Localization of RNA in incubated rat liver nuclei

JOURNAL OF ULTRASTRUCTURE RESEARCH

81, 145-157 (1982)

Localization of RNA in Incubated Rat Liver Nuclei JACQUES P A I E M E N T AND MOYSE BENDAYAN

From the Ddpartement d' anatomie, Facultd de mddecine, Universitd de Montrial, CP 6128, Succursale A, Montrdal, Quebec H3C 3J7, Canada Received February 1, 1982, and in revised form May 21, 1982 When isolated rat liver nuclei are subjected to specific conditions of incubation they undergo morphological transformation. Most notably electron-dense granules and an associated diffuse fibrillar network become prominent throughout the nucleoplasm. The dense granules become evident during incubation when Mn 2+ is present. Cytochemical studies revealed that this system of granules and fibrils contained RNA. Incubation of nuclei with [aH]UTP led to an incorporation of the label into trichloroacetic acid precipitable material, and radioautography showed that the label was mainly associated with nucleoli and regions rich in dense granules. These results suggest that dense granules and associated fibrillar network comprise the main site of extranucleolar RNA synthesis and/or processing.

Perichromatin granules and associated perichromatin fibrils have been described as common nuclear components in mammalian cells. These structures are of a ribonucleoprotein (RNP) nature (Monneron and Bernhard, 1969) and have been implicated in RNA synthesis and processing (Petrov and Bernhard, 1971; Bachellerie et al., 1975; Palombi and Viron, 1977; Fakan and Odartchenko, 1980; Puvion and Lange, 1980). Recently while studying the effect of varying incubation conditions on nuclear membrane fusion (Paiement, 1981) striking internal changes were observed within the incubated nuclei. Under certain incubation conditions a system of fibrillar and granular particles became prominent throughout the nucleoplasm. Although several studies have been carried out on the structure (Monneran and Maul6, 1968; Laval and Bouteille, 1973) and function (Pogo et al., 1967; Maul and Hamilton, 1967) of isolated nuclei, little attention has been given to the network of granules and fibrils contained within such nuclei. In the present study we describe the morphological properties of a similar net-

work within incubated nuclei and show by cytochemical and radioautography techniques that both the granules and the fibrillar network contain RNA some of which is

newly synthesized during incubation. MATERIALS AND METHODS Sprague-Dawly rats (150200 g) were used in these studies. Glutaraldehyde was from Polysciences (Warrington, Pa.) and osmium tetroxide from Meca Lat~oratories (Pointe-aux-Trembles, Quebec). Tetrachloroauric acid (HAuC14) was purchased from BDH Chemicals, Toronto, Canada. Adenosine triphosphate (ATP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosine monophosphate (GMP), guanosine 3', 5'-mono p h o s p h a t e (cGMP), g u a n y l y l - i m i d o d i p h o s p h a t e (GppNp), guanylyl-(fl,y-methylene)-diphosphonate (GppCp), inosine triphosphate (ITP), uridine triphosphate (UTP), dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), phosphoenolpyruvate, pyruvate kinase, and RNase A (EC 3.1.4.22) purified from bovine pancreas (Type XII-A) were all from Sigma Chemical Company (St. Louis, Mo.).

Preparation of Nuclear Fractions Nuclear fractions were prepared from rat liver homogenates in 250 mM sucrose, 50 mM Tris-HC1, pH 7.5, 25 mM KC1, and 5 mM MgC12 (250 mM sucroseTKM medium), according to the procedure of Blobel and Potter (1966). The nuclei were subsequently washed

145 0022-5320/82/110145-13502.00/0 Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

146

PAIEMENT AND BENDAYAN

once in 250 mM sucrose-TKM, centrifuged at 1000 g (av) for 10 min, and resuspended in varying amounts of the same buffer.

was performed before examination in the electron microscope. Incorporation o f [3H]UTP

Incubation o f Nuclei

The standard medium consisted of 0.2 ml of nuclear fraction (4-9 x l0 t nuclei in 250 mM sucrose-TKM, employed always immediately after resuspension) and 0.3 ml of a buffer containing 40 mM Tris-HC1, pH 7.4, 30 mM KC1, 7.5 rnM MgC12, 4.17 mM MnC12, 4.17 mM DTT, 1.67 mM ATP, 0.84 mM GTP, 16.67 mM phosphoenolpyruvate, and 25/zg pyruvate kinase. Assays for RNA synthesis (scintillation counting and radioautography) were done as follows, 0.2 ml of nuclear fraction was added to 0.3 ml of the medium defined above except that ATP was present at a concentration of 0.67 mM, GTP, at 0.33 mM, CTP at 0.33 raM, UTP at 0.08 mM and [3H]UTP (New England Nuclear, 26.1 Ci/mmol) at 2.5/zCi. Morphological Procedures

