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The production of ribosomal RNA from high molecular weight precursors I. Factors which influence the ability of isolated nucleoli to process 45-S RNA
196
BIOCHIMICAET BIOPHYSICAACTA
BBA 96032
T H E P R O D U C T I O N OF RIBOSOMAL RNA FROM H I G H MOLECULAR WEIGHT PRECURSORS I. FACTORS W H I C H I N F L U E N C E T H E A B I L I T Y OF ISOLATED N U C L E O L I TO PROCESS 45-S RNA
M. C. LIAU, N. C. CRAIG AND R. l:). PERRY The Institute /or Cancer Research, Fox Chase, Philadelphia, Pa. I 9 z H (U.S.A.)
(Received May 27th, 1968)
SUMMARY A search was made for conditions which would allow isolated nucleoli to convert 45-S RNA in vitro to 28-S and IS-S ribosomal RNA or to other natural derivatives. The nuclease activity of nucleoli purified from L cells m a y be altered or controlled as follows: (a) if incubation of nucleoli in vitro is preceded by a wash with relatively high concentrations (about IOO/,g/ml) of polyvinyl sulfate or with 0.5 M NaC1 and deoxyribonuclease, nuclease activity is almost totally suppressed; (b) for extensive degradation of 45-S R N A to occur Mg 2+ must be present in the incubation medium; and (c) inclusion of bulk quantities of R N A in the incubation medium allows the selective breakdown of 45-S RNA without the production of heterogeneous fragments. After incubation for 30 rain at 3 °o in a medium containing 0.5 mM Mg 2+ and IOO200/~g RNA per ml, 45-S nucleolar RNA was degraded, and a limited quantity of fragments which migrated in acrylamide gels like the 32-S component and another intermediate were produced. Under these conditions extrinsic 45-S RNA which was incubated together with the nucleoli was also degraded, but extrinsic 32-S, 28-S and I8-S RNA underwent little or no degradation. The fragments produced during incubation in vitro were enriched in their methyl group/uridine ratio as compared to the 45-S component, although the enrichment was not as great as that observed after conversion in vivo. These results suggest a tentative model for the conversion process whereby 45-S RNA undergoes a single scission with an endonuclease followed b y a stepwise trimming with exonuclease.
INTRODUCTION Ribosome synthesis in higher organisms occurs in the nucleolus. It is a complex process involving: (a) transcription of a set of rRNA genes to produce high molecular weight precursor molecules (45-S R N A in mammalian cells), (b) cleavage of the 45-S RNA to form intermediates, including a relatively long lived 32-S component Abbreviation: rRNA, ribosomal RNA. Biochim. Biophys. Acta, I69 (1968) 196-2o 5
PROCESSING OF
45-S R N A BY ISOLATED NUCLEOLI
197
and (c) conversion of the intermediates to the 28-S and I8-S R N A ' s of ribosomes. Concomitant with these transformations the RNA becomes associated with protein including most, if not all, of the ribosomal protein (c[. review I for references). The mechanism of RNA cleavage involves a high degree of specificity. Assuming that certain enzymes convert the 45-S component to appropriate derivatives, one would like to know how such enzymes recognize the particular sites on the covalently continuous 45-S RNA molecules where they are to catalyze a cleavage. Moreover, how are the products formed in these reactions spared from further degradation? It seems fairly obvious that an affective means for studying this problem would be provided b y preparations of isolated nucleoli capable of carrying out the proper RNA conversions in vitro. For this reason we have looked for ways of regulating the nuclease activity of isolated nucleoli, and in particular *or a means of suppressing non-specific degradative reactions while allowing the specific conversion reactions to occur. Our studies, although still at an early stage, suggest that such a regulated activity is attainable, and that under certain conditions a limited amount of conversion in vitro m a y be observed. Recently C. VESCO AND S. PENMAN (personal communication) have obtained similar results.
MATERIALS AND METHODS
For each series of experiments about 3.2" lO s L cells, grown in suspension culture at 37 ° (ref. 2) were labeled for 15 rain with [SH]uridine (3/~C/ml, 11. 4 C/mmole). At the end of this brief incubation, when most of the radioactive RNA in nucleoli consists of 45-S R N A 1, the cells were chilled, washed in balanced salts solution, and used for isolation of nucleoli as described below (c/., also ref. 3). In the experiments in which the 45-S RNA was doubly labeled with [14C]uridine and [Me-~H]methionine the cells were incubated for 15 min in a medium containing I/IO the normal concentration of methionine, 5 #C/ml [Me-3H]methionine (3.1 C/mmole) and o.o5/~C/ml [14C]uridine (51.5 mC/mmole). The isolation of nucleoli was performed at 0-3 °. The cell pellet (about I ml) was dispersed in 20 ml of o.oi M Tris-acetate buffer (pH 7.0) containing 0.25 M sucrose and 5 mM MgCI~ in a P o t t e r - E l v e h i e m homogenizer equipped with a motordriven Teflon pestle. This treatment does not cause extensive cell breakage, but is used simply to insure that the nuclei are well equilibrated with 5 mM Mg ~+. The cells were then pelleted, resuspended in IO ml of Tris-acetate buffer containing 5 mM MgC12 and 0.2 mM CaC12, and passed through a French pressure cell at 5ooo=t=_1ooo lb/inch 2. Upon emergence from the pressure cell the brei was collected in a sufficient volume of a concentrated sucrose solution to give a final sucrose concentration of 0.25 M. The brei was centrifuged for 5 rain at 600 × g, the supernatant withdrawn and discarded, and the pellet packed more tightly b y recentrifugation for IO rain at 800 ×g. The pellet was suspended in Tris-acetate buffer containing 1.5 mM Mg ~+ and 2.2 M sucrose and centrifuged for IO rain at 88 700 ×g. After draining the tube carefully, the pellet, containing morphologically intact nucleoli with little chromatin or cytoplasmic contamination, was suspended in 8 ml of Tris-acetate buffer containing 1.5 mM MgC12 and 0.25 M sucrose, and divided into eight equal portions which conBiochim. Biophys. Acta, 169 (1968) 196-2o5
198
M.C. LIAU, N. C. CRAIG, R. P. PERRY
stituted an experimental series. The nucleoli were resedimented b y centrifugation for 5-1o min at 8oo × g. Each portion of isolated nucleoli (derived from about 4" lO7 cells) assayed approximately 2-3 A260 ma units if dissolved in o.3 M KOH. In cases where the nucleoli were given an additional wash before incubation, they were suspended in a few ml of the wash medium and recovered by centrifugation for IO min at 20 ooo × g. For the incubations in vitro a portion of nucleoli was suspended in 0.5 ml of 0.02 M triethanolamine buffer (pH 7.4) containing 0.05 M KC1, 0.5 mM MgC12 and o.oi M dithiothreJtol (I medium) and kept at 3 °o for 30 rain. In the experiments so designated the incubation medium also contained lOO-2OO #g/ml of unlabeled R N A extracted with phenol from rat liver ribosomes. In some experiments high specific activity Ea~pIRNA was also added to the incubation mixtures as a probe for the ability of nucleolar enzymes to discriminate between R N A precursor within the structure of the nucleolus (3H-labeled) and free RNA. This ~3~pIRNA was obtained from cells labeled for 24 h with 2.5 #C/ml Na332po4. It was prepared either b y phenol-sodium dodecyl sulfate extraction of pelleted ribosomes ([32pIrRNA) or b y phenol-sodium dodecyl sulfate extraction of nucleoli isolated b y the procedure of PENMAN et al. 4 (32p-labeled 45-S and 32-S RNA). After incubation, or after an equivalent period at 0-3 ° in the case of non-incubated controls, RNA was extracted from the nucleoli with 0.5 % sodium dodecyl sulfate and phenol at room temperature. After ethanol precipitation the RNA was dissolved in 30-50 #1 of electrophoresis buffer (0.04 M Tris (pI-[ 7.2)-0.02 M sodium a c e t a t e - I mM EDTA) containing IO °/o sucrose, and analyzed b y acrylamide-gel electrophoresis 5. In experiments where [32p~RNA was not added to the incubation mixture a trace amount of [3~plrRNA was layered on the gel as a mobility marker. The 5-cm gels, composed of 2.7 % (w/v) acrylamide and 0.25 % (v/v) ethylene diacrylate, were run for 3.5 to 5 h at 5 mA per gel in electrophoresis buffer containing 0.2 °/o sodium dodecyl sulfate. After completion of a run, the gels were frozen in hexane at --7 °o and sectioned at I m m with a maaifold of razor blades. One or two sections were put into each of a series of vials, dissolved in 0.5 ml of concentrated NH4OH, and counted in Bray's solution with a liquid-scintillation counter.
