Nucleases in hela cell nucleoplasm and nucleoli

Nucleases in hela cell nucleoplasm and nucleoli

428 Bioehimica et Biophysica Acta, 349 (1974) 428--441 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 97998 ...

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428

Bioehimica et Biophysica Acta, 349 (1974) 428--441 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 97998

NUCLEASES IN HELA CELL NUCLEOPLASM AND NUCLEOLI

CATHERINE NING KWAN, S A D A O G O T O H and DAVID SCHLESSINGER

Department of Microbiology Washington University School of Medicine, St. Louis, Mo. 63110 (U.S.A.) (Received December 12th, 1973)

Summary HeLa cell nucleoplasmic and nucleolar nucleases were fractionated on DEAE-cellulose columns. Each fractionation showed at least three activity peaks that produced acid-soluble fragments from [SH]poly(A); but different enzyme activities seemed to be differentially concentrated in nucleoli and nucleoplasm. The most active ribonuclease in nucleoplasm (called "Nu-B") was identified as a processive exonuclease, while the most active ribonuclease in the nucleolar fraction (called "Nu-A") was shown to be an endonuclease that produced oligonucleotides bearing 5'-phosphate groups. Crude and partially purified nuclease fractions were all tested for their capacity to cleave pre-rRNA in nucleoli and preribosomes. With both substrates, the sizes and the specific methyl content of the fragments resembled those made in vivo to some extent; but all the column fractionsalso degraded purified 18 S rRNA, 28 S rRNA, and artificialpolynucleotides. Thus, previous reports of "processing" activity from nucleoli were apparently measuring the effects of a number of enzymatic activitiesof weak specificity.In spite of the apparent localizationof enzymes in nucleoli and nucleoplasm, there is as yet no clear correlation with specificphysiological functions.

Introduction At least three distinct forms of RNA polymemse occur in the eucaryotic cell, each with a unique function and location [1]. Since the production of mature forms of rRNA involves degradation as well as synthesis [2], it seemed possible that this diversity also extended to ribonucleases. Our starting question was whether there are nucleolar nucleases distinct from nucleoplasmic ones. Here w e report preliminary fractionation of nucleoplasmic and nucleolar nuclease activities on DEAE cellulose columns. Each profile showed at least three degradative activities when poly(A) was used as sub6trate. The action of material from two of these peaks was further studied. Also, the ability of

429 nuclease fractions to process pre-ribosomal RNA was assayed. Tests for "processing" seemed reasonable, since the formation of ribosomal RNA in HeLa cells involves the stepwise cleavage of a 45 S precursor RNA into smaller RNA appearing in the cytoplasm. The in vivo steps for this process, involving intermediate 41, 32, and 20-S RNA species complexed with protein, have been elucidated from many laboratories [2,3]. Since processing of rRNA occurs mainly in the nucleoli, one would specifically expect that there migl~t be ribonuclease concentrated in the nucleoli and involved in the processing. Endonuclease activity has earlier been obtained from nucleoli by Mirault and Scherrer [4] which tends to convert the preribosomal RNA (pre-rRNA) in isolated preribosomes to sizes comparable to nucleolar and cytoplasmic RNA in vivo. Such endonuclease activity has also been observed by Winicov and Perry (personal communication) from mouse L-cells. Here we report more extensive results, obtained primarily with the crude and DEAE-fractionated nucleolar endonuclease activities. Evidence has been obtained that the nuclease fraction from nucleoli contains several activities that can attack free RNA or RNA in preribosomes. Although none of the fractions showed a specificity limited to these substrates, their localization and endonucleolytic action suggest a possible function in rRNA processing. Materials and Methods

(A) Materials [3 H] Uridine (27 #Ci/#mole), ['4C]uridine (54 mCi/mmole), and [Me-3H]methionine (2.3 Ci/mmole) were from Schwarz Bioresearch, Inc. Heparin was from Organon, Inc., W. Orange, N.J., DEAE cellulose from Whatman; Deoxyribonuclease, spleen phosphodiesterase, and snake venom phosphodiesterase from Worthington Biochemicals; pancreatic ribon (ribon A) from Sigma Chemical Co. Essentially homogeneous purified alkaline phosphatase was kindly provided by Dr M. Schlesinger. [3 H] Poly(A) (83.6 pCi/pmole P); [3 H]poly(C) {51.5 #Ci/pmole P); and [3H]poly(U)(82.2 pCi/pmole P ) w e r e from Schwarz Bioresearch, Inc. Thin layer chromatogram plates were from Brinkmann Instruments, Inc. MEM Joklik medium was purchased from Grand Island Biological Co.

