Studies on RNA metabolism in Acetabularia mediterranea

12

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96069

STUDIES ON RNA METABOLISM IN A C E T A B U L A R I A M E D I T E R R A N E A II. T H E LOCALIZATION AND STABILITY OF RNA SPECIES; T H E EFFECTS ON RNA METABOLISM OF DARK PERIODS AND ACTINOMYCIN D

F L O R E N C E E. F A R B E R *

Laboratoire de Morphologie Animale, Facultd des Sciences, Universitd Libre de Bruxelles, Brussels (Belgium) and International Laboratory o] Genetics and Biophysics, Naples (Italy) (Received J u l y 26th, 1968)

SUMMARY

I. Slowly labelled I5-S RNA of Acetabularia is synthesized in the chloroplasts. The predominant radioactive species of mitochondria and other cellular constituents is a 5-S type of RNA. 2. The exposure of plants to long dark periods markedly inhibits the incorporation of RNA precursors into chloroplast RNA. A new RNA species appears in sedimentation profiles following this treatment. 3. The labelling of very young plants results in the retention of the slowly labelled I5-S component after a 35-day chase. Sedimentation profiles of RNA isolated following a 58-day chase are polydisperse, and indicate that the I5-S peak has been split into several fractions. Plants possess caps at this stage. When plants at the same stage of development are labelled, the I5-S component is still the predominantly labelled species. 4- Actinomycin D chase experiments do not result in any changes in the relative proportions of labelled RNA species in whole algae or plant fragments. However, pretreatment with the antibiotic causes the suppression of all RNA fractions with exception of the 5-S component.

INTRODUCTION

In the first article of this series 1, a method has been described for isolating RNA from the unicellular alga Acetabularia mediterranea by precipitating plant homogenates with 2 M LiC1. The metabolic fate of various RNA species extracted by this procedure was investigated. Isotopic labelling with [3H]uridine for specific time intervals disclosed the existence of a rapidly labelled RNA component, which sediAbbreviations: m R N A , messenger RNA; t R N A , transfer RNA; rRNA, ribosomal RNA. * Present address: D e p a r t m e n t of Physiology-anatomy, University of California, Berkeley, Calif., U.S.A.

Biochim. Biophys. Acta, 174 (1969) 12-22

RNA

METABOLISM IN ACETABULARIA.

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mented at 5 S, and a I5-S species, which incorporated the radioactive precursor at a much slower rate. In this paper, the results of studies dealing with the localization and stability of these RNA species, and the effects on RNA metabolism of exposure to dark periods and actinomycin D are presented.

METHODS

Isolation o/RNA #ore sub-cellular #actions Conditions for the cultivation of Acetabularia have been reported elsewhereL Approx. 2000 plants (2.5 cm average length) were labelled with [3H]uridine for 48 h as previously described 1. The plants were then homogenized at o ° in 5 ml of o.oi M Tris-HC1 buffer (pH 7.4) containing I mg/ml of naphthalene disulfonate, i mM EDTA, and 0.54 M glucose. The mixture was filtered through bolting silk, and the filtrate was centrifuged at 800 rev./min for 2 min at 4 °. All subsequent operations were carried out at 4 °. The supernatant was recentrifuged at 3000 rev./min for IO min in order to sediment the chloroplast fraction. The resulting supernatant was further fractionated by centrifugation at 14 500 rev./min for io min. This step brought down the mitochondrial fraction and left the cytoplasmic fraction in the supernatant. I t should be noted that this method of differential centrifugation yields fractions which consist mainly of chloroplasts and mitochondria. But the possibility of cross contamination m a y not be excluded. The chloroplast and mitochondrial pellets were each suspended in 2 ml of o.oi M sodium acetate buffer (pH 5.0) containing I mg/ml naphthalene disulfonate, I mM EDTA, and 0.54 M glucose. An equal volume of 4 M LiC1 was added to the suspension, and it was allowed to stand for 16 h. Since Acetabularia contains very little cytoplasm in comparison to other cellular constituents, the cytoplasmic fraction was combined with the pellet obtained from the first centrifugation step. The mixture, which contained nuclear material, was brought to p H 5.0 with I M sodium acetate buffer (pH 5.o), and an equal volume of 4 M LiC1 was added as before. RNA was extracted from all fractions according to the method described previously 1.

