- Email: [email protected]
Ultrastructural and biochemical studies on ribonucleoprotein particles from isolated nucleoli of thioacetamide-treated rat liver
Experimtvtal Cell Research68 (191I) 235-246
ULTRASTRUCTURAL ON RIBONUCLEOPROTEIN
AND
BIOCHEMICAL
PARTtCLES
FROM
THIOACETAMIDE-TREATED K. KOSHIBA, Electronmicroscopy
C. THIRUMALACHARY,
STUDIES
ISOLATED RAT
Y. DASKAL
NUCLEOLI
OF
LlVER and H. BUSCH
Laboratory and Tumor By-Products Laboratory, Department Baylor College of Medicine, Houston, Tex. 77 025, USA
of Pharmacology,
SUMMARY The morphology of isolated nucleolar ribonucleoprotein particles from thioacetamide-treated rat livers was found to be very similar to those in situ. The sedimentation profiles of these nucleolar ribonucleoprotein particles in sucrose density gradients showed the presence of three components. The particles in these peaks were electron opaque spherical particles that were quite homogeneous in size (200-250 A). The ultrastructure of these RNP particles from thioacetamide-treated livers is similar to that of both ribosomes and intranucleolar RNP particles inasmuch as at high magnifications a convoluted, linear strand of RNA was observed to be present in each of the particles. In each peak of the sedimentation profile, the average diameter of the RNP particles was also 210 A. The diameters of the nucleolar granules in situ were essentially the same as those of the isolated ribonucleoprotein particles averaging 210 8, and ranging from 190-250 A. The RNA in the isolated ribonucleoprotein particles was mainly 28s RNA. Quantitative analysis indicates that the RNP particles in the most rapidly sedimenting RNP peak had a higher RNA to protein ratio than those in the less rapidly sedimenting peaks.
The nucleolus contains granular RNP elements in varying concentrations depending upon its activity, i.e. the less active nucleoli of lymphocytes, normal or actinomycin D-treated livers have fewer granular RNP particles than those of tumor cells or TAtreated livers [I]. Recently, several studies have been made on methods for isolation of the nucleolar ribonucleoprotein particles from L cell nucleoli and normal liver nucleoli [2, 31. Thus far the ultrastructure of these isolated particles has not been satisfactorily defined and it was not clear whether that of the isolated particles was identical to that of the nucleolar RNP particles in situ. Although some analyses have been made of the behavior of their RNA on sucrose density I fi
illXI4
gradients, little information is available on the composition and type of RNA in the particles or of the numbers and types of associated proteins. In part, this problem has arisen because of the relatively small amount of these ribonucleoprotein particles that could be isolated from the cells employed thus far. It has been shown in this and other laboratories [I] that the weak hepatocarcinogen, thioacetamide, causes a marked increase in the mass of liver nucleoli and, in addition, a marked increase in the number of granular elements of the nucleolus. Because of these changes [l], it seemed possible that these nucleoli might afford a richer source of ribonucleoprotein particles for isolation and Exptl Cell Rrs 68
236
K. Koshiba et al.
further studies of their roles in maturation of ribosomal precursors. The present series of experiments provides electron microscopic evidence for the similarity of these isolated RNP particles with those of the nucleolus and further, of the characteristics of their RNA. In addition, the present studies indicate that the nucleoli of thioacetamide-treated cells are a five-fold richer source of these RNP particles than are those of normal liver.
MATERIALS
AND METHODS
Animals Male Holtzman rats weighing approx. 1755225 g were used. Thioacetamide was administered intraperitoneally in a dose of 50 mg/kg body weight daily for 8 days as a 2g,, solution in 0.15 M sodium chloride.
Isolation of nucleoli from thioacetatnidetreated rats and normal rats Nuclei were isolated from livers of thioacetamidetreated rats and normal rats by a modification of the Chauveau procedure [I, 21. In some experiments, 5 mM magnesium acetate was used in place of the 3 mM calcium acetate usually employed. The nucleoli were isolated by the sonication procedure [I, 41.
Preparation of nucleolar ribonucleoprotein particles These particles were isolated by a modification of the method of Liau & Perry [3]. The nucleolar pellet was suspended in 5 ml of a buffer containing 0.25 M sucrose. 0.01 M sodium acetate, 0.05 M KC], 0.002 M MgCI, and 0.01 % diethylpyrocarbonate (pH 6.0). To provide an indication of mass, its 260 nm absorbance was determined by solubilizing an aliquot in 0.5 0,) SDS solution. After the concentration of the nucleolar suspension was adjusted to approx. I mg/ml (IO OD.,, ~ I mg) with the same buffer, the suspension was centrifuged at 13 000 rpm for 10 min. The pellet was washed with a similar buffer lacking diethylpyrocarbonate to remove traces of diethylpyrocarbonatc in the pellet. The washed nucleolar pellet was treated with DNase (IO /Ag/mg) for 20 min in ice. Polyvinylsulfate (PVS) was added to a final concentration of 40 //g/ml [8]. After standing 10 min in ice, the suspension was recentrifuged at 13 000 rpm for 10 min. The pellet was homogenized with a loose Teflon homogenizer in 3-5 ml of TrisHCI buffer (pH 8.5) containing 0.5 mM M&I,, 0.01 M potassium chloride and 0.02 M dithiothreitol. Exptl Cdl Rrs 68
The ribonucleoprotein particles were extracted by incubation of the suspension at room temperature for 15 min. The suspension was then centrifuged at I3 000 rpm for I5 min [3, 61.
Density gradient centr(fugation Approx. 1-I .5 ml of the solution of ribonucleoprotein particles (2.5-7.5 mg/ml) was carefully layered over a 34 ml linear 15-55”; (w/w) sucrose gradient containing 0.5 mM MgCI,, 0.01 M potassium chloride, 0.001 M dithiothreitol, 0.01 M triethanolamine HCI (pH 7.4) and centrifuged at 22 000 rpm for I5 h in the SW 27 Spinco rotor. The gradients were analysed by recording the optical density at 254 nm in an ISCO ultraviolet analyser [3].
