Characterization of ribonucleoprotein particles from the protozoa Astasia longa

57

Biochimica et Biophysica Acta, 4 7 6 ( 1 9 7 7 ) 5 7 - - 6 4 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 98911

C H A R A C T E R I Z A T I O N OF R I B O N U C L E O P R O T E I N PARTICLES FROM THE P R O T O Z O A ASTASIA LONGA

R. M O R O S O L I * a n d J.G. L A F O N T A I N E

Laboratoire de Biologie ceUulaire et moldculaire, D$partment de Biologie, Facultd des Sciences et de G$nie, Universitd Laval, Quebec, GiK 7P4 (Canada) (Received November 10th, 1976)

Summary Ribonucleoprotein particles released from nucleoli of Astasia longa b y treatment with heparin were characterized biochemically. When centrifuged in a sucrose gradient containing an appropriate buffer (Tris/KC1/Mg2+), four populations of particles were obtained, sedimenting at 90 S, 75 S, 60 S, and 44 S, respectively. The first t y p e of particles contained the high molecular weight (3.5 • 106) ribosomal RNA precursor. The RNAs present as the major components in the 75-S and 44-S particles had molecular weights of 1.35 • 10 + and 0.85 • 106, respectively, whereas the 60-S particles contained a mixture of 0.85 • 106 and 1.35 • 106 RNA. After a brief labeling, the radioactivity was found in the R N A constituent of the 90-S particles; following a 90 min chase, the label disappeared from this latter fraction and accumulated in the 75-S, 60-S and 44-S particles. This indicates a precursor-product relationship between the R N A of the 90-S particles and that of the three other ribonucleoprotein particles, consistent with the conversion: 3.5 • 106 R N A -* 1.35 • 106 RNA + 0.85 • 106 RNA.

Introduction It is now well established that, in eukaryotic cells, ribosomal RNA is synthesized in the nucleolus in the form of a high molecular weight precursor which is sequentially processed to give finally two mature ribosomal RNA species [ 1,2 ]. This precursor R N A is associated with proteins and constitutes a distinct ribonucleoprotein complex which has been characterized in several animal cell types [3--5], as well as in plants [6] and yeast [7]. In L-cells' nucleoli, the 110--62-S ribosome precursor and intermediate particles were * Present address: G6netique Humaine, CHUL, 2705 Boul. Laurier, Ste. Foy, Qu6bec, G I V 4G2, Canada.

