Isolation of the outer layer of the nuclear envelope

Experimental

ISOLATION

OF THE

OUTER

Cell Research 55 (1969) 185-197

LAYER

OF THE

NUCLEAR

ENVELOPE

Composition of the RNA S. J. SMITH,’

H. R. ADAMS,

K. SMETANA

and H. BUSCH

Department of Pharmacology, Baylor University College of Medicine, Houston, Tex. 77025, USA

SUMMARY A new method is presented for the isolation of the outer layer of the nuclear envelope and its attached ribosomes. Nuclei isolated from rat livers by the method of Chauveau et al. were treated with 2.5 % citric acid to separate the outer layer of the nuclear envelope from the remainder of the nucleus. Morphological evidence showed that the precipitate obtained by high speed centrifugation of the citric acid extract contained both membranes and ribosomes. Evidence that the RNA of the outer layer of the nuclear envelope is a mixture of ribosomal and other RNAs was obtained by kinetic studies, UV and 82P nucleotide analyses, and the effects of actinomycin D. The kinetics of labeling of the 18s and 28s RNA of the outer layer of the nuclear envelope with erotic W-2-acid were intermediate between those of cytoplasmic ribosomal RNA and nuclear RNA. Maximal labeling was found in 6 h for RNA of the nuclear envelope, 46 h for ribosomal RNA, and 40 min for nuclear RNA. The half-lives of the 18s and 28s RNA of the outer layer of the nuclear envelope were shorter than those of ribosomal RNA and longer than those of nuclear RNA. Although the UV base compositions of nuclear RNA and RNA of the outer layer of the nuclear envelope were similar, the 18s RNA of the latter had a higher content of uridylic acid and a lower content of guanylic acid than nuclear 18s RNA. The 28s RNA of the nuclear envelope had a higher content of adenylic and uridylic acids and a lower content of cytidylic and guanylic acids than nuclear 28s RNA. The distribution of szP in the nucleotides of 18s RNA of the outer layer of the nuclear envelope was similar to that of nuclear 18s RNA. For the 28s RNA of the nuclear envelope, the differences were more marked. The 28s RNA of the nuclear envelope had a lower saP content in uridylic acid and a higher saP content in guanylic and cytidylic acids than nuclear 28s RNA. Actinomycin D inhibited the labeling of nuclear 18s and 28s RNA equally but in the outer layer of the nuclear envelope the labeling of 18s RNA was inhibited by more than 50 % with no significant effect on labeling of the 283 RNA.

The nuclear envelope consists of two layers. The outer layer of the nuclear envelope (NE-OL) is continuous with the endoplasmic reticulum and apparently there are ribosomes on its surface [37, 381. Studies on the formation of the nuclear envelope in telophase have shown that part of it is formed by elements of the endoplasmic reticulum [3, 17, 281.Although the nuclear envelope has long been known to be a permeable interface between the nucleus and cytoplasm, its role in nucleo-cytoplasmic interchanges is just beginning to be evaluated [ll, 12, 331. Thus far, it has been suggested that the components of the 1 Predoctoral

trainee on the USPHS grant GM/OO670.

nuclear envelope may be involved in regulation of the transport of materials through the nuclear pores [ 12, 33, 381,in the alteration of transported substances as they enter the nuclear pores [4, 26, 271, and in the formation of new polysomes from nascent messengerRNA [l , 291. Recently, Sadowski & Howden [29] reported that they isolated the outer layer of the nuclear envelope with its attached ribosomes by washing liver nuclei with the detergent Triton-X-100. They found that the polysomes contained in this membrane fraction had a higher specific activity 30 min following labeling with 14C erotic acid than cytoplasmic polysomes. Bach & Johnson [2], using viscous solutions of sonicated chromaExptl CeN Res 55

186

S. J. Smith et al.

tin, obtained a membrane fraction from HeLa cell nuclei that also contained ribosomal aggregates. The newly synthesized RNA in this fraction was labeled at a rate that was intermediate between that of microsomal RNA and the nuclear residue that remained after extraction of the membrane fraction. The present study was designed to determine the composition and rates of formation of RNA in the outer layer of the nuclear envelope of liver. Liver nuclei were isolated by the sucrose method of Chauveau, MoulC & Rouillier [8] and were treated with 2.5 % citric acid to remove the outer layer of the nuclear envelope [15]. It was found that the rates of labeling of the 18s and 28s RNA with erotic 14C-acid were different in this membrane fraction from that of the nuclear RNA or that of cytoplasmic ribosomal RNA. The half-life time of the RNA contained in the outer layer of the nuclear envelope was longer than that of nuclear RNA and shorter than that of cytoplasmic ribosomal RNA. Interestingly, actinomycin D in a dose of 800 ,ug/kg decreased the labeling of the 185 RNA of the outer layer of the nuclear envelope by more than 50% without significantly affecting labeling of the 28s RNA of this fraction.

Preparation of nuclei Nuclei were prepared by a modification [6, 231 of the procedure of Chauveau, Moult & Rouillier [8]. Minced liver tissue was suspended in 2.4 M sucrose (1: 14 w/v> containing 0.0033 M calcium acetate and was homogenized with 7 strokes of a Teflon pestle (9-12 x 1O-3 inch clearance) rotating at 2000 rpm. The suspension was filtered through 4 layers of cheesecloth and was centrifuged at 40,000 g for 75 min.

