Nucleolar RNA synthesis in the liver of partially hepatectomized and cortisol-treated rats

244

Biochimica et Biophysica Acta, 402 (1975) 244--252 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98364

N U C L E O L A R R N A SYNTHESIS IN THE L I V E R OF P A R T I A L L Y HEPATECTOMIZED AND CORTISOL-TREATED RATS

W. SCHMID and C.E. SEKERIS

Institut fiir Zellforschung am Deutschen Krebsforschungszentrum Heidelberg, Heidelberg (G.F.R.) (Received January 27th, 1975)

Summary RNA synthesis by isolated nucleoli from rat liver is significantly enhanced 12--14 h after partial h e p a t e c t o m y and 4 h after cortisol administration. The increased RNA synthetic capacity is demonstrable also in the respective high salt nucleolar extracts and in Biogel A-1.5 filtration fractions of the nucleolar extracts. DNA saturation experiments using nucleoli and Biogel fractions from control and treated animals as R N A polymerase source, have demonstrated, that independent of the extent of RNA synthesis, saturation of transcription is reached at the same concentration of exogenous template. We conclude that the activity and not the a m o u n t of nucleolar R N A polymerase is increased as a result of partial h e p a t e c t o m y or cortisol administration. Parallel to the effects on RNA polymerase, the activity of RNA-degrading enzymes present in nucleoli is also enhanced by the same treatment.

Introduction One of the crucial events in the control of anabolism and cell growth is the formation of ribosomes. The synthesis of ribosomal precursor R N A in the nucleolus is influenced by a number of agents e.g. triiodothyronine, growth hormone [1], glucocorticostero.ids [2,3], cell loss [4] and fastening [5]. Nucleolar RNA synthesis is also underlying diurnal rhythms [6,7 ]. In a previous work [8], we have presented evidence that the increase in liver ribosomal R N A synthesis in rats treated with cortisol is preceded by a discrete stimulation of extranucleolar R N A synthesis, and we concluded that the enhancement of nucleolar R N A synthesis seems to be a secondary effect in hormone action. The increased rate of ribosomal R N A synthesis is n o t due to an increase of the amount of template available, as hormonal effects are still evident after extraction of nucleolar R N A polymerase from isolated nucleoli and assaying it in an in vitro system using exogenous DNA as template [ 9--11 ].

245 In this paper we will demonstrate that this is also the case in regenerating rat liver. Further, evidence will be presented favoring a change in the activity and n o t in the amount of nucleolar R N A polymerase as the principle control element in nucleolar gene transcription in partially hepatectomized or cortisoltreated rats. Methods Male Wistar BR ll rats (120--200 g) were obtained commercially and fed ad libitum. Partial h e p a t e c t o m y was performed under ether anaesthesia according to Higgins and Anderson [12] 12--14 h before sacrifice. Adrenalectomy was performed via the dorsal route 3--5 days before sacrifice. To minimize the influence of diurnal rhythmicity of nucleolar R N A polymerase activity, the animals were sacrificed between 9.00 and 10.00 a.m. Cortisol (30 mg/kg) was applied intraperitoneaUy in isotonic sucrose solution. [3H] UTP (spec. act. 30 Ci/mmol) was obtained from N.E.N., Dreieichenhain; nucleoside triphosphates from P.W.A. Waldhof, Aschaffenburg; calf thymus DNA from Serva, Heidelberg; and the other chemicals from Merck, Darmstadt.

