Synthesis and maturation of chloroplast and cytoplasmic ribosomal RNA in Chlamydomonas reinhardi

35

Biochimica et Biophysica Acta, 366 (1974) 35--44 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98O97

SYNTHESIS AND MATURATION OF CHLOROPLAST AND CYTOPLASMIC RIBOSOMAL RNA IN CHLAMYDOMONAS R E I N H A R D I

MARK J. MILLER and DANIEL McMAHON

Division of Biology, California Institute of Technology, Pasadena, Calif. 91109 (U. S. A,) (Received March 19th 1974)

Summary The work of a large n u m b e r of people has shown that the ribosomes of the chloroplast differ from those of the cytoplasm in the spectrum of their sensitivity to inhibitors of protein synthesis, the size of their rRNAs, and in the sensitivity of chloroplast rRNA synthesis to rifamycin SV. The genes for chloroplast rRNAs reside on the chloroplast DNA while those for cytoplasmic rRNAs are found on the nuclear DNA. We have characterized the synthesis and processing of cytoplasmic and chloroplast rRNAs of the alga Chlamydomonas reinhardi in order to further elucidate the difference between chloroplast and nuclear genetic systems. Cytoplasmic rRNAs of C. reinhardi are cleaved from a single precursor of molecular weights 2.4" 106 to a mature rRNA ( 0 . 6 9 . 106 mol. wt) and a 1 . 4 . 106-mol. wt precursor of the mature 1 . 3 . 106-mol. wt rRNA. The kinetics of incorporation of radioactive label into the rRNAs suggest that the 0.69 • 106-mol. wt rRNA gene is located closer to the p r o m o t o r than is the gene for the 1.4 • 106 -mol. wt rRNA. The synthesis of cytoplasmic rRNAs is extremely sensitive to camptothecin, an inhibitor of nuclear rRNA synthesis, but synthesis of chloroplast rRNA is quite resistant to the inhibitor. This has allowed us to demonstrate that chloroplast rRNAs are processed from precursors which resemble those of blue-green algae. A 1.14 • 106 -mol. wt precursor is processed to the 1 . 0 7 . 106-mol. wt mature chloroplast rRNA, and a 0.64 • 106 -mol. wt precursor is cleaved to a 0.56 • 106 -mol. wt species and then to the mature 0 . 5 4 . 1 0 6 - m o l . wt rRNA. This study demonstrates two new ways in which the function of the chloroplast genome resemble those of prokaryotes more than those of the nucleus.

Introduction

Chlamydomonas reinhardi, and other plant species, contain two major classes of ribosomes. Those found in the cytoplasm resemble the cytoplasmic