RESULTS In preliminary experiments we encountered extensive degradation of the 45-S R N A component when isolated nucleoli were incubated for 3o rain at 3 o°. Essentially all ( > 99 %) of the 45-S component disappeared. Only a heterogeneous array of fragments, amounting to less than 25 % of the radioactivity originally present, could be recovered (Figs. I a and b). When a small amount of high specific activity free [32p]rRNA was added to the incubation mixture, it was degraded to a similar extent (Fig. Ib'). A search was then made for possible ways of reducing the non-specific degradation of RNA. The effect of polyvinyl sulfate, a compound which has been used fairly extensively as a ribonuclease inhibitor, was studied. Polyvinyl sulfate added to the incubation medium had a marked inhibitory effect on the degradation process. Moreover, when nucleoli were washed in a medium containing varying amounts of Biochim. Biophys. Acta, 169 (1968) 196-2o5
45-S
PROCESSING OF
RNA
199
B Y ISOLATED NUCLEOLI
(o)
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i 4000
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Fig. I. E f f e c t of v a r i o u s w a s h i n g p r o c e d u r e s o n t h e n u c l e a s e a c t i v i t y of i s o l a t e d nucleoli. Cells w e r e l a b e l e d for 15 rain w i t h [ a H ] u r i d i n e a n d n u c l e o l i w e r e i s o l a t e d as d e s c r i b e d i n t e x t . D i a g r a m s i l l u s t r a t e t h e r a d i o a c t i v e R N A as c h a r a c t e r i z e d b y a c r y l a m i d e - g e l e l e c t r o p h o r e s i s , a. N u c l e o l i w e r e w a s h e d o n c e a t o - 3 ° in o . o i M T r i s - a c e t a t e b u f f e r (pH 7.0) c o n t a i n i n g 0.25 M s u c r o s e a n d 1. 5 m M M g z+, a n d R N A e x t r a c t e d i m m e d i a t e l y . T h e s a m e p a t t e r n s w e r e o b t a i n e d f r o m n u c l e o l i w h i c h w e r e i n c u b a t e d for 3 ° m i n a t 0 - 3 ° a f t e r w a s h i n g . D o t t e d c u r v e r e p r e s e n t s [ 3 2 P ] r R N A w h i c h w a s a d d e d to t h e gel as a m o b i l i t y m a r k e r . T h e s a m e m a r k e r w a s also a d d e d to t h e gels of (b), (c), a n d (d) b u t o n l y tile p o s i t i o n s of t h e 28-S a n d I8-S c o m p o n e n t s are m a r k e d for c l a r i t y . • , c o u n t s / m i n SH. b. N u c l e o l i w a s h e d as ill (a) a n d t h e n i n c u b a t e d in I m e d i u m a t 3 °° for 3 ° min. b'. C o n d i t i o n s s a m e as (b) e x c e p t t h a t a b o u t 2o00 c o u n t s / m i n [ 3 2 P ] r R N A ( a b o u t 2/2g) w a s p r e s e n t d u r i n g t h e 3 °o i n c u b a t i o n . O , c o u n t s / m i n 32p. c. C o n d i t i o n s s a m e as (b) e x c e p t t h a t b e f o r e i n c u b a t i o n t h e n u c l e o l i w e r e g i v e n a n a d d i t i o n a l w a s h in m e d i u m c o n t a i n i n g I o o # g / m l p o l y v i n y l s u l f a t e in 0.25 M sucrose, 2 m M MgC1 v 0.05 M KC1, o . o i M s o d i u m a c e t a t e (pH 6.0). d. C o n d i t i o n s s a m e as (b) e x c e p t t h a t b e f o r e i n c u b a t i o n t h e n u c l e o l i w e r e g i v e n a n a d d i t i o n a l w a s h i n m e d i u m c o n s i s t i n g of 0.5 M NaCI, 50 m M MgCt2, o . o i M Tris (pH 7.4) a n d 5 ° / ~ g / m l of e l e c t r o p h o r e t i c a i l y purified deoxyribonuclease. Fig. 2. E f f e c t s of d i f f e r e n t i n c u b a t i o n c o n d i t i o n s o n t h e d e g r a d a t i o n of n u c l e o l a r 45-S R N A . L a b e l i n g , n u c l e o l a r i s o l a t i o n , a n d w a s h a s i n Fig. in. • , c o u n t s / m i n 8H; . . . O • -., c o u n t s / r a i n [ 3 ~ P ] r R N A m o b i l i t y m a r k e r , a. N o i n c u b a t i o n , b. I n c u b a t i o n 3 ° m i n a t 3 °0 i n m e d i u m c o n t a i n i n g i o m M e a c h of E D T A , NaC1, a n d d i t h i o t h r e i t o l , c. I n c u b a t i o n 3 ° rain a t 3 °0 i n I m e d i u m minus M g 2+ a n d c o n t a i n i n g i o o / , g / m l r R N A . d. I n c u b a t i o n 3 ° m i n a t 3 °0 i n I m e d i u m c o n t a i n i n g i o o / , g / m l r R N A . - • • • -, scale e n l a r g e d b y f a c t o r of 4.
polyvinyl sulfate in o.oi M sodium acetate (pH 6.0) and then incubated as previously described, considerable reduction in the extent of degradation was also observed. The inhibitory power of polyvinyl sulfate was proportional to the concentration employed. After washes with IOO pg/ml polyvinyl sulfate and subsequent incubation, there was no detectable degradation of 45-S R N A (Fig. ic). Washing the nucleoli with polyvinyl sulfate also served to completely inhibit the degradation of free [s2P]RNA which was added to the incubation mixture in certain experiments. The nuclease activity could not be restored by raising the Mg *+ concentration of the incubation medium from 0.5 to 2 mlV[. In earlier experiments it was noted that the 45-S R1VA in nucleoli isolated by the procedure of PENMAN e~ al. 4 was more stable than that in the nucleoli isolated Biochim. Biophys. Acta, 169 (1968) 196--2o5
200
M . C . LIAU, N. C. CRAIG, R. P. PERRY
b y our method. Therefore we investigated the effect of washing our nucleoli in o.5 M NaC1 and IOO #g/ml deoxyribonuclease, the medium used in the other isolation procedure. As seen in Fig. Id, such a wash did, indeed, greatly reduce the nuclease activity, although the inhibition was not as great as with IOO #g/rot polyvinyl sulfate. It would seem, therefore, that the polyvinyl sulfate and the high salt deoxyribonuclease probably remove and/or inactivate a high proportion of the non-specific nucleolar nuclease activity. However, since very little of the 45-S RNA is cleaved after treatment with these agents, it appears that the nucleolytic enzymes responsible for conversion are also inhibited. Next we investigated whether more control over the nuclease activity could by achieved by altering the composition of the incubation medium. When the incubations were performed in a medium consisting of IO mM dithiothreitol, NaC1, and E D T A (a medium used for the extraction of ribosomal precursor particles from nucleoli e) degradation patterns such as that shown in Fig. 2b were obtained. In this case about half of the 45-S R N A was degraded, producing relatively large fragments which
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Fig. 3. Effect of i n c u b a t i o n in vitro of isolated nucleoli on intrinsic 45-S nucleolar R N A a n d extrinsic R N A ' s p r e s e n t in the i n c u b a t i o n m e d i u m . Nucleoli i n c u b a t e d for 3 ° m i n at 3 °° in I m e d i u m c o n t a i n i n g 2o0/zg/ml unlabeled r R N A and a few/zg of [n2P]rRNA (a and b) or 32P-labeled 45-S and 32-S R N A (c and d). a and c, before incubation, b and d, after incubation. O , 32P-labeled extrinsic R N A ; O , 3H-labeled nucleolar RNA. Note t h a t for SH c o u n t s / r a i n the o r d i n a t e scales of (b) and (d) axe 1/2 t h o s e of (a) and (c). F o r [35P]counts/min the o r d i n a t e scales of (b) a n d (d) axe respectively equal to those of (a) and (c). Fig. 4. C o m p a r i s o n of intrinsic and extrinsic R N A before and after in vitro i n c u b a t i o n of nucleoli. I n c u b a t i o n in I m e d i u m containing 2 0 0 / , g / m l unlabeled R N A and a few/zg of 35P-labeled 45-S and 32-S RNA.
