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Ribosome formation in rat liver: Evidence for post-transcriptional control
Io9
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96329
RIBOSOME FORMATION IN RAT LIVER: TRANSCRIPTIONAL CONTROL
EVIDENCE FOR POST-
A R T U R O J. RIZZO AND THOMAS E. W E B B
McGill University Cancer Research Unit, Mclntyre Medical Sciences Building, Montreal zo9, Quebec (Canada) (Received J u n e I6th, 1969)
SUMMARY
An investigation of the amount of label incorporated from [6-~*C]orotic acid into the nuclear and cytoplasmic ribosomal RNA of non-proliferating rat liver, during a I-h treatment with several inhibitors of protein biosynthesis, gave the following results: I. The incorporation into nuclear 45-S, 28-S and I8-S RNA of liver was increased during a i-h treatment with anisomycin and cycloheximide by 5° %, and with puromycin by 25 %; 8-azaguanine showed little effect. 2. The appearance of label in newly synthesized ribosomes increased approx. 25 % after cycloheximide or anisomycin treatment; in contrast, this parameter was reduced to 25 % that of the controls after puromycin treatment, and to 50 % that of the controls after 8-azaguanine treatment. This differential effect of the inhibitors on ribosome transport was also observed in the slow growing Hepatoma 7800. 3. The size of the ribosomal subunit (60 S and 40 S) pools were not significantly altered by the inhibitors, even though puromycin and 8-azaguanine caused extensive conversion of the polyribosomes to monomers and dimers. The results suggest that the differential effect of the inhibitors on ribosome formation and polyribosome structure are related. They further suggest that this feed-back control, which may involve monomeric ribosomes, acts at a post-transcriptional step. There appears to be a pool of ribosomal proteins in non-proliferating rat liver and in the slow growing hepatoma which is sufficient to support ribosome synthesis for at least I h.
INTRODUCTION
In a previous study 1, evidence was presented that the rate of transport of newly synthesized ribosomes to the cytoplasm and the size of the pool of monomeric ribosomes were coupled in the rat liver system. It was proposed that such a relationship has a bearing on the problem of the control of ribosome formation, e.g. the monomeric ribosomes may be involved in the control of ribosome synthesis through a mechanism involving negative feedback. This possibility was further supported by the finding that inhibitors of protein biosynthesis which cause an increase in the size of the monomer pool (e.g. puromycin) reduced the incorporation of [6-14C]orotic Biochim. Biophys. Acta, 195 (1969) lO9-122
IiO
A . J . RIZZO, T. E. WEBB
acid into cytoplasmic ribosomes of regenerating rat liver to a greater extent than those which maintain the polyribosome structure (e.g. cycloheximide). The monomeric ribosomes have also been implicated in the control of RNA synthesis in bacterial cells ~,3. The latter studies showed that starvation of a stringent strain of Escherichia coli for an amino acid caused extensive polyribosome breakdown and an inhibition oi RNA synthesis; however, polyribosomes were maintained and RNA synthesis continued in a relaxed strain. It was not established in the previous investigation 1 whether the rate of labelling of the cytoplasmic ribosomal RNA was a function of the rate of transcription of the ribosomal RNA, a function of the rate of utilization (e.g. transport to the cytoplasm) of newly synthesized ribosomal RNA, or both. The results of the present investigation, which represents a study of the differential effects of various inhibitors of protein biosynthesis on ribosomal RNA synthesis and ribosome formation, support the hypothesis that the rate of ribosome synthesis in non-proliferating rat liver is controlled, at least in part, at the level of utilization or transport of the RNA. They also provide further support for the possibility that monomeric ribosomes, but not ribosomal proteins, are involved in this post-transcriptional control. MATERIALS AND METHODS
Materials Male Sprague-Dawley rats weighing 35o-4oo g were maintained in plastic cages on a Purina chow diet and were fasted in wire bottom cages for 16 h prior to use. The Morris H e p a t o m a 7800 was carried* intramuscularly in rats of the Buffalo strain. Lighting was from 6 a.m. to 6 p.m.; the experiments were routinely started at i o - i i a.m. to minimize variations due to diurnal rhythms. The livers and tumors were removed under light ether anaesthesia. [6-1*C~Orotic acid hydrate (specific activity 4.8 mC/mmole), L-[I-laClleucine (specific activity 20 mC/mmole) and DL-E3-1*C~tryptophan (specific activity IO mC/ mmole) were purchased from the New England Nuclear Corp., Boston. Cycloheximide, puromycin and 8-azaguanine were purchased from the Nutritional Biochemicals Corporation Cleveland. Anisomycin was a gift from Chas. Pfizer and Co., Conn. The isotopes and drugs were administered intraperitoneally.
Size distribution analysis o/ribosomal components The relative concentration of the monomeric ribosomes and polyribosomes were determined as previously described 1,5. A portion (3 g) of the liver from control and treated rats was used for the size distribution analysis of the ribosomal components and for the preparation of the ribosome fraction; the purified nuclei were prepared from the remainder of the liver (see below). Immediately prior to removal, the liver was perfused with IO ml of cold 0.25 M sucrose in 5 ° mM Tris-25 mM KC15 mM MgCI~ buffer p H 7.5 (Tris-KC1-MgCl~-buffer). After treating the post-mitochondrial supernatant (IO rain, 13 ooo ×g) with deoxycholate, it was diluted with an equal volume of buffer and o.7-ml aliquots were analyzed on lO-35 % sucrose gradients as before 1. For the separation and estimation of the relative concentrations of the ribosomal monomers and ribosomal subunits, the deoxycholate-treated post-mitochonBiochim. Biophys. Act*, 195 (1969) lO9-122
RIBOSOME FORMATION IN RAT L I V E R
III
drial supernatant was diluted as before, then layered over exponential 7-47 % sucrose gradients which were centrifuged at 5 ° ooo x g av. for 18. 5 h in a SW 25.1 rotor of a Beckman centrifuge. The gradients were analyzed as described previously 1.
Preparation of ribosomal fraction Approx. 4 ml of the deoxycholate-treated post-mitochondrial supernatant prepared for the size distribution analysis, was centrifuged at lO5 ooo x g for I h to obtain a ribosome pellet; the ribosomes were resuspended in aqueous medium and the RNA was extracted as outlined below.
Preparation o/ nuclei The purified nuclei were prepared according to MURAMATSU AND BUSCH~ with minor modifications. Briefly, a portion of the liver (5~6 g) was finely minced with scissors, then suspended in 2.4 M sucrose (1:15, w/v) containing 3-3 mM calcium acetate and homogenized with 4 strokes of a teflon pestle. The clearance between the pestle and the wall of the glass P o t t e r - E l v e h j e m homogenizer was 0.02 mm; the pestle rotated at 800 rev./min. Under our conditions a tighter pestle rotating at the same speed caused extensive breakdown of the 45 S RNA. The homogenate was filtered through cheesecloth and centrifuged at 40 ooo x g for I h, then the nuclear pellet was resuspended in I.O M sucrose-I.O mM calcium acetate and re-centrifuged at 3000 x g for 5 rain to give a pellet of purified nuclei.
Extraction o / R N A The purified nuclei and ribosomal pellets were resuspended in o.14 M NaC10.05 M sodium acetate buffer, p H 5.0, containing 0.3 % sodium dodecyl sulfate (1. 5 ml/g of liver). The suspension was treated with an equal volume of redistilled aq. phenol at 65 and 25 ° according to the procedure of MURAMATSU AND BUSCH~. The RNA was precipitated from the reextracted aqueous phases b y the addition of 2.5 vol. of 95 % ethanol containing 2 % potassium acetate at --15 °. The RNA was dissolved in a small volume of 0.02 M sodium acetate, p H 5.0.
Separation o/ RNA on density gradients Aliquots of the purified RNA (approx. 2oo fig in o. 4 ml) were layered over linear sucrose (lO-4O %) gradients e containing o.I M NaC1, 0.02 M sodium acetate and o.ooi M E D T A (pH 5.0). The gradients were centrifuged for 16 h at 63 ooo x g av. in a SW 25.1 rotor of a Beckman centrifuge. Ribonuclease-free sucrose (Mann Research Corporation) was used in the preparation of the sucrose density gradients. Following the separation of the various species of RNA according to size, the effluent from the bottom of the gradient tube was monitored at 260 m# in a flow cell. A drop counter activated an event marker on the recorder to register each milliliter of effluent. To determine the radioactivity in each fraction the effluent was collected in pre-cooled centrifuge tubes, then the acid-insoluble material was precipitated with 5 % trichloroacetic acid, using serum albumin as co-precipitant. The precipitate was dissolved in solubilizer (NCS, Nuclear Chicago) then counted in liquid scintillant at an efficiency of 80 %. The specific radioactivity of the various RNA species was calculated directly from the absorbance and radioactivity profiles using the conversion factor 20 abBiochim. Biophys. Acta, 195 (1969) l O 9 - 1 2 2
112
A . J . RIZZO, T. E. WEBB
sorbance units = I m g of RNA. The absorbance of a blank gradient was subtracted from each of the absorbance and radioactivity profiles.
Incorporation o/amino acids into soluble proteins A mixture of L-[/J4C]leucine (I2~C) and DL-[3-14C]tryptophan (I2/~C) was administered to 400 g control or treated rats 15 min prior to removal of the livers. The latter were homogenized in 0.25 M sucrose in Tris-KC1-MgC12 buffer (I.O g/ 2.0 ml) and the homogenate was centrifuged at lO5 ooo ×g for I h. The proteins of the postmicrosomal supernatant were precipitated with 5 % trichlororacetic acid at 4 °. The precipitates were extracted consecutively with 95 % ethanol and ethanolether (i:I,V/V) at 4 o°. The residue was suspended in solubilizer and counted in liquid scintillant. Protein was determined in duplicate samples using biuret reagent 7.