Extraction of RNA The RNA was extracted by the cold SDS-phenol method [4, 81.
Enzymatic digestions The specimens were fixed in IO (lo formaldehyde containing I M phosphate buffer (pH 7.4) for 10 min at 4”C, and then washed twice with 0.1 M phosphate buffer at the same pH. Digestion procedures’were carried out after washing with 0. I M phosphate buffer and were terminated by treatment with trichloroacetic acid or osmium tetroxide fl. 71. The following enzymatic digestion procedures were used: pepsin twice crvstallized. 0.01 or 0.05 me/ml. in 0.1 N HCI for 10 *min; pancreatic ribonucieask, crystallized, I mg/ml at pH 6.5 for 2 or 3 h; deoxyribonuclease electrophoretically purified, free of ribonuclease, 1 mg/ml in 0.003 M magnesium acetate; pepsin followed by ribonuclease; ribonuclease followed by pepsin. The specimens were washed three times with 0.1 M phosphate buffer at pH 7.4 before treatment with the second enzyme. Ribonuclease dissolved in acetate buffer at pH 5 was heated at 80°C for IO min to remove deoxyribonuclease activity and then the pH was adjusted to 6.5 with 0.01 N NaOH. The enzymes used in the present study were obtained from Worthington Biochemical Corp., Freehold, N.J.
Electron microscopy The specimens were fixed in I o,, glutaraldehyde in 0.1 M sodium cacodvlate buffer at aH 7.4 for 20 min at 4°C washed twice with the same buffer and postfixed in a solutioncontaining % osmium tetroxide and 0.14 M Verona] acetate buffer at pH 7.4 for 1 h [I, 71. The specimens were then dehydrated through a series of ethanol solutions of graded concentration, embedded in Epon or Epon-Araldite, and sectioned with a glass knife using a Porter Blum microtome (MT-2). The sections were mounted on copper grids and stained with uranyl acetate and lead citrate. Specimens were examined with a Philips EM-200 electron microscope.
Ultrastructure and biochemistry of ribanuclein particles
237
Fig. 1. Electron micrograph of a nucleus isolated from a thioacetamide-treated rat liver. The largt : nucleolus
(N) is almost completely composed of granular elements. The nucleoplasm contains pet-ichromati n granules (hluck UWOW)and interchromatin granules (point) which are well demarcated. Nucleolus associated chromatin (C). Glutaraldehyde-osmium fixation; uranyl acetate-lead citrate stain. :i 39 000. Fig. 2. Higher magnification electron micrograph of a portion of a nucleolus isolated frc ,rn a thio acetamidetreated rat liver. The isolated nucleoli were digested with DNase (10 {(g/ml) for 40 min at 0°C and then fixed with osmium tetroxide. The nucleolar ribonucleoprotein particles (points) are irregular in skIape; thei r diameters range from 200-250 A. x 120 000.
238
K. Koshiba et al.
Fig. 3. Electron micrograph of an isolated nucleolus from a thioacetamide-treated rat liver, fixed w ith formalin and then digested with DNase (IO ,trg/ml for 40 min) and then by pepsin, 0.05 mg/ml in 0.01 N HCI for IO min. After these digestions, the nucleolar ribonucleoprotein particles have not disaf speared completely. .120000. Fig.-i. Electron micrograph of isolated nucleolar RNP particles of thioacetamide-treated rat liver from peak I of the sucrose gradient pattern shown in fig. 5. They consist of electron-opaque spherical particles 200-250 8, in diameter. Some thread-like extensions appear to be joined to some of these particles (h/a<:k cwrow ). Glutaraldehyde-osmium fixation; uranyl acetate-lead citrate stain. I20 000. Expfl
Cell Rrs 68
Ultrastructure
239
particles
Nuclwlar RNP Thioacetamldelreated
Rat Liver
RNP particles
As noted earlier [I], the nucleoli of thioacetamide-treated rat liver contain dense concentrations of granular elements (fig. I). Fig. 2 shows the appearance of such nucleoli after digestion with DNase (10 ,ug/ml) for 50 min at O’C. The nucleolar ribonucleoprotein particles are closely packed, round or oval in shape and range in size from 200 to 250 A. The average diameter of the particles analysed was 210 A. The diameters of the larger particles were about 250 A and those of the smaller particles were about 175 A. After treatment with DNase followed by mild digestion with pepsin, the density of these particles diminished, but they are visible as distinct entities (fig. 3). At these high magnifications ( 7 120 000), the nucleolar ribonucleoprotein (RNP) particles appear to be somewhat heterogeneous in shape. RNase digestion (1 mg/ml) resulted in destruction of the fine filamentous elements of the RNP particles and a more diffuse appearance which suggests that the filaments in these particles contain RNA. In addition, the fine filaments emerging from the surfaces of the particles also disappeared (fig. 4). Morphology particles
of rihonuclein
0.5 IT
RESULTS Morphology of liver nucleolar of thioacetan7ide-treated rats
and biochemistry
of isolated nucleolar
R NP
When the nucleolar RNP particles were isolated from thioacetamide-treated rat liver (fig. 4) as described in Materials and Methods and subjected to sucrose density gradient centrifugation, three distinct peaks were found (fig. 5). As shown in fig. 4, the particles in these peaks were electron-opaque, spherical and 200 to 250 A in diameter. The average diameters of these isolated RNP particles were 207 A. The diameters of the larger particles were about 250 A.
/ I15W
i
10
15
LO
25
30
i55Cl
(li%l
5
10
15
20
25
10
i55W
0 5 0 4
Fig. 5. Ahscissu: fraction number; ordinate:
A,,,,. Sucrose density gradient profile of RNP particles extracted as described in Materials and Methods. Approx. I to IO mg of RNP particles were placed on I5555 “,, (w/w) sucrose gradients containing 0.01 M triethanolamine-HCI, pH 7.4, 0.5 mM MgCI,, 0.01 M KCI, 0.001 M dithiothreitol and centrifuged in the SW 27 rotor at 22 000 rpm for I5 h.