36 ribosomes of eukaryotes in general while those in the chloroplast resemble the ribosomes of prokaryotes [1--4]. Although C. reinhardi contain mitochondria, mitochondrial ribosomes form an insignificant fraction of cellular ribosomes. Examination of thin sections shows that the frequency of ribosomes within mitochondria is less than 1% of the frequency of those within the chloroplast and purified mitochondria are quite poor in ribosomes (Goodenough, U., personal communication). C. reinhardi is an organism, therefore, where the synthesis and processing of cytoplasmic and chloroplast RNAs can be compared without serious interference from mitochondrial rRNA s:yntheses. The rRNAs of chloroplast and cytoplasm differ in a number of respects including their size [1,2,5] and the location of the genes which encode their primary structure [7--9]. The synthesis of chloroplast but not nuclear RNAs is sensitive to rifamycin SV [10], again resembling RNA synthesis in prokaryotes. Rifamycin SV also seems to be an agent which specifically disrupts the chloroplast genetic system as opposed to that of the mitochondrion. When C. reinhardi is treated with rifamycin SV, the structure of the chloroplast becomes disrupted and its content of ribosomes becomes severely depleted while the structure and ribosomal content of the mitochondria is unaffected [11] (Goodenough, U., personal communication). We have examined the synthesis and processing of chloroplast and cytoplasmic rRNAs in C. reinhardi for several reasons. First, to re-investigate a report that its cytoplasmic rRNAs were not made from a single high molecular weight precursor [12]. If this were true it would be unique among the eukaryotes. In addition we wished to determine whether camptothecin, an agent which inhibits RNA synthesis in eukaryotes, but n o t in prokaryotes [13,14] was a more potent inhibitor of nuclear than chloroplast rRNA synthesis. This antibiotic then allowed us to demonstrate that precursors of chloroplast rRNA which resemble those of blue-green algae are made in C. reinhardi. Finally, McMahon and Langstroth [15] have demonstrated total RNA synthesis of C. reinhardi is under "stringent" control b u t that this control is regulated in a manner which differs substantially from prokaryotes. In order to investigate the precise locus of this control it is important to determine the pathways of RNA synthesis. Materials and Methods Chemicals Radioactive chemicals were purchased from Schwarz/Mann. Diethyl oxydiformate was the product of Eastman Kodak Co., Rochester, N.Y. Sodiump-aminosalicylate was from K and K laboratories, Hollywood, Calif. Sodium dodecylsulfate was purchased from Alcolac Chemical Co., Baltimore, Md. Rifamycin SV was purchased from Calbiochem, San Diego, Calif. and ribonucleasefree deoxyribonuclease was purchased from Worthington Biochemical Corp., Freehold, N.J. Both phenol and m-cresol were obtained from Mallinckrodt Chemical Works, St. Louis, Mo. The phenol was distilled over powdered zinc before use. The m-cresol was repeatedly distilled over zinc in vacuo until it was colorless and then stored under N2 at --20°C. Actinomycin D and camptothecin were the gifts of Dr Walter Gall (Merck and Co., Rahway, N.J.) and Dr Harry Wood (National Cancer Institute, Bethesda, Md.), respectively.

37

Cells and culture conditions Two strains of C. reinhardi were used: arg 2, a mutant-lacking arginosuccinate synthetase [16] was obtained from Dr W. Ebersold, Department of Biology, University of California, Los Angeles; CW-15, a m u t a n t which lacks a normal cell wall [17] was obtained from Dr D. Roy Davies, John Innes Institute, Norwich, England. All cells were grown in HSM medium [18] containing in addition 24 mM sodium acetate. For arg 2 the medium was supplemented with 0.57 mM arginine. When cells were labeled with 32po3- the medium was prepared with only 1% of its normal c o n t e n t of phosphate (0.133 mM PO2-). In this case the medium was buffered with 20 mM Tris--HC1 (pH 7.2). Cells grew at normal rates for more than 24 h in this medium. Cells were always grown at 22°C and a light intensity of 1500 lux to a concentration of 1 " 10 6 - 2 • 10 6/ml. Extraction of R N A Method A: 25 ml of CW-15 cells were poured over 6--7 g of ice and then centrifuged at 1000 X g for 2 min. This and all subsequent steps were performed at 0--4°C. The lysis buffer was prepared just prior to centrifugation by adding 0.6 g of sodium-p-aminosalicylate, 0.2 ml of diethyl oxydiformate and 1 ml of 20% sodium dodecylsulfate to 10 ml of Buffer I of Brown and Haselkorn [19]. It was poured onto the cell pellet and the cells were disrupted by Vortex mixing. 10 ml of phenol--m-cresol--8-hydroxyquinoline (90:10:0.1, v/v/w) solution saturated with Buffer I were added and the mixture was shaken intermittently for 15 min. It was then centrifuged for 20 min at 900 X g. The lower, organic layer was removed and the aqueous layer re-extracted with phenol--m-cresol--8-hydroxyquinoline and centrifuged. After removal of the phenol--m-cresol--8-hydroxyquinoline solution, the aqueous layer was extracted with a solution of 4% iso-amyl alcohol in chloroform. Finally, 25 ml of ethanol were added to the aqueous layer and nucleic acids were allowed to precipitate for several hours at --25°C. The pellet was collected by centrifugation (11 000 X g for 20 min) and treated with deoxyribonuclease by the method of Brown and Haselkorn [19], with the exception that the final extraction was done twice with two volumes of a 1:1 mixture of phenol--m-cresol-8-hydroxyquinoline and chloroform--iso-amyl alcohol. Method B: Cells with normal walls were harvested as above and the pellet was resuspended in 10 ml of Buffer I. The cells were disrupted using a French pressure cell operated at a pressure of less than 1600 lb/inch 2 and collected into a tube containing 0.2 ml diethyl oxydiformate. Sodium dodecylsulfate and sodium-p-aminosalicylate were added to the same concentrations as in Method A and the lysate extracted with phenol--m-cresol--hydroxyquinoline and chloroform--iso-amyl alcohol as in Method A. Gel electrophoresis Polyacrylamide-agarose gels were made by the m e t h o d of Simmons and Strauss [20]. Electrophoresis was performed at 0--4°C using a buffer consisting of 0.04 M Tris, 0.02 M sodium acetate, 0.001 M EDTA (pH 7.8) and containing 0.1% sodium dodecylsulfate. After electrophoresis (2.2% acrylamide, 0.4% agarose) t h e y were sliced on a Mickle gel slicer into 1.0-mm thick slices. The RNA