RESULTS
E//ectiveness o/inhibitors o/protein biosynthesis Much of the work to be reported involves a comparison of the differential effects of various inhibitors of protein biosynthesis on ribosomal RNA synthesis and transport to the cytoplasm. It was therefore necessary to confirm that the dosages employed effectively inhibited protein biosynthesis over the period of time during which measurements were made. The effect of three inhibitors of protein biosynthesis in rat liver was tested and the results are shown in Table I. Anisomycin s TABLE I EFFECT
OF A N I S O M Y C I N , C Y C L O H E X I M I D E
AND PUROMYCIN ON PROTEIN BIOSYNTHESIS
R a t s weighing 400 g were injected w i t h 20 m g / k g of anisomycin, I m g / k g of cycloheximide, or i o o m g / k g of p u r o m y c i n and the livers were r e m o v e d 30 or 60 m i n later. 15 m i n prior to the rem o v a l of the liver; a m i x t u r e of L-[i--!*C]leucine (12/~C) and [3--14C]tryptophan (i2/zC) was administered. The specific radioactivity of the soluble p r o t e i n s was determined as described u n d e r MATERIALS AND METHODS.
Inhibitor
Anisomycin Cycloheximide Puromycin
Inhibition (%)during the interval* I5-3o rain
3o-6o rain
85 9o 95
6o-85 94 96
* The time i n t e r v a l over which the soluble p r o t e i n s were labelled, m e a s u r e d from the time of a d m i n i s t r a t i o n of the inhibitor.
(2o mg/kg), cycloheximide9 (I mg/kg) and puromycin 1° (IOO mg/kg) inhibited protein biosynthesis by 85-95 % within 15 min of administration. This level of inhibition was maintained for at least an additional 45 min by cycloheximide and puromycin. In the case of anisomycin, however, the level of inhibition varied from 60 to 85 % in four separate experiments during the interval of 45-60 min. During the time interval 45-60 rain after the administration of anisomycin, cycloheximide or puromycin at the dosages outlined above, protein biosynthesis in Hepatoma 7800 was inhibited by 7 ° , 85 and 88 % respectively. Biochim. Biophys. Acta, 195 (1969) lO9-122
113
RIBOSOME FORMATION IN RAT LIVER
EHect o/ inhibitors on polyribosomes, monomers and subunits Further attempts were made to establish the inverse relationship between the size of the inactive monomer pool and the rate of transport of newly synthesized ribosomes to the cytoplasm. In the present series of experiments the duration of treatment with the inhibitors of protein biosynthesis was I h. Shown in Fig. I a is the effect of anisomycin on the size of the monomer and polyribosome pools. The results indicate that there is only a slight shift of the polyribosome size distribution to the lighter species with no significant change in the size of the monomer pool as compared to the controls. The size distribution of the ribosomal components prepared from the liver of rats treated with cycloheximide were similar to those of anisomycin-treated rats. However, as shown in earlier studies 5,9, marked polyribosome breakdown, with consequent increases in the inactive monomer and dimer pools, occurs in rat liver following the administration of 8-azaguanine or puromycin (Fig. Ib). It should be noted that only one-half the amount of post-mitochondrial supernatant was layered on the gradient in the case of the puromycin-treated rat. I.I I.C 0.9
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Fig. I(a) Size d i s t r i b u t i o n profiles of the p o l y r i b o s o m e s and the relative size or the m o n o m e r pool in the deoxycholate-treated s u p e r n a t a n t (0. 7 ml) p r e p a r e d from the liver of a control r a t ( - - ) a n d of a r a t w h i c h w a s t r e a t e d for I h w i t h 20 m g / k g of anisomycin (. . . . ). The m o n o m e r p e a k is centered a t F r a c t i o n 2o and the t o p of the g r a d i e n t is to the f i g h t of the figure. (b) Similar p a t t e r n s of ribosomal c o m p o n e n t s in o. 7 ml of deoxycholate-treated s u p e r n a t a n t p r e p a r e d f r o m the liver of a r a t t r e a t e d w i t h IOO m g / k g of 8-azaguanine for 4 h (--), or in o.35 nil of deoxycholatet r e a t e d s u p e r n a t a n t p r e p a r e d f r o m the liver of a r a t t r e a t e d w i t h i o o m g / k g of p u r o m y c i n for I h ( . . . . ). Fig. 2. Size d i s t r i b u t i o n profile s h o w i n g the relative c o n c e n t r a t i o n of ribosomal m o n o m e r s a n d s u b u n i t s in the liver from control ( - - ) a n d a n i s o m y c i n - t r e a t e d (. . . . ) rats. The m o n o m e r p e a k a n d the 6o-S and 4o-S s u b - u n i t peaks are centered a t F r a c t i o n s 12, 15 and 2o, respectively.
The relative concentrations of the monomeric ribosomes and the 6o-S and 4o-S ribosomal sub-units in control and anisomycin-treated rats are shown in Fig. 2. Neither the monomer pool, nor the subunit pools appear to be affected by the drug. It is concluded that anisomycin, the action of which has not been previously studied Biochim. Biophys. Acta, 195 (1969) lO9-122
114
A . J . RIZZO, T. E. WEBB
in the rat liver system, does not affect the pool size of the various ribosomal components. The effects of the various inhibitors of protein biosynthesis on the size of the monomer and subunit pools are tabulated in Table II. Since the dimers, in particular
TABLE II EFFECT OF ANISOMYCIN, CYCLOHEXlMIDE AND PUROMYCIN ON THE SIZE OF THE RIBOSOMALMONOMER AND SUBUNIT POOLS R a t s weighing 25o-3oo g were injected w i t h 20 m g / k g of anisomycin, I m g / k g of cycloheximide, or IOO m g / k g of p u r o m y c i n and the livers were r e m o v e d I h later. The polyribosomes, m o n o m e r s a n d s u b u n i t s p r e s e n t in the deoxycholate t r e a t e d p o s t - m i t o c h o n d r i a l s u p e r n a t a n t were separated on d e n s i t y gradients. The relative heights (A 2e0 mr*) of the peaks of these c o m p o n e n t s in the size d i s t r i b u t i o n profiles are recorded; the combined heights of the m o n o m e r and dimer peaks are also given in parentheses. Inhibitor
Control Anisomycin Cycloheximide Puromycin 8-Azaguanine
Monomer
0.52 0.55 0.62 2.2 0.70
(Monomer+dimer)
(0.80) (0.85) (0.90) (3.2) (1.2)
Subunits 6o s
4° s
0.35 0.33 0.32 0.38 0.34
o. i8 o. 15 o. 15 0.20 o.16
those derived from polyribosome breakdown in vivo, are presumed to belong to the monomer pool in vivo 11, the combined heights of the monomer and dimer pool are given in parentheses. As in the case of anisomycin, cycloheximide has little effect on the monomer or subunit pools. Although puromycin causes the conversion of a substantial portion of the polyribosomes to monomers and dimers, only a slight increase in the subunit pools is observed. The subunit pools were corrected for a portion of the 6o-S subunits which tended to aggregate with the monomers at high concentrations of the latter. This aggregate sediments as a peak on the receding edge of the dimer peak in the size distribution profiles 5. It is concluded that the subunit pools remain relatively constant following the inhibition of protein biosynthesis, with or without concommittant polyribosome breakdown.
E//ect o/inhibitors on R N A synthesis Typical absorbance and radioactivity profiles of nuclear and cytoplasmic RNA prepared from the livers of rats injected with saline (controls), or with one of several inhibitors of protein biosynthesis, are shown in Fig. 3 and Fig. 4. In these experiments !6-14Clorotic acid was injected into the rat 5 rain after the administration of saline or the inhibitor and the liver was removed I h later. The sedimentation constants assigned to the various species of RNA, are those which have been established under the conditions employed 6. Shown in Fig. 3 are the absorbance and radioactivity patterns of nuclear RNA from the liver of (a) control, (b) anisomycin-treated, (c) cycloheximide-treated and (d) puromycin-treated rats. The pattern of labelling was similar in the livers from all animals; however, the extent of labelling was slightly greater in the 45-S, I8-S Biochim. Biophys. Acta, I95 (I969) IO9-I22
RIBOSOME FORMATION IN RAT LIVER
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Fig. 3. A b s o r b a n c e ( - - ) a n d r a d i o a c t i v i t y (. . . . ) profiles of n u c l e a r 1RNA e x t r a c t e d f r o m t h e livers of r a t s w h i c h received [6-14C]orotic acid (32/zC/4oo g) 5 rain a f t e r t h e a d m i n i s t r a t i o n of (a) saline (control), (b) a n i s o m y c i n (20 m g / k g ) , (c) c y c l o h e x i m i d e (i m g / k g ) , or (d) p u r o m y c i n (IOO m g i k g ) . T h e labelling period w a s I h. T h e 45-S, 28-S, I8-S, a n d 7-S species are c e n t e r e d a t F r a c t i o n s 9, 14, 19 a n d 25, respectively.