Following incubation, nuclei were fixed overnight with phosphate-buffered (50 raM, pH 7.4) 1.5% icecold glutaraldehyde. The nuclei were recovered by filtration onto Millipore membranes (0.45-/xm pores, Millipore Corp., Bedford, Mass.) using the Baudhuin procedure (Baudhuin et al., 1967) as modified by Wibo et al. (1971). The nuclear pellicle was postfixed in osmium tetroxide stained en bloc with uranyl acetate, dehydrated, and embedded in Epon. Thin sections mounted on copper grids were stained with lead citrate and examined in a Siemens 1A electron microscope. For cytochemistry, postfixation in osmium tetroxide and staining en bloc was omitted; embedding was performed either in Epon or in glycol methacrylate (Leduc and Bernhard, 1967). Thin sections were mounted on nickel grids and processed for cytochemical labeling. RNP-containing structures were revealed using the EDTA regressive technique of Bernhard (1969). The thin sections were stained with 3% aqueous solution of uranyl acetate for 5 min, rinsed with distilled water, and floated on a drop of 200 mM EDTA solution for 15 min. Counter staining with lead citrate (5 rain) was performed before examination in the electron microscope. RNA was localized using the enzyme-gold cytochemical technique (Bendayan, 1981). RNase-gold complex was prepared according to methods already described (Bendayan, 1981). The thin sections mounted on nickel grids were incubated for 5 rain on a drop of 10 mM phosphate-buffered saline (PBS, composed of 10 mM phosphate at pH 7.2 and 150 mM sodium chloride) and then transferred on a drop containing the RNase-gold complex. Incubation was performed at room temperature for 30 min. The sections were then thoroughly washed with PBS, rinsed in distilled water and dried. Staining with uranyl acetate and lead citrate

The reaction was started with the addition of the nuclear suspension and incubations were carried out for varying lengths of time at 25 or 37°C. The amount of [~H]UTP incorporated was measured using the acidinsoluble fraction of incubated nuclei. The reaction was stopped by adding 5 ml of ice-cold 10% trichloroacetic acid (TCA). The precipitates were then washed three times with cold 5% TCA, dried under vacuum, and dissolved in 1 ml of 1 N NaOH at 80°C for 30 min. After neutralization with 0.9 ml 1 N acetic acid, 0.7 ml was counted using a Beckman Model LS-8100 liquid scintillation spectrometer. The sites of incorporated [3H]UTP were studied using electron microscope radioautography. Nuclei were incubated for 120 min at 37°C, fixed, and embedded as mentioned above. Thin sections were transferred to celloidin-coated glass slides and covered with an Ilford L4 emulsion by the dipping technique (Kopriwa, 1973). The radioautographs were exposed for periods up to 100 days and then developed in Kodak D-19B (Kopriwa, 1975). After photographic processing, sections were stained with uranyl acetate and lead citrate.

RESULTS

Morphology

Immediately after isolation the purified nuclei appeared as shown in Fig. 1 and displayed morphological characteristics similar to those isolated by Blobel and Potter (1966) and Monneron et al. (1972). Besides some damaged nuclei the majority had structural features recognized in nuclei in situ, including nucleoli, dense chromatin, and nuclear envelopes with their two membranes and pores (Fig. 1). Ribosomes were evident along the cytoplasmic surface of the outer membrane of almost all nuclei. After incubation for 120 rain at 37°C in the standard medium the nuclei showed morphological transformations. In particular, conspicuous electron-dense granules were evident in the nucleoplasm (Fig. 2). The granules were often observed aligned in clusters forming arcs which often extended across the nucleoplasm from regions near the nucleoli to the periphery near the envelope. The chromatin presented variable densities. Some nuclear ghosts re-

