Eur. J. Biochem. 56, 117-122 (1975)
Rapidly Labelled RNA in Tetrahymena pyriformis Claudina R. POUSADA, Lise MARCAUD, Marie-Madeleine PORTIER, and Dona1 H. HAYES Institut de Biologie Physico-Chimique, Fondation Edmond de Rothschild, Paris (Received February 28/April 15, 1975)
39-54 29-S and 18-S nuclear rRNA precursors of Tetrahymena pyriformis have been characterized by acrylamide gel electrophoresis. Evidence is presented for the presence of high-molecular-weight non-methylated RNA species (28 - 39 S, M , range 1.9 - 2.7 x lo6) in the cytoplasm of this protozoa.
The detailed sequence of events in ribosomal RNA maturation in mammalian cells is now well established [l]. The largest precursor species observed in most studies of RNA biosynthesis in mammalian cells has a molecular weight of 4.1 x lo6 (45 S) but the existence of a family of still larger precursors with slightly higher molecular weights (46 S, 47 S) has been reported in studies of L5178 Y cells [2]. Precursor species of smaller size have been detected in other eukaryotic cells, e.g. 40-S precursor-rRNA in Xenopus laevis [3]. By utilizing an electrophoretic analysis of nucleolar RNAs, Weinberg and Penman [4] found that the 45-S precursor is cleaved to a 41-S molecule, which in turn is split into 32-S and 20-S RNA fragments. The 32-S molecule then matures into 28-S rRNA and the 20-S molecule matures into 18-S rRNA. However, in the unicellular eukaryote, Tetrahymena pyriformis, the pathway leading to the mature ribosomal RNAs is not yet very well known. Besides, the results published by several authors are not in agreement with each other. By sucrose gradient analysis of extracts of cells labelled with [Me-3H]methionine and [3H]uridine, Leick [5] and Kumar [6] respectively have detected the presence of precursor rRNAs sedimenting at about 45 S and 32 S and at about 35 S. In agreement with Kumar, Prescott and coworkers [7] have estimated a sedimentation coefficient of about 35 S for precursor rRNA utilizing the same analysis technique. These authors also report an intermediate precursor rRNA species with a sedimentation coefficient of about 30 S but their studies do not lead to a clear definition of multiple steps in the maturation process. In this paper we present additional information about the pattern of ribosomal RNA biosynthesis based on electrophoretic analysis of RNA extracted from cells labelled with [Me-3H]methionine and Eur. J. Biochem. 56 (1975)
[14C]uracil. Our results show that the mechanism of maturation is much less complex in Tetrahymena pyriformis than in higher metazoan cells. We also observe the presence of several kinds of non-methykdted RNA, in the nucleus as well as in the cytoplasm; in particular, we demonstrate the presence of a highmolecular-weight non-methylated RNA (39 s, 2.7 x lo6) in the nucleus which is transferred to the cytoplasm without significant reduction in size. However, our analytical procedures do not exclude the possibility that this transfer is accompanied by a small change in molecular weight. Evidence for the presence of giant RNA in the cytoplasm of several types of cell has also been presented by other authors [8 - 101.
MATERIALS AND METHODS Culture Conditions Cultures of Tetrahymena pyriformis GL were grown aerobically at 28 "C in a medium containing 0.075 % protease peptone, 0.075 % yeast extract, 0.15% glucose, 0.1 mM MgSO,, 5 pM CaCl,, 10 pM ferric citrate, 45 mM NaC1, 0.9 mM MgCI,, 4.5 mM sodium phosphate pH 6.8. This medium is limiting for growth (doubling time, 5.5 h, as compared to 3 h in a rich medium) but is used in order to reduce isotope dilution as far as possible. For incorporation studies, the cells were labelled in late log phase at a density of 1.5 x lo5 cell/ml. Labelling Cells were labelled with [3H]uridine or with a mixture of [14C]uracil and [Me-3H]methionine and were collected by centrifugation. RNA was then
118