and 28-S species of nuclear RNA from the cycloheximide and anisomycin-treated rats as compared to the corresponding species in the control and puromycin-treated animals. The corresponding absorbance and radioactivity profiles of the cytoplasmic ribosomal RNA are shown in Fig. 4. The results indicate that the specific activities Biochirn. Biophys. Acta, 195 (1969) lO9-122
116
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Fig. 4. A b s o r b a n c e ( - - ) a n d r a d i o a c t i v i t y (. . . . ) profiles of c y t o p l a s m i c R N A e x t r a c t e d f r o m t h e livers of r a t s w h i c h were t r e a t e d w i t h (a) saline (control), (b) a n i s o m y c i n , (c) cyclohexirnide a n d (d) p u r o m y c i n as for Fig. 3. T h e 28-S a n d I8-S species are c e n t e r e d a t F r a c t i o n s 16 a n d 20, respectively.
of the I8-S and 28-S cytoplasmic ribosomal RNA's are markedly higher in the livers from (a) control, (b) anisomycin and (c) cycloheximide-treated animals, than from the (d) puromycin-treated rats. The effect of 8-azaguanine on the labelling pattern of nuclear and cytoplasmic ribosomal RNA was also tested. In this experiment the [6-z4C]orotic acid was adminisBiochim. Biophys. Mcta, 19,5 (1969) lO9-122
117
RIBOSOME FORMATION IN RAT LIVER
tered 4 h after the administration of the base analog and I h before removal of the liver. As shown previously 5,x3 polyribosome breakdown is maximal during this period. The inhibition of protein biosynthesis which appears to parallel polyribosome breakdown, is approx. 60 ~'o at 4.5 h after the administration of the base analog (S.-W. KWAN AND T. E. WEBB, unpublished observations). The absorbance and radioactivity profiles of the nuclear RNA (Fig. 5a) are very similar to that of the control rat; however, the corresponding labelling pattern of the cytoplasmic RNA (Fig. 5b) resembles that from the puromycin-treated rats. 1.5 1.2
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Fig. 5. A b s o r b a n c e ( - - ) a n d r a d i o a c t i v i t y (. . . . ) profiles of (a) n u c l e a r a n d (b) c y t o p l a s m i c R N A e x t r a c t e d f r o m t h e liver of a r a t w h i c h received E6-zaC]orotic acid (32 # C / 4 o o g) 4 h a f t e r t h e a d m i n i s t r a t i o n of i o o m g / k g of 8 - a z a g u a n i n e . T h e labelling period w a s i h.
The specific activities of the various species of nuclear and cytoplasmic ribosomal RNA estimated from a number of size distribution and radioactivity profiles similar to those shown in Figs. 3 and 4 are tabulated in Table III. The results indicate that the labelling of the 45-S ribosomal RNA is increased 25-50 % above that of the controls after treatment with anyone of the inhibitors, with the exception of 8-azaguanine which caused little change. The specific activity of the I8-S and 28-S nuclear RNA is correspondingly increased after treatment of the rats with anisomycin, cycloheximide or puromycin; this suggests that the 45-S precursor molecule does not accumulate after drug treatment, but is converted to its products with the same efficiency as in the control livers. Despite the increased labelling of the nuclear ribosomal RNA in puromycintreated rats, the specific activity of the corresponding cytoplasmic ribosomal RNA was only 25 % that of the controls. In contrast, the specific activities of these species in the anisomycin or cycloheximide-treated rats were, in general, 25-4 ° % higher Biochim. Biophys. Acta, 195 (1969) lO9-122
118
A . J . RIZZO, T. E. WEBB
TABLE III EFFECT OF INHIBITORS OF THE INCORPORATION AND CYTOPLASMIC RNA
OF LABEL
FROM
[6-14C]OROTIC
ACID INTO NUCLEAR
The rats received [6--x4C]orotic acid (32/zC]4oo g) 5 min after the administration of anisomycin (20 mg/kg), cycloheximide (I.O mg/kg), or puromycin (ioo mg/kg), or 4 h after the administration of 8-azaguanine (ioo mg/kg per dose) in 2 doses 3 ° rain apart. The livers were removed I h later and the nuclear and cytoplasmic RNA was extracted.The specific radioactivities of the various species were estimated from the central region of each peak in the absorbance and radioactivity profiles. The number of rats used in each experiment is shown in parentheses. Treatment
Specific radioactivity (counts~rain per i~g R N A )* Nuclear R N A
Control Anisomycin Cycloheximide Puromycin 8 azaguanine
(4) (2) (2) (2) (I)
Cytoplasmic R N A
45 S
28 S
18 S
28 S
I8 S
880 13oo 132o ilOO 800
34 ° 55 ° 580 480 300
33 ° 560 57 ° 520 280
2.8 3-5 4 .0 o. 7 1.2
3.6 5 .0 3.5 0.8 2. 4
* The standard error was in all cases less than IO % of the mean.
t h a n t h a t of t h e c o n t r o l s . A l t h o u g h t h e s p e c i f i c a c t i v i t i e s of t h e v a r i o u s s p e c i e s of nuclear RNA were essentially unaffected by 8-azaguanine treatment, the appearance of n e w r i b o s o m a l R N A in t h e c y t o p l a s m w a s m a r k e d l y i n h i b i t e d . T h e d a t a in T a b l e I I I also c o n f i r m t h a t in c o n t r a s t t o a n i s o m y c i n a n d c y c l o h e x i m i d e , p u r o m y c i n a n d 8a z a g u a n i n e ( w h i c h a p p e a r t o i n h i b i t t h e t r a n s p o r t of r i b o s o m a l R N A t o t h e c y t o p l a s m ) c a u s e a m a r k e d i n c r e a s e in t h e c o n c e n t r a t i o n of i n a c t i v e m o n o m e r s ( a n d d i m e r s ) . T h e d i f f e r e n t i a l e f f e c t of s o m e i n h i b i t o r s of p r o t e i n b i o s y n t h e s i s o n t h e t r a n s p o r t of n e w l y f o r m e d r i b o s o m a l R N A t o t h e c y t o p l a s m w a s also t e s t e d u s i n g t h e r a t h e p a t o m a s y s t e m . T h e s p e c i f i c a c t i v i t i e s of t h e I 8 - S a n d 28-S r i b o s o m a l R N A i s o l a t e d f r o m t h e c y t o p l a s m of H e p a t o m a 7800 a r e r e c o r d e d in T a b l e IV. (The s p e c i f i c a c t i v i t y
"FABLE IV EFFECT OF INHIBITORS ON THE FORMATION OF RIBOSOMES IN HEPATOMA 7800 Rats bearing Hepatoma 7800 received [6-14C]orotic acid (4°/zC) 0. 5 h before the administration of 0.9 % saline (controls), 20 mg/kg of anisomycin, i mg/kg of cycloheximide, or ioo mg/kg of puromycin; the tumors were removed and processed 1. 5 h after the administration of the labelled precursor. The RNA was extracted from the cytoplasmic ribosomes and the I8-S and 28-S species were separated on sucrose gradients. The specific radioactivities were estimated from the center of the peaks in the combined absorbance-radioactivity profiles. Inhibitor
Control Anisomycin Cycloheximide Puromycin
Height o[ monomer ( +dimer) * peak (A26o mll)
Speci[ic activity (counts/rain I~g per R N A ) 18 S
28 S
0.68 0.64 0.49 1.5o
2.8 2.2 3-4 1. 4
1.2 1.2 1.9 0.6
(i.22) (1.18) (0.85) (2. 7 )
* Refers to combined height of monomer and dimer peaks as for Table II. Biochim. Biophys. $cta, 195 (1969) lO9-122
RIBOSOME FORMATION IN RAT L I V E R
119
of the nuclear RNA was not determined.) In this series of experiments the [6-14C2orotic acid (4 ° #C/25o g) was administered 30 rain before the inhibitors since the incorporation of orotic acid into ribosomal RNA was much lower in the hepatoma as compared to liver. However, orotic acid was superior to either cytosine or uracil as a RNA precursor in this hepatoma. The tumors were removed 1.5 h after the administration of [6-1~Cjorotic acid. Since the inhibitors were injected 30 min after the isotope, any effects observed probably result from a modified rate of transport rather than a modified rate of ribosomal RNA synthesis. The results show that in neoplastic liver anisomycin and cycloheximide have only slight effects on the labelling of cytoplasmic ribosomal RNA; in contrast, puromycin inhibits the appearance of newly formed ribosomes by approx. 50 %. As in normal liver, H e p a t o m a 7800 appears to possess a pool of ribosomal proteins sufficient to permit normal ribosome synthesis over a period of I h.