(B) Preparation of crude ribonuclease fraction Nucleases were obtained from crude nucleoplasmic and nucleolar fractions. Nucleoplasm and nucleoli were made by following the methods of Penman [5] with some modification. 2 • 109 cells were swollen in 40 ml (0.01 M Tris--HC1 (pH 7.50)--0.01 M NaC1--0.15 mM MgC12) for 10 min at 0°C and then 2 M sucrose in 0.01 M Tris--HC1 (pH 7.5)--0.01 M NaC1--0.15 mM MgC12 was added to a final sucrose concentration of 0.25 M and Triton X-100 to a final concentration of 0.25%. This mixture was given 10 strokes in a Dounce Homogenizer. It was then layered on an equal volume of 0.5 M sucrose in 0.01 M Tris--HC1 (pH 7.5)--0.01 M NaC1--0.15 mM MgCI2, and centrifuged at 2000 × g for 5 minutes to yield a pellet of nuclei. The nuclear pellet was resuspended with 10 ml of 0.01 M Tris--HC1 (pH 7.5)--0.01 M NaC1--0.15 mM MgC12,1.0 ml Tween 80 (10%), and 0.5 ml deoxycholate (10%) and mixed rapidly for 30 s

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on a Vortex shaker. The suspension was immediately centrifuged. The resulting nuclear pellet was free of cytoplasmic tabs when observed in a phase contract microscope. The purified nuclei were disrupted by resuspending in 40 ml of high salt buffer (0.01 M Tris--HC1 (pH 7.5)--0.05 M MgC12--0.5 M NaC1) + 800 /~g deoxyribonuclease. The mixture was mixed vigorously on a Vortex mixer and pipetted repeatedly until there were no viscous clumps. The mixture was then layered on 0.88 M sucrose in 0.01 M Tris--HC1 (pH 7.5)--0.01 M NaC1--0.15 mM MgC12 and centrifuged for 20 min at 500 × g. The procedure was repeated once on the pelleted nucleoli. The final pellet was clean nucleoli, judged by microscopic examination after staining with Azure C. The first supernatant was nucleoplasm, and was saved for further enzyme preparation. To prepare crude nucleolar enzyme the pellet of clean nucleoli was extracted with 5 ml of preribosome extracting buffer (0.01 M Tris--HC1 (pH 7.5)--0.01 M dithiothreitol--0.1% Brij 58--0.01 M KC1--0.1 mM MgC12 ) for 10 min at 25 ° C with gentle homogenization. Then 1 ml of 1 M Tris buffer (pH 8.0) was added, followed by 6 ml of 2 M NH4 C1 containing 0.02 M EDTA. The total mixture (12 ml) was further homogenized for 5 min and centrifuged (1500 × g, 10 min). The supernatant was crude nucleolar enzyme. Both nucleoplasmic and nucleolar crude enzyme were dialyzed against 0.01 M Tris--HCl (pH 7.5)-0.01 M NaC1--0.15 mM MgCl2 overnight. To purify the enzyme further, 10 to 20 mg protein of either nucleoplasmic or nucleolar crude enzyme was loaded on a 1 cm × 10 cm DEAE cellulose column. The column was eluted first with 10 ml of 0.01 M Tris--HC1 (pH 7.5)--0.01 M NaC1--0.15 mM MgC12, then with a linear gradient of 30 ml of 0.01 M Tris--HC1 (pH 7.5)--0.01 M NaCI--O.15 mM MgC12 and 30 ml of 0.01 M Tris--HC1 (pH 7.5)--0.01 M NaC1--0.15 mM MgC12 plus 0.4 M NaCl. 1.5 ml fractions were collected, and the enzymaticaUy active fractions were pooled and dialyzed against 0.01 M Tris--HCl.(pH 7.5)--0.01 M NaCI-0.15 mM MgC12. 1 mg/ml of bovine serum albumin was added to each pooled ribonuclease fraction to stabilize the enzyme.

