Copvright All rights
Q 1975 by Academic Press, of reproduction in any form
Inc.
rescrwd
Experimental
RIBOSOMAL
PRECURSOR J. TRAPMAN,
Biochemisch
Laboratorium,
SO (1975) 95-104
Cell Research
PARTICLES
J. RETeL Vrue
FROM
YEAST
and R. J. PLANTA
Universiteit,
Amsterdam,
The Netherlands
SUMMARY Ribosomal precursor particles were extracted from the yeast Saccharomyces carlsbergensis and analysed. After a brief labelling of yeast protoplasts with 3H-uridine, three basic ribonucleoprotein components were detected, sedimenting at approx. 9OS, 66s and 43 S in sucrose gradients containing magnesium. The 90s particles contained the 37s ribosomal precursor RNA as a major component and a small though variable amount of 29s ribosomal precursor RNA. The 66s and 43 S particles contained 29s and 18s ribosomal precursor RNA, respectively. Kinetic data indicate a precursorrproduct relationship between the 90s particles and the two other ribonucleoprotein components, consistent with the conversion: 90s --f66s + 43 S. The 90s and 66s preribosomes appeared to be present exclusively in the nucleus, whereas the 43 S particles were mainly present in the cytoplasmic fraction. Apparently, the final maturation step in the formation of the 40s ribosomal subunits takes place in the cytoplasm. The 90s and 66s precursor particles have a relatively higher ratio of protein to RNA than the mature large ribosomal subunits, as judged from their buoyant densities in CsCl gradients. This finding suggests that also in a primitive eukaryotic organism, like yeast, ribosome maturation involves, in addition to a decrease in the size of the RNA components, an even stronger decrease in the amount of associated protein. In contrast, the 43s particles appeared to have the same buoyant density as the 40s ribosomal subunits.
The process of ribosome formation has been the subject of considerable investigation in the last few years. In eukaryotic cells this complex process starts in the nucleolus with the transcription of the two major speciesof ribosomal RNA (rRNA) in the form of a single large precursor RNA molecule (for a review seerefs [ 1,2]). This ribosomal precursor RNA contains, besidesone copy of each of the two rRNA components, also considerable stretches of non-ribosomal RNA, which are removed in a number of specific cleavage steps. In mammalian cells the first ribosomal precursor RNA becomesalready associatedwith proteins, including most, if not all, of the ribosomal proteins and also some nonribosomal proteins [3-91. In this way a preribosome is formed, which contains a higher 7-741803
proportion of associated protein than the mature ribosomal subunits. In the subsequent maturation steps the non-ribosomal proteins are removed together with the non-ribosomal RNA stretches, resulting in the formation of at least two ribonucleoprotein intermediates. The ribonucleoprotein particles, which finally appear in the cytoplasm are completed or almost completed ribosomal subunits, which after a lag period can become part of the polysomes [l, lo]. A similar schemeseemsto apply for the formation of ribosomes in higher plant cells [l 11. In prokaryotic cells the formation of ribosomesseemsto follow another scheme,which differs from the one described above in two main respects. First, usually the two major rRNA components do not exist initially as Exptl
Cell Res 90 (1975)
96
Trapman, Ret21 and Planta
parts of one common large precursor RNA, but appear directly as two separate precursor RNA molecules [12]. Secondly, only a part of the ribosomal proteins are associated with rRNA early in the process of ribosome formation. The other ribosomal proteins are attached at later stages of the maturation process, resulting in the formation of ribosoma1 precursor particles with an increasing protein content [13, 141. Only in:the final stage of the maturation process the ribosomal subunits get their;full complement of ribosomal proteins. Relatively little is known so far about the mechanism of ribosome assembly in primitive eukaryotic organisms. However, it has been suggested that lower eukaryotes would occupy an intermediate place in this respect, in that the mode of ribosome assembly in these organisms at the initial stages of maturation is rather similar to that found in higher eukaryotic cells, but at the final stages of maturation resembles that observed in bacteria
[151. Therefore, we decided to investigate in more detail the process of ribosome assembly in a primitive eukaryotic microorganism like yeast. We previously reported that rRNA formation in this organism occurs in a manner similar to that in higher eukaryotes [16, 171. In this paper we describe the isolation and some properties of three ribosomal precursor particles of yeast. Our data indicate that the process of ribosome assembly in yeast is very similar to that observed in mammalian cells at all stages of maturation.
MATERIALS
AND METHODS
Isolation of ribonucleoprotein and RNA
particIes
The strain of Saccharomyces carlsbergensis used in this study was no. 74 of the British National Collection Exptl
Cell Res 90 (1975)
of Yeast Cultures. Conditions of growth. preparation of yeast protoplasts and conditions of p&e labelling of protoplasts with 3H-uridine (27 Ci/mmole, Radiochemical Centre, Amersham) or steady-state labelling with W-uracil (50 mCi/mmole, Radiochemical Centre) were essentially the same as described previously 1181.Before starting a labelling experiment the protoplasts were incubated for 45 mm at 23°C under gentle shaking, in order to permit recovery from the treatment with hemicellulase. Nuclei were prepared according to the method of Sillivis Smitt et al. -[19] as modified by Ret&l & Planta [20] with one further modification. Instead of collecting the nuclei in the interphase layer between 70 and 10 % sucrose solutions [20], the homogenate obtained from protoplasts was first centrifuged at 1 000 g for 5 min at 0°C in order to remove whole cells. cell debris and membranes. The nuclei were then collected by centrifugation of the 1000 g supernatant for 10 min at 20 000 g at 0°C. For the preparation of the cytoplasmic fraction, the homogenate of protoplasts was immediately centrifuged at 20 000 g for 10 min at O”C, in order to minimize the leakage of material from the nuclei and degradation of RNA. The 20 000 g supernatant represents the cytoplasmic fraction. Ribonucleoprotein particles were prepared from nrotonlasts and nuclei bv susnendina them in 1 ml of a Tris-HCl buffer (0.01 M: pH 7.4) containing 5 mM MgCl,, 10 mM DTT, 0.2 % Brij-58 and 0.01 M, or 0.2 M, or 0.5 M KCI, and a RNAse inhibitor isolated from calf lens (500 U/ml). After stirring for 5 min in ice the suspension was centrifuged at 20 000 g for 10 min at 0°C. The ribonucleoorotein aarticles are present in the 20 000 g supernatant. . RNA was prepared from the various fractions by phenol-O.5 % SDS extraction at 30°C in essentially the same way as previously described [18].
Analysis of ribonucleoprotein particles and RNA Analysis of ribonucleoprotein particles was performed bv centrifugation throuah a linear 15-30% sucrose gradient, made in a TrisrHCl buffer (0.01 M, pH 7.4) containing 5 mM MgCl,, 1 mM DTT and 0.01 M, or 0.2 M, or 0.5 M KCI. The sucrose solutions were pretreated with Macaloid (5 . ma/ml) -, , to inactivate ribonucleases. Buoyant densities of ribonucleoprotein particles were determined by sedimentation to equilibrium in CsCl gradients essentially according to the method of Perry-& Kelley [21]. RNA was analysed by electrophoresis on 2.6 % polyacrylamide gels [22] to which glycerol (7.5 %, v/v) was added in order to facilitate subsequent slicing of the gels. The Tris-phosphate electrophoresis buffer [23] contained 2.5 % glycerol. The gels were sliced in a frozen state into 1 mm slices with a Joyce-Loebl gel slicer, and subsequently assayed for 3H- and Wradioactivity in a Mark II Liauid Scintillation Counter (Nuclear Chicago) after incubation overnight at 37°C in 10 ml of a toluene-based scintillation fluid containing the usual scintillators (PPO and POPOP) and 2.5 % (v/v) NCS (Nuclear Chicago Solubilizer).
