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Studies on the metabolism of RNA of free and membrane-bound polysomes from rat liver
507
BIOCHIMICA ET BIOPHYSICA ACTA BBA 96642
S T U D I E S ON T H E METABOLISM OF RNA OF F R E E AND MEMBRANEBOUND POLYSOMES FROM R A T L I V E R TATSUO TANAKA, MASAMICHI TAKAGI* AND K I K U O OGATA
Department o[ Biochemistry, Niigata University School o/Medicine, Niigata (Japan) (Received May i9th, 197 o)
SUMMARY
I. Through the employment of the microsomal deoxycholate-soluble fraction from rat liver, conditions were found in which messenger RNA of the rat liver polysomes were degraded, while the ribosomal structural RNA remained intact. This method made it possible to compare the rate of labeling of ribosomal structural RNA and m R N A between free and membrane-bound polysomes from rat liver. 2. The rate of incorporation of [14C~orotic acid into messenger RNA of free polysomes was higher than that into messenger RNA of bound polysomes up to 4 h after an intraperitoneal iniection of L14Clorotic acid. 3. CsC1 equilibrium centrifugation of EDTA-treated polysomes from rat liver, labeled in vivo with I14C~orotic acid for 50 rain, also showed that the rate of incorporation into messenger RNA-containing particles 1,~ was higher in free polysomes t h a n in bound polysomes. 4. On the contrary, the rates of labeling of ribosomal structural RNA of these two classes of polysome were shown to be similar. These results are discussed in relation to the biosynthesis of different kinds of protein between two classes of liver polysome.
INTRODUCTION
Many reports 8-1°, including those of the authors 3,4, have been published showing that the membrane-bound polysomes synthesize serum proteins, and that the free polysomes synthesize non-serum liver proteins in the hepatic cell. The most reasonable explanation of the functional difference between these two classes of polysome may be that these particles have different species of messenger RNA. Therefore, it seems important to obtain direct evidence for such differences. Concerning the metabolic differences of RNA between these two classes of liver polysome, the data available are very few and seem to be contradictory n-l*. The contradictions m a y be the result of the complexity of polysomal RNA which includes ribosomal structural RNA (rRNA), messenger RNA (mRNA), transfer RNA (tRNA) and 5-S RNA. In short pulseAbbreviations: rRNA, ribosomal structural RNA; mRNA, messenger RNA; tRNA, transfer RNA. " Present address: Laboratory of Radiation Genetics, Faculty of Agriculture, University of Tokyo, Tokyo, Japan.
Biochim. Biophys. Acta, 224 (I97 o) 5o7-517
508
T. TANAKA et al.
labeling experiments, it is particularly difficult to distinguish mRNA from rRNA. In fact, in the above-mentioned papers n-l* they were not well differentiated. In the present experiments rRNA and mRNA of rat liver polysomes were distinguished by treatment of polysomes with the liver microsomal deoxycholatesoluble fraction containing mild ribonuclease activity, and the difference in the kinetics of the labeling in vivo of these RNA's between two classes of polysome was investigated. The results of CsC1 equilibrium centrifugation of EDTA-treated polysomes, pulse-labeled with lil*Clorotic acid in vivo, are also described.
MATERIALS AND METHODS
Chemicals E6-14C]Orotic acid (60.8/zC/#mole) and ~5-~HJorotic acid 6240 #C//~mole) were purchased from the Radiocbemical Centre, Amersham, England. Sodium deoxycholate was obtained from Difco Lab. Co., sodium dodecyl sulfate from Nakarai Chemicals Ltd., Kyoto, and bovine pancreatic ribonuclease (EC 2.7.7.16) from Worthington Biochemical Corp. Animals Rats of the Wistar strain weighing 13o-16o g were starved for about IS h before the experiments. !l*ClOrotic acid was administered by intraperitoneal injection. At the end of the time specified, the animals were killed by decapitation, and their livers were excised and chilled in ice-cold medium A (0.25 M sucrose, 5 mM MgCl~, 25 mM KCI, 50 mM Tris-HC1, pH 7.6). Fraclionalion o/liver cell The livers were weighed, minced and homogenized in 2.5 volumes of medium A in a glass Potter-Elvehjem type homogenizer with a loosely-fitted Teflon pestle with 8 strokes at Iooo rev./min. The postmitochondrial supernatant after centrifugation of the homogenate at io ooo ~
METABOLISM OF R N A
o r POLYSOMES
509
to all the solutions used throughout the procedures of polysomal preparation as described previously 3-5.
Treatment of polysomes with microsomal deoxyeholate-soluble fraction The microsomal pellet, prepared by centrifugation of the postmitochondrial supernatant from 5 g of rat liver at lO5 o o o × g for I h, was suspended in 2 ml of Medium A'. Deoxycholate was added to the microsomal suspension to give a final concentration of 1. 3 °/o, and centrifuged at 59 3 o o × g for I h. The upper half of the supernatant was used as a microsomal deoxycholate-soluble fraction. IOO #1 of the microsomal deoxycholate-soluble fraction containing about 2.5 mg of protein was added to I ml of the polysomal suspension. The mixture stood for 5 rain at o °, and was centrifuged at 59 3 o o × g for 9 ° min to sediment ribosomes. As the control, polysomes were subjected to the same procedures as described above, except that the microsomal deoxycholate-soluble fraction was not added before centrifugation. Recentrifugation and resuspension had little effect on the specific activity of the total polysomal RNA.
Sucrose density-gradient centri/ugation o] polysomal RNA and determination o/ radioactivity The ribosomal pellet thus obtained was suspended in I ml of 25 mM Tris-HC1 (pH 7.6) containing 1 % sodium dodecyl sulfate. The resulting solution was layered over 28 ml of 5-2o % linear sucrose density gradient containing 25 mM Tris-HC1 (pH 7.6), and spun in a RPS- 25A rotor of a Hitachi 65 P ultracentrifuge at 51 5o5 × g at 6 ° for 15 h. The centrifugal tube was then punctured at the bottom and the ultraviolet absorbance at 260 nm (A~e0 nm) of the effluent fraction of each 15 drops was measured by a Hitachi Perkin-Elmer model 139 spectrophotometer. From each fraction the acid-insoluble material was precipitated with 5 % trichloroacetic acid at o ° overnight, retained on a Millipore filter (pore size 0.45 #) and washed with 5 °/o trichloroacetic acid. The radioactivity was then determined by an automatic lowbackground gas-flow counter of Nihonmusen Ltd. (background counts of about 1. 5 counts/rain). In the double-labeling experiments with I14C~- and I3H]orotic acid, the acidinsoluble material of each fraction from density-gradient centrifugation was sedimented with cold 5 % trichloroacetic acid, after the addition of I m g of serum protein as a carrier, and was washed with ethanol-ether (3 : I, by vol.). The final pellet was dissolved into 0.2 ml of hyamine at 5°° for 30-60 rain. After the addition to each of IO ml of toluene scintillator, 3H- and 14C radioactivities were determined with a Beckman LS 15o liquid scintillation counter.
RESULTS
Treatment o] liver polysomes with the microsomal deoxycholate-soluble [raction The total polysomes prepared from the rat liver labeled with [14C]orotic acid for 5° rain in vivo were treated with the microsomal deoxycholate-soluble fraction (1.5 mg or 3 mg protein per ml) and were sedimented by centrifugation at 59 3oo ×g for 9° rain. As a control, polysomes were sedimented as above without treatment Biochim. Biophys. Acta, 224 (197 o) 507 517