(C) Assay of ribonuclease using synthetic polynucleotides as substrates A reaction mixture of 50/~1 contained 0.08 to 0.16/~g of [3 H] poly(A), [3 H] poly(C) or [3 H] poly(U); 0.01 M Tris--HC1 (pH 7.8)--0.01 M NaCI--0.005 M MgC12 and enzyme. After incubation at 37°C, 200 #1 of 10% trichloracetic acid was added together with 100/~g of carrier yeast RNA [6]. After 30 min on ice, the mixture was centrifuged. 150 #1 of supernatant was pipetted into 10 ml of Bray's scintillation fluid and counted. To observe the extent of fzagmentation of the substrates, 0.2 ml of 95% ethanol was added to a reaction mixture together with carrier yeast RNA. Ethanol precipitated polynucleotide was dissolved in buffer and analyzed by polyacrylamide gel electrophoresis [7]. Ethanol soluble radioactivity was determined by adding scintillation fluid to the supernatant.

(D) Preparation of nucleoli and preribosomal particles as substrates $3 HeLa cells were grown in spinners in MEM Joklik modified medium plus 5% horse serum. Cells were labeled at a concentration of 2 • 10 4 cells/ml,

431 with [3 H] uridine (5 gCi/ml) for 15 min. For double labeling with [Me-3H] methionine (10 pCi/ml) and [14C]uridine (0.5 #Ci/ml), cells were concentrated in medium minus methionine plus 2 • 10 -5 M adenosine and guanosine, and incubated in growing conditions for 15 min before the addition of the labeled compounds. 10 s cells were then collected, washed and broken by hypotonic swelling in 6 ml 0.01 Tris-HC1 (pH 7.5)--0.01 M NaC1--0.015 mM MgC12. Nuclei were prepared as described and washed with 2 ml of 0.01 TrisHC1 (pH 7.5)--0.01 M NaC1--0.015 mM MgC12 containing 0.2 ml Tween 80 (10%) and 0.1 ml deoxycholate (10%). To prepare nucleoli, two methods were used: (1) Modified Penman method [5] : Clean nuclei were resuspended in 6 ml high salt buffer with 50 #g/ml of deoxyribonuclease and 100 #g/ml polyvinyl sulfate. The mixture was layered on 0.88 M sucrose in 0.01 Tris--HC1 (pH 7.5)--0.01 M NaC1--0.015 mM MgCl~ plus 100 /~g/ml polyvinyl sulfate and centrifuged. The procedure was repeated at least once. The final pellet was clean nucleoli. (2) Alternatively, detergent cleaned nuclei were lysed and nucleoli prepared by following the method of Scherrer [8]. Nuclei were lysed with deoxycholate in the presence of heparin and deoxyribonuclease. Nucleoli were pelleted by centrifugation. Preribosomal particles were prepared from nucleoli prepared by either of the above two methods by extracting them with preribosome extracting buffer (see above) at 25°C for 10 min by gently mixing in a homogenizer. The mixture was centrifuged (1500 . g 10 min), and the supernatant contained the preribosomes.

(E) In vitro cleavage of RNA in nucleoli and preribosomes In 1 ml of reaction mixture, nucleoli or preribosomes were incubated with enzyme in the presence of 0.01 M Tris--HC1 (pH 7.5)--0.01 M NaC1--0.0015 M MgC12 and 100 #g yeast RNA. Since preribosomes were extracted with "preribosome extracting buffer", reactions with preribosomes always contained 0.005 M dithiothreitol--0.005 M KC1--0.05% Brij 58. Reactions were stopped with sodium dodecylsulfate added to a final concentration of 0.5% and EDTA to 0.002 M final concentration. RNA from nucleoli was extracted by hot phenol following Penman's method [5], and precipitated with ethanol. RNA from preribosomes was directly precipitated with ethanol. RNA was examined by agarose polyacrylamide gel electrophoresis [7]. Results

(1) Partial purification of nucleoplasmic ribonuclease activities When nuclei were fractionated, 63% of the total nuclear protein, as detected by the biuret reaction [9], was recovered in the nucleoplasm. Synthetic polynucleotides were readily hydrolyzed to alcohol-soluble material during incubation with the crude nucleoplasmic fraction. Preribosomes incubated with crude nucleoplasmic fraction also released alcohol soluble material, ~though after a lag (Fig. 1). RNA extracted from preribosomes after 5 min and 30 min of incubation showed that extensive endonucleolytic degradation had occurred even after 5 min of incubation when almost no alcohol-soluble mate~al (3%) had been released (Fig. 2). It thus seemed likely that the crude nucleoplasmic

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Fig. I. Kinetics o f soinbilizationof preribosomes by crude nucleoplasmic fraction, l~eribosomes (0.03 A 2 6 0 n m units, 3000 cpm) were incubated with 34 #g protein of crude nucleoplasmic extract in a I ml reaction mixture, as in Materials and Methods. The reaction mixture was preincubated without enzyme at 37°C for 5 m i n to bring it to temperatuxe; enzyme was then added (0 min). At the times indicated, 200/~I of ethanol was added to stop the reaction in replicate samples. Ethanol soluble radioactivitieswere determined.

