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RNA associated with the outer membrane of rat liver nuclei
BIOCHIMICA ET BIOPHYSICA ACTA
41
BBA 95917
RNA ASSOCIATED WITH THE OUTER MEMBRANE OF RAT LIVER NUCLEI ELIZABETH D. WHITTLE*, DONALD E. BUSHNELL AND VAN R. POTTER McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisc. (U.S.A.)
(Received February 9th, 1968)
SUMMARY
R a t liver nuclei still possessing a double m e m b r a n e were p r e p a r e d in a high s t a t e of p u r i t y b y a p u b l i s h e d sucrose m e t h o d . The o u t e r nuclear m e m b r a n e a n d its associated ribosomes-like p a r t i c l e s were r e m o v e d in a wash w i t h T r i t o n X - I o o . The R N A from the s e d i m e n t a b l e fraction of the T r i t o n wash was studied. I t r e p r e s e n t e d o n l y 0.2 % of t o t a l cellular R N A . The t i m e course of its labelling in vivo w i t h E~H]orotie acid was e x a m i n e d in r e l a t i o n to t h e labelling p a t t e r n s of nuclear R N A a n d t o t a l c y t o p l a s m i c r i b o s o m a l R N A . W h e n t h e results of an a p p r o p r i a t e control were t a k e n into account, it a p p e a r e d to h a v e t h e labelling kinetics of t o t a l c y t o p l a s m i c r i b o s o m a l R N A . I t gave a s e d i m e n t a t i o n profile in a sucrose d e n s i t y g r a d i e n t c h a r a c t e r i s t i c of r i b o s o m a l R N A . I t is c o n c l u d e d t h a 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 from nucleus to c y t o p l a s m either does not involve an i n t e r m e d i a t e association with the o u t e r nuclear m e m b r a n e , or t h a t , if it does, t h e association is so t r a n s i t o r y t h a t it was not detected.
INTRODUCTION E l e c t r o n m i c r o g r a p h s of tissue sections show t h a t nuclei possess a double m e m b r a n e 1-3, a n d suggest t h a t ribosomelike particles are a t t a c h e d to the o u t e r m e m b r a n e 4. I t is g e n e r a l l y a c c e p t e d t h a t t h e nucleolus is the site of synthesis of c y t o p l a s m i c r i b o s o m a l 28-S a n d I8-S R N A (ref. 5). There h a v e r e c e n t l y 6 been d e t e c t e d in a nuclear s u p e r n a t a n t fraction b o t h large a n d small r i b o n u c l e o p r o t e i n particles containing t h e n e w l y m a d e 28-S a n d I8-S R N A , respectively. E v i d e n c e has been p r o v i d e d t h a t t h e nuclear p a r t i c l e s are t h e precursors of n e w l y synthesized c y t o p l a s m i c ribos o m a l subunits. The s u b u n i t s m u s t therefore pass from nucleus to c y t o p l a s m t h r o u g h t h e double nuclear m e m b r a n e , Hence, it is of i n t e r e s t to e x a m i n e the n a t u r e of t h e R N A a s s o c i a t e d w i t h t h e o u t e r nuclear m e m b r a n e in relation to this p r o b l e m of t r a n s p o r t of r i b o s o m a l R N A . R e c e n t l y , BLOBEL AND POTTER 7 h a v e described a modified sucrose m e t h o d for o b t a i n i n g r a t liver nuclei in a high s t a t e of p u r i t y . E l e c t r o n m i c r o g r a p h s show t h e m still to possess a double m e m b r a n e . On t r e a t m e n t with t h e non-ionic d e t e r g e n t , T r i t o n X-IOO, the nuclei lose none of t h e i r D N A , b u t a b o u t 5 % of their R N A a n d t h e o u t e r m e m b r a n e w i t h its a t t a c h e d ribosomelike particles are removed. * Fellow of the Jane Coffin Childs Memorial Fund for Medical Research, 1965-1966. Present address: Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester 2o, Great Britain. Biochim. Biophys. Acta, 161 (1968) 41 5°
42
E.D.
W H I T T L E , D. E. B U S H N E L L , V. R. P O T T E R
In the present studies, the above procedures v were used with slight modifications so that RNA associated with the outer nuclear membrane could be recovered in a sedimentable fraction from the Triton wash with a minimum of nuclear and cytoplasmic contamination. The kinetics of its labelling with [3Hlorotic acid was examined in relation to the labelling of nuclear RNA and total cytoplasmic ribosomal RNA. It was characterized by its sedimentation profile in a sucrose density gradient. MATERIALS AND METHODS
[3H~orotic acid (14o mC/mmole) was obtained from the New England Nuclear Corporation, and [6-14Clorotic acid (34.7 mC/mmole) from Schwarz BioResearch, Inc. Triton X-Ioo and sodium lauryl sulphate were purchased from Mann Research Laboratories, Inc. Medium A consisted of o.o5 M Tris-HC1 (pH 7.7 at 2o°), o.o25 M KC1, and o.oo5 M MgCI2. Medium B was o.25 M sucrose in medium A. A high-speed supernatant fraction from rat liver, ($3), used as the source of a ribonuclease inhibitor 8,9, was obtained by the method of BLOBEL AND POTTERs. Two additional 4-h centrifugations at lO5 ooo x g were performed to reduce the possibility of contamination. The final $3 fraction was diluted with an equal volume of medium B and stored for up to 3 weeks in aliquots at --2o °. Male albino rats were obtained from the Holtzman Co., Madison, Wisc. On arrival they were housed in a windowless room with the lighting automatically regulated to provide I 2 h of light (9 p.m. to 9 a.m.) and i 2 h of darkness (9 a.m. to 9 p.m.). They were fed a 60 ~o protein dieO ° ad libitum for 3 days before the experiment. Rats were injected intraperitoneally with [aH]- or [6-14C~orotic acid. At given times after injection they were killed by decapitation without anaesthesia.
Cell/ractionalion procedure The liver was quickly removed and chilled in ice-cold medium B. All subsequent operations were carried out at temperatures near o °. The liver was cleaned and homogenized in 2 vol. of medium B as fully described by BLOBEL .aND POTTER7. Two 7-ml aliquots of the filtered homogenate were taken for the preparation of nuclei:. Each aliquot was mixed with 14 ml of 2.3 M sucrose in medium A, and the mixture was underlaid with 7 ml of 2.3 M sucrose in medium A. The tubes were spun in a Spinco No. SW 25.1 rotor at 25 ooo rev./min (63 o o o x g , average) for I h. The supernatant was poured off and the material adhering to the walls of the tube was added to the supernatant (post-nuclear supernatant fraction). The walls of the tube were wiped dry with absorbent paper wrapped around a spatula, then carefully washed with drops of medium B, and finally wiped dry. The nuclear pellet was taken up in 4 ml of medium B containing 5 ~o of Sa fraction and resedimented by centrifugation at 6oo × g for 15 rain. The nuclei were washed a second time with medium B containing5%ofS3. The two nuclear fractions from the same liver were then treated differently, as summarized in Fig. I. One nuclear fraction was suspended in 3.8 ml of medium B containing IO O//oof S3. o.2 ml of 2o % (w/v) Triton X-Ioo was added, and the suspension was mixed on the w~rtex mixer. It was spun for 15 rain at 6oo x g to give a pellet (T-nuclei) and supernatant (T-wash). The other nuclear fraction was suspended Biochim. I3iophys. Ac/a, a6~ (1908) 4 I - 5 °
OUTER NUCLEAR MEMBRANE R N A
43
Nuclei (Sucrose-prepared)
Washed once
with I% Triton
600 xg for IS min
600 xg forl 5 rain
I
I
Pellet T - nuclei
Supernatant T-wash
I ]
Pellet C-nuclei
Supernatant
1fo054000 x g
Triton added to
I%
C-wash II 05 000 x g 4h
for
I T-pellet
l T-supernatant
I C-pellet
l C-supernatant
Fig. i. Plan of f r a c t i o n a t i o n procedure whick is described more fully in METHODS.