extracted either from the whole cells or from the cell fractions. Cell Fractionation After different times of labelling, growth was quickly stopped by addition of iced medium, and the cells were collected by centrifugation at low speed (3000 x g for 5 min) and washed twice in 10 mM TrisHCl pH 7.5, 3 mM CaCl,, 1 mM MgCl,, and 0.25 M sucrose. After washing, the cells were resuspended in the same buffer, and lysed using the non-ionic detergent, nonidet P40 (0.23%) which leaves the nuclei intact [I 11. The lysate was immediately centrifuged at 10000 x g for 5 min and the supernatant was recentrifuged under the same conditions and used as the cytoplasmic fraction. The pellet was resuspended in the same buffered medium and recentrifuged at 3000 x g for 10 min; this operation was repeated once more and the recovered nuclei were used for RNA extraction, after resuspension in 10 mM Tris-HC1 pH 7.5. R N A Extraction
RNA was extracted from whole cells, from the cytoplasmic fractions and from isolated nuclei by the dodecylsulfate/phenol procedure and further deproteinised by treatment with isoamyl alcohol/chloroform [12]. After separation of the phases by centrifugation, RNA was precipitated from the aqueous phase, in the presence of 0.2 M NaCl and 2 vol. of ethanol. The precipitation was performed overnight at -20 "C; RNA was then spun down at 10000 x g for 30 min and when DNA was present the pellet was dissolved in 10 mM Tris-HC1 pH 7.5 containing 5 mM MgCl, and treated with DNase (30 pg of RNase-free DNase per ml, 10 min, 0 "C). Deproteinisation with phenol and RNA precipitation was then repeated as described above and the final RNA pellet was washed once with 95% ethanol and dissolved in 10 mM Tris-HC1 pH 7.5. Electrophoretic Analysis Acrylamide gels (2.7 %) were prepared in a buffer containing 0.040 M Tris-HC1, 0.020 M sodium acetate, 2 mM EDTA, 10% glycerol adjusted to pH 7.8 with acetic acid and polymerised with 0.1 %, tetramethylethylenediamine and 1 %, ammonium persulfate [13]. Electrophoresis was carried out for 6 h with a current of 7 mA per gel, in the cold room. After completion of electrophoresis the gels were frozen and cut into 2-mm slices. Each slice was eluted overnight at 62 "C with 1 ml of 0.6 M NaCl, 0.06 M
Rapidly Labelled RNA in Tetrahymena pyriformis
sodium citrate pH 7.2 [14]. The ultraviolet absorbance of the eluates was measured in order to determine the positions of 17-S and 25-S rRNA and 0.5 ml of each fraction was deposited in a glass filter, dried and counted in a scintillation fluid containing 4 g PPO and 100mg POPOP per 1 of toluene. Radioactivity was then counted in a Intertechnique SL-30 liquid scintillation spectrometer. When double labelling was performed corrections for the incomplete separation of 14C and 3H spectra were applied.
RESULTS The results of electrophoretic analysis of RNA isolated from whole cells labelled with [3H]uridine for 10 min are shown in Fig. 1. Several peaks can be seen, whose sedimentation coefficients by reference to mature 25-S and 1 7 3 rRNA are 39 S, 29 S, 23 S, and 13 S. These sedimentation coefficients were estimated from log M , versus relative mobility plots, constructed for the well-characterised ribosomal RNA markers [15]. It is worth noting that the 39-S and 29-S peaks are broad, suggesting that these species are not homogeneous and may contain heterodisperse RNA. To determine which of the RNA species shown in Fig. 1 were intermediates in rRNA maturation, we performed several experiments in which we labelled a culture of Tetrahymena pyriformis simultaneously for 10 rnin with [14C]uraciland [Me-3H]methionine. The results of a typical double-labelling experiment are presented in Fig. 2 which shows the presence of three main peaks of methylated RNA, corresponding to sedimentation coefficients of 39 S , 29 S and 17 S and reveals that many of the species present in the rapidly labelled RNA are not methylated, e.g. peaks at 33 S, 23 S , 18 S and polydisperse components with sedimentation coefficients lower than 17 S and higher than 39 S. These results have proved to be reproducible in a series of experiments. In further experiments the two main cellular compartments (nucleus and cytoplasm) were studied separately. Fig. 3 shows the electrophoretic separation of nuclear RNA isolated from cells labelled for 2, 5, 10 and 20 min. After 2-min labelling (Fig. 3A) only the 39-S ribosomal RNA precursor molecule and all the classes of non-methylated RNA, including species heavier than 39 S are seen. The rapidly sedimenting non-methylated RNA is comparable in size to the main bulk of rapidly labelled heterogeneous nuclear RNA in higher metazoans [16]. After 5 rnin of incorporation (Fig. 3 B) the same general pattern is seen but with an increased amount of RNA sedimenting faster than 39 s, and with distinct peaks at 29 S, and 17 S of which the former is a precursor of 25-S Eur. J . Biochem. 56 (1975)