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Fig. 3. D E A E - c e l l u l o s e c o l u m n c h r o m a t o g r a p h y o f c r u d e e n z y m e f r a c t i o n s . (a) 3 6 . 0 m g p r o t e i n o f c r u d e n u c l e o p l a s m i c e x t r a c t w a s p u t o n t h e c o l u m n (see M a t e r i a l s a n d M e t h o d s ) . (h) 1 0 . 5 m g p r o t e i n o f c r u d e n u c l e o l i e x t r a c t w a s p u t o n t h e c o l u m n . A f t e r t h e a b s o r b ~ n c e a t 2 8 0 rum w a s d e t e r m i n e d f o r e a c h f r a c t i o n ( . . . . -), 40/~I o f e a c h w a s a s s a y e d w i t h [ 3 H ] p o l y ( A ) as s u b s t r a t e , a n d t h e release o f a c i d - s o l u b l e r a d i o a c t i v i t y w a s m e a s u r e d (o----o) a f t e r a d d i t i o n o f 2 0 0 pl o f 1 0 % t r i c h l o r o a c e t i c a c i d . Fig. 4. Gel e l e c t r o p h o r e t i c a n a l y s i s o f [ 3 H ] p o l y ( A ) a n d its p r o d u c t s a f t e r d i g e s t i o n b y N u - B e n z y m e . [ 3 H ] P o l y ( A ) ( 0 . 1 6 /~g, 1 4 0 0 0 c p m ) w a s i n c u b a t e d f o r 3 0 r a i n w i t h (a) n o e n z y m e (b) 8 0 ~I o f N u - B enzyme. To stop the reaction 1 ml of ethanol was added together with I00/~g of carrier RNA. After I h a t - - 2 0 ° C , R N A w a s c o l l e c t e d b y c e n t r i f u g a t i o n a n d a n a l y z e d b y gel e l e c t r o p h o r e s i s .

fraction contained both endonuclease and exonuclease activity. Fractionation on a DEAE cellulose column with a linear salt gradient resolved several minor and one major ribonuclease activity peak (Fig. 3a), as measured by the acid-solubilization of poly(A). The major activity peak, labeled Nu-B, was investigated further. It gave 5'-AMP as the only alcohol soluble product during incubation with [3 HI poly(A). It was shown to act as a processive exonuclease by a diagnostic test: when 30% of the input [3 H] poly(A) was alcohol soluble, the remaining poly(A) still had the same size as the unincubated control (Figs 4a and 4b).

(2) Partial purification of nucleolar ribonuclease activities Crude nucleoli contained 13% of the total nuclear proteins. When synthetic polynucleotides or purified 45, 28 or 18 S RNA were incubated with crude nucleolar fraction, very little alcohol or acid soluble material was released during short term incubations. However, when the RNAs were examined by polyacrylamide gel electrophoresis after incubation they were somewhat fragmented, indicating that during these trials, the nucleolar ribonuclease activity detected was mainly endonucleolytic in nature. This interpretation was supported by assays with fractionated material. By a fractionation on DEAE cellulose similar to that applied to the nucleoplasmic fraction, three ribonuclease activities were detected (Fig. 3b). These are referred to below as Peak Nos A, B, and C. All three enzyme fractions fragmented poly(A) and 28 S + 18 S RNA extensively without producing appreciable alcohol soluble materials (Fig. 5). Thus, no fraction corresponding

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Fig. 5. Gel e l e c t r o p h o r e t i c analysis of [ 3 H ] p o l y ( A ) , 28 S + 18 S [ 1 4 C ] R N A a n d their p r o d u c t s after d i g e s t i o n b y various n u c l e o l a r e n z y m e fractions. [ 3 H ] P o l y ( A ) 45813 cpm, 10 pg) w a s i n c u b a t e d for 1 h w i t h 4a) n o e n z y m e ; 4b) 40 /~1 of No. A enzyme; 4c) 40 #1 of No. B e nz yme ; and (d) 40 ~] of No. C enzyme. 14C 28 S + 18 S [ 1 4 C ] R N A 47417 epm, 8.8 #g) w a s i n c u b a t e d for 1 h w i t h (e) no e nz yme ; (f) 40 pl of No. A e n z y m e ; (g) 40 pl of No. B e n z y m e ; and 4h) 40 pl of No. C enzyme. T o s t o p t h e r e a c t i o n 20 #1 of e t h a n o l w a s a d d e d t o g e t h e r w i t h 1 0 0 ~tg o f carrier R N A . A f t e r 1 h at --20°C R N A w a s c o l l e c t e d by c e n t r i f u g a t i o n and a n a l y z e d b y gel e l e c t r o p h o r e s i s . E t h a n o l soluble c p m w e r e also d e t e r m i n e d ; t h e y were (as percentage o f input); (a) 0; (b) 0.9; (c) 5.9; (d) 0.6; (e) 0; (f) 2.0; (g) 2.9; and (h) 0.6.