Biochim. Biophys. Acta, 169 (1968) 196-2o5
PROCESSING OF
45-S
RNA
BY ISOLATED NUCLEOLI
201
migrated in the gels between the 45-S and 28-S components. A similar result was obtained when incubations were carried out in I medium minus Mg2+. If fairly large quantities (about IOO/zg/ml) of unlabeled RNA were added to an incubation medium lacking Mg 2+, the suppression of nuclease activity was almost complete (Fig. 2c). However, incubation with the same quantity of unlabeled RNA and 0.5 mM Mg2+ resulted in a substantial degradation of the 45-S component (Fig. 2d). These results suggest that our nucleolar preparations contain two types of nucleolytic enzyme: one which does not require Mg ~+ and is suppressed by bulk RNA, and another which requires Mg 2+ and is active in the presence of bulk RNA. The type of degradation observed in the presence of large quantities of unlabeled RNA and Mg2+ appeared interesting from two points of view. First, in spite of the fact that two thirds or more of the 45-S component was degraded, most of the surviving fragments were not small and highly heterogeneous such as those shown in Figs. Ib and Ib', but rather seemed restricted to specific sizes resembling the 28-S, 32-S and 36-4I-S components (Figs. 2d, 3b, 3d, and 4a). Second, if free 3zP-labeled rRNA was added to the incubation mixture most of it remained intact (Figs. 3a and b), and if a mixture of free 32P-labeled 45-32-S RNA's are added, only the 45-S component experienced an appreciable amount of degradation (Figs. 3c, 3d and 4b). In Table I the extent of degradation of the RNA within the nucleolar structure (intrinsic RNA) is quantitatively compared with the degradation of the admixed TABLE
I
INHIBITORY EFFECT OF R N A ON NUCLEASE ACTIVITIES OF ISOLATED NUCLEOLI
Amount o[ Number Nucleolar E3H~RNA unlabeled of (% change)* rRNA experiadded to ments Total*** 45 S 32 S 0.5 ml o[ incubation mixture (/~g)
Type
Admixed/tee [3,p] R N A ** (% change) Total*" 45 S
32 S
--34
None 5° 5°
2 2 4
--75 --35 --22
--99 --72 --61
--83 +67 +42
45S-32S rRNA
--38 --24
--67
IOO IOO ioo
3 5 4t
--Ii -- I --15
--46 --25 --51
+45 +44 +13
45S-32S rRNA rRNA
-- 6 -- 2 --14
--35
28 S
--38 +
3 --13 --21
* C a l c u l a t e d f r o m t h e a m o u n t s of r a d i o a c t i v i t y s u m m e d o v e r i n d i v i d u a l p e a k s a n d e n t i r e l e n g t h s of a c r y l a m i d e gels s u c h as t h o s e u s e d for Figs. l - 3. P e r c e n t c h a n g e e q u a l s IOO × (c ount s / m i n of i n c u b a t e d s a m p l e / c o u n t s / m i n of n o n - i n c u b a t e d c o n t r o l ) . -- or + s i g n i n d i c a t e s , r e s p e c t i v e l y , a n e t d e c r e a s e or i n c r e a s e as a r e s u l t of i n c u b a t i o n . ** U s e d h e r e to m e a s u r e t h e e x t e n t of n u c l e a s e a c t i v i t y on e x t r i n s i c R N A , i.e., R N A w h i c h is n o t p a r t of t h e n u c l e o l a r s t r u c t u r e . T h e specific a c t i v i t y is s u f f i c i e n t l y g r e a t so t h a t t h e t o t a l a m o u n t of [32P]RNA a d d e d to t h e i n c u b a t i o n m i x t u r e is n e g l i g i b l e c o m p a r e d t o t h e a m o u n t s of u n l a b e l e d r R N A u s e d as n u c l e a s e i n h i b i t o r . *** R N A ' s of m o l e c u l a r w e i g h t less t h a n t h e I8-S c o m p o n e n t m i g r a t e off t h e gel u n d e r t h e c o n d i t i o n s used, a n d t h e r e f o r e t h e p e r c e n t loss of t o t a l R N A s t r i c t l y r e p r e s e n t s t h e a m o u n t of d e g r a d a t i o n to f r a g m e n t s s m a l l e r t h a n t h e I8-S c o m p o n e n t . t F o r t h i s s e t of e x p e r i m e n t s t h e R N A ' s w e r e e x t r a c t e d f r o m t h e i n c u b a t i o n m i x t u r e , prec i p i t a t e d w i t h e t h a n o l , d i s s o l v e d in 25 m M s o d i u m a c e t a t e b u f f e r (pH 5.1) c o n t a i n i n g 5 mM E D T A a n d 0. 5 % s o d i u m d o d e c y l s u l f a t e , a n d e x p o s e d to 5 °0 for 5 m i n or t o 5 M u r e a for 3 h. A f t e r rep r e c i p i t a t i o n w i t t l e t h a n o l t h e y w e r e a n a l y z e d on a c r y l a m i d e gels i n t h e u s u a l m a n n e r .
Biochim. Biophys. Acta, 169 (1968) 196-2o5
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M. C. L I A U , N. C. C R A I G , R. P. P E R R Y
free R N A (extrinsic RNA). The nuclease action on all free RNA's except the 45-S component was almost completely suppressed when the concentration of unlabeled RNA in the incubation mixture was raised to 200/zg/ml. Under these conditions there is a 25-45 % loss of both the intrinsic and extrinsic 45-S RNA. A somewhat greater amount of degradation is detected when the RNA's are analyzed after denaturation with heat or urea, indicating that some scissions occur during incubation which are normally not expressed because of intramolecular hydrogen bonding. Nevertheless, under these conditions of analysis, the loss of 45-S component still appears to be appreciably greater than that of the other species, suggesting that the nucleases have a preference for 45-S RNA. A more detailed study of the reaction was made in another experiment shown in Fig. 4. In this case I-ram sections of the acrylamide gels were counted in order to afford better resolution of the products. The electrophoretic profiles representing the R N A before and after incubation are superimposed to facilitate comparison. The intrinsic nucleolar RNA suffered a substantial loss of 45-S RNA and of one of the intermediate sized components, I1, and exhibited a small net increase in component 12 and in 32-S RNA. Concomitantly, the extrinsic RlgA exhibited a loss of 45-S component, but no detectable change in the 3 2 - S component. From comparisons such as that made in Fig. 4a it was estimated that about 20-50 % of the loss in 3H-labeled 45-S RNA appears as an observed net increase in fragments migrating faster than the 45-S component. This should be compared to a m a x i m u m expected recovery of 66 % which is estimated on the basis of the approximate molecular weights of the 45-S, 32- and 18-5 components (see DISCUSSION). Although the fragments produced during the incubations in vitro seem to occur in specific sizes resembling 32-S RNA and the other intermediate components, the net increase in these fragments is usually too small to allow one to decide conclusively whether they represent the natural degradation products encountered in the conversion of 45-S RNA to rRNA. One criterion which m a y be used to distinguish a true intermediate from a fragment produced b y a non-specific scission of 45-S RNA is the methyl group enrichment which accompanies the processing of 45-S RNA in vivo 7. If one labels L cells briefly with E14C~uridine and IMe-aH~methionine it can be shown TABLE
II
METHYL GROUP ENRICHMENT ACCOMPANYING THE CONVERSION OF 4 5 - S R N A TO 3 2 - S R N A in vivo AND in vitro In all case RNA was extracted
from isolated nucleoli and resolved in acrylamide gels.
counts~rain [Me-3H]melhionine/ counts/min E14C]uridine
in vivo in vitro
I J
45-S R N A
j2-S RNA
°'58"
0 . 9 7 "* o . 7 1 ***
% methyl enrichment
66 23
* Cells i n c u b a t e d f o r 15 m i n w i t h [ 1 4 C ] u r i d i n e a n d [ M e - a H ] m e t h i o n i n e . ** C e l l s l a b e l e d a s i n f i r s t n o t e a n d t h e n f u r t h e r i n c u b a t e d f o r 3 0 m i n w i t h z / , g / m l a c t i n o m y cin D and 5°/zg/ml unlabeled methionine. *** C e l l s l a b e l e d a s i n f i r s t n o t e t h e n n u c l e o l i i s o l a t e d a n d i n c u b a t e d in vitro 3 ° n l i n a t 3 °0 in I medium containing 200/~g/ml unlabeled RNA.
