, but this was accompanied by insignificant changes in both the nuclear and cytosol “free” forms. Quantitatively similar results for the inhibition of “engaged” enzyme activity were ob-

500

THOMAS C. DFMBINSIU and Table 1. Effects of treatment

Assay conditions, pl.CCUKSOr

Nuclear “engaged”, [‘H]UTP [‘HfGTP Nuclear “free” (‘H]UTP ’ CytosoI “free” [‘H]UTP

PHILIP

A. BELL

of rat thymocytes with dexamethasone aolvmerase A activities

RNA polymerase A activity (pm01 precursor/mg DNA/l5 min) ___._..~ Control

+ Dex

on “free” and “engaged”

Dex-treated, Y0 of control - ___.--._. _

RNA

No. of ~. expts

34.4 & 12.4 42.1 * 5.1

24.3 i 9.5 28.3 & 3.5

70.0 * 5.4 67.2 + 0.3

8 2

36.3 f 9.5

32.4 + IO.1

88.8 + 9.0

8

33.2 & 4.5

30.5 f 4.9

91.7 f 2.4

2

Suspensions of rat thymocytes were incubated for 3 h at 37°C in the presence or absence of I pM dexamethasone. Nuclei were then prepared and assayed for RNA polymerase A activity. “Free” activity was determined simultaneously with that of the “engaged” form in the presence of poly[d(A-T)] (SOfig/ml) and actinomycin D (160 fig/ml). The MgCI, supematant from the preparation of nuclei was the source of cytosol “free” enzyme. Results are expressed as the mean value rf. 1 SD.

tained from assays with labelled GTP in place of UTP, and the U : G ratios were similar in the absence or presence of dexamethasone. EJficcts of r;~~yc~ AFjO-13 on controi and dexameth~one-inhibited RNA polymerase A activity in rat thymocytes Investigations of the effect of glucocorticoids on RNA polymerase A polyribonucleotide elongation rates were performed using rifamycin AF/O-13 to suppress re-initiation of the enzyme in the nuclear assay in vitro. On titration of endogenous activity with the inhibitor a plateau level of UMP incorporation (approx 75% of control activity) was observed between 100 and 250 pg/ml, confirming the utility of rifamycin AF/O-13 as an in~bitor of re-i~tiation of eukaryotic RNA polymerase A. The effect of incubation of thymus cells with 1 FM dexamethasone on the activity of the “‘engaged” form of polymerase A was then studied (Fig. 2). In the absence of rifamycin, the initial enzyme activity in nuclei from steroid-treated cells was 62.5% of that in

Incubation

time (mm1

Fig. 2. Effects of dexamethasone

and rifamycin AF/O-13 on RNA polymerase A activity in rat thymocyte nuclei. Rat thymocytes were incubated for 3 h at 37°C in the absence (m. 0) or presence (m. 0) of t pM dexamcthasone; nuclei were then isolated and assayed for RNA polymerase A activity in the absence (m, l ) or presence (a, 0) of rifamycin AFjO-13 (200 ~g~rni).

nuclei from control cells. The inhibitor did not affect initial synthetic rates, but prevented re-initiation by binding enzyme released after te~ination of the pol~ucleotide chains. In the presence of ~famycin AF/O-13 (2~~g/ml), sensitivity to the inhibitor developed earlier in time in nuclei from control cells than in nuclei from steroid-treated cells, but at similar extents of incorporation of UMP. Furthermore, the effect of dexamethasone on the plateau value of incorporation of UMP in the presence of rifamycin AF/O-13 was much less than the effect on the initial synthetic rate; the mean inhibition by dexamethasone at 15 min was 7.3 + 1.4% (mean * SD, PI = 3).

~~a~t~t~t~o~ of tr~s~r~bing~orm A RNA polymerases in rat thyme nuclei Steroid-induced changes in RNA synthesis were quantitated in terms of numbers of transcribing enzyme molecules and their polyribonucleotide elongation rates. Determinations of the numbers of elongating RNA polymerases depend upon the assumption that only these enzymes generate 3’-termini in RNA chains. In practice this may not be true, since some termini can be produced by the combined action of ribonuclease and phosphatase activity. An assessment was therefore made of these enzyme activities in isolated thymus nuclei. The combined action of ribonuclease and phosphatase was assayed by allowing incubations to proceed for longer times in the presence af actinomycin D; actinomycin D inhibits RNA synthesis in nuclei without impeding degradation by ribonucleases and phosphatases. The activity of these enzymes did not appear to be significant, since further incubation (13-26 min) in the presence of actinomycin D did not appreciably affect levels of UMP incorporation in nuclei from control or steroid-treated cells (Table 2). The separation by TLC of uridine and phosphorylated nudeosides obtained by alkaline hydrolysis of RNA is shown in Fig. 3. Since UT’P was removed by extensive perchloric acid washings, only UMP and U were present in RNA hydrolysis products. The lack of

501

Glucocorticoids and RNA polymerase A Table 2. Effect of prolonged incubation in the presence of actinomycin D on RNA oolvmerase A-directed RNA svnthesis UMP incorporated, pmol/mg DNA Assay duration, conditions 13 min (No actinomycin D) 26 min (Actinomycin D from 13 min)

Control

+DeF.