119
C. R. Pousada, L. Marcaud, M.-M. Portier, and D. H. Hayes
39 s
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Fig. 1. Electrophoresis of whole cell RNA in 2.7% acrylamide gels. A culture of cells was labelled for 10 min with [3H]uridine (10 pCi/ ml; spec. act. 10 mCi/mmol) and total RNA was then extracted and
39 s
h ,
I
analysed as described in Materials and Methods. The gel was loaded with 550 pgofRNA(1000counts3Hmin-' pg-'). ( 0 d ) in gel slice eluates (mature rRNA) 3H in gel slice; (----)
25 S
1
6000
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E
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-s I 0
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Fig. 2. Electrophoresis of total R N A prepared from doubly labelled cells. A culture of cells was labelled for 10 min with a mixture of ~-[Me-~H]methionine (100 pCi/ml; spec. act. 4.5 Ci/mmol) and ['4C]uracil (4 pCi/ml; spec. act. 42 mCi/mmol) and total RNA was
extracted and analysed as in Fig. 1. The gel was loaded with 250 pg of RNA (1000 counts 3H and 50 counts 14Cmin-' pg-I); (M) 3H in gel slice; (O---o) 14C in gel slice. Arrows indicate the positions of mature 2 5 3 and 1 7 3 rRNA
rRNA. In some experiments the 17-S peak shows a slightly lower electrophoretic mobility than the marker of mature 17-S rRNA, suggesting that it corresponds to a precursor of this RNA. Further analysis will,
however, be needed to determine whether this rapidly labelled RNA has the same primary structure as the mature 17-S RNA. No other kinds of methylated RNA have been found even after labelling periods of
Eur. J. Biochem. 56 (1975)
120
Rapidly Labelled RNA in Tctrahymenu pyriformis
'4c
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- 2000
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Fig. 3. Eieectroplioreiic anulysis of nucleur RNA iubrlled f o r 2, 5, 10 und 20 rnin. h 60-in1 cell culture was incubated with a mixture of ['4C]uracil (8 pCi/ml; spec. act. 53.3 mCi/mmol) and L - [ M ~ - ~ H ] methionine (83 pCi/InI; spec. act. 7.3 Cijmmol); after (A) 2, (B) 5, (C) 10, and ( D ) 20 min incorporation, samples (30, 15, 8 and 5 ml respectively) were taken, chilled and mixed at 0 "C with varying volumes (20. 35, 42 and 45 ml respectively) of a parallel unlahelled culture in order to produce mixed samples containing approximately
equal quantities of cells. Cells were then collected and nuclcar and cytoplasmic RNA fractions were prepared as described in Materials and Methods. Electrophoretic analysis was carried out as described in the legend to Fig. 1 and 2. Gels were loaded with the whole of 'H; (0 -0) ''C. each preparation of nuclear RNA. (O---o) (Radioactivity in each sample has been normalised to 60 ml of labelled cells.) Arrows indicate the positions of mature rRNAs
10 or 20 min (Fig. 3, C and D). RNAs isolated after these longer periods of incorporation give the same profiles for the non-methylated RNA species as RNA labelled for 2 or 5 min although the proportion of heavy RNA ( > 39 S) is decreased. The same electrophoretic analysis was carried out for the KNA isolated from the cytoplasmic fractions. After 2-min labelling incorporation of isotopes into cytoplasmic RNA is low (Fig. 4A) but the profile of methylated RNA contains a distinct 17-S peak. Several small peaks of non-methylated RNA can also be seen. Leick [51] has also observed that the first ribosomal RNA exported to the cytoplasm is 17-S RNA. However, after 5-min labelling a small peak of 25-S rRNA as well as a very-well-defined peak of 17-S RNA are observed. The 2 5 3 component becomes
more important at longer incorporation times (Fig. 4, C and D). Several classes of heavy non-methylated RNA with sedimentation coefficients of 39 S, 35 S, 23 S and 15 S (Fig. 4B, C, D) appear clearly in cytoplasmic RNA after 10 and 20-min labelling times.
