Release of rRNA from liver nuclei during the early stages of the acute-phase reaction

Biochimica et Bi,~phvsica Acta. 783 (1984) 179 182 Elsevier

179

BBA Report BBA 90034

RELEASE OF rRNA FROM LIVER NUCLEI DURING T H E EARLY STAGES OF T I l E A C U T E - P H A S E REACTION MARIA G I O V A N N A ALF.TTI. ROBERTA PI('('OLETI'I and ALDO B E R N E L L I - Z A Z Z E R A

lstituto di Patologia Generale dell'Unit,ersiti¢ degli Stmh di Mikmo, ('entro di Studio per la Patol.~ia ('c/luk~re de/( :\ R, c;a Mangiagalli 31. 20133 Milano (ltalv) (Received August 7th. 1984)

Key wordw rRNA," Inflanlmation; RNA transport," RN.4 methvh*lion; .4cute-phase reaction," (Rat lirer;

Liver nuclei isolated from rats 5 h after turpentine injection show an increased release of rRNA, of the transport-related nucleoside-triphosphatase activity and of the amount of nuclear RNA; RNA methylation is also likely to undergo some activation. These changes occur when RNA synthesis is still normal.

The acute-phase reaction, which accompanies the presence of an inflammatory process, is characterized by the increase in concentration of a set of plasma proteins, known as acute-phase reactants [1,2]. The synthesis of these proteins, which occurs in the liver, depends on the increased availability of their specific mRNAs [3 5], and an expanded ribosome population, both related to an activated transcription [6,7]. Some evidence suggests that under conditions of increased or decreased growth rate, post-transcriptional mechanisms of ribosome production operate more promptly and efficiently than transcriptional control [8,9]. Since nucleocytoplasmic transport of RNA molecules represents one of the key steps in the biogenesis of the ribosomes, we have now investigated the rate of rRNA efflux from the nuclei of rat liver early (5 h) after the onset of an acute-phase reaction induced by turpentine injection, at a time when rRNA transcription is not yet stimulated [6]. To confirm and interpret the results of these experiments we have also measured other parameters related to processing and transport of rRNA molecules, such as methylase and ATPase activities. Inflammation was induced by a single subcutaneous injection of 0.5 ml of sterile turpentine/100 0167-4781/84/$03.00 :':~ 1984 Elsevier Science Publishers B.V.

g body wt. in the back of male albino rats (200 g, Wistar strain). Control rats received the same volume of sterile saline. The release of rRNA from isolated prelabeled liver nuclei [10,11] was studied in a cell-free system, containing either homologous or heterologous cytosol, and fortified with ATP and an energy-regenerating system [12,13]. Nuclei were obtained from animals injected intraperitoneally with 20 /~Ci of [14C]orotate 120 rain before death; this time of labeling does not give the highest absolute values, but permits the best discrimination between rRNA and m R N A transport. Cytosol consisted of the 105000 × g supernatant of liver homogenate dialysed 18 h at 4°C against buffer containing 50 mM Tris-HC1 (pH 7.5)/25 mM KCI/2.5 mM MgC12. Incubations were performed for 40 rain at 0 or 350( `. The reaction was stopped by immersion of the incubation vessels in ice and nuclei were removed by centrifugation at 800 × g for 15 rain. Aliquots of the supcrnatant were precipitated with cold 5% trichloroacetic acid. The pellet was washed and the alkali-soluble radioactivity was measured in a Tri-Carb Packard liquid-scintillation spectrometer. The characterization of transport nucleoproteim [12,14] was performed on a linear 10 30% sucrose gradient, prepared in 30 mM Tris-HC1 (pH 7.5)/25