Preribosomes
cl
15"
b
23'
from yeast
97
T 29s
375
! I A6
b . Fig. I. Abscissa: distance (cm); ordinate: (left) 3H dpm x 1OW (O-O); (right) 14C dpm x IOW’ (O---O). Polyacrylamide gel electrophoresis of RNA extracted from yeast protoplasts pulse-labelled at the indicated temperatmes. After preincubation of the protoplasts for 45 min at 23°C the temperature of the protoplast suspension was shifted to (a) 15°C; (b) 23°C; or (c) 29°C and then the protoplasts were labelled with 3H-uridine (5 @/ml) for 5 min. The RNA was extracted and analysed by electrophoresis on 2.6 % polyacrylamide gels as described in Materials and Methods. An appropriate amount of 14C-uracil-labelled 26s and 17s rRNA was used as a reference in the electrophoresis. The arrows (b, c) indicate the position of 32s ribosomal precursor RNA.
RESULTS Effect of the incubation temperature on the processing of ribosomal precursor RNA
In our previous studies [16] we showed that the process of rRNA synthesis in yeast can be described as follows:
Under optimal conditions of growth the various precursors of rRNA in yeast are present in rather low cellular amounts. This applies particularly for the 42s and the 32s RNA species, which are minor components and normally difficult to detect. Only under special conditions, viz. methionine-starvation of a methionine-requiring mutant, which interferes with the methylation of ribosomal precursor RNA and rRNA, a pronounced accumulation of these precursor molecules can be provoked [16]. However, methioninestarvation does not only interfere with the methylation process of rRNA, but also with
the synthesis of proteins. Since we want to study the assembly process of rRNA and ribosomal proteins, and since it might be expected that under conditions of methioninestarvation precursor particles shall accumulate with an aberrant protein composition, the application of methionine-starvation was considered as less appropriate. Therefore, we looked for another method to slow down the process of ribosome formation in yeast. In fig. 1 the effect of the incubation temperature on the rRNA synthesis is shown. Yeast protoplasts were incubated at the indicated temperatures and subsequently labelled with 3H-uridine for a very short time (5 min). The RNA was extracted and analysed by electrophoresis on 2.6 % polyacrylamide gels. After a 5 min pulse at 29°C radioactivity is already found in all the main ribosomal precursor RNA species, and in 26 S and 17s rRNA (fig. 1c), whereas at the suboptimal temperature of 15°C only 37s precursor RNA is detectably labelled (fig. 1a). An intermediate situation is found at 23°C Exptl Cell Res 90 (1975)
98
Trapman, Rettl and Planta n-l” b
CUISO
3 i
-2
(fig. lb). Extended pulse-labelling at 15°C produced similar labelling-patterns as those shown in fig. 1b and c. Therefore, we chose pulse-labelling at 15°C as the best method to study the process of ribosome assembly in yeast. Sedimentation analysis of ribonucleoprotein particles from yeast protoplasts
265 1
-10
~20
30
Fig. 2. Abscissa: (a, c, e) fraction no.; (6, d,f) distance (cm); ordinate: (left) $H dpm x 1OV (O-O); (right) 14C dpm x 1OV (O---O). Sucrose gradient centrifugation of ribonucleoprotein particles (a, c, e) and polyacrylamide gel electrophoresis of RNA (b, d, f) of yeast protoplasts pulse-labelled for the indicated periods of time. Appropriate portions of yeast protoplasts were labelled with 3H-uridine (10 &i/ml) for 5 min (a, b), 10 min (c, d) and 5 min followed by a 10 min chase with a very large excess of unlabelled uridine (e, f). Each portion of protoplasts was then divided into Exptl
Cell Res 90 (1975)
Protoplasts were labelled for 5 and 10 min with 3H-uridine at 15°C and the ribonucleoprotein particles were extracted in a low ionic strength buffer, containing 10 mM KC1 (see Materials and Methods). The sedimentation patterns obtained by centrifugation through linear 15-30 % sucrosegradients are shown in fig. 2a, c. After labelling for 5 min radioactivity is mainly found in a particle with an approximate sedimentation coefficient of 90s (fig. 2a). The predominantly labelled RNA species under these conditions is 37s ribosomal precursor RNA as can be inferred from fig. 2b, where the corresponding electrophoretic analysis is shown of the RNA extracted from the sameprotoplasts. After labelling for 10min, two other radioactive particles appear, sedimenting at about 66S and 43 S as compared with the 60s and 40s ribosomal subunits (fig. 2~). In this case, besidesthe 37s two parts. From one part the ribonucleoprotein particles were extracted, from the other part the RNA as described in Materials and Methods. The ribonucleoprotein particles were analysed by sedimentation through a 15-30 % sucrose gradient and the RNA by electrophoresis on 2.6 % polyacrylamide gels (see Materials and Methods). Appropriate amounts of 14C-uracil-labelled 8OS, 60s and 40s ribosomal particles, and 14C-uracil-labelled 26s and 17s rRNA were added as markers in the sedimentation and electrophoretic analysis, respectively. Centrifugation was for 4 h at 34 000 rpm at 4°C in the Spinco SW41 rotor (a) or for 16 h at 18 000 rpm in the Spinco SW27 rotor (c, e). Fractions of 0.3 ml (SW41 rotor) or 0.9 ml (SW27 rotor) were collected, and assayed for radioactivity. To this end 100 @ampIes were digested for 2 h at 37°C with pancreatic RNAse (20 y/ml), and then mixed with 10 ml of a dioxane-based scintillation liquid (containing 90 g naphthalene, 7 g PPO and 50 mg POPOP per litre).
Preribosomes
from yeast
99
c
b 29s
/665/
3. A6scis.r~ distance (cm); ordinate: (left) 3H dpm x 10e2 (O-O); (right) IT dpm / 1OV’ (O---O). Polyacrylamide gel electrophoresis of pulse-labelled RNA from the (a) 90s; (b) 66s; and (c) 43s ribonucleoprotein particles. Fractions from the regions indicated in fig. 2a, e were pooled, the RNA extracted and then analysed by electrophoresis on 2.6 % polyacrylamide gels as described in Materials and Methods. T-labelled 26s and IIS rRNA was used as a reference. Fig.
