Turnover of ribosomal RNA in cells in culture

Printed in Sweden Copyright ~ 1977 by Academic Press, Inc. All rights of reproduction in any form reserved 1SSN 0014-4827

Experimental Cell Research 108 (1977) 207-219

TURNOVER

OF RIBOSOMAL RNA IN CELLS IN CULTURE J. F. SCOTT

Huntington Laboratories, Massachusetts General Hospital, Boston, M A 02114, USA

SUMMARY BALB/c 3T3 cells have been used to study the turnover of ribosomal RNA. It has been found that as the cells become confluent there ensues a stochastic degradation of ribosomal RNA (rRNA) which had been isotopically labeled by exposure of the cells to methyl-labeled methionine during the growth phase of the cultures. The rate of loss of label from the 18S RNA was slower than that from the 28S RNA when the RNA was fractionated by centrifugation in aqueous sucrose gradients, but not when the RNA was fractionated in dimethylsulfoxide-sucrose gradients. The RNA of sedimentation velocities intermediate to the 18S and 28S appears to arise from the 28S by a time-dependent chain cleavage. Results of studies with 3T3 cells labeled to equilibrium with 3~p show that there are at least two kinds of endonucleolytic chain scissions particulayly in the 28S RNA: (1) Those which lead to fragmentation of the RNA on deproteinization of the ribosomes at room temperature; and (2) those which only appear when the isolated RNA is fractionated in the presence of dimethylsulfoxide. No significant difference in the level of such scissions could be established in RNA derived from native ribosomal subunits, monomeric ribosomes, or polysomes. Neither did the results support the conclusion that the endonucleolytic process which gives rise to these chain scissions was connected with the mechanism which results in the degradation of ribosomes in these cells at confluence.

It has been known for a number of years that the RNA of ribosomes (and the proteins as well) of the livers of normal, adult rats and kidneys of mice is in a steady state of balanced synthesis and degradation [1--4]. (This simultaneous synthesis and degradation was termed "turnover" by Schoenheimer [5] and that term will be used in the same sense in this paper.) Such turnover can also be inferred for tissues in which labeled precursors are incorporated into rRNA in the absence of net increase in the amount of rRNA during the period of labeling [6]. There is evidence that as non-growing tissues are stimulated to increase cell number or cell size an early event is the increase in the ribosomal content of the tissues [713]. For this and other reasons considerable 14-771801

emphasis has been given to the details of the synthesis and processing rRNA and controls thereof. Recently evidence has appeared which indicates that in some tissues so stimulated the accumulation of ribosomes is due to alteration in the rate of degradation of the organelle [ 14-16, 51]. Studies on cells in culture have clearly shown that in growing cells there is no discernable degradation of rRNA while in cells which slow and stop growing as the cell concentration increases to confluence the rate of synthesis slows and a stochastic degradation of existing ribosomes ensues [17-23]. These phenomena are quickly reversed when such cells are stimulated to grow by the addition of fresh serum [1819]. It thus appears that both in vivo and in cells in culture the concentration of riExp Cell Res 108 (1977)

208

J. F. Scott

bosomes is regulated both by changes in the rate of synthesis and changes in the rate of degradation. Our own interest in the phenomenon of ribosome degradation was stimulated by our observations [24-26] and those of others [27] that in some transplantable tumors there is conservation of rRNA. While the degradation of ribosomes appears to be a regulated process, there is little evidence as to how ribosomes are disposed of. A number of investigators [2837] have noted that in addition to the 18S and 28S RNA species of the ribosomes there are a number of species smaller than 28S which appear to arise in vivo by degradation of the 28S and some [28-29, 3637] have suggested that the endonucleolytic process which gives rise to these fragments may be involved in the process of disposal of ribosomes in the turnover. We wish here to report on studies using BALB/c 3T3 cells to determine whether there was a correlation between the level of "nicking" of the rRNA and the onset of turnover of ribosomes in such cells. No such correlation could be established. The results do not support the conclusion that the endonucleolytic process(es) which give rise to the "nicking" is connected with the mechanism by which ribosomes are degraded in these cells at confluence. We further show that about 30% of the 28S rRNA isolated from native large subunits, monomeric ribosomes, or polysomes from both growing and confluent cells contain nicks which are revealed by fractionation under disaggregating conditions. The 18S rRNA is much more stable, particularly that from the native small ribosomal subunits. METHODS Cells Two kinds of cells were used in the experiments reported here: (1) a BALB/c3T3 line [38] (Clone A31, Exp Cell Res 108 (1977)

Aaronson obtained from Dr R. Roblin); and (2) a secondary BALB/c mouse embryo fibroblast culture which was established by us by allowing cells to migrate from small bits of subepithelial connective tissue from an embryo. Cells of either kind were stored at 77 K and brought up for each of the experiments. Cells were grown in Dulbecco's modification of Eagles medium (Grand Island Biological Co.) containing 15 % undialysed calf serum (Flow Laboratories). This medium with 7.5% (v/v) dimethylsulfoxide (Me2SO) (Spectrophotometric Grade, Aldrich Chemical Co.) added was used for the storage of the frozen cells. During the experiments the medium contained 50 tzg/ ml of Gentamycin (Schering Corp.). For the most part ceils were grown in plastic dishes of appropriate size. The cell cultures used as stock for the experiments were tested for mycoplasma contamination by culture through the courtesy of Mrs S. Annenberg in the laboratory of the late Dr Louis Dienes. Details of the seeding densities, growth kinetics, and modifications in the medium are noted in connections with the individual experiments.

Cell labeling Cells which were exposed to [3H]methyl-methionine or [3H]uridine for short periods were incubated in the presence of 10 4 M thymidine to reduce the flow of radioactivity into DNA. For longer periods of incubation the concentration of thymidine was reduced to I0 -5 M. For long term labeling with 32p, carrier-free 32p was added to unmodified medium containing the usual (0.9 raM) concentration of phosphate. It was found that 1-2/xCi 32P/ml of medium did not alter the growth rate or morphology of the cells. At 3 /xCi 32P/ml the growth rate was reduced and bizarre cells were found after 5-7 days of exposure. Further details of the labeling of the cells with radioactive tracers are given in connection with the various experiments.