Isolation of the outer layer of the nuclear envelope The supernate from the 40,000 g centrifugation was discarded. The precipitated nuclei were suspended in 2.5 % citric acid (1 :5 w/v) and were homogenized using a Teflon pestle (lo-12 x l0-a inch clearance) at low speed with 3 stokes. The nuclear suspension in citric acid was then centrifuged in an RC-3 Sorvall refrigerated centrifuge at 700 g for 10 min to sediment the nuclei which were used for studies on nuclear RNA. The 700 g supernate was centrifuged at 100,000 g for 60 min (4°C) to precipitate the fragments in the outer layer of nuclear envelope and its attached ribosomes.

Preparation of nucleoli Purified liver nucleoli were isolated as described previously [7, 201. Nuclei prepared in 2.4 M sucrose as described above were resuspended in 0.25 M sucrose (1 ml/g original wet wt of tissue) and sonicated for 40 to 50 set in a Branson sonifier (0.9-1.0 amp). Twenty ml of sonicated suspension were layered over 20 ml of 0.88 M sucrose and centrifuged at 2000 g for 20 min to sediment the nucleoli. The purified nucleoli contained no nuclei and little other contamination [20, 221.

Preparation of cytoplasmic ribosomesand microsomes MATERIALS

AND

METHODS

Animals and isotope The male rats used in these experiments(Cheek-Jones Co., Tomball, Texas) weighed 220-250 g and were fed ad libitum on Purina laboratory chow. For experiments on the RNA of the outer layer of the nuclear envelope, 4-6 rats were used for each time point. For isolation of nucleoli, 5-6 rats were used in each experiment; and for isolation of cvtoolasmic ribosomes, 1 rat was used in each experime&-For each time peiiod, 3-6 expts were carried out. Each animal was injected intraperitoneally with 3 PC of erotic W-2-acid (25 mc/pM, Nuclear Chicago) or 2 mc of **P-orthophosphate (carrier-free orthophosphate, code P-I, Union Carbide Nuclear Co., Oak Ridge, Tenn.). Actinomycin D, a gift of Merck, Sharp & Dohme. was iniected intraueritoneally into rats in doses of 150,800 and-1600 ,&/kg body weight 1 h after the isotope and the animals were sacrificed 6 h later. The rats were killed under diethyl ether anesthesia by severing the abdominal aorta. The livers were perfused through the portal vein with ice-cold 0.25 M sucrose (40 ml), then rapidly removed, placed in ice-cold 0.25 M sucrose and transferred to a cold laboratory (4”C), where all further steps were carried out. ExptC Cell Res 55

Ten g of minced tissue (1 liver) were homogenized with a loose-fitting Teflon pestle with 3 strokes in an ice-cold solution of 0.05 M Tris-HCl (pH 7.6) containing 0.005 M magnesium acetate, 0.25 M potassium chloride and 0.25M sucrose (I : 10 w/v). After filtration through 4 layers of cheesecloth, the suspension was centrifuged twice at 12,000 g for 20 min to remove nuclei, whole cells and mitochondria. The remaining supemate was centrifuged at 100,000 g for 2 h to sediment the cytoplasmic ribosomes and microsomes.

Extraction of RNA The various cell fractions were homogenized in a solution containing 0.3 % sodium dodecylsulfate [32], 0.14 M NaCl, and 0.05 M sodium acetate at pH 5.1. The homogenization was carried out for 1 min (15 strokes) witha loose-fitting Teflon pestle. An equal volume of 90 % phenol, containing 0.1 % hydroxyquinoline (saturated with 0.05 M sodium acetate, pH 5.1), was then added 1311. and the samole was homogenized again for 1 min. ?he suspension was incubated while shaking in a water bath at 65°C for 10 min and then was shaken in an “Equipose” shaker at room temperature (25°C) for 20 min [31, 321. For the fractions treated with citric acid, i.e., nuclei and NE-OL fractions, the sodium dodecylsulfate-

Outer layer oj’ the nuclear envelope

187

Fig. 1. Liver nuclei isolated in hypertonic sucrose. The outer layer of tbe nuclear envelope is largely intact. Some adherent vesicles were also present. x 6400.

phenol solution was adjusted to pH 5.1 with 1.0 N NaOH before heating at 65°C. After the mixture was centrifuged at 17,000 g for 10 min in a Sorvall centrifuge, the aqueous phase was removed and fresh phenol (2/3 vol.) was added. After shaking for 10 min, the layers were again separated by centrifugation at 17,000 g for 10 min. Fresh phenol was added a 3rd time to the separated aqueous phase; after shaking for 5 min and centrifugation as described above, the aqueous layer was removed and the RNA was precipitated overnight at - 20°C with 2.0-2.5 vol. of ethanol containing 2 % potassium acetate 1231. The precipitated RNA was washed once with 75 %-ethanol. The RNA was then dissolved and stored at - 20°C in 2.0-4.0 ml of 0.01 M sodium acetate buffer, pH 5.1.

Sucrose density gradient centrifugation For sucrose density gradient centrifugation, a volume of 0.5 ml to 1 .O ml of sodium acetate buffer containing l-2 mg of RNA was layered over 26.5 ml of a 1040 % &ear gradient of sucrose solution containing 0.1 M NaCl, 1.0 mM EDTA, and 0.01 M sodium acetate, pH 5.1 [30]. For experiments on 82P-labeled RNA, the RNA was purified with Sephadex G-25 to remove contaminating phosphate before preparation of sucrose density gradients. The gradient was centrifuged in a Spinco SW 25.1 rotor at 25,000 rpm for 16 h at 5°C. Fractionation of the gradient was carried out with the aid of an Isco automatic fractionator (obtained from Instrument Specialties Co., Lincoln. Neb.). To obtain 18s and 28s RNA free of contamination’from other sedimentation classes of RNA, fractions containing RNA with approx. the same sedimentation coefficient from initial gradient runs were pooled and layered over a linear gradient of 10-40% sucrose; the conditions for repurification of peaks were the same as described above for the initial gradient runs.