rRNA precursor, also the 29 S and 18 S rRNA precursors are clearly labelled (fig. 2d). The appearance of the 3H-uridine marker into the 66s and 43 S particles, and the 29s and 18s precursor RNAs, is also observed, when a 5 min pulse is followed by a 10 min chase with a large excess of unlabelled uridine (fig. 2e, f). This indicates the existence of a precursor-product relationship between the 90s particles, and the 66s and 43 S particles. These results strongly suggest, that the 37 S, 29 S and 18 S rRNA precursors in yeast are present in the form of ribonucleoprotein particles with sedimentation coefficients of approx. 9OS, 66 S and 43 S, respectively. RNA componentsof ribonucleoprotein particles
In order to characterize more definitely the RNA constituents of the rapidly labelled ribonucleoprotein particles, the regions indicated in fig. 2a and e were pooled, the RNA was extracted by phenol-O.5 % SDS treatment,
and analysed by electrophoresis on 2.6 % polyacrylamide gels (fig. 3). The 90s particles contain 37s ribosomal precursor RNA as well as some 29s RNA (fig. 3a). The 66s and 43s particles appear to contain almost exclusively 29s and 18 S ribosomal precursor RNA, respectively (fig. 3 b, c). The presence of 29s RNA in the 90s particle is most probably due to cleavage of 37s precursor RNA taking place during the isolation of the ribonucleoprotein particles, since it was found that the RNA-constituent of the 90s particle is rather sensitive to specific and unspecific degradation. Patterns as shown in fig. 3a can only be obtained by isolating the particles in the presence of a RNAse inhibitor prepared from calf lens [24]. In the absence of this inhibitor no 37s precursor RNA can be extracted from the 90s particles. These results give additional evidence, that the 9OS, 66s and 43 S particles are indeed preribosomal particles. Exptl
Cell Res 90 (1975)
100 Trapman, Retbl and Planta Table 1. Some properties of the preribosomes as compared with those of the mature ribosomal subunits at low ionic strength (0.01 M KU)
Particle
RNA component
Buoyant density k/ml)
90s 66s 60s 43s 40s
37 S pre-rRNA 29 S pre-rRNA 26s rRNA 18 S pre-rRNA 17s rRNA
1.46 1.50 1.57 1.54 1.54
% protein 58 51 41 45 45
Percentage protein was calculated from the buoyant densities of the particles according to Perry & Kelley
WI. Protein content of ribosomal precursor particles In order to obtain information on the RNA/ protein ratio of the various precursor particles their buoyant densities in CsCl gradients were determined in essentially the same way as described by Perry & Kelley [21]. The results are summarized in table 1. The buoyant densities of the 90 S and 66 S particles appeared to be considerably lower than those of the
60s mature ribosomal subunits, whereas the 43s particle has the same buoyant density in CsCl as the 40s ribosomal subunit. These results indicate that the protein content of the 90s and 66s particle is substantially higher than that of the ribosomal subunits. From the buoyant densities one can calculate that the 9OS, 66 S and 43 S preribosomal particles have a protein content of 58, 51 and 45%, respectively. The corresponding values for the 60s and 40 S ribosomal subunits are 41 and 45 %, respectively. One possible explanation for the relatively high percentage of protein in the 90s and 66 S preribosomes is that the excess protein is derived from non-specific adsorption of cellular non-ribosomal proteins taking place during the extraction of the ribonucleoprotein particles. Therefore, we investigated the effect of high salt concentrations (0.2 or 0.5 M KCl) on both the sedimentation coefficients and the buoyant densities of the particles. From the data, summarized in table 2, it can be concluded that extraction and sedimentation of the ribonucleoprotein particles
Table 2. Effect of the KC1 concentration on the sedimentation densities of the preribosomal particles
coefficients and the buoyant
Ribonucleoprotein particles were prepared and analysed as described in Materials and Methods concentrations (0.01 M KCl, or 0.2 M KCl, or 0.5 M KCl). The sedimentation coefficients of preribosomes (containing 37s and 29s RNA, respectively) were compared with that of the large units, and the sedimentation coefficient of the smallest preribosome (containing 18s RNA) with that of the small subunit. Results are expressed as *’
= Smeribosome
- Sribosomal
at various KC1 the two largest ribosomal subwas compared
subunit
Buoyant densities in CsCl-gradients
were determined according to Perry & Kelley 1211
S-value of particles at 0.01 M KC1
0.2 M KC1 RNA-constituent
AS
Buoyant density k/ml)
AS
Buoyant density k/ml)
90s 66s 60s 43s 40s
37s pre-rRNA 29s pre-rRNA 26s rRNA 18 S pre-rRNA 17s rRNA
+23 +3 0 +3 0
1.49a 1.52 1.58 1.55 1.55
+2 -5 0 +3 0
1.53= 1.54 1.59 1.56 1.56
a The “90s”-particles Exptl
0.5 M KC1
appeared to be rather unstable at high ionic strength.
Cell Res 90 (1975)
Preribosomes
under conditions of high ionic strength resulted in a rather dramatic reduction of the sedimentation coefficients, in particular for the 37 S and 29 S RNA containing particles as compared with the large ribosomal subunit, analysed under the same conditions. On the other hand, the changes in sedimentation rate of the 18 S RNA containing particle are rather similar to those of the small ribosomal subunit. There is also some loss of protein from all particles, including the ribosomal subunits, at higher KC1 concentrations, as judged from the change in buoyant densities (table 2). However, these losses are far too small to explain the rather large changes in sedimentation rates. The reduction of the sedimentation coefficients, in particular those of the 37s and 29s containing particles, must be largely due to conformational changes induced by high salt concentrations. In addition, it appeared that even at the high, non-physiological, KC1 concentration of 0.5 M, the protein content of the particles containing 37s and 29s ribosomal precursor RNA is still higher than that of the 60s ribosomal subunit under the same conditions. This makes it unlikely that non-specific adsorption of proteins is wholly responsible for the higher protein content of these preribosomes. Cellular location of the various preribosomal particles
The cellular location of the preribosomal particles was studied by preparing from yeast protoplasts a nuclear and cytoplasmic fraction, after a short pulse with 3H-uridine (10 min) at 15°C. Both the cytoplasmic and the nuclear fraction were divided into two parts. From one part the ribonucleoprotein particles were extracted and analysed by sedimentation through a 15-30 % sucrose gradient (fig. 4a, c). From the other part the RNA was extracted by phenol-O.5 % SDS treatment, and sub-
’ I~-I 15
1 NUCLEUS 605
101
from yeast
r b
NUCLEUS
i
405
605
,.
5
605 605
405 6-
-6
,O
K)
20
30
1
2
3
4
Fig. 4. Abscissa: (a, c) fraction number; (b, d) distance (cm); ordinate: (left) 3H dpm x 1OW (O-O); (right) 14C dpm x 1OP (o---e). (a, c) Sucrose gradient centrifugation of pulselabelled nuclear (a) and cytoplasmic (c) ribonucleoprotein particles; (b, d) polyacrylamide gel electrophoresis of pulse-labelled nuclear (b) and cytoplasmic (d) RNA. An appropriate portion of yeast protoplasts was labelled with 3H-uridine (10 pCi/ml) at 15°C for 10 min. The nuclear and cytoplasmic fractions were. prepared as described in Materials and Methods. Each fraction was divided into two parts. From one part the ribonucleoprotein particles were extracted, from the other part the RNA. The ribonucleoprotein particles were analysed by sedimentation through a 1530% sucrose gradient (a, c), as described for fig. 2c, e, the RNA by electrophoresis on a 2.6 % polyacrylamide gel (b, d). Appropriate amounts of Wuracil labelled SOS, 60s and 40s ribosomal particles, and ‘*C-uracil labelled 26s and 17s rRNA were used as a reference in the sedimentation and electrophoretic analysis, respectively.