58 reported to contain 45-S, 36-S and 32-S RNA [4]. These different particles correspond to the early maturation stages of ribosomal RNA. In plants, although 70-S, 60-S, 46-S and 32-S particles were isolated containing 33-S, 25-S and 16-S RNAs, the various intermediate products of ribosomal RNA processing have not yet been fully characterized, presumably due to the high RNAase c o n t e n t of this material [6]. In the case of the protozoa, Euglena gracilis, the precursor RNA has a molecular weight of 3.5 • 106 while that of the larger (25-S) mature ribosomal RNA is 1.35 • 106; the sedimentation constant (20-S) of the other ribosomal species {0.85 • 106) is slightly higher than in other organisms [8]. So far, no work has been done on the precursor ribosomal particles in protozoa. The present paper describes the isolation and properties of ribosomal precursor particles from purified nucleolar preparations of a bleached protozoa, Astasia longa, a species closely related to E. gracilis. Materials and Methods Cultures of A. longa (Jahn strain) were grown axenically at constant temperature (27°C) in a modified Cramer-Meyer medium with acetate (0.02 M) as sole source of carbon and energy [9]. The cells were maintained in a 12 1 culture vessel as described by Blum and Padilla [10], in the presence of a continuous flow of air. Cell counts were performed with a Coulter Counter (Coulter Co. Hialeah, Fla. U.S.A.) after appropriate dilution of the culture with 0.5% NaC1. Buffers. Buffer I (used to isolate nucleoli): 0.3 M sucrose/10 mM Tris • HC1 (pH 7.8)/5 mM MgC12. Buffer II: 1.2 M sucrose/10 mM Tris • HC1 (pH 7.8)/ 5 mM MgC12. Buffer III (used to extract ribonucleoprotein particles): 10 mM Tris • HC1 (pH 7.4)/2 mM MgC12/20 mM KC1/10 pg per ml polyvinyl sulfate. Buffer IV (used to isolate ribosomes): 0.7 M sucrose/0.1 M Tris • HC1 (pH 7.9)/ 0.25 M NaC1/4 mM MgC12/5 mM 2-mercaptoethanol. Buffer V (used to deproteinize ribosomes and nucleoli): 0.1 M Tris • HC1 (pH 7.9), 0.25 M NaC1/ 4 mM MgC12/5 mM 2-mercaptoethanol/2 mM EDTA. Buffer VI (used for gel electrophoresis): 40 mM Tris-acetate (pH 7.8)/0.1 M sodium acetate. Isolation ofnucleoli. The cells (2 • l 0 s cells/ml) were harvested by centrifugation in a continuous flow system (Sorvall Inc), the pellets obtained were washed once in buffer I, and, after a brief centrifugation, the cells were resuspended in the same buffer to a density of 30 • 106 cells/ml. Cells were broken by passing them twice through a French pressure cell at 3000 lb/inch 2. Most of the pellicular fragments were removed by passing the homogenate through a column (1.5 × 10 cm) filled with glass wool [11]. The nucleoli were separated from solubilized cytoplasm by centrifugation through a 40% (w/v) sucrose layer (containing: 0.01 M Tris • HC1, pH 7.8, and 5 mM MgC12) at 1000 X g for 10 min. The nucleolar pellet thus obtained was suspended in the extraction buffer, layered onto 30 ml of 40% sucrose solution and centrifuged in Sorvall HS-4 buckets at 50 X g for 10 min. The supernatant was removed and centrifuged at 1000 X g for 10 min. The pellet contained nucleoli, paramylon bodies and some pellicular fragments. All operations were carried out at 4 ° C. The final pellet containing the nucleoli was dispersed in buffer I and layered onto a 2 ml solution of 70% (w/v) RNAase-free sucrose. The 30-ml tubes

59 placed in a SW-25.1 rotor were centrifuged at 20 000 rev./min (57 000 X g) for 10 min at 5°C. The pellet thus obtained was used for extraction of ribonucleoprotein particles. Isolation of ribonucleoprotein particles. The procedure employed for the extraction of nucleolar ribonucleoprotein particles was essentially that of Simard et al. [12]. Nucleoli were washed in 2 ml of buffer III and centrifuged 5 min at 5000 rev./min (3000 X g). Following resuspension of the pellet in buffer III, heparin (0.5%) (158 units/mg) (Sigma) and DNAase (50 pg/ml) (Worthington) were added. The dispersed nucleoli were centrifuged at 15 000 rev./min (27 000 X g) for 10 min, and the supernatant was then layered onto 12 ml of 15--30% (w/w) sucrose gradient containing buffer III. To study the sedimentation behaviour of the ribonucleoprotein particles, preparations were centrifuged at 5°C with a Beckman L2-65B instrument, for 5 h at 40 000 rev./ min in a SW-41 rotor. Fractions (0.5 ml) were collected and monitored at 260 nm with a Gilford Model 240 spectrophotometer. Labeling of rRNA. Cultures of A. longa (2 • l 0 s cells/ml)were harvested by centrifugation at 1200 X g for 1 min at 23°C and rapidly washed with distilled water. The cells were transferred into a 250 ml flask containing fresh medium deprived of phosphate at a concentration of 2 • 106 cells/ml. Since this organism does n o t incorporate pyrimidine precursors [ 1 3 ] , labeling was achieved by adding 40 pCi/ml of 32p (New England Nuclear). Extraction ofRNA. A. longa ribosomal RNA was isolated as described for E. gracilis [14]. The ribosomes were suspended in buffer V, sodium dodecyl sulfate was added (final concentration of 2%), and the solution was shaken for 15 min in an equal volume of phenol/10% m-cresol/0.1% 8-hydroxyquinoline (v/v/w). The extraction was repeated as above, and the aqueous phase was precipitated with two volumes of ethanol a t - - 2 0 ° C , overnight. Extraction of RNA from the various ribonucleoprotein particles was carried out by first pooling the appropriate fractions and precipitating them with t w o volumes of ethanol at --20°C, overnight. After centrifugation of the latter fractions at 10 000 X g for 10 min, the pellets were suspended in 0.1 ml of buffer V to which dodecyl sulfate was added to a final concentration of 2%. Aliquots were finally analyzed by polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of RNA was performed according to Loening [15] using 2.4% gels prepared in buffer VI. Electrophoresis was usually for 2.5 or 3 h at 4°C with 10 mA/gel. The gels were analyzed using a Gilford Model 2410 Linear Transport System. Calculations were carried o u t using Loening's [16] linear relationship between the log of the molecular weight and the distance traveled in the gel. For experiments with labeled RNA, gels were prepared with 10% glycerol. Electrophoresis was carried o u t in buffer VI also containing 10% glycerol in order to facilitate slicing of the gels. The latter were cut in slices 1 mm thick using a Mickel gel slicer (The Mickel Laboratory Engineering Co.), and radioactivity was counted according to Cverenkov [ 17 ] in a Beckman LS-355 liquid scintillation system. Determination of base composition. Following the analysis of radioactivity on the intact gel, the radioactive bands were sliced from their indicated positions, and the RNA was hydrolyzed by incubating the gel slices in 0.3 M KOH at 37°C for 18 h. The gel was removed by centrifugation and nucleotides were