Cell harvest and fractionation In some experiments the cells were harvested by a modification of the method described by Weber [18]. The dishes were chilled, rinsed three times with cold BSS, and the cells lysed in a solution containing 1% (w/v) SDS, 0.5 % (v/v) mercaptoethanol, 0.1 M EDTA, 0.1 M LiCI, 0.01 M Tris:HC1 (pH 7.5). The lysate was collected from the dish with the aid of a rubber policeman and removed to a centrifuge tube. The dish was rinsed with half a volume of the lysing medium. Approx. 0.5 mg of carrier tRNAco,i was added together with self-digested pronase (100 /zg]ml). Self-digested pronase was prepared by incubating the crude enzyme at a concentration of 1 mg/ml in 1/10 0.1 M NaC1, 0.01 M Na citrate (pH 7.0) (SSC) for 3 h at 37°C. This mixture was stirred and incubated for 2 h at 37°C. At the end of the incubation the mixture was extracted two times at room temperature with an equal volume of freshly dissolved phenol which had been equilibrated against the lysing medium. The nucleic

Turnover of rRNA acids were then precipitated from the upper phase by addition of 2.5 vol of cold ethanol. The precipitate was collected by centrifugation after storage at -17°C overnight. Control experiments in which labeled 18S and 28S RNA were incubated under the above conditions and then fractionated by centrifugation in MezSO as described below showed no detectable endonucleolytic activity of the self-digested pronase. In experiments in which the ribosomal components were to be separated, cycloheximide (50/zg/ml of medium) was added to the dishes 30 min prior to harvest. The cells were then rinsed twice with BSS lacking Mg 2÷ and Ca 2÷ and released by exposure to 0.5/zg/ml trypsin, EDTA 2 mg/ml. The action of trypsin was stopped with culture medium. An aliquot of the cell suspension was removed for counting and the remainder was packed by centrifugation in the cold. The cell pellet was then treated by a modification of the method described by Gielkens et al. [39]. The supernatant was removed and the cells suspended in 0.02 M Tris:HC1 (pH 7.3), 0.13 M KC1, 0.005 M MgCI~ containing 5 mg/ml bovine serum albumin, fraction V. The cells were centrifuged and suspended in 300/xl of 0.05 M Tris : HC1 (pH 8.5), 0.3 M KC1, 0.01 M MgC12, 0.001 M dithiothreitol. To the suspension was added 300 p.l of the same medium containing 2 % (v/v) Triton X-100 and 50/zg/ml dextran sulfate (Type 500-5, Sigma Chemical Co.). The cell suspension was mixed with a vortex mixer at 2 min intervals five times and was held in ice between mixings. The nuclei and unbroken cells were removed by centrifuging the lysate at 700 g for 5 min. The supernatant was removed for further fractionation as described below.

Sucrose gradient fractionation Ribosomalfractionation. Native subunits, monomers, and polysomes were obtained by centrifugation through a non-linear gradient in the Spinco SW41 rotor. The gradient was formed by feeding 42 % (w/w) sucrose in 0.08 M KC1, 0.005 M MgCI~, 0.05 M Tris : HC1 (pH 8.0) into a constant volume (10 ml) mixing vessel containing at the start 20% (w/w) sucrose in the above solvent. Eleven and one-half ml of gradient were pumped into the centrifuge tube and 1 ml of 60 % sucrose in the above solvent was delivered onto the bottom of the tube as a "cushion". The postnuclear supernatant was layered onto the gradient and centrifuged at 41000 rpm at 4 ° for various times (90400 min) depending on the portion of the gradient to be resolved. For the longer centrifugation times the "cushion" delayed or prevented pelleting of the polysome fraction. In some experiments the total ribosomal content of the post-nuclear material was converted to subunits by layering the post-nuclear fraction on a linear gradient formed from 15-30% (w/w) sucrose in 0.01 M LiC1, 0.01 M EDTA, 0.01 M Tris : HCI (pH 7.6). The gradients were centrifuged in the 10.16 cm tubes of the SW27 rotor at 4°, 26000 rpm for 18 h. The desired portions of such gradients were fractionated by observation of the ultraviolet absorbance monitor of an ISCO fractionator (for example, see fig. 2). The ribonucleoprotein material of each fraction was precipitated by addition of 2.5 vol of ethanol.

