Localisation of rapidly and slowly labelled nuclear RNA as visualized by high resolution autoradiography

Experimental

LOCALISATION

cell Research 67 (1971) 129-141

OF RAPIDLY RNA

AND

AS VISUALIZED

SLOWLY BY HIGH

LABELLED

NUCLEAR

RESOLUTION

AUTORADIOGRAPHY S. FAKAN’ Institut

de Recherches

and W. BERNHARD

Scientifiques

sur le Cancer,

94-Villejuif,

France

SUMMARY Autoradiography combined with electron microscopy is used to visualize the incorporation of 3H-uridine into the nuclei of cultured BSC, monkey kidney cells. A preferential RNP stain is applied to differentiate between chromatin and nuclear ribonucleoproteins. It is demonstrated that the incorporation of the radioactive precursor can be localized after pulses of only 2 min., on the one hand at the limit of the chromatin and of fibrillar areas of the nucleolus, and on the other hand in the proximity of condensed chromatin throughout the nucleoplasm where perichromatin fibrils are present: Pulses of 5 to 15 min show a considerable increase of labelling. The number of silver grains over the fibrillar areas of the nucleolus increases. The pattern of extranucleolar incorporation is not changed. If these pulses are followed by a chase varying from 15 min to 3 h, some radioactivity can always be demonstrated throughout the nucleolus, with a considerable amount in the RNP containing interchromatin area as well. However, interchromatin granules are not, or only weakly, labelled even after a labelling of 1 h followed bv a chase of UD to 3 h. -The results are discussed in view of recent biochemical findings of a rapidly labelled and locally metabolized nuclear RNA. It is suggested that the early-labelled RNA visualized in the vicinity of the condensed chromatin may at least partially correspond to the DNA-like RNA or HnRNA fraction.

There has been an increasing interest in recent years in a biochemically isolated, rapidly labelled nuclear, but extranucleolar RNA fraction which, in addition, has other remarkable properties: (1) its base composition reveals a DNA-like character; (2) most of it does not leave the nucleoplasm and is metabolized in situ. The size variation of its molecules is very wide, ranging between 20s to 100s [2, 6, 10, 30, 31, 341. This type of RNA has been isolated from HeLa and duck erythroblast cell nuclei but also is present in single puff areas of giant chromosomes of 1 Present address: Institut Suisse de Recheches Experimentales sur le Cancer, 21 rue de Bugnon, Lausanne, Suisse. 9-

711811

Diptera aspolydisperse “chromosomal” RNA [8]. It seemed of interest to find out which type of morphological structure, carrying RNP and being visible in the electron microscope, would correspond to this rapidly labelled RNA species.Possibly good candidates for it were expected to be the perichromatin fibrils, recently described new components of the nucleoplasm which are visualized after bleaching of the chromatin with the EDTA staining technique [4] at the periphery of the condensed chromatin area throughout the nucleoplasm [ 181. The most suitable approach for an electron microscopic study of this problem seemedto be a combined application of high resolution Exptl

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130 S. Fakan & W. Bernhard

Figs l-8. BSC, cell cultures, labelled with 3H-UdR, fixed 1 h in phosphate-buffered glutraraldehyde 1.6 %. Embedding in Epon. Gold latensification-Elon-ascorbic acid development. EDTA method for preferential RNP stain. Figs 6-7, Ilford L4 emulsion; the others, Gevaert NUC 3.07. Fig. 1. Nucleolus (nu) after 2 min of SH-UdR labelling. The radioactivity is localized at the periphery of the bleached portion of intranucleolar chromatin (-). The granular portion (g) is unlabelled. x 23 000.

autoradiography and the preferential RNPstaining method. One of us has already demonstrated that the technical problems linked with the simultaneous use of both can be solved [9]. In a previous paper, Granboulan & Granboulan [12], using short pulses of 3H-uridine and a classical stain, have shown that the precursor was incorporated within 5 min in the fibrillar part of the nucleolus and also in the interchromatin area where euchromatin was supposed to be localized. Extranucleolar label was also found by other workers [l, 171to be localized outExptl

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side the condensed chromatin. However, up to the present it was not possible to distinguish the diffuse chromatin fibrils from ribonucleoprotein fibrils as the classical staining techniques did not allow differentiation between RNA- and DNA-carrying structures. In this study, we have paid particular attention to the extranucleolar incorporation sites of 3H-uridine after short times of labelling, with and without a chase of variable duration with the cold precursor. The behaviour of the nucleolus will be reexamined,

Rapidly and slowly labelled nuclear RNA

Fig. 2. Extranucleolar portion of a nucleoplasm after high. The majority of the silver grains are localized and the RNP of the interchromatin area. No cytoplasmic

I3 I

2 min of $H-UdR labelling. Incorporation unusually over the border zone of the bleached chromatin (chr) label. x 23 000. Exptl

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132 S. Fakan & W. Bernhard although this organelle has not been the main object of our investigation.