Exposure o! plants to dark periods 3oo plants were placed in the dark for IO and 2o days, respectively. The algae were manipulated in the presence of a small light source supplied with a green filter (480 m/~). On the Ioth and 2oth day of the dark period, plants were labelled with [aH]uridine for 4 h. They were then homogenized together with 300 unlabelled carrier algae. The mixture was filtered through bolting silk, and chloroplasts were isolated as described above. The supernatant arising from sedimentation of the chloroplast fraction was combined with the pellet from the first centrifugation step, membranes, and other cellular material. This mixture was designated as "non-chloroplastic" material. RNA was extracted from chloroplasts and non-chloroplastic material b y the method described above. RNA was isolated from whole plant controls in the usual manner. Biochirn. Biophys. Acta, 174 (1969) 12-22

14

F . E . FARBER

Pulse-chase studies Young plants (2 m m average length) were labelled with ~*H]uridine for 48 h, and were then placed in non-radioactive complete medium 1 for periods of 35 and 58 days. During the first week after administration of the radioactive isotope, the medium was changed daily, and every 4 days, thereafter. RNA was isolated from 300 plants following 35- and 58-day intervals.

RNA extraction/rom algae at di//erent cap devdopmental stages For each developmental stage, 300 plants were labelled with IaH]urldine for 48 h. RNA was extracted in the usual manner from algae which possessed caps with average diameters of 2 and 5 mm, respectively.

Preparation o/Escherichia coli RNA The strain of E. coli B-B (a gift of Prof. R.

THOMAS) w a s grown in aqueous medium containing 1 % tryptone and 0.5 % NaC1. Exponentially-growing cells were harvested by centrifugation and washed twice with 0.9 % NaC1. Cells were suspended in o.I M Tris-HC1 buffer (pH 7.4) containing o.I M KCI and I mM EDTA. Deoxyribonuclease I (electrophoretically pure, EC 3.I.4.6; Worthington Biochemical Corp.) and lysozyme (EC 3.2.1.17; Worthington Biochemical Corp.) were added to the cell suspension in final concentrations of 15/~g/ml and 2oo/zg/ml, respectively. The cells were lysed by one freezing and thawing cycle. The mixture was then taken up in a Io-ml pipet and expelled several times. Aqueous phenol was added, and the mixture was shaken for 30 min at 4 °. All further purification steps were carried out at this temperature. Following centrifugation at IO ooo rev./min for IO min, the aqueous phase was re-extracted with phenol until protein was no longer visible at the interface. The final aqueous layer was made 1 % in NaC1, and 2 vols. of cold, abs. ethanol were added. Nucleic acids were allowed to precipitate overnight at --20 ° . The precipitate was collected by centrifugation at 3000 rev./min for IO min, and washed twice with ethanol. The sediment was dissolved in o.I M Tris-HC1 buffer (pH 7-4) containing 3 mM MgC12. Deoxyribonuclease was added to a final concentration of IOO/~g/ml, and the mixture was incubated for 30 min at 37 °. Deproteinization and subsequent precipitation of RNA were carried out as described earlier. RNA was further purified by three reprecipitation steps. Purified RNA was dissolved in o.oi M sodium acetate buffer (pH 5.0) containing o.I M KCI, divided into 2 ml portions, and stored at - - 4 °0 .

Actinomycin D experiments 25 whole algae (2 cm average length) were incubated for 24 h with 5o pC of E~H]uridine in 4 ml of complete medium. The cells were washed, and incubated for additional 7 or 48 h periods in 4 ml of complete medium containing IO, 20, or 40/~g/ml of actinomycin D (a gift of Merck, Sharp, and Dohme). In a reversal of this procedure, 25 plants were first exposed for 7 or 48 h to the antibiotic, and were then placed in radioactive medium for 24 h. 25 anucleate or nucleate plant fragments 1 were labelled with [SH]uridine for 24 h, and chased for 7 h with 20/~g/ml actinomycin. Another Biochim. Biophys. Acta, 174 (1969) 12-22

R N A METABOLISM IN ACETABULARIA. II

15

set of anucleate and nucleate fragments was first pretreated with actinomycin for 7 h, and the fragments were then transferred to radioactive medium for 2 4 h. Whole algae and plant fragments were homogenized in 2 ml of buffer, and R N A was extracted in the presence of 18 7 #g of unlabelled E. coli carrier RNA. RESULTS

RNA species o/ sub-cellular/factions The sedimentation pattern in Fig. I illustrates the fate of labelled RNA in subcellular fractions of Acetabularia. It may be seen that the slowly labelled I5-S component is found predominantly in the chloroplast fraction (Fig. Ia). In other cellular constituents, the major radioactive species is located in the 5-S region of sucrose gradients (Fig. Ib, c). The absorbance pattern of chloroplasts differs markedly from the profiles of other sub-cellular fractions. This difference is, once again, observable in the I5-S fraction. 0.5

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Fig. I. S e d i m e n t a t i o n profiles of R N A e x t r a c t e d from sub-cellular fractions of Acetabula~ria. (a) chloroplasts; (b) mitochondria; (c) c y t o p l a s m c o m b i n e d w i t h o t h e r cellular constituents. For details see METHODS. Q - Q , A25o my; O - - - O , radioactivity.