209

Sucrose gradient fractionation of RNA components of ribosomal fractionsl The ethanol-precipitated ribonucleoprotein fractions from the gradients described above were packe d by centrifugation, drained, and washed once with 70 % ethanol. To the well-drained pellets was added 400/zl of 1% SDS (w/v) in 0.1 M LiC1, 0.01 M EDTA, 0.01 M Tris : HC1 (pH 7.5). The suspended material was heated to 45°C for 5 min and layered on a linear 15-30% (w/w) sucrose gradient in 0.1 M LiC1, 0.01 M EDTA, 0.01 M Tris:HC1 (pH 7.5) containing 0.2 % (w/v) SDS. The gradients were centrifuged for 21 h at 27000 rpm at 23°C in the 10.16 cm tubes of the Spinco SW27 rotor. The gradients were removed from the top by means of an ISCO fractionator collecting samples of either 0.3 or 0.4 ml. An example is shown in fig. 3. Me2SO gradient fractionation. Linear gradient of 0-20 % (w/w) sucrose dissolved in buffered 85 % (v/v) MezSO were formed in polyallomer tubes of the Spinco SW56 rotor [40]. The buffer for the gradient was 0.028 M EDTA in the free acid form and 0.28 M Tris. The pH of the buffer was adjusted to 7.5 with HC1. Fifteen ml of this buffer was mixed with 85 ml of Me2SO (Aldrich Chemical Co., Spectrophotometric Grade) and the apparent pH of the mixture was adjusted to 7.5 by the careful addition of 1 N HC1 before using. The use of the free acid form of EDTA is necessary to avoid formation of a precipitate when the pH of the MeaSO solution is finally adjusted. The dried RNA samples were taken up in 200 /zl of the buffered 85 % Me2SO solution, heated to 50°C for 3 min, cooled and layered on the gradients. Centrifugation was at 23°C, 55000 rpm for 21-28 h depending on the samples. After 28 h the 28S RNA had migrated 66 % of the distance from the miniscus to the bottom of the gradient. The gradients were removed by means of an ISCO fractionator and 50 p~l fractions were collected manually. An example of such a gradient is shown in fig. 4. All sucrose solutions were treated with diethylpyrocarbonate [41] (Naftone, Inc., Park Avenue, New York), all glassware was heated in hot air to 275°C for 2 h, gloves were worn and plugged sterile pipettes were used. Apparatus which could not be heat-treated was treated with 1% SDS followed by 1% diethylpyrocarbonate in 70 % ethanol, rinsed with 95 % ethanol and dried. Counting. For samples containing [3H] and [14C] the nucleic acids were precipitated by the addition of excess cold 10% (w/v) TCA and the precipitate collected on 13 mm glass fiber filters. The sample tubes and filters were rinsed twice with 10 % TCA and twice with 95 % ethanol and dried. The dried filters were placed in counting vials and wetted with 400 /zl of a mixture of NCS (Amersham/Searle Corp., Arlington Heights, Ill.) solubilizer and water (20:3). The vials were capped and incubated at room temperature overnight, 2.5 mlPi50" t'O-P0~P : toluene Scintillation counting solution was added and the samples stirred and counted. Samples containing a2p as sole isotope were (where necessary) diluted to 2 ml with water and counted for Cherenkov radiation in a scintillation counter. All samples were counted for sufficient time to yield a standard error of counting of <3 %. Cell growth and related changes in RNA and DNA

Exp Cell Res 108 (1977)

210

J. F. Scott

"

A •

i

i

~

I

r

[

op

6~

J

RESULTS

tt~z48 h

C

o

20

40

60

ao

too

~ao t4o

Fig. l. Abscissa: time (hours); ordinate: (B, C) log cpm X102/m26onm . (A) + - - + , cells/6 cm dish x105; © - - G , total radioactivity in RNA, cpm x 104; A - - A , total rRNA/dish, /,g; A - - A , total radioactivity in DNA, cpm × 104; (B) specific activities of RNA from SDS:sucrose gradients (G--G, 18S; 0 - - 0 , 28S; x - - x , 18-28S); (C) specific activities of RNA from MezSO gradients (G--G, 18S; 0 - - 0 , 28S). The straight segments of all lines which include more than two points are least square lines.

radioactivity, RNA content, and specific activities of rRNA species. Fifty dishes 6 cm in diameter were plated with 3x105 cells/dish. After the cells were established the medium was replaced with medium lacking methionine and containing 15 % undialysed calf serum. To twenty dishes was added [3H]methylmethionine (ll Ci/mmole) to the level of 5 /zCi/ml and unlabeled thymidine to 10-4 M. To a second set of twenty was added [6-aH]thymidine, 1 /,Ci/ml and no radioactivity was added to the remaining ten dishes. Twenty-four hours later the medium was removed and all dishes were rinsed three times with 5 ml of warm BSS and fresh complete medium was added. At that time two dishes of each of the labeled sets were withdrawn and two unlabeled dishes were taken for cell count. Labeled cells were harvested in duplicate morning and afternoon at the times indicated. Cell counts were obtained from replicate dishes in the morning of each harvest day. The labeled cells were harvested by lysis in SDS medium and the nucleic acids isolated by phenol extraction as described under Exp Cell Res 108 (1977)

Methods. The isolated nucleic acids were taken up in 0.01 M LiC1 containing 1% (w/v) SDS and aliquots were taken for radioactivity determinations. The individual samples for each morning and afternoon harvest were pooled and again precipitated with ethanol. The pooled samples were then subjected to S D S ' s u crose and Me2SO:sucrose gradient fractionation as described under Methods. The rRNA fractions were collected, 1/9 vol of 10 M LiC1 and 2.5 vol of ethanol were added to precipitate the RNA. The precipitates were collected by centrifugation at 3 000 g for 30 rain, drained, and dried. They were then dissolved in 0.01 M LiC1 and the A~60nm was measured and aliquots were taken for scintillation counting.

For a number of reasons we wished to utilize the well characterized 3T3 line of mouse cells in these studies. For purposes of comparison with the data on primary chick fibroblast cultures [16, 17] it was first necessary to investigate rRNA turnover in these cells with respect to the growth conditions. Cells were grown, labeled, and analysed as described in fig. 1. From these data the total radioactivity in the rRNA, the total amount of rRNA, the specific activities of the 18S, the 28S, and the RNA in the interval between these two rRNA peaks was determined. From the label in the nucleic acid from the cells exposed to [3H]thymidine, the degree of retention of cells labeled during the period of exposure to isotope could be estimated. The results presented in fig. 1 show: (1) that the DNA radioactivity is stable within 10% (A); (2) that there is turnover of the total RNA as well as the rRNAs (A); (3) that the loss of radioactivity is essentially first order except for the "18-28S" fraction 03); (4) that there is an almost two-fold difference in the half-time of turnover between the 18S and 28S rRNAs in the aqueous gradient separation 03); (5) that the rate of loss of label from the 18S which is slower in the aqueous gradient fractionation is increased to nearly that of the 28S R N A in