Dex-treated, “/, of control

26.0

18.8

72.3

24.4

18.4

IS.5

Suspensions of rat thymocytes were incubated for 3 h at 37°C in the presence or absence of 1 pM dexamethasone. Nuclei were then prepared and assayed for RNA polymerase A activity as described in the experimental section, except that assays were either terminated after 13 min or were allowed to proceed for 26 min after the addition of actinomycin D (3OOgg/ml) at 13 min. Results are mean values for duplicate nuclear preparations.

cross-contamination of separated uridine and UMP zones allowed accurate quantitation of labelled uridine and UMP in RNA hydrolysis products. The results obtained for nuclear RNA synthesis directed by the A enzyme in rat thymocytes incubated in the presence or absence of dexamethasone and cycloheximide for 3 h are presented in Table 3. A

Origin UMP U --

significant dexamethasone-induced decrease in UMP incorporation occurred concomitantly with a reduction in the elongation rate, while the number of transcribing enzyme molecules was not affected by the steroid. The presence of cycloheximide in the incubation did not significantly affect control UMP incorporation, but restored the activity of the dexamethasone-treated nuclei to the control value. However, in both control and dexamethasone-treated cells incubated in the presence of cycloheximide, an altered transcriptional response was observed. Decreases in the polyribonucleotide elongation rates were accompanied by compensatory increases in the numbers of form A enzyme molecules active in transcription. Eflect of mild proteolysis on nuclear RNA polymerase A activity in rat thymocytes

Fraction

no.

Fig. 3. Chromatography of [‘Hluridine and [3H]UMP in alkaline hydrolysates of RNA. Portions (0.03 ml) of the hydrolysate were subjected to thin-layer chromatography and the radioactivity determined. The migration areas of non-radioactive carriers, visible under U.V. light (260 nm), are marked.

Conditions for the trypsin treatment of nuclei were optimized with regard to the maximum enzyme concentration and duration of enzyme exposure that nuclei could withstand without rupturing (Table 4). These conditions allowed the effective removal of the enzyme by dilution and redispersion of pelleted nuclei in TKMS buffer for polymerase assay. If exposed to higher trypsin concentations or longer treatment times, the nuclei formed chromatin gels, indicative of membrane rupture, and could not be redispersed for assay of RNA polymerase activity. Following the treatment of rat thymus cells with dexamethasone for

Table 3. Parameters of RNA polymerase A-directed transcription in nuclei from rat thymocytes incubated in the nresence or absence of dexamethasone or cvcloheximide Cells incubated with: Dex

Cyclohex

_ _ + +

+ +

UMP incorporated, pmol/mg DNA/7 min (% control) 12.6 + 11.1 * 8.8 It 12.6 +

0.4 0.9 0.7 0.2

(100) (88) (70) (100)

IO-’ x No. of transcribing enzyme molecules/cell

(% control) 2.06 f 3.66 k 2.02 5 3.60 +

0.35 0.32 0.25 0.32

(100) (179) (99) (176)

Polyribonucleotide elongation rate, nucleotides/min

(% control) 90.7 + 51.2 f 66.8 * 59.1 +

1.9 1.7 4.5 0.9

(100) (56) (74) (651

Suspensions of rat thymocytes were incubated for 3 h at 37°C in the presence or absence of 1 PM dexamethasone, with or without 0.1 mM cycloheximide (cycloheximide was added I5 min before dexamethasone). Nuclei were prepared, and total UMP incorporation was determined in the presence of a-amanitin in a ‘I-min incubation as previously described. RNA was hydrolysed and numbers of transcribing RNA polymerase molecules and elongation rates were determined after chromatography of the hydrolysis products. Results are expressed as mean values of duplicate experiments f 1 SD, with values as percentages of the control in the absence of both dexamethasone and cycloheximide given in parentheses.