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Fig. 2. S e d i m e n t a t i o n profiles of R N A e x t r a c t e d from algae w h i c h have been subjected to long dark periods. On t h e i o t h and 2oth d a y of t h e dark period, p l a n t s were labelled with [SH]uridine for 4 h. (a) whole p l a n t s a n d a i o - d a y dark period; (b) non-chloroplastic material and a I o - d a y dark period; (c) chloroplasts and a i o - d a y dark period; (d) whole p l a n t s and a 2ooday dark period; (e) non-chloroplastic material and a 2o-day dark period; (f) chloroplasts and a 2o-day dark period. For det&ils see METHODS. O - - D , A260 m/~; ( ~ - - - ( ~ , radioactivity.

Biochim. Biophys. Hcta, 174 (1969) I2-22

R N A METABOLISM IN ACETABULARIA. II

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Exposure o/ plants to long dark periods Fig. 2 depicts the effects of long dark periods on whole plants and sub-cellular fractions. As might be expected, chloroplasts are affected most by this treatment. After a Io-day dark exposure and a 4-h label with [~H]uridine, the 5-S component in chloroplasts (Fig. 2c) was 50 % of the 5-S fraction in whole plants (Fig. 2a). Following a 2o-day dark period, the 5-S RNA of chloroplasts (Fig. 2f) was reduced to 70 % of the control value (Fig. 2d). A striking feature of whole plant and non-chloroplastic RNA absorbance profiles was the disparity between the 24-S peak and that of the nearest radioactive species (Fig. 2a, b, d, e). This difference in coincidence of absorbance and radioactivity profiles was not found in chloroplast fractions. F i

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Fig. 4. S e d i m e n t a t i o n profiles of R N A e x t r a c t e d from algae t r e a t e d witli a c t i n o m y c i n D. (a) p l a n t s were labelled for 24 h, and were t h e n placed in m e d i u m containing a c t i n o m y c i n (io/~g/ ml) for 7 or 48 tl; (b) p l a n t s were p r e t r e a t e d w i t h a c t i n o m y c i n (io Hg/ml) for 7 or 48 h, and were t h e n labelled for 24 11; (c) algae were labelled for 24 Ix, and were t h e n placed in m e d i u m containing a c t i n o m y c i n (20 or 4 ° Hg/ml) for 7 or 48 li; (d) p l a n t s were p r e t r e a t e d witli a c t i n o m y c i n (20 or 4o/zg/ml) for 7 or 48 li, and were t h e n labelled for 24 li. For details, see METHODS. 0 - - 0 , A26o mtJ of E. coli carrier R N A ; O - - - O , radioactivity.

Pulse-chase studies

When young plants (2 mm average length) were incubated for 48 h in the presence of [SH]uridine, and then transferred to non-radioactive medium for long chase periods, the I5-S component was the major radioactive peak after a 35-day chase (Fig. 3a). Moreover, there was evidence of RNA heterogeneity in the heavy region of the gradient. Following a chase of 58 days, the radioactive peaks were more heterogeneous, exhibiting a polydisperse sedimentation profile (Fig. 3b). The 5-S peak remained proportionally unchanged, while the I5-S peak appeared to have been split into several fractions. Algae at this stage of development possessed caps with average diameters of 2 mm. Whole plants at the same developmental stage, as well Biochim. Biophys. Acta, 174 (1969) 12-22

RNA METABOLISMIN ACETABULARIA. II [

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Fig. 5. Sedimentation profiles of RNA extracted from plant fragments treated with actinomycin D (20/zg/ml). (a) anucleate algae were labelled for 24 h, and were then placed in medium containing actinomycin for 7 h; (b) anucleate fragments were pretreated with actinomycin for 7 h, and were then labelled for 24 h; (c) nucleate cells were labelled for 24 h, and were then placed in medium containing actinomycin for 7 h; (d) nucleate fragments were pretreated with actinomycin for 7 h, and were then labelled for 24 h. For details, see METHODS. O - O , -426o mr* of E. coli carrier RNA; O- - - O , radioactivity.