DISCUSSION
The nucleus of eukaryotic cells contains most of the DNA and is the site of synthesis of a variety of RNA molecules. Most of the latter are transferred to the cytoplasm where they participate in protein biosynthesis. Evidence from a number of sources 1~-18 suggests that the transport is selective, and that a portion of the RNA transcribed in the nucleus is also degraded. In view of this evidence which suggests that there is selective control over the types of RNA which m a y leave the nucleus, the question m a y be asked whether there is control over the amount of a n y one species which is transported to the cytoplasm. In earlier experiments l, it was shown that the reduction in the incorporation of radioactive label into the RNA of cytoplasmic ribosomes with increasing time of liver regeneration was directly proportional to the size of the inactive monomeric ribosome pool. Furthermore, evidence was obtained that inhibitors of protein biosynthesis which cause the breakdown of polyribosomes to monomers and dimers inhibit ribosome formation in regenerating liver to a greater extent than inhibitors which maintain the polyribosome structure. It was not established whether the former class of inhibitors affected RNA synthesis directly, or whether the inhibition resulted from a depletion of ribosomal proteins or interference with the transport of newly synthesized ribosomal RNA to the cytoplasm in the form of subunits. However, the observation 1 that ribosome formation was inhibited only 40 % in the 42-h regenerating liver during a 2-h treatment with cycloheximide, while under similar conditions puromycin caused a 65 °/o inhibition, together with the findings of other laboratories 19-21 strongly suggests that factors other than ribosomal proteins are involved in the control of ribosome synthesis. In the present investigation a I-h labelling period was selected as a compromise, since according to the experimental procedure, a longer labelling period necessitates a longer treatment with the inhibitors of protein biosynthesis. Since the results of an earlier study 1 indicated that cycloheximide inhibited ribosome synthesis in regenerating liver within 2 h, it was anticipated that extended periods of near total inhibition of protein biosynthesis would seriously complicate the interpretation of the data in the present study. Inhibitory effects observed only after extended treatBiochim. Biophys. Acta, 195 (1969) l O 9 - 1 2 2
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A . J . RIZZO, T. E. WEBB
merit m a y be due to depletion of ribosomal protein pools, decreased RNA polymerase activity or non-specific effects due to depletion of other cellular enzymes. On the other hand, a labelling period significantly less than I h would not permit measurable amounts of newly synthesized ribosomal RNA to enter the cytoplasm. It is also important to note that the dosages of the inhibitors used were the minimal amounts required to achieve approximately their maximal effects, and thus to avoid excessive toxicity. This factor m a y be important, since in the present investigation cycloheximide was administered at a dosage of I mg/kg and no inhibition of ribosomal RNA biosynthesis was observed over a period of I h. However, when the cycloheximide dosage employed is 5o-fold higher, a significant inhibition of ribosome synthesis in rat liver is observed 2j, although it is also clear from these data that the ribosomal proteins are not limiting under these conditions. The results of the present study indicate that the rate of incorporation of label from I6-1*C~orotic acid into nuclear RNA during a I-h treatment with several inhibitors of protein biosynthesis is either increased, or remains unchanged as compared to the controls. Part of the increase observed m a y be due to increased production of corticosteroids in response to stress, since an increased synthesis of 45-S RNA after hydrocortisone treatment has been observed in the livers of adrenalectomized rats 22. In view of these results any decrease in the rate of appearance of labelled RNA in the cytoplasm in response to any of these inhibitors cannot be due to a decreased labelling of nuclear precursors, due in turn to an increase in the size of nucleotide pools, or to a decreased rate of RNA biosynthesis. Although cycloheximide caused an almost complete inhibition of protein biosynthesis within 5 rain after administration, it did not decrease the transport of ribosomal RNA to the cytoplasm during the I-h labelling period. Anisomycin s, which has a mechanism of action similar to that of cycloheximide 9 produced a similar effect on the transport of RNA to the cytoplasm, although the inhibition of protein biosynthesis was variable during the interval 45-60 rain (c/. Table I). The results suggest that the ribosomal proteins are present in excess, since a fraction of the nuclear RNA synthesized over and above the control level after ahnost total inhibition of protein synthesis with cycloheximide and anisomyein, was transported to the cytoplasm in the form of ribosomal subunits. It would appear that the pool of ribosomal protein is limiting in regenerating liver, since ribosomes are synthesized at only 60 % of the control level after cycloheximide treatment in the 42-h regenerating liver 1. This phenomenon could be a characteristic of proliferating tissue in general, since a decreased synthesis of ribosomal RNA was recently observed in H e L a cells after cycloheximide treatment 23. In contrast to cycloheximide and anisomycin which maintain the structure of the polyribosome, puromycin 1° and 8-azaguanine 5,1~, which cause the conversion of a portion of the polyribosomes to inactive monomers (and dimers) produced a marked inhibition of the appearance of newly formed ribosomal RNA in the cytoplasm. The mechanism by which 8-azaguanine induces polyribosome breakdown is not firmly established, although it appears to act through the formation of defective messenger RNA 5,~2. Puromycin induces polyribosome breakdown b y accelerating translation and b y causing the premature release of ribosomes from the messenger RNA 1°. It seems probable that the inhibition of ribosome formation by puromycin or 8-azaguanine, two drugs which have quite different mechanisms of action, is related to the ability of both to induce polyribosome breakdown. As in an earlier study Biochim. Biophys. Acta, 195 (1969) lO9-122
RIBOSOME FORMATION IN RAT LIVER
121
employing H e L a cells 24, the present results indicate that there is a normal formation of 45-S precursor RNA in the presence of 8-azaguanine, although the appearance of newly formed ribosomal RNA in the cytoplasm is markedly inhibited. However, the present data also indicate that the initial processing in the nucleus of the precursor RNA to I8-S and 28-S RNA is also normal and therefore do not support the suggestion ~ that the inhibition of ribosome formation is due to an inability of the cell to process 45-S RNA which contains the base analog. However, the possibility that the presence of 8-azaguanine in the ribosomal RNA interferes with later steps in ribosome assembly cannot be ruled out. Both 8-azaguanine and puromycin also cause an increase in the dimer pool in addition to the monomer pool. Since these dimers are inactive in vivo 4 and since there is a tendency for monomeric ribosomes to dimerize in vitro in extracts from rat liver 11, it seems probable that the dimers should be included in the monomer pool. The results of this study support the hypothesis that the inactive ribosomal components are involved in the control of ribosome synthesis and that this control is exerted, at least in part, post-transcriptionally. Similar studies, which do not involve the use of inhibitors, on the transitional increase in RNA synthesis which occurs after partial hepatectomy, also suggest that at least a portion of the control of ribosome biosynthesis operates at this level (A. J. Rlzzo ANn I. E. W E B B , unpublished observations). In view of the finding that the ribosomal subunit pools are not affected b y puromycin, it would appear that the monomeric ribosomes are involved in the postulated feed-back control. Subunit pools in bacteria and mouse ascites tumor ceils also remain essentially constant during a number of treatments which affect the monomer pool ~5,26. A dissociation factor similar to the protein factor of E. coli which has been shown to be present in limiting concentrations and to cause the dissociation of 7o-S ribosomes into subunits ~7, appears to be operative in mammalian cells ~s,~. An inhibition of ribosome synthesis b y puromycin has also been observed in H e L a cells3°. This inhibition was attributed to interference of puromycin peptides with ribosome maturation. However, in the absence of any correlation between the production of such peptides and an inhibition of protein biosynthesis and in view of the apparent normal processing of nuclear ribosomal RNA shown in the present study, it is unlikely that the decreased transport of newly synthesized ribosomes to the cytoplasm after puromycin treatment is due to an interference with normal processing of the 45-S precursor. The results obtained with the neoplastic liver (Hepatoma 7800) were qualitatively similar to those of normal liver. However, if monomeric ribosomes are involved in the control of ribosome synthesis, this regulation m a y be modified, since tumors invariably contain high concentrations of inactive ribosomes 2~,~1, at least under conditions where the growth rate is relatively slow. In summary, the results suggest that ribosome synthesis is controlled, at least in part, after gene transcription in resting (non-proliferating) tissue. Furthermore, in agreement with evidence from the bacterial system s,a, there appears to be a relationship between the rate of transport of ribosomal RNA to the cytoplasm and the concentration of inactive ribosomes in the cytoplasm. Non-proliferating or slow growing tissues appear to contain pools of ribosomal proteins sufficient to permit continued ribosome synthesis for at least I h. Biochim. Biophys. Acta, 195 fI969) io9-122
122
A . J . RIZZO, T. E. WEBB
ACKNOWLEDGEMENTS
This work was supported by Grant MA 1949 from the Medical Research Council of Canada and by a grant from the National Cancer Institute of Canada. One of us (A.J.R.) is the holder of a Fellowship from the National Cancer Institute of Canada. The authors are grateful to Dr. H. P. Morris, Howard University, Washington, D.C., for supplying the tumor-bearing rats used in this study. REFERENCES i 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3° 31
A. J. Rizzo AND T. E. WEBB, Biochim. Biophys. Acta, I69 (1968) 163. D. W. MORRIS AND J. A. DEMOSS, Proc. Natl. Acad. Sci. U.S., 56 (1966) 262. E. Z. RON, R. E. KOHLER AND ]3. E. DAVIS, Proc. Natl. Acad. Aci. U.S., 56 (1966) 471. H. P. MORRIS, Advan. Cancer Res., 9 (1965) 227. S.-W. ]~WAN AND T. E. WEBB, J. Biol. Chem., 242 (1967) 5542. M. MURAMATSU AND H. BUSCH, in H. ]DuscH, Methods in Cander Research, Vol. 2, Academic Press, N e w York, 1967, p. 303 . A. G. GORNALL, C. J. ]DARDAWlLL AND M. M. DAVID, J. Biol. Chem., 177 (1949) 751. A. P. GROLLMAN, J. Biol. Chem., 242 (1967) 3226. A. C. TRAKATELLIS, M. MONTJAR AND A. E. AXELROD, Biochemistry, 4 (1965) 2o65. S. VILLA-TREVINO, E. FARBER, T. STAEHELIN, F. O. WETTSTEIN AND H. NOLL, J. Biol. Chem., 239 (1964) 3826. R. W. READER AND C. P. STANNERS, J. Mol. Biol., 28 ~I967) 211. T. E. WEBB, Biochim. Biophys. Acta, 138 (1967) 307 . J. W. WATTS, Biochem. J., 93 (1964) 3 °6. H. HARRIS, Nature, 202 (1964) 249. J. DREWS, G. ]DRAWERMAN AND H. P. MORRIS, Naturwissenscha/ten, 23 (1967) 619. R. CHURCH AND 13. J. MCCARTHY, Proc. Natl. Acad. Sci. U.S., 58 (1967) 1548. S. PENMAN, J. Mol. Biol., 17 (1966) 117. R. W. SHEARER AND ]3. J. McCARTHY, Biochemistry, 6 (1967) 283. J. R. ~VARNER, M. GIRARD, H. LATHAM AND J. E. DARNELL, J. Mol. Biol., 19 (1966) 373. P. S. COHEN AND E. L. ENNIS, Biochim. Biophys. Acta, 145 (1967) 300. S. CHAUDHURI AND I. LIEBERMAN, J. Biol. Chem., 243 (1968) 29. S. T. JACOB, E. SAJDEL AND H. N. MUNRO, European J. Biochem., 7 (1969) 449. M. WILLEMS, M. PENMAN AND S. PENMAN, J. Cell Biol., 41 (1969) 177. R. P. PERRY, Natl. Cancer Inst. Monograph, 14 (1964) 73R. E. KOHLER, E. Z. RON AND ]3. D. DAVIS, J. Mol. Biol., 36 (1968) 71 . ]3. L. M. HOGAN AND A. I~ORNER, Biochim. Biophys. Acta, 169 (1968) 129. A. R. SUBRAMANIAN, E. Z. RON AND D. DAVIS, Proc. Natl. Acad. Sci. U.S., 61 (1968) 761. ]3. S. BALIGA, A. W. PRONCZUK AND H. N. MUNRO, ft. Mol. Biol., 34 (1968) 199" ]3. COLOMBO, C. VESCO AND C. BAGLIONI, Proc. Natl. Acad. Sci. U.S., 61 (1968) 651. R. SOEIRO, M. H. VAUGHAN AND J. E. DARNELL, J. Cell Biol., 36 (1968) 91. S.-VV. KWAN, T. E. WEBB AND H. P. MORRIS, Biochem. J., lO9 (1968) 617.