60 separated by thin-layer chromatography on precoated analytical TLC plastic sheets PE1-Cellulose F (Merck) using 1.0 M LiC1 as solvent [18]. The spots of separated nucleotides were cut from the sheet and counted as above. Results

RNA components o f the nucleolar fraction Electrophoresis patterns of RNA extracted from cytoplasmic ribosomes and from nucleoli are presented in Figs. l a and l b , respectively. The rRNA fractionated into two peaks with molecular weights of 1.35 • 106 and 0.85 • 106, respectively. The nucleolar RNA fraction, on the other hand, separated into four peaks corresponding to molecular weights of 3.5 • 106, 2.2 • 106, 1.35 • 10 s and 0.85 • 106. The latter two of these molecular weights correspond to those o f the mature ribosomal RNAs. The 3.5 • 106 peak most likely represents precursor ribosomal RNA judging from its molecular weight and its base composition (Table I). The peak at 2.2 • 106 was present in very small quantities, suggesting that it has a high turnover rate; it could be an intermediary product of the maturation of ribosomal RNA in the nucleolus. If the 1.35 • 106 and 0.85 • 106 fractions were formed in equal amounts, their absorbance ratio (260 nm) would be expected to be 1.6. A ratio close to this value was observed in the case of RNA extracted from ribosomes, but for the RNA obtained from nucleoli, a value of 0.6 was calculated. Ribonucleoprotein particles released by heparin in the presence of magnesium The sedimentation profiles of particles released by heparin in the presence of magnesium are shown in Fig. 2a. Four populations of particles were obtained sedimenting at 90 S, 75 S, 60 S and 44 S, respectively. The higher molecular weight peaks were lost in the presence of high concentrations of polyvinyl sulfate and the remaining ribonucleoprotein particles fractions sedimented at 51 S and 44 S. No sedimentation profile was obtained when polyvinyl sulfate was omitted from the extraction medium, all the ultraviolet-absorbing material and the radioactivity remaining at the top of the gradient as a result of degradation of the ribonucleoprotein particles. This last observation suggests that our nucleolar preparations were n o t contaminated by cytoplasmic ribosomes which, as judged by their sedimentation behaviour, remained intact during this procedure. RNA con ten t o f ribonucleopro rein particles The fractions obtained on sucrose gradients were collected and their c o n t e n t in RNA was analyzed by electrophoresis (Fig. 3). The 90 S region of the gradient contained RNA having a molecular weight of 3.5 • 10 s (Fig. 3a). The 75-S particles contained the 1.35 • 106 RNA with small amounts of 0.85 • 106 RNA (Fig. 3b), and the 44-S particles contained as major c o m p o n e n t the 0.85 • 106 RNA with only a small a m o u n t of 1.35 • 106 RNA (Fig. 3d). The 60-S particles contained both species of ribosomal RNA 1.35 • 106 and 0.85 • 106 (Fig. 3c). The 51-S and 44-S particles, obtained when nucleoli were exposed to high concentration of polyvinyl sulfate, yielded RNAs having molecular weights of 1.35 • 106 and 0.85 • 106, respectively. The high molecular weight