RNA CYTOCHEMISTRY OF INCUBATED NUCLEI

147

F~G. 1. Micrograph showing a rat liver nucleus after isolation in 250 mM sucrose-TKM. Condensed chromatin is conspicuous along the nuclear envelope as well as surrounding the nucleolus (Nu). Ribosomes can also be seen along the outer nuclear membrane (arrows). Glutaraldehyde fixation, Epon embedding, ua-lead, x 20 000.

suiting f r o m the lysis of d a m a g e d nuclei were also o b s e r v e d (Fig. 2). The effect of various incubation parameters on the nuclear structure was checked. In the a b s e n c e of pyruvate kinase and phosp h o e n o l p y r u v a t e (a nucleotide regenerating system, Boyer, 1962) some of the chromatin a p p e a r e d condensed along the nuclear envelope (Fig. 3). Nucleoli displayed normal structure and the dense granules were distinct, often distributed at the periphery of the c o n d e n s e d chromatin or across the nucleoplasm (Fig. 3).

The dense granules varied in size from 10 to 90 nm in diameter (Fig. 4). The average size of the granules after incubation in the standard m e d i u m (40.1 _+ 7.2 nm) was :not statistically different from that of the granules (35.5 +_ 3.5 nm) o b s e r v e d after incubation in the standard medium minus the nucleotide regenerating system (Student's t test, 0.5 > P > 0.2). At high magnification the dense granules a p p e a r e d linked together b y a weakly electron-dense fibrillar network (Figs. 5 and 6). A n a l y s i s o f nuclei following different

148

PAIEMENT AND BENDAYAN

r

®

i~~ . ~ " ~ : ~ . ~

~ ' , ' L ~ ' ? > ~ ' ~ ~ ~ . ~"i ~ : ':

~

.:'

"~!~.

®

RNA CYTOCHEMISTRY OF INCUBATED NUCLEI 50

40 ¸ >-

C) Z Ld 0 bd nIt.

3o ¸

:i::i::i::i:! .......:,.....,

20

I0 ¸

o ,o 20 30 40 so 60 zo so 9'0 iGo DENSE-GRANULE DIAMETER (nm)

FIG. 4. Distribution of dense-granules diameters in nuclei after incubation for 120 rain at 37°C in standard medium (open histogram) or in standard medium minus the nucleotide regenerating system (stippled histogram). Measurements were made on electron micrographs at a final magnification of 45 000 × ; 341 granules were measured in nuclei after incubation in standard medium and 400 granules were measured in nuclei after incubation in standard medium lacking the nucleotide regenerating system.

times of incubation at 37°C in standard medium revealed that these dense granules were conspicuous as early as 15 min (not shown). Nuclei incubated for periods of 30, 60, and 120 rain contained dense granules with the characteristics described in Figs. 2-6 and these were consistently associated with the fibrillar network. Incubations were also carried out for 120 min at different temperatures. At 4 and 10°C nuclear chromatin was diffuse and dense granules were small and inconspicuous (Fig. 7). At 20°C the chromatin granules were

14!9

more evident and resembled those observed at 37°C. Complete absence of nucleotides or the presence of ATP, CTP, GTP, and UTP alone (at 0.5 raM) or in combination (at 0.2 raM) had no effect on the structure of the dense granules. ITP, GMP, GDP, GTP, GppCP, cAMP, and cGMP present alone and at concentrations of 0.5 raM, also had no significant effect on the granules. The effect on nuclear structure of the presence of Mg 2+ and Mn 2+ were also analyzed. Omission of both cations caused rupture and agglutination of the nuclei (not shown). Nuclei incubated in the presence of MnClz (2.5 raM) and MgC12 (2 mM) had dispersed chromatin and prominent dense granules, whereas nuclei incubated in the presence of MgC12 (6.5 raM) alone contained no prominent dense granules (Fig. 8).

Cytochemistry In an effort to better understand the changes in nuclear structure after incubation, thin sections were processed for cytochemical analysis using the EDTA regressive stain for ribonucleoproteins and the enzyme-gold technique for the localization of nuclear RNA. The application of tlhe EDTA staining method resulted in bleaching the chromatin and in contrasting the dense granules and parts of the nucleoli (Figs. 9 and 10). The granules appeared as dense cores surrounded by or aligned along a less dense fibrillar material (Figs. 11 and 12). The association of the granules with the periphery of nucleoli and along nucleo-