Fractions under each optical density peak were pooled separately and the RNA was precipitated at -4°C with 2.0 to 2.5 vol of ethanol containing 2 % potassium acetate. The precipitated RNA was washed with 75 % ethanol and stored in 0.01 M sodium acetate, pH 5.1, at -20°C. The peak fractions of optical density in the 18s and 28s RNA of each cell fraction were collected from the gradients, precipitated with ethanol and analyzed directly for specific activity.

Determination

of specific activity

RNA which had been precipitated from the sucrose density gradients was dissolved in 2.0 ml of distilled water. The optical densities were determined in a Beckman DU spectrophotometer at a wavelength of 260 m,u. For determination of radioactivity, 0.1 ml of 5 N perchloric acid was added to each 0.2 ml aliquot and the samples were hydrolyzed for 20 min at 70°C; 8 ml of fluor-containing solution was added [5] and the samples were counted in an automatic liquid scintillation spectrometer. To obtain the labeling patterns for the sucrose density gradients, aliquots of 0.1 ml were taken from each gradient tube and radioactivity was determined in the same manner.

Base analyses RNA fractions were hydrolyzed at 37°C for 18 h [9]. The hydrolysate was adjusted to pH 3-4 with 5.0 N perchloric acid in the cold and centrifuged. The supernate was adjusted to pH 67 with 0.5 N KOH and centrifuged. The supemate was chromatographed on a Dowex l-formate column using a linear gradient of formic acid. For *ZP analysis of nucleotides, yeast RNA hydrolysate was added as a carrier to the purified peaks. After desiccation, the nucleotides were dissolved in 0.1 N HCl 1191. Expfl

Cell

Res 55

188

S. J. Smith et al.

Fig. 2. Higher magnification

of a liver nucleus isolated in sucrose showing the outer layer of the nucleai envelope (white arrow). The black ar;ow shows denseintranuclear granules. Small adherent portions of cytoplasm were occasionally observed.

An aliquot was taken from each nucleotide fraction for analysis of optical density and for determination of radioactivity.

Electron microscopy The specimens were fixed in 2 % osmium tetroxide [24]; after dehydration in graded concentrations of ethanol containing uranyl acetate, the specimens were embedded in Epon-araldite [18]. The sections cut with an LKB ultramicrotome were stained with 2.5 % uranvl acetate in 50 % ethanol and lead citrate [36]. The stained ultrathin sections were observed with a Philips 200 electron microscope.

RESULTS Electron microscopy Isolated nuclei. The ultrastructural appearance of nuclei isolated in sucrose was described in previous reports from this laboratory [6, 201. Numerous ribosomes were bound to the outer layer of the nuclear envelope (figs 1, 2, 3). Isolated nuclei after citric acid treatment. The morphology of nuclei treated with citric acid was described previously [15]. In most of the nuclei, the outer layer of the nuclear envelope and its associated ribosomes was removed (figs Exptl Cell Res55

4, 5). The adjacent membranes of endoplasmic reticulum and cytoplasm were also removed. Small fragments of adjacent perinuclear amorphous material were occasionally found (fig. 5). Isolated outer layer of the nuclear envelope. After treatment of the nuclei with citric acid, the suspension was centrifuged at 700 g for 10 min at 4°C. The precipitate contained nuclei as shown in figs 4 and 5. The supernate containing the outer layer of the nuclear envelope and its associated ribosomes was centrifuged at 100,000g for 60 min at 4°C. The pellet contained mainly membranes and ribosomes (figs 6, 7). The supernate obtained from this 100,000 g centrifugation contained small quantities of unlabeled low molecular weight RNA amounting to 2 % of the total RNA of the NE-OL fraction. Nuclear RNA As a control for studies on the RNA of the outer layer of the nuclear envelope, studies were made on whole nuclear RNA. Liver nuclear RNA contains 4-7’S, 18S, 28S, 35s and 45s RNA

Outer layer of the nuclear envelope

Fig. 3. Higher magnification layer of the nuclear envelope particles (arrows). x 52,900.

Fig. 4. Nuclei treated somes and adherent

which shows that the outer contains many ribosomal

with citric acid showing that the cytoplasm was almost completely

189

(fig. 8); frequently a shoulder of 55s RNA is seen [Wj. Citric acid treatment did not change the nucleotide compositions of these RNAs [I 61. Fig. 8 shows that after 20 min of labeling with erotic 14C-acid in nuclear RNA, the main peak of radioactivity was in the 4% RNA. By 60 min the 35s RNA had become labeled. After 6 h of labeling, a peak of radioactivity was found in the 28s RNA. In addition, a labeled shoulder was found in the 18s RNA and there was some radioactivity in the 4-7s region. At 46 h of labeling there was a considerable decrease of radioactivity in the higher RNA sedimentation classes. Kinetics of labeling of whole nuclear 18s and 28s RNA. Fig. 9 shows the repurification on sucrose density gradients of 18s and 28s RNA precipitated from the initial sucrose density gradients seen in fig. 8 in order to remove contamination from other sedimentation classes of RNA. Fig. 10 shows that kinetics of labeling of nuclear 18s and 28s RNA were first order in the

outer layer of the nuclear removed. Phase contrast.

envelope x 1100.

with

its attached Exptl

Cell

riboRes 5.5

190

S. J. Smith et al.

Fig. 5. Electron micxograph of a nucleus treat ed with citric acid showing that the outer layer of the nuclear envelope is largely r ,emoved.