~
DISCUSSION We have studied ribosomal RNA processing in Tetrahymena pyriformis by kdbelling exponentidly growing cells with [Me-3H]methionine and [14C]uracil. Only two kinds of high-molecular-weight rRNA precursors (39-S and 29-S) are revealed by our experiments. A rapidly labelled methylated 17-S RNA which we observe in both nuclear and total RNA Eur. J . Hiochem. 56 (1975)
C. R. Pousada, L. Marcaud, M.-M. Portier, and D. H. Hayes
preparations may be a precursor form of mature 17-S rRNA but our analytical procedures are not powerful enough to establish this conclusion. Using acrylamide gel electrophoresis instead of sucrose gradient sedimentation we have not observed the existence of RNA species intermediate between these precursors and the mature rRNAs. Our results therefore indicate a much less complex mechanism of maturation of rRNA in Tetrahymena pyrformis than that which has been found in mammalian cells. The pathway in Tetrahymena pyrformis seems in fact to resemble the mechanism observed in E. coli (30-S precursor RNA + 25-S 17-S + 23-S 16-S RNAs [17,18]. We have also observed the existence of heavy non-methylated RNA with a sedimentation coefficient of 35-45 S and in particular peaks sedimenting at 35 S and 39 S in cytoplasmic RNA prepared after 10-min and 20-min pulse labelling. Several authors have demonstrated the existence of giant RNA in the cytoplasm of eukaryotic cells. Daneholt
+
Eur. J. Biochem. 56 (1975)
+
121
and Hosick [8] reported the existence of 75-S RNA in the cytoplasm of salivary gland cells of Chironomus tentans, Suzuki and Brown [19] found that the cytoplasmic messenger RNA for silk fibroin sedimented at 45-65 S and Guidice [9] found a giant RNA of nuclear origin in the cytoplasm of sea urchin embryos. Several observations show that the 35- 39-S species of non-methylated cytoplasmic RNA which we observe is probably not derived from artefactual nuclear leakage during the cell fractionation. If this were the case, we should expect to observe leakage of the large (39-S) methylated rRNA precursor molecule into the cytoplasm also and this does not occur. Furthermore nuclear leakage would be expected to be dependent on the method of preparation of nuclei and not on the duration of pulse labelling and it can be seen by inspection of Fig. 3 and 4 that the 39-S species are present in the nucleus at all labelling times but in the cytoplasm only after long (> 5 min) labelling periods. Finally the method of cell fractionation
122
C. R. Pousada, L. Marcaud, M.-M. Portier, and D. H. Hayes: Rapidly Labelled RNA in Teirahymena pyriformis
we use is very mild and has been reported to yield intact macronuclei from T. pyriformis [ll]. At present, we have no information concerning the cytoplasmic location or function of the 35- 39-S non-methylated RNA species. Experiments are in progress which are aimed at further characterization ofthis RNA and investigation of its transport, processing and function in the cytoplasm. This work has been supported by grants to D. H. Hayes from the Centre National de la Recherche Scientifique (Equipe de Recherche no 101 ; A T P d@renciaiion cellulaire, contract number 419905) the Fonds de la Recherche Mtdicale Francaise, and the Commissariat a I’Energie Atomique. One of us, C.R.P., thanks the French Ministry of Foreign affairs for a fellowship (1973- 1974).
REFERENCES 1. Burdon, R. H. (1971) Progr. Nucleic Acid Res. 11, 33-79. 2. Tiollais, P.. Galibert, F. & Boiron, M. (1971) Proc. Nail Acad. Sci. U . S . A .68, 1117-1120. 3. Loening, LJ. E., Jones, K. W. & Birnstiel, M. L. (1969) J . Mol. Biol. 45, 353 - 366.
4. Weinberg, R. A. & Penman, S. (1970) J . Mol. Biol. 47, 169178. 5. Leick, V. (1969) Eur. J . Biochem. 8, 221-228. 6. Kumar, A. (1970) J . Cell B i d . 45, 623-634. 7. Prescott, D. M., Bostock, C., Gamow, E. & Lauth, M. (1971) Exp. Cell Res. 67, 124- 128. 8. Daneholt, B. & Hosick, H. (1973) Proc. Nail Acad. Sci. U . S . A . 70,442 - 446. 9. Giudice, G., Sconzo, G., Ramirez, F. & Albanese, I. (1972) Biochim. Biophys. Acia, 262, 401 -403. 10. Kumar, A. & Lindberg, U. (1972) Proc. Natl Acad. Sci. U.S.A. 69,681 -685. 11. Mita, T., Shiomi, H. & Iwai, K. (1966) Exp. Cell Res. 43, 696 - 698. 12. Imamoto, F., Morikawa, N., Sato, K., Mishima, S., Nishimura, T. & Matsushiro, A. (1965) J . Mol. Biol. 13,157- 168. 13. Weinberg, R. A,, Loening, U . E., Willems, M. & Penman, S. (1967) Proc. Nail Acad. Sci. U.S.A. 58, 1088-1095. 14. Marcaud, L., Portier, M. M., Kourilsky, P., Barrel, B. G. & Gros, F. (1971) J. Mol. Biol. 57, 247-261. 15. Kurland, C. G. (1960) J . Mol. Biol. 2, 83-91. 16. Darnell, J. E. (1968) Bacieriol. Rev. 32, 262-290. 17. Nikolaev, N., Silengo, L. & Schlessinger, D. (1973) Proc. Nail Acad. Sci. U . S . A . 70, 3361-3365. 18. Dunn, J. J. & Studier, F. W. (1973) Proc. Natl Acad. Sci. U . S . A . 70,3296-3300. 19. Suzuki, Y . & Brown, D. D. (1972) J . Mol. Biol. 63,409-429.