Preparation of nucleoli and extraction of the DNA-dependent RNA polymerase Nucleoli were prepared as described by Busch [ 1 3 ] . To minimize aggregations of the sonified material, nuclei from one control rat or two hepatectomized rats were suspended in 4 ml isotonic sucrose solution. Care was taken that no more than 0.1% intact nuclei were detectable after sonication. Extraction of nucleolar R N A polymerase was performed as described by Seifart et al. [14]. Nucleoli from two, and in the case of partial hepatectomy, from four rats, were extracted in 10 ml 1 M (NH4)2 SO4 buffer after short sonication, stirred 30 min at 4°C, and proteins were precipitated b y addition of solid (NH4)2 SO4 (final concentration 3.5 M) for 4--5 h. The protein sediment obtained after centrifugation was dissolved in 5 ml dialysis buffer (10 mM Tris, pH 7.9, 20% glycerol, 5 mM mercaptoethanol), dialyzed 12 h against 100 vol. dialysis buffer and insoluble nucleoprotein complexes removed by centrifugation. The clear supernatant was taken as crude R N A polymerase extract. R N A polymerase activity in isolated nucleoli and in the extracts were assayed as described elsewhere [ 1 5 ] . Synthesis time was 5 min for nucleoli and 30 min for the extracts, as during these time intervals incorporation was linear in the respective preparation. Mg 2÷ concentration was 10 mM in the nucleolar system and 20 mM when calf thymus DNA was present. All values are expressed as cpm/mg nucleolar DNA. DEAE-cellulose chromatography was either performed as batch chromatography and the polymerase eluted with 1.5 ml of 0.2 M NH4 C1 in dialysis buffer or as gradient chromatography in Pasteur pipettes filled with 0.5 ml of DEAE-cellulose. Biogel A-1.5 (200--400 mesh) chromatography was performed on a 1.6 × 68 cm column. The volume of the applied extract was 2--4 ml, flow rate about 2 ml/h, fraction size 1.9 mt. Nucleoli of 10 control and 20 partially hepatectomized rats were extracted for the gel filtration experiments.

246

Ribonuclease activity was tested by incubating 120 #l of the material to be tested with boiled D N A • R N A hybrid synthesized on single-stranded D N A by Escherichia coli RNA polymerase with [a H] UTP as labeled precursor as described by Roewekamp and Sekeris [ 1 6 ] . A b o u t 10 000 cpm corresponding to a RNA concentration of 1.4 pg RNA/ml were present in a 150-pl assay. Mg 2÷ concentration was 10 mM. After 30 min incubation at 37°C, the acid-precipitable radioactivity was measured. DNAase activity was determined by incubating calf thymus D N A with the nucleolar extracts under the conditions of the RNA polymerase assay and subsequent alkaline sucrose gradient centrifugation. D N A and protein concentration were determined according to Burton [17] and Lowry et al. [ 1 8 ] , respectively, after precipitation of the material with 5%HCIO4. Results

Yield of nucleoli and of nucleolar RNA polymerase activity About 3.5% (-+0.5%) of nuclear D N A was obtained in the nucleolar sediment independent of the previous functional state of the animal. The amount of RNA polymerase activity recovered from control animals, determined as dpm/mg nucleolar DNA, was reproducible in the _+15% range regardless if assayed in isolated nucleoli or after extraction from nucleoli. However, the degree of stimulation of RNA polymerase activity after the various treatments showed major fluctuations. The RNA polymerase activity was completely resistant to a-amanitin. TABLE

I

EFFECT OF PARTIAL HEPATECTOMY O N R N A S Y N T H E S I S IN I S O L A T E D NUCLEI, NUCLEOLI AND ON RNA POLYMERASE ACTIVITY EXTRACTED FROM NUCLEOLI BEFORE AND AFTER BATCH CHROMATOGRAPHY ON DEAE-CELLULOSE Nuclei, nucleoli and R N A polymerase extracts were obtained from control rats and 12 h after partial hepatectomy as described under Methods. Synthesis time was 5 rain in the case of nuclei and nucleoli, 30 rain in the extracts.

cpm

DNA mg/ml

Chauveau nuclei (Mg 2+ 5 raM) 1210 Control 2210 12 h regenerating liver

0.9 1.22

Nucleoli (Mg 2+ 5 raM) Control 12 h regenerating liver

0.075 0.100

1000 4350

Crude R N A polymerase extracts (Mg 2+ 20 raM) 880 Control 2860 12 h r e g e n e r a t i n g liver Crude R N A p o l y m e r a s c e x t r a c t b a t c h e l u t i o n from D E A E - c e l l u l o s e (Mg 2+ 20 r a M ) 860 Control 2820 12 h r e g e n e r a t i n g