to Nu-B was detected. No. C was more active against 28 S + 18 S RNA while it was very inactive in degrading [3 H]poly(A); an effect on poly(A) was detected only after prolonged incubation with the low substrate concentration used (data not shown). The major peak of activity (No. A) has been characterized more extensively, as its origin seemed predominantly nucleolar (c.f. Fig. 3a). The products of peak No. A action were oligonucleotides with 5'-phosphate end groups. This was shown by the results in Table I. The test design was based on the use of spleen phosphodiesterase, which produces 3'-XMP from substtates (oligonucleotides or RNA) that bear a 5'-OH end group [ 1 0 ] . Oligonucleotides bearing 5'-phosphate groups are resistant to the enzyme. As shown in Table I,

435 TABLE I THIN LAYER [3H] POLY(A)

CHROMATOGRAPHIC

ANALYSIS

OF PRODUCTS

OF PEAK

No. A ACTION

ON

Each column entry shows the percentage of total input 3H-label found at that position on a thin-layer c h r o m a t o g r a p h i c p l a t e . E x p t 1. I n 1 5 0 DI, 0 . 3 6 # g o f [ 3 H I p o l y ( A ) w a s i n c u b a t e d w i t h (a) n o e n z y m e (b) 9 0 //I o f P e a k N o . A e n z y m e a n d (c) 0 . 6 Dg o f p a n c r e a t i c r i b o n u e l e a s e . A f t e r 1 h a t 3 7 ° C e a c h w a s s e p a r a t e d i n t o t h r e e 5 0 DI p o r t i o n s . T h e f i r s t w a s p r e c i p i t a t e d w i t h e t h a n o l t o r e c o v e r t h e p o l y ( A ) a n d e x a m i n e d b y gel e l e c t r o p h o r e s i s ( r e s u l t , see Fig. 7). T h e s e c o n d w a s i n c u b a t e d f o r 2 0 m i n w i t h o u t a n y a d d i t i o n . T h e t h i r d 5 0 / ~ l p o r t i o n w a s i n c u b a t e d f o r 2 0 r a i n w i t h 1 0 ~tg o f s p l e e n p h o s p h o d i e s t e r a s e . T h e second and third portions were spotted on thin-layer chromatographic plates and developed together with u l t r a v i o l e t m a r k e r s f o r 9 0 r a i n i n 0 . 3 M LiCI. T h e l o c a t i o n s c o r r e s p o n d i n g t o t h e o r i g i n a n d 2 ' ( 3 t ) - A M P a n d a d e n o s i n e w e r e c u t o u t a n d t h e i r r a d i o a c t i v i t i e s d e t e r m i n e d . E x p t 2. 1 5 0 D1 e a c h o f t w o r e a c t i o n s c o n t a i n i n g 0 . 3 6 /~g o f [ 3 H ] p o l y ( A ) w e r e c a r r i e d o u t w i t h (a) n o a d d e d e n z y m e (b) w i t h 9 0 #l o f N o . A e n z y m e . A f t e r 6 0 r a i n a t 3 7 ° C e a c h w a s s e p a r a t e d i n t o t h r e e e q u a l p o r t i o n s . To t h e f i r s t n o t h i n g w a s a d d e d . T o t h e s e c o n d 2 0 /Jg o f s p l e e n p h o s p h o d i e s t e r a s e w a s a d d e d . T o t h e t h i r d 1 0 /~g o f a l k a l i n e p h o s p h a t a s e w e r e a l s o a d d e d . All p o r t i o n s w e r e i n c u b a t e d f o r a n a d d i t i o n a l 1 5 r a i n b e f o r e s p o t t e d o n thin-layer chromatographic plates. Radioactivities at the origin, 2'(3')-AMP and adenosine locations were determined. Expt I Locations on thin-layer chromatographic plate