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F i g . 1. A n a l y s i s of R N A i s o l a t e d f r o m c y t o p l a s m i c r i b o s o m e s a n d f r o m n u c l e o l i in p o l y a c r y l a m i d e gels. Cells (2 • 1 0 6 / m l ) w e r e l a b e l e d f o r 30 r a i n w i t h 32p ( 4 0 ~ C i / m l ) in f r e s h m e d i u m d e p r i v e d o f p h o s p h a t e . T h e l a b e l e d cells w e r e w a s h e d a n d s o a k e d in t h e b u f f e r I a n d t h e n u c l e o l i w e r e i s o l a t e d . T h e n u c l e o l i w e r e s u s p e n d e d in b u f f e r V a n d R N A w a s e x t r a c t e d a t 4 ° C w i t h t h e d o d e c y l s u l f a t e - p h e n o l t r e a t m e n t . T h e n u c l e o l a r R N A w a s t h e n a n a l y z e d o n p o l y a c r y l a m i d e gel ( 2 . 4 % ) . E l e c t r o p h o r e s i s c o n d i t i o n : 3 h, 10 m A / gel, at 4 ° C in b u f f e r V1. 20 ~ g o f r R N A i s o l a t e d f r o m r i b o s o m e s as r e p o r t e d e a r l i e r [ 1 4 ] w e r e a d d e d as m a r k e r to t h e n u c l e o l a r e x t r a c t s . (a) r R N A o f r i b o s o m e s ( ). (b) R N A f r o m i s o l a t e d n u c l e o l i (o-------o) and rRNA marker ( ). F i g . 2. S e d i m e n t a t i o n p r o f i l e s o f r i b o n u c l e o p r o t e i n p a r t i c l e s e x t r a c t e d f r o m n u c l e o l i in T r i s / H C 1 / M g 2+ b u f f e r . Ceils at a c o n c e n t r a t i o n o f 2 • 106 c e i l s / m l w e r e i n c u b a t e d in a f r e s h m e d i u m d e p r i v e d o f p h o s p h a t e . L a b e l i n g w a s a c h i e v e d b y a d d i n g 4 0 btCi/ml o f 32p. T h e l a b e l e d cells w e r e s o a k e d in b u f f e r I a n d s u b s e q u e n t l y t h e n u c l e o l i w e r e i s o l a t e d . T h e n u c l e o l i w e r e s u s p e n d e d in 2 m l b u f f e r I I I , a n d b o t h h e p a r i n ( 0 . 5 % ) a n d D N A a s e ( 5 0 /~g/ml) w e r e a d d e d . T h e s o l u t i o n w a s c e n t r i f u g e d a t 27 0 0 0 X g f o r 10 r a i n a t 4 ° C . T h e s u p e r n a t a n t w a s t h e n l a y e r e d o n a 12 m i 1 5 - - 3 0 % ( w / w ) s u c r o s e g r a d i e n t in b u f f e r I I I c o n t a i n i n g 20 ~ g / m l o f p o l y v i n y l s u l f a t e a n d c e n t r i f u g e d at 41 5 0 0 r e v . / m i n in a B e c k m a n SW-41 r o t o r f o r 5 h at 4 ° C . F r a c t i o n s w e r e c o l l e c t e d a n d m o n i t o r e d a t 2 6 0 n m w i t h a G i l f o r d M o d e l 2 4 0 s p e c t r o p h o t o m e t e r . T h e r a d i o a c t i v i t y w a s c o u n t e d in a B e c k m a n L S - 3 5 5 l i q u i d s c i n t i l l a t i o n s y s t e m . S e d i m e n t a t i o n p r o f i l e s o f r i b o n u c l e o p r o t e i n p a r t i c l e s e x t r a c t e d a f t e r a l a b e l i n g p e r i o d o f (a) 30 r a i n , ( b ) 10 m i n , a n d (c) a 9 0 r a i n c h a s e . Cells w h i c h w e r e l a b e l e d f o r 10 rain w e r e s u b s e q u e n t l y c h a s e d f o r 9 0 r a i n in a c u l t u r e medium containing phosphate. , absorbance at 260 nm; o o, r a d i o a c t i v i t y ; . . . . . . , Escherichia coil r i b o s o m e s u s e d as r e f e r e n c c w e r e s e d i m e n t e d in s e p a r a t e g r a d i e n t s .