Biochim. Biophys. Acta. 1 6 9 ( 1 9 6 8 ) 1 9 6 - 2 o 5
PROCESSING OF 45-S
RNA BY ISOLATED NUCLEOLI
2o3
that conversion in vivo of 45-S to 32-S RNA is accompanied by an increase in the aH/14C ratio of about 66 °/o (Table II). When this same measurement was made on the products in vitro, the aH/14C ratio was found to increase about 23 %. Thus, although the incubations in vitro produce a 32-S product which is significantly enriched in methyl group, the enrichment is only about one third that observed with natural 32-S RNA.
DISCUSSION
(a) Factors in/luencing the nuclease activities o~ isolated nucleoli It is evident that nucleoli isolated by our procedure contain active nucleases which are capable of degrading RNA's intrinsic to the nucleolus as well as RNA's added to the incubation medium. After washing the nucleoli in relatively high concentrations of polyvinyl sulfate (about Ioo#g/ml) or in a high salt-deoxyribonuclease solution there is little or no degradation of intrinsic or extrinsic RNA by these nucleoli during a subsequent incubation in vitro. Since IOO/~g of polyvinyl sulfate may bind as much as 0.8/,mole of Mg~+, one could suppose that the effect of polyvinyl sulfate is simply due to its removal of Mg 2+, a certain level of which is required for enzymatic activity. This does not seem likely, however, since activity could not be restored by incubation in media containing 2/~mole of Mg2+. Thus, it is reasonable to suppose that treatment with a polyanion such as polyvinyl sulfate, or with high salt removes or irreversibly inactivates the nucleolar nucleases. Although such treatments are quite valuable for preserving the integrity of large rRNA precursors in isolated nucleoli, they seem to be too drastic for use in purifying an active in vitro system. Two other factors were found to exert marked effects on the nuclease activity of isolated nucleoli: (i) the presence of Mg 2+ in the incubation medium, and (2) the presence in the incubation medium of relatively large quantities of rRNA which presumably act competitively as nuclease inhibitors. The degradation which occurs when lOO-2OO #g/ml rRNA is present in the incubation medium is such that the quantity of 45-S component may be reduced by 2/3 or more without the production of significant amounts of heterogeneous RNA fragments. Those new RNA components which are formed under these conditions migrate in acrylamide gels similar to the 32-S and 12 components. The production of heterogenous fragments which occurs in the absence of bulk RN'A is characteristic of endonucleolytic activity. Yet, an incomplete degradation of 45-S RNA without the production of heterogeneous fragments, such as occurs in the presence of bulk RNA,is characteristic of exonucleolytic activity. Therefore the results described above might be interpreted to mean that nucleoli, as we isolate them, possess both endo- and exonucleolytic activities, and that the endonucleases are effectively suppressed by the addition of competitive quantities of rRNA. These endoand exonucleases could conceivably correspond respectively to the ribonuclease and polynucleotide phosphorylase previously found in nucleoli isolated from rat liver cells s. The results of the Mg ~+ deprivation experin-.ents which were carried out in the presence of bulk RNA (Figs. 2c, d) indicate that the presumed exonuclease acBiochim. Biophys. Acta, 169 (I968) I96-2o 5
204
M.C. LIAU, N. C. CRAIG, R. P. PERRY
tivity clearly requires Mg2+. By comparison, in the absence of bulk RNA there is a detectable amount of degradation when Mg2+ is removed by chelation (Fig. 2b). This suggests that the presumed endonucleolytic activity may not be dependent on Mg 2+ even though in this case the extent of degradation is not very extensive. These relative dependencies on Mg 2+ are consistent with the known behavior of polynucleotide phosphorylase (requires Mg 2+) and ribonuclease (does not usually require Mg 2+).
(b) Specificity o~ nuclease activity In the presence of Mg2+ and sufficient amounts of RNA the nuclease activity displays a remarkable degree of specificity with regard to the type of RNA attacked. When extrinsic 28-S, I8-S or 32-S RNA is incubated together with nucleoli it undergoes almost no detectable degradation, although both extrinsic 45-S RNA and the intrinsic 45-S RNA of the nucleolus are markedly degraded. The effective degradation of the extrinsic 45-S RNA suggests that the specificity of enzyme attack may be attributable solely to the structural characteristic of the RNA, e.g. end groups, base sequences, sites of methylation, or conformation. In terms of an exonuclease activity one might speculate that the critical determinant is the attachment of enzyme to an appropriate RNA terminus. It is not difficult to imagine how such an attachment would depend on specific properties of the 45-S molecule. The methyl/uridine enrichment observed after incubation in vitro was about 1/3 that associated with conversion in vivo. This suggests that under the present conditions our system in vitro does not yield solely the products which are found in vivo, and therefore that only a portion of the reaction may yield biologically significant products.
(c) Relation o/reactions in vitro to the system in vivo which cleaves 45-S RNA In vivo a 45-S molecule with a molecular weight of approx. 4-4 million is degraded in such a way to eventually yield 32-S and I8-S components of aggregate molecular weight about 3.0 million (refs. 7, 9; E. H. McCONKEY, personal communication). The remaining one-third of the 45-S molecule, which seems to have a relative abundance of guanine residues 1° and is deficient in methylated ribose 7, has not been accounted for. One possible scheme for this reaction, which would be consistent with the apparent presence of both endo- and exonuclease activity, is that the first step in conversion entails an endonuclease which cleaves the 45-S molecule into two segments, one containing the I8-S component, and the other the 32-S component (which contains a sequence identical to the 28-S molecule within it). These fragments would then be trimmed to their appropriate sizes by the stepwise action of exonuclease. The components, 11 and 12, would presumably represent intermediate stages of degradation of the segment which eventually yields the 32-S component. Such a model presupposes mechanisms for determining the site of the endonuclease attack and for producing the proper end groups for the exonuclease. For example polynucleotide phosphorylase would require a 3'-OH terminus, thus necessitating removal of a 3'-phosphate after a ribonuclease cleavage. Moreover, a mechanism would also have to be provided for terminating the exonuclease activity at the appropriate places in the polynucleotide chain. Some of these controlling factors Biochim. Biophys. Acta, 169 (1968) 196-2o5
PROCESSING OF 4 5 - S
RNA
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205
could conceivably reside in the large amount of protein which is known to be complexed with the 45-S RNA TM.
ACKNOWLEDGEMENTS
This research was supported by a Grant from the National Science Foundation (formerly No. GB 4137, new No. GB 7o5I), a Grant from the National Institutes of Health (No. CA o6927), and an Appropriation from the Commonwealth of Pemlsylvania.
REFERENCES i 2 3 4 5 6 7 8 9 io Ii
R. P. PERRY, Progr. Nucleic Acid Res. Mol. Biol., 6 (1967) 219. R. P. PERRY AND D. E. I~ELLI~Y, J. Mol. Biol., 16 (1966) 255. M. C. LIAU, L. S. HNILICA AND R. B. HURLBERT, Proc. Natl. Acad. Sci. U.S., 53 (1965) 626. S. PENMAN, I. SMITH, E. HOLTZMAN AND H. GREENBERG, Natl. Cancer Inst. Monograph, 23 (1966) 489 . U. LOEI~ING, Biochem. J., lO2 (1967) 251. J. R. WARNER AND ]~. SOEIRO, Proc. Natl, Acad. S d . U.S., 58 (1967) 1984. R. A. WEINBERG, U. LOENING, M. WILLEMS AND S. PENMAN, Proc. Natl. Acad. Sci. U.S., 58 (1967) lO88. G. SIEBERT, J. VILLALOBOS, JR., T. S. RO, W. J. STEELE, G. LINDENMAYER, H. ADAMS AND H. BUSCH, J. Biol. Chem., 241 (1966) 71. D. D. BROWN AND C. S. WEBER, J. Mol. Biol., 34 (1968) 661. M. WILLEMS, E. WAGNER, R. LAING AND S. PENMAN, J. Mol. Biol., 32 (1968) 211. M. C. LIAU AND R. P. PERRY, J. Cell Biol., in t h e press.