in 3.8 ml of medium B containing IO ~o of S3, and spun for 15 rain at 6oo × g to give a pellet (C-nuclei) and a supernatant. 0.2 ml of 20 % Triton was added and mixed with the supernatant (C-wash). The T - a n d C-washes were treated similarly. 6 ml of medium B were added, and the mixture spun in a Spinco No. 40 rotor at 40 ooo rev./min (lO5 ooo x g, average) for 4 h to give a sediment (T- or C-pellet, respectivel y ), and supernatant (T- or C-supernatant, respectively).
Total cytoplasmic ribosomal [raction This was prepared from the post-nuclear supernatant essentially as described by BLOBEL AND POTTER 11, but a preliminary spin at 600 × g was included to remove quantitatively the remaining nuclei that contaminated this fraction 12.
Sedimentation pro/ile o / R N A in a sucrose gradient The pellet was suspended in 0.2 ml of 0.05 M sodium acetate (pH 5.o), 0.02 ml of io % (w/v) sodium lauryl sulphate, and o.oi ml of 0.5 M EDTA. The whole was mixed on a vortex mixer till solution was obtained, and then layered on top of a 4.5 ml linear gradient (15 to 30 % (w/w) sucrose in o.I M LiC1, o.oi M sodium acetate (pH 5.o), 0.25 % sodium lauryl sulphate, and o.ooi M EDTA). The tube was centrifuged in a Spinco No. SW 39 rotor at 39 ooo rev./min (124 o o o x g , average) for 4 h at 20 °. The gradient was eluted from the bottom of the tube at constant flow rate through a Gilford Model 200 continuously recording spectrophotometer measuring the absorbance at 260 In/,, and collected in fractions (12 drops). The fractions were cooled in ice and I m g of bovine serum albumin was added. Cold trichloroacetic acid was added to a concentration of IO %. The tubes were left in ice for a further 15 rain. The precipitates were collected on membrane filters, 0.45 # pore size. The dried filters were transferred to counting vials for determination of radioactivity. Biochim. Biophys. Acta, 161 (I968) 41-5 °
44
E.D.
WHITTLE, D. E. BUSHNELL, V. R. POTTER
Determination o / R N A RNA-containing fractions were acidified with perchloric acid to a final concentration of 0.2 M and washed twice with 0.2 M perchloric acid to remove all contaminating acid-soluble radioactivity and Triton. Tubes were inverted to drain. RNA was determined by the method of FLECK AND MUNRO13. The acidified alkaline digest of RNA was analysed for absorbance at 260 m/, and radioactivity. An absorbance of I.OOO at 260 m# for a i-cm light path was taken to be equivalent to 32/~g RNA per ml (ref. 14). Tubes containing residues for DNA determination were inverted to drain.
Determination o/radioactivity To o.5-ml aliquots in 0.2 1V[perchloric acid were added io ml of a scintillation solution ~5. Vials were counted in a Packard Tri-Carb scintillation counter, and quenching was corrected for by external standardization. IO ml of toluene-PPO were added to the membrane disks, and these vials were counted in the scintillation counter adjusted to have a wide window and high amplification.
Determination o~ DNA The pellets produced by acidification of the alkaline hydrolysates were dissolved in o. I M KOH and aliquots taken for DNA determination by the method of CERIOTTI~6 slightly modified 7. RESULTS
The results of a preliminary experiment to determine the time course of incorporation of [aHlorotic acid into RNA from Triton-washed nuclei and total cytoplasmic ribosomes (Fig. 2) showed that, at 3o min after injection, the specific activ-
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Fig. 2. Specific a c t i v i t y of R N A from T r i t o n - w a s h e d n u c l e i ( Q - Q ) a n d t o t a l c y t o p l a s m i c ribos o m e s ((2) O ) l a b e l l e d i~ vivo w i t h [3H~orotic acid. 17 r a t s ( w e i g h t r a n g e , i 5 5 - i 6 5 g) w e re i n j e c t e d b e t w e e n 9.2o a.in. a n d io.oo a.m. w i t h I ml of a s o l u t i o n c o n t a i n i n g IOo/~C of [3H~orotic a c i d (14o m C / m m o l e ) . R a t s were killed in g r o u p s of 3 (2 for t h e 3o-nlin t i m e p o i n t ) . N u c l e i w a s h e d w i t h 1 % T r i t o n X - I o o a n d t o t a l c y t o p l a s m i c r i b o s o m e s w e r e o b t a i n e d a n d a n a l v s e d as d e s c r i b e d in METHODS. A v e r a g e v a l u e s are given.
Biochim. B i o p h y s . . 4 c t a ,
i61 (1968) 41 5 °
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TABLE
I
I 2 3
Rat No.
42.0 29.6 31.9
O.225 O.172 O.175
o] homog, RNA 395 ° 2360 2800
288 288 33 °
1.38 1.70 2.14
~g
A mount
Activity (disint./min)
A clivily (disint./min)
A mount
%
C-pelletRNA
T-supernatant RNA
T-pelletRNA
~g
Control-wash
Triton-wash
O.OO7 O.OIO O.O12
o/ homog. RNA
%
2710 2075 3320
A ctivily (disint./min)
288 150 216
A ctivity (disint./min)
C-supernatant RNA
3 r a t s ( w e i g h t , 1 8 7 - 1 8 8 g) w e r e i n j e c t e d a t i o . o o a . n l . w i t h I m l of a s o l u t i o n c o n t a i n i n g i o o p C of [ D H ] o r o t i c a c i d (14o m C / p e r m m o l e ) . R a t s w e r e k i l l e d 3 ° n i i n a f t e r i n j e c t i o n a n d f r a c t i o n s w e r e p r e p a r e d a s g i v e n in METHODS a n d Fig. I. T h e r e s u l t s a r e e x p r e s s e d p e r 7 m l of l i v e r h o m o g e n a t e .