180 mM NaC1. The sample was centrifuged for 16 h at 25 000 rpm in an SW27 rotor: fractions were then collected, and monitored for radioactivity and absorbance at 260 nm. For the determination of ATPase activity unlabeled liver nuclei were suspended in 1 M sucrose and incubated for 15 rain at 35°C in 50 mM Tris-HCl (pH 7.8)/5 mM MgC12/5 mM A T P / [ y - 3 2 p ] A T P (1 /~Ci per each sample). The reaction was stopped by 5% trichloroacetic acid addition. The supernatant was absorbed to charcoal and the amount of 7-32p released was quantitated by measuring Cerenkov radiation. The rRNA synthesis and methyltransferase activity were determined with isolated nucleoli [15]. A final volume of 150 ~1 contained: 0.25 M sucrose, 100 mM Tris-HC1 (pH 7.8), 2 mM MgC12, 40 mM NH4F, o~-amanitin (2 p,g/ml), 0.4 mM ATP, 0.2 mM each of GTP, CTP and UTP, 10 >M S-adenosylmethionine, and an amount of nucleoli containing approx. 20 >g of DNA. 0.2 mM of [~H]UTP (spec. act. 0.133 C i / m m o l ) and 10 mM S[methyl--~H]adenosylmethionine (spec. act. 1.2 C i / m m o l ) replaced the corresponding unlabeled compounds in the assays for RNA synthesis and R N A methylation, respectively. The assays were carried out for 20 min at 30°C. The reaction was terminated by adding 10% trichloroacetic acid containing 40 mM Na4P20 v. The acid-insoluble material was then collected on Whatman G F / A filters, washed and counted in a Tri-Carb liquidscintillation spectrometer with an efficiency of 20%. The DNA content of nuclei and nucleoli was estimated by the diphenylamine procedure [16], the RNA was measured by a modified orcinol method [17], and protein determination was performed by the biuret method [181. Ample evidence from the literature shows that the cell-free systems used in vitro can support rRNA processing and transport with a mechanism essentially comparable to the in vivo situation (see Refs. 12, 19, 20). To verify some aspects of our experimental conditions, we performed a group of experiments (results not shown): (1) to ascertain that the release of RNA was linear over the 40-rain period of our incubation, and was a temperatureand energy-dependent active process: (2) to rule out the possibility of a differential stability of the transported RNA: when the media used in trans-

port experiments were freed of nuclei and incubated for a further 40 rain no appreciable changes were found in the specific activities of rRNA released from both control and turpentinetreated rats; (3) to demonstrate that rRNA is transported as 40 S and 60 S subunits; the labeled RNA released from both control and turpentinetreated rats sedimenting in the 40 S and 60 S regions of a sucrose-density gradient accounted for 60% of the acid-precipitable counts released in vitro. The ratio of the 60 S : 40 S radioactivity was the same m turpentine-treated as in normal animals. To assess the rate of rRNA transport during the acute-phase reaction we performed cross-mixing experiments so that nuclei from normal and turpentine-treated rats were incubated in turn with either homologous or heterologous cell sap. This experimental design was based on the observations that cell sap components are essential for RNA transport [12], and are responsible for changes in the rate of transport in particular states of the cell [14,21]. The rate of rRNA transport by nuclei isolated from acute-phase livers is higher than the normal, irrespective of the source of cell sap (Table I): moreover, the rate of efflux by normal nuclei does not increase even when the latter are incubated with cytosol from acute-phase livers. Therefore, this increase in nucleocytoplasmic translocation of rRNA is entirely ascribable to nuclear mechanisms, without any cooperative effort of cytosolic protein factors. The nuclear envelope of eukaryotes appears to act as a selective barrier to the release of RNA, and it has been shown that the species of RNA released in vitro from isolated liver nuclei are transported through the mediation of a nuclear pore nucleoside triphosphatase [22,23]. In particular, it has been suggested that ATP hydrolysis by ATPase is a prerequisite for RNA efflux from hepatic nuclei [24]. In keeping with these observations, and in agreement with our results on RNA release, the activity of an Mg 2~ -dependent ATPase is significantly increased in nuclei from turpentine-treated rats (Table I). Chemical and subcellular evidence indicates the role played by rRNA methylases in regulating maturation and assuring ribosome stability [25]. The methylation pattern obtained with isolated nucleon resembles closely the events occurring in

181 TABLE 1 EFFECT OF 5-h T U R P E N T I N E TREATMENT ON rRNA RELEASE FROM ISOLATED LIVER NUCLEI A N D ATPase ACTIVITY OF THE N U C L E A R ENVELOPE Liver nuclei, labeled in vivo with [3H]orotic acid. were incubated in vitro with either homologous (normal + normal: treated + treated) or heterologous cytosol as described in the text. The results (means of 10 experiments_+ S.E.) represent the differences between metabolic (35°C) and passive (0°C) release and are expressed as the percentage of radioactivity initially present in the nuclei. For the measurement of ATPase. unlabeled liver nuclei were incubated with [¥-32p]ATP for 15 rain at 35°C. The latter results are the means of 5 experiments +_S.E. All the results have been subjected to Student's t test. Normal Percentage of radioactivity released in the presence of: Homologous cell sap Heterologous cell sap ATPase activity (nmol ~2p released 15 rain per mg DNA)

