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The synthesis and processing of ribosomal RNA precursor molecules in yeast
BIOCHIMICAET BIOPHYSICAACTA
317
BBA 96255
T H E S Y N T H E S I S AND PROCESSING OF RIBOSOMAL RNA P R E C U R S O R MOLECULES IN YEAST R. L. TABER, JR. AND W. S. VINCENT Department of Anatomy and Cell Biology, University o[ Pittsburgh, School of Medicine, Pittsburgh, Pa. 15213 (U.S.A.) (Received March I8th, 1969)
SUMMARY Experiments are presented which firmly establish the ribosomal precursor nature of the 38-S RNA molecule found in the yeast Schizosaccharomyces pombe by showing that (I) the kinetics of the 38-S molecule are consistent with a precursorproduct relationship with ribosomal RNA, (2) the molecule is methylated, (3) the kinetics of methylation are similar to those of uracil labeling, (4) the methyl label can be chased into ribosomal RNA and (5) the cistrons coding for 38-S RNA are homologous to those coding for ribosomal RNA.
INTRODUCTION When the yeast Schizosaccharomyces pombe is treated with cycloheximide, an inhibitor of protein synthesis, a large (38-S) RNA molecule accumulates 1,.1. Preliminary evidence suggested that this molecule was a ribosomal precursor. If this were true, then the accumulation of 38-S RNA in the absence of protein synthesis strongly implies that a post-transcriptional control mechanism in the maturation of ribosomal RNA exists. The purpose of this paper is to show conclusively that the 38-S molecule is indeed a ribosomal precursor. MATERIALSAND METHODS
Culture and labeling conditions Pulse labeling experiments were done with cultures of S. pombe, strain NCYC 132, that were grown to an absorbance at 595 m# of 0.2 (1.6. lO 6 cells per ml) in an Edinburgh minimal medium 2. At this time cells from I 1 were collected by Millipore filtration and were resuspended in either 50 or IOO ml of fresh media before the addition of the radioactive precursor. At the end of the pulse, cells were again collected by Millipore filtration and were rapidly frozen at --20 ° in the presence of Macaloid 3. The cells were completely frozen within 15 sec of pulse termination. Further processing, including sucrose gradient analysis, was as previously described 1. For uptake and incorporation studies, the radioactive precursor was added to cultures of exponentially growing cells (0.2 A 595ms)" I-ml samples were taken at appropriate intervals and were suspended at o ° in i ml of IO % trichloroacetic acid for Biochim. Biophys. Acta, 186 (1969) 317-325
318
R . L . TABER, JR., v¢. S. VINCENT
incorporation or in I ml of water for uptake studies. The cells were then collected on Millipore filters (HAWP) and were washed with either 5 °/o trichloroacetic acid or water, dried and counted as previously described 1.
Reagents Radioactive compounds, obtained from the New England Nuclear Corp. (Boston), had the following specific activities: [Me-SH]methionine, 182.8 mC/mmole; Jail]uracil, 5.61 C/mmole; [a~P]phosphate, carrier free. Bovine pancreatic ribonuclease A was obtained from Worthington Biochemicals (Freehold, N.J.); T 1 ribonuclease from Calbiochemicals (Los Angeles, Calif.); Macaloid from the Baroid Division, National Lead Co., (Houston, Texas) and cycloheximide (actidione) from Upjohn (Kalamazoo, Mich.).
Isolation o/DNA and RNA DNA was isolated from S. pombe according to the technique of SMITH AND HALVORSON4. RNA was isolated as previously described 1.
Hybridization Hybridization was carried out essentially according to GILLESPIE AZqDSPIEGELMAN5. Yeast DNA was denatured by adding NaOH to 0.2 M in 0. 9 M NaCl-o.o 9 M sodium citrate (pH 7.0). It was neutralized with HC1 and made to o.I M with Tris (pH 7.5). A solution of this DNA sufficient to give lO-2O #g/cm 2 of a H A W P Millipore filter was allowed to flow by gravity through a circular filter (142 mm in diameter) from a straight-sided container. The filter was then washed with 0. 9 M NaC10.09 M sodium citrate, blotted, allowed to dry at room temperature for 2 h and baked overnight in a 60 ° oven. About 7 ° I-cm square filters were cut from the large filter. Each had a highly reproducible amount of DNA bound 5. Hybridization was carried out in 0. 9 M NaCl-o.o9 M sodium citrate with 0.2 °/o sodium dodecyl sulfate in a scintillation vial at 63 ° for 20 h, a time which we found sufficient to produce saturation of ribosomal cistrons. After hybridization, filters were thoroughly washed with 0.3 M NaCl-o.o3 M sodium citrate (pH 7.0) and were placed in a solution of 0.3 M NaC1-0.03 M sodium citrate containing 4 o/zg/ml of pancreatic ribonuclease (electrophoretieally pure) and io/~g/ml T 1 ribonuclease for I h at room temperature. The filters were then washed again with 0. 9 M NaCl-o.o 9 M sodium citrate and were dried and counted to a minimum of 5 % reproducibility in a Beckman DPM-Ioo liquid scintillation spectrometer. The DNA bound to each filter was determined, following counting, according to BROWN AND W E B E R 6.
RESULTS
Kinetics o/labeling o] 38-S molecule with RNA precursor In order to establish the kinetics of labeling of the 38-S molecule, cells were labeled for I, 3, 5 and 15 rain. Gradient analyses of the RNA are shown in Fig. I. After I rain of labeling with [3H]adenine (Fig. IA), it can be seen that there are considerable counts in a region heavier than the mature larger ribosomal RNA molecule. The only distinct peak is found at the 38-S region and is superimposed over polydisperse RNA. At 3 rain (Fig. IB) using [3H]uracil, there are peaks at 38 S, 30 S and Biochim. Biophys. ,4cta, i86 (1969) 317-325.
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F i g . I. Sucrose g r a d i e n t a n a l y s i s a f t e r l a b e l i n g for t h e t i m e s i n d i c a t e d . T h e g r a d i e n t s w e r e of 1 5 - 3 o % sucrose m a d e i n o . I M E D T A , o . o i M NaC1, o . o o i M T r i s ( p H 7.4) a n d o. 5 % s o d i u m d o d e c y l s u l f a t e a n d w e r e s e d i m e n t e d for 16 h a t 25 o o o r e v . / m i n at 23 ° i n a S p i n c o S W - 2 5 rotor. O---Q, 3H; - - , A ~ 0 m#. F i g . 2. U p t a k e - i n c o r p o r a t i o n d a t a of a u r a c i l c h a s e e x p e r i m e n t . [ 3 H J U r a c i l : 0 - 0 , i n c o r p o r a t i o n . Ea4C]Uracil: A - - - A , u p t a k e ; A - . -- A , i n c o r p o r a t i o n .