5IO
T. TANAKA el al.
with the microsomal deoxycholate-soluble fraction. The RNA of these polysomes. was then analyzed by sucrose density-gradient centrifugation. Although the pattern of absorbance at 26o nm of 28-S and I8-S rRNA's remained unchanged after treatment with the microsomal deoxycholate-soluble fraction, the distribution of radioactive R N A shifted to a smaller S region (Fig. I). As the concentration of the microsomal deoxycholate-soluble fraction was increased from 1.5 mg to 3.o mg protein per ml, a larger part of the rapidly labeled R N A was decomposed to acid-soluble materials, but the profiles of ultraviolet absorbance and radioactivity that ran parallel with ultraviolet absorbance in the I8-S and 28-S. region remained unchanged. In accordance with these results, the specific activities of I8-S and 28-S RNA's were ahnost unchanged between the two cases in which different concentrations of the microsomal deoxycholate-soluble fraction were employed, although the specific /^, 200
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I
' 100
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Fig. i. T h e effect of t h e m i c r o s o m a l d e o x y c h o l a t e - s o l u b l e fraction on profiles of density-gradient centrifugation of polysomal RNA. One r a t r e c e i v e d a n i n t r a p e r i t o n e a l injection of 3.3 #C of [x4Clorotic acid per ioo g of b o d y weight. After 5o rain, t h e r a t w a s killed, and t h e t o t a l l i v e r polysomes w e r e p r e p a r e d b y t h e m e t h o d of SUGANO et a l. 16 e x c e p t t h a t d i s c o n t i n u o u s s u c r o s e g r a d i e n t centrifugation at 264 500 × g and suspending in Medium A' were employed. The polysomal s u s p e n s i o n w a s divided into t h r e e e q u a l aliquots: (I) no t r e a t m e n t , (2), (3) t r e a t e d w i t h I. 5 m g / m l and 3 m g / m l protein of the microsomal deoxycholate-soluble fraction, r e s p e c t i v e l y . E a c h a l i q u o t w a s c e n t r i f u g e d a t 59 3 °0 × g for 90 rain. T h e r e s u l t i n g pellet w a s suspended in I ml of 25 mM Tris-HC1 (pH 7.6}, containing i °/o s o d i u m dodecyl sulfate, and analyzed b y t h e s u c r o s e density-gradient centrifugation as d e s c r i b e d in MATERIALSAND MI~THODS. O - - O , A260 n m ; × - - - × , radioactivity.
Biochim. Biophys. Acta, 224 (197 o) 5o7-517
METABOLISM OF
RNA
o F POLYSOMES
51I
TABLE I EFFECT OF THE MICROSOMKL DEOXYCHOLATE-SOLUBLE FRACTION ON POLYSOMAL l ~ ' ~ WITH EI4C~OROTIC ACID FOR 5 ° rain
LABELED
A~60 nm a n d r a d i o a c t i v i t i e s of t h e t o t a l p o l y s o m a l , 28-S a n d I8-S R N A ' s were c a l c u l a t e d from t h e a r e a u n d e r each c u r v e of A ~n0 nm a n d of r a d i o a c t i v i t y in t h e c o r r e s p o n d i n g re gi ons in Fig. i.
Microsomal deoxycholate-soluble ]raction
1. 5 m g p r o t e i n
3 mg protein
;8 S
28 S
Total
A2e0 nm Counts/min C o u n t s / r a i n per Az60 nm
2.11 336.2 159.3
3.61 213.7 59.2
5.72 1447.3 253
A2,0 nm Counts/rain C o u n t s / m i n per A2, 0 nm
2.22 106. i 47.7
3-53 173.3 49.1
5.75 9o2.4 157
(62 %)
A 260 nm Counts/rain C o u n t s / r a i n per A2n0 nm
2.20 lO4.2 47.4
3.81 173.5 45.5
6.01 747.6 124
(49 %)
(IOO %)
activity of RNA especially in the I8-S region was very low in these cases, as compared with the case in which untreated polysomes were employed (Table I). These findings indicate that treatment of liver polysomes with an adequate concentration (I.5-3 mg protein per ml) of the microsomal deoxycholate-soluble fraction degraded mRNA, while rRNA remained intact.
Kinetics o/labeling polysomal RNA's From the results described above, it became possible to determine the rate of labeling of m R N A as the difference between the radioactivity of the total polysomal RNA and that of rRNA at various times after the administration of [14C]orotic acid. These radioactivities could be calculated from the area under the curves of radioactivity in sucrose density-gradient centrifugation of polysomal RNA, as shown in Fig. 2. Furthermore, it was possible to compare the rate of labeling of r R N A between two types of polysome since that of the polysomal RNA was insensitive to treatment with the microsomal deoxycholate-soluble fraction. The time courses of the incorporation of E14C]orotic acid into mRNA, rRNA and the total RNA of both classes of polysome are shown in Fig. 3. While the rate of labeling of rRNA was the same between two kinds of polysome, there was a great difference in that of mRNA. That is, the labeling rate of m R N A of free polysomes was about twice as high as that of bound polysomes.
The e//ect o/deoxycholate treatment on the ratio o/ the labeling o] m R N A between/ree and bound polysomes It might be possible that the rate of labeling of m R N A of free polysomes was higher than that of bound polysomes when it was actually shown that the degradation of m R N A did not occur during the preparation, especially during deoxycholate treatment of the membrane fraction containing bound polysomes. The findings that the ultracentrifugal pattern and the incorporating activity of E14C~amino acid into protein in the cell-free system were nearly the same in the two kinds of polyBiochim. Biophys. Acta, 224 (197 o) 5o7-517
R. TANAKA g[ (ll.
512
Totalpolysomal RNA r //I //~ 1000
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Time (h)
Fig. 2. Sucrose density-gradient centrifugation of R N A from b o u n d polysomes with and w i t h o u t t r e a t m e n t with the microsomal deoxycholate-soluble fraction. B o u n d polysomes from r a t liver labeled with [14C]orotic acid (3.3 ItC/I oo g) for 5 ° min were suspended in Medium A'. The suspension was divided into t w o equal aliquots; (i) no t r e a t m e n t , (2) treated with 2.5 m g / m l protein of the microsomal soluble fraction. The suspension was centrifuged at 59 3o0 × g for i h. The pellet was suspended in sodium dodecyl sulfate, as described in the legend to Fig. I, and subjected to sucrose density-gradient centrifugation. @-(2), Au0 nm; × - - - × , radioactivity. Fig. 3. Nineties of the labeling of R N A ' s of free and b o u n d polysomes. The two rats employed at each time, received intraperitoneal injections of [14C]orotic acid (3.3/~C/ioo g). At 35 rain, 5o rain and 4 h after injection, the r a t s were killed and free and b o u n d polysomes were prepared as described in MATERIALS AND METHODS. E a c h polysomal suspension in Medium A' was divided into two equal aliquots, and density-gradient centrifugation of polysomal RNA, with or w i t h o u t t r e a t m e n t with the microsomal deoxycholate-soluble fraction, was carried o u t as described in the legend to Fig. 2. The radioactivity of the total polysomal R N A was the s u m of the radioactivities in all the fractions t h r o u g h o u t the sucrose gradient w i t h o u t deoxycholate t r e a t m e n t (e.g. T u b e s 1--26 in Fig. 2 (i)) : The radioactivity of r R N A was the s u m of the radioactivities distributed from the 28-S to the I8-S region (for example, Tubes 2-16 in Fig. 2 (2)). The radioactivities of the total polysomal R N A and r R N A are expressed as radioactivity per IO A,,onm, and t h a t of m R N A was calculated as the difference between these two corrected radioactivities, x - • - ~ , free polysomes; Q - O , b o u n d polysomes.
some 3'5 m a y exclude the possibility of the degradation of m R N A during the preparation of polysomes. However, because this point was important, the double-labeling experiment was performed as outlined schematically in Fig. 4. The finding that the radioactivity of m R N A of free polysomes was markedly higher than that of bound polysomes whether treated or not (Table II), clearly Biochim. Biophys. Acla, 224 (i97 o) 5o7-517
METABOLISM OF R N A
513
o r POLYSOMES
Rat liver labeled with [SH]orotic acid for 50 rain
Rat liver labeled with [14C]orotic acid for 50 rain
First discontinuous sucrose density-gradient centrifugation of the postrnitochondrial supernatant fraction Turbid interohase
Precipitate
Deoxycholate treatment
Mixture ~
IContro114C-labeled [ free po ysomes J
Deoxycholate treatment
l
Second discontinuous sucrose density-gradient centrifugationt ] Precipitate [Control 3H-labeled l
Ibound polysomes I I
Precipitate Deoxycholatetreated [14C]-'polysomes[SIN]-
1 Control i-14C3_' [3H]_ po ysornes
Fig. 4. A s c h e m e for t h e double-labeling e x p e r i m e n t .