LABELLING OF RD-N~AFRACTIONS FROM TRITON- AND CONTROL-XVASHES OF RAT-LIVER NUCLEI 30 r a i n AFTER INJECTION OF [DH~OROTIC ACID
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40
E.D. WHITTLE, D. E. BUSHNELL, V. R. POTTER
ity of nuclear RNA was over 2oo times that of total cytoplasmic ribosomal RNA, but b y 24 h this ratio had been reduced to 1.8. In this experiment with 17 rats, 77.7-k 0.5 % of total homogenate DNA was recovered in the nuclear pellet. The percentage of total homogenate R N A recovered in the nuclear pellet and total cytoplasmic ribosomal RNA was 3 . 3 ~ o . o and 84.6±o.1, respectively. Since the amount of RNA present in the sedimentable fraction released b y Triton treatment of nuclei was found to represent only approx, o.2 % of total homogenate RNA (see also Table I), the danger of its contamination b y other cellular RNA components, especially the highly labelled nuclear RNA at early time points, was immediately apparent. A procedure which included a control was therefore required as an a t t e m p t to distinguish between the RNA that was specifically released from nuclei by Triton treatment and unspecific material present in a wash not containing Triton. The amount of sedimentable RNA in the control wash was reduced b y two prior washes of the nuclei with medium B containing 5 % of Sa fraction. Its amount in the control wash was found to be sensitive to the pH of the medium: this was increased if the p H were lowered to 7-35. Thus, a nuclear washing procedure was developed as summarized in Fig. I. Detailed results with 3o-min labelled livers are shown in Table I. Both Tand C-washes contained less than o.5 % of the DNA present in the nuclear fractions. A small amount of RNA appeared still to be present in the C-pellet, 3-7 ~'o of that in the T-pellet. The total radioactivity in both fractions was of the same order of magnitude, slightly higher in the T-pellet for rats I and 2, but lower for rat 3. Thus at this 3o-min time point it would seem that the activity in T-pellet RNA can be accounted for mainly b y a contamination with a non-membrane-bound fraction of very low RNA content but high activity. The activities in both T- and C-supernatant RNA were of the same order of magnitude and represented only 6-11 °/o of the activities of RNA in these washes. It was shown also for the 24-tl labelled liver that the activities in T- and C-supernatant RNA were low and similar (3o4 and 333 disint./min, respectively); however, the activity in T-pellet RNA (IO 9oo disint./min) had much increased, whereas it remained about the same in C-pellet RNA (275o disint./min). Therefore it would seem that labelled RNA that was specifically released b y Triton treatment of nuclei was sedimentable under the conditions used.
Time course o/incorporation o/ [3H]orotic acid into T-pellet R N A An experiment was then cairied out to relate the specific activity of T-pellet RNA to that of nuclear and total cytoplasmic ribosomal RNA at 3 time points, 40 rain, 6 tl, and 24 11 after a single injection of [3H~orotic acid (Table II). The corresponding specific activities of T- and C-nuclear RNA were especially close at tile 24-h time point, but at 4 ° min and 6 h the specific activity of T-nuclear RNA was slightly higher (3-8 °/o) as would be expected if the nuclei had lost through Triton treatment a small fraction of RNA of considerably lower specific activity. The specific activity of T-pellet RNA has been expressed in two ways: uncorrected, and corrected for activity and RNA content of the corresponding C-pellet. Considering first the specific activity of the uncorrected T-pellet RNA: at the 4o-min time point, it was approx. 6 times higher than that of total cytoplasmic ribosomal RNA, whereas at 6 and 24 h it was similar. It increased for each of the time intervals chosen, in particular, the 24-h value was considerably higher than the 6-h Biochim. Biophys. Acla, i6i (1908) 41 -50
OUTER NUCLEAR MEMBRANE R N A TABLE
47
II
SPECIFIC ACTIVITY OF R N A FRACTIONS FROM RAT LIVER AFTER INJECTION OF E3H~OROTIC ACID 8 r a t s ( w e i g h t r a n g e , 17o-18o g) w e r e i n j e c t e d b e t w e e n 9.20 a.m. a n d 9.4 ° a.m. w i t h I m l of a s o l u t i o n c o n t a i n i n g 1 I5 pC of [3H]orotic acid (14o m C / m m o l e ) . T h e y w e r e k i l l e d a t t h e t i m e s s h o w n a f t e r i n j e c t i o n . T h e l i v e r s w e r e r e m o v e d a n d f r a c t i o n s o b t a i n e d a n d a n a l y s e d as g i v e n i n METHODS a n d Fig. I.
Rat No.
Specific activity (disint./min per ttg R N A ) Time of killing
C-nuclear RNA
T-n~clear RNA
Total cytoplasmic ribosomal RNA
T-pellet RNA
Corrected T-pellet RNA *
3520 3050 4380 373 ° 375 ° 142o 133o 165o
375 ° 3260 461o 385 ° 404 ° 142o 136o 169o
24.8 19. 9 643 688 514 119o 994 lO6O
167 118 656 717 542 117o 989 lO4O
-- I3.6 --42.3 349 475 339 932 808 79 °
(h)
I 2 3 4 5 6 7 8
4omin 4omin 6 6 6 24 24 24
* C o r r e c t e d for R N A c o n t e n t a n d a c t i v i t y of c o r r e s p o n d i n g C-pellet.
value, as for cytoplasmic ribosomal RNA and very unlike the situation for nuclear RNA. In contrast to the uncorrected value for T-pellet RNA, the specific activity of C-pellet RNA (not shown) was very similar for all time points. Hence, the effect of correcting T-pellet RNA for RNA content and activity of the C-pellet was most marked
0.5
2BS
500
Q4
~-0.2 0 04
400
;00 18S
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100
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15 210 2L5 310 Froction No.
Fig. 3. S e d i m e n t a t i o n c h a r a c t e r i s t i c s of T - p e l l e t R N A l a b e l l e d in vivo w i t h [6-14C]orotic a c i d for 24 h. 3 r a t s (weight, 147 g) were i n j e c t e d w i t h i ml of a s o l u t i o n c o n t a i n i n g 18.6 ffC of [6-14C]o r o t i c acid (34.7 m C / m m o l e ) . A f t e r 24 h t h e r a t s w e r e k i l l e d a n d t h e i r l i v e r s w e r e r e m o v e d , hom o g e n i z e d a n d pooled. T h r e e 7-ml a l i q u o t s of t h e f i l t e r e d h o m o g e n a t e w e re t a k e n for p r e p a r a t i o n of t w o T - p e l l e t s a n d one C-pellet. The R N A in e a c h p e l l e t w a s s u b j e c t e d to s e d i m e n t a t i o n a n a l y s i s in a sucro se d e n s i t y g r a d i e n t as d e s c r i b e d in METHODS. T h e 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 b o t h T - p e l l e t R N A ' s w e r e t h e same. --, A260 my; © - - © , 14C r a d i o a c t i v i t y .
Biochim. Biophys. Acta, 161 (1968) 41-5 °
48
E.D.
W H I T T L E , D. E. B U S H N E L L , V. R. P O T T E R
at the early 4o-min time point. When the correction was made, the specific activity of T-pellet RNA was calculated to be negative, i.e., for this experiment there was less total activity in T-pellet RNA than in C-pellet RNA. This result should be compared with the results shown in Table I and, taken together, they indicate some variability, which could be due in part to experimental error. In addition, the possibility has to be borne in mind that the control m a y not have been ideal. Nevertheless, the results lead us to question the validity of the high value for the specific activity of the uncorrected T-pellet RNA at the early time point, and suggest that its true value at all 3 time points was nearly the same as that of total cytoplasmic ribosomal RNA.