cpm/mg DNA

Proteinmgperml

DNA/protein

20200 36800

1.71 2.55

1:1.8 1:2.1

200000 650000

0.39 0.86

1:5.2 1:8.6

176000 430000

172O0O 423000

247

Effect of partial hepatectomy and cortisol application on nucleolar RNA synthesis in vitro and on solubilized RNA polymerase activity Nuclei, nucleoli and RNA polymerase extracts were prepared from control and 12 h regenerating liver as described under Methods. The DNA concentration in the assays of the extracts was 200 #g/ml. The results of a typical experiment are given in Table I. Partial h e p a t e c t o m y results in a large increase of RNA synthesis in isolated nuclei, when tested at low ionic strength and in the presence of Mg :÷, conditions optimal for ribosomal R N A synthesis. The stimulation is more pronounced if R N A synthesis is performed with isolated nucleoli and can also be observed, to a similar degree, in the high ionic strength extract derived from isolated nucleoli. The increased R N A synthesis is still evident in the nucleolar extract after its adsorption on DEAE-cellulose and elution in one step by 0.2 M NH4 C1. The range of increase of nucleolar R N A polymerase activity observed was between 80 and 300% with an average of about 100%. It should be noted that the protein content of the nucleoli is increased in regenerating liver. A b o u t 20--30% of total nucleolar proteins are solubilized by the high ionic extraction procedure. Due to a variable loss of R N A polymerase activity after gradient chromatography on DEAE-cellulose, varying between 20 and 90% of the starting activity, a comparison between preparations derived from differentially treated animals was impossible. However, chromatography of the nucleolar extracts on Biogel-A-1.5 columns at low ionic strength led to a recovery of approx. 100--130% of the applied polymerase activity. The R N A polymerase activity appears immediately after the void volume, demonstrating that the enzyme has aggregated under the low ionic conditions of the chromatography. Some nonaggregated activity is occasionally seen as a slight shoulder, its position in Fig. 1 is indicated by an arrow. Although at higher ionic strength (0.3 M NH4C1)

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Fig. 1. Biogel A - 1 . 5 gel c h r o m a t o g r a p h y o f R N A p o l y m e r a s e e x t r a c t s d e r i v e d f r o m liver n u c l e o l i f r o m c o n t r o l a n d 12 h p a r t i a l l y h e p a t e c t o m i z e d rats. R N A p o l y m e r a s e w a s e x t r a c t e d f r o m n u c l e o l i derived f r o m t h r e e c o n t r o l a n d six h e p a t e c t o r n i z e d a n i m a l s as d e s c r i b e d u n d e r M e t h o d s . 2 m l o f e x t r a c t , d e r i v e d f r o m the s a m e a m o u n t o f n u c l e o l i in r e s p e c t t o D N A c o n t e n t , w e r e applied o n t w o 1.6 X 6 8 c m c o l u m n s , 1.9-rnl f r a c t i o n s w e r e c o l l e c t e d and t e s t e d for R N A p o l y m e r a s e ( A ) a n d n u c l e a s e (B) a c t i v i t y . • --, control; o o, r e g e n e r a t i n g liver. T h e t h i c k a r r o w i n d i c a t e s the p o s i t i o n o f R N A p o l y m e r a s e in a r u n u n d e r high i o n i c s t r e n g t h (0.3 M N H 4 C I ) c o n d i t i o n s .

248

2000

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500

5

10

15

Fig. 2. Biogel A - I . 5 gel c h r o m a t o g r a p h y o f n u c l e o l a r R N A p o l y m e r a s e e x t r a c t derived f r o m t h e livers o f a d r e n a l e c t o m i z e d and c o r t i s o l - t r e a t e d rats. R N A p o l y m e r a s e w a s e x t r a c t e d f r o m n u c l e o l i o f three c o n t r o l and t h r e e c o r t i s o l - t r e a t e d rats. C o r t i s o l ( 3 0 m g / k g ) w a s applied 4 h b e f o r e s a c r i f i c e . C o n d i t i o n s o f t h e c h r o m a t o g r a p h y as in Fig. 1. • =, c o n t r o l ; o o, c o r t l s o l t r e a t m e n t .