As shown in figs 6 and 7, peaks 2 and 3 also contained RNP particles with diameters ranging from 200 to 250 A. The average diameters of these particles was 210 8, (fig. 7). In each of the peaks, some of the particles had thread-like extensions. At high magnifications, these RNP particles appear to consist of fine granular structures and filaments about 25 A in diameter (fig. 8). These filaments appear to be cross sections of long strands of electrondense fibrils which have been shown to be the unit component of the ribosomal subunits and nucleolar RNP particles [I]. When negatively stained with uranyl acetate, the particles appear to have a round or slightly elongated form (fig. 9). When the particles were extracted from nucleoli isolated in media containing Ca” I, they had a more compact appearance (figs 10. 11) than those isolated in media contain-
240
K. Koshiba et al.
Fig. 6. Electron micrograph of nucleolar RNP particles of thioacetamide-treated rat liver showing particles of peak 2 of the sucrose density gradient pattern (fig. 5). They consist of electron-opaque spherical particles 200-250 A in diameter. Thread-like extensions of many particles are noted (bluck a~uw). Glutaraldehydeosmium fixation; uranyl-acetate-lead citrate stain. 120 000. Fig. 7. Electron micrograph of nucleolar RNP particles of thioacetamide-treated rat liver. This photograph shows particles of peak 3 of the sucrose density gradient pattern (fig. 5). They consist of electron-opaque spherical particles which measure 200-250 ti in diameter. This fraction shows aggregation of some particles. “Thread-like” extensions are noted (black anow). Glutaraldehyde-osmium fixation: uranyl acetate-lead citrate stain. 120 000. Exptl Cell Res 68
Ultrastructure and biochemistry of ribondein
particles
241
Fig. 8. High magnification electron micrograph of nucleolar RNP particles of fig. 4 These uarti cles consist of fine granular structures about 25 8, in diameter (black auvow). Some particles c ontain small thread-like structures in their centers (large black arrow) which are probably RNA strands (point) *218000. Fig. 9. High magnification electron micrograph of unfixed nucleolar RNP particles, negatively stained with 1 0,) uranyl acetate. This preparation is the same as that of fig. 4. The particles have either almoz ;t round or slightly elongated polygonal forms. * 390 000. Exptl Cell Rrs 68
242 K. Koshiba et al.
Fig. 10. Electron micrograph of nucleolar RNP particles of thioacetamide-treated rat liver from peak I of the sucrose density gradient pattern (fig. 5). Glutaraldehyde-osmium fixation; uranyl acetate-citrate stain. These nucleoli were isolated in the presence of Ca2-. 120 000. Fig. II. Electron micrograph of nucleolar RNP particles of normal rat liver isolated in the presence of Ca”’ from peak 2 of the sucrose density gradient (fig. 5). Glutaraldehyde-osmium fixation; many1 acetate-lead citrate stain. h. 120 000.
Ultrastructure and biochemistry of ribonuclein particles
243
F&. 12. Electron micrograph of nucleolar RNP particles of normal rat liver. These nucleoli were isolated in the presence of Mg”+. This electron micrograph shows the particles in a pooled preparation of peaks l-3 from the sucrose density gradients (fig. 5). These particles are electron-opaque and spherical and range in diameter from 200&250 A. Glutaraldehyde-osmium fixation; uranyl acetate-lead citrate stain. _’ 119 600. Fig. 13. Electron micrograph of nucleolar RNP particles of normal rat liver. These nucleoli were isolated in the presence of CaZ’ . This electron micrograph shows the particles in a pooled preparation of peaks 1-3 from the sucrose density gradients (fig. 5). Appearance of these particles is essentially the same as those in fig. II. Glutaraldehyde-osmium fixation; uranyl acetate-lead citrate stain. :i I19 600. E.uptl Cdl Res 68
244
K. Koshiba
et al.
Table 1, Yield of nucleolar
RNP particles Tissue yield (mg/lOO g wet wt)
Normal rat liver Thioacetamide-treated rat liver Novikoff hepatoma
(260 nm) in various nucleolar
7.76 -t 0.42 9.08 + I .07
ing Mg2+. When the nucleolar RNP particles were extracted from normal liver nucleoli isolated in media containing either Mg”+ (fig. 12) or Ca 2+ (fig. 13), their appearance was the same as those obtained from thioacetamide-treated rat liver. The sucrose density gradient centrifugation patterns were essentially the same as those obtained from thioacetamide-treated livers (fig. 5). of nucleolav R NP
To determine the relative yields of nucleolar RNP particles, the nucleolar extract was subjected to sucrose density gradient centrifugation (fig. 5) and the quantities of RNP in the peaks was determined for normal rat liver, TA-treated rat liver and Novikoff hepatoma (tables I, 2). The percentages of E,,,, absorbing substances extracted with the procedures employed were 33, 51 and 64?, for normal rat liver, TA-treated rat liver and Novikoff hepatoma, respectively. Of the total extracted, 12, 43 and 18 0o were in the RNP peaks of the normal rat liver, TAtreated rat liver and Novikoff hepatoma, respectively. Accordingly, the yield of RNP particles was almost 30 times higher in the TA-treated rat liver than in the normal rat liver (table I). Thus, the TA-treated rat liver Exprl Cell Res 68
absorbancy fractions
0.26 x 0.01
In these experiments, peaks I-3 of gig. 5 were collected from the gradients and their optical density was determined. The values are the averages of the recoveries for 4, 8 and 6 separate experiments for normal thioacetamide and Novikoff, respectively and are based on the formula I mg 10 OD,,, units. The standard errors are shown.