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18 S*. At 5 rain with uracil (Fig. IC), the relative amount of counts at 38 S is much less. A shoulder is seen at 30 S with most of the counts in 25- and I8-S RNA. These results are consistent with the flow of label from the 38 S to the 30 S and 18 S and finally to the 25 S. Although the 30-25 S transition is somewhat uncertain b y our methods, as the two represent only a 1-2 tube difference on the gradients, LOENING~ has demonstrated the difference more clearly using gel electrophoresis. * In this p a p e r w e shall refer to t h e "28 S" as "25 S" as s u g g e s t e d b y LOENING T for fungi. T h e p r e c u r s o r m o l e c u l e s w i l l be r e f e r r e d to as 38 a n d 3 ° S; it m u s t b e r e m e m b e r e d t h a t t h e s e v a l u e s are o n l y a p p r o x i m a t i o n s f r o m t h e g r a d i e n t s .
Biochim. Biophys Acta, 186 (1969) 317-325
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R. L. TABER, JR., W. S. VINCENT
If the precursor-product relationship above were true, it should be possible to demonstrate with a chase experiment the rapid disappearance of label from the 38 S and 3o S and its concomitant appearance in the 25 and 18 S. As shown in Fig. 2, such an experiment with uracil proved to be unfeasible. In this experiment cells were labeled with [3H]uracil, and 8 min later an I8-fold excess of [14Cluraeil was added. Samples were taken at 2-min intervals, and both the uptake and incorporation were measured. It can be seen that the incorporation of [3H]uracil remained linear for IO min after the El*C]uracil chase. It is apparent that the [3H]uracil pool continued to support the 3H incorporation in spite of the fact that 14C rapidly penetrated the cells and was incorporated in a linear fashion from I min after its addition. As would be expected from these results, a Io ooo-fold excess of unlabeled uracil did not affect the io-min incorporation period after the chase.
Kinetics o/methylation o] 38-S molecule Since the above pulse-chase experiment could not be done, we examined the methylation of the 38-S molecule. It is known that the mammalian precursor is methylated s, 9 and we have shown previously that the 38-S molecule in cycloheximide-
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Fig. 3. Sucrose g r a d i e n t a n a l y s i s of R N A a f t e r l a b e l i n g w i t h [ M e - 3 H l m e t h i o n i n e for t h e t i m e s indicated. O - - - 0 , 8H; - - , A280 m/~. Fig. 4' Sucrose g r a d i e n t a n a l y s i s of R N A e x t r a c t e d a f t e r a d d i t i o n of EMe-3H]methionine followed a t 30 sec b y a i o o - f o l d excess of u n l a b e l e d L-methionine. The cells were c o l l e c t e d a n d frozen a t 3 min. O - - - 0 , all; - - , A 260 m/~.
Biochim. Biophys. Acta, 186 (I969) 317-3~5
RIBOSOMAL
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321
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treated cells is also methylated 1. In order to examine the kinetics of methylation of the rapidly labeled 38-S RNA component, cells were pulsed with [Me-SHlmethionine for various times. When the cells were pulsed for 30 sec (Fig. 3A), three distinct peaks were found, one at 38 S, another at 30 S and a third at 18 S. This pattern of labeling is very similar to the 3-min uracil pulse shown in Fig. IB. With a 3-rain [Me-SH3methi onine pulse, the results shown in Fig. 3B were obtained. This shows some label at 38 S with relatively more counts at 30 and 18 S than were found at 30 sec. Fig. 3C is the result of a 5-rain pulse and demonstrates that at this time labeling and absorbance patterns are nearly identical. That the labeling by EMe-3Hlmethionine of the 38-S fraction can be chased is shown in Fig. 4. In this experiment cells were pulsed with [Me-SH]methionine and then chased at 30 sec with a ioo-fold excess of unlabeled L-methionine for 2.5 min. We found that, unlike uracil, methionine incorporation stopped upon a chase within I rain (Fig. 5). No label appears in the 3o-S region after this short chase. When this figure is considered in conjunction with Fig.3A and 3B, it further supports the idea that the 38-S molecule is a precursor for ribosomal RNA.
Homology o/ base sequences between 38-S R N A and 25- and zS-S R N A To establish that the 38-S molecule contained the identical base sequences as the 25-S and I8-S RNA, molecular hybridization experiments were done. To obtain a sufficient quantity of 38-S RNA, cells were treated with Ioo #g/ml of cycloheximide. I min later 2 mC of s~p were added, and at 20 rain the cells were collected and frozen. RNA was extracted from these cells and was analyzed on sucrose gradients as described previously 1. The gradient is shown in Fig. 5. The fraction indicated was collected and dialyzed against o.i M EDTA, o.oi M Tris (pH 7.4) and 0.2 ~o sodium dodecyl sulfate overnight before being precipitated with ethanol. This RNA was then rerun on a similar gradient in the SW-39 Spinco rotor, and tile tubes containing
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Fig. 5. I n c o r p o r a t i o n of EMe-14C]methionine a n d t h e effect of t h e a d d i t i o n of a Ioo-fold excess of u n l a b e l e d L - m e t h i o n i n e . Q - Q , c o n t r o l ; O - Q , chase. Fig. 6. Sucrose g r a d i e n t a n a l y s i s of R N A t r e a t e d w i t h c y c l o h e x i m i d e as d e s c r i b e d a n d t h e n l a b e l e d for 20 rain w i t h ? S P ] p h o s p h a t e . The t u b e s i n d i c a t e d w i t h s t i p p l i n g w e r e us e d as a source of l a b e l e d 38-S R N A for h y b r i d i z a t i o n e x p e r i m e n t s . (•)-Q, s2p; _ . _ , A 26~ m/*.
Biochim. Biophys. Acta, 186 (1969) 317-325
322
R . L . TABI,-'R, JR., W. S. VINCI.;NI
t h e R N A were collected, d i a l y z e d a n d p r e c i p i t a t e d as before. The a m o u n t of m a t e r i a l collected was insufficient to o b t a i n A 260 m~ values. The 38-S R N A fraction was t a k e n u p in 3 ml of o. 9 M N a C l - o . o 9 M sodium citrate, a n d its annealing characteristics to D N A were d e t e r m i n e d . C o m p e t i t i o n for i d e n t i c a l sequences in 38-S R N A were carried out b y preh y b r i d i z i n g t h e D N A w i t h u n l a b e l e d R N A l°,lx. One set of filters was i n c u b a t e d for 2o h at 63 ° in u n l a b e l e d 18- plus 25-S R N A (2//g/ml) t h a t was purified using sucrose g r a d i e n t s e d i m e n t a t i o n . A n o t h e r set was i n c u b a t e d u n d e r identical conditions except t h a t t h e R N A was o m i t t e d . A f t e r i n c u b a t i o n b o t h sets were w a s h e d with o.3 M N a C l - o . o 3 M s o d i u m c i t r a t e a n d were i n c u b a t e d as described with ribonuclease. A f t e r a t h o r o u g h washing, t h e filters were p l a c e d in 3 ml of o. 9 M N a C l - o . o 9 M s o d i u m cit r a t e c o n t a i n i n g 23o0 c o u n t s / m i n per ml of 38-S R N A . Filters c o n t a i n i n g no D N A were a d d e d as blanks, a n d i n c u b a t i o n was allowed to proceed at 63 ° for 20 h. A t the e n d of this time, t h e filters were again t r e a t e d w i t h ribonuclease, dried a n d c o u n t e d to 5 % r e p r o d u c i b i l i t y for the b l a n k s a n d 3 % for filters with a p p r e c i a b l e b o u n d R N A . T h e results are shown in T a b l e I. I t can be seen t h a t the p r e s a t u r a t i o n of ribos o m a l sites on the D N A c o m p l e t e l y inhibits the b i n d i n g of the 38-S R N A fraction. TABLE I C O M P E T I T I O N FOR 3 8 - S B I N D I N G TO D N A
B Y I 8 - AND
25-S RIBOSOMAL ] ~ . N A
H y b r i d i z a t i o n c a r r i e d o u t as d e s c r i b e d in t h e t e x t . The R N A s a m p l e s were p r e p a r e d from t w o d i f f e r e n t 32P-labeled p r e p a r a t i o n s p r e p a r e d in i d e n t i c a l fashion. E a c h f i l t e r c o n t a i n e d 9 # g of D N A . T h e n u m b e r s r e p r e s e n t c o u n t s / r a i n b o u n d per filter.