38 in the slices was h y d r o l y z e d for at least 2 h at 70°C in 0.5 ml concentrated NH4OH. The samples were cooled and, after evaporation of the ammonia, dioxane-based scintillation fluid was added and the samples were counted. The molecular weights of the species of RNA detected on the gel were assigned on the basis of the k n o w n proportionality between distance migrated and log molecular weight [21,22] using the molecular weights of the internal markers of C. reinhardi rRNAs as standards. These were assumed to be 1 . 3 0 . 1 0 6 , 0 . 6 9 . 1 0 6 , 1 . 0 7 . 1 0 6 and 0 . 5 4 - 1 0 6 molecular weights for the cytoplasmic 25-S and 18-S rRNAs and the chloroplast 23-S and 16-S rRNAs, respectively [23]. Results

Isolation o f R N A The rRNAs and their precursors of C. reinhardi are extremely labile. Several techniques for the isolation of RNA [1,19,23,24] failed to extract precursors; even the mature rRNAs were often degraded. A wide variety of other techniques and buffers also failed to extract a possible precursor to rRNAs. The extreme difficulty we encountered in developing an isolation procedure probably explains the failure of Wilson and Chiang [12] to isolate a precursor of the cytoplasmic rRNAs. Our initial success occurred using a m u t a n t of C. reinhardi which lacks a cell wail, CW-15. In contrast to wild-type cells, these cells lysed instantaneously in the detergent, and polyacrylamide gel electrophoresis revealed potential precursors of the rRNAs. The presence of the nuclease inhibitor, diethyl oxydiformate, in the isolation buffer is critical for success, as is the requirement for rapid cooling before centrifugation. Omission of sodiump-aminosalicylate from the lysis buffer reduces the yield of precursor. Precursors could also be isolated from wild-type cells by disrupting the wall in the French pressure cell (Method B) demonstrating that the presence of precursor is indeed a characteristic of normal cells and n o t a secondary mutation of CW-15. Synthesis and processing o f cytoplasmic rRNAs Fig. 1 illustrates the pattern of incorporation of label into RNA as a function of the time of labeling. A molecule of molecular weight 2.4 • 106 (+ 0.1 • 106 ) is the first discrete species to be labeled by 3zpo2-. Incorporation of 32PO43- into a 1 . 4 . 106-mol. wt species occurs almost as rapidly. Label accumulates in a 2.2 • 106 -mol. wt (-+ 0.1 • 106 ) molecule (difficult to resolve from the 2.4 • 106 -mol. wt molecule) and in molecules close to the molecular weight of the mature rRNAs as the period of labeling continues. A minor peak of molecular weight 1.14 • 106 (+ 0.05 • 106 ) is also labeled. This will be shown later to be a precursor of the 1.07 • 106 -mol. wt chloroplast rRNA. The pattern of incorporation of label into RNAs is very similar to that found in several other plant species [25--28]. The molecular weights of the molecules and their order of appearance are in accord with the scheme for synthesis of cytoplasmic rRNA presented in Fig. 2. The position occupied by the 2.2 • 106 -mol. wt molecule is ambiguous and will be discussed later. The scheme in Fig. 2 is supported by the experiments illustrated in Fig. 3