Yield and distribution particles
Table 2. Percentage of total nucleolar
Nucleolar extract Top fraction of the gradient RNP Fraction 1 11 111 Nucleolar residue
Normal rat liver
TAtreated rat liver
Novikoff hepatoma
33.3
51.3
64.0
29.3
28.3
53.0
1.3 0.7 2.1 66.6
5.6 4.5 12.2 48.7
3.8 3.2 5.2 36.0
The nucleolar extract (20 000 g supernatant) was prepared as described in Materials and Methods. The total absorbancy of whole nucleoli was determined after the nucleoli were solubilized in 0.5 ‘Y, SDS (sodium dodecyl sulfate). The absorbancy of the nucleolar extract was subtracted to give the value for the nucleolar residue. Each value is the average of experiments in which the absorbancy of a given fraction was determined at 260 nm.
is a much better source of RNP particles than normal rat liver. The RNP particles are composed only of protein and RNA (table 3). Some differences were found in the composition of the RNP particles of peak 3 and peaks 1 and 2, i.e., peak 3 contained somewhat more RNA and somewhat less protein than peaks I and 2. However, the results for peaks 1 and 2 were Table 3. Composition
of nucleolar RNP peaks
Fraction
Treated
Protein’ ql,I
Protein’ /111 (3)
RNA o<,
RNA Protein
I II III I II III
TA TA TA Nor Nor Nor
45.4 45.5 39.7 39.4 41.4 32.0
44.2 53.0 57.0c
54.6 54.5 60.3 60.6 58.6 68.0
1.2 1.2 1.5 I.5 I.4 2.1
u Extimated by folin-BSA as standard. ’ Estimated by orcinol-ribose and yeast RNA as standards. ’ Calculated from estimation of buoyant densities of protein 1.250 and RNA ~; I .900 [3].
Ultrastructure and biochemistry 0,s 1
particles
245
DISCUSSION 285 A
5%)
of ribonuclein
5
10
15
20
!
25
30
I40961
fraction number; ordinate: A,,,,. Sucrose density. gradient profile of RNA of the nucleolar RNP particles of thioacetamide-treated rat livers. The sucrose density gradient (5-40”,,) containing I mM EDTA, 0.1 M NaCI, 0.02 M sodium acetate buffer, pH 5.1 was centrifuged for I6 h at 26 000 rpm in a SW-27 rotor. Fig. 14. Abscissa:
virtually the same. The data of Liau et al. [3] which are shown for comparison differ in that they found a higher protein content of the more rapidly sedimenting peaks. The reasons for this discrepancy are not entirely clear but may be related to the fact that their particles were obtained from L cells and their analytical procedure was different. RNA of the nucleolar RNP particles In the RNA extracted from either whole RNP particles (fig. 14) or from the three RNP peaks, 28s RNA was the predominant species. The shoulder with an approximate sedimentation coefficient of 32-35s may result from aggregation of the 28s RNA [9]. More rapidly sedimenting nucleolar RNA could not be detected. In studies in progress, Prestayko et al. [5] have found that the 4-8s RNA of these nucleolar particles contains U3 RNA [I], 8s RNA and other low molecular weight RNA species. Since these RNAs as well as the 28s RNA are present in all of the particles with the different sedimentation coefficients. it seems possible that these particles represent various stages of maturation of nucleolar RNP particles leading to the formation of cytoplasmic ribosomes.
Although the granular elements of the nucleolus have been extensively studied in recent years [I], the ribonucleoprotein particles which have been isolated by a number of authors [3, 6, IO] have not been examined in detail by electron microscopy. The only paper which has presented some structural information is that of Shankar Narayan & Birnstiel [6] in which negative staining of the particles was employed to demonstrate their irregular shape and the presence of some fibrillar structures. In addition, as shown in this study, the overall appearance of the particles was found to be roughly spherical or oblate. Since positive staining was not employed, it was difficult to evaluate precisely the components of these particles. The present studies have indicated that the constitution and ultrastructure of these particles is similar to that of ribosomal subunits. particularly the 60s ribosomal subunit and the nucleolar RNP particles. These particles contain a ribonucleoprotein filament that appears to be twisted and turned on itself at a number of points. This subcoiling gives a multifaceted appearance to the RNP particle. Treatment with pepsin or RNase did not destroy the integrity of the particle. but decreased its electron microscopic definition and the electron density of the internal filamentous structures. The nucleoli of thioacetamide-treated liver exhibit two major differences from those of normal liver, i.e., a marked increase in the relative number of RNP particles so that the overall appearance of these nucleoli is far more granular than those of normal liver cells, and secondly, their size is greater. In addition, the same methods for extraction of the RNP particles produces a 30-fold increase in the yield of the nucleolar RNP particles per 100 g of liver. These higher yields will Exptl Cell Res 6X
246
K. Koshiba et al.
permit a more satisfactory approach to analysis of the types of high and low molecular weight RNA in these particles and a more detailed study of their protein composition that has hitherto been possible. It is not clear why the nucleolar RNP particles sediment in several, usually 3, major peaks. The components of two of the peaks do not differ markedly from one another either in sedimentation patterns for the RNA or in the relative content of RNA and protein. On the other hand, the protein content of the slower sedimenting peaks seems to be somewhat greater than that of the more rapidly sedimenting peaks. It is possible that the RNP particles in these peaks differ in their constituent proteins, but it remains for future studies to determine whether these proteins can be isolated in a satisfactorily pure form and yield to permit detailed characterization and studies on their structure.
Exptl Cell Rr.r 68
This work was supported in part by Cancer Center Grant CA-10893 P.5 and American Cancer Society Grant P-339.
REFERENCES I. Busch, H & Smetana, K, The nucleolus. Academic Press, New York (1970). 2. Chauveau, J, MoulC, Y & Rouiller, C, Exptl cell res I I (1956) 317. 3. Liau, M C & Perry, R F, J cell biol 42 (1969) 212. 4. Muramatsu, M & Busch, H, Methods in cancer
research, p. 303. Academic Press, New York (I 967).
5. Prestayko, A W. In manuscript. 6. Shankar Narayan, K & Birnstiel, M L, Biochim biophys acta 190 (1969) 470. 7. Smetana, K, Methods in cancer research, p. 362. Academic Press, New York (1967). 8. Steele, W J & Busch, H, Methods in cancer research, p. 61. Academic Press, New York ( 1967). 9. Wagner, E K, Katz, L & Penman, S, Biochem biophys res commun 28 (1967) 152. IO. Warner, J R & Soeiro, R, Proc natl acad sci US 58 (1967) 1984.