Fig. 6. Ribosomes and membranes of the outer layer of the nuclear envelope isolated after the nuclei obtained in sucrose were treated with citric acid. The whole preparation was centrifuged at 700 g for 10 min to sediment the nuclei. The supemate was centrifuged at 100,000 g to sediment the fraction shown in this figure. This fraction is composed mainly of membranes and ribosomes (arrow). x 30,000. Fig. 7. The

same specimen as in fig. 6 at higher magnification showing the ribosomes attached to portions of memx 64,500. brane (arrow).

Outer layer of the nuclear envelope 20

BOO0

50,000 40,000

1.0

4000

30,000

a

20,000

20

8000

10

4000

I

3000

10

F

i 10.000 i

b 29

191

1500 c

20

IO00

10

500

0 TOP

5

10

15

20

d 25 BOTTOM

Fig. 8. Density gradient sedimentation patterns of liver nuclear RNA after labeling of nuclear RNA for various times with erotic 14C-2-acid. The approximate sedimentation coefficients are shown above the optical density peaks. RNA was extracted from citric acid treated nuclei and layered on l&40 % linear sucrose density gradients as described in the text. RNA under the 18s and 28s RNA optical density peaks was collected from the gradient and precipitated at -20°C with 2 vol of ethanol containing 2 % potassium acetate. Radioactivity is indicated by the dashed line and optical density is indicated by the solid line. Abscissa: Tube number; ordinate: (left) optical density (254 mp); (right) dpm/fraction. a, 20 min; b, 1 h; c, 6 h; d, 46 h.

period of 20 to 40 min after injection of the isotope. Fig. 11 shows that the decrease in labeling of nuclear 18s and 28s RNA was much slower. The specific activity of the nuclear 28s RNA was higher than that of nuclear 18s RNA

L

I

I

0

20

30

I

40

Fig. 10. Kinetics of labeling of nuclear 18s and 28s RNA at various times after injection with erotic W-2acid. Nuclear 18s and 28s RNA was purified on linear sucrose density gradients and precipitated from the gradients with 2 vol. of ethanol containing 2 % potassium acetate as described in the text and was dissolved in 2.0 to 3.0 ml of distilled H,O. Optical density was determined by liquid scintillation counting. Each point on the figure is the average of 3 to 5 expts. Standard errors are indicated on the figures and were determined by the equation S.E. = 1/x x2/n(n - 1). Straight lines were drawn by subjecting the data to linear regression analysis of the log y vs x on an IBM 7094 computer. Abscissa: Mitt; ordinate: dpmimg RNA.

at all times studied. The labeling reached halfmaximal levels at approximately 25 min for both 18s and 28s RNA (table 1). The half-life of nuclear 18s and 28s RNA was approx. 1 day. Similar studies showed that the half-life of nucleolar 18s and 28s RNA was 14 h. By contrast, the half-life of cytoplasmic ribosomal 18s and 28s RNA was 180 and 140 h, respectively (table 1).

2.0 [

‘OF-J-0

5

IO

15

20

25

Fig. 9. Purification of nuclear 185 and 28s RNA on linear l&40% sucrose density gradients. Nuclear RNA under the optical density peaks of 18s and 28s as shown in fig. 8 was collected from the gradient, precipitated with 2 vol. of ethanol containing 2 % potassium acetate and redissolved in 1.0 ml of 0.02 M sodium acetate, pH 5.1 as described in the text. Reruns for purification were carried out as indicated for the initial gradients in fig. 8. The shadowed portion was collected from the gradient and precipitated as described above. Abscissa: Tube number: 0, top; 25, bottom; ordinate: optical density (254 mp).

Fig. II. Kinetics of decrease in specific activity of nuclear 18s and 28s RNA at various times after injection with erotic W-2-acid. Specific activities were calculated as indicated for fig. 10. Each point on the figure is the average of 4-6 expts. Standard errors and straight lines were determined as indicated for fig. 10. Abscissa: Hours; ordinate: dpm/mg RNA. Exptl Cell Res 55

192 S. .J. Smith et al. Table 1. Labeling of 18s and 28s RNA of various cell fractions

Nuclear RNA

Time of half-maximal labeling Time of maximal labeling Half-life time

RNA of the outer RNA of layer of the nuclear cytoplasmic envelope ribosomes

Nucleolar RNA --

18s

28s

18s

28s

22 min 40 min 24 h

26 min 40 min 23 h

28 min lh 14h

32 min

18s U-4

28s (h)

2

i

1::

146

10:

18s G-4

28s (h)

9 46

46

180

140

11

The half-maximal labeling time and half-life were calculated for the nuclear and NE-OL 18s and 28s RNA from the data shown in figs 10, 11, 13 and 14. For nucleolar and cytoplasmic ribosomal RNA, these values were determined in similar manner. The half-maximal labeling and half-life times were determined from the straight lines determined by an IBM 7094 computer as indicated for fig. 10.