Turnover of rRNA

211

The discrepancy between the turnover rates of 18S and 28S as measured by fractionation in aqueous solvent was puzzling but it appears that when covalently intact OIL ~ _ r--h i ~ i t. I0 molecules were fractionated in Me2SO gra- ~- 2 3 4 5 6 ~ s 9 ~o ~ Fig. 2. Abscissa: effluent (mi); ordinate: A254nm; (left) dients the discrepancy is essentially eli- - ; (right)---. minated. Graph of the ultraviolet absorbance of a sucrose The time-dependent flow of radioactivity gradient used to fractionate the postnuclear supernatant fraction. Growing cells labeled with ~2p were har- into the 18-28S region of the gradients has vested, lysed, and the postnuclear supernatant fraction was applied to a non-linear sucrose gradient as de- been observed by others [28, 29, 32, 34, 43, scribed under Methods. In this case the centrifugation 52] and has been found to arise from the was for 300 min at 41000 rpm in the SW41 rotor at 4°C. Arrows indicate the points at which fractions in- 28S RNA. Melvin et al. [51] have demondicated by Roman numerals were separated. The let- strated more generally that these intermediters S, L, and M indicate the small subunits, large subunits, and monomeric ribosomes, respectively. ate RNA fractions can be shown to arise Pool V contains the polysomes which are not well from the larger ribosomal subunit. We condisplayed in this longer run designed to separate better the lighter ribosomal elements. Arrows indicate the di- firmed this for 3T3 cells (data not shown). rection of sedimentation in this and subsequent figIt thus appeared that if we wished to exures. plore the possible relation between nicks in the covalent structure of 18S and 28S rRNA the MezSO gradient fractionation (C vs B); and ribosomal turnover it would be necesand (6) the time course of the specific radio- sary to utilize cells containing uniformly laactivity of the RNA in the 18-28S region is beled RNA in order to achieve a quanticonsistent with a time-dependent degrada- tative relation between radioactivity and tion of 28S in which more recently synthesized 28S RNA is less likely to appear 28S in that fraction than "older" 28S RNA 03). In experiments not shown it was found that the label in rRNA derived from [3H]methyl- 20 methionine was conserved during expo18S nential growth. Points (2) and (3) are consistent with the I0 results of Emerson [17], Weber [18], and Abelson et al. [20] and to some extent those of Kolodny [23]. The discrepancy between 4O 10 20 30 the rates of loss of label from 18S and 28S Fig. 3. Abscissa: fraction no.; ordinate: cpm × 103. Distribution of radioactivity from an SDS : sucrose in 3T3 at confluence (point (4)) was also gradient of RNA from polysomes from growing cells. pointed out by Abelson et al. [20] in a paper Cells labeled with 3~p were harvested and polysomes which appeared while this study was in were isolated as described in the text and illustrated in fig. 2. The polysome fraction was collected, preprogress. Point (6) is consistent with the cipitated, treated with SDS, and fractionated on an evidence and conclusions of Kokileva et al. SDS:sucrose gradient as described under Methods. The gradient was fractionated from the top and 0.4 [28], and of Nair & Knight [29]. It thus ap- ml fractions were collected and counted directly by pears that certain aspects of the degradation Cherenkov radiation. The arrows indicate the positions of 22S and 25S RNA. The dotted lines under of rRNA in various cells and tissues have the envelopes of 18S and 28S indicate the boundaries set for summing the radioactivity in those species. some features in common. Exp Cell Res 108 (1977)

212

J. F. Scott

Table 1. SDS : sucrose gradient fractionation o f R N A from different cell fractions at different stages o f growth Cells were labeled with 3~Pi, harvested, lysed, and fractionated. The RNA from the indicated ribosomal fractions was separated on an SDS : sucrose gradient. The procedures are described in Methods. The gradient regions referred to in the top row of the table are those indicated in fig. 3 of a representative gradient. The values in the columns are the percentage of the total radioactivity found in the respective regions recovered from each gradient. The sample numbers refer to the points in the cell growth at which cells were harvested. Cell densities at points 1, 2, 3, and 4 were, respectively, 0.94, 1.64, 3.2, and 7.5 all × 104 cells/cmL The last point represents confluence. The mole ratios of 28S/18S were calculated from the radioactivity assuming molecular weights of 1.65× 106 and 0.7× l0 s D, respectively. S.E. = 1/~,(x-~)z.[1/(n - 1)] Gradient region (% recovered radioactivity)

Large subunits

Sample

<18S

18S

18-28S

28S

>28S

1 2 3 4

19.6 16.8 16.7 15.0

10.7 7.2 9.7 7.4

7.1 8.7 7.0 9.5

57.6 62.4 61.7 62.9

4.9 4.9 4.9 5.2

61.2 _+2.42

±S.E. Monomers

Polysomes

1 2 3 4

12.8 13.5 13.0 15.2

20.1 21.4 23.6 21.5

6.7 7.2 7.3 9.9

54.1 53.2 52.8 50.0

6.3 4.7 3.3 3.4

±S.E.

13.6 1.09

21.6 1.45

7.8 1.44

52.5 1.77

4.4 1.40

1 2 3 4

9.8 9.3 9.4 12.7

25.1 25.8 24.4 21.7

7.0 4.6 6.2 6.1

52.2 56.8 56.6 55.3

5.9 3.5 3.4 4.2

~? ±S.E.