MATERIAL

AND

METHODS

A stabilised monkey kidney cell strain, BSC, [15] was used in these experiments. The cells were cultivated as monolayers in prescription bottles in Eagle minimal essential medium __ 171 suonlemented with __ 10% calf serum. For labelling of the cells, 3H-5-uridine (spec. act. 18 Ci/mM, CEA) was added to the cultures at the end of exuonential growth. The cells were exvosed or for 1 h for either-2, 5 or 15 min to 80 &i/ml, to 60 &i/ml, of 3H-uridine. After short pulses, some cultures .were briefly washed in cold medium containing 1 mg/ml of non-radioactive uridine and immediately fixed. Another group of cultures, after a pulse of 5 min and 15 min, was washed and postincubated in a medium containing 100 &ml of nonradioactive uridine for 15 min, 30 min; 1’ h and 3 h and fixed. The cells labelled for 1 h with 3H-UdR were fixed after 1 and 3 h of chase. The fixation was carried out in 1.6 % glutaraldehyde in Sorensen’s phosphate buffer for 1 h at 4°C. The cells were then rinsed for about 20 h in frequent changes of cold phosphate buffer with 0.2 M sucrose in order to eliminate the non-incorporated soluble RNA precursor molecules [20]. They were then scraped off the glass with a rubber policeman, centrifuged at low speed for 10 min to form a pellet, dehydrated with acetone and embedded in Epon according to the usual procedure. Ultrathin sections of silver-gold interference colour were cut with an LKB ultramicrotome equipped with a diamond knife. We did not use glycol methacrylate embedding for digestion of the preparations with RNAse, as controls for suecific incorooration into RNA have been carried ‘out repeatedly in earlier work by Granboulan & Granboulan 1121 ._nublished from this laboratory. The sections were deposited on microscopic glass slides covered with a thin collodion membrane [13]. The slides were dipped in either Gevaert NUC 3.07 or Ilford L4 emulsion, diluted to form a monolayer of silver halide crystals. After exposure for 25 days to 4 months. the maioritv of ureuaration were developed by the gold Iatensificat-ionlElon ascorbic acid method according to Salpeter & Bachmann [27] modified by Wisse & Tates [38]. This technique increases considerably the sensitivity, and therefore, shortens the exposure time. Furthermore, it also increases the resolution, especially when the NUC 3.07 emulsion is used. In order to localize more exactly the radioactive source corresponding to reduced silver grains, we have calculated the error limiting the resolution according to Bachmann & Salpeter [3]. Thus, the radius determining the distance around the develoned grain where the radioactive source is located with 50% probability, or the radius of the circle within which 50% of developed grains fall is, for an average section thickness of 850 A and with gold latensification-Elon ascorbic acid Exptl

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development, 1 120 8, for NUC 3.07 emulsion and 1 510 8, for L4 emulsion. Another imnortant advantage of the small grains (300-500 A)^ developed by this technique is that the underlying structures remain easilv visible. For comoarison. the remaining group of preparations was deieloped’in D-19 develoner for 4 min at 18-19°C. The background of the autoradiographs was very low for both techniques of development. After development and photographic fixation, the slides are washed and grids are slipped under the partially detached collodion membrane in the region of the sections, and then dried at 37°C. The staining is carried out on the slides either with uranvl acetate 5 % 10 min, followed by lead citrate 5 min or with the regressive EDTA stain [4]: uranyl acetate 5 % 10 min, rinsing in distilled water, drying 10 min at 37°C. differentiation in 0.2 M EDTA, nH 7. between 10 and 20 min followed by rinsing in distilled water, drying 10 min at 37°C and poststaining with lead citrate 5 min. Onlv at this stage are the grids detached from the glass slide with a steel needle and examined with the E.M. We used a Philins EM200 at 80 kV with a 50 p objective aperture. -