THOMASC. DEMBINSKI and

502

PHILIP

A. BELL

Table 4. Effect of treatment of nuclei with trvasin on RNA wlvmerase

A activitv

RNA polymerase A activity, pmol UMP/mg DNA/l 5 min (% control) Dexamethasone

Trypsin

_

_

_ + +

+ _ +

Expt 1 28.1 27.3 14.9 27.2

Expt 2 (100) (97) (53) (97)

23.0 23.0 12.7 20.0

(100) (100) (57) (87)

Suspensions of rat thymocytes were incubated for 3 h at 37°C in the presence or absence of 1 PM dexamethasone. Nuclei were then prepared and duplicate portions were incubated for 4 min at 22°C in the presence or absence of trypsin (1.7 pgjml). TKMS buffer (2 ml) was then added and the nuclei harvested by centrifugation at 8OOg for 2 min. The nuclei were redispersed in TKMS buffer and assayed for RNA polymerase A activity as described. Results from two separate experiments are shown, expressed as mean values for duplicate nuclear preparations with values as percentages of untreated controls given in parentheses.

3 h, RNA polymerase A activity was inhibited by 4347% in trypsin-untreated nuclei. Mild proteolysis had no apparent effect on control nuclear RNA polymerase activity, but restored dexamethasoneinhibited activity to control values (Table 4).

DISCUSSION

Previous investigations of the control of ribosomal RNA synthesis have revealed the presence of two discrete pools of RNA polymerase A in eukaryotic cell nuclei [ 181. One pool of enzyme exists as a tightly bound transcription complex, and is probably RNA polymerase AI1 [19], while the other pool is “free” with respect to its ability to transcribe an exogenous poly[d(A-T)] template in the presence of actinomycin D [18] and is considered to be RNA polymerase AI [19]. Both the “free” and “engaged” forms of the enzyme display a similar ability to transcribe purified DNA and synthetic templates [20,21]. Since the activity of the enzyme in the form of the transcription complex appears to be regulated via protein synthesis [22], the intranuclear redistribution of RNA polymerase A between “free” and “engaged” pools has been proposed as a mechanism for the modulation of the rate of nucleolar 45s rRNA synthesis [23]. The relevance of this hypothesis to the inhibitory effects of glucocorticoids on RNA polymerase A activity in rat thymus cells was investigated in experiments in which the activity of the “free” form of RNA polymerase A was determined, simultaneously with that of the “engaged” form, in rat thymus nuclei in the presence of an exogenous poly[d(A-T)] template and actinomycin D. Poly[d(A-T)] addition, in the absence or presence of actinomycin D, resulted in increased RNA polymerase A activity which was additive to, and did not inhibit or compete with, transcription of the endogenous template. Similar results have been described for rat hepatocytes [24,25]. In contrast to the findings of Kellas et a1.[19] with rat liver nuclei, rat thymus nuclei prepared by hypo-osmotic lysis of cells contained a substantial pool of “free” RNA polymerase A activity capable of transcribing a poly[d(A-T)] template in the presence of high concentrations of actinomycin D (Fig. 1).

However, it has been observed that this pool of enzyme can diffuse from nuclei with comparative ease under iso- and hypo-osmotic conditions [19]. Therefore “free” enzyme activity was also determined in the cytosol remaining following the preparation of nuclei. Treatment of rat thymus cells with 1 PM dexamethasone for 3 h resulted in a significant inhibition of the activity of the “engaged” form of RNA polymerase A, while the activities of both the nuclear and cytosol “free” forms were not significantly affected. Since no increase in “free” pool RNA polymerase A activity was detected after steroid treatment, these results suggested that the glucocorticoid-induced inhibition of RNA polymerase A activity was not simply a consequence of a decrease in the average number of initiated enzyme molecules. Although the glucocorticoid appeared to have little effect on the level of “free” activity in rat thymocytes under these conditions, there is evidence that changes in the size of the “free” pool can occur in liver in circumstances where the rate of ribosomal RNA synthesis is changed, such as partial hepatectomy [26], the administration of glucocorticoid hormones [24] or cycloheximide in uiuo [23,25]. Further support for the conclusion that the change in “engaged” RNA polymerase A activity is not due to enzyme redistribution was obtained from experiments using rifamycin AF/O-13 to suppress reinitiation of RNA polymerase A in the assay in vitro. Rifamycin AF/O-13 functions as an initiation inhibitor by binding to the released enzyme and rendering it inactive in eukaryotic transcription [27]. In agreement with the postulated mode of action of the inhibitor, rifamycin did not affect initial synthetic rates in nuclei from either control or steroid-treated cells, but prevented re-initiation by binding enzyme released after termination of polynucleotide chains. Sensitivity to the effect of rifamycin developed earlier in time in nuclei from control cells than in nuclei from steroid-treated cells, although similar plateau levels of incorporation of UMP were attained in both conditions. Together with the earlier findings, these results suggest that glucocorticoid treatment of rat thymocytes inhibits 4% rRNA synthesis primarily by decreasing the polyribonucleotide elongation rate of RNA polymerase A.