(a) N o e n z y m e

(b) + N o . A e n z y m e

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14.5% of untreated poly(A) was degraded by spleen phosphodiesterase. However, when poly(A) was first fragmented with Peak No. A enzyme, the product was completely resistant to spleen phosphodiesterase. This result implies that the end groups produced by Peak No. A enzyme bear 5'-PO4 groups. The apparent disappearance of even the basal (14.5%) level of susceptibility could result from several causes. For example, 5'-PO4 end groups might competitively inhibit enzymatic action on the small number of pre-existing 5'-OH ends. Alternatively, or in addition, the fraction of polynucleotide chains originally sensitive to spleen phosphodiesterase may have become inactive or quantitatively insignificant as they were shortened by cleavage during the pretreatment with No. A enzyme. In support of the first interpretation, it was seen that removal of the 5'-PO4 group from the products formed by peak No. A enzyme with purified alkaline phosphatase rendered them completely sensitive to spleen phosphodiesterase (Expt II, Table I). As a control, poly(A) was treated with a relatively high level of pancreatic ribonuclease (4 pg/ml) that produced oligonucleotides of similar size, as

436

detected by gel electrophoresis (pancreatic ribonuclease is relatively selective for pyrimidine sites, but will slowly cleave poly(A) [11] ). Although only 8% of the products of ribonuclease A appear as 2'(3')-AMP, further treatment by spleen phosphodiesterase results in an 8-fold increase in this product. This is expected, since pancreatic ribonuclease produces oligonucleotides that bear 5'-OH end groups [11], and are therefore susceptible to degradation by the spleen phosphodiesterase. Each of the 3 pooled enzyme fractions was also tested for its ability to degrade poly(C) and poly(U). Fractions No. A and No. C degraded poly(C) and poly(U) to acid-soluble products at a rate 2 to 4 times faster than the highest rates observed with poly(A), while No. B showed a similar activity with all the substrates.

Cleavage of R N A in nucleoli and preribosomes by nucleolar enzyme fractions. To obtain undegraded 45 S RNA from nucleoli purified by the high salt method of Penman [12], we found it necessary to wash the nucleoli with high salt buffer and polyvinylsulfate, and to include yeast RNA in the test incubations [13]. In addition, nucleoli were purified by centrifugation through layers of sucrose containing polyvinylsulfate (see methods), and 100 pg/ml of yeast RNA was also included in the incubation mixture. The electrophoretic pattern of RNA from such nucleoli showed no change after incubations of up to 20 min at 37°C without enzyme. However, when these nucleoli were incubated with increasing amounts of crude nucleolar enzyme (Fig. 6), a decrease of 45 S RNA was detected, with a concomitant increase in RNA in the 28 S and 18 S regions. RNAs of intermediate sizes (about 41 S, 32 S, 20 S) were also found, but very little RNA smaller than 18 S was detected. (It may be significant that in contrast, Fig. 5 shows that purified 18 S and 28 S RNA were extensively fragmented by similar amounts of crude enzyme; see Discussion). Incubation of these reaction mixtures yielded no measurable alcohol soluble radioactivity, consistent with the observations that such preparations contain little if any exonuclease activity (Part I above). Comparable results were obtained with preribosomes prepared by either of two methods (see Methods). There was no apparent processing of RNA when it was incubated without added enzyme in assay mixtures for 30 min or more, but in confirmation of the results of Mirault and Scherrer [4], much of the 45 S RNA was again cleaved by the crude nucleolar enzymes to material with higher mobilities on gels. An additional criterion to gauge whether nuclease action mimics processing in vivo is provided by the distribution of methyl groups in the products. Pre-rRNA is already methylated, and the dispensable portion of the molecule contains no methyl groups [14,15]. Therefore, during processing in vivo, the ratio of methyl groups to uridine increases. Nucleoli doubly labeled with [Me .3H] methionine and [i 4 C] uridine (see Materials and Methods) were also used as substrate in the in vitro assay system. As shown in Fig. 7, in the control with no enzyme added, the major R N A species was 45 S R N A , with a ratio in c p m of 3 H/Z 4 C of 0.25. W h e n enzyme was added, R N A of the 28 S and 18 S size class began to appear; the ratio in the 28 S region was 0.35, while for 18 S it was about 0.40. The results thus