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DN A control
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38 S
136 I3I 127 135
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29 27 17 17
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The p o s s i b i l i t y r e m a i n e d t h a t t h e 38-S region r e p r e s e n t e d aggregates of 18-, 25-S or o t h e r R N A . I n order to e x a m i n e this, we p r e s a t u r a t e d t h e D N A w i t h 3Hl a b e l e d 25-S R N A alone. These filters were t r e a t e d as above. T h e y were then h y b r i dized with a ~2P-labeled 38-S fraction o b t a i n e d s i m i l a r l y to the one used a b o v e a n d a 6 - I o - S R N A fraction from t h e same g r a d i e n t as t h e 38 S. The results of this experiT A B L E 1I COMPETITION FOR 38-S
AND
6--IO-S BINDING BY 25-S R N A
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3zP-labeled R N A sample
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79 80 86 81
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Biochim. Biophys. Acta, 186 (1969) 317-325
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ment are shown in Table I I where it can be seen that 25-S RNA appreciably inhibits 38-S binding, while 6-1o S was not inhibited at all. As NYGAARD AND HALL12 have shown that R N A - D N A complexes in solution were subject to dissociation upon prolonged incubation, the possibility existed that the binding demonstrated in Table U could have been the result of the dissociation of the prehybridized RNA. T h a t this is not so is seen in Table I I I . In this experiment, filter-bound DNA was incubated for 20 h with 3H-labeled RNA and was processed as described. No significant change in the binding of RNA p e r / , g of DNA was found. TABLE
III
STABILITY OF R N A - D N A FILTER COMPLEX ON PROLONGED INCUBATION F i l t e r s w e r e i n c u b a t e d w i t h 8 H - l a b e l e d 2 5 - S R N A f o r 2 0 h, w a s h e d , t r e a t e d w i t h r i b o n u c l e a s e a n d w e r e r i n s e d a s d e s c r i b e d i n t h e t e x t . T h e f i l t e r s w e r e t h e n i n c u b a t e d i n o . 9 M N a C l - o . o 9 RI s o d i u m c i t r a t e 4 - o . 2 °/o s o d i u m d o d e c y l s u l f a £ e f o r t h e t i m e s i n d i c a t e d , r e p r o c e s s e d a s d e s c r i b e d a b o v e a n d counted. The DNA content of each filter was determined chemically after counting*.
Incubation time (h)
Counts bound per #g DNA
o 2 9 20
lO 3 lO2 92 lO2
DISCUSSION
We have previously demonstrated the presence of a 38-S RNA molecule in yeast which accumulates after cycloheximide treatment 1,21. This molecule had characteristics which suggested that it might be a ribosomal RNA precursor. RETlkL AND PLANTA13 have shown that there is a high molecular weight RNA in Saccharomyces carlsbergensis which m a y also be a ribosomal precursor. However, in neither of the above reports could definitive relationships be established. In this paper we have shown b y three independent procedures that the 38-S molecule is a precursor of ribosomal RNA. The results from a short term labeling with E3H]uracil and [Me-3H]methionine strongly suggest a precursor-product relationship between the 38-S and mature 25-S and I8-S RNA molecules. The results from R N A - D N A hybridization establish the identity of nucleotide sequences between the 38-S and the 25- and I8-S molecules. In Tables I and I I I , data are given which provide convincing evidence that the nucleotide sequences in the 38-S RNA are those contained in the 25-S RNA, and b y implication the I8-S RNA as well. A mixture of 18- and 25-S RNA completely inhibits subsequent binding of the 38-S molecule to DNA. 25-S RNA alone, prehybridized to DNA, suppresses 38-S binding b y approx. 75 %. This is somewhat greater than would be expected on the basis of the relative molecular weights ascribed to the 38-, 25- and I8-S molecules 7, where the expected interference would be only 57 °o. This discrepancy could be due to an interference in the binding of the 38-S RNA preparation by the prehybridized 25-S RNA, particularly if the 25- and I8-S cistrons are in alternating sequences as has been suggested for other forms 14,15. Tile failure of the 6-Io-S material to be competed for b y unlabeled 25-S RNA is significant, as it suggests that (a) the 38-S region does not contain aggregates of Biochim. Biophys. Acta, 186 ( 1 9 6 9 ) 3 1 7 - 3 2 5
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R . L . TABER, JR., W. S. VINCENT
precursors of messenger-type RNA, and (b) the ribosomal RNA fractions have not been fragmented appreciably by the extraction and isolation processes. The data from pulse labeling also provide information as to the speed of synthesis and processing of the ribosomal RNA molecules. First, with respect to the time for production of mature ribosomal RNA, the data in Figs. i, 3 and 4 suggest 2-3 min. This is much more rapid than for mammalian cells where the time is on the order of 2o min 16,17. However, it is interesting to note that in HeLa cells, this represents 1. 3 °/o of a generation time (24 h generation time), whereas in yeast (3 tl) it is I.I°~,, i.e. roughly the same proportion of the cell cycle being required for processing in both forms. The 38-S molecule proceeds through at least one intermediate during maturation to the 25-S RNA which we have arbitrarily designated as the 3o-S stage. I8-S RNA appears simultaneously with this step as is shown in Figs. IB and 3A. The 3o S then apparently becomes the 25 S. These steps are similar to those of mammalian cells is. A major difference is in the size of the precursor. LOENING7estimates molecular weights of 2.3" IO6 for the 38 S and 2.o. lO6 for the 18-25 S. This suggests an ahnost conservative processing, whereas in HeLa cells close to 4 ° o~, of the precursor is lost in the transition to mature ribosomal RNA 18. The methylation experiments show that processing of the 38 S is very rapid, much more so than the synthesis of the molecule. One can see from Fig. 3A that within 3 ° sec, the 18 and 3o S as well as the 38 S are methylated. The fact that the 3o-S precursor is labeled, while the 25 S appears not to be, suggests that methylation has occurred in the 38-S stage with subsequent cleavage to 3o- and I8-S molecules. It is also evident from comparing Figs. I and 3 that the time required for the synthesis of the 38-S molecule is considerably longer than the time required for the methylation and processing of the 3o 25-S and I8-S stages. In Fig. 3A it is likely that the processing of the 38-S molecule through methylation and cleavage to 3oand I8-S stages has occurred in 3o sec. From Fig. IB the time of synthesis of the 38-S molecule with processing to the stages demonstrated by Fig. 3 A is about 3 min. If one assumes that the rate of nucleotide synthesis determined by BREMER AND YUAN19 for bacteria (55 nucleotides per sec) holds for yeast, the time required to synthesize the 38-S molecule (2.3"1o 6 daltons, or approx. 