sequently analysed by electrophoresis on 2.6 % polyacrylamide gels (fig. 4b, d). The predominantly labelled particles in the nuclear fraction are the 90s and 66s preribosomes Exptl Cell Res 90 (1975)
102
Trapman, Ret61 and Planta
(fig. 4a, b). These particles are completely absent in the cytoplasm, indicating a high degree of purity of this fraction (fig. 4~0, d). The only labelled particle, detected in the cytoplasmic fraction, is the 43 S preribosome, containing the 18 S Ii-RNA precursor, the direct precursor of mature 17s rRNA. The relatively small amount of 43s in the nucleus is most probably due to a slight cytoplasmic contamination of the nuclear fraction. This result strongly suggests that the final maturation step, which converts the 43s particle into the 40s ribosomal subunit, takes place in the cytoplasm. On the other hand, the labelled 29s rRNA precursor, or the corresponding 66s preribosome, was never found in the cytoplasmic fraction, even when pulselabelling with 3H-uridine was performed for longer periods of time, suggesting that the final maturation of the large subunit takes place in the nucleus. DISCUSSION The object of this study was to examine the mode of ribosome assembly in yeast. Three ribonucleoprotein particles with sedimentation coefficients of approx. 9OS, 66s and 43 S were detected by pulse-labelling with 3Huridine at relatively low temperature. These particles were found to contain 37S, 29s and 18 S ribosomal precursor RNA, respectively. Evidence was obtained that there exists a precursor-product relationship between the 90s particles and the two other kinds of particles. Thus it may be concluded that the 43 S particles are precursors to the 40 S subunits, the 66s particles precursors to the 60s subunits, whereas the 90s particles represent precursors to both the ribosomal subunits. Particles containing the previously [ 161 described 42 S and 32 S rRNA precursors have not been found in the present study. This is not surprising, since these precursor molecules Exptl
Cell Res 90 (197.5)
can hardly be detected in the wild type of S. carlsbergensis under the usual and applied labelling conditions (see fig. 1 b, c). Only the 32s rRNA precursor is sometimes visible in the RNA-fractionation pattern as a minor component (e.g. in fig. 2d, f). An accumulation of the rather transient 42s and 32s rRNA precursors can only be brought about under special conditions, such as methioninestarvation of a methionine-requiring mutant [16], or by inhibiting protein synthesis by cycloheximide [25, 261. For the present investigations we considered the application of such conditions as less appropriate, since it is known that artificial ribonucleoprotein complexes of rRNA can be formed upon inhibition of protein synthesis [27]. Though we do not yet know whether the first ribosomal transcription product (the 42 S precursor RNA) is already associated with protein, it can be said that the mode of ribosome assembly resembles that observed in mammalian cells [3-51 and plants [ll] in the main respects. For these higher eukaryotic organisms, however, the existence of a direct precursor to the small ribosomal subunits, comparable to the 43s particles of yeast, has not been reported. In the present study we found that the 43s particles, containing the direct precursor (18 S RNA) of 17 S rRNA, are mainly located in the cytoplasmic fraction. This finding indicates that the final maturation step in the formation of the small ribosomal subunit, which converts 18 S rRNA precursor into 17s rRNA, takes place in the cytoplasm. This confirms earlier observations following a different approach (see [2]), and the very recent results obtained by Udem & Warner [28] for the yeast Saccharomyces cerevisiue. On the other hand, the 66s preribosomal particles appeared to be exclusively present in the nuclear fraction, indicating that the formation of the large ribosomal subunits is largely completed in the nucleus.
Preribosomes from yeast No indication was obtained for the occurrence of a precursor particle of the 60s subunit similar to that observed by Taber et al. in the yeast Schizosaccharomyces pombe, which sediments at 55 S in high ionic strength sucrose gradients and contains mature rRNA [29]. However, if we assume that under the conditions of isolation and analysis used by these authors the 29s precursor RNA is largely processed to mature rRNA this “55 S” particle might be equivalent to the 66s preribosome in Saccharomyces carlsbergensis, which has a comparable sedimentation rate at high ionic strength (table 2). Our finding, that in yeast both the 90s and the 66s preribosomes contain more protein than the 60s ribosomal subunit, whereas the 43s preribosome and the 40s subunit have about the same protein content, is at complete variance with the suggestion that the late steps of ribosome formation in lower eukaryotic organisms resemble bacterial ribosome assembly [15, 291. In bacteria preribosomal particles have a lower protein/ RNA ratio than the ribosomal subunits, which acquire their full complement of ribosomal proteins only at a late stage of ribosome formation [13, 141. In this respect the situation in yeast is highly similar to that in mammalian cells, where the preribosomes were found to contain a large number of excess proteins, not present in mature ribosomes [4, 51. The extra proteins of the mammalian preribosomes differ from the ribosomal proteins with respect to size and labelling kinetics. When the nucleolar precursor particles mature into cytoplasmic ribosomes, these, non-ribosomal, proteins are discarded and remain in the nucleolus [30]. The function of this special class of proteins is not yet clear, but it can be speculated that among other proteins it contains the enzymes responsible for the processing of the ribosomal precursor RNAs. The fact, that the 90s pre-
103
ribosomal particles always contain a small amount of 29s RNA, besides 37s precursor RNA, is in agreement with this suggestion. At the moment we cannot exclude the possibility that some of the ribosomal proteins are missing from the precursor particles. It was recently reported that in mammalian cells one or more proteins, present in the large ribosomal subunit, are lacking from its direct precursor [31-331. Experiments, in which the proteins of the yeast precursor particles are compared with those of the cytoplasmic ribosomes by twodimensional polyacrylamide gel electrophoresis are in progress now. The present study was supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). The authors are indebted to Mr E. Veltkamp, Mr A. Vlug, Miss G. M. Zijlstra and Mrs E. C. Koper-Zwarthoff for skilful technical assistance; to Dr W. G. M. van den Broek (Biochemisch Laboratorium, University of Nijmegen, The Netherlands) who kindly provided the RNAse inhibitor of calf lens.
REFERENCES 1. Maden, B E H, Progr biophys mol biol 22 (1971) 127. 2. Planta, R J, van den Bos, R C & Klootwijk, J, Ribosomes: Structure, function and biogenesis (ed H Bloemendal & R J Planta) p. 183. NorthHolland, Amsterdam (1972). 3. Warner, J R & Soeiro, R, Proc natl acad sci US 58 (1967) 1984. 4. Liau, M C & Perry, R J, J cell bio142 (1969) 272. 5. Kumar, J & Warner, J R, J mol bio163 (1972) 233. 6. Mirault, M E & Scherrer, K, Eur j biochem 23 (1971) 372. 7. Tamaoki, T, J mol biol I5 (I 966) 624. 8. Narayan, K S & Birnstiel, M L, Biochim biophys acta 190 (1969) 470. 9. Shepherd, J & Maden, B E H, Nature new biol 236 (1972) 211. IO. Darnell, J E, Bact rev 32 (1968) 262. II. Takahashi, N, Shimada, T, Higo, M, Higo, S & Tanifuji, S, Biochim biophys acta 262 (1972) 502. 12. Pace, N R, Bact rev 37 (1973) 562. 13. Osawa, S, Ann rev biochem 37 (1968) 109. 14. Nierhaus, K H, Bordasch, K & Homann, H E, J mol biol 74 (1973) 587. 15. Mizukami, Y & Iwabuchi, M, Biochim biophys acta 272 (1972) 81. 16. Retkl, J & Planta, R J, Biochim biophys acta 224 (1970) 458. Exptl
Cell Res 90 (1975)
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Trapman, Ret21 and Planta
17. van den Bos, R C & Planta, R J, Biochim biophys acta 294 (1973) 464. 18. Ret&l, J & Planta, R J, Em j biochem 3 (1967) 248. 19. Sillevis Smitt, W W, Vlak, J M, Schiphof, R & Rozijn, Th H, Exptl cell res 71 (1972) 33. 20. Ret&l, J & Planta, R J, Biochim biophys acta 281 (1972) 299. 21. Perry, R P & Kelley, P E, J mol biol 16 (1966) 255. 22. Loening, U E, Biochem j 102 (1967) 251. 23. -Ibid 113 (1969) 131. 24. van den Broek, W G M, Koopmans, M A G & Bloemendal, H, Mol biol reports 1 (1974) 295. 25. Mayo, V S, Andrean, B A G & de Kloet, S R, Biochim biophys acta 169 (1968) 297. 26. Taber, R L & Vincent, W S, Biochim biophys acta 186 (1969) 317.
Exptl
Cell Res 90 (197.5)
27. Craig, N C & Perry, R P, J cell bioI45 (1970) 554. 28. Udem, S A &Warner, J R, J biol them 248 (1973) 1412. 29. Taber. R L. Vincent. W S & Coetzee. M L. Biochim biophys acta 195 (1969) 99. ’ ’ Soeiro. R & Basile. C. J mol biol 79 (1973) 507. ::: Tsurugi, K, Morita; T’& Ogata, K, Eur j b&hem 25 (1972) 117. 32. Tsurugi, K, Morita, T & Ogata, K, Eur j biochem 32 (1973) 555. 33. Higashinakagawa, T & Muramatsu, M, Eur j biochem 42 (1974) 245.