C. R. Pousada, L. Marcaud, M.-M. Portier, and D. H. Hayes, Institut de Biologie Physico-Chimique, 13 Rue Pierre-et-Marie-Curie, F-75005 Paris, France
Eur. J. Biochem. 56 (1975)
Rapidly Labelled RNA in Tetrahymena pyriformis Claudina R. POUSADA, Lise MARCAUD, Marie-Madeleine PORTIER, and Dona1 H. HAYES Institut de Biologie Physico-Chimique, Fondation Edmond de Rothschild, Paris (Received February 28/April 15, 1975)
39-54 29-S and 18-S nuclear rRNA precursors of Tetrahymena pyriformis have been characterized by acrylamide gel electrophoresis. Evidence is presented for the presence of high-molecular-weight non-methylated RNA species (28 - 39 S, M , range 1.9 - 2.7 x lo6) in the cytoplasm of this protozoa.
The detailed sequence of events in ribosomal RNA maturation in mammalian cells is now well established [l]. The largest precursor species observed in most studies of RNA biosynthesis in mammalian cells has a molecular weight of 4.1 x lo6 (45 S) but the existence of a family of still larger precursors with slightly higher molecular weights (46 S, 47 S) has been reported in studies of L5178 Y cells [2]. Precursor species of smaller size have been detected in other eukaryotic cells, e.g. 40-S precursor-rRNA in Xenopus laevis [3]. By utilizing an electrophoretic analysis of nucleolar RNAs, Weinberg and Penman [4] found that the 45-S precursor is cleaved to a 41-S molecule, which in turn is split into 32-S and 20-S RNA fragments. The 32-S molecule then matures into 28-S rRNA and the 20-S molecule matures into 18-S rRNA. However, in the unicellular eukaryote, Tetrahymena pyriformis, the pathway leading to the mature ribosomal RNAs is not yet very well known. Besides, the results published by several authors are not in agreement with each other. By sucrose gradient analysis of extracts of cells labelled with [Me-3H]methionine and [3H]uridine, Leick [5] and Kumar [6] respectively have detected the presence of precursor rRNAs sedimenting at about 45 S and 32 S and at about 35 S. In agreement with Kumar, Prescott and coworkers [7] have estimated a sedimentation coefficient of about 35 S for precursor rRNA utilizing the same analysis technique. These authors also report an intermediate precursor rRNA species with a sedimentation coefficient of about 30 S but their studies do not lead to a clear definition of multiple steps in the maturation process. In this paper we present additional information about the pattern of ribosomal RNA biosynthesis based on electrophoretic analysis of RNA extracted from cells labelled with [Me-3H]methionine and Eur. J. Biochem. 56 (1975)
[14C]uracil. Our results show that the mechanism of maturation is much less complex in Tetrahymena pyriformis than in higher metazoan cells. We also observe the presence of several kinds of non-methykdted RNA, in the nucleus as well as in the cytoplasm; in particular, we demonstrate the presence of a highmolecular-weight non-methylated RNA (39 s, 2.7 x lo6) in the nucleus which is transferred to the cytoplasm without significant reduction in size. However, our analytical procedures do not exclude the possibility that this transfer is accompanied by a small change in molecular weight. Evidence for the presence of giant RNA in the cytoplasm of several types of cell has also been presented by other authors [8 - 101.