T A B L E II THE EFFI~CT OF DEOXYCHOLATE TREATMENT ON THE LABELING OF m R N A Of 4 rats, t w o received a n i n t r a p e r i t o n e a l injection of [14C]orotic acid (3 /~C p e r i o n g of b o d y weight) a n d t h e o t h e r 2 t h a t of [aHlorotic acid (I5 #C p e r i o n g of b o d y weight). A f t e r 5 ° m i n , t h e r a t s were killed, a n d free a n d b o u n d p o l y s o m e s were p r e p a r e d as described in MATERIALS AND METHODS. T h e outline of e x p e r i m e n t a l p r o c e d u r e s is s c h e m a t i c a l l y s h o w n in Fig. 4. Free p o l y s o m e s labeled w i t h [14C]orotic acid were divided into t w o p a r t s . O n e p a r t w a s m i x e d w i t h t h e t u r b i d i n t e r p h a s e c o n t a i n i n g b o u n d p o l y s o m e s labeled w i t h [SH]orotic acid, a n d t h e m i x t u r e w a s t r e a t e d w i t h 1.3 % d e o x y c h o l a t e in t h e presence of ribonuclease inhibitor. T h e doublelabeled p o l y s o n l e m i x t u r e w a s t h e n p r e c i p i t a t e d t h r o u g h 2 M sucrose solution a n d s u s p e n d e d in M e d i u m A'. T h i s p r e p a r a t i o n is referred to as d e o x y c h o l a t e t r e a t e d [14C]-, [SH]polysomes. T h e o t h e r p a r t of t h e free p o l y s o m e s w a s directly m i x e d w i t h b o u n d p o l y s o m e s labeled w i t h [aH]orotic acid a n d u s e d as t h e control. B o t h k i n d s of double-labeled p o l y s o m e were s u b j e c t e d to t h e s a m e p r o c e d u r e s for a n a l y s i s of r R N A a n d m R N A as described for Fig. 3 . 3 H - a n d ~4C radioactivities of t h e s e R N A ' s were t h e n d e t e r m i n e d as described in MATERIALS AND METHODS. T h e r a t i o of 14C to SH r a d i o a c t i v i t y of m R N A w a s corrected b y a factor w h i c h m a d e radioaetivities of r R N A of free a n d b o u n d p o l y s o m e s equal.
Polysomes
Counts/rain rRNA
mRNA
Bound/#ee o/mRNA
Control [14C~-, ESH]polysomes
Free [xaC~B o u n d [3H]-
128o 128o
4417 2119
I/2.I
Deoxycholate-treated Et4C]-, [SH]polysomes
Free [14C~B o u n d [3H~ -
687 687
3872 1484
1/2.6
Biochim. Biophys. Acta, 224 (197 o) 5 o 7 - 5 1 7
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T. TANAKAel al.
indicated that the m R N A of polysomes remained intact during deoxycholate treatmerit under our experimental conditions. Therefore, the difference in the labeling of m R N A between free and bound polysomes was not due to the degradation of m R N A of bound polysomes during the preparation. The effect of deoxycholate treatment on the specific activity of free polysomes was also examined, since the existence of membrane fragments in this fraction was reported TM, so that the possibility of the existence of rapidly labeled RNA in these fragments could not be ruled out completely. Deoxycholate treatment of free polysomes had no effect on the specific activity of the total RNA of free polysomes.
Purity o/ two classes o/ polysome It is well known that 45-S ribonucleoprotein particles, which contain rapidly turning over RNA, exist in the cytoplasm of mammalian cells 19,2°. These particles exist only in the free state 21. On the other hand, PENMAN el al. 22 demonstrated the existence of heterodisperse RNA, which co-sediments with, but is not attached to, polysomes in H e L a cells. This RNA was not affected by EDTA, which altered the sedimentation of polysomes in sucrose gradients and was broadly distributed from IO to 7 ° S. There is a possibility that the difference in labeling between two classes of polysome described above m a y be due to the contamination of these components. Therefore, two classes of polysome labeled for 50 rain with E14Clorotic acid were analyzed by sucrose density-gradient centrifugation with and without the addition of EDTA. The results are shown in Fig. 5. The centrifugal patterns without E D T A treatment showed that only about 4 % of the total radioactivity was distributed in the region smaller than monomers in both classes of polysome. This radioactivity m a y be attributable to partial degradation of polysomes during the preparation rather t h a n contamination of 45-S particles, for the proportion of radioactivity in this light region was similar between two classes of polysome. In the sedimentation pattern after the addition of EDTA, no significant radioactivity could be detected in the region larger than 47-S sub-units. These results together indicate that the two polysomal preparations are satisfactorily pure for RNA turnover analysis, and that the difference in the rate of labeling between two classes of polysome is not due to contamination of RNA of 45-S particles or heterodisperse RNA. CsCl equilibrium centri[ugation analysis o/polysomes Recently HENSHAW 2, employing liver cells, and PERRY AND KELLEY1, employing L cells, showed that E D T A treatment of polysomes resulted in the release of m R N A in association with protein and that this mRNA-protein complex was separated from ribosomal subunits by subsequent CsC1 equilibrium centrifugation. In order to obtain more direct evidence for the metabolic difference of m R N A between two kinds of polysome, the above method was employed: two classes of polysome labeled for 50 min with I14C]orotic acid were subjected to CsC1 equilibrium centrifugation with and without EDTA treatment. As shown in Fig. 6, free and bound polysomes banded at the density about 1.59 g/cm 3, and the distribution of the radioactivity was coincident with that of ultraviolet absorbing material. The specific radioactivity at the peak of free polysomes was 1. 5 times higher than that of bound polysomes. The centrifugal pattern Biochim. Biophys. Acta, 224 (197o) 5o7-517
METABOLISM OF R N A
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Fig. 5. Sucrose density-gradient centrifugation of b o t h classes of liver polysome. Free and b o u n d polysomes p r e p a r e d f r o m r a t livers labeled w i t h [l*C]orotic acid (5/~C/ioo g) as described in MATERIALS AND METHODS, except t h a t the lO 5 o o o × g s u p e r n a t a n t fraction freshly p r e p a r e d f r o m r a t liver was used instead of ribonuclease inhibitor according to BLOBEL AND POTTER~1. Free and b o u n d polysomes were suspended in I ml of Medium A'. The polysome suspension w a s layered on 28 ml of a 13.5-28. 5 % linear sucrose density-gradient containing 25 mM Tris HC1 buffer (pH 7.6), 5 ° mM KC1 and 5 mM MgCI v and s p u n in a RPS-25 r o t o r of a Hitachi 4 ° P ultracentrifuge at 25 ooo revolutions per miD for 15o miD. F o r E D T A - t r e a t e d polysomes, o.I nil of 15 ° m M E D T A was added to I ml of the polysomal suspensi6n and MgCI~ was o m i t t e d from the m e d i u m of sucrose gradient. After centrifugation, the gradients were divided into i ml fractions b y an ISCO density gradient fractionator m o n i t o r i n g ultraviolet absorbance. The radioact i v i t y of each fraction was determined as described in MATERIALS AND M E T H O D S . (I) Free polysomes; (2) b o u n d polysomes; (3) free polysomes t r e a t e d w i t h E D T A ; (4) b o u n d p o l y s o m e s treated with EDTA. - - , A254nm; × - - - × , radioactivity. Fig. 6. CsC1 equilibrium centrifugation of polysomes. Two r a t s received intraperitoneal injections of 5/zC of [14C~orotic acid per IOO g of b o d y weight. After 5 ° rain t w o classes of p o l y s o m e were p r e p a r e d from livers b y the procedures described in MATERIALS AND METHODS, except t h a t Tris HC1 buffer was replaced b y the same concentration of triethanolamine-HC1 buffer (pH 7.6), The pellets of two kinds of polysome were suspended in Medium A' containing t r i e t h a n o l a m i n e HC1, and E D T A was added to a final concentration of 15 mM (IO mM excess over the Mg 2+ concentration). After the m i x t u r e had stood at o ° for 2 h, 3 ° % formaldehyde neutralized with t r i e t h a n o l a m i n e was added to a final concentration of 6 %, and the m i x t u r e was kept at o ° for 20 h. After being dialyzed against 0.02 M triethanolamine-HC1 buffer containing 5 ° mM KC1 for 2 4 h at o °, the polysomes fixed with formaldehyde were subjected to CsC1 equilibrium centrifugation, which was carried o u t according to the m e t h o d of BRUNK AND LEICK ~s as described below. The lower p a r t of a B e c k m a n SW-39 t u b e contained 2. 5 ml of 5 ° % (w/w) CsC1 in o.o2 M triethanolamine HC1 buffer containing 50 mM KC1. Then 2. 5 ml of 3 6 % CsC1 in the same buffer, which contained fixed polysomes, was layered onto the lower portion. Centrifugation was carried out in a Hitachi 1RPS-4o rotor at 35 ooo r e v . / m i n for 18 h. After centrifugation the t u b e s were p u n c t u r e d at the b o t t o m and 6 drops were collected f r o m each. The d e t e r m i n a t i o n of A 260 nm and radioactiv i t y of each fraction were carried o u t b y the same m e t h o d as for the sucrose gradient centrifugation. Control polysomes w i t h o u t E D T A t r e a t m e n t were p r e p a r e d f r o m 2 r a t livers labeled w i t h [14Clorotic acid u n d e r the same experimental conditions and applied to CsC1 equilibrium centrifugation as described above. (i) Free polysomes; (2) b o u n d polysomes; (3) free polysomes t r e a t e d w i t h E D T A ; (4) b o u n d polysomes t r e a t e d with E D T A . G - Q , 3 ~ 0 nm; × - - - × , radioactivity.