Biochim. Biophys. Acta, 195 (1969) lO9-122
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96329
RIBOSOME FORMATION IN RAT LIVER: TRANSCRIPTIONAL CONTROL
EVIDENCE FOR POST-
A R T U R O J. RIZZO AND THOMAS E. W E B B
McGill University Cancer Research Unit, Mclntyre Medical Sciences Building, Montreal zo9, Quebec (Canada) (Received J u n e I6th, 1969)
SUMMARY
An investigation of the amount of label incorporated from [6-~*C]orotic acid into the nuclear and cytoplasmic ribosomal RNA of non-proliferating rat liver, during a I-h treatment with several inhibitors of protein biosynthesis, gave the following results: I. The incorporation into nuclear 45-S, 28-S and I8-S RNA of liver was increased during a i-h treatment with anisomycin and cycloheximide by 5° %, and with puromycin by 25 %; 8-azaguanine showed little effect. 2. The appearance of label in newly synthesized ribosomes increased approx. 25 % after cycloheximide or anisomycin treatment; in contrast, this parameter was reduced to 25 % that of the controls after puromycin treatment, and to 50 % that of the controls after 8-azaguanine treatment. This differential effect of the inhibitors on ribosome transport was also observed in the slow growing Hepatoma 7800. 3. The size of the ribosomal subunit (60 S and 40 S) pools were not significantly altered by the inhibitors, even though puromycin and 8-azaguanine caused extensive conversion of the polyribosomes to monomers and dimers. The results suggest that the differential effect of the inhibitors on ribosome formation and polyribosome structure are related. They further suggest that this feed-back control, which may involve monomeric ribosomes, acts at a post-transcriptional step. There appears to be a pool of ribosomal proteins in non-proliferating rat liver and in the slow growing hepatoma which is sufficient to support ribosome synthesis for at least I h.
INTRODUCTION
In a previous study 1, evidence was presented that the rate of transport of newly synthesized ribosomes to the cytoplasm and the size of the pool of monomeric ribosomes were coupled in the rat liver system. It was proposed that such a relationship has a bearing on the problem of the control of ribosome formation, e.g. the monomeric ribosomes may be involved in the control of ribosome synthesis through a mechanism involving negative feedback. This possibility was further supported by the finding that inhibitors of protein biosynthesis which cause an increase in the size of the monomer pool (e.g. puromycin) reduced the incorporation of [6-14C]orotic Biochim. Biophys. Acta, 195 (1969) lO9-122
IiO
A . J . RIZZO, T. E. WEBB
acid into cytoplasmic ribosomes of regenerating rat liver to a greater extent than those which maintain the polyribosome structure (e.g. cycloheximide). The monomeric ribosomes have also been implicated in the control of RNA synthesis in bacterial cells ~,3. The latter studies showed that starvation of a stringent strain of Escherichia coli for an amino acid caused extensive polyribosome breakdown and an inhibition oi RNA synthesis; however, polyribosomes were maintained and RNA synthesis continued in a relaxed strain. It was not established in the previous investigation 1 whether the rate of labelling of the cytoplasmic ribosomal RNA was a function of the rate of transcription of the ribosomal RNA, a function of the rate of utilization (e.g. transport to the cytoplasm) of newly synthesized ribosomal RNA, or both. The results of the present investigation, which represents a study of the differential effects of various inhibitors of protein biosynthesis on ribosomal RNA synthesis and ribosome formation, support the hypothesis that the rate of ribosome synthesis in non-proliferating rat liver is controlled, at least in part, at the level of utilization or transport of the RNA. They also provide further support for the possibility that monomeric ribosomes, but not ribosomal proteins, are involved in this post-transcriptional control. MATERIALS AND METHODS
Materials Male Sprague-Dawley rats weighing 35o-4oo g were maintained in plastic cages on a Purina chow diet and were fasted in wire bottom cages for 16 h prior to use. The Morris H e p a t o m a 7800 was carried* intramuscularly in rats of the Buffalo strain. Lighting was from 6 a.m. to 6 p.m.; the experiments were routinely started at i o - i i a.m. to minimize variations due to diurnal rhythms. The livers and tumors were removed under light ether anaesthesia. [6-1*C~Orotic acid hydrate (specific activity 4.8 mC/mmole), L-[I-laClleucine (specific activity 20 mC/mmole) and DL-E3-1*C~tryptophan (specific activity IO mC/ mmole) were purchased from the New England Nuclear Corp., Boston. Cycloheximide, puromycin and 8-azaguanine were purchased from the Nutritional Biochemicals Corporation Cleveland. Anisomycin was a gift from Chas. Pfizer and Co., Conn. The isotopes and drugs were administered intraperitoneally.
Size distribution analysis o/ribosomal components The relative concentration of the monomeric ribosomes and polyribosomes were determined as previously described 1,5. A portion (3 g) of the liver from control and treated rats was used for the size distribution analysis of the ribosomal components and for the preparation of the ribosome fraction; the purified nuclei were prepared from the remainder of the liver (see below). Immediately prior to removal, the liver was perfused with IO ml of cold 0.25 M sucrose in 5 ° mM Tris-25 mM KC15 mM MgCI~ buffer p H 7.5 (Tris-KC1-MgCl~-buffer). After treating the post-mitochondrial supernatant (IO rain, 13 ooo ×g) with deoxycholate, it was diluted with an equal volume of buffer and o.7-ml aliquots were analyzed on lO-35 % sucrose gradients as before 1. For the separation and estimation of the relative concentrations of the ribosomal monomers and ribosomal subunits, the deoxycholate-treated post-mitochonBiochim. Biophys. Act*, 195 (1969) lO9-122
RIBOSOME FORMATION IN RAT L I V E R
III
drial supernatant was diluted as before, then layered over exponential 7-47 % sucrose gradients which were centrifuged at 5 ° ooo x g av. for 18. 5 h in a SW 25.1 rotor of a Beckman centrifuge. The gradients were analyzed as described previously 1.
Preparation of ribosomal fraction Approx. 4 ml of the deoxycholate-treated post-mitochondrial supernatant prepared for the size distribution analysis, was centrifuged at lO5 ooo x g for I h to obtain a ribosome pellet; the ribosomes were resuspended in aqueous medium and the RNA was extracted as outlined below.
Preparation o/ nuclei The purified nuclei were prepared according to MURAMATSU AND BUSCH~ with minor modifications. Briefly, a portion of the liver (5~6 g) was finely minced with scissors, then suspended in 2.4 M sucrose (1:15, w/v) containing 3-3 mM calcium acetate and homogenized with 4 strokes of a teflon pestle. The clearance between the pestle and the wall of the glass P o t t e r - E l v e h j e m homogenizer was 0.02 mm; the pestle rotated at 800 rev./min. Under our conditions a tighter pestle rotating at the same speed caused extensive breakdown of the 45 S RNA. The homogenate was filtered through cheesecloth and centrifuged at 40 ooo x g for I h, then the nuclear pellet was resuspended in I.O M sucrose-I.O mM calcium acetate and re-centrifuged at 3000 x g for 5 rain to give a pellet of purified nuclei.
Extraction o / R N A The purified nuclei and ribosomal pellets were resuspended in o.14 M NaC10.05 M sodium acetate buffer, p H 5.0, containing 0.3 % sodium dodecyl sulfate (1. 5 ml/g of liver). The suspension was treated with an equal volume of redistilled aq. phenol at 65 and 25 ° according to the procedure of MURAMATSU AND BUSCH~. The RNA was precipitated from the reextracted aqueous phases b y the addition of 2.5 vol. of 95 % ethanol containing 2 % potassium acetate at --15 °. The RNA was dissolved in a small volume of 0.02 M sodium acetate, p H 5.0.