RESULTS The combined application of an autoradiographic technique based on physical development to reduce the grain size and to increase the sensitivity, with a staining technique which allows the bleaching of chromatin were indispensablefor obtaining the information which we needed. In the controls where classical development with Kodak D-19 was used and where the usual uranyl-lead stain was employed, the limit between chromatin and interchromatin areas could not be determined in most casesand the large grain size obscured the underlying nucleoplasmic structures. The examination of the cultures labelled with H3-UdR followed or not by chasesof various durations gave the following results. (1) Two-minute label In cells fixed immediately after 2 min labelling with 3H-UdR nearly all radioactivity is clearly localized over the limiting zone between EDTA-resistant RNP structures and the bleached chromatin. This is particularly well seen in the nucleolus where the silver

Rapidly and slowly labelled nuclear RNA

133

Fig. 3. Portion of a nucleolus (nu) and extranucleolar region, after a 5 min pulse of 3H-UdR. The drawing indicates the interface between the bleached chromatin (A) and the RNP-containing interchromatin area. Some silver grains are surrounded by a circle of radius 1 120 8, indicating the resolution of this technique. Most of the label is found over the nucleolus and the border zone between the condensed chromatin and interchromstin which is usually rich in perichromatin fibrils. x 23 000. Exptl

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134

S. Fakan & W. Bernhard

Fig. 4. Nucleoplasm after 5 min labelling with 3H-UdR. More silver grains zone, and many are still localized in the vicinity of the condensed chromatin 3 and 4 represent an average intensity of labelling. x 23 000.

are visible on the interchromatin (chr) which is not labelled. Figs

Rapidly and slowly labelled nuclear RNA

135

Fig. 5. Intensely labelled nucleoplasm after a 5 min pulse and a chase with cold uridine of 15 min. Practically all radioactivity is found in the RNP-containing interchromatin area and in the nucleolus (nu). x 23 000.

grains are localized predominantly at the limit of the fibrillar portions, where intranucleolar nucleohistones are supposed to be present (fig. 1). The same character of labelling is also found in the nucleoplasm, which however, seems relatively less labelled than the nucleolus (fig. 2). Nevertheless it is clearly shown that the large dense chromatin areas are not labelled whereas the majority of the grains are localized over the adjacent zone where RNP carrying structures are visible (fig. 2). (2) Five-minute label After 5 min of labelling the 3H-UdR incorporated in the nucleolus is seen both on the

limiting zone between the bleached intranucleolar chromatin and over the fibrillar part of the nucleolonema as well. The granular portion remains practically unlabelled. The extranucleolar nucleoplasm has the same pattern of labelling as after 2 min of pulse; however, the labelling is more intense and the preferential localization of the radioactivity on the border between dense chromatin and RNP containing interchromatin zone is accentuated (figs 3,4, table 1). After a 15 min chase, the labelling of the granular portions of the nucleolus has started, but the radioactivity is still predominantly in the fibrillar zones. In the nucleoplasm, there is a relative increase of labelling over the EDTA-resisExptl

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136 S. Fakan & W. Bernhard

Fig. 6. Nucleoplasm with weakly labelled intcrchromatin granules (ig) after a 5 min pulse with 3H-UdRanda chase of 1 h. The label is found predominantly at the periphery of the cluster. Compare intense labelling of the nucleolus (nu). x 65 000.

tant RNP structures in comparison with the border zones where the labelling decreases (fig. 5). With a prolonged chase the labelling of the interchromatin area increases still further and the distribution of silver grains, over both areas of the nucleolus, becomes more regular. Weak cytoplasmic label has appeared after the 15 min chase and reaches approximately the nuclear level after 3 h. Unlike the intensively labelled perichromatin fibrils, the aggregates of interchromatin granules are weakly labelled. Some radioactivity is observed at the periphery of the Exptl

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clusters, while their interior remains mostly unlabelled even after a 1 h chase (fig. 6).

(3) Fifteen-minute

label

After a pulse of 15 min, the radioactivity in the nucleolus is distributed more regularly over its components than after shorter periods of labelling. In the nucleoplasm, there is a relatively high amount of silver grains over the limiting region between RNP and chromatin, and the total labelling of the interchromatin area is higher than after shorter pulses. Again, after chasesof increasing duration, the

Fig. 7. Nucleoplasm with weakly labelled interchromatin granules (ig) after a 15 min pulse with 3H-UdR and a chase of 1 h. All the radioactivity is found at the periphery of the cluster. x 33 000. Fig. 8. Nucleoplasm with nucleolus (nu) and interchromatin granules (ig), after labelling of 1 h with SH-UdR and a chase of 1 h. Compare the very strong nucleolar labelling with the practically absent radioactivity of the cluster of interchromatin granules. x 38 000. Exptl