39 (a)

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Fi~ 1. Synthesis and processing of rRNAs. C W - 1 5 cells were incubated in 0.1 ~Ci/ml [3H]adenine (22 Ci/mM) for 3 h to label mature rRNAs. They were washed twice and resuspended in 0.133 m M phosphate- 20 m M Tris--HCI--HSM (pH 7.2) m e d i u m and then divided into four equal cultures. The cultures were pulse labeled with 32po45" (20 ~Ci/ml) for: a, 5 rain; b, 10 min; c, 20 rain;d, 30 rain. The pattern of incorporation of [3H] adenine is drawn with thin lines,the 32PO43- with thick ones. The 3 H c p m scale is omitted in subsequent figures,but is of similarmagnitude.

in which actinomycin D was used to stop incorporation of 32po43- into RNA and the flow of precursor RNAs into mature rRNA observed. In this and similar experiments the a2p does not appear to chase from the 2.4 • 106 -mol. wt species through the 2.2 • 106 -mol. wt species of RNA but instead appears to directly enter the pools of 0.69 • 106 and 1.4 • 106 -mol. wt RNAs. Actinomycin D does not inhibit chloroplast rRNA synthesis very effectively. This may be the result of a permeability barrier for actinomycin D at the chloroplast membrane, or it may reflect a lower affinity of chloroplast DNA for actinomycin D because of its low content of guanine plus cytosine [ 2 9 ] . Cytoplasmic

rRNA / - - - ~ 1.4 M ~ I . 3 M

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Fig. 2. Maturation pathway of cytoplasmic and chloroplast ribosomal RNA'.

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Fig. 3. Processing o f r R N A p r e c u r s o r s in t h e p r e s e n c e of a c t i n o m y c i n D. Cells of CW-15 w e r e l a b e l e d for 2 h w i t h 0,5 #Ci/ml [ 3 H ] a d e n i n e (27 C i / m M ) . T h e i r R N A was e x t r a c t e d a n d m i x e d w i t h R N A e x t r a c t e d f r o m cells w h i c h h a d b e e n l a b e l e d for 10 m i n w i t h 3 2 p o 4 3 - ( 4 0 p C i / m l ) a n d c h a s e d f o r v a r y i n g l e n g t h s of t i m e using a e t i n o m y c i n D ( 4 0 p g / m l ) , a, 10 rain 3 2 p o 4 3 - pulse; b, pulse f o l l o w e d b y a 10-rain actinom y c i n D chase; c~ 2 0 - r a i n chase. T h e 3 2 p o 4 3 - c p m are i n d i c a t e d b y the t h i c k lines.

R i f a m y c i n SV, an inhibitor of chloroplast r R N A polymerase [ 1 0 ] reduces i n c o r p o r a t i o n of 3 2 p o 2 - i n t o the chloroplast r R N A by 80--90%. It has n o effect on the synthesis of the 2.4 • 106 , 1.4 • 106 , 1.3 • 106 or 0 . 6 9 • 106 -mol. w t r R N A s (Fig. 4). Similarly, the absence o f these m o l e c u l e s in cells labeled in the presence of c a m p t o t h e c i n (Fig. 5) also supports their nuclear origin.

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Fig. 4. T h e e f f e c t of r i f a m y c i n SV u p o n the p a t t e r n of i n c o r p o r a t i o n of 3 2 p o 4 3 - i n t o R N A . CW-15 cells w e r e l a b e l e d o v e r n i g h t in 0.5 # C i / m l [ 3 H ] a d e n i n e ( 0 . 8 4 C i / m M ) t h e n w a s h e d a n d r e s u s p e n d e d in 0 . 1 3 3 m M p h o s p h a t e - - 2 0 m M T r i e - H C 1 - - H S M ( p H 7.2) m e d i u m + 500 p g / m l r i f a m y c i n SV a n d d i v i d e d i n t o f o u r e q u a l aliquots. 3fiPO43- (30 p C i / m l ) w a s a d d e d a f t e r 1 h a n d t h e cells w e r e i n c u b a t e d for: a, 5 rnin; b, 15 min; e, 30 rain; d 45 rain. T h e 3 2 p o 4 3 - c p m are i n d i c a t e d b y the t h i c k lines.