enzyme aggregation can be avoided, we have not performed gel filtration under these ionic conditions, as the enzyme activity thus recovered is significantly reduced. In order to explain the increased yield of R N A polymerase observed after the gel filtration step, we determined in the column fractions nuclease activity, which is present in addition to the polymerase in the original nucleolar extracts. As evident in Figs 1A and 1B part of the nuclease activity can be separated from the R N A polymerase, resulting in an apparent increase in R N A polymerase activity of the Biogel fractions. 12 h after h e p a t e c t o m y there is a significant increase in the R N A polymerase A activity recovered in the Biogel fractions in comparison to control animals {Fig. 1A). Furthermore, nuclease activity is also enhanced in the hepatectomized animals {Fig. 1B). No significant DNAase activity could be detected in the extracts by alkaline sucrose gradient centrifugation. The effects of cortisol treatment on R N A polymerase and nuclease activity is similar to the effects of hepatectomy. R N A polymerase activity is increased approx. 2--3-fold in isolated nucleoli, in nucleolar extracts, in batch eluates from DEAE-cellulose as well as after Biogel chromatography {Fig. 2A). Nuclease activity is also considerably enhanced (Fig. 2B).

Effect of addition of exogenous DNA on RNA synthesis in nucleoli isolated from control and hepatectomized rats and on the respective preparations from Biogel columns The described effects of h e p a t e c t o m y and cortisol treatment on nucleolar R N A synthesis could be due to an increased amount or an increased activity of RNA polymerase A. As at the present time no direct m e t h o d exists for the titration of the a m o u n t of R N A polymerase A we have attempted indirectly to achieve this by DNA saturation experiments. In a first experiment, nucleoli were incubated in the presence and absence of exogenous DNA. In the absence of exogenous DNA, R N A synthesis is linear in the first 5 min and reaches a plateau after 10--20 min. In the presence of

249

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Fig. 3. R N A s y n t h e s i s b y i s o l a t e d n u c l e o l i d e r i v e d f r o m c o n t r o l a n d p a r t i a l l y h e p a t e c t o m i z e d r a t s in t h e p r e s e n c e o f e x o g e n o u s D N A . N u c l e o l i d e r i v e d f r o m c o n t r o l (e "-) a n d h e p a t e c t o m i z e d (o c) r a t s w e r e i n c u b a t e d f o r 5 m i n in t h e p r e s e n c e o f e x o g e n o u s D N A a t t h e i n d i c a t e d c o n c e n t r a t i o n s a n d t h e i n c o r p o r a t i o n o f [ 3 H ] U T P i n t o a c i d - p r e c i p i t a b l e m a t e r i a l e v a l u a t e d (see M e t h o d s ) .

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20

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Fig. 4. D e p e n d e n c e o f c o n c e n t r a t i o n o f D N A n e e d e d t o r e a c h t h e s a t u r a t i o n p o i n t o f R N A s y n t h e s i s o n t h e a m o u n t o f n u c l e o l i i n c u b a t e d . V a r y i n g a m o u n t s o f n u c l e o l i , d e r i v e d f r o m c o n t r o l (o) a n d p a r t i a l l y h e p a t e c t o m i z e d (~) r a t s , w e r e i n c u b a t e d w i t h i n c r e a s / n g a m o u n t s o f D N A u n d e r c o n d i t i o n s o f R N A synthee/s. The concentration of DNA needed to reach the saturation point of synthesis was then plotted against the amount of incubated nucleoli.

250 exogenous DNA, R N A synthesis is linear for at least 10 min, reaching a plateau at 20--30 min. The increment of R N A synthesis, due to the addition of exogenous DNA, is linear in the first 20 min, decreasing thereafter. In the following experiments therefore, we have chosen a time period of incubation of 20 min. Isolated nucleoli from control and partially hepatectomized rats were then incubated in the presence of increasing amounts of exogenous DNA. As seen from Fig. 3, nucleoli from partially hepatectomized animals synthesize at least twice as much R N A as control nucleoli (see also Table I). The a m o u n t of additional RNA synthesis in the presence of DNA is also much higher after partial hepatectomy. Furthermore, and independent of previous treatment, a saturation point is reached with increasing amounts of DNA. This saturation point is attained at the same concentration of DNA for both nucleolar preparations from control and partially hepatectomized animals, independent of the height of the plateau reached. These results are consistent with the presence of similar amounts of R N A polymerase in the nucleoli isolated from control and hepatectomized animals, independent of the degree of stimulation of R N A synthesis. That this conclusion is correct is corroborated by the findings shown in Fig. 4 in which the concentration of DNA needed to reach the saturation point of RNA synthesis is plotted against the amount of added nucleoli. The same linear relationship is found for both nucleoli from control and hepatectomized rats. Using as RNA polymerase preparation fractions derived from the Biogel chromatography of the nucleolar extracts, similar results have been obtained as described above for isolated nucleoli. Although R N A synthesis is greater with fractions obtained from hepatectomized animals (Fig. 5) saturation is reached at the same DNA concentration for both preparations. cDm