Sedimentation analysis o/T-pellet RNA The sedimentation profile of T-pellet RNA labelled in vivo with I6-1aC]orotic acid for 24 h is shown in Fig. 3. The absorbance profile can be compared with that of RNA from the total cytoplasmic ribosomal fraction n. The two peaks corresponding to 28 S and 18 S and their relative magnitudes (2 : I) are characteristic of cytoplasmic ribosomal RNA (ref. 17). Furthermore, the radioactivity and absorbance profiles coincided, indicating that most of the radioactivity in T-pellet RNA at the 24-h time point was associated with ribosomal RNA. No significance could be attached to the sedimentation profile obtained with the corresponding C-pellet RNA on account of the small amount of material present such that values were not significantly above background.
DISCUSSION
In this study, the time course of labelling of the sedimentable fraction of RNA that is released b y Triton treatment of purified sucrose-prepared nuclei has been examined. The bulk of this RNA was shown b y sedimentation analysis to be ribosomal. However, if messenger RNA were present on the outer nuclear membrane we should expect it to be included in the sedimentable fraction, as the washing procedure was carried out in the presence of Sa fraction, which has been shown to inhibit ribonuclease activity 8,9. In addition, it was shown that 89 % or more of the RNA radioactivity in the Triton wash was associated with sedimentable RNA even at the early time point. Total cytoplasmic ribosomal RNA was prepared in a similar way to the sedimentable RNA fraction from the Triton wash, and would also contain messenger RNA. Hence the two fractions, T-pellet RNA and total cytoplasmic ribosomal RNA, would seem to be comparable in terms of classes of RNA present. Great care was taken to reduce cytoplasmic contamination of sucrose-prepared nuclei. We confirmed that the method 7 gave minimal contamination of nuclei with cytoplasmic endoplasmic reticulum and ribosomes, and we further reduced the possibility of contamination by washing the nuclei twice before treating with Triton. Furthermore, the RNA content of the C-pellet was consistently small, i.e., only 3-7 9/0 of that of the T-pellet. It is therefore considered that the total o.2 °/o of cellular RNA present in the T-pellet is accounted for mainly by the ribosome-like particles attached to the outer nuclear membrane. Assuming that this RNA is all of ribosomal origin, the number of ribosomes associated with the outer membrane of a cell nucleus can be estimated. Approx. 8o °/o of total liver RNA is ribosomal and there are approxiBiochim. Biophys..qcta, 161 ( I 9 0 8 ) 4 1 - 5 °
OUTER NUCLEAR MEMBRANE
RNA
49
mately 6. lO 6 ribosomes per average liver cell n. Thus there would be of the order of 15 ooo ribosomes on the outer membrane of a liver cell nucleus. <hough the C-pellet RN~A had low RNA content, it had considerable activity, which was similar for all time points investigated, and had a specific activity of the same order but less than that of total nuclear RN&. This radioactivity could not be removed b y repeated washing with 0.2 M perchloric acid. It probably represents a small fraction of highly labelled RNA that is not membrane bound but is released from broken nuclei b y washing. <hough the exact origin of the activity in C-pellet RNA is unknown, the necessity for taking it into account has been demonstrated when considering the significance of the specific activity of T-pellet RNA in relation to that of total cytoplasmic ribosomal RN& at the early time point. This correction reduces the specific activity of T-pellet RNA more nearly to that of cytoplasmic ribosomal RNA and suggests that the two are most probably not significantly different. At the 6- and 24-h time points the effect of the activity of the control was not so marked, and the specific activity of T-pellet RNA, whether corrected or not, was similar to that of cytoplasmic ribosomal RNA, being higher at the 24-h than at the 6-h time point. Newly-formed ribosomal RN& must pass relatively rapidly from nucleus to cytoplasm. Thus, if its transport were to involve an intermediate association with the outer nuclear membrane, we might expect that the specific activity of the small pool of T-pellet RNA would follow more closely the pattern of labelling of nuclear RNA, i.e., reach a m a x i m u m soon after the m a x i m u m specific activity of nuclear RN& and decline similarly (Fig. 2). Our data at the 6- and 24-h time points show clearly that this was not the case. We therefore conclude that, if the outer nuclear membrane is involved in the transport of newly synthesized ribosomal RN& from nucleus to cytoplasm, the association is so transitory that no evidence for it could be detected. Since it has been suggested ls,2° that a nucleus-associated polysome fraction is involved in the transport of rapidly labelled RN& from nucleus to cytoplasm, differences in experimental approach should be noted. LAWFORD, SADOWSKIAND SCHACHTERTM treated sucrose-prepared nnclei with the anionic detergent, sodium deoxycholate, which disrupts the inner as well as the outer membrane of rat liver nuclei causing fragmentation of nuclei. More recently, SADOWSKI AND ALCOCK 19 have noted in a brief report that RNA in a polysome fraction obtained by treating purified sucrose-prepared nuclei with Triton X-Ioo was higher in specific activity than RN& in a cytoplasmic polysome fraction 30 rain after injection of E14Clorotic acid. Our results would be in agreement if no correction is made for labelled material that is washed from the nuclei in the presence of ribonuclease inhibitor but in the absence of Triton X-Ioo. The highly labelled polysome fraction studied by BACH AND JOHNSON2°'21 was obtained by treating H e L a cell nuclei with DNA a]ter washing the nuclei with 0.25 % Triton X-Ioo. Thus, if the Triton treatment had been sufficiently vigorous, (it was repeated several times), the DN& extract could not include the ribosome-like particles on the outer nuclear membrane. In the passage of ribosomal RN& from nucleus to cytoplasm, the nuclear pore m a y be implicated, but no direct evidence for this has yet been obtained 22. Our data would be consistent with the hypothesis that subribosomal particles pass through the nuclear pores into the cytoplasm in a free (i.e., non-membrane-bound) form, there to form a small pool of newly formed subribosomal particles 23,6, before subsequent assembly into polysomes, both free and membrane bound. Biochim. Biophys. Acta,
161 (1968) 4 1 - 5 °
5°
E.D.
WHITTLE, D. E. BUSHNELL, V. R. POTTER
ACKNOWLEDGEMENTS
This work was supported by Grant CA-o7125 and Training Grant CRTY-5oo2 from the National Cancer Institute, U.S. Public Health Service, and a grant from the Jane Coffin Childs Memorial Fund for Medical Research.
REFERENCES i 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19 20 21 22 23
J. F. HARTMANN, J. Comp. Neurol., 99 (1953) 2Ol. D. W. FAWCETT, J. Natl. Cancer Inst., 15 (1955) 1475. M. L. WATSON, J. Biophys. Biochem. Cytol., i (1955) 257. G. E. PALADE, J. Biophys. Biochem. Cytol., i (1955) 59. R. P. PERRY, in J. N. DAVIDSON AND W. E. COHN ,Progress in Nucleic Acid Research and Molecular Biology, Vol. 6, Academic Press, New York, 1967, p. 219. M. H. VAUGHAN, J. R. WARNER AND J. E. DARNELL, J. Mol. Biol., 25 (1967) 235. G. BLOBEL AND V. R. POTTER, Science, 154 (1966) 1662. G. BLOBEL AND V. R. POTTER, Proc. Natl. Acad. Sci. U.S., 55 (1966) 1283. G. R. LAWFORD, P. LANGFORD AND H. SCHACHTER, J. Biol. Chem., 241 (1966) 1835. FI. C. PITOT, V. R. POTTER AND H. P. MORRIS, Cancer Res., 21 (1961) IOOi. G. BLOBEL AND V. R. POTTER, J. Mol. Biol., 26 (1967) 279. D. E. BUSHNELL, E. D. WHITTLE AND V. R. POTTER, in p r e p a r a t i o n . A. FLECK AND H. N. MUNRO, Biochim. Biophys. Acta, 55 (1962) 571. H. N. MUNRO AND A. FLECK, Analyst, 91 (1966) 78. F. E. KINARD, Rev. Scient. Inst., 28 (1957) 293. G. CERIOTTI, J. Biol. Chem., 198 (1952) 297. K. S. KIRBY, Biochem. J., 96 (1965) 266. G. R. LAWFORD, P. SADOWSKI ANn H. SCHACHTER, J. Mol. Biol., 23 (1967) 81. P. D. SADOWSKI AND J. ALCOCK, Federation Proc., 26 (1967) 514 . M. K. BACH AND ]7I. G. JOHNSON, Nature, 2o9 (1966) 893. M. K. BACH AND H. G. JOHNSON, Biochemistry, 6 (1967) 1916. W. BERNHARD, Natl. Cancer Inst. Monograph, 23 (1966) 13. M. GIRARD, H. LATHAM, S. PENMAN AND J. E. DARNELL, J. Mol. Biol., i i (1965) 187.