Received April 20, 1971
ULTRASTRUCTURAL ON RIBONUCLEOPROTEIN
AND
BIOCHEMICAL
PARTtCLES
FROM
THIOACETAMIDE-TREATED K. KOSHIBA, Electronmicroscopy
C. THIRUMALACHARY,
STUDIES
ISOLATED RAT
Y. DASKAL
NUCLEOLI
OF
LlVER and H. BUSCH
Laboratory and Tumor By-Products Laboratory, Department Baylor College of Medicine, Houston, Tex. 77 025, USA
of Pharmacology,
SUMMARY The morphology of isolated nucleolar ribonucleoprotein particles from thioacetamide-treated rat livers was found to be very similar to those in situ. The sedimentation profiles of these nucleolar ribonucleoprotein particles in sucrose density gradients showed the presence of three components. The particles in these peaks were electron opaque spherical particles that were quite homogeneous in size (200-250 A). The ultrastructure of these RNP particles from thioacetamide-treated livers is similar to that of both ribosomes and intranucleolar RNP particles inasmuch as at high magnifications a convoluted, linear strand of RNA was observed to be present in each of the particles. In each peak of the sedimentation profile, the average diameter of the RNP particles was also 210 A. The diameters of the nucleolar granules in situ were essentially the same as those of the isolated ribonucleoprotein particles averaging 210 8, and ranging from 190-250 A. The RNA in the isolated ribonucleoprotein particles was mainly 28s RNA. Quantitative analysis indicates that the RNP particles in the most rapidly sedimenting RNP peak had a higher RNA to protein ratio than those in the less rapidly sedimenting peaks.
The nucleolus contains granular RNP elements in varying concentrations depending upon its activity, i.e. the less active nucleoli of lymphocytes, normal or actinomycin D-treated livers have fewer granular RNP particles than those of tumor cells or TAtreated livers [I]. Recently, several studies have been made on methods for isolation of the nucleolar ribonucleoprotein particles from L cell nucleoli and normal liver nucleoli [2, 31. Thus far the ultrastructure of these isolated particles has not been satisfactorily defined and it was not clear whether that of the isolated particles was identical to that of the nucleolar RNP particles in situ. Although some analyses have been made of the behavior of their RNA on sucrose density I fi
illXI4
gradients, little information is available on the composition and type of RNA in the particles or of the numbers and types of associated proteins. In part, this problem has arisen because of the relatively small amount of these ribonucleoprotein particles that could be isolated from the cells employed thus far. It has been shown in this and other laboratories [I] that the weak hepatocarcinogen, thioacetamide, causes a marked increase in the mass of liver nucleoli and, in addition, a marked increase in the number of granular elements of the nucleolus. Because of these changes [l], it seemed possible that these nucleoli might afford a richer source of ribonucleoprotein particles for isolation and Exptl Cell Rrs 68
236
K. Koshiba et al.
further studies of their roles in maturation of ribosomal precursors. The present series of experiments provides electron microscopic evidence for the similarity of these isolated RNP particles with those of the nucleolus and further, of the characteristics of their RNA. In addition, the present studies indicate that the nucleoli of thioacetamide-treated cells are a five-fold richer source of these RNP particles than are those of normal liver.
MATERIALS
AND METHODS
Animals Male Holtzman rats weighing approx. 1755225 g were used. Thioacetamide was administered intraperitoneally in a dose of 50 mg/kg body weight daily for 8 days as a 2g,, solution in 0.15 M sodium chloride.
Isolation of nucleoli from thioacetatnidetreated rats and normal rats Nuclei were isolated from livers of thioacetamidetreated rats and normal rats by a modification of the Chauveau procedure [I, 21. In some experiments, 5 mM magnesium acetate was used in place of the 3 mM calcium acetate usually employed. The nucleoli were isolated by the sonication procedure [I, 41.
Preparation of nucleolar ribonucleoprotein particles These particles were isolated by a modification of the method of Liau & Perry [3]. The nucleolar pellet was suspended in 5 ml of a buffer containing 0.25 M sucrose. 0.01 M sodium acetate, 0.05 M KC], 0.002 M MgCI, and 0.01 % diethylpyrocarbonate (pH 6.0). To provide an indication of mass, its 260 nm absorbance was determined by solubilizing an aliquot in 0.5 0,) SDS solution. After the concentration of the nucleolar suspension was adjusted to approx. I mg/ml (IO OD.,, ~ I mg) with the same buffer, the suspension was centrifuged at 13 000 rpm for 10 min. The pellet was washed with a similar buffer lacking diethylpyrocarbonate to remove traces of diethylpyrocarbonatc in the pellet. The washed nucleolar pellet was treated with DNase (IO /Ag/mg) for 20 min in ice. Polyvinylsulfate (PVS) was added to a final concentration of 40 //g/ml [8]. After standing 10 min in ice, the suspension was recentrifuged at 13 000 rpm for 10 min. The pellet was homogenized with a loose Teflon homogenizer in 3-5 ml of TrisHCI buffer (pH 8.5) containing 0.5 mM M&I,, 0.01 M potassium chloride and 0.02 M dithiothreitol. Exptl Cdl Rrs 68
The ribonucleoprotein particles were extracted by incubation of the suspension at room temperature for 15 min. The suspension was then centrifuged at I3 000 rpm for I5 min [3, 61.
Density gradient centr(fugation Approx. 1-I .5 ml of the solution of ribonucleoprotein particles (2.5-7.5 mg/ml) was carefully layered over a 34 ml linear 15-55”; (w/w) sucrose gradient containing 0.5 mM MgCI,, 0.01 M potassium chloride, 0.001 M dithiothreitol, 0.01 M triethanolamine HCI (pH 7.4) and centrifuged at 22 000 rpm for I5 h in the SW 27 Spinco rotor. The gradients were analysed by recording the optical density at 254 nm in an ISCO ultraviolet analyser [3].
Extraction of RNA The RNA was extracted by the cold SDS-phenol method [4, 81.