MATERIALS AND METHODS Culture Conditions Cultures of Tetrahymena pyriformis GL were grown aerobically at 28 "C in a medium containing 0.075 % protease peptone, 0.075 % yeast extract, 0.15% glucose, 0.1 mM MgSO,, 5 pM CaCl,, 10 pM ferric citrate, 45 mM NaC1, 0.9 mM MgCI,, 4.5 mM sodium phosphate pH 6.8. This medium is limiting for growth (doubling time, 5.5 h, as compared to 3 h in a rich medium) but is used in order to reduce isotope dilution as far as possible. For incorporation studies, the cells were labelled in late log phase at a density of 1.5 x lo5 cell/ml. Labelling Cells were labelled with [3H]uridine or with a mixture of [14C]uracil and [Me-3H]methionine and were collected by centrifugation. RNA was then
118
extracted either from the whole cells or from the cell fractions. Cell Fractionation After different times of labelling, growth was quickly stopped by addition of iced medium, and the cells were collected by centrifugation at low speed (3000 x g for 5 min) and washed twice in 10 mM TrisHCl pH 7.5, 3 mM CaCl,, 1 mM MgCl,, and 0.25 M sucrose. After washing, the cells were resuspended in the same buffer, and lysed using the non-ionic detergent, nonidet P40 (0.23%) which leaves the nuclei intact [I 11. The lysate was immediately centrifuged at 10000 x g for 5 min and the supernatant was recentrifuged under the same conditions and used as the cytoplasmic fraction. The pellet was resuspended in the same buffered medium and recentrifuged at 3000 x g for 10 min; this operation was repeated once more and the recovered nuclei were used for RNA extraction, after resuspension in 10 mM Tris-HC1 pH 7.5. R N A Extraction
RNA was extracted from whole cells, from the cytoplasmic fractions and from isolated nuclei by the dodecylsulfate/phenol procedure and further deproteinised by treatment with isoamyl alcohol/chloroform [12]. After separation of the phases by centrifugation, RNA was precipitated from the aqueous phase, in the presence of 0.2 M NaCl and 2 vol. of ethanol. The precipitation was performed overnight at -20 "C; RNA was then spun down at 10000 x g for 30 min and when DNA was present the pellet was dissolved in 10 mM Tris-HC1 pH 7.5 containing 5 mM MgCl, and treated with DNase (30 pg of RNase-free DNase per ml, 10 min, 0 "C). Deproteinisation with phenol and RNA precipitation was then repeated as described above and the final RNA pellet was washed once with 95% ethanol and dissolved in 10 mM Tris-HC1 pH 7.5. Electrophoretic Analysis Acrylamide gels (2.7 %) were prepared in a buffer containing 0.040 M Tris-HC1, 0.020 M sodium acetate, 2 mM EDTA, 10% glycerol adjusted to pH 7.8 with acetic acid and polymerised with 0.1 %, tetramethylethylenediamine and 1 %, ammonium persulfate [13]. Electrophoresis was carried out for 6 h with a current of 7 mA per gel, in the cold room. After completion of electrophoresis the gels were frozen and cut into 2-mm slices. Each slice was eluted overnight at 62 "C with 1 ml of 0.6 M NaCl, 0.06 M
Rapidly Labelled RNA in Tetrahymena pyriformis
sodium citrate pH 7.2 [14]. The ultraviolet absorbance of the eluates was measured in order to determine the positions of 17-S and 25-S rRNA and 0.5 ml of each fraction was deposited in a glass filter, dried and counted in a scintillation fluid containing 4 g PPO and 100mg POPOP per 1 of toluene. Radioactivity was then counted in a Intertechnique SL-30 liquid scintillation spectrometer. When double labelling was performed corrections for the incomplete separation of 14C and 3H spectra were applied.
RESULTS The results of electrophoretic analysis of RNA isolated from whole cells labelled with [3H]uridine for 10 min are shown in Fig. 1. Several peaks can be seen, whose sedimentation coefficients by reference to mature 25-S and 1 7 3 rRNA are 39 S, 29 S, 23 S, and 13 S. These sedimentation coefficients were estimated from log M , versus relative mobility plots, constructed for the well-characterised ribosomal RNA markers [15]. It is worth noting that the 39-S and 29-S peaks are broad, suggesting that these species are not homogeneous and may contain heterodisperse RNA. To determine which of the RNA species shown in Fig. 1 were intermediates in rRNA maturation, we performed several experiments in which we labelled a culture of Tetrahymena pyriformis simultaneously for 10 rnin with [14C]uraciland [Me-3H]methionine. The results of a typical double-labelling experiment are presented in Fig. 2 which shows the presence of three main peaks of methylated RNA, corresponding to sedimentation coefficients of 39 S , 29 S and 17 S and reveals that many of the species present in the rapidly labelled RNA are not methylated, e.g. peaks at 33 S, 