Biochim. Biophys. Acta, 161 (1968) 41-5 °
41
BBA 95917
RNA ASSOCIATED WITH THE OUTER MEMBRANE OF RAT LIVER NUCLEI ELIZABETH D. WHITTLE*, DONALD E. BUSHNELL AND VAN R. POTTER McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisc. (U.S.A.)
(Received February 9th, 1968)
SUMMARY
R a t liver nuclei still possessing a double m e m b r a n e were p r e p a r e d in a high s t a t e of p u r i t y b y a p u b l i s h e d sucrose m e t h o d . The o u t e r nuclear m e m b r a n e a n d its associated ribosomes-like p a r t i c l e s were r e m o v e d in a wash w i t h T r i t o n X - I o o . The R N A from the s e d i m e n t a b l e fraction of the T r i t o n wash was studied. I t r e p r e s e n t e d o n l y 0.2 % of t o t a l cellular R N A . The t i m e course of its labelling in vivo w i t h E~H]orotie acid was e x a m i n e d in r e l a t i o n to t h e labelling p a t t e r n s of nuclear R N A a n d t o t a l c y t o p l a s m i c r i b o s o m a l R N A . W h e n t h e results of an a p p r o p r i a t e control were t a k e n into account, it a p p e a r e d to h a v e t h e labelling kinetics of t o t a l c y t o p l a s m i c r i b o s o m a l R N A . I t gave a s e d i m e n t a t i o n profile in a sucrose d e n s i t y g r a d i e n t c h a r a c t e r i s t i c of r i b o s o m a l R N A . I t is c o n c l u d e d t h a 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 from nucleus to c y t o p l a s m either does not involve an i n t e r m e d i a t e association with the o u t e r nuclear m e m b r a n e , or t h a t , if it does, t h e association is so t r a n s i t o r y t h a t it was not detected.
INTRODUCTION E l e c t r o n m i c r o g r a p h s of tissue sections show t h a t nuclei possess a double m e m b r a n e 1-3, a n d suggest t h a t ribosomelike particles are a t t a c h e d to the o u t e r m e m b r a n e 4. I t is g e n e r a l l y a c c e p t e d t h a t t h e nucleolus is the site of synthesis of c y t o p l a s m i c r i b o s o m a l 28-S a n d I8-S R N A (ref. 5). There h a v e r e c e n t l y 6 been d e t e c t e d in a nuclear s u p e r n a t a n t fraction b o t h large a n d small r i b o n u c l e o p r o t e i n particles containing t h e n e w l y m a d e 28-S a n d I8-S R N A , respectively. E v i d e n c e has been p r o v i d e d t h a t t h e nuclear p a r t i c l e s are t h e precursors of n e w l y synthesized c y t o p l a s m i c ribos o m a l subunits. The s u b u n i t s m u s t therefore pass from nucleus to c y t o p l a s m t h r o u g h t h e double nuclear m e m b r a n e , Hence, it is of i n t e r e s t to e x a m i n e the n a t u r e of t h e R N A a s s o c i a t e d w i t h t h e o u t e r nuclear m e m b r a n e in relation to this p r o b l e m of t r a n s p o r t of r i b o s o m a l R N A . R e c e n t l y , BLOBEL AND POTTER 7 h a v e described a modified sucrose m e t h o d for o b t a i n i n g r a t liver nuclei in a high s t a t e of p u r i t y . E l e c t r o n m i c r o g r a p h s show t h e m still to possess a double m e m b r a n e . On t r e a t m e n t with t h e non-ionic d e t e r g e n t , T r i t o n X-IOO, the nuclei lose none of t h e i r D N A , b u t a b o u t 5 % of their R N A a n d t h e o u t e r m e m b r a n e w i t h its a t t a c h e d ribosomelike particles are removed. * Fellow of the Jane Coffin Childs Memorial Fund for Medical Research, 1965-1966. Present address: Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester 2o, Great Britain. Biochim. Biophys. Acta, 161 (1968) 41 5°
42
E.D.
W H I T T L E , D. E. B U S H N E L L , V. R. P O T T E R
In the present studies, the above procedures v were used with slight modifications so that RNA associated with the outer nuclear membrane could be recovered in a sedimentable fraction from the Triton wash with a minimum of nuclear and cytoplasmic contamination. The kinetics of its labelling with [3Hlorotic acid was examined in relation to the labelling of nuclear RNA and total cytoplasmic ribosomal RNA. It was characterized by its sedimentation profile in a sucrose density gradient. MATERIALS AND METHODS
[3H~orotic acid (14o mC/mmole) was obtained from the New England Nuclear Corporation, and [6-14Clorotic acid (34.7 mC/mmole) from Schwarz BioResearch, Inc. Triton X-Ioo and sodium lauryl sulphate were purchased from Mann Research Laboratories, Inc. Medium A consisted of o.o5 M Tris-HC1 (pH 7.7 at 2o°), o.o25 M KC1, and o.oo5 M MgCI2. Medium B was o.25 M sucrose in medium A. A high-speed supernatant fraction from rat liver, ($3), used as the source of a ribonuclease inhibitor 8,9, was obtained by the method of BLOBEL AND POTTERs. Two additional 4-h centrifugations at lO5 ooo x g were performed to reduce the possibility of contamination. The final $3 fraction was diluted with an equal volume of medium B and stored for up to 3 weeks in aliquots at --2o °. Male albino rats were obtained from the Holtzman Co., Madison, Wisc. On arrival they were housed in a windowless room with the lighting automatically regulated to provide I 2 h of light (9 p.m. to 9 a.m.) and i 2 h of darkness (9 a.m. to 9 p.m.). They were fed a 60 ~o protein dieO ° ad libitum for 3 days before the experiment. Rats were injected intraperitoneally with [aH]- or [6-14C~orotic acid. At given times after injection they were killed by decapitation without anaesthesia.