Biochim. Biophys. Acta, 224 (197 o) 5o7-517
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T. TANAKAet al.
after EDTA treatment indicates that large ribosomal sub-units banded at the density about 1.61 g/cm 3, and mRNA-containing particles with high specific activity were released from ribosomal subunits and distributed heterogeneously in a broad density range extending from 1.35 to 1.55 g/cm 3. It is noticeable that the specific activities of these ribonucleoprotein particles were markedly higher throughout this density range in free polysomes than in bound polysomes. On the contrary, the specific activity of ribosomal sub-units was similar between two classes of polysome.
DISCUSSION
Treatment of liver polysolnes with the microsomal deoxycholate-soluble fraction resulted in the degradation of mRNA, although rRNA remained intact. Ribonuclease is present in hepatic microsomal fraction 2a,24 and deoxycholate released this ribonuclease which causes the breakdown of liver polysomes into monomeric and dimeric ribosomes 16. Results of treatment of polysomes with dilute pancreatic ribonuclease were not reproducible, sometimes showing sedimentation patterns of the degradation of rRNA, in agreement with the results of FENWlCK25. Therefore, it is preferable to employ microsomal deoxycholate-soluble ribonuclease for the purpose of selective degradation of mRNA. The incorporation of I14CJorotic acid into mRNA, calculated as the difference in radioactivity between the total polysomal RNA and rRNA, was found to be higher in free polysomes than in bound polysomes. It was further shown by double-labeling experiments that the higher incorporation of I14Clorotic acid into mRNA of free polysomes was not due to the degradation of mRNA of bound polysomes during the preparative procedures including recentrifugation and resuspension of polysomes before treatment with sodium dodecyl sulfate. The radioactivity of mRNA calculated as above may include that of tRNA and 5-S RNA. However, methylated albumin column chromatography of the whole RNA of the total polysomes of rat liver, labeled with ElaC~orotic acid for 50 min, showed that the proportion of radioactivity of tRNA and 5 S RNA to that of the whole RNA was very small 26. The incorporation into these RNA's can be negligible, especially in early labeling periods. Furthermore, the difference in the rate of labeling between the two classes of polysome was shown to be neither due to the contamination of 45-S ribonucleoprotein particles with free polysomes nor to the existence of heterodisperse RNA which co-sediment with but are not attached to polysomes. Therefore, it seems reasonable to contend that the labeling rate of mRNA of free polysomes is more rapid than that of bound polysomes. More direct support for this contention was given by CsC1 equilibrium centrifugation of EDTAtreated polysomes pulse-labeled with ~l*C]orotic acid in vivo, since the specific activity of the mRNA-protein complex from free polysomes was markedly higher than that from bound polysomes. If our contention be accepted, three possibilities may exist; (I) Newly synthesized mRNA passes through a stage in which it is associated with free polysomes, later becoming bound to membranes. (2) mRNA in free polysomes turns over more rapidly. (3) There is more mRNA per ribosome in free polysomes. The third possibility is probably unlikely, since the activity in protein synthesis in vivo and in vitro was Biochim. Biophys. Acta, 224 (i97 o) 5o7-517
METABOLISM OF R N A
OE POLYSOMES
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shown to be similar between two classes of polysome. It is impossible from this kind of experiment to distinguish between the first two possibilities, and further study must be done to elucidate these points. Recently, however, SARMA et al. 27 reported that the administration of actinomycin D to mouse resulted in marked disaggregation of hepatic free polysomes leaving bound polysomes intact. These findings suggest that m R N A in free polysomes turns over more rapidly. Recently, several reports 3-1° including those of the authors, have indicated differences in the kinds of protein synthesized by the two classes of polysome. As reported in our previous paper 3, these results suggest the possibility that the distribution of m R N A is different between the two kinds of polysome. The results of the present experiments provide evidence for a metabolic difference between the two classes of liver polysome. Our experiments showed that there is no difference in the turnover rate of rRNA between the two classes of liver polysome, in agreement with those of LOEB et al. 13. These results suggest that liver ribosomes are capable of changing between free and membrane-bound states or that there is no intrinsic difference in metabolism between them. 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
]:{. P. PERRY AND D. E. KELLEY, J. Mol. Biol., 35 (1968) 37E. C. HENSHAW, J. Mol. Biol., 36 (1968) 4Ol. 3/[. TAKAGI AND K. OGATA, Biochem. Biophys. Res. Commun., 33 (1968) 55. iV[. TAKAGI, T. TANAKA AND K. OGATA, J. Biochem., 65 (1969) 651. 1V[. TAKAGI, T. TANAKA AND K. OGATA, Biochim. Biophys. Acta, 217 (197o) 148. C. IV[. REDMAN, Biochem. Biophys. Res. Commun., 31 (1968) 845. T. HALLINAN, C. N. MURTV AND J. H. GRAND, Li/e Sci., 7 (1968) 225. S. J. HICKS, J. W. D. DRYSDALE AND H. ~'. MUNRO, Science, 164 (1969) 584 . C. ]~,{. REDMAN, J. Biol. Chem., 244 (1969) 43o8. M. C. GANOZA AND C. A. WILLIAMS, Proc. Natl. Acad. Sci. U.S., 63 (1969) 137 o. Y. MOULE AND G. DELHUMEAU DE ONGAY, Biochim. Biophys. Acta, 91 (1964) 113. T. HALLINAN AND H. MUNRO, Biochim. Biophys. Acta, lO8 (1965) 285. J. N. LOEB, R. R. HOWELL AND G. M. TOMKINS, J. Biol. Chem., 242 (1967) 2069. C. N. MURTY AND T. HALLINAN, Biochim. Biophys. Acta, 157 (1968) 414 . F. O. WETTSTEIN, T. STAEHELIN AND H. NOLL, Nature, 197 (1963) 778. H. SUGANO, I. WATANABE AND K. OGATA, J. Biochim., 61 (1967) 778. K. SHORTMAN, Biochim. Biophys. Acta, 51 (1961) 37. C. N. 1VIURTYAND T. HALLINAN, Biochem. J., 112 (1969) 269. E. C. I-[ENSHAW, M. REVEL AND n . H. HIATT, J. Mol. Biol., 14 (1965) 241. R. V. PERRY AND D. E. KELLEY, J. Mol. Biol., 16 (1966) 255. G. BLOBEL AND V. R. POTTER, J. Mol. Biol., 28 (1967) 539. S. PENMAN, C. VESCO AND IV[. PENMAN, J. Mol. Biol., 34 (1968) 49. C. DE I)UVE, B. C. PRESSMAN, R. GIANETTO, R. WATTIAUX AND F. APPLEMANS, Biochem. J., 6o (1955) 604. R. MORRIS AND G. D. LAMIRANDE, Biochim. Biophys. Acta, 95 (1965) 4 °. M. L. FENWlCK, Biochem. J., lO 7 (1968) 481. T. TANAKA AND I{. OGATA, u n p u b l i s h e d result. D. S. R. SARMA, I. M. REID AND H. SIDRANSKY, Biochem. Biophys. Res. Commun., 36 (1969) 582. C. F. BRUNK and V. LEICK, Biochim. Biophys. Acta, 179 (1969) 136.