40000

3 0 OOC

20000

10 000

y 260 pg ONA / ml

Fig. 5. R N A s y n t h e s i s b y f r a c t i o n s o f Biogel A - 1 . 5 c h r o m a t o g r a p h y o f n u c l e o l a r e x t r a c t s derived f r o m c o n t r o l and h e p a t e c t o m i z e d rats. F r a c t i o n s f r o m the Biogel c o l u m n s w e r e i n c u b a t e d in the p r e s e n c e o f the i n d i c a t e d a m o u n t s o f e x o g e n o u s D N A and t h e i n c o r p o r a t i o n o f [ 3 H ] U T P i n t o acid-precipitable m a t e r i a l e v a l u a t e d (see M e t h o d s ) . • e cpm incorporated by control preparations; o o, c p m i n c o r p o r a t e d b y p r e p a r a t i o n s d e r i v e d f r o m partially h e p a t e c t o m l z e d a n i m a l s .

251

Discussion

Among the possible mechanisms postulated for the increased production of ribosomal RNA in eucaryotic organisms, increased transcription or increased amount {amplification) of ribosomal genes as well as an increase in the activity or the a m o u n t of RNA polymerase have received considerable attention. The results presented in this paper are in favour of regulation of ribosomal RNA synthesis by modulation of RNA polymerase A activity in the case of partial h e p a t e c t o m y and cortisol administration. This is in full agreement with earlier findings of Sajdel and Jacob [9], Yu and Feigelson [19], Smuckler and Tata [10] and Cooke and Brown [11]. It seems that this type of regulation of nucleolar RNA synthesis is universal in eucaryotes, with the one exception of extrachromosomal RNA synthesis in amphibian oocytes. In this case, amplification of the template by a factor of 1000 [20] is responsible for the increased ribosomal RNA formation. Unfortunately, due to the limitations of the experimental procedures currently available, no direct answer can be given to the question of whether an increased a m o u n t of polymerase A or an increased activity of the enzyme is responsible for the increased transcriptional rate. This has been achieved in the case of the extranucleolar RNA polymerase B [21] using radioactive-labeled a-amanitin, which binds stoichiometrically to the polymerase B [22]. We have therefore attempted an indirect titration of the a m o u n t of RNA polymerase A by DNA saturation experiments. Such experiments were performed with isolated nucleoli as well as with fractions from Biogel chromatography of nucleolar extracts. The main findings of these experiments was that independent of the extent of nucleolar RNA synthesis, which was higher in regenerating than in control livers, saturation in isolated nucleoli from both groups of animals is reached at the same concentration of exogenous DNA. Furthermore, this saturation is dependent solely on the a m o u n t of nucleoli assayed. This clearly indicated that not the amount, but the activity of the nucleolar RNA polymerase has increased in the regenerating liver. Similar results were obtained with Biogel fractions of nucleolar extracts, corroborating the findings with isolated nucleoli. Comparable results have been obtained with preparations from cortisol-treated animals. There is no evidence that the observed increase in RNA synthetic capacity is due to increased breaks in the DNA by endogenous DNAase activity (see Butterworth et al. [23] ). The problem of the activation of RNA polymerase A is receiving currently great attention. Among others, protein and RNA regulatory factors [24] have been implicated. Analysis of the nucleolar RNA polymerases from animals of differing metabolic status will give further insight into the mechanism of this regulation, a formidable task, demanding among others large amounts of starting material, avoidance of polymerase denaturation seen at low protein concentrations (Muramatsu, personal communication} and extensive purification of the enzyme. The effect of partial h e p a t e c t o m y and cortisol administration on the intranuclear ribonuclease is an interesting finding and should be briefly discussed. Barnabei and Ottolenghi [25] reported that glucocorticosteroids inhibit cyto-