Cell/ractionalion procedure The liver was quickly removed and chilled in ice-cold medium B. All subsequent operations were carried out at temperatures near o °. The liver was cleaned and homogenized in 2 vol. of medium B as fully described by BLOBEL .aND POTTER7. Two 7-ml aliquots of the filtered homogenate were taken for the preparation of nuclei:. Each aliquot was mixed with 14 ml of 2.3 M sucrose in medium A, and the mixture was underlaid with 7 ml of 2.3 M sucrose in medium A. The tubes were spun in a Spinco No. SW 25.1 rotor at 25 ooo rev./min (63 o o o x g , average) for I h. The supernatant was poured off and the material adhering to the walls of the tube was added to the supernatant (post-nuclear supernatant fraction). The walls of the tube were wiped dry with absorbent paper wrapped around a spatula, then carefully washed with drops of medium B, and finally wiped dry. The nuclear pellet was taken up in 4 ml of medium B containing 5 ~o of Sa fraction and resedimented by centrifugation at 6oo × g for 15 rain. The nuclei were washed a second time with medium B containing5%ofS3. The two nuclear fractions from the same liver were then treated differently, as summarized in Fig. I. One nuclear fraction was suspended in 3.8 ml of medium B containing IO O//oof S3. o.2 ml of 2o % (w/v) Triton X-Ioo was added, and the suspension was mixed on the w~rtex mixer. It was spun for 15 rain at 6oo x g to give a pellet (T-nuclei) and supernatant (T-wash). The other nuclear fraction was suspended Biochim. I3iophys. Ac/a, a6~ (1908) 4 I - 5 °
OUTER NUCLEAR MEMBRANE R N A
43
Nuclei (Sucrose-prepared)
Washed once
with I% Triton
600 xg for IS min
600 xg forl 5 rain
I
I
Pellet T - nuclei
Supernatant T-wash
I ]
Pellet C-nuclei
Supernatant
1fo054000 x g
Triton added to
I%
C-wash II 05 000 x g 4h
for
I T-pellet
l T-supernatant
I C-pellet
l C-supernatant
Fig. i. Plan of f r a c t i o n a t i o n procedure whick is described more fully in METHODS.
in 3.8 ml of medium B containing IO ~o of S3, and spun for 15 rain at 6oo × g to give a pellet (C-nuclei) and a supernatant. 0.2 ml of 20 % Triton was added and mixed with the supernatant (C-wash). The T - a n d C-washes were treated similarly. 6 ml of medium B were added, and the mixture spun in a Spinco No. 40 rotor at 40 ooo rev./min (lO5 ooo x g, average) for 4 h to give a sediment (T- or C-pellet, respectivel y ), and supernatant (T- or C-supernatant, respectively).
Total cytoplasmic ribosomal [raction This was prepared from the post-nuclear supernatant essentially as described by BLOBEL AND POTTER 11, but a preliminary spin at 600 × g was included to remove quantitatively the remaining nuclei that contaminated this fraction 12.
Sedimentation pro/ile o / R N A in a sucrose gradient The pellet was suspended in 0.2 ml of 0.05 M sodium acetate (pH 5.o), 0.02 ml of io % (w/v) sodium lauryl sulphate, and o.oi ml of 0.5 M EDTA. The whole was mixed on a vortex mixer till solution was obtained, and then layered on top of a 4.5 ml linear gradient (15 to 30 % (w/w) sucrose in o.I M LiC1, o.oi M sodium acetate (pH 5.o), 0.25 % sodium lauryl sulphate, and o.ooi M EDTA). The tube was centrifuged in a Spinco No. SW 39 rotor at 39 ooo rev./min (124 o o o x g , average) for 4 h at 20 °. The gradient was eluted from the bottom of the tube at constant flow rate through a Gilford Model 200 continuously recording spectrophotometer measuring the absorbance at 260 In/,, and collected in fractions (12 drops). The fractions were cooled in ice and I m g of bovine serum albumin was added. Cold trichloroacetic acid was added to a concentration of IO %. The tubes were left in ice for a further 15 rain. The precipitates were collected on membrane filters, 0.45 # pore size. The dried filters were transferred to counting vials for determination of radioactivity. Biochim. Biophys. Acta, 161 (I968) 41-5 °
44
E.D.
WHITTLE, D. E. BUSHNELL, V. R. POTTER
Determination o / R N A RNA-containing fractions were acidified with perchloric acid to a final concentration of 0.2 M and washed twice with 0.2 M perchloric acid to remove all contaminating acid-soluble radioactivity and Triton. Tubes were inverted to drain. RNA was determined by the method of FLECK AND MUNRO13. The acidified alkaline digest of RNA was analysed for absorbance at 260 m/, and radioactivity. An absorbance of I.OOO at 260 m# for a i-cm light path was taken to be equivalent to 32/~g RNA per ml (ref. 14). Tubes containing residues for DNA determination were inverted to drain.
Determination o/radioactivity To o.5-ml aliquots in 0.2 1V[perchloric acid were added io ml of a scintillation solution ~5. Vials were counted in a Packard Tri-Carb scintillation counter, and quenching was corrected for by external standardization. IO ml of toluene-PPO were added to the membrane disks, and these vials were counted in the scintillation counter adjusted to have a wide window and high amplification.
Determination o~ DNA The pellets produced by acidification of the alkaline hydrolysates were dissolved in o. I M KOH and aliquots taken for DNA determination by the method of CERIOTTI~6 slightly modified 7. RESULTS
The results of a preliminary experiment to determine the time course of incorporation of [aHlorotic acid into RNA from Triton-washed nuclei and total cytoplasmic ribosomes (Fig. 2) showed that, at 3o min after injection, the specific activ-
z
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g o
O
~
Ln O 6 Time atter
•
i
i
12 18 i n j e c t i o n (h)
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i
24
Fig. 2. Specific a c t i v i t y of R N A from T r i t o n - w a s h e d n u c l e i ( Q - Q ) a n d t o t a l c y t o p l a s m i c ribos o m e s ((2) O ) l a b e l l e d i~ vivo w i t h [3H~orotic acid. 17 r a t s ( w e i g h t r a n g e , i 5 5 - i 6 5 g) w e re i n j e c t e d b e t w e e n 9.2o a.in. a n d io.oo a.m. w i t h I ml of a s o l u t i o n c o n t a i n i n g IOo/~C of [3H~orotic a c i d (14o m C / m m o l e ) . R a t s were killed in g r o u p s of 3 (2 for t h e 3o-nlin t i m e p o i n t ) . N u c l e i w a s h e d w i t h 1 % T r i t o n X - I o o a n d t o t a l c y t o p l a s m i c r i b o s o m e s w e r e o b t a i n e d a n d a n a l v s e d as d e s c r i b e d in METHODS. A v e r a g e v a l u e s are given.
Biochim. B i o p h y s . . 4 c t a ,
i61 (1968) 41 5 °
kaa O
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TABLE
I
I 2 3
Rat No.
42.0 29.6 31.9
O.225 O.172 O.175
o] homog, RNA 395 ° 2360 2800
288 288 33 °
1.38 1.70 2.14
~g
A mount
Activity (disint./min)
A clivily (disint./min)
A mount
%
C-pelletRNA
T-supernatant RNA
T-pelletRNA
~g
Control-wash
Triton-wash
O.OO7 O.OIO O.O12
o/ homog. RNA
%
2710 2075 3320
A ctivily (disint./min)
288 150 216
A ctivity (disint./min)
C-supernatant RNA
3 r a t s ( w e i g h t , 1 8 7 - 1 8 8 g) w e r e i n j e c t e d a t i o . o o a . n l . w i t h I m l of a s o l u t i o n c o n t a i n i n g i o o p C of [ D H ] o r o t i c a c i d (14o m C / p e r m m o l e ) . R a t s w e r e k i l l e d 3 ° n i i n a f t e r i n j e c t i o n a n d f r a c t i o n s w e r e p r e p a r e d a s g i v e n in METHODS a n d Fig. I. T h e r e s u l t s a r e e x p r e s s e d p e r 7 m l of l i v e r h o m o g e n a t e .