23 S , 18 S and polydisperse components with sedimentation coefficients lower than 17 S and higher than 39 S. These results have proved to be reproducible in a series of experiments. In further experiments the two main cellular compartments (nucleus and cytoplasm) were studied separately. Fig. 3 shows the electrophoretic separation of nuclear RNA isolated from cells labelled for 2, 5, 10 and 20 min. After 2-min labelling (Fig. 3A) only the 39-S ribosomal RNA precursor molecule and all the classes of non-methylated RNA, including species heavier than 39 S are seen. The rapidly sedimenting non-methylated RNA is comparable in size to the main bulk of rapidly labelled heterogeneous nuclear RNA in higher metazoans [16]. After 5 rnin of incorporation (Fig. 3 B) the same general pattern is seen but with an increased amount of RNA sedimenting faster than 39 s, and with distinct peaks at 29 S, and 17 S of which the former is a precursor of 25-S Eur. J . Biochem. 56 (1975)
119
C. R. Pousada, L. Marcaud, M.-M. Portier, and D. H. Hayes
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Fig. 1. Electrophoresis of whole cell RNA in 2.7% acrylamide gels. A culture of cells was labelled for 10 min with [3H]uridine (10 pCi/ ml; spec. act. 10 mCi/mmol) and total RNA was then extracted and
39 s
h ,
I
analysed as described in Materials and Methods. The gel was loaded with 550 pgofRNA(1000counts3Hmin-' pg-'). ( 0 d ) in gel slice eluates (mature rRNA) 3H in gel slice; (----)
25 S
1
6000
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E
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Fig. 2. Electrophoresis of total R N A prepared from doubly labelled cells. A culture of cells was labelled for 10 min with a mixture of ~-[Me-~H]methionine (100 pCi/ml; spec. act. 4.5 Ci/mmol) and ['4C]uracil (4 pCi/ml; spec. act. 42 mCi/mmol) and total RNA was
extracted and analysed as in Fig. 1. The gel was loaded with 250 pg of RNA (1000 counts 3H and 50 counts 14Cmin-' pg-I); (M) 3H in gel slice; (O---o) 14C in gel slice. Arrows indicate the positions of mature 2 5 3 and 1 7 3 rRNA
rRNA. In some experiments the 17-S peak shows a slightly lower electrophoretic mobility than the marker of mature 17-S rRNA, suggesting that it corresponds to a precursor of this RNA. Further analysis will,
however, be needed to determine whether this rapidly labelled RNA has the same primary structure as the mature 17-S RNA. No other kinds of methylated RNA have been found even after labelling periods of
Eur. J. Biochem. 56 (1975)
120
Rapidly Labelled RNA in Tctrahymenu pyriformis
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Fig. 3. Eieectroplioreiic anulysis of nucleur RNA iubrlled f o r 2, 5, 10 und 20 rnin. h 60-in1 cell culture was incubated with a mixture of ['4C]uracil (8 pCi/ml; spec. act. 53.3 mCi/mmol) and L - [ M ~ - ~ H ] methionine (83 pCi/InI; spec. act. 7.3 Cijmmol); after (A) 2, (B) 5, (C) 10, and ( D ) 20 min incorporation, samples (30, 15, 8 and 5 ml respectively) were taken, chilled and mixed at 0 "C with varying volumes (20. 35, 42 and 45 ml respectively) of a parallel unlahelled culture in order to produce mixed samples containing approximately
equal quantities of cells. Cells were then collected and nuclcar and cytoplasmic RNA fractions were prepared as described in Materials and Methods. Electrophoretic analysis was carried out as described in the legend to Fig. 1 and 2. Gels were loaded with the whole of 'H; (0 -0) ''C. each preparation of nuclear RNA. (O---o) (Radioactivity in each sample has been normalised to 60 ml of labelled cells.) Arrows indicate the positions of mature rRNAs
10 or 20 min (Fig. 3, C and D). RNAs isolated after these longer periods of incorporation give the same profiles for the non-methylated RNA species as RNA labelled for 2 or 5 min although the proportion of heavy RNA ( > 39 S) is decreased. The same electrophoretic analysis was carried out for the KNA isolated from the cytoplasmic fractions. After 2-min labelling incorporation of isotopes into cytoplasmic RNA is low (Fig. 4A) but the profile of methylated RNA contains a distinct 17-S peak. Several small peaks of non-methylated RNA can also be seen. Leick [51] has also observed that the first ribosomal RNA exported to the cytoplasm is 17-S RNA. However, after 5-min labelling a small peak of 25-S rRNA as well as a very-well-defined peak of 17-S RNA are observed. The 2 5 3 component becomes
more important at longer incorporation times (Fig. 4, C and D). Several classes of heavy non-methylated RNA with sedimentation coefficients of 39 S, 35 S, 23 S and 15 S (Fig. 4B, C, D) appear clearly in cytoplasmic RNA after 10 and 20-min labelling times.