Biochim. Biophys. Acta, 169 (1968) 196-2o 5
BIOCHIMICAET BIOPHYSICAACTA
BBA 96032
T H E P R O D U C T I O N OF RIBOSOMAL RNA FROM H I G H MOLECULAR WEIGHT PRECURSORS I. FACTORS W H I C H I N F L U E N C E T H E A B I L I T Y OF ISOLATED N U C L E O L I TO PROCESS 45-S RNA
M. C. LIAU, N. C. CRAIG AND R. l:). PERRY The Institute /or Cancer Research, Fox Chase, Philadelphia, Pa. I 9 z H (U.S.A.)
(Received May 27th, 1968)
SUMMARY A search was made for conditions which would allow isolated nucleoli to convert 45-S RNA in vitro to 28-S and IS-S ribosomal RNA or to other natural derivatives. The nuclease activity of nucleoli purified from L cells m a y be altered or controlled as follows: (a) if incubation of nucleoli in vitro is preceded by a wash with relatively high concentrations (about IOO/,g/ml) of polyvinyl sulfate or with 0.5 M NaC1 and deoxyribonuclease, nuclease activity is almost totally suppressed; (b) for extensive degradation of 45-S R N A to occur Mg 2+ must be present in the incubation medium; and (c) inclusion of bulk quantities of R N A in the incubation medium allows the selective breakdown of 45-S RNA without the production of heterogeneous fragments. After incubation for 30 rain at 3 °o in a medium containing 0.5 mM Mg 2+ and IOO200/~g RNA per ml, 45-S nucleolar RNA was degraded, and a limited quantity of fragments which migrated in acrylamide gels like the 32-S component and another intermediate were produced. Under these conditions extrinsic 45-S RNA which was incubated together with the nucleoli was also degraded, but extrinsic 32-S, 28-S and I8-S RNA underwent little or no degradation. The fragments produced during incubation in vitro were enriched in their methyl group/uridine ratio as compared to the 45-S component, although the enrichment was not as great as that observed after conversion in vivo. These results suggest a tentative model for the conversion process whereby 45-S RNA undergoes a single scission with an endonuclease followed b y a stepwise trimming with exonuclease.
INTRODUCTION Ribosome synthesis in higher organisms occurs in the nucleolus. It is a complex process involving: (a) transcription of a set of rRNA genes to produce high molecular weight precursor molecules (45-S R N A in mammalian cells), (b) cleavage of the 45-S RNA to form intermediates, including a relatively long lived 32-S component Abbreviation: rRNA, ribosomal RNA. Biochim. Biophys. Acta, I69 (1968) 196-2o 5
PROCESSING OF
45-S R N A BY ISOLATED NUCLEOLI
197
and (c) conversion of the intermediates to the 28-S and I8-S R N A ' s of ribosomes. Concomitant with these transformations the RNA becomes associated with protein including most, if not all, of the ribosomal protein (c[. review I for references). The mechanism of RNA cleavage involves a high degree of specificity. Assuming that certain enzymes convert the 45-S component to appropriate derivatives, one would like to know how such enzymes recognize the particular sites on the covalently continuous 45-S RNA molecules where they are to catalyze a cleavage. Moreover, how are the products formed in these reactions spared from further degradation? It seems fairly obvious that an affective means for studying this problem would be provided b y preparations of isolated nucleoli capable of carrying out the proper RNA conversions in vitro. For this reason we have looked for ways of regulating the nuclease activity of isolated nucleoli, and in particular *or a means of suppressing non-specific degradative reactions while allowing the specific conversion reactions to occur. Our studies, although still at an early stage, suggest that such a regulated activity is attainable, and that under certain conditions a limited amount of conversion in vitro m a y be observed. Recently C. VESCO AND S. PENMAN (personal communication) have obtained similar results.
MATERIALS AND METHODS
For each series of experiments about 3.2" lO s L cells, grown in suspension culture at 37 ° (ref. 2) were labeled for 15 rain with [SH]uridine (3/~C/ml, 11. 4 C/mmole). At the end of this brief incubation, when most of the radioactive RNA in nucleoli consists of 45-S R N A 1, the cells were chilled, washed in balanced salts solution, and used for isolation of nucleoli as described below (c/., also ref. 3). In the experiments in which the 45-S RNA was doubly labeled with [14C]uridine and [Me-~H]methionine the cells were incubated for 15 min in a medium containing I/IO the normal concentration of methionine, 5 #C/ml [Me-3H]methionine (3.1 C/mmole) and o.o5/~C/ml [14C]uridine (51.5 mC/mmole). The isolation of nucleoli was performed at 0-3 °. The cell pellet (about I ml) was dispersed in 20 ml of o.oi M Tris-acetate buffer (pH 7.0) containing 0.25 M sucrose and 5 mM MgCI~ in a P o t t e r - E l v e h i e m homogenizer equipped with a motordriven Teflon pestle. This treatment does not cause extensive cell breakage, but is used simply to insure that the nuclei are well equilibrated with 5 mM Mg ~+. The cells were then pelleted, resuspended in IO ml of Tris-acetate buffer containing 5 mM MgC12 and 0.2 mM CaC12, and passed through a French pressure cell at 5ooo=t=_1ooo lb/inch 2. Upon emergence from the pressure cell the brei was collected in a sufficient volume of a concentrated sucrose solution to give a final sucrose concentration of 0.25 M. The brei was centrifuged for 5 rain at 600 × g, the supernatant withdrawn and discarded, and the pellet packed more tightly b y recentrifugation for IO rain at 800 ×g. The pellet was suspended in Tris-acetate buffer containing 1.5 mM Mg ~+ and 2.2 M sucrose and centrifuged for IO rain at 88 700 ×g. After draining the tube carefully, the pellet, containing morphologically intact nucleoli with little chromatin or cytoplasmic contamination, was suspended in 8 ml of Tris-acetate buffer containing 1.5 mM MgC12 and 0.25 M sucrose, and divided into eight equal portions which conBiochim. Biophys. Acta, 169 (1968) 196-2o5
198
M.C. LIAU, N. C. CRAIG, R. P. PERRY
stituted an experimental series. The nucleoli were resedimented b y centrifugation for 5-1o min at 8oo × g. Each portion of isolated nucleoli (derived from about 4" lO7 cells) assayed approximately 2-3 A260 ma units if dissolved in o.3 M KOH. In cases where the nucleoli were given an additional wash before incubation, they were suspended in a few ml of the wash medium and recovered by centrifugation for IO min at 20 ooo × g. For the incubations in vitro a portion of nucleoli was suspended in 0.5 ml of 0.02 M triethanolamine buffer (pH 7.4) containing 0.05 M KC1, 0.5 mM MgC12 and o.oi M dithiothreJtol (I medium) and kept at 3 °o for 30 rain. In the experiments so designated the incubation medium also contained lOO-2OO #g/ml of unlabeled R N A extracted with phenol from rat liver ribosomes. In some experiments high specific activity Ea~pIRNA was also added to the incubation mixtures as a probe for the ability of nucleolar enzymes to discriminate between R N A precursor within the structure of the nucleolus (3H-labeled) and free RNA. This ~3~pIRNA was obtained from cells labeled for 24 h with 2.5 #C/ml Na332po4. It was prepared either b y phenol-sodium dodecyl sulfate extraction of pelleted ribosomes ([32pIrRNA) or b y phenol-sodium dodecyl sulfate extraction of nucleoli isolated b y the procedure of PENMAN et al. 4 (32p-labeled 45-S and 32-S RNA). After incubation, or after an equivalent period at 0-3 ° in the case of non-incubated controls, RNA was extracted from the nucleoli with 0.5 % sodium dodecyl sulfate and phenol at room temperature. After ethanol precipitation the RNA was dissolved in 30-50 #1 of electrophoresis buffer (0.04 M Tris (pI-[ 7.2)-0.02 M sodium a c e t a t e - I mM EDTA) containing IO °/o sucrose, and analyzed b y acrylamide-gel electrophoresis 5. In experiments where [32p~RNA was not added to the incubation mixture a trace amount of [3~plrRNA was layered on the gel as a mobility marker. The 5-cm gels, composed of 2.7 % (w/v) acrylamide and 0.25 % (v/v) ethylene diacrylate, were run for 3.5 to 5 h at 5 mA per gel in electrophoresis buffer containing 0.2 °/o sodium dodecyl sulfate. After completion of a run, the gels were frozen in hexane at --7 °o and sectioned at I m m with a maaifold of razor blades. One or two sections were put into each of a series of vials, dissolved in 0.5 ml of concentrated NH4OH, and counted in Bray's solution with a liquid-scintillation counter.