RNA of the outer layer of the nuclear envelope The sedimentation pattern of the NE-OL RNA showed that by contrast with the nuclear RNA, this fraction contained mainly 18s and 28s RNA; only small amounts of 4-7s RNA were present (fig. 12). The gradient pattern closely resembled that of cytoplasmic ribosomal RNA. Fig. 12 shows that there was little labeling in this fraction after 20 min of labeling with erotic 20

20 MIN 185

IO

1200

285

* **I HOUR 2.0 w

600 -

I 1200

l*C-acid as compared with nuclear RNA which was highly labeled, particularly in the 35s and 45s RNA (fig. 8). These data indicate that little, if any, nuclear RNA was present in this fraction. At 60 min there was relatively low labeling of the 18s and 28s RNA of the NE-OL fraction, which increased at 6 h. The 4-7s RNA contained little isotope at early times, but at 46 h a discrete peak of label was present. Kinetics of labeling of the NE-OL RNA. Fig. 13 shows that the kinetics of labeling of 18s and 28s NE-OL RNA were first order for the period of 1 to 6 h after injection of erotic l*C-acid. Halfmaximal labeling for both 18s and 2&S NE-OL RNA was reached in 2 h (table 1). The kinetics of decrease in labeling were first order for 6 to 239 h (fig. 14). The labeling of l&S NE-OL RNA decreased more slowly than that of 28s NE-OL

8,000 6,000

Fig. 12. Density gradient sedimentation patterns of RNA of the outer layer of the nuclear envelope after labeling for various times with erotic W-2-acid. The approximate sedimentation coefficients are shown above the optical density peaks. RNA extracted from the ribosomes and membranes of the outer nuclear envelope was layered on l&40 % linear sucrose density gradients as described in the text. The RNA under the 18s and 28s optical density peaks was precipitated in the same manner as in fig. 8. Abscissa: Tube number; 0, top; 25, bottom; ordinate: (left) optical density (254 m,u); (right) dpm/fraction. Exptl

Cell

Res 55

0

I2

3

6

Fig. 13. Kinetics of labeling of NE-OL 18s and 28s RNA at various times after injection with erotic W-2-acid. Specific activities were calculated as indicated for fig. 10. Each point on the figure is the average of 46 expts. Standard errors and straight lines were determined as indicated for fig. 10 . Abscissa: Hours; ordinate: dpm/mg RNA.

Outer layer of the nuclear envelope Table 3. Distribution

d

0620

46

239

NE-OL

Fig. 14. Kinetics of decrease in specific activity of NE-OL 18s and 28s RNA at various times after injection with erotic W-2-acid. Specific activities were calculated as indicated for fig. 10. Each point on the figure is the average of 3-6 expts. Standard errors and straight lines were determined as indicated for fig. 10. Abscissa: Hours; ordinate: dpm/mg RNA.

RNA. Table 1 shows that the half-life of the 18s and 28s NE-OL RNA was 146 h and 106 h, respectively. In the NE-OL fraction, the specific activity of the 28s RNA was greater than that of the 18s RNA at most times studied. The UV and 32P base composition of the 18s and 28s RNA of the outer layer of the nuclear envelope. Table 2 shows that the UV base comTable 2. UV analysis of nucleotides 9 2

9 r!

9 s

z 3

Cell fraction

NE-OL Nuclei [16] Cytoplasmic ribosomes [20] Nucleoli NE-OL Nuclei [ 161 Cytoplasmic ribosomes [20] Nucleoli [21]

18s RNA 20.9 24.6 +0.4 io.2 20.8 22.8

30.2 24.3 kO.1 +0.5 33.2 23.2

0.83

20.7 -

21.4 -

32.2 -

25.7 -

0.73 -

28s RNA 19.3 20.5 kO.6 +O.l 17.3 18.1

34.2 +0.6 35.7

26.0 +0.2 28.9

0.66

17.2 15.0

34.9 34.9

28.7 30.2

0.57 0.54

19.2 19.9

0.79

0.55

The values for base composition are mole percents of the given nucleotide as determined by ultraviolet analysis of nucleotides obtained by ion-exchange chromatography. For the 18s and 28s NE-OL RNA, 2 experiments were carried out; variation of the means is indicated (SE. A). For the nuclear, cytoplasmic ribosomal and nucleolar RNA data, the references are shown in parentheses.

Nuclei [21] Cytoplasmic ribosomes [14] Nucleoli [21] NE-OL Nuclei [21] Cytoplasmic ribosomes [14] Nucleoli [21]

193

of 32P in nucleotides of RNA

18s RNA 26.0 21.8 +0.4 +0.5 25.6 23.9

27.1 25.1 kO.1 +0.1 26.2 24.3

0.92

22.4 20.7

31.2 35.8

25.7 26.8

0.76 0.60

28s RNA 24.8 19.9 +0.2 +0.3 25.2 23.5

30.5 24.8 kO.2 +0.3 27.6 23.7

0.81

21.2 20.5

34.0 36.3

0.64 0.61

20.7 16.7

18.0 17.2

26.8 26.0

0.98

0.95

The values for nucleotide composition of labeled NE-OL RNA are percentages of the total 8aP in the nucleotides obtained by ion-exchange chromatography. For the 18s and 28s NE-OL RNA, two experiments were carried out; the variation of the means is indicated (SE. +). For the nuclear, cytoplasmic ribosomal and nucleolar RNA data, the references are shown in parentheses. The duration of the oulse was 2 h for NE-OL RNA. 30 min for nuclear RNk [21], 3 h for cytoplasmic ribosbmal RNA [14], and 30 min for nucleolar RNA 1211. These labeling times approximated the half-maximal labeling times f& these RNAs except for cytoplasmic ribosomal RNA.