LABELLING OF RD-N~AFRACTIONS FROM TRITON- AND CONTROL-XVASHES OF RAT-LIVER NUCLEI 30 r a i n AFTER INJECTION OF [DH~OROTIC ACID
z.n
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40
E.D. WHITTLE, D. E. BUSHNELL, V. R. POTTER
ity of nuclear RNA was over 2oo times that of total cytoplasmic ribosomal RNA, but b y 24 h this ratio had been reduced to 1.8. In this experiment with 17 rats, 77.7-k 0.5 % of total homogenate DNA was recovered in the nuclear pellet. The percentage of total homogenate R N A recovered in the nuclear pellet and total cytoplasmic ribosomal RNA was 3 . 3 ~ o . o and 84.6±o.1, respectively. Since the amount of RNA present in the sedimentable fraction released b y Triton treatment of nuclei was found to represent only approx, o.2 % of total homogenate RNA (see also Table I), the danger of its contamination b y other cellular RNA components, especially the highly labelled nuclear RNA at early time points, was immediately apparent. A procedure which included a control was therefore required as an a t t e m p t to distinguish between the RNA that was specifically released from nuclei by Triton treatment and unspecific material present in a wash not containing Triton. The amount of sedimentable RNA in the control wash was reduced b y two prior washes of the nuclei with medium B containing 5 % of Sa fraction. Its amount in the control wash was found to be sensitive to the pH of the medium: this was increased if the p H were lowered to 7-35. Thus, a nuclear washing procedure was developed as summarized in Fig. I. Detailed results with 3o-min labelled livers are shown in Table I. Both Tand C-washes contained less than o.5 % of the DNA present in the nuclear fractions. A small amount of RNA appeared still to be present in the C-pellet, 3-7 ~'o of that in the T-pellet. The total radioactivity in both fractions was of the same order of magnitude, slightly higher in the T-pellet for rats I and 2, but lower for rat 3. Thus at this 3o-min time point it would seem that the activity in T-pellet RNA can be accounted for mainly b y a contamination with a non-membrane-bound fraction of very low RNA content but high activity. The activities in both T- and C-supernatant RNA were of the same order of magnitude and represented only 6-11 °/o of the activities of RNA in these washes. It was shown also for the 24-tl labelled liver that the activities in T- and C-supernatant RNA were low and similar (3o4 and 333 disint./min, respectively); however, the activity in T-pellet RNA (IO 9oo disint./min) had much increased, whereas it remained about the same in C-pellet RNA (275o disint./min). Therefore it would seem that labelled RNA that was specifically released b y Triton treatment of nuclei was sedimentable under the conditions used.
Time course o/incorporation o/ [3H]orotic acid into T-pellet R N A An experiment was then cairied out to relate the specific activity of T-pellet RNA to that of nuclear and total cytoplasmic ribosomal RNA at 3 time points, 40 rain, 6 tl, and 24 11 after a single injection of [3H~orotic acid (Table II). The corresponding specific activities of T- and C-nuclear RNA were especially close at tile 24-h time point, but at 4 ° min and 6 h the specific activity of T-nuclear RNA was slightly higher (3-8 °/o) as would be expected if the nuclei had lost through Triton treatment a small fraction of RNA of considerably lower specific activity. The specific activity of T-pellet RNA has been expressed in two ways: uncorrected, and corrected for activity and RNA content of the corresponding C-pellet. Considering first the specific activity of the uncorrected T-pellet RNA: at the 4o-min time point, it was approx. 6 times higher than that of total cytoplasmic ribosomal RNA, whereas at 6 and 24 h it was similar. It increased for each of the time intervals chosen, in particular, the 24-h value was considerably higher than the 6-h Biochim. Biophys. Acla, i6i (1908) 41 -50
OUTER NUCLEAR MEMBRANE R N A TABLE
47
II
SPECIFIC ACTIVITY OF R N A FRACTIONS FROM RAT LIVER AFTER INJECTION OF E3H~OROTIC ACID 8 r a t s ( w e i g h t r a n g e , 17o-18o g) w e r e i n j e c t e d b e t w e e n 9.20 a.m. a n d 9.4 ° a.m. w i t h I m l of a s o l u t i o n c o n t a i n i n g 1 I5 pC of [3H]orotic acid (14o m C / m m o l e ) . T h e y w e r e k i l l e d a t t h e t i m e s s h o w n a f t e r i n j e c t i o n . T h e l i v e r s w e r e r e m o v e d a n d f r a c t i o n s o b t a i n e d a n d a n a l y s e d as g i v e n i n METHODS a n d Fig. I.
Rat No.
Specific activity (disint./min per ttg R N A ) Time of killing
C-nuclear RNA
T-n~clear RNA
Total cytoplasmic ribosomal RNA
T-pellet RNA
Corrected T-pellet RNA *
3520 3050 4380 373 ° 375 ° 142o 133o 165o
375 ° 3260 461o 385 ° 404 ° 142o 136o 169o
24.8 19. 9 643 688 514 119o 994 lO6O
167 118 656 717 542 117o 989 lO4O
-- I3.6 --42.3 349 475 339 932 808 79 °
(h)
I 2 3 4 5 6 7 8
4omin 4omin 6 6 6 24 24 24
* C o r r e c t e d for R N A c o n t e n t a n d a c t i v i t y of c o r r e s p o n d i n g C-pellet.
value, as for cytoplasmic ribosomal RNA and very unlike the situation for nuclear RNA. In contrast to the uncorrected value for T-pellet RNA, the specific activity of C-pellet RNA (not shown) was very similar for all time points. Hence, the effect of correcting T-pellet RNA for RNA content and activity of the C-pellet was most marked
0.5
2BS
500
Q4
~-0.2 0 04
400
;00 18S
0.2
._c E
~00 i
i:: o
100
mo 0.1 L] <
%
15 210 2L5 310 Froction No.
Fig. 3. S e d i m e n t a t i o n c h a r a c t e r i s t i c s of T - p e l l e t R N A l a b e l l e d in vivo w i t h [6-14C]orotic a c i d for 24 h. 3 r a t s (weight, 147 g) were i n j e c t e d w i t h i ml of a s o l u t i o n c o n t a i n i n g 18.6 ffC of [6-14C]o r o t i c acid (34.7 m C / m m o l e ) . A f t e r 24 h t h e r a t s w e r e k i l l e d a n d t h e i r l i v e r s w e r e r e m o v e d , hom o g e n i z e d a n d pooled. T h r e e 7-ml a l i q u o t s of t h e f i l t e r e d h o m o g e n a t e w e re t a k e n for p r e p a r a t i o n of t w o T - p e l l e t s a n d one C-pellet. The R N A in e a c h p e l l e t w a s s u b j e c t e d to s e d i m e n t a t i o n a n a l y s i s in a sucro se d e n s i t y g r a d i e n t as d e s c r i b e d in METHODS. T h e 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 b o t h T - p e l l e t R N A ' s w e r e t h e same. --, A260 my; © - - © , 14C r a d i o a c t i v i t y .