76oo nucleotides) would be about 2 min. This is in line with the data given here. The finding that the time required to process the ribosomal RNA precursor is short, relative to the time required for its synthesis, may represent a significant difference between yeast and mammalian cells for in the latter the processing time is far longer than the time for synthesis 16A8. This could possibly be due to a limiting step in the post-transcriptional cleavage of the molecule in mammalian cells that does not exist in yeast. DE KLOET et al. 2°-~2 was the first to observe that cycloheximide affected RNA synthesis in yeast and has since investigated this phenomenon extensively. He concluded that the high molecular weight RNA which accumulates in yeast after cycloheximide treatment contains two components, a DNA-like RNA and a ribosomallike RNA. This conclusion was based primarily on a base ratio analysis of the RNA which sediments at greater than 12 S in a sucrose gradient. From our results we conclude that better than 95 °Jo of the high molecular weight RNA is homologous in sequence to ribosomal RNA. Our "high molecular weight" fraction, however, was Biochim. Biophys. Acta, 186 (1969) 317-325
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325
taken only from the 38-S region. This would suggest that the DNA-like R N A is found primarily in the I2-35-S area. Also, our cells were labeled for 20 min after cycloheximide treatment, whereas DE KLOET'S were labeled for 9 ° min. It is likely that after longer labeling, a greater proportion of the R N A made is DNA-like. The establishment of the precursor nature of the 38-S molecule makes its accumulation in the presence of cycloheximide significant. The continuation in the synthesis of the molecule in the absence of protein synthesis indicates that the control does not reside solely at the level of transcription of the R N A precursor molecule. A similar phenomenon has previously been observed in HeLa cells by VAUGHAN et al. 23,~4 who showed that methionine and valine deprivation resulted in a failure to process the precursor. A failure of processing to the complete ribosomes has also been shown in HeLa cells treated with puromycin ~5 and in stationary lens epithelium cells 26. These findings further indicate that in eukaryotes post-transcriptional control mechanisms in ribosomal processing are available to the cell. This is in contrast to bacteria in which the two large types of ribosomal R N A (16 S and 23 S) appear to be synthesized directly .7. ACKNOWLEDGMENTS
This work was supported in part by U.S. Public Health Service grant GM 11480 to W . S . V . R . L . T . was supported in part by U.S. Public Health Service grant 5 ToI HD ooo-4o-i2. REFERENCES i R. I,. TABER AND W. S. VINCENT, Biochem. Biophys. Res. Commun., 34 (1969) 488. 2 J. M. MITCHISON, in D. M. PRESCOTT, Methods in Cell Physiology, Vol. 4, A c a d e m i c Press, N e w York, 1969, in t h e press. 3 W. M. STANLEY, JR. AND R. M. BOCK, Biochemistry, 4 (1965) 13°2. 4 J. D. SMITH AND H. O. HALVORSON, Methods Enzymol., 12 (1967) 538. 5 D. GILLESPIE AND S. SPIEGELMAN, J . Mol. Biol., 12 (1965) 829. 6 D. D. BROWN AND C. S. WEBER, J. Mol. Biol., 34 (1968) 611. 7 U. E. LOENING, J. Mol. Biol., 38 (1968) 355" 8 E. WTAGNER, S. PENMAN AND XV. M. INGRAM, J. Mol. Biol., 23 (1967) 149. 9 C. F. ZIMMERMAN AND B. HOLLER, J. Mol. Biol., 23 (1967) 149. io T. KASAI AND E. BAUTZ, in H. J. VOGEL, J. O. LAMPEN AND V. BRYSON, Organizational Biosw2thesis, A c a d e m i c Press, N e w York, 1966, p. I I I. I I H. C. BIRNBOIM, J. j. PENE AND J. E. DARNELL, Proc. Natl. Acad. Sci. U.S., 58 (1967) 32o12 A. P. X~YGAARDAND ]3. D. HALL, J. Mol. Biol., 9 (1964) 125. 13 J. RETlkL AND R. J. PLANTA, European J. Bioehem., 3 (1967) 248. 14 D. D. BROWN, Current Develop. Biol., 2 (1967) 47. 15 M. BIRNSTIEL, J. SPEIRS, I. PURDOM, K. JONES AND U. E. LOENING,Nature, 219 (1968) 45416 J. E. DARNELL, Bacteriol. Rev., 32 (1968) 262. 17 S. CHAUDHURI AND I. LIEBERMAN, dr. Mol. Biol., 33 (1968) 323 . 18 l~. A. WEINBERG, 1.~. E. LOENING, M. WILLEMS AND S. PENMAN, Proc. Natl. Acad. Sci. U.S.~ 58 (1967) lO88. 19 H. BREMER AND D. YUAN, dr. Mol. Biol., 38 (1968) 163. 20 S. R. DE KLOET, Biochem. Biophys. Res. Commun., 19 (1965) 582. 21 S. R. DE KLOET, Biochem. J., 99 (1966) 566. 22 V. S. MAYO, B. A. G. ANDREAN AND S. R. DE I(LOET, Biochim. Biophys. Acta, 169 (1968) 29723 M. H. VAUGHAN, JR., R. SOIERO, J. R. WARNER AND J. E. DARNELL, JR., Proc. Natl. Acad. Sci. U.S., 58 (1967) 1527 . 24 B. E. H. MADEN AND M. H. VAUGHAN, JR., Biophys. J. Abstr., (1968) A-88. 25 R. SOIERO, M. H. VAUGHAN,JR., J. R. WARNER AND J. E. DARNELL, J. Cell Biol., 39 (1968) II2. 26 J. PAPACONSTANTINOU AND E. M. JOLKU, J. Cell Physiol., 72, Suppl. I (1968) 161. 27 S. OSAV~'A, Ann. Rev. Biochem., 37 (1968) lO9-
Biochim. Biophys. Acta, 186 (1969) 317-325
317
BBA 96255
T H E S Y N T H E S I S AND PROCESSING OF RIBOSOMAL RNA P R E C U R S O R MOLECULES IN YEAST R. L. TABER, JR. AND W. S. VINCENT Department of Anatomy and Cell Biology, University o[ Pittsburgh, School of Medicine, Pittsburgh, Pa. 15213 (U.S.A.) (Received March I8th, 1969)
SUMMARY Experiments are presented which firmly establish the ribosomal precursor nature of the 38-S RNA molecule found in the yeast Schizosaccharomyces pombe by showing that (I) the kinetics of the 38-S molecule are consistent with a precursorproduct relationship with ribosomal RNA, (2) the molecule is methylated, (3) the kinetics of methylation are similar to those of uracil labeling, (4) the methyl label can be chased into ribosomal RNA and (5) the cistrons coding for 38-S RNA are homologous to those coding for ribosomal RNA.