10.3 1.61

24.2 i.79

6.0 1.00

55.2 2.12

4.2 1.16

mass of RNA which would not be confused by time-dependent migration of radioactivity from one class to RNA to smaller classes and possible effects of recycling of degradation products [18]. Growing 3T3 cells were labeled with ~2p as described in Methods for three to four generations. The labeled cells were harvested and dispersed into a number of 10 cm dishes and the exposure to a2p was continued. The cells were harvested in groups while growing and after confluence as described in table 1. Unlabeled cells from confluent cultures were mixed with each batch of labeled cells to provide carrier amounts of material. The ribosomal components were separated and the RNA from the cornExp Cell Res 108 (1977)

Mole ratio 28S/18S

1.14 1.05 0.95 0.99

0.88 0.95 0.98 1.08

ponents was displayed by centrifugation as described under Methods. The results of this experiment are shown in table 1. From the data presented it does not appear that there is a significant increase in the RNA in the portions of the gradients outside the 18S and 28S bands as the cells begin to degrade the rRNA when growth is inhibited by population density. Indeed, making allowances for 4S and 5S RNA, one can account for 80-90% of the expected radioactivity in the 18S and 28S pools from the monomer and polysome fractions. Because we had uniformly mixed the labeled cells with unlabeled cells from confluent cultures we considered the possibility

Turnover of rRNA

213

Table 2. Comparison of the distribution of radioactivity in the RNA from native ribosomal subunits, monomers, and polysomes from growing and confluent cells fractionated in aqueous and Me2SO gradients Cells were labeled, harvested, and fractionated as described under Methods. Gradient regions and the numbers in the columns are as defined in table 1. The values are means _+S.E.M. The number of samples derived from separate experiments included in the mean is given in parentheses at the end of each row. Where only two samples were analysed the _+ gives the range around the mean. In the case of the small subunits, all radioactivity sedimenting faster than 18S was pooled Gradient region (% recovered radioactivity) Culture conditions

< 18S

18S

18-28S

28S

>28S

Polysomes Growing cells gradient solvent Aqueous Me2SO

9.5-+0.15 22.6_+ 1 . 4 1

22.6+0.61 22.6_+0.93

10.9+0.26 10.5_+ 1.32

52.8_+0.65 34.6+ 1.71

4.2_+0.15(3) 9.7_+0.23 (3)

Confluent cells Aqueous Me2SO

8.1_+ 1.59 16.8_+0.8

23.1_+ 0.64 24.5_+ 1.4

11.3_+0.61 10.5_+0.4

52.4_+ 1.50 39.9+0.9

5.1 +0.55 (3) 8.3+0.1 (2)

Monomers Growing cells Aqueous Me2SO

10.6+ 1.36 19.6-+0.92

21.6+ 1.11 20.9+ 1.37

8.3_+ 1.07 12.7-+ 1 . 5 5

54.5+ 1 . 0 1 38.6+0.60

5.0+0.47 (3) 8.2_+0.45(3)

Confluent cells Aqueous Me2SO

8.0+0.72 17.0+ 1 . 2 8

21.6+0.82 21.2+0.82

9.6+0.66 14.2+0.60

56.8+ 1 . 1 2 39.2_+ 1 . 3 8

4.0_+0.92(4) 8.4+0.87 (4)

Large subunits Growing cells Aqueous Me2SO

17.9+2.7 20.4+ 1.6

8.4+ 1.5 10.8-+ 1.7

7.7+0.2 13.2_+ 1.5

61.2+0.8 48.0+ 1.8

4.8+0.1 7.6_+0.3

(2) (2)

Confluent cells Aqueous Me2SO

10.8+0.1 18.6-+ 1.0

9.4+0.8 12.8_+ 1.6

8.0+0.8 14.0-+1.2

67.4+0.4 47.6+3.3

4.4-+0.3 7.0+0.8

(2) (2)

Small subunits Growing cells Aqueous Me2SO

23.2_+0.4 25.0+0.2

67.0+0.2 63.8+0.7

9.8+0.5 10.2+ 1.5

-

-

(2) (2)

Confluent cells Aqueous Me2SO

18.8_+0.5 22.0+ 1.7

71.4_+0.4 67.8+ 1.4

9.8+_0.1 10.2___0.1

-

-

(2) (2)

that the uniformity of the results illustrated in table 1 might be a reflection of some effect of the cytoplasmic material from the unlabeled confluent cells which was present in all samples. However, control experiments showed no differences when labeled cells were mixed with growing or confluent unlabeled cells before processing (data not shown).

In other experiments we had noted that there were chain scissions in RNA which were revealed only when the RNA was treated under conditions which greatly reduced the secondary structure of the molecule. To minimize the possibility the fragments produced by disaggregating conditions might reassociate during fractionation we utilized centrifugation in 85 % Me2SO Exp Cell Res 108 (1977)

214

J. F. Scott •

04 I

0.2

0. t

i

\

t8s

20

Fig. 4. Abscissa:

50

f r a c t i o n no. ;

40

28S

fi t~

50

ordinate: A254n m.

Absorbance curve of Me2SO:sucrose gradient of rRNA derived from monomeric ribosomes. Monomeric ribosomes from confluent cells labeled with 32p were isolated as described under Methods and illustrated in fig. 2. The RNA was isolated by phenol extraction in the presence of SDS and the aqueous phase was layered on a Me2SO : sucrose gradient prepared and run as described under Methods. Fifty/zl fractions were taken from the top of the gradient. The light line under the peaks represents an optical blank from a parallel gradient without added RNA. The region of the final twenty fractions is not shown.

at low ionic strength as described in Methods. The cells were labeled with z2p as in the previous experiment and seeded at 2/10 and 9/10 saturation. One day later they were harvested and processed as in the previous experiment save that separate aliquots of RNA were fractionated through SDS : sucrose and Me2SO : sucrose gradients. The results are given in table 2. The data for the aqueous gradients are in reasonable agreement with those of table 1. Again there was no marked effect of the confluent state of the cells on the integrity of the rRNAs. Two other facts stand out: (1) of the 28S RNA only 70% of that isolated by aqueous centrifugation is covalently intact in the presence of Me2SO regardless of the ribosomal fraction from which it is isolated; and (2) the 18S RNA from the polysomes, monomers, and native small subunits is apparently intact. Again there was no marked difference between the results for rRNA from growing and confluent cells. Exp Cell Res 108 (1977)