Biochim. Biophys. Acta, 224 (197 o) 5o7-517
BIOCHIMICA ET BIOPHYSICA ACTA BBA 96642
S T U D I E S ON T H E METABOLISM OF RNA OF F R E E AND MEMBRANEBOUND POLYSOMES FROM R A T L I V E R TATSUO TANAKA, MASAMICHI TAKAGI* AND K I K U O OGATA
Department o[ Biochemistry, Niigata University School o/Medicine, Niigata (Japan) (Received May i9th, 197 o)
SUMMARY
I. Through the employment of the microsomal deoxycholate-soluble fraction from rat liver, conditions were found in which messenger RNA of the rat liver polysomes were degraded, while the ribosomal structural RNA remained intact. This method made it possible to compare the rate of labeling of ribosomal structural RNA and m R N A between free and membrane-bound polysomes from rat liver. 2. The rate of incorporation of [14C~orotic acid into messenger RNA of free polysomes was higher than that into messenger RNA of bound polysomes up to 4 h after an intraperitoneal iniection of L14Clorotic acid. 3. CsC1 equilibrium centrifugation of EDTA-treated polysomes from rat liver, labeled in vivo with I14C~orotic acid for 50 rain, also showed that the rate of incorporation into messenger RNA-containing particles 1,~ was higher in free polysomes t h a n in bound polysomes. 4. On the contrary, the rates of labeling of ribosomal structural RNA of these two classes of polysome were shown to be similar. These results are discussed in relation to the biosynthesis of different kinds of protein between two classes of liver polysome.
INTRODUCTION
Many reports 8-1°, including those of the authors 3,4, have been published showing that the membrane-bound polysomes synthesize serum proteins, and that the free polysomes synthesize non-serum liver proteins in the hepatic cell. The most reasonable explanation of the functional difference between these two classes of polysome may be that these particles have different species of messenger RNA. Therefore, it seems important to obtain direct evidence for such differences. Concerning the metabolic differences of RNA between these two classes of liver polysome, the data available are very few and seem to be contradictory n-l*. The contradictions m a y be the result of the complexity of polysomal RNA which includes ribosomal structural RNA (rRNA), messenger RNA (mRNA), transfer RNA (tRNA) and 5-S RNA. In short pulseAbbreviations: rRNA, ribosomal structural RNA; mRNA, messenger RNA; tRNA, transfer RNA. " Present address: Laboratory of Radiation Genetics, Faculty of Agriculture, University of Tokyo, Tokyo, Japan.
Biochim. Biophys. Acta, 224 (I97 o) 5o7-517
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T. TANAKA et al.
labeling experiments, it is particularly difficult to distinguish mRNA from rRNA. In fact, in the above-mentioned papers n-l* they were not well differentiated. In the present experiments rRNA and mRNA of rat liver polysomes were distinguished by treatment of polysomes with the liver microsomal deoxycholatesoluble fraction containing mild ribonuclease activity, and the difference in the kinetics of the labeling in vivo of these RNA's between two classes of polysome was investigated. The results of CsC1 equilibrium centrifugation of EDTA-treated polysomes, pulse-labeled with lil*Clorotic acid in vivo, are also described.
MATERIALS AND METHODS
Chemicals E6-14C]Orotic acid (60.8/zC/#mole) and ~5-~HJorotic acid 6240 #C//~mole) were purchased from the Radiocbemical Centre, Amersham, England. Sodium deoxycholate was obtained from Difco Lab. Co., sodium dodecyl sulfate from Nakarai Chemicals Ltd., Kyoto, and bovine pancreatic ribonuclease (EC 2.7.7.16) from Worthington Biochemical Corp. Animals Rats of the Wistar strain weighing 13o-16o g were starved for about IS h before the experiments. !l*ClOrotic acid was administered by intraperitoneal injection. At the end of the time specified, the animals were killed by decapitation, and their livers were excised and chilled in ice-cold medium A (0.25 M sucrose, 5 mM MgCl~, 25 mM KCI, 50 mM Tris-HC1, pH 7.6). Fraclionalion o/liver cell The livers were weighed, minced and homogenized in 2.5 volumes of medium A in a glass Potter-Elvehjem type homogenizer with a loosely-fitted Teflon pestle with 8 strokes at Iooo rev./min. The postmitochondrial supernatant after centrifugation of the homogenate at io ooo ~
METABOLISM OF R N A
o r POLYSOMES
509
to all the solutions used throughout the procedures of polysomal preparation as described previously 3-5.
Treatment of polysomes with microsomal deoxyeholate-soluble fraction The microsomal pellet, prepared by centrifugation of the postmitochondrial supernatant from 5 g of rat liver at lO5 o o o × g for I h, was suspended in 2 ml of Medium A'. Deoxycholate was added to the microsomal suspension to give a final concentration of 1. 3 °/o, and centrifuged at 59 3 o o × g for I h. The upper half of the supernatant was used as a microsomal deoxycholate-soluble fraction. IOO #1 of the microsomal deoxycholate-soluble fraction containing about 2.5 mg of protein was added to I ml of the polysomal suspension. The mixture stood for 5 rain at o °, and was centrifuged at 59 3 o o × g for 9 ° min to sediment ribosomes. As the control, polysomes were subjected to the same procedures as described above, except that the microsomal deoxycholate-soluble fraction was not added before centrifugation. Recentrifugation and resuspension had little effect on the specific activity of the total polysomal RNA.
Sucrose density-gradient centri/ugation o] polysomal RNA and determination o/ radioactivity The ribosomal pellet thus obtained was suspended in I ml of 25 mM Tris-HC1 (pH 7.6) containing 1 % sodium dodecyl sulfate. The resulting solution was layered over 28 ml of 5-2o % linear sucrose density gradient containing 25 mM Tris-HC1 (pH 7.6), and spun in a RPS- 25A rotor of a Hitachi 65 P ultracentrifuge at 51 5o5 × g at 6 ° for 15 h. The centrifugal tube was then punctured at the bottom and the ultraviolet absorbance at 260 nm (A~e0 nm) of the effluent fraction of each 15 drops was measured by a Hitachi Perkin-Elmer model 139 spectrophotometer. From each fraction the acid-insoluble material was precipitated with 5 % trichloroacetic acid at o ° overnight, retained on a Millipore filter (pore size 0.45 #) and washed with 5 °/o trichloroacetic acid. The radioactivity was then determined by an automatic lowbackground gas-flow counter of Nihonmusen Ltd. (background counts of about 1. 5 counts/rain). In the double-labeling experiments with I14C~- and I3H]orotic acid, the acidinsoluble material of each fraction from density-gradient centrifugation was sedimented with cold 5 % trichloroacetic acid, after the addition of I m g of serum protein as a carrier, and was washed with ethanol-ether (3 : I, by vol.). The final pellet was dissolved into 0.2 ml of hyamine at 5°° for 30-60 rain. After the addition to each of IO ml of toluene scintillator, 3H- and 14C radioactivities were determined with a Beckman LS 15o liquid scintillation counter.
RESULTS
Treatment o] liver polysomes with the microsomal deoxycholate-soluble [raction The total polysomes prepared from the rat liver labeled with [14C]orotic acid for 5° rain in vivo were treated with the microsomal deoxycholate-soluble fraction (1.5 mg or 3 mg protein per ml) and were sedimented by centrifugation at 59 3oo ×g for 9° rain. As a control, polysomes were sedimented as above without treatment Biochim. Biophys. Acta, 224 (197 o) 507 517
5IO
T. TANAKA el al.
with the microsomal deoxycholate-soluble fraction. The RNA of these polysomes. was then analyzed by sucrose density-gradient centrifugation. Although the pattern of absorbance at 26o nm of 28-S and I8-S rRNA's remained unchanged after treatment with the microsomal deoxycholate-soluble fraction, the distribution of radioactive R N A shifted to a smaller S region (Fig. I). As the concentration of the microsomal deoxycholate-soluble fraction was increased from 1.5 mg to 3.o mg protein per ml, a larger part of the rapidly labeled R N A was decomposed to acid-soluble materials, but the profiles of ultraviolet absorbance and radioactivity that ran parallel with ultraviolet absorbance in the I8-S and 28-S. region remained unchanged. In accordance with these results, the specific activities of I8-S and 28-S RNA's were ahnost unchanged between the two cases in which different concentrations of the microsomal deoxycholate-soluble fraction were employed, although the specific /^, 200
(I)
/ i
1.C
x"
100
,
I
E
I
' 100
u
,< x
:
u
i i
i
1.0k (3)
100
bottom
Fraction No.
top~
Fig. i. T h e effect of t h e m i c r o s o m a l d e o x y c h o l a t e - s o l u b l e fraction on profiles of density-gradient centrifugation of polysomal RNA. One r a t r e c e i v e d a n i n t r a p e r i t o n e a l injection of 3.3 #C of [x4Clorotic acid per ioo g of b o d y weight. After 5o rain, t h e r a t w a s killed, and t h e t o t a l l i v e r polysomes w e r e p r e p a r e d b y t h e m e t h o d of SUGANO et a l. 16 e x c e p t t h a t d i s c o n t i n u o u s s u c r o s e g r a d i e n t centrifugation at 264 500 × g and suspending in Medium A' were employed. The polysomal s u s p e n s i o n w a s divided into t h r e e e q u a l aliquots: (I) no t r e a t m e n t , (2), (3) t r e a t e d w i t h I. 5 m g / m l and 3 m g / m l protein of the microsomal deoxycholate-soluble fraction, r e s p e c t i v e l y . E a c h a l i q u o t w a s c e n t r i f u g e d a t 59 3 °0 × g for 90 rain. T h e r e s u l t i n g pellet w a s suspended in I ml of 25 mM Tris-HC1 (pH 7.6}, containing i °/o s o d i u m dodecyl sulfate, and analyzed b y t h e s u c r o s e density-gradient centrifugation as d e s c r i b e d in MATERIALSAND MI~THODS. O - - O , A260 n m ; × - - - × , radioactivity.