INTRODUCTION When the yeast Schizosaccharomyces pombe is treated with cycloheximide, an inhibitor of protein synthesis, a large (38-S) RNA molecule accumulates 1,.1. Preliminary evidence suggested that this molecule was a ribosomal precursor. If this were true, then the accumulation of 38-S RNA in the absence of protein synthesis strongly implies that a post-transcriptional control mechanism in the maturation of ribosomal RNA exists. The purpose of this paper is to show conclusively that the 38-S molecule is indeed a ribosomal precursor. MATERIALSAND METHODS
Culture and labeling conditions Pulse labeling experiments were done with cultures of S. pombe, strain NCYC 132, that were grown to an absorbance at 595 m# of 0.2 (1.6. lO 6 cells per ml) in an Edinburgh minimal medium 2. At this time cells from I 1 were collected by Millipore filtration and were resuspended in either 50 or IOO ml of fresh media before the addition of the radioactive precursor. At the end of the pulse, cells were again collected by Millipore filtration and were rapidly frozen at --20 ° in the presence of Macaloid 3. The cells were completely frozen within 15 sec of pulse termination. Further processing, including sucrose gradient analysis, was as previously described 1. For uptake and incorporation studies, the radioactive precursor was added to cultures of exponentially growing cells (0.2 A 595ms)" I-ml samples were taken at appropriate intervals and were suspended at o ° in i ml of IO % trichloroacetic acid for Biochim. Biophys. Acta, 186 (1969) 317-325
318
R . L . TABER, JR., v¢. S. VINCENT
incorporation or in I ml of water for uptake studies. The cells were then collected on Millipore filters (HAWP) and were washed with either 5 °/o trichloroacetic acid or water, dried and counted as previously described 1.
Reagents Radioactive compounds, obtained from the New England Nuclear Corp. (Boston), had the following specific activities: [Me-SH]methionine, 182.8 mC/mmole; Jail]uracil, 5.61 C/mmole; [a~P]phosphate, carrier free. Bovine pancreatic ribonuclease A was obtained from Worthington Biochemicals (Freehold, N.J.); T 1 ribonuclease from Calbiochemicals (Los Angeles, Calif.); Macaloid from the Baroid Division, National Lead Co., (Houston, Texas) and cycloheximide (actidione) from Upjohn (Kalamazoo, Mich.).
Isolation o/DNA and RNA DNA was isolated from S. pombe according to the technique of SMITH AND HALVORSON4. RNA was isolated as previously described 1.
Hybridization Hybridization was carried out essentially according to GILLESPIE AZqDSPIEGELMAN5. Yeast DNA was denatured by adding NaOH to 0.2 M in 0. 9 M NaCl-o.o 9 M sodium citrate (pH 7.0). It was neutralized with HC1 and made to o.I M with Tris (pH 7.5). A solution of this DNA sufficient to give lO-2O #g/cm 2 of a H A W P Millipore filter was allowed to flow by gravity through a circular filter (142 mm in diameter) from a straight-sided container. The filter was then washed with 0. 9 M NaC10.09 M sodium citrate, blotted, allowed to dry at room temperature for 2 h and baked overnight in a 60 ° oven. About 7 ° I-cm square filters were cut from the large filter. Each had a highly reproducible amount of DNA bound 5. Hybridization was carried out in 0. 9 M NaCl-o.o9 M sodium citrate with 0.2 °/o sodium dodecyl sulfate in a scintillation vial at 63 ° for 20 h, a time which we found sufficient to produce saturation of ribosomal cistrons. After hybridization, filters were thoroughly washed with 0.3 M NaCl-o.o3 M sodium citrate (pH 7.0) and were placed in a solution of 0.3 M NaC1-0.03 M sodium citrate containing 4 o/zg/ml of pancreatic ribonuclease (electrophoretieally pure) and io/~g/ml T 1 ribonuclease for I h at room temperature. The filters were then washed again with 0. 9 M NaCl-o.o 9 M sodium citrate and were dried and counted to a minimum of 5 % reproducibility in a Beckman DPM-Ioo liquid scintillation spectrometer. The DNA bound to each filter was determined, following counting, according to BROWN AND W E B E R 6.
RESULTS
Kinetics o/labeling o] 38-S molecule with RNA precursor In order to establish the kinetics of labeling of the 38-S molecule, cells were labeled for I, 3, 5 and 15 rain. Gradient analyses of the RNA are shown in Fig. I. After I rain of labeling with [3H]adenine (Fig. IA), it can be seen that there are considerable counts in a region heavier than the mature larger ribosomal RNA molecule. The only distinct peak is found at the 38-S region and is superimposed over polydisperse RNA. At 3 rain (Fig. IB) using [3H]uracil, there are peaks at 38 S, 30 S and Biochim. Biophys. ,4cta, i86 (1969) 317-325.
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F i g . I. Sucrose g r a d i e n t a n a l y s i s a f t e r l a b e l i n g for t h e t i m e s i n d i c a t e d . T h e g r a d i e n t s w e r e of 1 5 - 3 o % sucrose m a d e i n o . I M E D T A , o . o i M NaC1, o . o o i M T r i s ( p H 7.4) a n d o. 5 % s o d i u m d o d e c y l s u l f a t e a n d w e r e s e d i m e n t e d for 16 h a t 25 o o o r e v . / m i n at 23 ° i n a S p i n c o S W - 2 5 rotor. O---Q, 3H; - - , A ~ 0 m#. F i g . 2. U p t a k e - i n c o r p o r a t i o n d a t a of a u r a c i l c h a s e e x p e r i m e n t . [ 3 H J U r a c i l : 0 - 0 , i n c o r p o r a t i o n . Ea4C]Uracil: A - - - A , u p t a k e ; A - . -- A , i n c o r p o r a t i o n .
uptake; C)-O,
18 S*. At 5 rain with uracil (Fig. IC), the relative amount of counts at 38 S is much less. A shoulder is seen at 30 S with most of the counts in 25- and I8-S RNA. These results are consistent with the flow of label from the 38 S to the 30 S and 18 S and finally to the 25 S. Although the 30-25 S transition is somewhat uncertain b y our methods, as the two represent only a 1-2 tube difference on the gradients, LOENING~ has demonstrated the difference more clearly using gel electrophoresis. * In this p a p e r w e shall refer to t h e "28 S" as "25 S" as s u g g e s t e d b y LOENING T for fungi. T h e p r e c u r s o r m o l e c u l e s w i l l be r e f e r r e d to as 38 a n d 3 ° S; it m u s t b e r e m e m b e r e d t h a t t h e s e v a l u e s are o n l y a p p r o x i m a t i o n s f r o m t h e g r a d i e n t s .