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138 S. Fakan & W. Bernhard Table 1. Counts of silver grains over three different regions of the nucleoplasm of 5 cells, fixed after 5 min label&g with 3H-uridine Cell no. 1 2 3 4 5 Total %

Border line of condensed chromatin

Interchromatin area

51 36 47 19 2;:

27 7 9 8 ;:

72

23.5

Condensed chromatin 1 3 t 6 14 4.5

Emulsion NUC 3.07, Gold latensification Elon ascorbic acid development. The results clearly show that the main radioactivity is found in proximity of the border line between condensed chromatin and the interchromatin area. Very few silver grains are seen over the bleached chromatin. The multiple grains whose centers were closer than 700 L%(mean diameter of silver halide crystals) were counted as a single grain.

total labelling of nuclear RNP structures becomes more intense. However, the clusters of interchromatin granules remain practically unlabelled or show some radioactivity at the periphery (fig. 7). (4) One-hour label

We have examined only cells fixed after 1 and 3 h of chase following 1 h of incubation with 3H-UdR. Our interest was mainly focused on the interchromatin granules. After both chases these constituents showed principally the same character of labellingas mentioned above for short pulses. The aggregates of such granules are labelled mostly over their periphery, while their centre remains practically unlabelled (fig. 8). The rest of the interchromatin area appears very heavily and rather randomly labelled. DISCUSSION The results of this investigation clearly show that 3H-uridine incorporation in cells of the BSC, strain can be demonstrated in well Exptl

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characterized areas of the nucleoplasm after a pulse of only 2 min. The two sites of radioactivity are:

(1) the fibrillar portion of the nucleolonema at the periphery of intranucleolar chromatin and; (2) The borderline between the condensed and EDTA-bleached chromatin and the EDTA-resistant interchromatin area. Concerning the labelling of the nucleolus the original work done by Granboulan & Granboulan [12] is confirmed and, by using the EDTA staining method, it can be shown that the early-appearing silver grains are localized at the peripheral parts of bleached regions of the nucleolonema, where DNA matrixes are believed to be present. The question arises where the DNA used for transcription is localized. We originally assumed that it corresponds to the intranucleolar dense chromatin lamellae [5]. However, according to Recher et al. [26] some DNA might also be present inside the fibrillar RNP zones. After about 15 min of chase, the now well known migration of radioactivity into the granular portions of the nucleolus is demonstrated morphologically. We also noticed the striking fact that there is much residual label in the nucleolus even after 15 min or 1 h of labelling and 3 h of chase. It is interesting that the label, whether nucleolar or extranucleolar, generally still increases after a chase of 15 min up to 1 h. This phenomenon has also been observed by other investigators [24]. One might hypothesize that a certain amount of radioactive precursor is somehow bound to structural elements and cannot be chased. Concerning the extranucleolar labelling, we can distinguish very rapidly labelled areas of RNP and, on the contrary, very slowly labelled or unlabelled regions believed to contain RNP.

Rapidly and slowly labelled nuclear RNA

As mentioned above, the very rapidly appearing label is seen at the border of the condensed chromatin, approximately in the region where perichromatin fibrils have been localized. However, in the cell strain we employed these newly described nuclear components are not as clearly found immediately adjacent to the clumped chromatin, and the resolution of our autoradiographic technique is not adequate to localize fibrils whose diameter varies between 50 and 200 A. Although we have no direct proof that the perichromatin fibrils are indeed the carriers of the radioactivity after very brief labelling, there is indirect evidence for this assumption. Petrov & Bernhard [25] were able to induce perichromatin fibrils in liver within 15 min after cortisone injection, a procedure known to stimulate rapid RNA synthesis. These fibrils were all localized at the periphery of the dense chromatin clumps. In later stages, the fibrils of RNP material are more irregularly dispersed in the interchromatin region. It was concluded that the newly synthesized RNA is visualized in the electron microscope in the form of these fibrillar components. If, in our experimental system, pulses are followed by chases of various intervals, one would expect the labelling to be more diffuse within the interchromatin area, and this is indeed the case. Earlier findings on localized RNA synthesis in the nucleoplasm are based on light or electron microscopic autoradiography the resolution of which was below that of our present method. Nevertheless, the results obtained can be put in line with our observations. Hsu [16] observed a very weak 3H-UdR incorporation in nuclear (heterochromatin) chromocentres. Littau et al. [17] and Allfrey et al. [l] found after 30 min of uridine labelling that there was considerable radioactivity present in the diffuse chromatin outside the condensed areas in isolated thymus cell nuclei,