We wished to determine the distribution of the RNA which disappeared from the 28S band in the Me2SO gradient fractionation. In an experiment similar in design to those which yielded the data of table 2 the 28S RNA was isolated from the SDS:sucrose gradients from four samples and the 18S RNA from two samples from the polysome fractions. The isolated RNA of each fraction was taken up and run on Me2SO : sucrose gradients. A representative gradient of 28S RNA is shown in fig. 5 and the data are presented in table 3. The data suggest that, at least for the polysome fraction, the apparent stability of the 18S may be the result of loss of about 15% of the 18S RNA to lighter fractions and a compensatory gain from RNA arising from the 28S RNA fraction. DISCUSSION We have shown here that in cultures of BALB/c cells which have been exposed to methyl-labeled methionine during growth t2~-

8

4~

,,I

~0

20

5O

40

Fig. 5. Abscissa: fraction no.; ordinate: cpm × 10~. Distribution of radioactivity from isolated 28S RNA fractionated in a Me2SO : sucrose gradient. 28S RNA labeled with 3zp was isolated from an SDS:sucrose gradient as described under Methods and illustrated in fig. 3. The precipitated 28S RNA was taken up in 85% Me2SO and fractionated on a Me2SO:sucrose gradient as described under Methods. In this experiment 0.1 ml fractions were collected, diluted, and counted by Cherenkov radiation. The bared line indicates the half-maximum band width and position of 18S RNA from parallel gradients. The dotted line under the 28S RNA envelope indicates the estimated area of the 28S proper from which the radioactivity in that species was calculated.

Turnover o f r R N A Table 3. Distribution of radioactivity in Me2SO gradients of l8S and 28S isolated by aqueous sucrose gradient fractionation of polysomal rRNA The culture conditions, labeling, harvest, and fractionation are as described under Methods. The gradient regions are those indicated in fig. 5. The values in the "<28S" column include those given in the "18S" column Gradient region (% recovered radioactivity) 28S Culture conditions

<28S

"18S"

28S

Growing cells

29.8 28.2

17.0 22.6

66.2 68.2

Confluent cells

20.5 29.3

17.8 15.8

76.2 65.7

27.0 2.18

18.3 1.49

69. I 2.44

±S.E.M.

18S <18S

18S

>18S

Growing cells

11.6

85.7

2.7

Confluent cells

15.0

81.4

3.6

the label is lost from the 18S and 28S rRNA when the growth of the cells slows and ceases as the layer becomes confluent. In experiments which are not presented here we have found that in such 3T3 cells as well as secondary cultures of BALB/c mouse embryo fibroblasts which have been similarly labeled the rRNA is stable during growth phase of the cultures. This stability of the rRNA during growth and subsequent turnover at confluence is consistent with what has been previously shown for chick embryo fibroblasts by Emerson [17] and by Weber [18]. These data are also consistent with those of Abelson et al. [20] on 3T3 and 3T6 cells which appeared while this work was in progress. For reasons which are not clear we did not observe the bi-

215

phasic decay kinetics found by Kolodny [23]. An unusual aspect of the results presented here and those reported by Abelson et al. [20] is the difference in the rates at which the labeled 18S and 28S rRNA are lost during turnover when the RNA is fractionated in aqueous solvent. A similar finding has been reported for the rRNA from mouse kidney by Melvin et al. [51] and by Barka [53] for the rat salivary gland. However, we find that the discrepancy vanishes when the rRNA is fractionated in the presence of 85 % Me2SO and the slower rate of loss of 18S increased to that of 28S which in turn is little changed. In the discussion below we propose an explanation for this finding. For the purposes of discussion let us say that there are at least three kinds of chain scissions of the convalent structure of the rRNA which can occur in the ribosome in vivo. (1) A scission which occurs in a portion of the molecule which is not stabilized by secondary structure which is then revealed when the ribosome is deproteinized and the RNA fractionated at room temperature. (2) A scission which occurs in a portion of the molecule which is stabilized by secondary structure which is therefore not detected unless the isolated rRNA molecule is exposed to disaggregating conditions prior to or during fractionation. (3) A scission which causes the breakdown of the ribosome structure and which would leave the rRNA fragments in the form of fragmented ribosomes. We propose that "nicks" of the first two types occur at relatively few sites and more frequently in the 28S rRNA in both growing and in non-growing cells and further that ribosomes so nicked continue to function in protein synthesis. The likelihood that a Exp Cell Res 108 (1977)

216

J. F. Scott

given site will be nicked increases with time polysomal rRNA, allowing for the associbut such chain scissions occur at random ated 4S RNA and the 5S RNA component among the available sites. Among the nicks of the large ribosomal subunit, we can acof type 2 the stability may vary. Nicks of count for 80-90 % of the radioactivity which type 3 are hypothetical and limiting facts should be present in the 18S and 28S (tables 1, 2). In the presence of Me2SO there is will be discussed later. With regard to the question as to whether little apparent change in the proportion of the nicking which we have reported here the RNA in the 18S band, but that in the arose during the isolation procedures or 28S region decreases to 57% of what one existed in vivo the following points can be would expect if all the 28S were intact (almade. When the RNA was isolated by di- lowing, in this case, for the loss of the 5.8S rect lysis of the cell layers with SDS buffer RNA [42] which is associated with the 28S (Methods), followed by phenol extraction, in aqueous media). The loss of radioacand the rRNA estimated either by absorb- tivity in the 28S region can be accounted ance or by 3~p content after equilibrium for by an increase in the <18S and the 18labeling, essentially the same pattern of 28S regions. There are no significant difchain scission was found as that found by ferences between growing and confluent the protocol described above (Methods). cells nor between polysomal-RNA and moFurther, as shown in table 2, the 18S RNA nomer-RNA. The 28S RNA from the native from the small subunits shows a very low large subunit shows slightly fewer nicks of level of nicking of the second type. These the second kind, though the difference is observations support the interpretation that not significant. The data of table 3 indicates the observed level of nicking is a reflec- that the nicks of the second kind estimated tion of the state of the rRNA in the cell. from the fractionated 28S is essentially the We have shown here that in growing 3T3 same as that estimated from the mixed cells in which the rRNA is conserved and RNA. There is also the suggestion in this in such cells in confluent layers in which data that the stability of the 18S may be the rRNA is being degraded the rRNA con- overestimated by fractionation of the mixed tains nicks of both the first and second type RNA since approx. 15% of the radioactivand further there is no significant change ity isolated as 18S migrates elsewhere in the in the level of such nicking when growing presence of Me2SO while approx. 18% of cultures are compared with confluent ones. the radioactivity lost from the 28S in Me2SO We find two bands of RNA between the would have been counted as 18S in a frac18S and 28S with an estimated sedimenta- tionation of mixed RNA (fig. 5, table 3). tion velocity of 22S and 25S corresponding The 18S RNA examined in table 3 was to estimated molecular weights of 1.00× 106 derived from polysomes. The data in table and 1.35× 106 D, respectively, which agree 2 indicates that the 18S from the small subwith the results of Aaij et al. [37] and Boedt- units is less nicked than that of the polyker et al. [32]. These estimated sedimenta- somes. tion velocities are unchanged in Me, SO. Others have presented evidence that There is also apparently other less specific nicks of types 1 and 2 occur in different nicking which leads to a loss of RNA from cell types in growing as well as non-growing the 18S and 28S bands which is found in states [28-37, 51, 52] and some [28, 29, 36, the low mol. wt region. In the case of the 37] have suggested that the occurrence of Exp Cell Res 108 (1977)