Biochim. Biophys Acta, 186 (1969) 317-325
320
R. L. TABER, JR., W. S. VINCENT
If the precursor-product relationship above were true, it should be possible to demonstrate with a chase experiment the rapid disappearance of label from the 38 S and 3o S and its concomitant appearance in the 25 and 18 S. As shown in Fig. 2, such an experiment with uracil proved to be unfeasible. In this experiment cells were labeled with [3H]uracil, and 8 min later an I8-fold excess of [14Cluraeil was added. Samples were taken at 2-min intervals, and both the uptake and incorporation were measured. It can be seen that the incorporation of [3H]uracil remained linear for IO min after the El*C]uracil chase. It is apparent that the [3H]uracil pool continued to support the 3H incorporation in spite of the fact that 14C rapidly penetrated the cells and was incorporated in a linear fashion from I min after its addition. As would be expected from these results, a Io ooo-fold excess of unlabeled uracil did not affect the io-min incorporation period after the chase.
Kinetics o/methylation o] 38-S molecule Since the above pulse-chase experiment could not be done, we examined the methylation of the 38-S molecule. It is known that the mammalian precursor is methylated s, 9 and we have shown previously that the 38-S molecule in cycloheximide-
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Fig. 3. Sucrose g r a d i e n t a n a l y s i s of R N A a f t e r l a b e l i n g w i t h [ M e - 3 H l m e t h i o n i n e for t h e t i m e s indicated. O - - - 0 , 8H; - - , A280 m/~. Fig. 4' Sucrose g r a d i e n t a n a l y s i s of R N A e x t r a c t e d a f t e r a d d i t i o n of EMe-3H]methionine followed a t 30 sec b y a i o o - f o l d excess of u n l a b e l e d L-methionine. The cells were c o l l e c t e d a n d frozen a t 3 min. O - - - 0 , all; - - , A 260 m/~.
Biochim. Biophys. Acta, 186 (I969) 317-3~5
RIBOSOMAL
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321
PRECURSOR MOLECULES
treated cells is also methylated 1. In order to examine the kinetics of methylation of the rapidly labeled 38-S RNA component, cells were pulsed with [Me-SHlmethionine for various times. When the cells were pulsed for 30 sec (Fig. 3A), three distinct peaks were found, one at 38 S, another at 30 S and a third at 18 S. This pattern of labeling is very similar to the 3-min uracil pulse shown in Fig. IB. With a 3-rain [Me-SH3methi onine pulse, the results shown in Fig. 3B were obtained. This shows some label at 38 S with relatively more counts at 30 and 18 S than were found at 30 sec. Fig. 3C is the result of a 5-rain pulse and demonstrates that at this time labeling and absorbance patterns are nearly identical. That the labeling by EMe-3Hlmethionine of the 38-S fraction can be chased is shown in Fig. 4. In this experiment cells were pulsed with [Me-SH]methionine and then chased at 30 sec with a ioo-fold excess of unlabeled L-methionine for 2.5 min. We found that, unlike uracil, methionine incorporation stopped upon a chase within I rain (Fig. 5). No label appears in the 3o-S region after this short chase. When this figure is considered in conjunction with Fig.3A and 3B, it further supports the idea that the 38-S molecule is a precursor for ribosomal RNA.
Homology o/ base sequences between 38-S R N A and 25- and zS-S R N A To establish that the 38-S molecule contained the identical base sequences as the 25-S and I8-S RNA, molecular hybridization experiments were done. To obtain a sufficient quantity of 38-S RNA, cells were treated with Ioo #g/ml of cycloheximide. I min later 2 mC of s~p were added, and at 20 rain the cells were collected and frozen. RNA was extracted from these cells and was analyzed on sucrose gradients as described previously 1. The gradient is shown in Fig. 5. The fraction indicated was collected and dialyzed against o.i M EDTA, o.oi M Tris (pH 7.4) and 0.2 ~o sodium dodecyl sulfate overnight before being precipitated with ethanol. This RNA was then rerun on a similar gradient in the SW-39 Spinco rotor, and tile tubes containing
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Fig. 5. I n c o r p o r a t i o n of EMe-14C]methionine a n d t h e effect of t h e a d d i t i o n of a Ioo-fold excess of u n l a b e l e d L - m e t h i o n i n e . Q - Q , c o n t r o l ; O - Q , chase. Fig. 6. Sucrose g r a d i e n t a n a l y s i s of R N A t r e a t e d w i t h c y c l o h e x i m i d e as d e s c r i b e d a n d t h e n l a b e l e d for 20 rain w i t h ? S P ] p h o s p h a t e . The t u b e s i n d i c a t e d w i t h s t i p p l i n g w e r e us e d as a source of l a b e l e d 38-S R N A for h y b r i d i z a t i o n e x p e r i m e n t s . (•)-Q, s2p; _ . _ , A 26~ m/*.
Biochim. Biophys. Acta, 186 (1969) 317-325
322
R . L . TABI,-'R, JR., W. S. VINCI.;NI
t h e R N A were collected, d i a l y z e d a n d p r e c i p i t a t e d as before. The a m o u n t of m a t e r i a l collected was insufficient to o b t a i n A 260 m~ values. The 38-S R N A fraction was t a k e n u p in 3 ml of o. 9 M N a C l - o . o 9 M sodium citrate, a n d its annealing characteristics to D N A were d e t e r m i n e d . C o m p e t i t i o n for i d e n t i c a l sequences in 38-S R N A were carried out b y preh y b r i d i z i n g t h e D N A w i t h u n l a b e l e d R N A l°,lx. One set of filters was i n c u b a t e d for 2o h at 63 ° in u n l a b e l e d 18- plus 25-S R N A (2//g/ml) t h a t was purified using sucrose g r a d i e n t s e d i m e n t a t i o n . A n o t h e r set was i n c u b a t e d u n d e r identical conditions except t h a t t h e R N A was o m i t t e d . A f t e r i n c u b a t i o n b o t h sets were w a s h e d with o.3 M N a C l - o . o 3 M s o d i u m c i t r a t e a n d were i n c u b a t e d as described with ribonuclease. A f t e r a t h o r o u g h washing, t h e filters were p l a c e d in 3 ml of o. 9 M N a C l - o . o 9 M s o d i u m cit r a t e c o n t a i n i n g 23o0 c o u n t s / m i n per ml of 38-S R N A . Filters c o n t a i n i n g no D N A were a d d e d as blanks, a n d i n c u b a t i o n was allowed to proceed at 63 ° for 20 h. A t the e n d of this time, t h e filters were again t r e a t e d w i t h ribonuclease, dried a n d c o u n t e d to 5 % r e p r o d u c i b i l i t y for the b l a n k s a n d 3 % for filters with a p p r e c i a b l e b o u n d R N A . T h e results are shown in T a b l e I. I t can be seen t h a t the p r e s a t u r a t i o n of ribos o m a l sites on the D N A c o m p l e t e l y inhibits the b i n d i n g of the 38-S R N A fraction. TABLE I C O M P E T I T I O N FOR 3 8 - S B I N D I N G TO D N A
B Y I 8 - AND
25-S RIBOSOMAL ] ~ . N A
H y b r i d i z a t i o n c a r r i e d o u t as d e s c r i b e d in t h e t e x t . The R N A s a m p l e s were p r e p a r e d from t w o d i f f e r e n t 32P-labeled p r e p a r a t i o n s p r e p a r e d in i d e n t i c a l fashion. E a c h f i l t e r c o n t a i n e d 9 # g of D N A . T h e n u m b e r s r e p r e s e n t c o u n t s / r a i n b o u n d per filter.