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showed that some o f the smaller RNA produced as degradation products had higher methyl content. The assays of cleavage of pre-rRNA and rRNA were extended to the partially purified fractions of nucleolar enzymes. In spite of the differences in relative activity of enzyme Peaks No.A, No. B and No. C toward poly(A), the same amounts of these three preparations showed equivalent activity against labeled nucleoli (Fig. 8) or purified 18 S or 28 S rRNA. However, all of them were totally inactive against comparable levels of rttNA in purified mature ribosomes, even during incubations of 90 min or more. Discussion Two enzyme activities have earlier been detected and partially purified from extracts of mammalian cell nuclei. One is a well characterized exonuclease

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439 that gives 5'-XMP as its products [16]. The activity we have partially characterized as Peak Nu-B (Fig. 3a) is probably the same enzyme. We found little if any of this activity in nucleoli; and it can tentatively be termed a nucleoplasmic (Nu) enzyme. The other previous relevant study was the description by Heppel [17] of a non-specific endonuclease from hog liver nuclei. While the techniques used were different, the enzyme was reported to produce oligonucleotides with 5'-PO4 end groups; no intranuclear localization was examined. Whether any of the activity peaks reported here are identical to this endonuclease is unknown, but Peak No. A could correspond. According to the fractionation trials like those of Fig. 3, at least 80% of the Peak No. A activity, and possibly all of it, was obtained from nucleoli, and it can tentatively be termed a nucleolar (No) enzyme. Very likely the enzyme activities No. A, No. B, and No. C were all included in the crude preparations in trials in earlier work using nucleolar enzyme to process 45 S pre-rRNA [4]. One might speculate that unique nucleolar endonucleases might function in rRNA formation, while others might be reserved for mRNA formation in the nucleoplasm. Pre-rRNA might first be cleaved endonucleolytically in the nucleolus. Dispensable fragments would then be released from the membrane-less nucleolus and further degraded by enzymes in the nucleoplasm. It has been suggested that the enzyme of Sporn et al. [16] may be involved in such a process [18,19]. However, the evidence that any of the activity obtained from nucleoli here or in previous trials is truly involved in processing pre-rRNA in vivo seems weak. Three positive indications from the trials reported here with crude nucleolar enzyme were that (1) RNA moved from the 45 S to the 28 S and to a lesser extent to the 18 S region (Figs 6--8); (2) Very little RNA smaller th~in 18 S was detected (Figs 6--8); (3) From RNA doubly labeled with [Me-3H]methionine and [ ' 4 C ] uridine, 28 S and 18 S RNA were produced with higher 3 H/14C ratios than that found in 45 S RNA (Fig. 7). In vivo, 45 S RNA has a relatively low ratio (0.63) that increases progressively in intermediate RNAs (41 S, 32 S, 20 S) and product 28 S (1.27) and 18 8 (1.82) species; fragments from the dispensable portions should have an extremely low ratio [3]. The results in vitro (Results, Section C) showed an enrichment from 0.25 to 0.35 and 0.40. While these results would be consistent with specific cleavage, Fractions No. A, No. B and No. C all degraded 45 S RNA in nucleoli (Fig. 8), but also attacked a variety of ribopolymers as well as purified 18 S and 28 8 rRNA. Also, the products obtained from 45 S pre-rRNA were somewhat similar but more polydisperse than those during processing as seen in RNA extracted from labeled cells. Furthermore, crude preparations of nucleoplasm also attacked preribosomal particles endonucleolyticaUy (Fig. 2); and even low levels of pancreatic ribonuclease gave similar products from 45 S RNA (unpublished results; also Mirault and Scherrer, personal communication). As for the enrichment of methyl groups in some cleavage products, the methyl label is known to be concentrated in those RNA segments destined to become the 28 S and 18 S rRNA species, and the bulk of the unmethylated,