FIG. 2. Survey micrograph showing liver nuclei after incubation at 37°C for 120 min in standard medium. Most nuclei are intact and contain homogeneously dispersed chromatin. Evident among the chromatin are nucleoli (Nu) and numerous electron-dense granules (dg). The latter can be seen clumped at the periphery of nucleoli (arrows) or in linear arrangements terminating near the nuclear envelope (curved arrow). Glutaraldehyde and osmium fixation, ua bloc staining, Epon embedding, lead. x 10 500. Fro. 3. Survey micrograph showing liver nuclei after incubation at 37°C for 120 rain in the standard medium lacking a nucleotide regenerating system. Many nuclei are intact and show condensed chromatin. The dense granules (dg) are evident within zones of the diffuse chromatin as well as along the edges of condensed perinucleolar chromatin near nucleoli (Nu) and along the edges of condensed extranucleolar chromatin. Glutaraldehyde and osmium fixation, ua bloc staining, Epon embedding, lead. × 15 000.

150

PAIEMENT AND BENDAYAN

plasmic arcs was conspicuous (Figs. 10-12). When thin sections of nuclei were incubated with the RNase-gold complex, a labeling by gold particles was observed mainly over the nucleoli and over regions rich in dense granules (Fig. 13). Few gold particles were present over the condensed peripheral chromatin (Fig. 13). Ribosomes still present on the outer membrane of the nuclear envelope were also labeled by gold particles (Fig. 13). At higher magnification the gold particles present over regions rich in dense granules, were found preferentially located at the periphery of the granules, displaying in certain cases crown-shaped figures around the granules (Fig. 14). In other instances the gold particles were arranged in linear patterns along the fibrillar

network associated with the dense granules (Fig. 15). These cytochemical results gave evidence for the presence of RNA in nucleoli and dense granules in incubated nuclei. This prompted us to wonder whether the RNA associated with these structures was newly synthesized during incubation. To answer this question, we first examined the incorporation of [sH]UTP within nuclei and then looked for the sites of this incorporation using electron microscope radioautography.

Incorporation of [3H]UTP Nuclei were incubated in the presence of [3H]UTP for 15 to 120 rain at either 25 or 37°C. After incubation nuclei were precipitated with TCA and the incorporated ra-

Fro. 5. High-magnification electron micrograph of a liver nucleus after incubation at 37°C for 120 rain in standard medium. Note the variation in size of the dense granules. Less electron-dense fibrillar material surrounds many of the granules and becomes continuous with the surrounding chromatin fibers (arrows). Glutaraldehyde and osmium fixation, ua bloc staining, Epon embedding, lead. x 45 000. FIr. 6. High magnification micrograph of a liver nucleus after incubation at 37°C for 120 min in standard medium lacking the nucleotide regenerating system. The dense granules are variable in electron density, size, and shape and appear interconnected by a fibrillar network of weaker electron density (arrows). Glutaraldehyde and osmium fixation, ua bloc staining, Epon embedding, lead. x 45 000. Fir. 7. Micrograph of a fiver nucleus incubated at 4°C in standard medium for 120 rain. The nuclear chromatin is dispersed and dense granules are small. Glutaraldehyde and osmium fixation, ua bloc staining, Epon embedding, lead. x 30 000. Fro. 8. Micrograph showing a liver nucleus after incubation at 37°C for 120 min in the standard medium minus MnCI2. The nuclear chromatin is dispersed and dense granules are indistinct. Glutaraldehyde and osmium fixation, ua bloc staining, Epon embedding, lead. x 30 000. Fro. 9. Nuclei incubated at 37°C for 120 min in standard medium and stained for RNP. The dispersed chromatin has been bleached, contrasting the dense granules (dg) that appear surrounded by a less electrondense fibrillar material. Glutaraldehyde fixation, Epon embedding, ua-EDTA-lead, x 32 000. Fro. 10. High magnification of an isolated liver nucleus after incubation at 37°C for 120 min in standard medium and stained for RNP. The nucleolus (Nu) appears highly contrasted, presenting regions of different densities. Large and contrasted dense granules (dg) are present at the periphery of the nucleolus and in the nucleoplasm. Glutaraldehyde fixation, GMA embedding, ua-EDTA-lead, x 55 000. Fins. 11 AND 12. High magnification of liver nuclei after incubation at 37°C for 120 rain in standard medium lacking the nucleotide regenerating system (Fig. 11) or in standard medium containing the nucleotide regenerating system (Fig. 12) and stained for RNP. The dense granules (dg) are surrounded by a less electron-dense fibrillar material (arrows). Glutaraldehyde fixation, GMA embedding, ua-EDTA-lead. Fig. 11, x 65 000; Fig. 12, x 55 000. Fins. 13-15. Nuclei incubated at 37°C for 120 min in standard medium lacking the nucleotide regenerating system and labeled with RNase-gold complex. The gold particles are present mainly over the nucleoli (Nu) and regions rich in dense granules (arrows) at the periphery of the dense chromatin. Ribosomes associated with the outer nuclear membrane are also labeled. Few gold particles are seen over the dense chromatin. At higher magnification (Figs. 14 and 15) the gold particles are seen at the periphery of the dense granules (dg). In some instances, the gold particles surround the granules forming crown-shaped figures (arrowheads); in others, they are seen aligned over fibrillar structures (curved arrows) closely associated with the dense granules. Glutaraldehyde fixation, Epon embedding, RNase-gold, ua-lead. Fig. 13, x 24 000; Fig. 14, x 50 000; Fig. 15, z 65 000.