Received May 10, 1974
Q 1975 by Academic Press, of reproduction in any form
Inc.
rescrwd
Experimental
RIBOSOMAL
PRECURSOR J. TRAPMAN,
Biochemisch
Laboratorium,
SO (1975) 95-104
Cell Research
PARTICLES
J. RETeL Vrue
FROM
YEAST
and R. J. PLANTA
Universiteit,
Amsterdam,
The Netherlands
SUMMARY Ribosomal precursor particles were extracted from the yeast Saccharomyces carlsbergensis and analysed. After a brief labelling of yeast protoplasts with 3H-uridine, three basic ribonucleoprotein components were detected, sedimenting at approx. 9OS, 66s and 43 S in sucrose gradients containing magnesium. The 90s particles contained the 37s ribosomal precursor RNA as a major component and a small though variable amount of 29s ribosomal precursor RNA. The 66s and 43 S particles contained 29s and 18s ribosomal precursor RNA, respectively. Kinetic data indicate a precursorrproduct relationship between the 90s particles and the two other ribonucleoprotein components, consistent with the conversion: 90s --f66s + 43 S. The 90s and 66s preribosomes appeared to be present exclusively in the nucleus, whereas the 43 S particles were mainly present in the cytoplasmic fraction. Apparently, the final maturation step in the formation of the 40s ribosomal subunits takes place in the cytoplasm. The 90s and 66s precursor particles have a relatively higher ratio of protein to RNA than the mature large ribosomal subunits, as judged from their buoyant densities in CsCl gradients. This finding suggests that also in a primitive eukaryotic organism, like yeast, ribosome maturation involves, in addition to a decrease in the size of the RNA components, an even stronger decrease in the amount of associated protein. In contrast, the 43s particles appeared to have the same buoyant density as the 40s ribosomal subunits.
The process of ribosome formation has been the subject of considerable investigation in the last few years. In eukaryotic cells this complex process starts in the nucleolus with the transcription of the two major speciesof ribosomal RNA (rRNA) in the form of a single large precursor RNA molecule (for a review seerefs [ 1,2]). This ribosomal precursor RNA contains, besidesone copy of each of the two rRNA components, also considerable stretches of non-ribosomal RNA, which are removed in a number of specific cleavage steps. In mammalian cells the first ribosomal precursor RNA becomesalready associatedwith proteins, including most, if not all, of the ribosomal proteins and also some nonribosomal proteins [3-91. In this way a preribosome is formed, which contains a higher 7-741803
proportion of associated protein than the mature ribosomal subunits. In the subsequent maturation steps the non-ribosomal proteins are removed together with the non-ribosomal RNA stretches, resulting in the formation of at least two ribonucleoprotein intermediates. The ribonucleoprotein particles, which finally appear in the cytoplasm are completed or almost completed ribosomal subunits, which after a lag period can become part of the polysomes [l, lo]. A similar schemeseemsto apply for the formation of ribosomes in higher plant cells [l 11. In prokaryotic cells the formation of ribosomesseemsto follow another scheme,which differs from the one described above in two main respects. First, usually the two major rRNA components do not exist initially as Exptl
Cell Res 90 (1975)
96
Trapman, Ret21 and Planta
parts of one common large precursor RNA, but appear directly as two separate precursor RNA molecules [12]. Secondly, only a part of the ribosomal proteins are associated with rRNA early in the process of ribosome formation. The other ribosomal proteins are attached at later stages of the maturation process, resulting in the formation of ribosoma1 precursor particles with an increasing protein content [13, 141. Only in:the final stage of the maturation process the ribosomal subunits get their;full complement of ribosomal proteins. Relatively little is known so far about the mechanism of ribosome assembly in primitive eukaryotic organisms. However, it has been suggested that lower eukaryotes would occupy an intermediate place in this respect, in that the mode of ribosome assembly in these organisms at the initial stages of maturation is rather similar to that found in higher eukaryotic cells, but at the final stages of maturation resembles that observed in bacteria
[151. Therefore, we decided to investigate in more detail the process of ribosome assembly in a primitive eukaryotic microorganism like yeast. We previously reported that rRNA formation in this organism occurs in a manner similar to that in higher eukaryotes [16, 171. In this paper we describe the isolation and some properties of three ribosomal precursor particles of yeast. Our data indicate that the process of ribosome assembly in yeast is very similar to that observed in mammalian cells at all stages of maturation.
MATERIALS
AND METHODS
Isolation of ribonucleoprotein and RNA
particIes
The strain of Saccharomyces carlsbergensis used in this study was no. 74 of the British National Collection Exptl
Cell Res 90 (1975)
of Yeast Cultures. Conditions of growth. preparation of yeast protoplasts and conditions of p&e labelling of protoplasts with 3H-uridine (27 Ci/mmole, Radiochemical Centre, Amersham) or steady-state labelling with W-uracil (50 mCi/mmole, Radiochemical Centre) were essentially the same as described previously 1181.Before starting a labelling experiment the protoplasts were incubated for 45 mm at 23°C under gentle shaking, in order to permit recovery from the treatment with hemicellulase. Nuclei were prepared according to the method of Sillivis Smitt et al. -[19] as modified by Ret&l & Planta [20] with one further modification. Instead of collecting the nuclei in the interphase layer between 70 and 10 % sucrose solutions [20], the homogenate obtained from protoplasts was first centrifuged at 1 000 g for 5 min at 0°C in order to remove whole cells. cell debris and membranes. The nuclei were then collected by centrifugation of the 1000 g supernatant for 10 min at 20 000 g at 0°C. For the preparation of the cytoplasmic fraction, the homogenate of protoplasts was immediately centrifuged at 20 000 g for 10 min at O”C, in order to minimize the leakage of material from the nuclei and degradation of RNA. The 20 000 g supernatant represents the cytoplasmic fraction. Ribonucleoprotein particles were prepared from nrotonlasts and nuclei bv susnendina them in 1 ml of a Tris-HCl buffer (0.01 M: pH 7.4) containing 5 mM MgCl,, 10 mM DTT, 0.2 % Brij-58 and 0.01 M, or 0.2 M, or 0.5 M KCI, and a RNAse inhibitor isolated from calf lens (500 U/ml). After stirring for 5 min in ice the suspension was centrifuged at 20 000 g for 10 min at 0°C. The ribonucleoorotein aarticles are present in the 20 000 g supernatant. . RNA was prepared from the various fractions by phenol-O.5 % SDS extraction at 30°C in essentially the same way as previously described [18].
Analysis of ribonucleoprotein particles and RNA Analysis of ribonucleoprotein particles was performed bv centrifugation throuah a linear 15-30% sucrose gradient, made in a TrisrHCl buffer (0.01 M, pH 7.4) containing 5 mM MgCl,, 1 mM DTT and 0.01 M, or 0.2 M, or 0.5 M KCI. The sucrose solutions were pretreated with Macaloid (5 . ma/ml) -, , to inactivate ribonucleases. Buoyant densities of ribonucleoprotein particles were determined by sedimentation to equilibrium in CsCl gradients essentially according to the method of Perry-& Kelley [21]. RNA was analysed by electrophoresis on 2.6 % polyacrylamide gels [22] to which glycerol (7.5 %, v/v) was added in order to facilitate subsequent slicing of the gels. The Tris-phosphate electrophoresis buffer [23] contained 2.5 % glycerol. The gels were sliced in a frozen state into 1 mm slices with a Joyce-Loebl gel slicer, and subsequently assayed for 3H- and Wradioactivity in a Mark II Liauid Scintillation Counter (Nuclear Chicago) after incubation overnight at 37°C in 10 ml of a toluene-based scintillation fluid containing the usual scintillators (PPO and POPOP) and 2.5 % (v/v) NCS (Nuclear Chicago Solubilizer).