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Fig. 5. I n c o r p o r a t i o n o f 3 2 p o 4 3 - i n t o R N A in t h e p r e s e n c e of c a m p t o t h e c i n . C W - 1 5 cells w e r e l a b e l e d o v e r n i g h t in 0 . 5 # C i / m l [ 3 H ] a d e n i n e ( 0 . 8 4 Ci/vaM). T h e y w e r e t h e n w a s h e d a n d r e s u s p e n d e d in 0 . 1 3 3 yaM p h o s p h a t e - 2 0 m M T r i s - - H C 1 - - H S M ( p H 7 . 2 ) v a e d i u v a a n d c a v a p t o t h e c i n (8 ~ g / m l ) . T h e c u l t u r e w a s d i v i d e d i n t o f o u r a l i q u o t s a n d p u l s e l a b e l e d w i t h 4 0 ktCi/val 3 2 P O 4 3 - . L e n g t h s o f 3 2 p o 4 3 - i n c u b a t i o n : a, 8 r a i n ; b, 1 5 r a i n ; c, 3 0 vain; d, 6 0 rain. T h e 3 2 p o 4 3 - epva are i n d i c a t e d b y t h e t h i c k lines.

Processing of chloroplast rRNA Camptothecin inhibits the incorporation of precursors into cytoplasmic rRNA in C. reinhardi at lower concentrations than those which inhibit chloroplast rRNA synthesis (Table I). Therefore, we examined the pattern of 32PO43incorporation into RNA in cells which had been inhibited with camptothecin. Fig. 5 shows that isotope initially appeared in species of RNA whose molecular weight was 1 . 1 4 . 1 0 6 (+ 0 . 0 5 . 1 0 6 ) and 0 . 6 4 . 1 0 6 ( + 0 . 0 2 . 1 0 6 ) a n d t h a t a TABLE I INCORPORATION INTO CYTOPLASMIC ENCE OF CAMPTOTHECIN Cavaptothecin concentration (~g/ml)

0 1 5 20

AND CHLOROPLAST

Cytoplasmic rRNA

RIBOSOMAL

R N A IN T H E P R E S -

Chloroplast rRNA

cpm/pg RNA layered

P e r c e n t of control

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Percent of control

5100 2000 0

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0

68

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Fig. 6. P r o c e s s i n g o f c h l o r o p l a s t r R N A . C W - 1 5 cells w e r e p r e l a b e l e d a n d r e s u s p e n d e d in 0 , 1 3 3 rnM p h o s p h a t e - 2 0 rnM T r i s - - H C I - - H S M ( p H 7 . 2 ) m e d i u m + c a m p t o t h e c i n as n o t e d in Fig. 5. A f t e r a 4 5 - r a i n p u l s e w i t h 3 2 p o 4 3 - ( 4 0 p C i / m l ) , s o l i d r i f a m y c i n S V w a s a d d e d to 5 0 0 p g / m l a n d t h e R N A w a s e x t r a c t e d a t i n t e r v a l s , a. 4 5 - r a i n 3 2 p o 4 3 - p u l s e ; b, p u l s e f o l l o w e d b y a 3 0 - m i n r i f a m y c i n S V c h a s e ; c, 6 0 - r a i n c h a s e ; d, 1 2 0 - r n i n c h a s e . T h e 3 2 p o 4 3 - c p m are i n d i c a t e d b y t h e t h i c k lines.

species of molecular weight 0 . 5 6 - 1 0 6 appears with longer labeling. After 60 min the label had accumulated primarily in mature chloroplast rRNAs. Although the background here appears high, its absolute magnitude is about the same as that found in uninhibited cells. This can be illustrated by comparing the backgrounds in Figs 1 and 5, noting that the camptothecin-treated cells were labeled with phosphate of twice the specific activity of that used to label the uninhibited cells. The molecular weights and order of appearance of RNAs suggested the processing scheme which is illustrated in Fig. 2. Since actinomycin D is n o t an efficient inhibitor of chloroplast rRNA synthesis we have used rifamycin SV to determine whether label incorporated in the 1 . 1 4 - 1 0 6 - , 0 . 6 4 - 1 0 6 - and the 0 . 5 6 - 1 0 6 - m o l . w t molecules would accumulate in chloroplast rRNAs in the absence of further synthesis of RNA. This is the case as Fig. 6 illustrates. Therefore these molecules appear to be precursors of chloroplast rRNAs. Definitive p r o o f of this will require hybridization competition experiments, however. Discussion The processing of C. reinhardi r R N A does n o t show any fundamental differences from that of other eukaryotes [ 2 5 - - 2 8 ] , although, compared to