~
DISCUSSION We have studied ribosomal RNA processing in Tetrahymena pyriformis by kdbelling exponentidly growing cells with [Me-3H]methionine and [14C]uracil. Only two kinds of high-molecular-weight rRNA precursors (39-S and 29-S) are revealed by our experiments. A rapidly labelled methylated 17-S RNA which we observe in both nuclear and total RNA Eur. J . Hiochem. 56 (1975)
C. R. Pousada, L. Marcaud, M.-M. Portier, and D. H. Hayes
preparations may be a precursor form of mature 17-S rRNA but our analytical procedures are not powerful enough to establish this conclusion. Using acrylamide gel electrophoresis instead of sucrose gradient sedimentation we have not observed the existence of RNA species intermediate between these precursors and the mature rRNAs. Our results therefore indicate a much less complex mechanism of maturation of rRNA in Tetrahymena pyrformis than that which has been found in mammalian cells. The pathway in Tetrahymena pyrformis seems in fact to resemble the mechanism observed in E. coli (30-S precursor RNA + 25-S 17-S + 23-S 16-S RNAs [17,18]. We have also observed the existence of heavy non-methylated RNA with a sedimentation coefficient of 35-45 S and in particular peaks sedimenting at 35 S and 39 S in cytoplasmic RNA prepared after 10-min and 20-min pulse labelling. Several authors have demonstrated the existence of giant RNA in the cytoplasm of eukaryotic cells. Daneholt
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and Hosick [8] reported the existence of 75-S RNA in the cytoplasm of salivary gland cells of Chironomus tentans, Suzuki and Brown [19] found that the cytoplasmic messenger RNA for silk fibroin sedimented at 45-65 S and Guidice [9] found a giant RNA of nuclear origin in the cytoplasm of sea urchin embryos. Several observations show that the 35- 39-S species of non-methylated cytoplasmic RNA which we observe is probably not derived from artefactual nuclear leakage during the cell fractionation. If this were the case, we should expect to observe leakage of the large (39-S) methylated rRNA precursor molecule into the cytoplasm also and this does not occur. Furthermore nuclear leakage would be expected to be dependent on the method of preparation of nuclei and not on the duration of pulse labelling and it can be seen by inspection of Fig. 3 and 4 that the 39-S species are present in the nucleus at all labelling times but in the cytoplasm only after long (> 5 min) labelling periods. Finally the method of cell fractionation
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C. R. Pousada, L. Marcaud, M.-M. Portier, and D. H. Hayes: Rapidly Labelled RNA in Teirahymena pyriformis
we use is very mild and has been reported to yield intact macronuclei from T. pyriformis [ll]. At present, we have no information concerning the cytoplasmic location or function of the 35- 39-S non-methylated RNA species. Experiments are in progress which are aimed at further characterization ofthis RNA and investigation of its transport, processing and function in the cytoplasm. This work has been supported by grants to D. H. Hayes from the Centre National de la Recherche Scientifique (Equipe de Recherche no 101 ; A T P d@renciaiion cellulaire, contract number 419905) the Fonds de la Recherche Mtdicale Francaise, and the Commissariat a I’Energie Atomique. One of us, C.R.P., thanks the French Ministry of Foreign affairs for a fellowship (1973- 1974).
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4. Weinberg, R. A. & Penman, S. (1970) J . Mol. Biol. 47, 169178. 5. Leick, V. (1969) Eur. J . Biochem. 8, 221-228. 6. Kumar, A. (1970) J . Cell B i d . 45, 623-634. 7. Prescott, D. M., Bostock, C., Gamow, E. & Lauth, M. (1971) Exp. Cell Res. 67, 124- 128. 8. Daneholt, B. & Hosick, H. (1973) Proc. Nail Acad. Sci. U . S . A . 70,442 - 446. 9. Giudice, G., Sconzo, G., Ramirez, F. & Albanese, I. (1972) Biochim. Biophys. Acia, 262, 401 -403. 10. Kumar, A. & Lindberg, U. (1972) Proc. Natl Acad. Sci. U.S.A. 69,681 -685. 11. Mita, T., Shiomi, H. & Iwai, K. (1966) Exp. Cell Res. 43, 696 - 698. 12. Imamoto, F., Morikawa, N., Sato, K., Mishima, S., Nishimura, T. & Matsushiro, A. (1965) J . Mol. Biol. 13,157- 168. 13. Weinberg, R. A,, Loening, U . E., Willems, M. & Penman, S. (1967) Proc. Nail Acad. Sci. U.S.A. 58, 1088-1095. 14. Marcaud, L., Portier, M. M., Kourilsky, P., Barrel, B. G. & Gros, F. (1971) J. Mol. Biol. 57, 247-261. 15. Kurland, C. G. (1960) J . Mol. Biol. 2, 83-91. 16. Darnell, J. E. (1968) Bacieriol. Rev. 32, 262-290. 17. Nikolaev, N., Silengo, L. & Schlessinger, D. (1973) Proc. Nail Acad. Sci. U . S . A . 70, 3361-3365. 18. Dunn, J. J. & Studier, F. W. (1973) Proc. Natl Acad. Sci. U . S . A . 70,3296-3300. 19. Suzuki, Y . & Brown, D. D. (1972) J . Mol. Biol. 63,409-429.
C. R. Pousada, L. Marcaud, M.-M. Portier, and D. H. Hayes, Institut de Biologie Physico-Chimique, 13 Rue Pierre-et-Marie-Curie, F-75005 Paris, France
Eur. J. Biochem. 56 (1975)