Preribosomes
cl
15"
b
23'
from yeast
97
T 29s
375
! I A6
b . Fig. I. Abscissa: distance (cm); ordinate: (left) 3H dpm x 1OW (O-O); (right) 14C dpm x IOW’ (O---O). Polyacrylamide gel electrophoresis of RNA extracted from yeast protoplasts pulse-labelled at the indicated temperatmes. After preincubation of the protoplasts for 45 min at 23°C the temperature of the protoplast suspension was shifted to (a) 15°C; (b) 23°C; or (c) 29°C and then the protoplasts were labelled with 3H-uridine (5 @/ml) for 5 min. The RNA was extracted and analysed by electrophoresis on 2.6 % polyacrylamide gels as described in Materials and Methods. An appropriate amount of 14C-uracil-labelled 26s and 17s rRNA was used as a reference in the electrophoresis. The arrows (b, c) indicate the position of 32s ribosomal precursor RNA.
RESULTS Effect of the incubation temperature on the processing of ribosomal precursor RNA
In our previous studies [16] we showed that the process of rRNA synthesis in yeast can be described as follows:
Under optimal conditions of growth the various precursors of rRNA in yeast are present in rather low cellular amounts. This applies particularly for the 42s and the 32s RNA species, which are minor components and normally difficult to detect. Only under special conditions, viz. methionine-starvation of a methionine-requiring mutant, which interferes with the methylation of ribosomal precursor RNA and rRNA, a pronounced accumulation of these precursor molecules can be provoked [16]. However, methioninestarvation does not only interfere with the methylation process of rRNA, but also with
the synthesis of proteins. Since we want to study the assembly process of rRNA and ribosomal proteins, and since it might be expected that under conditions of methioninestarvation precursor particles shall accumulate with an aberrant protein composition, the application of methionine-starvation was considered as less appropriate. Therefore, we looked for another method to slow down the process of ribosome formation in yeast. In fig. 1 the effect of the incubation temperature on the rRNA synthesis is shown. Yeast protoplasts were incubated at the indicated temperatures and subsequently labelled with 3H-uridine for a very short time (5 min). The RNA was extracted and analysed by electrophoresis on 2.6 % polyacrylamide gels. After a 5 min pulse at 29°C radioactivity is already found in all the main ribosomal precursor RNA species, and in 26 S and 17s rRNA (fig. 1c), whereas at the suboptimal temperature of 15°C only 37s precursor RNA is detectably labelled (fig. 1a). An intermediate situation is found at 23°C Exptl Cell Res 90 (1975)
98
Trapman, Rettl and Planta n-l” b
CUISO
3 i
-2
(fig. lb). Extended pulse-labelling at 15°C produced similar labelling-patterns as those shown in fig. 1b and c. Therefore, we chose pulse-labelling at 15°C as the best method to study the process of ribosome assembly in yeast. Sedimentation analysis of ribonucleoprotein particles from yeast protoplasts
265 1
-10
~20
30
Fig. 2. Abscissa: (a, c, e) fraction no.; (6, d,f) distance (cm); ordinate: (left) $H dpm x 1OV (O-O); (right) 14C dpm x 1OV (O---O). Sucrose gradient centrifugation of ribonucleoprotein particles (a, c, e) and polyacrylamide gel electrophoresis of RNA (b, d, f) of yeast protoplasts pulse-labelled for the indicated periods of time. Appropriate portions of yeast protoplasts were labelled with 3H-uridine (10 &i/ml) for 5 min (a, b), 10 min (c, d) and 5 min followed by a 10 min chase with a very large excess of unlabelled uridine (e, f). Each portion of protoplasts was then divided into Exptl
Cell Res 90 (1975)
Protoplasts were labelled for 5 and 10 min with 3H-uridine at 15°C and the ribonucleoprotein particles were extracted in a low ionic strength buffer, containing 10 mM KC1 (see Materials and Methods). The sedimentation patterns obtained by centrifugation through linear 15-30 % sucrosegradients are shown in fig. 2a, c. After labelling for 5 min radioactivity is mainly found in a particle with an approximate sedimentation coefficient of 90s (fig. 2a). The predominantly labelled RNA species under these conditions is 37s ribosomal precursor RNA as can be inferred from fig. 2b, where the corresponding electrophoretic analysis is shown of the RNA extracted from the sameprotoplasts. After labelling for 10min, two other radioactive particles appear, sedimenting at about 66S and 43 S as compared with the 60s and 40s ribosomal subunits (fig. 2~). In this case, besidesthe 37s two parts. From one part the ribonucleoprotein particles were extracted, from the other part the RNA as described in Materials and Methods. The ribonucleoprotein particles were analysed by sedimentation through a 15-30 % sucrose gradient and the RNA by electrophoresis on 2.6 % polyacrylamide gels (see Materials and Methods). Appropriate amounts of 14C-uracil-labelled 8OS, 60s and 40s ribosomal particles, and 14C-uracil-labelled 26s and 17s rRNA were added as markers in the sedimentation and electrophoretic analysis, respectively. Centrifugation was for 4 h at 34 000 rpm at 4°C in the Spinco SW41 rotor (a) or for 16 h at 18 000 rpm in the Spinco SW27 rotor (c, e). Fractions of 0.3 ml (SW41 rotor) or 0.9 ml (SW27 rotor) were collected, and assayed for radioactivity. To this end 100 @ampIes were digested for 2 h at 37°C with pancreatic RNAse (20 y/ml), and then mixed with 10 ml of a dioxane-based scintillation liquid (containing 90 g naphthalene, 7 g PPO and 50 mg POPOP per litre).
Preribosomes
from yeast
99
c
b 29s
/665/
3. A6scis.r~ distance (cm); ordinate: (left) 3H dpm x 10e2 (O-O); (right) IT dpm / 1OV’ (O---O). Polyacrylamide gel electrophoresis of pulse-labelled RNA from the (a) 90s; (b) 66s; and (c) 43s ribonucleoprotein particles. Fractions from the regions indicated in fig. 2a, e were pooled, the RNA extracted and then analysed by electrophoresis on 2.6 % polyacrylamide gels as described in Materials and Methods. T-labelled 26s and IIS rRNA was used as a reference. Fig.