positions of the 18s and 28s NE-OL RNA were similar to those of the nuclear and ribosomal 18s and 28s RNA. The 18s NE-OL RNA contained more uridylic acid and less guanylic acid than the nuclear and cytoplasmic ribosomal 18s RNAs (table 2). The 28s NE-OL RNA contained more adenylic and uridylic acids and less cytidylic acid than nuclear or cytoplasmic ribosomal 28s RNA. The 28s NE-OL RNA had a higher content of adenylic and a lower content of cytidylic acid than nucleolar 28s RNA. Table 3 shows that after injection of 32P, the nucleotide composition determined by distribution of 32P in the nucleotides of the newly synthesized 18s and 28s NE-OL RNA was different from

the nucleotide

composition

determined

by

UV analysis (table 2); in particular, the content of adenylic acid was significantly higher in both 18s and 28s NE-OL RNA. In the 18s NE-OL Exptl Cell Res 55

194 S. J. Smith et al. Table 4. Efsects of actinomycin D on labeling of 18s and 28s nuclear RNA and RNA of the outer layer of the nuclear envelope 28s

18s Control ,dkg

dpdmg

% inhibition

dpm/mg

?” inhibition

150 800 1600

Nuclear RNA 17,200f 1400 12,720+ 360 7,340i 560 7,560f 780

26 58 56

33,200 + 1940 26,900+ 860 14,600+_1940 11,960&1120

19 56 64

150 800 1600

NE-OL RNA 11,800~900 8,320 i- 680 5,520 i460 5,540+ 180

29 53 53

16,000~ 1040 13,600* 900 13,76O&ll18 12,000 f 1020

15 14 25

Actinomycin D was injected i.p. into rats one hour after the injection of 3 pc of erotic W-2-acid. Control animals were injected either with the isotope alone or with saline 1 h after the isotope. AI1 animals were sacrificed 6 h after the injection of the isotope. The specific activities were determined for the 18s and 28s RNA precipitated from density gradients. The values are averages of 3-5 experiments and standard errors were determined from the equation S.E.=vm

RNA, there was less uridylic and guanylic acid in the newly synthesized RNA as compared with the bulk RNA determined by UV base compositions, In the 28s NE-OL RNA, there was less guanylic acid. The distribution of 32P in the nucleotides showed that the 18s and 28s NE-OL RNA were considerably more AU-rich and correspondingly less GC-rich than nucleolar 18s and 28s RNA. In comparison with the

newly synthesized RNA of other cell fractions, the distribution of 32P in the nucleotides of the newly labeled 18s NE-OL RNA was similar to nuclear 18s RNA. Both differed from ribosomal 18s RNA in their higher adenylic acid and lower guanylic acid content. In both 18s and 28s NE-OL RNA, the 32P content of adenylic acid was similar to that of the nuclear fraction. The 32P was present in the other nucleotides in intermediate amounts. The 28s NE-OL RNA differed from nuclear 28s RNA in its higher percentage of label in guanylic acid and its lower percentage of label in uridylic acid. It had a higher percentage of label in adenylic and uridylic acids and a lower percentage of label in guanylic and cytidylic acids than ribosomal RNA. Effects of actinomycin D on labeling of 18s and 28s RNA of the outer layer of the nuclear envelope. Table 4 shows the selective effect of actinomycin D on the 18s RNA of the nuclear envelope. With low doses of the drug (150 ,ug/ kg), there was a small inhibition of the labeling of nuclear RNA and NE-OL RNA, but at higher doses (800 and 1600 pg/kg) the labeling of 18s and 28s nuclear RNA and 18s NE-OL RNA were decreased more than 50 %. However, the labeling of NE-OL 28s RNA was not markedly depressed. Eflects of actinomycin D on the UV base composition of the 18s and 28s RNA of the outer layer of the nuclear envelope. Table 5 shows that after treatment with actinomycin D for 5 h, the

Table 5. Efsects of actinomycin D on the UV base composition of 18s and 28s RNA of the outer layer of the nuclear envelope UV analysis of nucleotides

uv 18s Control Actinomycin 28s Control Actinomycin

PI

[3]

PI

[3]

AI-U

Adenylic acid (A)

Uridylic acid (U)

Guanylic acid (G)

Cytidylic acid (C)

c+c

20.9 k 0.4 21.5kO.3 19.3 LO.6 18.5 kO.2

24.6 lrO.2 26.2kO.l 20.5 20.1 22.2kO.8

30.2 25.5 34.2 28.3

24.3 26.7 26.0 30.9

0.83 0.91 0.66 0.69

+O.l kO.6 kO.6 *0.9

LO.5 k 0.3 k 0.2 + 0.5

In these experiments, a single dose of 800 pug/kg of actinomycin was injected i.p. into rats and the animals were treated with the drug 5 h before sacrifice. The number of experiments is presented in parentheses for each fraction. The variation of the mean values is indicated (+ s.E.). Exptl