32P-labeled R N A sample
DN A control
D N,4 prehybrid
Blank
38 S
136 I3I 127 135
3° 31 ~5 17
29 27 17 17
38 S
The p o s s i b i l i t y r e m a i n e d t h a t t h e 38-S region r e p r e s e n t e d aggregates of 18-, 25-S or o t h e r R N A . I n order to e x a m i n e this, we p r e s a t u r a t e d t h e D N A w i t h 3Hl a b e l e d 25-S R N A alone. These filters were t r e a t e d as above. T h e y were then h y b r i dized with a ~2P-labeled 38-S fraction o b t a i n e d s i m i l a r l y to the one used a b o v e a n d a 6 - I o - S R N A fraction from t h e same g r a d i e n t as t h e 38 S. The results of this experiT A B L E 1I COMPETITION FOR 38-S
AND
6--IO-S BINDING BY 25-S R N A
H y b r i d i z a t i o n c a r r i e d o u t as d e s c r i b e d in t h e t e x t . E a c h f i l t e r c o n t a i n e d 5 # g DNA. The n u m b e r s r e p r e s e n t c o u n t s / r a i n b o u n d p e r filter.
3zP-labeled R N A sample
DNA control
D N A with 25-S prehybrid
Blank
38 S
79 80 86 81
3° 32 85 80
15 17 18 18
6-1o S
Biochim. Biophys. Acta, 186 (1969) 317-325
RIBOSOMAL RNA
PRECURSOR MOLECULES
323
ment are shown in Table I I where it can be seen that 25-S RNA appreciably inhibits 38-S binding, while 6-1o S was not inhibited at all. As NYGAARD AND HALL12 have shown that R N A - D N A complexes in solution were subject to dissociation upon prolonged incubation, the possibility existed that the binding demonstrated in Table U could have been the result of the dissociation of the prehybridized RNA. T h a t this is not so is seen in Table I I I . In this experiment, filter-bound DNA was incubated for 20 h with 3H-labeled RNA and was processed as described. No significant change in the binding of RNA p e r / , g of DNA was found. TABLE
III
STABILITY OF R N A - D N A FILTER COMPLEX ON PROLONGED INCUBATION F i l t e r s w e r e i n c u b a t e d w i t h 8 H - l a b e l e d 2 5 - S R N A f o r 2 0 h, w a s h e d , t r e a t e d w i t h r i b o n u c l e a s e a n d w e r e r i n s e d a s d e s c r i b e d i n t h e t e x t . T h e f i l t e r s w e r e t h e n i n c u b a t e d i n o . 9 M N a C l - o . o 9 RI s o d i u m c i t r a t e 4 - o . 2 °/o s o d i u m d o d e c y l s u l f a £ e f o r t h e t i m e s i n d i c a t e d , r e p r o c e s s e d a s d e s c r i b e d a b o v e a n d counted. The DNA content of each filter was determined chemically after counting*.
Incubation time (h)
Counts bound per #g DNA
o 2 9 20
lO 3 lO2 92 lO2
DISCUSSION
We have previously demonstrated the presence of a 38-S RNA molecule in yeast which accumulates after cycloheximide treatment 1,21. This molecule had characteristics which suggested that it might be a ribosomal RNA precursor. RETlkL AND PLANTA13 have shown that there is a high molecular weight RNA in Saccharomyces carlsbergensis which m a y also be a ribosomal precursor. However, in neither of the above reports could definitive relationships be established. In this paper we have shown b y three independent procedures that the 38-S molecule is a precursor of ribosomal RNA. The results from a short term labeling with E3H]uracil and [Me-3H]methionine strongly suggest a precursor-product relationship between the 38-S and mature 25-S and I8-S RNA molecules. The results from R N A - D N A hybridization establish the identity of nucleotide sequences between the 38-S and the 25- and I8-S molecules. In Tables I and I I I , data are given which provide convincing evidence that the nucleotide sequences in the 38-S RNA are those contained in the 25-S RNA, and b y implication the I8-S RNA as well. A mixture of 18- and 25-S RNA completely inhibits subsequent binding of the 38-S molecule to DNA. 25-S RNA alone, prehybridized to DNA, suppresses 38-S binding b y approx. 75 %. This is somewhat greater than would be expected on the basis of the relative molecular weights ascribed to the 38-, 25- and I8-S molecules 7, where the expected interference would be only 57 °o. This discrepancy could be due to an interference in the binding of the 38-S RNA preparation by the prehybridized 25-S RNA, particularly if the 25- and I8-S cistrons are in alternating sequences as has been suggested for other forms 14,15. Tile failure of the 6-Io-S material to be competed for b y unlabeled 25-S RNA is significant, as it suggests that (a) the 38-S region does not contain aggregates of Biochim. Biophys. Acta, 186 ( 1 9 6 9 ) 3 1 7 - 3 2 5
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R . L . TABER, JR., W. S. VINCENT
precursors of messenger-type RNA, and (b) the ribosomal RNA fractions have not been fragmented appreciably by the extraction and isolation processes. The data from pulse labeling also provide information as to the speed of synthesis and processing of the ribosomal RNA molecules. First, with respect to the time for production of mature ribosomal RNA, the data in Figs. i, 3 and 4 suggest 2-3 min. This is much more rapid than for mammalian cells where the time is on the order of 2o min 16,17. However, it is interesting to note that in HeLa cells, this represents 1. 