252

plasmic ribonuclease activity, contributing also by such a mechanism to the increase of cytoplasmic RNA observed after glucocorticosteroid administration. In contrast to the cytoplasmic ribonuclease, however, the respective nucleolar enzymes show increased activity after similar treatment. The number and function of these enzymes in the nucleolus is still largely unknown. It is very probable that they are involved in the processing of newly synthesized RNA, so that under conditions of increased transcription an increase in their activity would be functionally meaningful. References 1 Tata, J.R. (1970) Biochemical Action of Hormones Vol 1, pp. 89--133, (Litwack, G., ed.), Academic Press, New York 2 Yu, F.-L. and Feigelson, P. (1969) Biochem. Biophys. Res. Commun. 35, 499--504 3 Jacob, S.T., Sajdel, E.M. and Munro, H.N. (1969) Eur. J. Biochem. 7 , 4 4 9 - - 4 5 3 4 Muramatsu, M. and Busch, H. (1965) J. Biol. Chem. 240, 3960--3966 5 Henderson, A.R. (1970) Biochem. J. 120, 205--214 6 Glasser, S.R. and Spelsberg, T.C. (1972) Biochem. Biophys. Res. Commun. 4 7 , 9 5 1 - - 9 5 8 7 Barbiroli, M.S., Moruzzi, M.G., Montl, B. and Tudolini, B. (1973) Biochem. Biophys. Res. C ommun. 54, 62--68 8 Schmid, W. and Sekeris, C.E. (1972) FEBS Lett. 26, 109--112 9 Sajdel, E.M. and Jacob, S.T. (1971) Biochem. Biophys. Res. Commun. 45, 707--715 10 Smuckler, E.A. and Tata, J.R. (1971) Nature 234, 37--39 11 Cooke, A. and Brown, M. (1973) Biochem. Biophys. Res. Commun. 51, 1042--1047 12 Higgius, G.M. and Anderson, R.M. (1931) Arch. Pathol. 12, 186 13 Busch, H. (1967) Methods of E n z y m o l o g y (Grosman, L. and Molder, K,, eds), VoL XII, pp. 448--464, Academic Press, New York 14 Seifart, K., Benecke, B. and Juhasz, P.P. (1972) Arch Biochem. Biophys. 151, 519--532. 15 Schmid, W. and Sekeris, C.E. (1973) Biochim. Biophys. Acta 312, 549--554 16 R o e w e k a m p , W. and Sekeris, C.E. (1974) Eur. J. Biochem. 4 3 , 4 0 5 - - 4 1 3 17 Burton, K. (1956) Biochem. J. 62, 315--323 18 Lowry, O.H., Roscbrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 1 9 3 , 2 6 5 - - 2 7 5 19 Yu, F.-L. and Feigelson, P. (1972) Proc. Natl. Acad. Sci. U.S. 69, 2833--2837 20 Birnstiel, M.L., Chipchase, M. and Speirs, J. (1971) Re c e nt Progress in Nucleic Acid Research and Molecular Biology (Dav~dson, J.N. and Cohn, W.E., eds), Vol. II, pp. 351--389, Academic Press. New York 21 Cochet-Meilhac, M., Nuret, P., Courvalin, J.C. and Chambon, P. (1974) Biochtm. Biophys. Acta 353, 185--192 22 Cochet-Meilhac, M. and Chambon, P. (1974) Biochtm. Biophys. Acta 3 5 3 , 1 6 0 - - 1 8 4 23 Butterworth, P.H.W., Flint, S.J. and Chesterton, C.J. (1973) Biochem. Soc. Trans. 1 , 6 5 0 - - 6 5 5 24 Stein, H. and Hausen, P. (1970) Eur. J. Biochem. 14, 270--277 25 Barnabci, O. and Ottolenghi, C. (1968) Advances in Enzyme Regulation (Weber, G.. ed.), Vol. VI, pp. 189--209, Pergamon Press, London-Oxford, New York