503

Glucocorticoids and RNA polymerase A To substantiate this finding, direct measurements were made of the numbers of transcribing enzyme molecules and of their elongation rates [15]. The method employed to measure these parameters is based on the fact that alkaline hydrolysis of an RNA molecule produces nucleoside monophosphates from internal residues and an unphosphorylated nucleoside from the 3’-hydroxyl end, where RNA synthesis was terminated [28]. The number of 3’-termini is a measure of the number of enzyme molecules active in transcription. Since degradative enzymes acting on RNA, such as ribonuclease and phosphatase, may generate additional nucleosides indistinguishable from those derived from 3’-termini, it appeared imperative to determine whether such activity was present. Such measurements showed the virtual absence of RNA degradative enzyme activities from the nuclei of rat thymocytes incubated in the absence or presence of dexamethasone. Parameters of RNA polymerase A-directed transcription were then determined. Control nuclei from rat thymocytes incubated for 3 h in the absence of steroid contained approx 2 x lo3 molecules of form A RNA polymerase actively transcribing per cell. This value remained essentially unchanged on dexamethasone treatment of cells and is similar to the number of transcribing RNA polymerase A molecules per diploid genome reported for chick oviduct nuclei [ 151.Glucocorticoid treatment, however, did significantly reduce the poly ribonucleotide elongation rate, this decrease entirely accounting for the hormone-induced inhibition of rRNA synthesis. These elongation rates are higher by an order of magnitude than the values reported by Cox [I 51 for chick oviduct or by Coupar et a1.[29] for rat liver. However, even our values are at least lOOO-fold lower than those expected from in vivo measurements [30]. It has been suggested that these low values may be attributable to the loss of elongation factors arising during the preparation of nuclei [ 151. In previous studies with rat thymus cells, cycloheximide did not affect control RNA polymerase A activity but restored the activity of glucocorticoidtreated cells to control levels [I]. Although this observation was confirmed in the present studies (Table 3), the measurement of transcriptional parameters revealed a change in the nature of the response after cycloheximide treatment. In the presence of cycloheximide, elongation rates were reduced below those of cells not treated with the inhibitor regardless of the presence or absence of dexamethasone. This reduction in elongation rate was accompanied by an increase in the number of transcribing enzyme molecules, which resulted in normal rates of RNA synthesis being maintained. These studies suggest that in rat thymus, as in liver [3 11,cycloheximide inhibits the synthesis of a rapidly-turning-over protein(s) which is involved in the regulation of the elongation rate of RNA polymerase A. Evidence for the existence of such factors has been obtained by a number of

investigators [32-361 but their precise nature and function remains obscure, as does the mechanism responsible for the compensatory increase in the number of enzyme molecules engaged in transcription. The reduction of the elongation rate to Z&60% of control values by cycloheximide may reflect the activity of the “core” enzyme [36]; this rate of elongation was not significantly affected by dexamethasone (Table 3) although the steroid would not be expected to have an affect in such circumstances, if its effects are indeed dependent on continuing protein synthesis. Other studies of RNA polymerase A activity in rat liver [37] have shown that glucocorticoid treatment increases the catalytic activity of the enzyme, and that this increased activity is sensitive to mild proteolysis. Although glucocorticoids inhibit rat thymic RNA polymerase A activity, the experimental approach of Todhunter et af.[37] was adopted in a study of the thymic enzyme activity. Mild treatment of nuclei with trypsin did not significantly affect control enzyme activity, while dexamethasoneinhibited activity was restored to a value similar to that of the control. Therefore, it is reasonable to suggest that a glucocorticoid-induced protein or peptide, labile to mild proteolysis, is the agent which regulates the activity of this enzyme. Such experiments with intact nuclei however do not locate the site of modification, which could be on the enzyme itself or on the transcribed gene. Nevertheless, it appears that this region is readily accessible to trypsin. In conclusion, it is apparent from the present studies that glucocorticoid treatment of rat thymus cells inhibits rRNA synthesis by decreasing the elongation rate of RNA polymerase A, possibly by modification of the enzyme, through a mechanism that requires continuing protein synthesis. Acknowledgement-The

authors are grateful to the Tenovus Organization for financial support. REFERENCES

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Young D. A., Barnard T., Mendelsohn S. and Giddings S.: An early cordycepin-sensitive event in the action of glucocorticoid hormones in rat thymus cells in vitro: evidence that synthesis of new mRNA initiates the

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5.

6.

7.

8.

9.

IO.

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