RNA CYTOCHEMISTRY OF INCUBATED NUCLEI

151

152

PAIEMENT AND BENDAYAN

2~

®

@

RNA CYTOCHEMISTRY OF INCUBATED NUCLEI

153

154

PAIEMENT AND BENDAYAN

dioactivity was counted. At 37°C, incorporation of labeled UTP into nuclei reached 80 ¸ a plateau already at 15 rain and remained 60 ¸ constant up to 120 min (Fig. 16). At 25°C, the plateau of incorporation was reached 40. later (Fig. 16). These results suggest that 20 ¸ the nuclei were able to sustain RNA synthesis during incubation. (5 3'0 4'5 6'0 7'5 9'0 I b5 i~)o In an attempt to locate the sites of incorTIME (rain) poration of [zH]UTP, nuclei incubated for FIG. 16. Time course of [aH]UTP incorporation at 120 rain at 37°C were processed for electron 25 and 37°C. Nuclei (3.96 x 10~, 25°C and 5.78 × l0 T, microscope radioautography. Silver grains 37~C) were incubated in 0.5 ml of medium containing 2.5 /~Ci [sH]UTP (see Materials and Methods). The were observed over nucleoli and over reradioactivity incorporated into acid-precipitable prod- gions rich in dense granules, little labeling ucts after different times of incubation was measured. was found over the diffuse or condensed Background radioactivity determined as the counts chromatin (Fig. 17). Quantitative assessfound at 0-rain incubation (nuclei + medium + ment of grain distribution confirmed the [zH]UTP + TCA in immediate succession) was subtracted from each measurement and values were nor- above observation and showed that the realized based on maximal incorporations (max at 25°C amount of label over zones rich in dense was 17 300 cpm, max at 37°C was 21 300 cpm). granules was highly significant (greater than I00-

/

FIG. 17. Radioautograph of a liver nucleus after incubation in the presence of [aH]UTP. Nuclei were incubated 120 rain at 37°C in medium containing 2.5/zCi [sH]UTP but lacking a nucleotide regenerating system. Grains are evident over dense regions of the nucleolns as well as over a region of the nucleoplasm containing numerous dense granules. Radioautographs were exposed 94 days. Glutaraldehyde and osmium fixation, ua bloc staining, Epon embedding, lead. × 30 000.

155

RNA CYTOCHEMISTRY OF INCUBATED NUCLEI TABLE 1 SITES OF INCORPORATION OF [3H]UTP WITHIN ISOLATED NUCLEI

Incubation conditions ~

No. of micrographs e x a m i n e & Total grains

Plus a nucleotide regenerating s y s t e m

68

210

Minus a nucleotide regenerating s y s t e m

15

75

Grain distribution (%) Over nucleoli c

Over chromatin

Over a d e n s e granules

Other ~

35

13.8

45.7

5.2

45.3

10.6

38.6

5.3

Nuclei were incubated as in Fig. 17, in the p r e s e n c e or a b s e n c e of p y r u v a t e kinase and p h o s p h o e n o l p y r u v a t e . b Photomicrographs of intact nuclei were taken at r a n d o m and e x a m i n e d at a final magnification of 30 000 x . c Includes all grains over the periphery of nucleoli irrespective of w h e t h e r the surrounding chromatin h a d d e n s e granules asociated with it or not. d This category includes all nuclear regions with two or more d e n s e granules in the immediate vicinRy (appendant) to the silver grains. e Includes grains over the nuclear envelope and the extranuclear spaces.