Separation o/ RNA on density gradients Aliquots of the purified RNA (approx. 2oo fig in o. 4 ml) were layered over linear sucrose (lO-4O %) gradients e containing o.I M NaC1, 0.02 M sodium acetate and o.ooi M E D T A (pH 5.0). The gradients were centrifuged for 16 h at 63 ooo x g av. in a SW 25.1 rotor of a Beckman centrifuge. Ribonuclease-free sucrose (Mann Research Corporation) was used in the preparation of the sucrose density gradients. Following the separation of the various species of RNA according to size, the effluent from the bottom of the gradient tube was monitored at 260 m# in a flow cell. A drop counter activated an event marker on the recorder to register each milliliter of effluent. To determine the radioactivity in each fraction the effluent was collected in pre-cooled centrifuge tubes, then the acid-insoluble material was precipitated with 5 % trichloroacetic acid, using serum albumin as co-precipitant. The precipitate was dissolved in solubilizer (NCS, Nuclear Chicago) then counted in liquid scintillant at an efficiency of 80 %. The specific radioactivity of the various RNA species was calculated directly from the absorbance and radioactivity profiles using the conversion factor 20 abBiochim. Biophys. Acta, 195 (1969) l O 9 - 1 2 2
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A . J . RIZZO, T. E. WEBB
sorbance units = I m g of RNA. The absorbance of a blank gradient was subtracted from each of the absorbance and radioactivity profiles.
Incorporation o/amino acids into soluble proteins A mixture of L-[/J4C]leucine (I2~C) and DL-[3-14C]tryptophan (I2/~C) was administered to 400 g control or treated rats 15 min prior to removal of the livers. The latter were homogenized in 0.25 M sucrose in Tris-KC1-MgC12 buffer (I.O g/ 2.0 ml) and the homogenate was centrifuged at lO5 ooo ×g for I h. The proteins of the postmicrosomal supernatant were precipitated with 5 % trichlororacetic acid at 4 °. The precipitates were extracted consecutively with 95 % ethanol and ethanolether (i:I,V/V) at 4 o°. The residue was suspended in solubilizer and counted in liquid scintillant. Protein was determined in duplicate samples using biuret reagent 7.
RESULTS
E//ectiveness o/inhibitors o/protein biosynthesis Much of the work to be reported involves a comparison of the differential effects of various inhibitors of protein biosynthesis on ribosomal RNA synthesis and transport to the cytoplasm. It was therefore necessary to confirm that the dosages employed effectively inhibited protein biosynthesis over the period of time during which measurements were made. The effect of three inhibitors of protein biosynthesis in rat liver was tested and the results are shown in Table I. Anisomycin s TABLE I EFFECT
OF A N I S O M Y C I N , C Y C L O H E X I M I D E
AND PUROMYCIN ON PROTEIN BIOSYNTHESIS
R a t s weighing 400 g were injected w i t h 20 m g / k g of anisomycin, I m g / k g of cycloheximide, or i o o m g / k g of p u r o m y c i n and the livers were r e m o v e d 30 or 60 m i n later. 15 m i n prior to the rem o v a l of the liver; a m i x t u r e of L-[i--!*C]leucine (12/~C) and [3--14C]tryptophan (i2/zC) was administered. The specific radioactivity of the soluble p r o t e i n s was determined as described u n d e r MATERIALS AND METHODS.
Inhibitor
Anisomycin Cycloheximide Puromycin
Inhibition (%)during the interval* I5-3o rain
3o-6o rain
85 9o 95
6o-85 94 96
* The time i n t e r v a l over which the soluble p r o t e i n s were labelled, m e a s u r e d from the time of a d m i n i s t r a t i o n of the inhibitor.
(2o mg/kg), cycloheximide9 (I mg/kg) and puromycin 1° (IOO mg/kg) inhibited protein biosynthesis by 85-95 % within 15 min of administration. This level of inhibition was maintained for at least an additional 45 min by cycloheximide and puromycin. In the case of anisomycin, however, the level of inhibition varied from 60 to 85 % in four separate experiments during the interval of 45-60 min. During the time interval 45-60 rain after the administration of anisomycin, cycloheximide or puromycin at the dosages outlined above, protein biosynthesis in Hepatoma 7800 was inhibited by 7 ° , 85 and 88 % respectively. Biochim. Biophys. Acta, 195 (1969) lO9-122
113
RIBOSOME FORMATION IN RAT LIVER
EHect o/ inhibitors on polyribosomes, monomers and subunits Further attempts were made to establish the inverse relationship between the size of the inactive monomer pool and the rate of transport of newly synthesized ribosomes to the cytoplasm. In the present series of experiments the duration of treatment with the inhibitors of protein biosynthesis was I h. Shown in Fig. I a is the effect of anisomycin on the size of the monomer and polyribosome pools. The results indicate that there is only a slight shift of the polyribosome size distribution to the lighter species with no significant change in the size of the monomer pool as compared to the controls. The size distribution of the ribosomal components prepared from the liver of rats treated with cycloheximide were similar to those of anisomycin-treated rats. However, as shown in earlier studies 5,9, marked polyribosome breakdown, with consequent increases in the inactive monomer and dimer pools, occurs in rat liver following the administration of 8-azaguanine or puromycin (Fig. Ib). It should be noted that only one-half the amount of post-mitochondrial supernatant was layered on the gradient in the case of the puromycin-treated rat. I.I I.C 0.9
0.6 i
i!
0.7
0.7 I ! i f
0.6
fi i
0.6
~ o.~
0.~
0.4 0.3
i
°'l
i i i
~0.4 j
1/"
l
\',
,.
"~, x
i/ t
t
0.3 Z
05'
~ 0.2
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,,
sJ
oJ 5
FRACTION NUMBER
~0 FRACTION
J5
20
25
NUMBER
Fig. I(a) Size d i s t r i b u t i o n profiles of the p o l y r i b o s o m e s and the relative size or the m o n o m e r pool in the deoxycholate-treated s u p e r n a t a n t (0. 7 ml) p r e p a r e d from the liver of a control r a t ( - - ) a n d of a r a t w h i c h w a s t r e a t e d for I h w i t h 20 m g / k g of anisomycin (. . . . ). The m o n o m e r p e a k is centered a t F r a c t i o n 2o and the t o p of the g r a d i e n t is to the f i g h t of the figure. (b) Similar p a t t e r n s of ribosomal c o m p o n e n t s in o. 7 ml of deoxycholate-treated s u p e r n a t a n t p r e p a r e d f r o m the liver of a r a t t r e a t e d w i t h IOO m g / k g of 8-azaguanine for 4 h (--), or in o.35 nil of deoxycholatet r e a t e d s u p e r n a t a n t p r e p a r e d f r o m the liver of a r a t t r e a t e d w i t h i o o m g / k g of p u r o m y c i n for I h ( . . . . ). Fig. 2. Size d i s t r i b u t i o n profile s h o w i n g the relative c o n c e n t r a t i o n of ribosomal m o n o m e r s a n d s u b u n i t s in the liver from control ( - - ) a n d a n i s o m y c i n - t r e a t e d (. . . . ) rats. The m o n o m e r p e a k a n d the 6o-S and 4o-S s u b - u n i t peaks are centered a t F r a c t i o n s 12, 15 and 2o, respectively.
The relative concentrations of the monomeric ribosomes and the 6o-S and 4o-S ribosomal sub-units in control and anisomycin-treated rats are shown in Fig. 2. Neither the monomer pool, nor the subunit pools appear to be affected by the drug. It is concluded that anisomycin, the action of which has not been previously studied Biochim. Biophys. Acta, 195 (1969) lO9-122
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A . J . RIZZO, T. E. WEBB
in the rat liver system, does not affect the pool size of the various ribosomal components. The effects of the various inhibitors of protein biosynthesis on the size of the monomer and subunit pools are tabulated in Table II. Since the dimers, in particular
TABLE II EFFECT OF ANISOMYCIN, CYCLOHEXlMIDE AND PUROMYCIN ON THE SIZE OF THE RIBOSOMALMONOMER AND SUBUNIT POOLS R a t s weighing 25o-3oo g were injected w i t h 20 m g / k g of anisomycin, I m g / k g of cycloheximide, or IOO m g / k g of p u r o m y c i n and the livers were r e m o v e d I h later. The polyribosomes, m o n o m e r s a n d s u b u n i t s p r e s e n t in the deoxycholate t r e a t e d p o s t - m i t o c h o n d r i a l s u p e r n a t a n t were separated on d e n s i t y gradients. The relative heights (A 2e0 mr*) of the peaks of these c o m p o n e n t s in the size d i s t r i b u t i o n profiles are recorded; the combined heights of the m o n o m e r and dimer peaks are also given in parentheses. Inhibitor
Control Anisomycin Cycloheximide Puromycin 8-Azaguanine
Monomer
0.52 0.55 0.62 2.2 0.70
(Monomer+dimer)
(0.80) (0.85) (0.90) (3.2) (1.2)
Subunits 6o s
4° s
0.35 0.33 0.32 0.38 0.34
o. i8 o. 15 o. 15 0.20 o.16
those derived from polyribosome breakdown in vivo, are presumed to belong to the monomer pool in vivo 11, the combined heights of the monomer and dimer pool are given in parentheses. As in the case of anisomycin, cycloheximide has little effect on the monomer or subunit pools. Although puromycin causes the conversion of a substantial portion of the polyribosomes to monomers and dimers, only a slight increase in the subunit pools is observed. The subunit pools were corrected for a portion of the 6o-S subunits which tended to aggregate with the monomers at high concentrations of the latter. This aggregate sediments as a peak on the receding edge of the dimer peak in the size distribution profiles 5. It is concluded that the subunit pools remain relatively constant following the inhibition of protein biosynthesis, with or without concommittant polyribosome breakdown.