Biochim. Biophys. Acta, 224 (197 o) 5o7-517
METABOLISM OF
RNA
o F POLYSOMES
51I
TABLE I EFFECT OF THE MICROSOMKL DEOXYCHOLATE-SOLUBLE FRACTION ON POLYSOMAL l ~ ' ~ WITH EI4C~OROTIC ACID FOR 5 ° rain
LABELED
A~60 nm a n d r a d i o a c t i v i t i e s of t h e t o t a l p o l y s o m a l , 28-S a n d I8-S R N A ' s were c a l c u l a t e d from t h e a r e a u n d e r each c u r v e of A ~n0 nm a n d of r a d i o a c t i v i t y in t h e c o r r e s p o n d i n g re gi ons in Fig. i.
Microsomal deoxycholate-soluble ]raction
1. 5 m g p r o t e i n
3 mg protein
;8 S
28 S
Total
A2e0 nm Counts/min C o u n t s / r a i n per Az60 nm
2.11 336.2 159.3
3.61 213.7 59.2
5.72 1447.3 253
A2,0 nm Counts/rain C o u n t s / m i n per A2, 0 nm
2.22 106. i 47.7
3-53 173.3 49.1
5.75 9o2.4 157
(62 %)
A 260 nm Counts/rain C o u n t s / r a i n per A2n0 nm
2.20 lO4.2 47.4
3.81 173.5 45.5
6.01 747.6 124
(49 %)
(IOO %)
activity of RNA especially in the I8-S region was very low in these cases, as compared with the case in which untreated polysomes were employed (Table I). These findings indicate that treatment of liver polysomes with an adequate concentration (I.5-3 mg protein per ml) of the microsomal deoxycholate-soluble fraction degraded mRNA, while rRNA remained intact.
Kinetics o/labeling polysomal RNA's From the results described above, it became possible to determine the rate of labeling of m R N A as the difference between the radioactivity of the total polysomal RNA and that of rRNA at various times after the administration of [14C]orotic acid. These radioactivities could be calculated from the area under the curves of radioactivity in sucrose density-gradient centrifugation of polysomal RNA, as shown in Fig. 2. Furthermore, it was possible to compare the rate of labeling of r R N A between two types of polysome since that of the polysomal RNA was insensitive to treatment with the microsomal deoxycholate-soluble fraction. The time courses of the incorporation of E14C]orotic acid into mRNA, rRNA and the total RNA of both classes of polysome are shown in Fig. 3. While the rate of labeling of rRNA was the same between two kinds of polysome, there was a great difference in that of mRNA. That is, the labeling rate of m R N A of free polysomes was about twice as high as that of bound polysomes.
The e//ect o/deoxycholate treatment on the ratio o/ the labeling o] m R N A between/ree and bound polysomes It might be possible that the rate of labeling of m R N A of free polysomes was higher than that of bound polysomes when it was actually shown that the degradation of m R N A did not occur during the preparation, especially during deoxycholate treatment of the membrane fraction containing bound polysomes. The findings that the ultracentrifugal pattern and the incorporating activity of E14C~amino acid into protein in the cell-free system were nearly the same in the two kinds of polyBiochim. Biophys. Acta, 224 (197 o) 5o7-517
R. TANAKA g[ (ll.
512
Totalpolysomal RNA r //I //~ 1000
H) !I
c
100
7RNA
×
g o o 500
~5 5
10
15
20
25
z /" z z • • / / /
i
,.i
/
c~ >.
(2)
>
0
I
2
3
4
mRNA _o ~x
/~
'.o I
5 bottom
loo
10
15 20 25top~
Fraction
No.
i
500
o
~
~
~
Time (h)
Fig. 2. Sucrose density-gradient centrifugation of R N A from b o u n d polysomes with and w i t h o u t t r e a t m e n t with the microsomal deoxycholate-soluble fraction. B o u n d polysomes from r a t liver labeled with [14C]orotic acid (3.3 ItC/I oo g) for 5 ° min were suspended in Medium A'. The suspension was divided into t w o equal aliquots; (i) no t r e a t m e n t , (2) treated with 2.5 m g / m l protein of the microsomal soluble fraction. The suspension was centrifuged at 59 3o0 × g for i h. The pellet was suspended in sodium dodecyl sulfate, as described in the legend to Fig. I, and subjected to sucrose density-gradient centrifugation. @-(2), Au0 nm; × - - - × , radioactivity. Fig. 3. Nineties of the labeling of R N A ' s of free and b o u n d polysomes. The two rats employed at each time, received intraperitoneal injections of [14C]orotic acid (3.3/~C/ioo g). At 35 rain, 5o rain and 4 h after injection, the r a t s were killed and free and b o u n d polysomes were prepared as described in MATERIALS AND METHODS. E a c h polysomal suspension in Medium A' was divided into two equal aliquots, and density-gradient centrifugation of polysomal RNA, with or w i t h o u t t r e a t m e n t with the microsomal deoxycholate-soluble fraction, was carried o u t as described in the legend to Fig. 2. The radioactivity of the total polysomal R N A was the s u m of the radioactivities in all the fractions t h r o u g h o u t the sucrose gradient w i t h o u t deoxycholate t r e a t m e n t (e.g. T u b e s 1--26 in Fig. 2 (i)) : The radioactivity of r R N A was the s u m of the radioactivities distributed from the 28-S to the I8-S region (for example, Tubes 2-16 in Fig. 2 (2)). The radioactivities of the total polysomal R N A and r R N A are expressed as radioactivity per IO A,,onm, and t h a t of m R N A was calculated as the difference between these two corrected radioactivities, x - • - ~ , free polysomes; Q - O , b o u n d polysomes.
some 3'5 m a y exclude the possibility of the degradation of m R N A during the preparation of polysomes. However, because this point was important, the double-labeling experiment was performed as outlined schematically in Fig. 4. The finding that the radioactivity of m R N A of free polysomes was markedly higher than that of bound polysomes whether treated or not (Table II), clearly Biochim. Biophys. Acla, 224 (i97 o) 5o7-517
METABOLISM OF R N A
513
o r POLYSOMES
Rat liver labeled with [SH]orotic acid for 50 rain
Rat liver labeled with [14C]orotic acid for 50 rain
First discontinuous sucrose density-gradient centrifugation of the postrnitochondrial supernatant fraction Turbid interohase
Precipitate
Deoxycholate treatment
Mixture ~
IContro114C-labeled [ free po ysomes J
Deoxycholate treatment
l
Second discontinuous sucrose density-gradient centrifugationt ] Precipitate [Control 3H-labeled l
Ibound polysomes I I
Precipitate Deoxycholatetreated [14C]-'polysomes[SIN]-
1 Control i-14C3_' [3H]_ po ysornes
Fig. 4. A s c h e m e for t h e double-labeling e x p e r i m e n t .