62 TABLE

I

NUCLEOTIDE

COMPOSITION

OF A. L O N G A

CYTOPLASMIC

RIBOSOMAL

RNA AND PRECURSOR

C y t o p l a s m i c r i b o s o m a l R N A w a s i s o l a t e d f r o m cells w h i c h h a d b e e n i n c u b a t e d in p h o s p h a t e - f r e e m e d i u m for 8 h with 1 pCi of 32p/ml. The RNA was then extracted from the ribosomes. Precursor RNA was e x t r a c t e d f r o m cell n u c l e o l i a f t e r 1 0 m i n p u l s e l a b e l i n g w i t h 4 0 p C i / m l o f 32p. R N A s w e r e l a y e r e d o n t o polyacrylamide gels ( 2 . 4 % ) . A f t e r e l e c t r o p h o r e s i s , the gels were sliced and the radioactive bands h y d r o l y z e d in 0 . 3 M K O H a t 3 7 ° C f o r 1 8 h. N u c l e o t i d e s w e r e s e p a r a t e d b y c h r o m a t o g r a p h y on PEIcellulose, and the radioactivity of the separated nuc]eotides was determined. The average values of three e x p e r i m e n t s w e r e e x p r e s s e d as t o o l % o f e a c h n u c l e o t i d e . Nucleotides

RNA

AMP GMP CMP UMP

3.5 - 106

1.35 - 106

0.85 • 106

20.9 31.5 26.6 21.0

21.9 29.8 25.2 23.1

22.5 30.1 26.1 21.3

± ± + +

0.5 0.6 0.8 0.9

± + ± 4

0.4 0.5 0.9 0.8

+- 0 . 6 ~ 0.4 :~ 0 . 8 4 0.9

R N A precursor (3.5 • 106), as well as its intermediary p r o d u c t (2.2 • 106) were n o t d e t e c t e d under this last experimental c o n d i t i o n in the s e d i m e n t a t i o n profile. The a m o u n t of r i b o n u c l e o p r o t e i n particles released from nucleoli by heparin

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F i g . 3. P o l y a c r y l a m i d e g e l a n a l y s i s o f R N A c o n t e n t o f r i b o n u c l e o P r o t e i n particles isolated from nucleoli. Fractions of a sucrose gradient corresponding to ribonucleoprotein peaks (Fig. 2a) obtained after a 30 min labeling period were precipitated with two volumes of ethanol and kept overnight at --20°C. The fractions were centrifuged at 10 000 X g for 10 rain at 4°C. The pellets were suspended in 0.1 ml of buffer V, sodium dodecyl sulfate was added to a final concentration of 2%. After a centrifugation at 10 000 × g for 10 rain at 4°C, aliquots of the supernatant were then layered on polyaerylamidc gels ( 2 . 4 % ) . 2 0 p g o f r R N A w a s a d d e d as m a r k e r t o e a c h f r a c t i o n . E l e c t r o p h o r e s i s c o n d i t i o n : 3 h, at 4°C, 10 m A / g e l , b u f f e r I V . a, R N A o f 9 0 - S p a r t i c l e s ( ~ ' ~);b, RNA of 75-Sparticles (~ ~);c, RNA of 6 0 - S p a r t i c l e s (() ~)); d , R N A o f 4 4 - S p a r t i c l e s ( o o); - - - - , rRNA marker.

63 magnesium was added to the extraction buffer. These particles formed a single broad peak in the 35--40 S region of the sucrose gradients. As shown by their electrophoretic pattern, the RNAs contained in these particles were extensively degraded and, as a result, the high molecular weight precursors, the 1.35 • 106 and the 0.85 • 106 RNAs, were n o t observed in the gradients.