E//ect o/inhibitors on R N A synthesis Typical absorbance and radioactivity profiles of nuclear and cytoplasmic RNA prepared from the livers of rats injected with saline (controls), or with one of several inhibitors of protein biosynthesis, are shown in Fig. 3 and Fig. 4. In these experiments !6-14Clorotic acid was injected into the rat 5 rain after the administration of saline or the inhibitor and the liver was removed I h later. The sedimentation constants assigned to the various species of RNA, are those which have been established under the conditions employed 6. Shown in Fig. 3 are the absorbance and radioactivity patterns of nuclear RNA from the liver of (a) control, (b) anisomycin-treated, (c) cycloheximide-treated and (d) puromycin-treated rats. The pattern of labelling was similar in the livers from all animals; however, the extent of labelling was slightly greater in the 45-S, I8-S Biochim. Biophys. Acta, I95 (I969) IO9-I22
RIBOSOME FORMATION IN RAT LIVER
115
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Fig. 3. A b s o r b a n c e ( - - ) a n d r a d i o a c t i v i t y (. . . . ) profiles of n u c l e a r 1RNA e x t r a c t e d f r o m t h e livers of r a t s w h i c h received [6-14C]orotic acid (32/zC/4oo g) 5 rain a f t e r t h e a d m i n i s t r a t i o n of (a) saline (control), (b) a n i s o m y c i n (20 m g / k g ) , (c) c y c l o h e x i m i d e (i m g / k g ) , or (d) p u r o m y c i n (IOO m g i k g ) . T h e labelling period w a s I h. T h e 45-S, 28-S, I8-S, a n d 7-S species are c e n t e r e d a t F r a c t i o n s 9, 14, 19 a n d 25, respectively.
and 28-S species of nuclear RNA from the cycloheximide and anisomycin-treated rats as compared to the corresponding species in the control and puromycin-treated animals. The corresponding absorbance and radioactivity profiles of the cytoplasmic ribosomal RNA are shown in Fig. 4. The results indicate that the specific activities Biochirn. Biophys. Acta, 195 (1969) lO9-122
116
A. j . RIZZO, T. E. WEBB
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Fig. 4. A b s o r b a n c e ( - - ) a n d r a d i o a c t i v i t y (. . . . ) profiles of c y t o p l a s m i c R N A e x t r a c t e d f r o m t h e livers of r a t s w h i c h were t r e a t e d w i t h (a) saline (control), (b) a n i s o m y c i n , (c) cyclohexirnide a n d (d) p u r o m y c i n as for Fig. 3. T h e 28-S a n d I8-S species are c e n t e r e d a t F r a c t i o n s 16 a n d 20, respectively.
of the I8-S and 28-S cytoplasmic ribosomal RNA's are markedly higher in the livers from (a) control, (b) anisomycin and (c) cycloheximide-treated animals, than from the (d) puromycin-treated rats. The effect of 8-azaguanine on the labelling pattern of nuclear and cytoplasmic ribosomal RNA was also tested. In this experiment the [6-z4C]orotic acid was adminisBiochim. Biophys. Mcta, 19,5 (1969) lO9-122
117
RIBOSOME FORMATION IN RAT LIVER
tered 4 h after the administration of the base analog and I h before removal of the liver. As shown previously 5,x3 polyribosome breakdown is maximal during this period. The inhibition of protein biosynthesis which appears to parallel polyribosome breakdown, is approx. 60 ~'o at 4.5 h after the administration of the base analog (S.-W. KWAN AND T. E. WEBB, unpublished observations). The absorbance and radioactivity profiles of the nuclear RNA (Fig. 5a) are very similar to that of the control rat; however, the corresponding labelling pattern of the cytoplasmic RNA (Fig. 5b) resembles that from the puromycin-treated rats. 1.5 1.2
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Fig. 5. A b s o r b a n c e ( - - ) a n d r a d i o a c t i v i t y (. . . . ) profiles of (a) n u c l e a r a n d (b) c y t o p l a s m i c R N A e x t r a c t e d f r o m t h e liver of a r a t w h i c h received E6-zaC]orotic acid (32 # C / 4 o o g) 4 h a f t e r t h e a d m i n i s t r a t i o n of i o o m g / k g of 8 - a z a g u a n i n e . T h e labelling period w a s i h.
The specific activities of the various species of nuclear and cytoplasmic ribosomal RNA estimated from a number of size distribution and radioactivity profiles similar to those shown in Figs. 3 and 4 are tabulated in Table III. The results indicate that the labelling of the 45-S ribosomal RNA is increased 25-50 % above that of the controls after treatment with anyone of the inhibitors, with the exception of 8-azaguanine which caused little change. The specific activity of the I8-S and 28-S nuclear RNA is correspondingly increased after treatment of the rats with anisomycin, cycloheximide or puromycin; this suggests that the 45-S precursor molecule does not accumulate after drug treatment, but is converted to its products with the same efficiency as in the control livers. Despite the increased labelling of the nuclear ribosomal RNA in puromycintreated rats, the specific activity of the corresponding cytoplasmic ribosomal RNA was only 25 % that of the controls. In contrast, the specific activities of these species in the anisomycin or cycloheximide-treated rats were, in general, 25-4 ° % higher Biochim. Biophys. Acta, 195 (1969) lO9-122
118
A . J . RIZZO, T. E. WEBB
TABLE III EFFECT OF INHIBITORS OF THE INCORPORATION AND CYTOPLASMIC RNA
OF LABEL
FROM
[6-14C]OROTIC
ACID INTO NUCLEAR
The rats received [6--x4C]orotic acid (32/zC]4oo g) 5 min after the administration of anisomycin (20 mg/kg), cycloheximide (I.O mg/kg), or puromycin (ioo mg/kg), or 4 h after the administration of 8-azaguanine (ioo mg/kg per dose) in 2 doses 3 ° rain apart. The livers were removed I h later and the nuclear and cytoplasmic RNA was extracted.The specific radioactivities of the various species were estimated from the central region of each peak in the absorbance and radioactivity profiles. The number of rats used in each experiment is shown in parentheses. Treatment
Specific radioactivity (counts~rain per i~g R N A )* Nuclear R N A
Control Anisomycin Cycloheximide Puromycin 8 azaguanine
(4) (2) (2) (2) (I)
Cytoplasmic R N A
45 S
28 S
18 S
28 S
I8 S
880 13oo 132o ilOO 800
34 ° 55 ° 580 480 300
33 ° 560 57 ° 520 280
2.8 3-5 4 .0 o. 7 1.2
3.6 5 .0 3.5 0.8 2. 4
* The standard error was in all cases less than IO % of the mean.
t h a n t h a t of t h e c o n t r o l s . A l t h o u g h t h e s p e c i f i c a c t i v i t i e s of t h e v a r i o u s s p e c i e s of nuclear RNA were essentially unaffected by 8-azaguanine treatment, the appearance of n e w r i b o s o m a l R N A in t h e c y t o p l a s m w a s m a r k e d l y i n h i b i t e d . T h e d a t a in T a b l e I I I also c o n f i r m t h a t in c o n t r a s t t o a n i s o m y c i n a n d c y c l o h e x i m i d e , p u r o m y c i n a n d 8a z a g u a n i n e ( w h i c h a p p e a r t o i n h i b i t t h e t r a n s p o r t of r i b o s o m a l R N A t o t h e c y t o p l a s m ) c a u s e a m a r k e d i n c r e a s e in t h e c o n c e n t r a t i o n of i n a c t i v e m o n o m e r s ( a n d d i m e r s ) . T h e d i f f e r e n t i a l e f f e c t of s o m e i n h i b i t o r s of p r o t e i n b i o s y n t h e s i s o n t h e t r a n s p o r t of n e w l y f o r m e d r i b o s o m a l R N A t o t h e c y t o p l a s m w a s also t e s t e d u s i n g t h e r a t h e p a t o m a s y s t e m . T h e s p e c i f i c a c t i v i t i e s of t h e I 8 - S a n d 28-S r i b o s o m a l R N A i s o l a t e d f r o m t h e c y t o p l a s m of H e p a t o m a 7800 a r e r e c o r d e d in T a b l e IV. (The s p e c i f i c a c t i v i t y
"FABLE IV EFFECT OF INHIBITORS ON THE FORMATION OF RIBOSOMES IN HEPATOMA 7800 Rats bearing Hepatoma 7800 received [6-14C]orotic acid (4°/zC) 0. 5 h before the administration of 0.9 % saline (controls), 20 mg/kg of anisomycin, i mg/kg of cycloheximide, or ioo mg/kg of puromycin; the tumors were removed and processed 1. 5 h after the administration of the labelled precursor. The RNA was extracted from the cytoplasmic ribosomes and the I8-S and 28-S species were separated on sucrose gradients. The specific radioactivities were estimated from the center of the peaks in the combined absorbance-radioactivity profiles. Inhibitor
Control Anisomycin Cycloheximide Puromycin
Height o[ monomer ( +dimer) * peak (A26o mll)
Speci[ic activity (counts/rain I~g per R N A ) 18 S
28 S
0.68 0.64 0.49 1.5o
2.8 2.2 3-4 1. 4
1.2 1.2 1.9 0.6
(i.22) (1.18) (0.85) (2. 7 )
* Refers to combined height of monomer and dimer peaks as for Table II. Biochim. Biophys. $cta, 195 (1969) lO9-122
RIBOSOME FORMATION IN RAT L I V E R
119
of the nuclear RNA was not determined.) In this series of experiments the [6-14C2orotic acid (4 ° #C/25o g) was administered 30 rain before the inhibitors since the incorporation of orotic acid into ribosomal RNA was much lower in the hepatoma as compared to liver. However, orotic acid was superior to either cytosine or uracil as a RNA precursor in this hepatoma. The tumors were removed 1.5 h after the administration of [6-1~Cjorotic acid. Since the inhibitors were injected 30 min after the isotope, any effects observed probably result from a modified rate of transport rather than a modified rate of ribosomal RNA synthesis. The results show that in neoplastic liver anisomycin and cycloheximide have only slight effects on the labelling of cytoplasmic ribosomal RNA; in contrast, puromycin inhibits the appearance of newly formed ribosomes by approx. 50 %. As in normal liver, H e p a t o m a 7800 appears to possess a pool of ribosomal proteins sufficient to permit normal ribosome synthesis over a period of I h.