Cell Res 55

Outer layer of the nuclear envelope

guanylic acid content decreased and the uridylic acid content increased in both 18s and 28s NEOL RNA. The cytidylic acid content increased more in the 28s RNA than in the 18s RNA. DISCUSSION A new method is presented for the isolation of the outer layer of the nuclear envelope based upon the previous finding [15] that citric acid removes the outer layer of the nuclear envelope from the main mass of the nucleus. Other methods reported for the isolation of the outer layer of the nuclear envelope and its attached ribosomes have used chromatin [2] and Triton-X100 [29, 391 for separation of the envelope layers. This present procedure utilizing dilute citric acid has been shown to provide nuclear RNA which is undegraded presumably because it is unaffected by RNase at the lower pH of the citric acid. It is logical that the outer layer of the envelope should contain a mixture of RNAs some of which may be in transit to the cytoplasm and others which may remain in the juxtanuclear region. Accordingly, the outer layer of the nuclear envelope should be that region of the cell most representative of the nuclear product that migrates to the cytoplasm. The evidence that the RNA of the outer layer of the nuclear envelope does not represent intranuclear RNA arises from several analyses including sucrose density gradient centrifugation patterns, kinetics of labeling of RNA, UV and 32P nucleotide compositions and differential effects of actinomycin D on labeling of RNA. Morphological evidence. Morphological evidence for the quality of the preparations of the outer layer of the nuclear envelope was obtained from electron microscopic studies which showed that the outer layer of the nuclear envelope was removed by citric acid and that the pellet obtained by high speed centrifugation (100,000 g) contained both membranes and ribosomes. Biochemical evidence. Biochemical evidence supports the morphological studies which suggest that there is little contamination of the fraction with intranuclear components: (1) Sedimen-

195

tation analysis of NE-OL RNA by sucrose density gradient centrifugation showed that it contained no 35s and 45s RNA. When nuclei were deliberately broken by excessive homogenization in 2.5 y0 citric acid, 45s nuclear RNA was found in the NE-OL fraction. Nuclei prepared with the citric acid procedure retained both rapidly labeled RNA as well as 4-7s RNA, including transfer RNA.l* 2 In the present study, the RNA of the NE-OL fraction accounted for about 15 Y0 of the total RNA of the nuclei obtained in sucrose. (2) The present study also demonstrated that the kinetics of labeling of the RNA in the outer layer of the nuclear envelope were different from the kinetics of labeling of nuclear RNA or cytoplasmic RNA. The 18s and 28s nuclear RNA were labeled maximally at 40 min after the injection of erotic 14C-2-acid; the 18s and 28s RNA of the outer layer of the nuclear envelope were not labeled maximally until 6 h. Halfmaximal labeling of these nuclear RNAs occurred in about 24 min, and in 2 h for the NE-OL RNAs. The half-life of 18s and 28s nuclear RNA was approx. 1 day and the half-lives of 18s and 28s NE-OL RNA were about 146 and 106 h, respectively. Cytoplasmic RNA was not labeled significantly at 2 h after injection of the isotope although NE-OL RNA was labeled at 1 h. For cytoplasmic ribosomal 18s and 28s RNA, halfmaximal labeling was found at approx. 10 h after the injection of the isotope and the maximal labeling was at 46 h. The half-lives of these RNAs were 180 h and 140 h, respectively. All these values for cytoplasmic ribosomal RNA were higher than those of NE-OL RNA (table 1). (3) Although the UV base compositions of nuclear and NE-OL RNA were similar, the 18s NE-OL RNA had a higher content of uridylic acid and a lower content of guanylic acid than nuclear 18s RNA. The 28s NE-OL RNA had a higher content of adenylic and uridylic acids and a lower content of cytidylic acid than nuclear 28s RNA. With the exception of a lower 32P content in uridylic acid, the distribution of 1 Kumar, S & Busch, H. Unpublished data. 2 Moriyama, Y & Busch, H. Unpublished data. Exptl Cell Res 55

196 S. J. Smith et al. 32P in the nucleotides of 18s NE-OL RNA was similar to that of nuclear 18s RNA. For the 28s NE-OL RNA, the differences were more marked. The 28s NE-OL RNA had a lower 32P content in uridylic acid and a higher 32P content in guanylic and cytidylic acids than nuclear 28s RNA. (4) Labeling of both nuclear 18s and 28s RNA were equally inhibited by actinomycin D; but this antibiotic had differential effects on NE-OL RNA in that the labeling of 18s NE-OL RNA was markedly inhibited without a significant effect on labeling of 28s NE-OL RNA. It is well established that nuclear and possibly nucleolar RNA are mixtures of variable amounts of ribosomal and heterogeneous DNA-like components. The present data concerning the UV and s2P base compositions of the RNA and the effects of actinomycin D suggest that the RNA in the outer layer of the nuclear envelope also consists of similar mixtures of RNA species. The turnover data is a reflection of the flux of label through various RNA species turning over at various rates. Although the specific activity values are not measures of metabolism of single molecular species of RNA, they do illustrate the relationship of the various NE-OL RNA species to other compartmentalized cellular species of RNA. After actinomycin D treatment, the bulk of the NE-OL RNA was more AU-rich. It is possible that abnormal products synthesized in the nucleus or broken fragments of newly synthesized nuclear RNA are released to the outer layer of the nuclear envelope. Recent work by Stewart and Farber [34] suggests that selective degradation of newly labeled RNA may be another mechanism of action of actinomycin D in high doses that is not directly related to inhibition of RNA synthesis. After 2 h of labeling with 32P orthophosphate, the newly synthesized 18s and 28s RNA of the nuclear envelope were more AU-rich than the bulk RNA determined by ultraviolet absorption suggesting a contribution of newly-labeled DRNA to the RNA of the nulear envelope. The functional significance of the juxtanuclear component remains to be answered. The various Exptl