as algae at more advanced stages (average cap diameter, 5 mm), were subjected to a long label: radioactivity patterns were identical for either stage of development, and the I5-S component was the predominant radioactive species. Heterogeneity was observed, as before, in the heavy region of the gradients. Absorbance profiles at all cap developmental stages indicated the disappearance of the 24-S peak and a reduction of the I5-S component (Fig. 3c). Studies with actinomycin D Fig. 4a depicts the radioactivity pattern of plants which were labelled for 24 h, and then placed in non-radioactive medium containing Io/,g/ml actinomycin. The Biochim. Biophys. Acta, z74 (i969) z2-22

20

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I5-S region contained the predominant radioactive peak. If plants were first placed in actinomycin for 7 or 48 h, and then labelled for 24 h in the absence of the antibiotic (Fig. 4b), the I5-S peak was reduced by 5 ° ~o. Fig. 4 c illustrates that a chase with 20/~g/ml actinomycin still does not change the proportions of the 15- and 5-S radioactive peaks. However, it may be seen that this actinomycin concentration suppresses 80 ~o of the I5-S fraction in plants which have been pretreated with the antibiotic (Fig. 4d). Actinomycin concentrations of 40/~g/ml afforded similar radioactivity profiles. An actinomycin chase (20/Jg/ml) did not result in any proportional differences between radioactive peaks of anucleate fragments (Fig. 5a). Pretreatment of anucleate halves with actinomycin caused the same inhibition as before (Fig. 5b c]. Fig. 4d). The situation was quite different in the case of nucleate fragments (Fig. 5c): the heights of the 15- and 5-S ~H-labelled peaks were comparable following an actinomycin chase. Pretreatment with the antibiotic (Fig. 5d) caused the same inhibition of the I5-S peak observed in anucleate fragments. It is unlikely that the reduction of the I5-S peak in nucleate radioactivity patterns can be ascribed to the effect of actinomycin. Nucleate fragments contain less chloroplastic material compared to anucleate halves. Since chloroplasts are the cytoplasmic organelles which account for major amounts of the I5-S peak, their absence could result in the diminution of the I5-S component in nucleate fragments. Since the relative amounts of the 23- and I6-S species of E. coli remain unaltered during the isolation of Acetabularia RNA, the LiC1 method appears to be an effective means of arresting enzymatic degradation of RNA. It is interesting to note that 4-S E. coli RNA is not precipitated by LiC1.

DISCUSSION Sub-cellular fractionation of Acetabularia homogenates, and subsequent extraction and sedimentation of RNA species from each fraction, establish that the synthesis of the slowly labelled I5-S component takes place in the chloroplasts. The predominant radioactive species of mitochondria and other cellular constituents, under these conditions, is a 5-S type of RNA. A comparison of the absorbance profiles of sub-cellular fractions reveals that chloroplasts contain a major portion of bulk, I5-S RNA. This could explain the diminution of the I5-S fraction in nucleate fragments which has been observed before1, since these halves contain fewer chloroplasts. Following long dark periods, the incorporation of [~Hluridine into RNA is inhibited primarily in the chloroplast fraction. These results are in keeping with studies made on isolated chloroplasts s. A new type of labelled RNA appears to be synthesized during prolonged dark exposures. This RNA sediments between the 24- and I5-S absorbance peaks. It is found in radioactivity profiles of whole plants and nonchloroplastic material, but not in chloroplasts themselves. Barring degradation, this RNA species might be one that diffuses out of the chloroplasts during dark intervals. Conversely, under conditions of reduced energy production, an abnormal RNA could be synthesized by another sub-cellular fraction of Acetabularia. Plants at early developmental stages were labelled, and the isotope was then chased for 35 and 58 days: after 35 days, the I5-S radioactive RNA species was still Biochim. Biophys. Acta, 174 (1969) 12-22