T A B L E II THE EFFI~CT OF DEOXYCHOLATE TREATMENT ON THE LABELING OF m R N A Of 4 rats, t w o received a n i n t r a p e r i t o n e a l injection of [14C]orotic acid (3 /~C p e r i o n g of b o d y weight) a n d t h e o t h e r 2 t h a t of [aHlorotic acid (I5 #C p e r i o n g of b o d y weight). A f t e r 5 ° m i n , t h e r a t s were killed, a n d free a n d b o u n d p o l y s o m e s were p r e p a r e d as described in MATERIALS AND METHODS. T h e outline of e x p e r i m e n t a l p r o c e d u r e s is s c h e m a t i c a l l y s h o w n in Fig. 4. Free p o l y s o m e s labeled w i t h [14C]orotic acid were divided into t w o p a r t s . O n e p a r t w a s m i x e d w i t h t h e t u r b i d i n t e r p h a s e c o n t a i n i n g b o u n d p o l y s o m e s labeled w i t h [SH]orotic acid, a n d t h e m i x t u r e w a s t r e a t e d w i t h 1.3 % d e o x y c h o l a t e in t h e presence of ribonuclease inhibitor. T h e doublelabeled p o l y s o n l e m i x t u r e w a s t h e n p r e c i p i t a t e d t h r o u g h 2 M sucrose solution a n d s u s p e n d e d in M e d i u m A'. T h i s p r e p a r a t i o n is referred to as d e o x y c h o l a t e t r e a t e d [14C]-, [SH]polysomes. T h e o t h e r p a r t of t h e free p o l y s o m e s w a s directly m i x e d w i t h b o u n d p o l y s o m e s labeled w i t h [aH]orotic acid a n d u s e d as t h e control. B o t h k i n d s of double-labeled p o l y s o m e were s u b j e c t e d to t h e s a m e p r o c e d u r e s for a n a l y s i s of r R N A a n d m R N A as described for Fig. 3 . 3 H - a n d ~4C radioactivities of t h e s e R N A ' s were t h e n d e t e r m i n e d as described in MATERIALS AND METHODS. T h e r a t i o of 14C to SH r a d i o a c t i v i t y of m R N A w a s corrected b y a factor w h i c h m a d e radioaetivities of r R N A of free a n d b o u n d p o l y s o m e s equal.
Polysomes
Counts/rain rRNA
mRNA
Bound/#ee o/mRNA
Control [14C~-, ESH]polysomes
Free [xaC~B o u n d [3H]-
128o 128o
4417 2119
I/2.I
Deoxycholate-treated Et4C]-, [SH]polysomes
Free [14C~B o u n d [3H~ -
687 687
3872 1484
1/2.6
Biochim. Biophys. Acta, 224 (197 o) 5 o 7 - 5 1 7
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T. TANAKAel al.
indicated that the m R N A of polysomes remained intact during deoxycholate treatmerit under our experimental conditions. Therefore, the difference in the labeling of m R N A between free and bound polysomes was not due to the degradation of m R N A of bound polysomes during the preparation. The effect of deoxycholate treatment on the specific activity of free polysomes was also examined, since the existence of membrane fragments in this fraction was reported TM, so that the possibility of the existence of rapidly labeled RNA in these fragments could not be ruled out completely. Deoxycholate treatment of free polysomes had no effect on the specific activity of the total RNA of free polysomes.
Purity o/ two classes o/ polysome It is well known that 45-S ribonucleoprotein particles, which contain rapidly turning over RNA, exist in the cytoplasm of mammalian cells 19,2°. These particles exist only in the free state 21. On the other hand, PENMAN el al. 22 demonstrated the existence of heterodisperse RNA, which co-sediments with, but is not attached to, polysomes in H e L a cells. This RNA was not affected by EDTA, which altered the sedimentation of polysomes in sucrose gradients and was broadly distributed from IO to 7 ° S. There is a possibility that the difference in labeling between two classes of polysome described above m a y be due to the contamination of these components. Therefore, two classes of polysome labeled for 50 rain with E14Clorotic acid were analyzed by sucrose density-gradient centrifugation with and without the addition of EDTA. The results are shown in Fig. 5. The centrifugal patterns without E D T A treatment showed that only about 4 % of the total radioactivity was distributed in the region smaller than monomers in both classes of polysome. This radioactivity m a y be attributable to partial degradation of polysomes during the preparation rather t h a n contamination of 45-S particles, for the proportion of radioactivity in this light region was similar between two classes of polysome. In the sedimentation pattern after the addition of EDTA, no significant radioactivity could be detected in the region larger than 47-S sub-units. These results together indicate that the two polysomal preparations are satisfactorily pure for RNA turnover analysis, and that the difference in the rate of labeling between two classes of polysome is not due to contamination of RNA of 45-S particles or heterodisperse RNA. CsCl equilibrium centri[ugation analysis o/polysomes Recently HENSHAW 2, employing liver cells, and PERRY AND KELLEY1, employing L cells, showed that E D T A treatment of polysomes resulted in the release of m R N A in association with protein and that this mRNA-protein complex was separated from ribosomal subunits by subsequent CsC1 equilibrium centrifugation. In order to obtain more direct evidence for the metabolic difference of m R N A between two kinds of polysome, the above method was employed: two classes of polysome labeled for 50 min with I14C]orotic acid were subjected to CsC1 equilibrium centrifugation with and without EDTA treatment. As shown in Fig. 6, free and bound polysomes banded at the density about 1.59 g/cm 3, and the distribution of the radioactivity was coincident with that of ultraviolet absorbing material. The specific radioactivity at the peak of free polysomes was 1. 5 times higher than that of bound polysomes. The centrifugal pattern Biochim. Biophys. Acta, 224 (197o) 5o7-517
METABOLISM OF R N A
,~ t 0.4t~~
0.~
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,
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,
,
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515
OF POLYSOMES
1°I1 1200
20
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100
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.~
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o
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15 20 Fraction No.
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17
16
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14
13
Fig. 5. Sucrose density-gradient centrifugation of b o t h classes of liver polysome. Free and b o u n d polysomes p r e p a r e d f r o m r a t livers labeled w i t h [l*C]orotic acid (5/~C/ioo g) as described in MATERIALS AND METHODS, except t h a t the lO 5 o o o × g s u p e r n a t a n t fraction freshly p r e p a r e d f r o m r a t liver was used instead of ribonuclease inhibitor according to BLOBEL AND POTTER~1. Free and b o u n d polysomes were suspended in I ml of Medium A'. The polysome suspension w a s layered on 28 ml of a 13.5-28. 5 % linear sucrose density-gradient containing 25 mM Tris HC1 buffer (pH 7.6), 5 ° mM KC1 and 5 mM MgCI v and s p u n in a RPS-25 r o t o r of a Hitachi 4 ° P ultracentrifuge at 25 ooo revolutions per miD for 15o miD. F o r E D T A - t r e a t e d polysomes, o.I nil of 15 ° m M E D T A was added to I ml of the polysomal suspensi6n and MgCI~ was o m i t t e d from the m e d i u m of sucrose gradient. After centrifugation, the gradients were divided into i ml fractions b y an ISCO density gradient fractionator m o n i t o r i n g ultraviolet absorbance. The radioact i v i t y of each fraction was determined as described in MATERIALS AND M E T H O D S . (I) Free polysomes; (2) b o u n d polysomes; (3) free polysomes t r e a t e d w i t h E D T A ; (4) b o u n d p o l y s o m e s treated with EDTA. - - , A254nm; × - - - × , radioactivity. Fig. 6. CsC1 equilibrium centrifugation of polysomes. Two r a t s received intraperitoneal injections of 5/zC of [14C~orotic acid per IOO g of b o d y weight. After 5 ° rain t w o classes of p o l y s o m e were p r e p a r e d from livers b y the procedures described in MATERIALS AND METHODS, except t h a t Tris HC1 buffer was replaced b y the same concentration of triethanolamine-HC1 buffer (pH 7.6), The pellets of two kinds of polysome were suspended in Medium A' containing t r i e t h a n o l a m i n e HC1, and E D T A was added to a final concentration of 15 mM (IO mM excess over the Mg 2+ concentration). After the m i x t u r e had stood at o ° for 2 h, 3 ° % formaldehyde neutralized with t r i e t h a n o l a m i n e was added to a final concentration of 6 %, and the m i x t u r e was kept at o ° for 20 h. After being dialyzed against 0.02 M triethanolamine-HC1 buffer containing 5 ° mM KC1 for 2 4 h at o °, the polysomes fixed with formaldehyde were subjected to CsC1 equilibrium centrifugation, which was carried o u t according to the m e t h o d of BRUNK AND LEICK ~s as described below. The lower p a r t of a B e c k m a n SW-39 t u b e contained 2. 5 ml of 5 ° % (w/w) CsC1 in o.o2 M triethanolamine HC1 buffer containing 50 mM KC1. Then 2. 5 ml of 3 6 % CsC1 in the same buffer, which contained fixed polysomes, was layered onto the lower portion. Centrifugation was carried out in a Hitachi 1RPS-4o rotor at 35 ooo r e v . / m i n for 18 h. After centrifugation the t u b e s were p u n c t u r e d at the b o t t o m and 6 drops were collected f r o m each. The d e t e r m i n a t i o n of A 260 nm and radioactiv i t y of each fraction were carried o u t b y the same m e t h o d as for the sucrose gradient centrifugation. Control polysomes w i t h o u t E D T A t r e a t m e n t were p r e p a r e d f r o m 2 r a t livers labeled w i t h [14Clorotic acid u n d e r the same experimental conditions and applied to CsC1 equilibrium centrifugation as described above. (i) Free polysomes; (2) b o u n d polysomes; (3) free polysomes t r e a t e d w i t h E D T A ; (4) b o u n d polysomes t r e a t e d with E D T A . G - Q , 3 ~ 0 nm; × - - - × , radioactivity.