RESULTS In preliminary experiments we encountered extensive degradation of the 45-S R N A component when isolated nucleoli were incubated for 3o rain at 3 o°. Essentially all ( > 99 %) of the 45-S component disappeared. Only a heterogeneous array of fragments, amounting to less than 25 % of the radioactivity originally present, could be recovered (Figs. I a and b). When a small amount of high specific activity free [32p]rRNA was added to the incubation mixture, it was degraded to a similar extent (Fig. Ib'). A search was then made for possible ways of reducing the non-specific degradation of RNA. The effect of polyvinyl sulfate, a compound which has been used fairly extensively as a ribonuclease inhibitor, was studied. Polyvinyl sulfate added to the incubation medium had a marked inhibitory effect on the degradation process. Moreover, when nucleoli were washed in a medium containing varying amounts of Biochim. Biophys. Acta, 169 (1968) 196-2o5
45-S
PROCESSING OF
RNA
199
B Y ISOLATED NUCLEOLI
(o)
(b)
6000
(b; 45s
i 4000
incuboled 30'.30"C
50
i
6')
I00
28S
2000
iBs
40
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: ! ! i
100
9
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285
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200
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28S elect*'ophoresis
18S
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Fig. I. E f f e c t of v a r i o u s w a s h i n g p r o c e d u r e s o n t h e n u c l e a s e a c t i v i t y of i s o l a t e d nucleoli. Cells w e r e l a b e l e d for 15 rain w i t h [ a H ] u r i d i n e a n d n u c l e o l i w e r e i s o l a t e d as d e s c r i b e d i n t e x t . D i a g r a m s i l l u s t r a t e t h e r a d i o a c t i v e R N A as c h a r a c t e r i z e d b y a c r y l a m i d e - g e l e l e c t r o p h o r e s i s , a. N u c l e o l i w e r e w a s h e d o n c e a t o - 3 ° in o . o i M T r i s - a c e t a t e b u f f e r (pH 7.0) c o n t a i n i n g 0.25 M s u c r o s e a n d 1. 5 m M M g z+, a n d R N A e x t r a c t e d i m m e d i a t e l y . T h e s a m e p a t t e r n s w e r e o b t a i n e d f r o m n u c l e o l i w h i c h w e r e i n c u b a t e d for 3 ° m i n a t 0 - 3 ° a f t e r w a s h i n g . D o t t e d c u r v e r e p r e s e n t s [ 3 2 P ] r R N A w h i c h w a s a d d e d to t h e gel as a m o b i l i t y m a r k e r . T h e s a m e m a r k e r w a s also a d d e d to t h e gels of (b), (c), a n d (d) b u t o n l y tile p o s i t i o n s of t h e 28-S a n d I8-S c o m p o n e n t s are m a r k e d for c l a r i t y . • , c o u n t s / m i n SH. b. N u c l e o l i w a s h e d as ill (a) a n d t h e n i n c u b a t e d in I m e d i u m a t 3 °° for 3 ° min. b'. C o n d i t i o n s s a m e as (b) e x c e p t t h a t a b o u t 2o00 c o u n t s / m i n [ 3 2 P ] r R N A ( a b o u t 2/2g) w a s p r e s e n t d u r i n g t h e 3 °o i n c u b a t i o n . O , c o u n t s / m i n 32p. c. C o n d i t i o n s s a m e as (b) e x c e p t t h a t b e f o r e i n c u b a t i o n t h e n u c l e o l i w e r e g i v e n a n a d d i t i o n a l w a s h in m e d i u m c o n t a i n i n g I o o # g / m l p o l y v i n y l s u l f a t e in 0.25 M sucrose, 2 m M MgC1 v 0.05 M KC1, o . o i M s o d i u m a c e t a t e (pH 6.0). d. C o n d i t i o n s s a m e as (b) e x c e p t t h a t b e f o r e i n c u b a t i o n t h e n u c l e o l i w e r e g i v e n a n a d d i t i o n a l w a s h i n m e d i u m c o n s i s t i n g of 0.5 M NaCI, 50 m M MgCt2, o . o i M Tris (pH 7.4) a n d 5 ° / ~ g / m l of e l e c t r o p h o r e t i c a i l y purified deoxyribonuclease. Fig. 2. E f f e c t s of d i f f e r e n t i n c u b a t i o n c o n d i t i o n s o n t h e d e g r a d a t i o n of n u c l e o l a r 45-S R N A . L a b e l i n g , n u c l e o l a r i s o l a t i o n , a n d w a s h a s i n Fig. in. • , c o u n t s / m i n 8H; . . . O • -., c o u n t s / r a i n [ 3 ~ P ] r R N A m o b i l i t y m a r k e r , a. N o i n c u b a t i o n , b. I n c u b a t i o n 3 ° m i n a t 3 °0 i n m e d i u m c o n t a i n i n g i o m M e a c h of E D T A , NaC1, a n d d i t h i o t h r e i t o l , c. I n c u b a t i o n 3 ° rain a t 3 °0 i n I m e d i u m minus M g 2+ a n d c o n t a i n i n g i o o / , g / m l r R N A . d. I n c u b a t i o n 3 ° m i n a t 3 °0 i n I m e d i u m c o n t a i n i n g i o o / , g / m l r R N A . - • • • -, scale e n l a r g e d b y f a c t o r of 4.
polyvinyl sulfate in o.oi M sodium acetate (pH 6.0) and then incubated as previously described, considerable reduction in the extent of degradation was also observed. The inhibitory power of polyvinyl sulfate was proportional to the concentration employed. After washes with IOO pg/ml polyvinyl sulfate and subsequent incubation, there was no detectable degradation of 45-S R N A (Fig. ic). Washing the nucleoli with polyvinyl sulfate also served to completely inhibit the degradation of free [s2P]RNA which was added to the incubation mixture in certain experiments. The nuclease activity could not be restored by raising the Mg *+ concentration of the incubation medium from 0.5 to 2 mlV[. In earlier experiments it was noted that the 45-S R1VA in nucleoli isolated by the procedure of PENMAN e~ al. 4 was more stable than that in the nucleoli isolated Biochim. Biophys. Acta, 169 (1968) 196--2o5
200
M . C . LIAU, N. C. CRAIG, R. P. PERRY
b y our method. Therefore we investigated the effect of washing our nucleoli in o.5 M NaC1 and IOO #g/ml deoxyribonuclease, the medium used in the other isolation procedure. As seen in Fig. Id, such a wash did, indeed, greatly reduce the nuclease activity, although the inhibition was not as great as with IOO #g/rot polyvinyl sulfate. It would seem, therefore, that the polyvinyl sulfate and the high salt deoxyribonuclease probably remove and/or inactivate a high proportion of the non-specific nucleolar nuclease activity. However, since very little of the 45-S RNA is cleaved after treatment with these agents, it appears that the nucleolytic enzymes responsible for conversion are also inhibited. Next we investigated whether more control over the nuclease activity could by achieved by altering the composition of the incubation medium. When the incubations were performed in a medium consisting of IO mM dithiothreitol, NaC1, and E D T A (a medium used for the extraction of ribosomal precursor particles from nucleoli e) degradation patterns such as that shown in Fig. 2b were obtained. In this case about half of the 45-S R N A was degraded, producing relatively large fragments which
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Fig. 3. Effect of i n c u b a t i o n in vitro of isolated nucleoli on intrinsic 45-S nucleolar R N A a n d extrinsic R N A ' s p r e s e n t in the i n c u b a t i o n m e d i u m . Nucleoli i n c u b a t e d for 3 ° m i n at 3 °° in I m e d i u m c o n t a i n i n g 2o0/zg/ml unlabeled r R N A and a few/zg of [n2P]rRNA (a and b) or 32P-labeled 45-S and 32-S R N A (c and d). a and c, before incubation, b and d, after incubation. O , 32P-labeled extrinsic R N A ; O , 3H-labeled nucleolar RNA. Note t h a t for SH c o u n t s / r a i n the o r d i n a t e scales of (b) and (d) axe 1/2 t h o s e of (a) and (c). F o r [35P]counts/min the o r d i n a t e scales of (b) a n d (d) axe respectively equal to those of (a) and (c). Fig. 4. C o m p a r i s o n of intrinsic and extrinsic R N A before and after in vitro i n c u b a t i o n of nucleoli. I n c u b a t i o n in I m e d i u m containing 2 0 0 / , g / m l unlabeled R N A and a few/zg of 35P-labeled 45-S and 32-S RNA.