43 some, it is made from a smaller primary precursor. As we noted earlier, there are two discrete species of very high molecular weight RNA ( 2 . 4 . 1 0 6 and 2.2 • 106 ), the larger of which is labeled first. Our results indicate that the peak of molecular weight 2.2 • 106 is n o t a normal intermediate between the 2.4 • 106-mol. wt precursor and the mature rRNAs since the material in the 2.4 • 106-mol. wt peak is n o t chased into the 2.2 • 106-mol. wt peak (Fig. 3), and because this species does not appear in any significant quantity until after the mature rRNAs have appeared. The 2.2 • 106 -mol. wt species may be another primary product of transcription which is more stable and which is transcribed at a slower rate. Alternatively, it may be a product of incorrect processing of the 2.4 • 106 -mol. wt molecule which is n o t processed into mature rRNA but which is eventually degraded. The existence of such defects in processing can be inferred from the results of WeUauer and Dawid [30] in HeLa cells. Assuming that there is no preferential extraction of precursors, the order of nucleolar rRNA genes within the unit of transcription can be determined by examining the relative rates of appearance of labeled 1.4 • 106 - and 0.69 • 106mol. wt RNAs. Since the first RNAs released from the transcriptional complex following the addition of label will be labeled predominantly at their 3' end, our data suggests that the rapidly labeled 1.4 • 106 -mol. wt RNA (precursor of 25-S rRNA) is located nearer the 3' end than is the 0.69 • 106 -tool. wt (18 S) rRNA. The gene order then would be: promotor, 18 S, 25 S. This is directly opposite the order of genes in HeLa [30] and rat [31] cells but agrees with the order of genes in Euglena gracilis [19]. It would be of interest to k n o w at what point in the evolution of the eukaryote this inversion in gene order occurred. Moreover, since the rRNA genes of C. reinhardi and of a variety of prokaryotes and eukaryotes are multiply re-iterated [9,23,32,33] the question of how such an inversion was accomplished is intriguing. This inversion of gene order appears to demand that an intermediate stage exists in the life cycle or existed in evolution where the number of replicated rRNA transcriptional units is one. There is also the alternative that inversion of gene order has resulted from inversion of the structural gene order with regard to a promotor, but this intermediate state would, of course, produce rRNAs which were the complements of the original rRNAs. Finally this might be the result of unequal crossing over or of a polyphyletic origin of the eukaryotes. Chloroplast rRNAs are synthesized from discrete precursors which resemble those of blue-green algae [34,35]. Although no RNA large enough to be a precursor for both rRNAs had been detected this does n o t exclude the possibility that such a c o m m o n precursor does exist but has n o t been detected because of the speed of its processing, etc. Recently, reports have appeared describing potential precursors of chloroplast rRNA in E. gracilis [36], and in spinach leaves [37,38]. In neither case was the possibility of mitochondrial rRNA contamination considered. A p r e c u r s o r - p r o d u c t relationship was not adequately demonstrated with pulse-chase experiments. The case of E. gracilis [36] was especially puzzling since label appeared in the mature 23-S chloroplast rRNA before becoming apparent in its presumed precursor. The chloroplast resembles prokaryotes in its relative resistance to camptothecin [13,14] and in the size of the precursors of its rRNAs. Thus, in two more ways the properties of the genetic system of the chloroplast resemble those of prokaryotes more than those of the rest of the cell.

44

Acknowledgements The authors gratefully acknowledge the fine technical assistance of Mr William Fry. This research was supported by U.S.P.H.S. grant GM 06965.

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