Biochim. Biophys. Acta, 161 (1968) 41-5 °
48
E.D.
W H I T T L E , D. E. B U S H N E L L , V. R. P O T T E R
at the early 4o-min time point. When the correction was made, the specific activity of T-pellet RNA was calculated to be negative, i.e., for this experiment there was less total activity in T-pellet RNA than in C-pellet RNA. This result should be compared with the results shown in Table I and, taken together, they indicate some variability, which could be due in part to experimental error. In addition, the possibility has to be borne in mind that the control m a y not have been ideal. Nevertheless, the results lead us to question the validity of the high value for the specific activity of the uncorrected T-pellet RNA at the early time point, and suggest that its true value at all 3 time points was nearly the same as that of total cytoplasmic ribosomal RNA.
Sedimentation analysis o/T-pellet RNA The sedimentation profile of T-pellet RNA labelled in vivo with I6-1aC]orotic acid for 24 h is shown in Fig. 3. The absorbance profile can be compared with that of RNA from the total cytoplasmic ribosomal fraction n. The two peaks corresponding to 28 S and 18 S and their relative magnitudes (2 : I) are characteristic of cytoplasmic ribosomal RNA (ref. 17). Furthermore, the radioactivity and absorbance profiles coincided, indicating that most of the radioactivity in T-pellet RNA at the 24-h time point was associated with ribosomal RNA. No significance could be attached to the sedimentation profile obtained with the corresponding C-pellet RNA on account of the small amount of material present such that values were not significantly above background.
DISCUSSION
In this study, the time course of labelling of the sedimentable fraction of RNA that is released b y Triton treatment of purified sucrose-prepared nuclei has been examined. The bulk of this RNA was shown b y sedimentation analysis to be ribosomal. However, if messenger RNA were present on the outer nuclear membrane we should expect it to be included in the sedimentable fraction, as the washing procedure was carried out in the presence of Sa fraction, which has been shown to inhibit ribonuclease activity 8,9. In addition, it was shown that 89 % or more of the RNA radioactivity in the Triton wash was associated with sedimentable RNA even at the early time point. Total cytoplasmic ribosomal RNA was prepared in a similar way to the sedimentable RNA fraction from the Triton wash, and would also contain messenger RNA. Hence the two fractions, T-pellet RNA and total cytoplasmic ribosomal RNA, would seem to be comparable in terms of classes of RNA present. Great care was taken to reduce cytoplasmic contamination of sucrose-prepared nuclei. We confirmed that the method 7 gave minimal contamination of nuclei with cytoplasmic endoplasmic reticulum and ribosomes, and we further reduced the possibility of contamination by washing the nuclei twice before treating with Triton. Furthermore, the RNA content of the C-pellet was consistently small, i.e., only 3-7 9/0 of that of the T-pellet. It is therefore considered that the total o.2 °/o of cellular RNA present in the T-pellet is accounted for mainly by the ribosome-like particles attached to the outer nuclear membrane. Assuming that this RNA is all of ribosomal origin, the number of ribosomes associated with the outer membrane of a cell nucleus can be estimated. Approx. 8o °/o of total liver RNA is ribosomal and there are approxiBiochim. Biophys..qcta, 161 ( I 9 0 8 ) 4 1 - 5 °
OUTER NUCLEAR MEMBRANE
RNA
49
mately 6. lO 6 ribosomes per average liver cell n. Thus there would be of the order of 15 ooo ribosomes on the outer membrane of a liver cell nucleus. <hough the C-pellet RN~A had low RNA content, it had considerable activity, which was similar for all time points investigated, and had a specific activity of the same order but less than that of total nuclear RN&. This radioactivity could not be removed b y repeated washing with 0.2 M perchloric acid. It probably represents a small fraction of highly labelled RNA that is not membrane bound but is released from broken nuclei b y washing. <hough the exact origin of the activity in C-pellet RNA is unknown, the necessity for taking it into account has been demonstrated when considering the significance of the specific activity of T-pellet RNA in relation to that of total cytoplasmic ribosomal RN& at the early time point. This correction reduces the specific activity of T-pellet RNA more nearly to that of cytoplasmic ribosomal RNA and suggests that the two are most probably not significantly different. At the 6- and 24-h time points the effect of the activity of the control was not so marked, and the specific activity of T-pellet RNA, whether corrected or not, was similar to that of cytoplasmic ribosomal RNA, being higher at the 24-h than at the 6-h time point. Newly-formed ribosomal RN& must pass relatively rapidly from nucleus to cytoplasm. Thus, if its transport were to involve an intermediate association with the outer nuclear membrane, we might expect that the specific activity of the small pool of T-pellet RNA would follow more closely the pattern of labelling of nuclear RNA, i.e., reach a m a x i m u m soon after the m a x i m u m specific activity of nuclear RN& and decline similarly (Fig. 2). Our data at the 6- and 24-h time points show clearly that this was not the case. We therefore conclude that, if the outer nuclear membrane is involved in the transport of newly synthesized ribosomal RN& from nucleus to cytoplasm, the association is so transitory that no evidence for it could be detected. Since it has been suggested ls,2° that a nucleus-associated polysome fraction is involved in the transport of rapidly labelled RN& from nucleus to cytoplasm, differences in experimental approach should be noted. LAWFORD, SADOWSKIAND SCHACHTERTM treated sucrose-prepared nnclei with the anionic detergent, sodium deoxycholate, which disrupts the inner as well as the outer membrane of rat liver nuclei causing fragmentation of nuclei. More recently, SADOWSKI AND ALCOCK 19 have noted in a brief report that RNA in a polysome fraction obtained by treating purified sucrose-prepared nuclei with Triton X-Ioo was higher in specific activity than RN& in a cytoplasmic polysome fraction 30 rain after injection of E14Clorotic acid. Our results would be in agreement if no correction is made for labelled material that is washed from the nuclei in the presence of ribonuclease inhibitor but in the absence of Triton X-Ioo. The highly labelled polysome fraction studied by BACH AND JOHNSON2°'21 was obtained by treating H e L a cell nuclei with DNA a]ter washing the nuclei with 0.25 % Triton X-Ioo. Thus, if the Triton treatment had been sufficiently vigorous, (it was repeated several times), the DN& extract could not include the ribosome-like particles on the outer nuclear membrane. In the passage of ribosomal RN& from nucleus to cytoplasm, the nuclear pore m a y be implicated, but no direct evidence for this has yet been obtained 22. Our data would be consistent with the hypothesis that subribosomal particles pass through the nuclear pores into the cytoplasm in a free (i.e., non-membrane-bound) form, there to form a small pool of newly formed subribosomal particles 23,6, before subsequent assembly into polysomes, both free and membrane bound. Biochim. Biophys. Acta,
161 (1968) 4 1 - 5 °
5°
E.D.
WHITTLE, D. E. BUSHNELL, V. R. POTTER
ACKNOWLEDGEMENTS
This work was supported by Grant CA-o7125 and Training Grant CRTY-5oo2 from the National Cancer Institute, U.S. Public Health Service, and a grant from the Jane Coffin Childs Memorial Fund for Medical Research.
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