Turnover of rRNA such nicks might be connected with the degradation of rRNA during the turnover of ribosomes. The data presented here indicates that this is not the case in 3T3 cells. In this connection Inoue et al. [36] also reported that there was a decrease in the amount of RNA in the intermediate bands during liver regeneration, and that the polysomal rRNA from fetal liver showed none of the intermediate bands [44]. This latter point is quite interesting and may be a reflection of the high erythropoietic activity of the fetal rat liver and points to possible differences in the nicking of rRNA in different cell types. Turning to the problem of ribosome turnover, certain facts should be noted in an attempt to devise a hypothetical scheme for the mechanism of ribosome degradation. (1) The attack on ribosomes which removes them from the counted pool is random since most studies with whole animals and tissue culture systems have shown the kinetics of loss to be first order [2-4, 18, 22, 52]. (2) Ribosomes may be disposed of as units since there is a coordinate loss of rRNA and proteins from both subunits. In most studies [3, 45] in which the loss of isotope from both ribosomal proteins and RNA have been measured there has been no significant differences between the rates. In the most recent study Tsurugi et al. [45] have compared the rates of loss of the various proteins of the large and small subunits as well as the larger RNA of each subunit. With the exception of protein $5 of the small subunit there were no differences in turnover among the ribosomal proteins or the RNAs. It seems unlikely that ribosomes would be disposed of from the functioning pool of polysomal ribosomes and since the rates of degradation are the same for both the large and small subunits it also seems unlikely

217

that the native subunits would be the pools from which candidates for disposal were drawn. The pool of monomeric ribosomes would appear to be most likely source for the coordinate disposal of both subunits. It may be that in such ribosomes the site at which an initial attack is made is among those occupied by mRNA, tRNAs, and initiation or elongation factors while the ribosome is participating in protein synthesis. Shine & Dalgarno [46] and Sprague & Steitz [47] have pointed out that the 3'terminal portion of the 16S RNA in prokaryotes possesses sequences in part complimentary to polypurine tracts of the 5'terminal region of some mRNAs. Thus an endonucleolytic attack directed against the 3'terminal portion of the 18S R N A might be the initial event which produces a pool of ribosomes incapable of participating in protein synthesis and thus exposed to further attack. It would appear, however, that the degradative system is not automatically active. Clearly the existence of a pool of monomers does not itself cause the' onset of degradation since even in growing 3T3 cells we have found that while the pool is small (containing about 10-15% of the total rRNA) it does exist and there is no turnover of the rRNA. Levine et al. [48] note that when labeled sparse cultures are overlain with sufficient cells to produce a confluent layer there is a rapid decrease in the proportion of the ribosomes in polysomes with compensatory increase in the pool of monomers. Only after 12-24 h, however, does degradation of labeled ribosomes ensue. Others [4%50] have shown that a large percentage of the monomeric ribosomes present in growth-arrested cells rapidly decreases after the arrest is released and there is a compensatory increase in the polysomes. Further, we have found [Scott, J. Exp Cell Res 108 (1977)