440 dispensable part occurs toward one end of the 45 S RNA chain [20]. As a result, even random initial cleavages of the precursor could tend to give enrichment for methyl groups in some fragments. If the observed localization of some endonuclease activity in nucleoli is indeed related to the mechanism of ribosome formation, then two alternatives seem possible at present: (1) The specificity of the observed activity is increased in vivo by an unknown regulatory element, or by the limited accessibility of substrate to enzyme. It seems of possible relevance that the RNA in finished ribosomes, in contrast to isolated 45 S pre-rRNA or preribosomes, was not cleaved by these nucleases. Also, the same amounts of enzyme that degraded 18 and 28 S RNA to small fragments (Fig. 5) produced no small fragments from equivalent amounts of RNA in preribosomes. The notion of cleavage sites selectively exposed free of protein has also been suggested by the reports that when protein synthesis was inhibited by cycloheximide [21] or puromycin [22], no mature rRNA was formed. 45 S RNA continued to be made; however, apparently due to a shortage of proteins, random ribonuclease degradation occurred. (2) The observed endonucleases might function in secondary, less specific cleavages, and an as yet uncharacterized activity could function to yield some of the specific first products observed in vivo. A strong hint for such an alternative has come from recent studies on the processing of an Escherichia coli 30 S pre-rRNA that is analogous to the large pre-rRNA in higher cells. In that case, a specific endonuclease, ribonuclease III, which gives site-specific cleavage at double-stranded RNA regions [23], has been shown to cleave the large prerRNA both in whole cells and with purified pre-rRNA [24--26]. In HeLa cells, double-stranded RNA sequences have been suggested as potential specific cleavage sites in 45 S pre-rRNA [27]. We will report separately on suggestive limited cleavages of 45 S pre-rRNA by E. coil ribonuclease III, and on activity from HeLa cell nuclei analogous to ribonuclease III of E. coll. Acknowledgements This work was supported by Grant No. CA12021 from the National Cancer Institute.W e are grateful to Heschel Raskas for helpful discussions. References 1 2 3 4 5 6 7 6 9 10

Roeder, R.G. and Ruttex, W.J. (1970) Proc. Natl. Aead. Sci. U.S. 65, 675---682 Darnell, J.E. (1968) Bacteriol. Rev. 32, 262--290 Maden, B.E.H. (1971) l~og. Biophys. Mol. Biol, 2 2 , 1 2 7 - - 1 7 7 Mirault, M.E. and Seherrer, K. (1972) FEBS Lett, 20, 288--238 Penman, S. (1966) J. Mol. Biol. 1 7 , 1 1 7 - - 1 3 0 Baltimore, D. and Smoler, D.F. (1972) J. Blol. Chem. 247, 7 2 6 2 - 7 2 8 7 Weiss, B. and Schleslnger, S. (1973) J. Virology, 12, 862--871 Scherrer, K. and Mirault, M.E. (1971) Eur, J, Biochem. 28, 372--386 Ellmann, G.L. (1962) Anal. Bioehem. 3, 40--48 RazzeU, W.E. (1968) Methods in E n z y m o l o g y (Colowick, S.P, and Kaplan, N.O., eds), Vol. VI, pp. 236--259, Academic Press, New Y o r k 11 Volkin, E. and Cohn, W.E. (1953) J. Biol. Chem. 2 0 5 , 7 6 7 - - 7 8 2 12 Vesco, C. and Penman, S. (1968) Bioch/m. Biophys. Acta 169, 186--195

441 13 Liau, M.C., Craig, N.C. and Perry, R.P. (1968) Biochim. Biophys. Acta 1 6 9 , 1 9 6 - - 2 0 5 14 Weinberg, R.A. and Penman, S. (1970) J. Mol. Biol. 47, 169--178 15 Weinberg, R.A., Loening, U., Williams, M. and Penman, S. (1967) Proc. Natl. Acad. Sci. U.S. 58, 1088--1095 16 Sporn, M.B., Lazarus, H.M., Smith, J.M. and Henderson, W.R. (1969) Biochemistry 8, 1698--1706 17 Heppel, L.P. in Procedures in Nucleic Acid Research (Cantoni, G.L. and Davies, D.R., eds), (1966) pp. 31--35, Harper and Row, New Y o r k 18 Kelley, D. and Perry, R. (1971) Biochim. Biophys. Acta 238, 357--362 19 Perry, R. and Kelley, D.E. (1972) J. Mol. Biol. 70, 265--279 20 Wellauer, P, and Dawid, I. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2827--2831 21 Wlllems, M., Penman, M., and Penman, S. (1969) J. Cell Biol. 4 1 , 1 7 7 - - 1 6 7 22 Soiero, R., Vaughan, M.H., and Darnell, J.E. (1968) J. Cell Biol. 36, 91--101 23 Ro bertson, H.D., Webster, R.D., and Zinder, N.D. (1968) J. Biol. Chem, 243, 82--91 24 Nikolaev, N., Silengo, L. and Schlessinger, D. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3361--3365 25 Dunn, J.J. and Studier, F.W. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3 2 9 6 - - 3 3 0 0 26 Nikolaev, N., Silengo, L. and Schlessinger, D. (1973) J. Biol. Chem. 248, 7967--7969 27 Snyder, A.L., Kann, H.E. and Kohn, K.W. (1971) J. Mol. Biol. 58, 555--565