45% of total nuclear grains, Table I). The grain distribution obtained from nuclei incubated in the absence of a nucleotide regenerating system was similar (Table I). DISCUSSION

Isolated nuclei incubated under varying conditions were studied using morphological and cytochemical approaches. Specific conditions of incubation induced pronounced effects on nuclear structure. In particular electron-dense granules appeared in the nucleoplasm in association with a less electron-dense fibrillar network. These granules were evident when Mn 2+ was present in the incubation medium, even at early times of incubation and were inconspicuous at 4°C. The cytochemical studies revealed the presence of RNA not only in nucleoli, but also within these dense granules and the associated fibrillar network. The incubated nuclei incorporated [3H]UTP within TCA-precipitable material and radioautography showed that this incorporation took place mainly in nucleoli and in regions rich in dense granules. These results suggest that the nuclei were able to sustain RNA synthesis while they underwent morphological change. It is well known that the composition of

the isolation and/or incubation media exer~ts considerable influence on the structural organization of incubated nuclei (Monneron and Moulr, 1968; Laval and Bouteille, 1973). In our studies we observed either diffuse distribution of nuclear chromatin in standard medium or its condensation in the absence of a nucleotide regenerating system. These changes might be a consequence of ionic flux occurring as a result of this omission. Incubation of the nuclei in standard medium also led to the appearance of prominent electron-dense granules. These dense granules resemble the perichromatin granules reported in situ (Swift, 1962; Watson, 1962; Monneron and Bernhard, 1969) based on their position and their RNP nature. Their association with the fibrillar network, which also stains for RNP, recalls the relationship described between perichromatin granules and perichromatin fibrils (Menneron and Bernhard, 1969; Puvion and Bernhard, 1975; Puvion-Dutilleul and lhavion, 1981). Although similarities can be recognized, some differences do exist. The electron-dense granules of incubated nuclei have a more irregular shape, they are observed in the nucleoplasm and thus not always associated with condensed chroma-

156

PAIEMENT AND BENDAYAN

tin. Furthermore the typical clear halo of in vivo and because their accumulation has perichromatin granules is absent. often been correlated with an impairment Three cytochemical techniques have been of the processing and/or transport of RNA applied that, together, have demonstrated it has been proposed that they represent the presence of RNA molecules in the dense sites of storage of R N A either incompletely granules and related fibrillar material of in- processed or nontransported (see Puvion cubated nuclei. The E D T A regressive tech- and Moyne, 1981, and Daskal, 1981, for denique revealing ribonucleoproteins (Bern- tailed discussions on this topic). The fact hard, 1969) has contrasted the nucleoli, the that the dense granules in incubated nuclei dense granules and the closely associated are already present and extensive at a time fibrillar network. Application of the R N a s e - (15 min) when [3H]UTP uptake is essengold complex resulted in a predominant la- tially stopped (Fig. 16) suggests that they beling over the periphery of the dense gran- also might be sites of RNA either incomules and the associated fibrillar network. pletely processed or nontransported. InThis e n z y m e - g o l d technique has allowed deed high concentrations of MnCI~ were simultaneous analysis of fine morphological used in our incubation medium. It has been details and specific localization of R N A previously suggested that such high Mn 2+ molecules. Although less precise in its res- can stimulate polymerase activity in an abolution, radioautography revealed the pres- normal manner leading to aberrant tranence of newly synthesized R N A molecules scription (Maclean and G r e g o r y , 1981). within the nucleoli and regions rich in dense Further studies on these intriguing particles may shed some light on certain aspects of granules. Previous electron microscope radioauto- R N A processing in vivo. graphic studies have shown that R N A synthesis occurs within nucleolar and extraWe thank Dr. V. Gisiger for constructive advice and nucleolar regions of isolated nuclei in vitro critical evaluation of the manuscript. This work was (Pogo et al., 1967; Maul and Hamilton, supported by grants from the Medical Research Coun1967). Our cytochemical and radioauto- cil of Canada and the Conseil de la Recherche en Sant6 du Qu6bec. graphic findings corroborate these results and suggest that regions rich in dense granREFERENCES ules constitute the main site of the newly BACHELLERIE, J. P., PUVION, E., AND ZALTA, J. P. synthesized extranucleolar RNA. (1975) Eur. J. Biochem. 58, 327-337. The electron-dense granules we describe BAUDHUIN,P., EVRARD,P., ANDBERTHET,J. (1967) J. Cell Biol. 32, 181-191. were particularly prominent in nuclei after incubation in the presence of MnC12. The BENDAVAN,M. (1981) J. Histochem. Cytochem, 29, 531-541. relationship between the presence of Mn 2+ BERNHARD, W. (1969) J. Ultrastruct. Res, 27, 250and the appearance of these granules is not 265. k n o w n . B e c a u s e Mn 2+ stimulates R N A BLOBEL,G., AND POTTER,V. R. (1966) Science 154, 1662-1665. polymerase activity in vitro (Widnell and Tata, 1964; Maul and Hamilton, 1967; Pogo BOYER,P. D. (1962)in BORER,P. D., LARDY,H., AND MYRBOCK,K. (Eds.), The Enzymes, Vol. 6, pp. 95et al., 1967), however, R N A synthesis and 113, Academic Press, New York. dense granule formation might be occurring DASKAL,Y. (1981) in BUSCHH. (Ed.), The Cell Nucoincidently at the same sites within the nucleus, Vol. VIII, Nuclear Particles, Part A, pp. 117137, Academic Press, New York. clei. The fact that newly synthesized R N A was revealed by our radioautographic stud- FAKAN, S., AND ODARTCrmNKO,N. (1980)Biol. cell. 37, 211-217. ies in association with dense granules is in KOPRrWA,B. M..(1973) Histochimie 37, 1-17. support of such a proposal. KOPRIWA,B. M. (1975) Histochemistry 44, 201-224. Perichromatin granules have been impli- LAVAL, M., AND BOUTEILLE, M. (1973)Exp. CellRes. 76, 337-348. cated in the storage of RNA within nuclei