rRNA precursor, also the 29 S and 18 S rRNA precursors are clearly labelled (fig. 2d). The appearance of the 3H-uridine marker into the 66s and 43 S particles, and the 29s and 18s precursor RNAs, is also observed, when a 5 min pulse is followed by a 10 min chase with a large excess of unlabelled uridine (fig. 2e, f). This indicates the existence of a precursor-product relationship between the 90s particles, and the 66s and 43 S particles. These results strongly suggest, that the 37 S, 29 S and 18 S rRNA precursors in yeast are present in the form of ribonucleoprotein particles with sedimentation coefficients of approx. 9OS, 66 S and 43 S, respectively. RNA componentsof ribonucleoprotein particles
In order to characterize more definitely the RNA constituents of the rapidly labelled ribonucleoprotein particles, the regions indicated in fig. 2a and e were pooled, the RNA was extracted by phenol-O.5 % SDS treatment,
and analysed by electrophoresis on 2.6 % polyacrylamide gels (fig. 3). The 90s particles contain 37s ribosomal precursor RNA as well as some 29s RNA (fig. 3a). The 66s and 43s particles appear to contain almost exclusively 29s and 18 S ribosomal precursor RNA, respectively (fig. 3 b, c). The presence of 29s RNA in the 90s particle is most probably due to cleavage of 37s precursor RNA taking place during the isolation of the ribonucleoprotein particles, since it was found that the RNA-constituent of the 90s particle is rather sensitive to specific and unspecific degradation. Patterns as shown in fig. 3a can only be obtained by isolating the particles in the presence of a RNAse inhibitor prepared from calf lens [24]. In the absence of this inhibitor no 37s precursor RNA can be extracted from the 90s particles. These results give additional evidence, that the 9OS, 66s and 43 S particles are indeed preribosomal particles. Exptl
Cell Res 90 (1975)
100 Trapman, Retbl and Planta Table 1. Some properties of the preribosomes as compared with those of the mature ribosomal subunits at low ionic strength (0.01 M KU)
Particle
RNA component
Buoyant density k/ml)
90s 66s 60s 43s 40s
37 S pre-rRNA 29 S pre-rRNA 26s rRNA 18 S pre-rRNA 17s rRNA
1.46 1.50 1.57 1.54 1.54
% protein 58 51 41 45 45
Percentage protein was calculated from the buoyant densities of the particles according to Perry & Kelley
WI. Protein content of ribosomal precursor particles In order to obtain information on the RNA/ protein ratio of the various precursor particles their buoyant densities in CsCl gradients were determined in essentially the same way as described by Perry & Kelley [21]. The results are summarized in table 1. The buoyant densities of the 90 S and 66 S particles appeared to be considerably lower than those of the
60s mature ribosomal subunits, whereas the 43s particle has the same buoyant density in CsCl as the 40s ribosomal subunit. These results indicate that the protein content of the 90s and 66s particle is substantially higher than that of the ribosomal subunits. From the buoyant densities one can calculate that the 9OS, 66 S and 43 S preribosomal particles have a protein content of 58, 51 and 45%, respectively. The corresponding values for the 60s and 40 S ribosomal subunits are 41 and 45 %, respectively. One possible explanation for the relatively high percentage of protein in the 90s and 66 S preribosomes is that the excess protein is derived from non-specific adsorption of cellular non-ribosomal proteins taking place during the extraction of the ribonucleoprotein particles. Therefore, we investigated the effect of high salt concentrations (0.2 or 0.5 M KCl) on both the sedimentation coefficients and the buoyant densities of the particles. From the data, summarized in table 2, it can be concluded that extraction and sedimentation of the ribonucleoprotein particles
Table 2. Effect of the KC1 concentration on the sedimentation densities of the preribosomal particles
coefficients and the buoyant
Ribonucleoprotein particles were prepared and analysed as described in Materials and Methods concentrations (0.01 M KCl, or 0.2 M KCl, or 0.5 M KCl). The sedimentation coefficients of preribosomes (containing 37s and 29s RNA, respectively) were compared with that of the large units, and the sedimentation coefficient of the smallest preribosome (containing 18s RNA) with that of the small subunit. Results are expressed as *’
= Smeribosome
- Sribosomal
at various KC1 the two largest ribosomal subwas compared
subunit
Buoyant densities in CsCl-gradients
were determined according to Perry & Kelley 1211
S-value of particles at 0.01 M KC1
0.2 M KC1 RNA-constituent
AS
Buoyant density k/ml)
AS
Buoyant density k/ml)
90s 66s 60s 43s 40s
37s pre-rRNA 29s pre-rRNA 26s rRNA 18 S pre-rRNA 17s rRNA
+23 +3 0 +3 0
1.49a 1.52 1.58 1.55 1.55
+2 -5 0 +3 0
1.53= 1.54 1.59 1.56 1.56
a The “90s”-particles Exptl
0.5 M KC1
appeared to be rather unstable at high ionic strength.
Cell Res 90 (1975)
Preribosomes
under conditions of high ionic strength resulted in a rather dramatic reduction of the sedimentation coefficients, in particular for the 37 S and 29 S RNA containing particles as compared with the large ribosomal subunit, analysed under the same conditions. On the other hand, the changes in sedimentation rate of the 18 S RNA containing particle are rather similar to those of the small ribosomal subunit. There is also some loss of protein from all particles, including the ribosomal subunits, at higher KC1 concentrations, as judged from the change in buoyant densities (table 2). However, these losses are far too small to explain the rather large changes in sedimentation rates. The reduction of the sedimentation coefficients, in particular those of the 37s and 29s containing particles, must be largely due to conformational changes induced by high salt concentrations. In addition, it appeared that even at the high, non-physiological, KC1 concentration of 0.5 M, the protein content of the particles containing 37s and 29s ribosomal precursor RNA is still higher than that of the 60s ribosomal subunit under the same conditions. This makes it unlikely that non-specific adsorption of proteins is wholly responsible for the higher protein content of these preribosomes. Cellular location of the various preribosomal particles
The cellular location of the preribosomal particles was studied by preparing from yeast protoplasts a nuclear and cytoplasmic fraction, after a short pulse with 3H-uridine (10 min) at 15°C. Both the cytoplasmic and the nuclear fraction were divided into two parts. From one part the ribonucleoprotein particles were extracted and analysed by sedimentation through a 15-30 % sucrose gradient (fig. 4a, c). From the other part the RNA was extracted by phenol-O.5 % SDS treatment, and sub-
’ I~-I 15
1 NUCLEUS 605
101
from yeast
r b
NUCLEUS
i
405
605
,.
5
605 605
405 6-
-6
,O
K)
20
30
1
2
3
4
Fig. 4. Abscissa: (a, c) fraction number; (b, d) distance (cm); ordinate: (left) 3H dpm x 1OW (O-O); (right) 14C dpm x 1OP (o---e). (a, c) Sucrose gradient centrifugation of pulselabelled nuclear (a) and cytoplasmic (c) ribonucleoprotein particles; (b, d) polyacrylamide gel electrophoresis of pulse-labelled nuclear (b) and cytoplasmic (d) RNA. An appropriate portion of yeast protoplasts was labelled with 3H-uridine (10 pCi/ml) at 15°C for 10 min. The nuclear and cytoplasmic fractions were. prepared as described in Materials and Methods. Each fraction was divided into two parts. From one part the ribonucleoprotein particles were extracted, from the other part the RNA. The ribonucleoprotein particles were analysed by sedimentation through a 1530% sucrose gradient (a, c), as described for fig. 2c, e, the RNA by electrophoresis on a 2.6 % polyacrylamide gel (b, d). Appropriate amounts of Wuracil labelled SOS, 60s and 40s ribosomal particles, and ‘*C-uracil labelled 26s and 17s rRNA were used as a reference in the sedimentation and electrophoretic analysis, respectively.