Biochim. Biophys. Acta, 169 (1968) 196-2o5
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45-S
RNA
BY ISOLATED NUCLEOLI
201
migrated in the gels between the 45-S and 28-S components. A similar result was obtained when incubations were carried out in I medium minus Mg2+. If fairly large quantities (about IOO/zg/ml) of unlabeled RNA were added to an incubation medium lacking Mg 2+, the suppression of nuclease activity was almost complete (Fig. 2c). However, incubation with the same quantity of unlabeled RNA and 0.5 mM Mg2+ resulted in a substantial degradation of the 45-S component (Fig. 2d). These results suggest that our nucleolar preparations contain two types of nucleolytic enzyme: one which does not require Mg ~+ and is suppressed by bulk RNA, and another which requires Mg 2+ and is active in the presence of bulk RNA. The type of degradation observed in the presence of large quantities of unlabeled RNA and Mg2+ appeared interesting from two points of view. First, in spite of the fact that two thirds or more of the 45-S component was degraded, most of the surviving fragments were not small and highly heterogeneous such as those shown in Figs. Ib and Ib', but rather seemed restricted to specific sizes resembling the 28-S, 32-S and 36-4I-S components (Figs. 2d, 3b, 3d, and 4a). Second, if free 3zP-labeled rRNA was added to the incubation mixture most of it remained intact (Figs. 3a and b), and if a mixture of free 32P-labeled 45-32-S RNA's are added, only the 45-S component experienced an appreciable amount of degradation (Figs. 3c, 3d and 4b). In Table I the extent of degradation of the RNA within the nucleolar structure (intrinsic RNA) is quantitatively compared with the degradation of the admixed TABLE
I
INHIBITORY EFFECT OF R N A ON NUCLEASE ACTIVITIES OF ISOLATED NUCLEOLI
Amount o[ Number Nucleolar E3H~RNA unlabeled of (% change)* rRNA experiadded to ments Total*** 45 S 32 S 0.5 ml o[ incubation mixture (/~g)
Type
Admixed/tee [3,p] R N A ** (% change) Total*" 45 S
32 S
--34
None 5° 5°
2 2 4
--75 --35 --22
--99 --72 --61
--83 +67 +42
45S-32S rRNA
--38 --24
--67
IOO IOO ioo
3 5 4t
--Ii -- I --15
--46 --25 --51
+45 +44 +13
45S-32S rRNA rRNA
-- 6 -- 2 --14
--35
28 S
--38 +
3 --13 --21
* C a l c u l a t e d f r o m t h e a m o u n t s of r a d i o a c t i v i t y s u m m e d o v e r i n d i v i d u a l p e a k s a n d e n t i r e l e n g t h s of a c r y l a m i d e gels s u c h as t h o s e u s e d for Figs. l - 3. P e r c e n t c h a n g e e q u a l s IOO × (c ount s / m i n of i n c u b a t e d s a m p l e / c o u n t s / m i n of n o n - i n c u b a t e d c o n t r o l ) . -- or + s i g n i n d i c a t e s , r e s p e c t i v e l y , a n e t d e c r e a s e or i n c r e a s e as a r e s u l t of i n c u b a t i o n . ** U s e d h e r e to m e a s u r e t h e e x t e n t of n u c l e a s e a c t i v i t y on e x t r i n s i c R N A , i.e., R N A w h i c h is n o t p a r t of t h e n u c l e o l a r s t r u c t u r e . T h e specific a c t i v i t y is s u f f i c i e n t l y g r e a t so t h a t t h e t o t a l a m o u n t of [32P]RNA a d d e d to t h e i n c u b a t i o n m i x t u r e is n e g l i g i b l e c o m p a r e d t o t h e a m o u n t s of u n l a b e l e d r R N A u s e d as n u c l e a s e i n h i b i t o r . *** R N A ' s of m o l e c u l a r w e i g h t less t h a n t h e I8-S c o m p o n e n t m i g r a t e off t h e gel u n d e r t h e c o n d i t i o n s used, a n d t h e r e f o r e t h e p e r c e n t loss of t o t a l R N A s t r i c t l y r e p r e s e n t s t h e a m o u n t of d e g r a d a t i o n to f r a g m e n t s s m a l l e r t h a n t h e I8-S c o m p o n e n t . t F o r t h i s s e t of e x p e r i m e n t s t h e R N A ' s w e r e e x t r a c t e d f r o m t h e i n c u b a t i o n m i x t u r e , prec i p i t a t e d w i t h e t h a n o l , d i s s o l v e d in 25 m M s o d i u m a c e t a t e b u f f e r (pH 5.1) c o n t a i n i n g 5 mM E D T A a n d 0. 5 % s o d i u m d o d e c y l s u l f a t e , a n d e x p o s e d to 5 °0 for 5 m i n or t o 5 M u r e a for 3 h. A f t e r rep r e c i p i t a t i o n w i t t l e t h a n o l t h e y w e r e a n a l y z e d on a c r y l a m i d e gels i n t h e u s u a l m a n n e r .
Biochim. Biophys. Acta, 169 (1968) 196-2o5
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free R N A (extrinsic RNA). The nuclease action on all free RNA's except the 45-S component was almost completely suppressed when the concentration of unlabeled RNA in the incubation mixture was raised to 200/zg/ml. Under these conditions there is a 25-45 % loss of both the intrinsic and extrinsic 45-S RNA. A somewhat greater amount of degradation is detected when the RNA's are analyzed after denaturation with heat or urea, indicating that some scissions occur during incubation which are normally not expressed because of intramolecular hydrogen bonding. Nevertheless, under these conditions of analysis, the loss of 45-S component still appears to be appreciably greater than that of the other species, suggesting that the nucleases have a preference for 45-S RNA. A more detailed study of the reaction was made in another experiment shown in Fig. 4. In this case I-ram sections of the acrylamide gels were counted in order to afford better resolution of the products. The electrophoretic profiles representing the R N A before and after incubation are superimposed to facilitate comparison. The intrinsic nucleolar RNA suffered a substantial loss of 45-S RNA and of one of the intermediate sized components, I1, and exhibited a small net increase in component 12 and in 32-S RNA. Concomitantly, the extrinsic RlgA exhibited a loss of 45-S component, but no detectable change in the 3 2 - S component. From comparisons such as that made in Fig. 4a it was estimated that about 20-50 % of the loss in 3H-labeled 45-S RNA appears as an observed net increase in fragments migrating faster than the 45-S component. This should be compared to a m a x i m u m expected recovery of 66 % which is estimated on the basis of the approximate molecular weights of the 45-S, 32- and 18-5 components (see DISCUSSION). Although the fragments produced during the incubations in vitro seem to occur in specific sizes resembling 32-S RNA and the other intermediate components, the net increase in these fragments is usually too small to allow one to decide conclusively whether they represent the natural degradation products encountered in the conversion of 45-S RNA to rRNA. One criterion which m a y be used to distinguish a true intermediate from a fragment produced b y a non-specific scission of 45-S RNA is the methyl group enrichment which accompanies the processing of 45-S RNA in vivo 7. If one labels L cells briefly with E14C~uridine and IMe-aH~methionine it can be shown TABLE
II
METHYL GROUP ENRICHMENT ACCOMPANYING THE CONVERSION OF 4 5 - S R N A TO 3 2 - S R N A in vivo AND in vitro In all case RNA was extracted
from isolated nucleoli and resolved in acrylamide gels.
counts~rain [Me-3H]melhionine/ counts/min E14C]uridine
in vivo in vitro
I J
45-S R N A
j2-S RNA
°'58"
0 . 9 7 "* o . 7 1 ***
% methyl enrichment
66 23
* Cells i n c u b a t e d f o r 15 m i n w i t h [ 1 4 C ] u r i d i n e a n d [ M e - a H ] m e t h i o n i n e . ** C e l l s l a b e l e d a s i n f i r s t n o t e a n d t h e n f u r t h e r i n c u b a t e d f o r 3 0 m i n w i t h z / , g / m l a c t i n o m y cin D and 5°/zg/ml unlabeled methionine. *** C e l l s l a b e l e d a s i n f i r s t n o t e t h e n n u c l e o l i i s o l a t e d a n d i n c u b a t e d in vitro 3 ° n l i n a t 3 °0 in I medium containing 200/~g/ml unlabeled RNA.