Enzymatic digestions The specimens were fixed in IO (lo formaldehyde containing I M phosphate buffer (pH 7.4) for 10 min at 4”C, and then washed twice with 0.1 M phosphate buffer at the same pH. Digestion procedures’were carried out after washing with 0. I M phosphate buffer and were terminated by treatment with trichloroacetic acid or osmium tetroxide fl. 71. The following enzymatic digestion procedures were used: pepsin twice crvstallized. 0.01 or 0.05 me/ml. in 0.1 N HCI for 10 *min; pancreatic ribonucieask, crystallized, I mg/ml at pH 6.5 for 2 or 3 h; deoxyribonuclease electrophoretically purified, free of ribonuclease, 1 mg/ml in 0.003 M magnesium acetate; pepsin followed by ribonuclease; ribonuclease followed by pepsin. The specimens were washed three times with 0.1 M phosphate buffer at pH 7.4 before treatment with the second enzyme. Ribonuclease dissolved in acetate buffer at pH 5 was heated at 80°C for IO min to remove deoxyribonuclease activity and then the pH was adjusted to 6.5 with 0.01 N NaOH. The enzymes used in the present study were obtained from Worthington Biochemical Corp., Freehold, N.J.
Electron microscopy The specimens were fixed in I o,, glutaraldehyde in 0.1 M sodium cacodvlate buffer at aH 7.4 for 20 min at 4°C washed twice with the same buffer and postfixed in a solutioncontaining % osmium tetroxide and 0.14 M Verona] acetate buffer at pH 7.4 for 1 h [I, 71. The specimens were then dehydrated through a series of ethanol solutions of graded concentration, embedded in Epon or Epon-Araldite, and sectioned with a glass knife using a Porter Blum microtome (MT-2). The sections were mounted on copper grids and stained with uranyl acetate and lead citrate. Specimens were examined with a Philips EM-200 electron microscope.
Ultrastructure and biochemistry of ribanuclein particles
237
Fig. 1. Electron micrograph of a nucleus isolated from a thioacetamide-treated rat liver. The largt : nucleolus
(N) is almost completely composed of granular elements. The nucleoplasm contains pet-ichromati n granules (hluck UWOW)and interchromatin granules (point) which are well demarcated. Nucleolus associated chromatin (C). Glutaraldehyde-osmium fixation; uranyl acetate-lead citrate stain. :i 39 000. Fig. 2. Higher magnification electron micrograph of a portion of a nucleolus isolated frc ,rn a thio acetamidetreated rat liver. The isolated nucleoli were digested with DNase (10 {(g/ml) for 40 min at 0°C and then fixed with osmium tetroxide. The nucleolar ribonucleoprotein particles (points) are irregular in skIape; thei r diameters range from 200-250 A. x 120 000.
238
K. Koshiba et al.
Fig. 3. Electron micrograph of an isolated nucleolus from a thioacetamide-treated rat liver, fixed w ith formalin and then digested with DNase (IO ,trg/ml for 40 min) and then by pepsin, 0.05 mg/ml in 0.01 N HCI for IO min. After these digestions, the nucleolar ribonucleoprotein particles have not disaf speared completely. .120000. Fig.-i. Electron micrograph of isolated nucleolar RNP particles of thioacetamide-treated rat liver from peak I of the sucrose gradient pattern shown in fig. 5. They consist of electron-opaque spherical particles 200-250 8, in diameter. Some thread-like extensions appear to be joined to some of these particles (h/a<:k cwrow ). Glutaraldehyde-osmium fixation; uranyl acetate-lead citrate stain. I20 000. Expfl
Cell Rrs 68
Ultrastructure
239
particles
Nuclwlar RNP Thioacetamldelreated
Rat Liver
RNP particles
As noted earlier [I], the nucleoli of thioacetamide-treated rat liver contain dense concentrations of granular elements (fig. I). Fig. 2 shows the appearance of such nucleoli after digestion with DNase (10 ,ug/ml) for 50 min at O’C. The nucleolar ribonucleoprotein particles are closely packed, round or oval in shape and range in size from 200 to 250 A. The average diameter of the particles analysed was 210 A. The diameters of the larger particles were about 250 A and those of the smaller particles were about 175 A. After treatment with DNase followed by mild digestion with pepsin, the density of these particles diminished, but they are visible as distinct entities (fig. 3). At these high magnifications ( 7 120 000), the nucleolar ribonucleoprotein (RNP) particles appear to be somewhat heterogeneous in shape. RNase digestion (1 mg/ml) resulted in destruction of the fine filamentous elements of the RNP particles and a more diffuse appearance which suggests that the filaments in these particles contain RNA. In addition, the fine filaments emerging from the surfaces of the particles also disappeared (fig. 4). Morphology particles
of rihonuclein
0.5 IT
RESULTS Morphology of liver nucleolar of thioacetan7ide-treated rats
and biochemistry
of isolated nucleolar
R NP
When the nucleolar RNP particles were isolated from thioacetamide-treated rat liver (fig. 4) as described in Materials and Methods and subjected to sucrose density gradient centrifugation, three distinct peaks were found (fig. 5). As shown in fig. 4, the particles in these peaks were electron-opaque, spherical and 200 to 250 A in diameter. The average diameters of these isolated RNP particles were 207 A. The diameters of the larger particles were about 250 A.
/ I15W
i
10
15
LO
25
30
i55Cl
(li%l
5
10
15
20
25
10
i55W
0 5 0 4
Fig. 5. Ahscissu: fraction number; ordinate:
A,,,,. Sucrose density gradient profile of RNP particles extracted as described in Materials and Methods. Approx. I to IO mg of RNP particles were placed on I5555 “,, (w/w) sucrose gradients containing 0.01 M triethanolamine-HCI, pH 7.4, 0.5 mM MgCI,, 0.01 M KCI, 0.001 M dithiothreitol and centrifuged in the SW 27 rotor at 22 000 rpm for I5 h.