RNA METABOLISMIN ACETABULARIA.I I

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the predominant fraction; following a 58-day chase, however, the I5-S peak appeared to have been split into several fractions. The breakdown of the I5-S component could, in some way, be correlated with cap formation. Absorbance profiles reveal the disappearance of the 24-S peak and a reduction of the I5-S fraction in algae at the cap stage of development. The behavior of bulk RNA would tend to support the theory of a depletion of certain RNA molecules once stalk elongation has ceased and cap differentiation has commenced. However, in long chase experiments, catabolic changes unrelated to cap formation might account for the fate of the tritiated I5-S fraction. A 48-h label of plants at the same developmental stage as those following a 58-day chase indicated that the I5-S component was still the major radioactive species. The difference in the amounts of I5-S RNA obtained from either type of experiment could be explained if, following administration of EaHJuridine to plants at late developmental stages, only newly synthesized RNA is labelled. Some stable RNA species necessary for stalk and cap formation may have been synthesized long before and, therefore, do not incorporate the isotope. In the latter studies, any disappearance of the I5-S RNA species is likely to be masked by newly synthesized I5-S RNA, which is associated with the chloroplast fraction. Actinomycin chase experiments did not result in any changes in the relative proportions of slowly labelled Acetabularia RNA species in whole algae or plant fragments. But pretreatment with actinomycin caused the suppression of all RNA species, with exception of the 5-S component. Transfer RNA is relatively resistant to actinomycin 4,5. This suggests that the 5-S RNA fraction isolated from Acetabularia may be tRNA, and it should be tested by measuring acceptor activity. It is known that low concentrations of actinomycin preferentially inhibit the synthesis of mammalian rRNA, while permitting the synthesis of mRNA n-s. Since the I5-S component of Acetabularia is probably ribosomal in nature 9, actinomycin could have interfered with rRNA production in the present studies. The salient feature of all radioactivity profiles following pretreatment with the antibiotic was an augmentation of the 5-S fraction. In the absence of RNA breakdown, there might be an accumulation of some form of low molecular weight RNA. Morphological studies 1°-12 have demonstrated that growth and cap formation are almost completely inhibited by actinomycin in nucleate fragments. Anucleate halves, on the other hand, are much less susceptible to the antibiotic. Actinomycin does not destroy pre-existing RNA molecules. Therefore, morphogenetic substances which have accumulated in anucleate cytoplasm should not be affected by the antibiotic. In the present studies, the effects of actinomycin are discernible in whole algae and plant fragments within a matter of hours following administration of the antibiotic. Morphological changes with comparable doses of actinomycin occur after much longer periods of treatment: 5-46 days. BRACHET,DEz~-ISAND DE VITRY1°observed that caps formed by anucleate fragments were smaller than in the controls and were often abnormal. It was suggested that this effect of actinomycin might be the result of its combination with chloroplastic DNA. Actinomycin inhibits the incorporation of radioactive precursors into RNA of isolated chloroplasts a. Since chloroplasts are the main site of the slowly labelled I5-S component, its rapid disappearance in these experiments lends further support to a secondary effect of the antibiotic. Although the precise nature of Acetabularia RNA species continues to remain obscure, the current investigation has yielded more information about the site, staBiochim. Biophys. Acta, 174 (1969) 12-22

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b i l i t y , a n d b e h a v i o r of t h e s e R N A c o m p o n e n t s u n d e r c o n d i t i o n s w h e r e n o r m a l cellular metabolism has been altered.

ACKNOWLEDGEMENTS This investigation was supported by Euratom (contract oo7.61.1o ABIB). The a u t h o r is a p o s t d o c t o r a l f e l l o w of t h e N a t i o n a l S c i e n c e F o u n d a t i o n .

REFERENCES F. E. FARBER, Biochim. Biophys, Acta, 174 (1969) I. L. LATEUR, Rev. Algologique, i (1963) 26. S. BERGER, Protoplasma, 64 (I967) 13. M. REVEL AND H. H. HIATT, Biochem. Biophys. Res. Commun., 17 (1964) 73 o. Y. MouLg AND R. M. LANDIN, Biochem. Biophys. Res. Commun., 20 (1965) 491. R. P. PERRY, Proc. Natl. Acad. Sci. U.S., 48 (1962) 2179. R. P. PERRY, P. R. SRINIVASAN AND D. E. KELLEY, Science, 145 (1964) 504 . W. K. ROBERTS AND J. F. E. NEWMAN, J. Mol. Biol., 20 (1966) 63. S. BONOTTO, 1V[. JANOWSKI AND M~. BOLOUKH~RE, Biochim. Biophys. Acta, submitted for publication. io J. BRACHET, H. DENIS AND F. DE VITRY, Develop. Biol., 9 (1964) 398. i i K. ZETSCHE, Naturwissenscha]ten, 51 (1964) 18. I2 K. ZETSCHE, Z. Natur]orsch., 19 B (1964) 751. i 2 3 4 5 6 7 8 9

Biochim. Biophys. Acta, 174 (1969) 12-22