Biochim. Biophys. Acta, 224 (197 o) 5o7-517
516
T. TANAKAet al.
after EDTA treatment indicates that large ribosomal sub-units banded at the density about 1.61 g/cm 3, and mRNA-containing particles with high specific activity were released from ribosomal subunits and distributed heterogeneously in a broad density range extending from 1.35 to 1.55 g/cm 3. It is noticeable that the specific activities of these ribonucleoprotein particles were markedly higher throughout this density range in free polysomes than in bound polysomes. On the contrary, the specific activity of ribosomal sub-units was similar between two classes of polysome.
DISCUSSION
Treatment of liver polysolnes with the microsomal deoxycholate-soluble fraction resulted in the degradation of mRNA, although rRNA remained intact. Ribonuclease is present in hepatic microsomal fraction 2a,24 and deoxycholate released this ribonuclease which causes the breakdown of liver polysomes into monomeric and dimeric ribosomes 16. Results of treatment of polysomes with dilute pancreatic ribonuclease were not reproducible, sometimes showing sedimentation patterns of the degradation of rRNA, in agreement with the results of FENWlCK25. Therefore, it is preferable to employ microsomal deoxycholate-soluble ribonuclease for the purpose of selective degradation of mRNA. The incorporation of I14CJorotic acid into mRNA, calculated as the difference in radioactivity between the total polysomal RNA and rRNA, was found to be higher in free polysomes than in bound polysomes. It was further shown by double-labeling experiments that the higher incorporation of I14Clorotic acid into mRNA of free polysomes was not due to the degradation of mRNA of bound polysomes during the preparative procedures including recentrifugation and resuspension of polysomes before treatment with sodium dodecyl sulfate. The radioactivity of mRNA calculated as above may include that of tRNA and 5-S RNA. However, methylated albumin column chromatography of the whole RNA of the total polysomes of rat liver, labeled with ElaC~orotic acid for 50 min, showed that the proportion of radioactivity of tRNA and 5 S RNA to that of the whole RNA was very small 26. The incorporation into these RNA's can be negligible, especially in early labeling periods. Furthermore, the difference in the rate of labeling between the two classes of polysome was shown to be neither due to the contamination of 45-S ribonucleoprotein particles with free polysomes nor to the existence of heterodisperse RNA which co-sediment with but are not attached to polysomes. Therefore, it seems reasonable to contend that the labeling rate of mRNA of free polysomes is more rapid than that of bound polysomes. More direct support for this contention was given by CsC1 equilibrium centrifugation of EDTAtreated polysomes pulse-labeled with ~l*C]orotic acid in vivo, since the specific activity of the mRNA-protein complex from free polysomes was markedly higher than that from bound polysomes. If our contention be accepted, three possibilities may exist; (I) Newly synthesized mRNA passes through a stage in which it is associated with free polysomes, later becoming bound to membranes. (2) mRNA in free polysomes turns over more rapidly. (3) There is more mRNA per ribosome in free polysomes. The third possibility is probably unlikely, since the activity in protein synthesis in vivo and in vitro was Biochim. Biophys. Acta, 224 (i97 o) 5o7-517
METABOLISM OF R N A
OE POLYSOMES
517
shown to be similar between two classes of polysome. It is impossible from this kind of experiment to distinguish between the first two possibilities, and further study must be done to elucidate these points. Recently, however, SARMA et al. 27 reported that the administration of actinomycin D to mouse resulted in marked disaggregation of hepatic free polysomes leaving bound polysomes intact. These findings suggest that m R N A in free polysomes turns over more rapidly. Recently, several reports 3-1° including those of the authors, have indicated differences in the kinds of protein synthesized by the two classes of polysome. As reported in our previous paper 3, these results suggest the possibility that the distribution of m R N A is different between the two kinds of polysome. The results of the present experiments provide evidence for a metabolic difference between the two classes of liver polysome. Our experiments showed that there is no difference in the turnover rate of rRNA between the two classes of liver polysome, in agreement with those of LOEB et al. 13. These results suggest that liver ribosomes are capable of changing between free and membrane-bound states or that there is no intrinsic difference in metabolism between them. 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
]:{. P. PERRY AND D. E. KELLEY, J. Mol. Biol., 35 (1968) 37E. C. HENSHAW, J. Mol. Biol., 36 (1968) 4Ol. 3/[. TAKAGI AND K. OGATA, Biochem. Biophys. Res. Commun., 33 (1968) 55. iV[. TAKAGI, T. TANAKA AND K. OGATA, J. Biochem., 65 (1969) 651. 1V[. TAKAGI, T. TANAKA AND K. OGATA, Biochim. Biophys. Acta, 217 (197o) 148. C. IV[. REDMAN, Biochem. Biophys. Res. Commun., 31 (1968) 845. T. HALLINAN, C. N. MURTV AND J. H. GRAND, Li/e Sci., 7 (1968) 225. S. J. HICKS, J. W. D. DRYSDALE AND H. ~'. MUNRO, Science, 164 (1969) 584 . C. ]~,{. REDMAN, J. Biol. Chem., 244 (1969) 43o8. M. C. GANOZA AND C. A. WILLIAMS, Proc. Natl. Acad. Sci. U.S., 63 (1969) 137 o. Y. MOULE AND G. DELHUMEAU DE ONGAY, Biochim. Biophys. Acta, 91 (1964) 113. T. HALLINAN AND H. MUNRO, Biochim. Biophys. Acta, lO8 (1965) 285. J. N. LOEB, R. R. HOWELL AND G. M. TOMKINS, J. Biol. Chem., 242 (1967) 2069. C. N. MURTY AND T. HALLINAN, Biochim. Biophys. Acta, 157 (1968) 414 . F. O. WETTSTEIN, T. STAEHELIN AND H. NOLL, Nature, 197 (1963) 778. H. SUGANO, I. WATANABE AND K. OGATA, J. Biochim., 61 (1967) 778. K. SHORTMAN, Biochim. Biophys. Acta, 51 (1961) 37. C. N. 1VIURTYAND T. HALLINAN, Biochem. J., 112 (1969) 269. E. C. I-[ENSHAW, M. REVEL AND n . H. HIATT, J. Mol. Biol., 14 (1965) 241. R. V. PERRY AND D. E. KELLEY, J. Mol. Biol., 16 (1966) 255. G. BLOBEL AND V. R. POTTER, J. Mol. Biol., 28 (1967) 539. S. PENMAN, C. VESCO AND IV[. PENMAN, J. Mol. Biol., 34 (1968) 49. C. DE I)UVE, B. C. PRESSMAN, R. GIANETTO, R. WATTIAUX AND F. APPLEMANS, Biochem. J., 6o (1955) 604. R. MORRIS AND G. D. LAMIRANDE, Biochim. Biophys. Acta, 95 (1965) 4 °. M. L. FENWlCK, Biochem. J., lO 7 (1968) 481. T. TANAKA AND I{. OGATA, u n p u b l i s h e d result. D. S. R. SARMA, I. M. REID AND H. SIDRANSKY, Biochem. Biophys. Res. Commun., 36 (1969) 582. C. F. BRUNK and V. LEICK, Biochim. Biophys. Acta, 179 (1969) 136.
Biochim. Biophys. Acta, 224 (197 o) 5o7-517