As shown in figs 6 and 7, peaks 2 and 3 also contained RNP particles with diameters ranging from 200 to 250 A. The average diameters of these particles was 210 8, (fig. 7). In each of the peaks, some of the particles had thread-like extensions. At high magnifications, these RNP particles appear to consist of fine granular structures and filaments about 25 A in diameter (fig. 8). These filaments appear to be cross sections of long strands of electrondense fibrils which have been shown to be the unit component of the ribosomal subunits and nucleolar RNP particles [I]. When negatively stained with uranyl acetate, the particles appear to have a round or slightly elongated form (fig. 9). When the particles were extracted from nucleoli isolated in media containing Ca” I, they had a more compact appearance (figs 10. 11) than those isolated in media contain-
240
K. Koshiba et al.
Fig. 6. Electron micrograph of nucleolar RNP particles of thioacetamide-treated rat liver showing particles of peak 2 of the sucrose density gradient pattern (fig. 5). They consist of electron-opaque spherical particles 200-250 A in diameter. Thread-like extensions of many particles are noted (bluck a~uw). Glutaraldehydeosmium fixation; uranyl-acetate-lead citrate stain. 120 000. Fig. 7. Electron micrograph of nucleolar RNP particles of thioacetamide-treated rat liver. This photograph shows particles of peak 3 of the sucrose density gradient pattern (fig. 5). They consist of electron-opaque spherical particles which measure 200-250 ti in diameter. This fraction shows aggregation of some particles. “Thread-like” extensions are noted (black anow). Glutaraldehyde-osmium fixation: uranyl acetate-lead citrate stain. 120 000. Exptl Cell Res 68
Ultrastructure and biochemistry of ribondein
particles
241
Fig. 8. High magnification electron micrograph of nucleolar RNP particles of fig. 4 These uarti cles consist of fine granular structures about 25 8, in diameter (black auvow). Some particles c ontain small thread-like structures in their centers (large black arrow) which are probably RNA strands (point) *218000. Fig. 9. High magnification electron micrograph of unfixed nucleolar RNP particles, negatively stained with 1 0,) uranyl acetate. This preparation is the same as that of fig. 4. The particles have either almoz ;t round or slightly elongated polygonal forms. * 390 000. Exptl Cell Rrs 68
242 K. Koshiba et al.
Fig. 10. Electron micrograph of nucleolar RNP particles of thioacetamide-treated rat liver from peak I of the sucrose density gradient pattern (fig. 5). Glutaraldehyde-osmium fixation; uranyl acetate-citrate stain. These nucleoli were isolated in the presence of Ca2-. 120 000. Fig. II. Electron micrograph of nucleolar RNP particles of normal rat liver isolated in the presence of Ca”’ from peak 2 of the sucrose density gradient (fig. 5). Glutaraldehyde-osmium fixation; many1 acetate-lead citrate stain. h. 120 000.
Ultrastructure and biochemistry of ribonuclein particles
243
F&. 12. Electron micrograph of nucleolar RNP particles of normal rat liver. These nucleoli were isolated in the presence of Mg”+. This electron micrograph shows the particles in a pooled preparation of peaks l-3 from the sucrose density gradients (fig. 5). These particles are electron-opaque and spherical and range in diameter from 200&250 A. Glutaraldehyde-osmium fixation; uranyl acetate-lead citrate stain. _’ 119 600. Fig. 13. Electron micrograph of nucleolar RNP particles of normal rat liver. These nucleoli were isolated in the presence of CaZ’ . This electron micrograph shows the particles in a pooled preparation of peaks 1-3 from the sucrose density gradients (fig. 5). Appearance of these particles is essentially the same as those in fig. II. Glutaraldehyde-osmium fixation; uranyl acetate-lead citrate stain. :i I19 600. E.uptl Cdl Res 68
244
K. Koshiba
et al.
Table 1, Yield of nucleolar
RNP particles Tissue yield (mg/lOO g wet wt)
Normal rat liver Thioacetamide-treated rat liver Novikoff hepatoma
(260 nm) in various nucleolar
7.76 -t 0.42 9.08 + I .07
ing Mg2+. When the nucleolar RNP particles were extracted from normal liver nucleoli isolated in media containing either Mg”+ (fig. 12) or Ca 2+ (fig. 13), their appearance was the same as those obtained from thioacetamide-treated rat liver. The sucrose density gradient centrifugation patterns were essentially the same as those obtained from thioacetamide-treated livers (fig. 5). of nucleolav R NP
To determine the relative yields of nucleolar RNP particles, the nucleolar extract was subjected to sucrose density gradient centrifugation (fig. 5) and the quantities of RNP in the peaks was determined for normal rat liver, TA-treated rat liver and Novikoff hepatoma (tables I, 2). The percentages of E,,,, absorbing substances extracted with the procedures employed were 33, 51 and 64?, for normal rat liver, TA-treated rat liver and Novikoff hepatoma, respectively. Of the total extracted, 12, 43 and 18 0o were in the RNP peaks of the normal rat liver, TAtreated rat liver and Novikoff hepatoma, respectively. Accordingly, the yield of RNP particles was almost 30 times higher in the TA-treated rat liver than in the normal rat liver (table I). Thus, the TA-treated rat liver Exprl Cell Res 68
absorbancy fractions
0.26 x 0.01
In these experiments, peaks I-3 of gig. 5 were collected from the gradients and their optical density was determined. The values are the averages of the recoveries for 4, 8 and 6 separate experiments for normal thioacetamide and Novikoff, respectively and are based on the formula I mg 10 OD,,, units. The standard errors are shown.