Maturation o f ribosomal precursor R N A After a pulse labeling of 10 min, the radioactivity appeared in the RNA constituent of the 90-S particles as shown in Fig. 2b. The 3.5 • 106 RNA was rapidly labeled and following a chase of 90 min the radioactivity disappeared from this species, with the concomitant accumulation of label in mature ribosomal RNAs present in 75-S, 60-S, and 44-S ribonucleoprotein particles, Fig. 2c. The intermediate products of the maturation of the 3.5 • 106 precursor RNA were n o t detected in these experiments. It can be concluded t h a t the 90-8 particles contained the precursor RNA of the RNA species present in the 75-S, 60-S, and 44-S particles. Discussion Our studies show that the processing of A. longa ribosomal RNA (Figs. 2b and 2c) involves the transformation of a high molecular weight precursor (3.5 • 106) into the two 1.35 • 106 and 0.85 • 106 mature ribosomal species. In this respect, therefore, our results are identical to those obtained with E. gracilis [8]. Although our electrophoretic profiles reveal the presence of a very small peak corresponding to a molecular weight of 2.2 • 106, the various other minor fractions (2.7 • 106, 2.5 • 106 and 2.0 • 106) believed to be involved in the maturation of the larger ribosomal subunits of E. gracilis, were n o t observed. These results can be explained by the much greater instability of the last three intermediates which most likely were degraded during the longer isolation procedure used in the present study for purifying nucleoli and ribonucleoprotein particles. The purity of the nucleolar preparations [11] and the base composition of the high molecular weight RNA (3.5 • 106), which was similar to that of the mature ribosomal RNAs (Table I), exclude a possible contamination of this 90 S fraction by heterogeneous nucleoplasmic particles. In A. longa the m o n o m e r ribosomes have a sedimentation coefficient of 82 S and are constituted of 60-S and 44-S subunits containing, respectively, 1.35 • 106 RNA and 0.85 • 106 RNA. Since 44-S particles containing 0.85 • 106 RNA can be isolated from nucleolar preparations, it seems reasonable to assume that they correspond to the small subunits of mature ribosomes. The presence of 1.35 • 106 and 0.85 • 106 RNA in the 60-S peak, requires an explanation. Firstly the 60-S peak is contaminated by the 75-S, and 44-8 peaks. In addition the 60-S peak could correspond to an accumulation of particles containing only the 1.35 • 106 RNA and therefore represent the large subunits of mature ribosomes. This implicates the transformation of the 75-S particles into 60-S particles although no evidence of this process has y e t been observed. It should be noted that the 1.35 • 106 RNA is known to be considerably unstable [14] and the degradation of appreciable quantities of this RNA species is observed

64 during subsequent procedure necessary for analyse. This could explain the poor recovery of the 1.35 • 106 RNA species in these experiments (Fig. 3c). In mammalian cells, the low molecular weight ribosomal RNA migrates rapidly into the cytoplasm whereas the larger one is known to remain longer in the nucleolus for additional processing [1]. Thus, in order to gain further information as to why the yield from our nucleolar preparations of the 1.35 • 106 RNA was much lower than that of the 0.85 • 106 RNA species, nucleoli were pelleted and the respective radioactivities of these RNAs in the supern a t a n t ribosomes were then analyzed. The fact that the specific activity of the 1.35 • 106 RNA was found to be 3-fold higher than that of the 0.85 • 10 ~ species could indicate that the particles containing the 1.35 • 106 RNA were either rapidly exported into both the nucleoplasm and cytoplasm or were liberated into the supernatant fraction artifactually during the isolation of the nucleoli. Since the present study with A. longa and an earlier one with E. gracilis [20], show that present methods for isolating nuclei from these protozoa involve degradation of nuclear RNA, it is difficult at this time to verify which of these two interpretations is valid.

Acknowledgments We thank M.S. Gugg for this excellent technical assistance and are also grateful to Dr. A. Gagnon for the use of the Coulter Counter. This investigation was supported by research grants from the Quebec Ministry of Education and the National Research Council of Canada.

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