sequently analysed by electrophoresis on 2.6 % polyacrylamide gels (fig. 4b, d). The predominantly labelled particles in the nuclear fraction are the 90s and 66s preribosomes Exptl Cell Res 90 (1975)
102
Trapman, Ret61 and Planta
(fig. 4a, b). These particles are completely absent in the cytoplasm, indicating a high degree of purity of this fraction (fig. 4~0, d). The only labelled particle, detected in the cytoplasmic fraction, is the 43 S preribosome, containing the 18 S Ii-RNA precursor, the direct precursor of mature 17s rRNA. The relatively small amount of 43s in the nucleus is most probably due to a slight cytoplasmic contamination of the nuclear fraction. This result strongly suggests that the final maturation step, which converts the 43s particle into the 40s ribosomal subunit, takes place in the cytoplasm. On the other hand, the labelled 29s rRNA precursor, or the corresponding 66s preribosome, was never found in the cytoplasmic fraction, even when pulselabelling with 3H-uridine was performed for longer periods of time, suggesting that the final maturation of the large subunit takes place in the nucleus. DISCUSSION The object of this study was to examine the mode of ribosome assembly in yeast. Three ribonucleoprotein particles with sedimentation coefficients of approx. 9OS, 66s and 43 S were detected by pulse-labelling with 3Huridine at relatively low temperature. These particles were found to contain 37S, 29s and 18 S ribosomal precursor RNA, respectively. Evidence was obtained that there exists a precursor-product relationship between the 90s particles and the two other kinds of particles. Thus it may be concluded that the 43 S particles are precursors to the 40 S subunits, the 66s particles precursors to the 60s subunits, whereas the 90s particles represent precursors to both the ribosomal subunits. Particles containing the previously [ 161 described 42 S and 32 S rRNA precursors have not been found in the present study. This is not surprising, since these precursor molecules Exptl
Cell Res 90 (197.5)
can hardly be detected in the wild type of S. carlsbergensis under the usual and applied labelling conditions (see fig. 1 b, c). Only the 32s rRNA precursor is sometimes visible in the RNA-fractionation pattern as a minor component (e.g. in fig. 2d, f). An accumulation of the rather transient 42s and 32s rRNA precursors can only be brought about under special conditions, such as methioninestarvation of a methionine-requiring mutant [16], or by inhibiting protein synthesis by cycloheximide [25, 261. For the present investigations we considered the application of such conditions as less appropriate, since it is known that artificial ribonucleoprotein complexes of rRNA can be formed upon inhibition of protein synthesis [27]. Though we do not yet know whether the first ribosomal transcription product (the 42 S precursor RNA) is already associated with protein, it can be said that the mode of ribosome assembly resembles that observed in mammalian cells [3-51 and plants [ll] in the main respects. For these higher eukaryotic organisms, however, the existence of a direct precursor to the small ribosomal subunits, comparable to the 43s particles of yeast, has not been reported. In the present study we found that the 43s particles, containing the direct precursor (18 S RNA) of 17 S rRNA, are mainly located in the cytoplasmic fraction. This finding indicates that the final maturation step in the formation of the small ribosomal subunit, which converts 18 S rRNA precursor into 17s rRNA, takes place in the cytoplasm. This confirms earlier observations following a different approach (see [2]), and the very recent results obtained by Udem & Warner [28] for the yeast Saccharomyces cerevisiue. On the other hand, the 66s preribosomal particles appeared to be exclusively present in the nuclear fraction, indicating that the formation of the large ribosomal subunits is largely completed in the nucleus.
Preribosomes from yeast No indication was obtained for the occurrence of a precursor particle of the 60s subunit similar to that observed by Taber et al. in the yeast Schizosaccharomyces pombe, which sediments at 55 S in high ionic strength sucrose gradients and contains mature rRNA [29]. However, if we assume that under the conditions of isolation and analysis used by these authors the 29s precursor RNA is largely processed to mature rRNA this “55 S” particle might be equivalent to the 66s preribosome in Saccharomyces carlsbergensis, which has a comparable sedimentation rate at high ionic strength (table 2). Our finding, that in yeast both the 90s and the 66s preribosomes contain more protein than the 60s ribosomal subunit, whereas the 43s preribosome and the 40s subunit have about the same protein content, is at complete variance with the suggestion that the late steps of ribosome formation in lower eukaryotic organisms resemble bacterial ribosome assembly [15, 291. In bacteria preribosomal particles have a lower protein/ RNA ratio than the ribosomal subunits, which acquire their full complement of ribosomal proteins only at a late stage of ribosome formation [13, 141. In this respect the situation in yeast is highly similar to that in mammalian cells, where the preribosomes were found to contain a large number of excess proteins, not present in mature ribosomes [4, 51. The extra proteins of the mammalian preribosomes differ from the ribosomal proteins with respect to size and labelling kinetics. When the nucleolar precursor particles mature into cytoplasmic ribosomes, these, non-ribosomal, proteins are discarded and remain in the nucleolus [30]. The function of this special class of proteins is not yet clear, but it can be speculated that among other proteins it contains the enzymes responsible for the processing of the ribosomal precursor RNAs. The fact, that the 90s pre-
103
ribosomal particles always contain a small amount of 29s RNA, besides 37s precursor RNA, is in agreement with this suggestion. At the moment we cannot exclude the possibility that some of the ribosomal proteins are missing from the precursor particles. It was recently reported that in mammalian cells one or more proteins, present in the large ribosomal subunit, are lacking from its direct precursor [31-331. Experiments, in which the proteins of the yeast precursor particles are compared with those of the cytoplasmic ribosomes by twodimensional polyacrylamide gel electrophoresis are in progress now. The present study was supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). The authors are indebted to Mr E. Veltkamp, Mr A. Vlug, Miss G. M. Zijlstra and Mrs E. C. Koper-Zwarthoff for skilful technical assistance; to Dr W. G. M. van den Broek (Biochemisch Laboratorium, University of Nijmegen, The Netherlands) who kindly provided the RNAse inhibitor of calf lens.
REFERENCES 1. Maden, B E H, Progr biophys mol biol 22 (1971) 127. 2. Planta, R J, van den Bos, R C & Klootwijk, J, Ribosomes: Structure, function and biogenesis (ed H Bloemendal & R J Planta) p. 183. NorthHolland, Amsterdam (1972). 3. Warner, J R & Soeiro, R, Proc natl acad sci US 58 (1967) 1984. 4. Liau, M C & Perry, R J, J cell bio142 (1969) 272. 5. Kumar, J & Warner, J R, J mol bio163 (1972) 233. 6. Mirault, M E & Scherrer, K, Eur j biochem 23 (1971) 372. 7. Tamaoki, T, J mol biol I5 (I 966) 624. 8. Narayan, K S & Birnstiel, M L, Biochim biophys acta 190 (1969) 470. 9. Shepherd, J & Maden, B E H, Nature new biol 236 (1972) 211. IO. Darnell, J E, Bact rev 32 (1968) 262. II. Takahashi, N, Shimada, T, Higo, M, Higo, S & Tanifuji, S, Biochim biophys acta 262 (1972) 502. 12. Pace, N R, Bact rev 37 (1973) 562. 13. Osawa, S, Ann rev biochem 37 (1968) 109. 14. Nierhaus, K H, Bordasch, K & Homann, H E, J mol biol 74 (1973) 587. 15. Mizukami, Y & Iwabuchi, M, Biochim biophys acta 272 (1972) 81. 16. Retkl, J & Planta, R J, Biochim biophys acta 224 (1970) 458. Exptl
Cell Res 90 (1975)
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17. van den Bos, R C & Planta, R J, Biochim biophys acta 294 (1973) 464. 18. Ret&l, J & Planta, R J, Em j biochem 3 (1967) 248. 19. Sillevis Smitt, W W, Vlak, J M, Schiphof, R & Rozijn, Th H, Exptl cell res 71 (1972) 33. 20. Ret&l, J & Planta, R J, Biochim biophys acta 281 (1972) 299. 21. Perry, R P & Kelley, P E, J mol biol 16 (1966) 255. 22. Loening, U E, Biochem j 102 (1967) 251. 23. -Ibid 113 (1969) 131. 24. van den Broek, W G M, Koopmans, M A G & Bloemendal, H, Mol biol reports 1 (1974) 295. 25. Mayo, V S, Andrean, B A G & de Kloet, S R, Biochim biophys acta 169 (1968) 297. 26. Taber, R L & Vincent, W S, Biochim biophys acta 186 (1969) 317.
Exptl
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27. Craig, N C & Perry, R P, J cell bioI45 (1970) 554. 28. Udem, S A &Warner, J R, J biol them 248 (1973) 1412. 29. Taber. R L. Vincent. W S & Coetzee. M L. Biochim biophys acta 195 (1969) 99. ’ ’ Soeiro. R & Basile. C. J mol biol 79 (1973) 507. ::: Tsurugi, K, Morita; T’& Ogata, K, Eur j b&hem 25 (1972) 117. 32. Tsurugi, K, Morita, T & Ogata, K, Eur j biochem 32 (1973) 555. 33. Higashinakagawa, T & Muramatsu, M, Eur j biochem 42 (1974) 245.
Received May 10, 1974