RNA CYTOCHEMISTRY OF INCUBATED NUCLEI LEDUC, E. H., AND BERNHARD, W. (1967) J. Ultrastruct. Res. 19, 196-199. MACLEAN, N., AND GREGORY, S. P. (1981) in BUSCH, H. (Ed.), The Cell Nucleus, Vol. VIII, Nuclear Particles, Part A, pp. 139-191, Academic Press, New York. MAUL, G. G., AND HAMILTON, T. H. (1967) Proc. Nat. Acad. Sci. USA 57, 1371-1378. MONNERON, A., AND BERNHARD, W. (1969) J. Ultrastruct. Res. 27, 266-288. MONNERON, A., AND MOULI~,Y. (1968) Exp. Cell Res. 51,531-554. MONNERON, A., BLOBEL, G., AND PALADE, G. E. (1972) J. Cell Biol. 55, 104-125. PAIEMENT, J. (1981) Exp. Cell Res, 134, 93-102. PALOMBI, F., AND VIRON, A. (1977) J. Ultrastruct. Res. 61, 10-20. PETROV, P., AND BERNHARD, W. (1971) J. Ultrastruct. Res. 35, 386--402. POGO, A. O., LITTAU, V. C., ALLFREY, V. G., AND

157

MIRSKY, A. E. (1967) Proc. Nat. Acad. Sci. USA 57, 743-750. PUVION, E., AND BERNHARD, W. (1975) J. Cell Bic,l. 67, 200-214, PUVlON, E., AND LANGE, M. (1980) Exp. Cell Res. 128, 47-58. PUVlON, E., AND MOVNE, G. (1981) in BUSCH, H. (Ed.), The Cell Nucleus, Vol. VIII, Nuclear Particles, Part A, pp. 59-115, Academic Press, New York. PUVlON-DUTILLEUL, F., AND PUVlON, E. (1981) J. Ultrastruct. Res. 74, 341-350. SWIFT, H. (1962) in HARRIS, R. J. C. (Ed.), Interpretation of Ultrastructure, Syrup. Intern. Soc. Cell Biol. Vol. 1, pp. 213-232, Academic Press, New York. WATSON, M. L. (1962) J. Cell Biol. 13, 162-167. WIBO, M., AMAR-COSTESEC,A., BERTHET, J., AND BEAUFAY, H. (1971) J. Cell Biol. 51, 52-71. WIDNELL, C. C., AND TATA, J. R. (1964) Biochim. Biophys. Acta 87, 531-533.