Biochim. Biophys. Acta. 1 6 9 ( 1 9 6 8 ) 1 9 6 - 2 o 5
PROCESSING OF 45-S
RNA BY ISOLATED NUCLEOLI
2o3
that conversion in vivo of 45-S to 32-S RNA is accompanied by an increase in the aH/14C ratio of about 66 °/o (Table II). When this same measurement was made on the products in vitro, the aH/14C ratio was found to increase about 23 %. Thus, although the incubations in vitro produce a 32-S product which is significantly enriched in methyl group, the enrichment is only about one third that observed with natural 32-S RNA.
DISCUSSION
(a) Factors in/luencing the nuclease activities o~ isolated nucleoli It is evident that nucleoli isolated by our procedure contain active nucleases which are capable of degrading RNA's intrinsic to the nucleolus as well as RNA's added to the incubation medium. After washing the nucleoli in relatively high concentrations of polyvinyl sulfate (about Ioo#g/ml) or in a high salt-deoxyribonuclease solution there is little or no degradation of intrinsic or extrinsic RNA by these nucleoli during a subsequent incubation in vitro. Since IOO/~g of polyvinyl sulfate may bind as much as 0.8/,mole of Mg~+, one could suppose that the effect of polyvinyl sulfate is simply due to its removal of Mg 2+, a certain level of which is required for enzymatic activity. This does not seem likely, however, since activity could not be restored by incubation in media containing 2/~mole of Mg2+. Thus, it is reasonable to suppose that treatment with a polyanion such as polyvinyl sulfate, or with high salt removes or irreversibly inactivates the nucleolar nucleases. Although such treatments are quite valuable for preserving the integrity of large rRNA precursors in isolated nucleoli, they seem to be too drastic for use in purifying an active in vitro system. Two other factors were found to exert marked effects on the nuclease activity of isolated nucleoli: (i) the presence of Mg 2+ in the incubation medium, and (2) the presence in the incubation medium of relatively large quantities of rRNA which presumably act competitively as nuclease inhibitors. The degradation which occurs when lOO-2OO #g/ml rRNA is present in the incubation medium is such that the quantity of 45-S component may be reduced by 2/3 or more without the production of significant amounts of heterogeneous RNA fragments. Those new RNA components which are formed under these conditions migrate in acrylamide gels similar to the 32-S and 12 components. The production of heterogenous fragments which occurs in the absence of bulk RN'A is characteristic of endonucleolytic activity. Yet, an incomplete degradation of 45-S RNA without the production of heterogeneous fragments, such as occurs in the presence of bulk RNA,is characteristic of exonucleolytic activity. Therefore the results described above might be interpreted to mean that nucleoli, as we isolate them, possess both endo- and exonucleolytic activities, and that the endonucleases are effectively suppressed by the addition of competitive quantities of rRNA. These endoand exonucleases could conceivably correspond respectively to the ribonuclease and polynucleotide phosphorylase previously found in nucleoli isolated from rat liver cells s. The results of the Mg ~+ deprivation experin-.ents which were carried out in the presence of bulk RNA (Figs. 2c, d) indicate that the presumed exonuclease acBiochim. Biophys. Acta, 169 (I968) I96-2o 5
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M.C. LIAU, N. C. CRAIG, R. P. PERRY
tivity clearly requires Mg2+. By comparison, in the absence of bulk RNA there is a detectable amount of degradation when Mg2+ is removed by chelation (Fig. 2b). This suggests that the presumed endonucleolytic activity may not be dependent on Mg 2+ even though in this case the extent of degradation is not very extensive. These relative dependencies on Mg 2+ are consistent with the known behavior of polynucleotide phosphorylase (requires Mg 2+) and ribonuclease (does not usually require Mg 2+).
(b) Specificity o~ nuclease activity In the presence of Mg2+ and sufficient amounts of RNA the nuclease activity displays a remarkable degree of specificity with regard to the type of RNA attacked. When extrinsic 28-S, I8-S or 32-S RNA is incubated together with nucleoli it undergoes almost no detectable degradation, although both extrinsic 45-S RNA and the intrinsic 45-S RNA of the nucleolus are markedly degraded. The effective degradation of the extrinsic 45-S RNA suggests that the specificity of enzyme attack may be attributable solely to the structural characteristic of the RNA, e.g. end groups, base sequences, sites of methylation, or conformation. In terms of an exonuclease activity one might speculate that the critical determinant is the attachment of enzyme to an appropriate RNA terminus. It is not difficult to imagine how such an attachment would depend on specific properties of the 45-S molecule. The methyl/uridine enrichment observed after incubation in vitro was about 1/3 that associated with conversion in vivo. This suggests that under the present conditions our system in vitro does not yield solely the products which are found in vivo, and therefore that only a portion of the reaction may yield biologically significant products.
(c) Relation o/reactions in vitro to the system in vivo which cleaves 45-S RNA In vivo a 45-S molecule with a molecular weight of approx. 4-4 million is degraded in such a way to eventually yield 32-S and I8-S components of aggregate molecular weight about 3.0 million (refs. 7, 9; E. H. McCONKEY, personal communication). The remaining one-third of the 45-S molecule, which seems to have a relative abundance of guanine residues 1° and is deficient in methylated ribose 7, has not been accounted for. One possible scheme for this reaction, which would be consistent with the apparent presence of both endo- and exonuclease activity, is that the first step in conversion entails an endonuclease which cleaves the 45-S molecule into two segments, one containing the I8-S component, and the other the 32-S component (which contains a sequence identical to the 28-S molecule within it). These fragments would then be trimmed to their appropriate sizes by the stepwise action of exonuclease. The components, 11 and 12, would presumably represent intermediate stages of degradation of the segment which eventually yields the 32-S component. Such a model presupposes mechanisms for determining the site of the endonuclease attack and for producing the proper end groups for the exonuclease. For example polynucleotide phosphorylase would require a 3'-OH terminus, thus necessitating removal of a 3'-phosphate after a ribonuclease cleavage. Moreover, a mechanism would also have to be provided for terminating the exonuclease activity at the appropriate places in the polynucleotide chain. Some of these controlling factors Biochim. Biophys. Acta, 169 (1968) 196-2o5
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205
could conceivably reside in the large amount of protein which is known to be complexed with the 45-S RNA TM.
ACKNOWLEDGEMENTS
This research was supported by a Grant from the National Science Foundation (formerly No. GB 4137, new No. GB 7o5I), a Grant from the National Institutes of Health (No. CA o6927), and an Appropriation from the Commonwealth of Pemlsylvania.
REFERENCES i 2 3 4 5 6 7 8 9 io Ii
R. P. PERRY, Progr. Nucleic Acid Res. Mol. Biol., 6 (1967) 219. R. P. PERRY AND D. E. I~ELLI~Y, J. Mol. Biol., 16 (1966) 255. M. C. LIAU, L. S. HNILICA AND R. B. HURLBERT, Proc. Natl. Acad. Sci. U.S., 53 (1965) 626. S. PENMAN, I. SMITH, E. HOLTZMAN AND H. GREENBERG, Natl. Cancer Inst. Monograph, 23 (1966) 489 . U. LOEI~ING, Biochem. J., lO2 (1967) 251. J. R. WARNER AND ]~. SOEIRO, Proc. Natl, Acad. S d . U.S., 58 (1967) 1984. R. A. WEINBERG, U. LOENING, M. WILLEMS AND S. PENMAN, Proc. Natl. Acad. Sci. U.S., 58 (1967) lO88. G. SIEBERT, J. VILLALOBOS, JR., T. S. RO, W. J. STEELE, G. LINDENMAYER, H. ADAMS AND H. BUSCH, J. Biol. Chem., 241 (1966) 71. D. D. BROWN AND C. S. WEBER, J. Mol. Biol., 34 (1968) 661. M. WILLEMS, E. WAGNER, R. LAING AND S. PENMAN, J. Mol. Biol., 32 (1968) 211. M. C. LIAU AND R. P. PERRY, J. Cell Biol., in t h e press.
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