Cell Res 5.5

RNA species of the nuclear envelope may be more representative of the total nuclear product than that obtained from cytoplasmic RNA because some of the nuclear RNA may stay at the nuclear envelope and function there in some specific way. It is possible that specific messenger RNAs or even regulatory RNAs are functional at the nuclear envelope for the synthesis of special proteins. In order to establish the functional role of the various RNA species of the nuclear envelope and to determine whether special messengers are limited to the confines of the juxtanuclear region, the 18s and 28s RNA of the nuclear envelope must be fractionated into their component subspecies. Unlike results previously reported for HeLa cells [25], these data show that in liver, cytoplasmic ribosomal 18s RNA is not labeled earlier than cytoplasmic ribosomal 28s RNA, and that newly labeled 28s RNA leaves the nucleus at virtually the same rate as 18s RNA. There are limitations to this interpretation of the kinetic data since the rapidly labeled RNA in these cell fractions do not appear to represent single molecular species of ribosomal RNA but probably contain various amounts of heterogeneous D-RNA. Moreover, higher doses of isotope and earlier time points than those used here may demonstrate the early labeling of 18s cytoplasmic ribosomal RNA. The isolation procedure used by Penman [25] and Girard et al. [13] involves the use of anionic and nonionic detergent treatment which has been used by Traub et al. [35] to extract ribosomes from the nucleus and has been found by Dingman and Peacock [lo] to disrupt nuclei. The findings by these authors of early labeling of 18s ribosomal RNA and the absence of 18s nuclear RNA may be an artifact of their isolation procedure. An alternative possibility is that there are differences between HeLa cells and other cells or differences between in vivo and tissue culture experiments. Penman [25] has claimed that there are no mature nuclear ribosomes on the basis of the finding that 18s RNA was not present in the HeLa cell nucleus. Recently, Sadowski and

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[29] reported the presence of intranuclear polysomes and ribosomes in rat liver nuclei and disputed Penman’s contention concerning the apparent absence of nuclear 18s RNA. In this present study, considerable 18s RNA was present in the isolated nuclei even after treatment with citric acid which removed almost completely perinuclear cytoplasmic contamination.

16. Higashi, K, Adams, H R & Busch, H, Cancer res 26 (1966) 2196. 17 ito, $, J biophys biochem cytol7 (1960) 433. 18. Mollenhauer. H H. Stain technol39 (1964) 111. Muramatsu, M & busch, H, J biol them &lO (1965) lg. 3960. 20. Muramatsu, M & Busch, H, Methods in cancer

Supported in part by the USPHS grant 8182, the American Cancer Society and the Jane Coffin Childs Fund.

24. 25. 26. 21.

Howden

21.

22.

REFERENCES 1. Bach, M K & Johnson, H G, Nature 209 (1966) 893. 2. - Biochem 6 (1967) 1919. 3. Barer, R, Joseph, S & Meek, G A, Exptl cell res 18 (1959) 179. 4. Bernhard, W, Nat1 cancer inst monogr 23 (1966) 13. 5. Bruno, G A & Christian, J E, Analyt them 33 (1961) 1216. 6. Busch, H, Methods in enzymol 12 (1967) 421. 7. - Ibid. 12 (1967) 448. 8. Chauveau, J, Mo1.116,Y & Rouillier, C H, Exptl cell res 11 (1956) 317. 9. Davidson, J N & Smellie, R M S, Biochem j 52 (1952) 594. 10. Dingman, C W & Peacock, A C, Biochem 7 (1968) 659. 11. Feldherr, C M, J cell bio125 (1965) 43. 12. Feldherr, C M & Harding, C V, Protoplasmatologia 5 (1964) 35. 13. Girard, M, Penman, S & Darnell, J E, Proc natl acad sci US 51 (1964) 205. 14. Harel, L, Harel, J; Boer, A, Imbenotte, J & Carpeni, N, Biochim biophys acta 87 (1964) 212. 15. Higashi, K, Shankar Narayanan, K, Adams, H R & Busch, H, Cancer res 26 (1966) 1582.

13 -

691819

29. 30. 31. 32. 33. 34. 35. 36. 31. 38. 39.

research (ed H Busch) vol. 2, p. 303. Academic Press, New York (1967). Muramatsu, M, Hodnett, J L & Busch, H, J biol them 241 (1966) 1544. Muramats;, MI Smetana, K & Busch, H, Cancer res 23 (1963) 510. Okamura, N & Busch, H, Cancer res 25 (1965) 693. Palade, G E, J Exptl med 95 (1952) 285. Penman, S, J mol biol 17 (1966) 117. Perry, R P, Nat1 cancer inst monogr 23 (1966) 527. - Progress in nucleic acid research (ed J N Davidson & W E Cohn) vol. 7, p. 220. Academic Press, New York (1967). Porter, K R & Machado, R D, J biophys biochem cytol 7 (1960) 167. Sadowski. P D & Howden. J A. J cell bio137 (1968) . I 163. ’ Scherrer, K & Darnell, J E, Biochem biophys res comm 7 (1962) 486. Steele, W J & Busch, H, Methods in cancer res (ed H Busch) vol. 3, p. 62. Academic Press, New York (1967). Steele, W J, Okamura, N & Busch, H, J biol them 240 (1965) 1742. Stevens, B J & Swift, H, J cell biol 31 (1966) 55. Stewart, G A & Farber, E, J biol them 243 (1968) 4479. Traub, A, Kaufmann, E & Ginzberg-Tietz, Y, Exptl cell res 34 (1964) 371. Venable, J H & Coggeshall, R, J cell biol 25 (1965) 401. Watson, M L, J biophys biochem cytol 1 (1955) 257. - Ibid. 6 (1959) 147. Whittle, E D, Bushnell, D E & Potter, V R, Biochim biophys acta 161 (1968) 41. I

I

Received October 22, 1968

ExptI Cell Res 55