3 °/o of a generation time (24 h generation time), whereas in yeast (3 tl) it is I.I°~,, i.e. roughly the same proportion of the cell cycle being required for processing in both forms. The 38-S molecule proceeds through at least one intermediate during maturation to the 25-S RNA which we have arbitrarily designated as the 3o-S stage. I8-S RNA appears simultaneously with this step as is shown in Figs. IB and 3A. The 3o S then apparently becomes the 25 S. These steps are similar to those of mammalian cells is. A major difference is in the size of the precursor. LOENING7estimates molecular weights of 2.3" IO6 for the 38 S and 2.o. lO6 for the 18-25 S. This suggests an ahnost conservative processing, whereas in HeLa cells close to 4 ° o~, of the precursor is lost in the transition to mature ribosomal RNA 18. The methylation experiments show that processing of the 38 S is very rapid, much more so than the synthesis of the molecule. One can see from Fig. 3A that within 3 ° sec, the 18 and 3o S as well as the 38 S are methylated. The fact that the 3o-S precursor is labeled, while the 25 S appears not to be, suggests that methylation has occurred in the 38-S stage with subsequent cleavage to 3o- and I8-S molecules. It is also evident from comparing Figs. I and 3 that the time required for the synthesis of the 38-S molecule is considerably longer than the time required for the methylation and processing of the 3o 25-S and I8-S stages. In Fig. 3A it is likely that the processing of the 38-S molecule through methylation and cleavage to 3oand I8-S stages has occurred in 3o sec. From Fig. IB the time of synthesis of the 38-S molecule with processing to the stages demonstrated by Fig. 3 A is about 3 min. If one assumes that the rate of nucleotide synthesis determined by BREMER AND YUAN19 for bacteria (55 nucleotides per sec) holds for yeast, the time required to synthesize the 38-S molecule (2.3"1o 6 daltons, or approx. 76oo nucleotides) would be about 2 min. This is in line with the data given here. The finding that the time required to process the ribosomal RNA precursor is short, relative to the time required for its synthesis, may represent a significant difference between yeast and mammalian cells for in the latter the processing time is far longer than the time for synthesis 16A8. This could possibly be due to a limiting step in the post-transcriptional cleavage of the molecule in mammalian cells that does not exist in yeast. DE KLOET et al. 2°-~2 was the first to observe that cycloheximide affected RNA synthesis in yeast and has since investigated this phenomenon extensively. He concluded that the high molecular weight RNA which accumulates in yeast after cycloheximide treatment contains two components, a DNA-like RNA and a ribosomallike RNA. This conclusion was based primarily on a base ratio analysis of the RNA which sediments at greater than 12 S in a sucrose gradient. From our results we conclude that better than 95 °Jo of the high molecular weight RNA is homologous in sequence to ribosomal RNA. Our "high molecular weight" fraction, however, was Biochim. Biophys. Acta, 186 (1969) 317-325
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taken only from the 38-S region. This would suggest that the DNA-like R N A is found primarily in the I2-35-S area. Also, our cells were labeled for 20 min after cycloheximide treatment, whereas DE KLOET'S were labeled for 9 ° min. It is likely that after longer labeling, a greater proportion of the R N A made is DNA-like. The establishment of the precursor nature of the 38-S molecule makes its accumulation in the presence of cycloheximide significant. The continuation in the synthesis of the molecule in the absence of protein synthesis indicates that the control does not reside solely at the level of transcription of the R N A precursor molecule. A similar phenomenon has previously been observed in HeLa cells by VAUGHAN et al. 23,~4 who showed that methionine and valine deprivation resulted in a failure to process the precursor. A failure of processing to the complete ribosomes has also been shown in HeLa cells treated with puromycin ~5 and in stationary lens epithelium cells 26. These findings further indicate that in eukaryotes post-transcriptional control mechanisms in ribosomal processing are available to the cell. This is in contrast to bacteria in which the two large types of ribosomal R N A (16 S and 23 S) appear to be synthesized directly .7. ACKNOWLEDGMENTS
This work was supported in part by U.S. Public Health Service grant GM 11480 to W . S . V . R . L . T . was supported in part by U.S. Public Health Service grant 5 ToI HD ooo-4o-i2. REFERENCES i R. I,. TABER AND W. S. VINCENT, Biochem. Biophys. Res. Commun., 34 (1969) 488. 2 J. M. MITCHISON, in D. M. PRESCOTT, Methods in Cell Physiology, Vol. 4, A c a d e m i c Press, N e w York, 1969, in t h e press. 3 W. M. STANLEY, JR. AND R. M. BOCK, Biochemistry, 4 (1965) 13°2. 4 J. D. SMITH AND H. O. HALVORSON, Methods Enzymol., 12 (1967) 538. 5 D. GILLESPIE AND S. SPIEGELMAN, J . Mol. Biol., 12 (1965) 829. 6 D. D. BROWN AND C. S. WEBER, J. Mol. Biol., 34 (1968) 611. 7 U. E. LOENING, J. Mol. Biol., 38 (1968) 355" 8 E. WTAGNER, S. PENMAN AND XV. M. INGRAM, J. Mol. Biol., 23 (1967) 149. 9 C. F. ZIMMERMAN AND B. HOLLER, J. Mol. Biol., 23 (1967) 149. io T. KASAI AND E. BAUTZ, in H. J. VOGEL, J. O. LAMPEN AND V. BRYSON, Organizational Biosw2thesis, A c a d e m i c Press, N e w York, 1966, p. I I I. I I H. C. BIRNBOIM, J. j. PENE AND J. E. DARNELL, Proc. Natl. Acad. Sci. U.S., 58 (1967) 32o12 A. P. X~YGAARDAND ]3. D. HALL, J. Mol. Biol., 9 (1964) 125. 13 J. RETlkL AND R. J. PLANTA, European J. Bioehem., 3 (1967) 248. 14 D. D. BROWN, Current Develop. Biol., 2 (1967) 47. 15 M. BIRNSTIEL, J. SPEIRS, I. PURDOM, K. JONES AND U. E. LOENING,Nature, 219 (1968) 45416 J. E. DARNELL, Bacteriol. Rev., 32 (1968) 262. 17 S. CHAUDHURI AND I. LIEBERMAN, dr. Mol. Biol., 33 (1968) 323 . 18 l~. A. WEINBERG, 1.~. E. LOENING, M. WILLEMS AND S. PENMAN, Proc. Natl. Acad. Sci. U.S.~ 58 (1967) lO88. 19 H. BREMER AND D. YUAN, dr. Mol. Biol., 38 (1968) 163. 20 S. R. DE KLOET, Biochem. Biophys. Res. Commun., 19 (1965) 582. 21 S. R. DE KLOET, Biochem. J., 99 (1966) 566. 22 V. S. MAYO, B. A. G. ANDREAN AND S. R. DE I(LOET, Biochim. Biophys. Acta, 169 (1968) 29723 M. H. VAUGHAN, JR., R. SOIERO, J. R. WARNER AND J. E. DARNELL, JR., Proc. Natl. Acad. Sci. U.S., 58 (1967) 1527 . 24 B. E. H. MADEN AND M. H. VAUGHAN, JR., Biophys. J. Abstr., (1968) A-88. 25 R. SOIERO, M. H. VAUGHAN,JR., J. R. WARNER AND J. E. DARNELL, J. Cell Biol., 39 (1968) II2. 26 J. PAPACONSTANTINOU AND E. M. JOLKU, J. Cell Physiol., 72, Suppl. I (1968) 161. 27 S. OSAV~'A, Ann. Rev. Biochem., 37 (1968) lO9-
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