DISCUSSION
The nucleus of eukaryotic cells contains most of the DNA and is the site of synthesis of a variety of RNA molecules. Most of the latter are transferred to the cytoplasm where they participate in protein biosynthesis. Evidence from a number of sources 1~-18 suggests that the transport is selective, and that a portion of the RNA transcribed in the nucleus is also degraded. In view of this evidence which suggests that there is selective control over the types of RNA which m a y leave the nucleus, the question m a y be asked whether there is control over the amount of a n y one species which is transported to the cytoplasm. In earlier experiments l, it was shown that the reduction in the incorporation of radioactive label into the RNA of cytoplasmic ribosomes with increasing time of liver regeneration was directly proportional to the size of the inactive monomeric ribosome pool. Furthermore, evidence was obtained that inhibitors of protein biosynthesis which cause the breakdown of polyribosomes to monomers and dimers inhibit ribosome formation in regenerating liver to a greater extent than inhibitors which maintain the polyribosome structure. It was not established whether the former class of inhibitors affected RNA synthesis directly, or whether the inhibition resulted from a depletion of ribosomal proteins or interference with the transport of newly synthesized ribosomal RNA to the cytoplasm in the form of subunits. However, the observation 1 that ribosome formation was inhibited only 40 % in the 42-h regenerating liver during a 2-h treatment with cycloheximide, while under similar conditions puromycin caused a 65 °/o inhibition, together with the findings of other laboratories 19-21 strongly suggests that factors other than ribosomal proteins are involved in the control of ribosome synthesis. In the present investigation a I-h labelling period was selected as a compromise, since according to the experimental procedure, a longer labelling period necessitates a longer treatment with the inhibitors of protein biosynthesis. Since the results of an earlier study 1 indicated that cycloheximide inhibited ribosome synthesis in regenerating liver within 2 h, it was anticipated that extended periods of near total inhibition of protein biosynthesis would seriously complicate the interpretation of the data in the present study. Inhibitory effects observed only after extended treatBiochim. Biophys. Acta, 195 (1969) l O 9 - 1 2 2
12o
A . J . RIZZO, T. E. WEBB
merit m a y be due to depletion of ribosomal protein pools, decreased RNA polymerase activity or non-specific effects due to depletion of other cellular enzymes. On the other hand, a labelling period significantly less than I h would not permit measurable amounts of newly synthesized ribosomal RNA to enter the cytoplasm. It is also important to note that the dosages of the inhibitors used were the minimal amounts required to achieve approximately their maximal effects, and thus to avoid excessive toxicity. This factor m a y be important, since in the present investigation cycloheximide was administered at a dosage of I mg/kg and no inhibition of ribosomal RNA biosynthesis was observed over a period of I h. However, when the cycloheximide dosage employed is 5o-fold higher, a significant inhibition of ribosome synthesis in rat liver is observed 2j, although it is also clear from these data that the ribosomal proteins are not limiting under these conditions. The results of the present study indicate that the rate of incorporation of label from I6-1*C~orotic acid into nuclear RNA during a I-h treatment with several inhibitors of protein biosynthesis is either increased, or remains unchanged as compared to the controls. Part of the increase observed m a y be due to increased production of corticosteroids in response to stress, since an increased synthesis of 45-S RNA after hydrocortisone treatment has been observed in the livers of adrenalectomized rats 22. In view of these results any decrease in the rate of appearance of labelled RNA in the cytoplasm in response to any of these inhibitors cannot be due to a decreased labelling of nuclear precursors, due in turn to an increase in the size of nucleotide pools, or to a decreased rate of RNA biosynthesis. Although cycloheximide caused an almost complete inhibition of protein biosynthesis within 5 rain after administration, it did not decrease the transport of ribosomal RNA to the cytoplasm during the I-h labelling period. Anisomycin s, which has a mechanism of action similar to that of cycloheximide 9 produced a similar effect on the transport of RNA to the cytoplasm, although the inhibition of protein biosynthesis was variable during the interval 45-60 rain (c/. Table I). The results suggest that the ribosomal proteins are present in excess, since a fraction of the nuclear RNA synthesized over and above the control level after ahnost total inhibition of protein synthesis with cycloheximide and anisomyein, was transported to the cytoplasm in the form of ribosomal subunits. It would appear that the pool of ribosomal protein is limiting in regenerating liver, since ribosomes are synthesized at only 60 % of the control level after cycloheximide treatment in the 42-h regenerating liver 1. This phenomenon could be a characteristic of proliferating tissue in general, since a decreased synthesis of ribosomal RNA was recently observed in H e L a cells after cycloheximide treatment 23. In contrast to cycloheximide and anisomycin which maintain the structure of the polyribosome, puromycin 1° and 8-azaguanine 5,1~, which cause the conversion of a portion of the polyribosomes to inactive monomers (and dimers) produced a marked inhibition of the appearance of newly formed ribosomal RNA in the cytoplasm. The mechanism by which 8-azaguanine induces polyribosome breakdown is not firmly established, although it appears to act through the formation of defective messenger RNA 5,~2. Puromycin induces polyribosome breakdown b y accelerating translation and b y causing the premature release of ribosomes from the messenger RNA 1°. It seems probable that the inhibition of ribosome formation by puromycin or 8-azaguanine, two drugs which have quite different mechanisms of action, is related to the ability of both to induce polyribosome breakdown. As in an earlier study Biochim. Biophys. Acta, 195 (1969) lO9-122
RIBOSOME FORMATION IN RAT LIVER
121
employing H e L a cells 24, the present results indicate that there is a normal formation of 45-S precursor RNA in the presence of 8-azaguanine, although the appearance of newly formed ribosomal RNA in the cytoplasm is markedly inhibited. However, the present data also indicate that the initial processing in the nucleus of the precursor RNA to I8-S and 28-S RNA is also normal and therefore do not support the suggestion ~ that the inhibition of ribosome formation is due to an inability of the cell to process 45-S RNA which contains the base analog. However, the possibility that the presence of 8-azaguanine in the ribosomal RNA interferes with later steps in ribosome assembly cannot be ruled out. Both 8-azaguanine and puromycin also cause an increase in the dimer pool in addition to the monomer pool. Since these dimers are inactive in vivo 4 and since there is a tendency for monomeric ribosomes to dimerize in vitro in extracts from rat liver 11, it seems probable that the dimers should be included in the monomer pool. The results of this study support the hypothesis that the inactive ribosomal components are involved in the control of ribosome synthesis and that this control is exerted, at least in part, post-transcriptionally. Similar studies, which do not involve the use of inhibitors, on the transitional increase in RNA synthesis which occurs after partial hepatectomy, also suggest that at least a portion of the control of ribosome biosynthesis operates at this level (A. J. Rlzzo ANn I. E. W E B B , unpublished observations). In view of the finding that the ribosomal subunit pools are not affected b y puromycin, it would appear that the monomeric ribosomes are involved in the postulated feed-back control. Subunit pools in bacteria and mouse ascites tumor ceils also remain essentially constant during a number of treatments which affect the monomer pool ~5,26. A dissociation factor similar to the protein factor of E. coli which has been shown to be present in limiting concentrations and to cause the dissociation of 7o-S ribosomes into subunits ~7, appears to be operative in mammalian cells ~s,~. An inhibition of ribosome synthesis b y puromycin has also been observed in H e L a cells3°. This inhibition was attributed to interference of puromycin peptides with ribosome maturation. However, in the absence of any correlation between the production of such peptides and an inhibition of protein biosynthesis and in view of the apparent normal processing of nuclear ribosomal RNA shown in the present study, it is unlikely that the decreased transport of newly synthesized ribosomes to the cytoplasm after puromycin treatment is due to an interference with normal processing of the 45-S precursor. The results obtained with the neoplastic liver (Hepatoma 7800) were qualitatively similar to those of normal liver. However, if monomeric ribosomes are involved in the control of ribosome synthesis, this regulation m a y be modified, since tumors invariably contain high concentrations of inactive ribosomes 2~,~1, at least under conditions where the growth rate is relatively slow. In summary, the results suggest that ribosome synthesis is controlled, at least in part, after gene transcription in resting (non-proliferating) tissue. Furthermore, in agreement with evidence from the bacterial system s,a, there appears to be a relationship between the rate of transport of ribosomal RNA to the cytoplasm and the concentration of inactive ribosomes in the cytoplasm. Non-proliferating or slow growing tissues appear to contain pools of ribosomal proteins sufficient to permit continued ribosome synthesis for at least I h. Biochim. Biophys. Acta, 195 fI969) io9-122
122
A . J . RIZZO, T. E. WEBB
ACKNOWLEDGEMENTS
This work was supported by Grant MA 1949 from the Medical Research Council of Canada and by a grant from the National Cancer Institute of Canada. One of us (A.J.R.) is the holder of a Fellowship from the National Cancer Institute of Canada. The authors are grateful to Dr. H. P. Morris, Howard University, Washington, D.C., for supplying the tumor-bearing rats used in this study. REFERENCES i 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3° 31
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Biochim. Biophys. Acta, 195 (1969) lO9-122