218

J. F. Scott

F., unpublished] that while non-confluent suggests that an understanding of this phe3T3 cells cease to grow when they are nomenon may well be quite important in shifted to conditioned medium containing an understanding of the maintenance of a 2 % undialysed serum, if such cells have dynamic controlled steady state in normal been exposed to methyl-labeled methionine tissues. during a previous growth phase the labeled rRNA is stable for 4-6 days during growth The skilled and enthusiastic participation of Vireinia Kuhns and Patricia Crawford is gratefully acknowlarrest in low serum. edged. The work reported here was supported by the ACS There are a number of indications that (grant no. NP-104), the NIH (grant no. CA1463003), the fragments produced by scissions of the and from General Research Support Grant (NIH) to Massachusetts General Hospital (allocation no. first and second kinds may differ in size the S.C. 1-1, C.S. 66). from one cell type to another [29, 31, 33, 36, This is publication No. 1522 of the Harvard Center 37]. It appears from the results which we Commission. have presented here and those of Abelson REFERENCES et al. [20] that there may also be differences in the manner of attack which yields 1. Takagi, Y, Hecht, L I & Potter, V R, Cancer res 16 (1956) 994. fragments from the type 3 nicks. We sug2. Loeb, J N, Howell, R R & Tompkins, G M, Science 149 (1956) 1093. gest that in the process of ribosome de3. Hirsch, C & Hiatt, H, J biol chem 241 (1966) 5936. gradation in 3T3 cells during confluent 4. Malt, R A & Lamaitre, D A, Am j physiol 214 growth arrest at least two independent scis(1968) 1041. 5. Schoenheimer, R, The dynamic state of body sions are introduced into the 18S RNA in constitutents. Harvard University Press, Camthe small subunit early in degradation. One bridge (1942). 6. Brown, G B & Roll, P M, Nucleic acids (ed E of these removes the 18S RNA from that Chargaff & J N Davidson) vol. 2, p. 341. Academic sedimentation class while a second, occurPress, New York (1955). 7. Tata, J R, Nature 219 (1968) 331. ring with approximately equal frequency, 8. Lieberman, I, Abrams, R & Ove, P, J biol them yields an 18S RNA which is stable follow238 (1963) 2141. 9. Baserga, R, Estensen, R D & Petersen, R O, Proc ing deproteinization but which dissociates natl acad sci US 54 (1965) 1141. in Me,SO. 10. Fujioka, M, Koga, M & Lieberman, I, J biol chem (1963) 3401. In connection with this phenomenon of a 11. 283 Ellem, K A O & Mironescu, S, J cell physiol 79 slower decay of prelabeled 18S relative to (1972) 389. 12. Zardi, L & Baserga, R, Exp mol pathol 20 (1974) 28S, the recent finding of Melvin et al. [51] 69. is of interest in that they show that while 13. Cooper, H L, Nature 227 (1970) 1105. J M, Ab, G & Malt, R A, Biochem j 144 the specific activities of the RNA contained 14. Hill, (1974) 447. in the intact large and small ribosomal sub- 15. Goodlad, G A J & Ma, G Y, Biochim biophys acta 378 (1975) 221. units show identical kinetics of decay, the 16. Hill, J M, J cell biol 64 (1975) 260. decay of the isolated 18S and 28S RNA 17. Emerson, C P, Jr, Nature new biol 232 (1971) 101. 18. Weber, M J, Nature new bio1235 (1972) 58. show a slower decay rate of 18S. 19. Johnson, L F, Abelson, H T, Green, H & Penman, We wish to emphasize the fact that while S, Cell 1 (1974) 95. H T, Johnson, L F, Penman, S & Green, the physiological basis for the turnover of 20. Abelson, H, Cell 1 (1974) 161. ribosomes in steady state as well as some 21. Horiuchi, R, Yaoi, Y & Amano, M, Dev growth differ 14 (1972) 185. growing tissues remains obscure, the sharp 22. Harris, H, Biochem j 73 (1959) 362. contrast between such tissue and those 23. Kolodny, G M, Exp cell res 91 (1975) 101. J F & Taft, E B, J natl cancer inst 15 (1955) transplantable cancers which have been 24. Scott, 1591. studied in which such turnover is absent 25. - - Biochim biophys acta 28 (1958) 45. Exp Cell Res 108 (1977)

Turnover of rRNA

26. Scott, J F, Taft, E B & Letourneau, N W, Biochim biophys acta 61 (1962) 62. 27. Bennett, L L, Skipper, H E, Simpson, L, Wheeler, G P & Wilcox, W S, Cancer res 20 (1960) 62. 28. Kokileva, L, Mladenova, I & Tsanev, R, FEBS lett 12 (1971) 313. 29. Nair, C N & Knight, E, Jr, J cell bio150 (1971) 787. 30. Leaver, C J & Ingle, J, Biochem j 123 (1971) 235. 31. Levin, S & Fausto, N, Biochemistry 12 (1973) 1282. 32. Boedtker, H, Crkvenjakov, R B, Dewey, K F & Lanks, K, Biochemistry 12 (1973) 4356. 33. Ishikawa, H, Biochem biophys res commun 54 (1973) 301. 34. Judes, C, Fuchs, J P & Jacob, M, Biochimie 54 (1972) 1031. 35. Peacock, A C & Dingman, C W, Biochemistry 6 (1967) 1818. 36. Inoue, T, Takagi, M, Umemura, Y & Yamaguchi, H, Agr biol chem 38 (1974) 2457. 37. Aaij, C, Agsteribbe, E & Borst, P, Biochim biophys acta 246 (1971) 233. 38. Todaro, G J & Green, H, J cell biol 17 (1963) 299. 39. Gielkens, A L J, Berns, T J M & Bloemendal, H, Eurj biochem 22 (1971) 478. 40. Strauss, J H, Kelly, R B & Sinsheimer, R L, Biopolymers 6 (1968) 793.

219

41. Solymosy, F, Fedorcgak, I, Gulyfis, A, Farkas, G L & Ehrenberg, L, Eurj biochem 5 (1968) 520. 42. Pene, J J, Knight, E, Jr & Darnell, J E, Jr, J mol biol 33 (1968) 609. 43. Miller, L & Knowland, J, J tool bio153 (1970) 329. 44. Inoue, T, Umemura, Y & Yamaguchi, H, J biochem 76 (1974) 205. 45. Tsurugi, K, Morita, T & Ogata, K, Eurj biochem 45 (1974) 119. 46. Shine, J & Dalgarno, L, Proc natl acad sci US 71 (1974) 1342. 47. Sprague, K U & Steitz, J A, Nucleic acids res 2 (1975) 787. 48. Levine, E M, Jeng, D-Y & Chang, Y, J cell physiol 84 (1974) 349. 49. Bandman, E & Gurney, T, Jr, Exp cell res 90 (1975) 159. 50. Noll, M & Burger, M M, J mol bio190 (1974) 215. 51. Melvin, W T, Kumar, A & Malt, R A, J cell bio169 (1976) 548. 52. Eliceiri, G L, Biochim biophys acta 447 (1976) 391. 53. Barka, T, Exp cell res 74 (1972) 439.

Received February 7, 1977 Accepted February 9, 1977

Exp Cell Res 108 (1977)