Yield and distribution particles
Table 2. Percentage of total nucleolar
Nucleolar extract Top fraction of the gradient RNP Fraction 1 11 111 Nucleolar residue
Normal rat liver
TAtreated rat liver
Novikoff hepatoma
33.3
51.3
64.0
29.3
28.3
53.0
1.3 0.7 2.1 66.6
5.6 4.5 12.2 48.7
3.8 3.2 5.2 36.0
The nucleolar extract (20 000 g supernatant) was prepared as described in Materials and Methods. The total absorbancy of whole nucleoli was determined after the nucleoli were solubilized in 0.5 ‘Y, SDS (sodium dodecyl sulfate). The absorbancy of the nucleolar extract was subtracted to give the value for the nucleolar residue. Each value is the average of experiments in which the absorbancy of a given fraction was determined at 260 nm.
is a much better source of RNP particles than normal rat liver. The RNP particles are composed only of protein and RNA (table 3). Some differences were found in the composition of the RNP particles of peak 3 and peaks 1 and 2, i.e., peak 3 contained somewhat more RNA and somewhat less protein than peaks I and 2. However, the results for peaks 1 and 2 were Table 3. Composition
of nucleolar RNP peaks
Fraction
Treated
Protein’ ql,I
Protein’ /111 (3)
RNA o<,
RNA Protein
I II III I II III
TA TA TA Nor Nor Nor
45.4 45.5 39.7 39.4 41.4 32.0
44.2 53.0 57.0c
54.6 54.5 60.3 60.6 58.6 68.0
1.2 1.2 1.5 I.5 I.4 2.1
u Extimated by folin-BSA as standard. ’ Estimated by orcinol-ribose and yeast RNA as standards. ’ Calculated from estimation of buoyant densities of protein 1.250 and RNA ~; I .900 [3].
Ultrastructure and biochemistry 0,s 1
particles
245
DISCUSSION 285 A
5%)
of ribonuclein
5
10
15
20
!
25
30
I40961
fraction number; ordinate: A,,,,. Sucrose density. gradient profile of RNA of the nucleolar RNP particles of thioacetamide-treated rat livers. The sucrose density gradient (5-40”,,) containing I mM EDTA, 0.1 M NaCI, 0.02 M sodium acetate buffer, pH 5.1 was centrifuged for I6 h at 26 000 rpm in a SW-27 rotor. Fig. 14. Abscissa:
virtually the same. The data of Liau et al. [3] which are shown for comparison differ in that they found a higher protein content of the more rapidly sedimenting peaks. The reasons for this discrepancy are not entirely clear but may be related to the fact that their particles were obtained from L cells and their analytical procedure was different. RNA of the nucleolar RNP particles In the RNA extracted from either whole RNP particles (fig. 14) or from the three RNP peaks, 28s RNA was the predominant species. The shoulder with an approximate sedimentation coefficient of 32-35s may result from aggregation of the 28s RNA [9]. More rapidly sedimenting nucleolar RNA could not be detected. In studies in progress, Prestayko et al. [5] have found that the 4-8s RNA of these nucleolar particles contains U3 RNA [I], 8s RNA and other low molecular weight RNA species. Since these RNAs as well as the 28s RNA are present in all of the particles with the different sedimentation coefficients. it seems possible that these particles represent various stages of maturation of nucleolar RNP particles leading to the formation of cytoplasmic ribosomes.
Although the granular elements of the nucleolus have been extensively studied in recent years [I], the ribonucleoprotein particles which have been isolated by a number of authors [3, 6, IO] have not been examined in detail by electron microscopy. The only paper which has presented some structural information is that of Shankar Narayan & Birnstiel [6] in which negative staining of the particles was employed to demonstrate their irregular shape and the presence of some fibrillar structures. In addition, as shown in this study, the overall appearance of the particles was found to be roughly spherical or oblate. Since positive staining was not employed, it was difficult to evaluate precisely the components of these particles. The present studies have indicated that the constitution and ultrastructure of these particles is similar to that of ribosomal subunits. particularly the 60s ribosomal subunit and the nucleolar RNP particles. These particles contain a ribonucleoprotein filament that appears to be twisted and turned on itself at a number of points. This subcoiling gives a multifaceted appearance to the RNP particle. Treatment with pepsin or RNase did not destroy the integrity of the particle. but decreased its electron microscopic definition and the electron density of the internal filamentous structures. The nucleoli of thioacetamide-treated liver exhibit two major differences from those of normal liver, i.e., a marked increase in the relative number of RNP particles so that the overall appearance of these nucleoli is far more granular than those of normal liver cells, and secondly, their size is greater. In addition, the same methods for extraction of the RNP particles produces a 30-fold increase in the yield of the nucleolar RNP particles per 100 g of liver. These higher yields will Exptl Cell Res 6X
246
K. Koshiba et al.
permit a more satisfactory approach to analysis of the types of high and low molecular weight RNA in these particles and a more detailed study of their protein composition that has hitherto been possible. It is not clear why the nucleolar RNP particles sediment in several, usually 3, major peaks. The components of two of the peaks do not differ markedly from one another either in sedimentation patterns for the RNA or in the relative content of RNA and protein. On the other hand, the protein content of the slower sedimenting peaks seems to be somewhat greater than that of the more rapidly sedimenting peaks. It is possible that the RNP particles in these peaks differ in their constituent proteins, but it remains for future studies to determine whether these proteins can be isolated in a satisfactorily pure form and yield to permit detailed characterization and studies on their structure.
Exptl Cell Rr.r 68
This work was supported in part by Cancer Center Grant CA-10893 P.5 and American Cancer Society Grant P-339.
REFERENCES I. Busch, H & Smetana, K, The nucleolus. Academic Press, New York (1970). 2. Chauveau, J, MoulC, Y & Rouiller, C, Exptl cell res I I (1956) 317. 3. Liau, M C & Perry, R F, J cell biol 42 (1969) 212. 4. Muramatsu, M & Busch, H, Methods in cancer
research, p. 303. Academic Press, New York (I 967).
5. Prestayko, A W. In manuscript. 6. Shankar Narayan, K & Birnstiel, M L, Biochim biophys acta 190 (1969) 470. 7. Smetana, K, Methods in cancer research, p. 362. Academic Press, New York (1967). 8. Steele, W J & Busch, H, Methods in cancer research, p. 61. Academic Press, New York ( 1967). 9. Wagner, E K, Katz, L & Penman, S, Biochem biophys res commun 28 (1967) 152. IO. Warner, J R & Soeiro, R, Proc natl acad sci US 58 (1967) 1984.
Received April 20, 1971