Turpentine-treated

1.64_+ 0.08 1.65 _+ 0.10

1354

+48

Percent difference

2.02 +_ 0.16 ~' 2.03 _+ 0.08 "

1857

+ 23 + 23

_+16 "

+37

Significantly different from the normal counterpart ( P < 0.05).

vivo, thus proving the efficacy and specificity of the nucleolar r R N A methylation systems [25]. Under the present experimental conditions, methylation occurring when nucleoli are incubated in the presence of ribonucleoside triphosphates measures the acceptor sites on both nascent pre-rRNA generated in vivo and those elongated in vitro, while methylation occurring in the absence of ribonucleoside triphosphates reflects only the number of the former ones. The efficiency of the methylation process is calculated from values of R N A synthesis and methylation in vitro. Table II summarizes the activities of R N A polymerase (form I) and r R N A methylase of nucleoli from control and turpentine-treated rats. The incorporation of [3H]UTP into nucleolar RNA confirms previous data obtained by us with isolated nuclei [6] and indicates that the rate of rRNA transcription does not change in the early stages of inflammation. Experimental results on RNA methylation do not reveal obvious differences and on this basis any statement would be unjustified. But when these data are used to calculate the ratio between the methyl groups and the UTP molecules incorporated into the R N A newly synthesized in vitro, as proposed [25,26], it can be conjectured that methylation occurs at a higher level in the nucleoli from rat livers cells during the early acute-phase response, when R N A synthesis is still normal. The

higher level of methylation, if real, would protect the rRNA sequences from being degraded during the maturation process, and decrease the nuclear wastage which has been considered as a means of regulating the accumulation of r R N A [27]. The hypothesis that such an event occurs is supported by the fact that the nuclear R N A content raises significantly from 197_+ 6 /~g/mg D N A in the

TABLE II EFFECT OF 5-h T U R P E N T I N E TREATMENT ON rRNA SYNTHESIS A N D METHYLATION RNA synthesis (incorporation of [3H]UTP into RNA) and RNA methylation (incorporation of [3H]CH~ from S-adenosylmethionine into RNA) were studied with isolated liver nucleoli and are expressed as picomoles incorporated per mg DNA (means of 5 experiments+-S.E.). - N T P designates the incubation medium without ribonucleoside triphosphates. Normal RNA synthesis (pmol U M P / m g DNA) RNA methylation ( p m o l / mg DNA) (a)+ NTP (b)-NTP (c) difference /~mol C H 3 / m o l UMP, incorporated into RNA in vitro

Turpentine-treated

1156+_135

82+ 72+ 10

8651

7.3 5.9

1259_+78

87_+ 4.9 71+ 7.3 16

12708

182

control liver to 217 + 7 btg/mg D N A in the liver of turpentine-treated rats (11 experiments: P < 0.005 ). The data presented in this paper, while confirming the role played by the nucleus, emphasize the importance of post-transcriptional mechanisms in priming the acute-phase response in the liver cell. Soon after the onset of an inflammatory reaction, the chain of events that will finally lead to an increased number of cytoplasmic ribosomes is started by a lowered rRNA wastage, leading to an intranuclear accumulation of RNA, and by' a modification of the nuclear envelope associated with an increased translocation rate of ribosomal subunits from nucleus to cytoplasm. We thank Miss. Maria Grazia Bombonato for typing the manuscript. The work was supported in part by a research grant MPI 8 1 / 8 2 - - Ric. 40%. References 1 Kqj, A. (1974) in Structure and Function of Plasma Proreins (Allison, A.C., ed.), Vol. t, pp. 73-125, Plenum Press, London 2 Kushner, 1. (19821 Ann. N.Y. Acad. Sci. 389, 39 3 Baglia. F.A,, Kw,am S.W. and Fuller, G.M. (1981} Biochim. Biophys. Acta 696, 107 4 Princen, J.M.G., Nieuwenhuizen, W., MoI-Backs, G.P.B.M. and Yap, S.H. (1981) Biochem. Biophys. Res. ('ommun. 102, 717 5 Ricca, G.A., Hamilton, R.W., McLean, J.W.. ('ann, A., Kalinyek, J.E. and Vajloij, M. (19811 J. Biol. Chem. 256. 10362

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