139

and Granboulan & Granboulan [12] could demonstrate silver grains in the interchromatin area after a 5 min pulse of 3H-UdR in BSC cells. Unuma et al. [37] also described a predominant labelling of the dispersed chromatin in hepatoma cells labelled in vivo with 3H-UdR. Recently, Goldstein [l I] showed by light microscopic autoradiography, that after 1 and 15 min of incubation of L cells with 3H-UdR no preferential site of labelling over the nuclear membrane can be detected. He thus demonstrated the non-validity for an eukaryote cell of Stent’s hypothesis, dealing with a possible coupling of the transcription and translation process [35]. Our results strongly confirm Goldstein’s conclusion, as we have never observed any preferential labelling in the proximity of the nuclear membrane. An important question is to know how the biochemical findings of rapidly labelled extranucleolar RNA can be integrated with our ultrastructural observations. It would seem to us logical to conclude that the heterogeneous RNA fraction isolated in density gradients corresponds to the RNA components which are adjacent to the chromatin, i.e. the perichromatin fibrils. The fibrils are also very heterogeneous from the morphological point of view, as far as their thickness and probably, their length is concerned. It is not yet clear what relationship, if any, exists between mRNA and HnRNA [6]. Both are DNA-like,but whereas the former leaves the nucleus and is associated with polysomes, the majority of the latter (up to 90 %) seems to be metabolized in the nucleoplasm [2, 6, 10, 23, 30, 31, 341, thus confirming an observation already mentioned by Harris in 1962 [14]. The rapidly labelled RNA seems to be continuously synthesized throughout the interphase [22]. Scherrer & Marcaud [30] assume that the process of continuous partial disintegration of this “giant nascent messenger-like Exptl

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140 S. Fakan & W. Bernhard RNA” might be linked with regulation on the translational level. Our finding that even after 3 h of chase, there is still a relatively intense labelling of the interchromatin space supports the idea that a good deal of the rapidly labelled RNA species described in this paper is indeed kept in the nucleus and probably to a great extent metabolized in situ. Thus the radioactive precursor may be locally reutilized to resynthesize a very unstable RNA. The times of labelling in our experiments were generally much shorter than those used in biochemical studies, where the shortest pulses were of the order of 10 min [34], but mostly longer, varying between 30 min and 1 h. We believe that for precise localisation of the sites of incorporation, very short pulses should be used to minimize the effects of subsequent migration of the synthesized product. Slight cytoplasmic radioactivity was found as early as 15 min after labelling, and was steadily increasing afterwards. After 3 h of chase, it reached about the same level as found in the nucleus. According to our observations, there also exists a very slowly labelled RNA species, present in the interchromatin granules. The biochemical nature of these components, universally present in all interphase nuclei is still poorly known since their discovery [36] and their function is totally obscure. They are thought to contain proteins and RNA [5, 18, 32, 331 ,but they are very resistant to RNAse digestion even after protease action and can be extracted only with difficulty by cold perchloric acid [18]. In our preparations of 3H-uridine-labelled BSC, cells, we have found that after all intervals of pulse or chase most of the interchromatin granules are weakly labelled, if at all, with the radioactivity localized rather at the periphery of the clusters. According to Exptl

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these observations our interchromatin granules do not seem to be the same type of RNP particles as those isolated from rat liver and sedimenting at 40s which were found to contain a rapidly labelled RNA [19, 211. These particles were suggested to represent the interchromatin granules visible in this sections [19]. It is also improbable that the interchromatin granules represent the 30s DNA-like RNA containing particles isolated by Samarina et al. [28, 291 which are also said to be labelled rapidly. Here is an important problem which remains unsolved. Experiments with the same cell strain are now in progress based on long labelling and chase periods in order to determine the metabolic activity of these rather mysterious components. The authors are grateful to Miss S. Estrade from the Department of Virology (Prof. Tournier) for the preparation of the tissue cultures, and to Mrs J. Fakanova for technical assistance. They wish to thank Dr M. Hill for helpful discussion and Mrs Ch. Taligault for preparing-the manuscript. This study was carried out thanks to a fellowship awarded to one of us (S. Fakan) by the French Government and profited from financial aid from the Centre de Recherches sur les Lymphomes Malins, Lausanne (Prof. F. Cardis).

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28. Samarina, 0 P, Krischevskaya, A A & Georgiev, G P, Nature 210 (1966) 1319. 29. Samarina, 0 P. Lukanidin, E M. Molnar, J, & Georgiev, G P, J mol biol33 (1968) 251. 30. Scherrer, K & Marcaud, L, J cell physiol 72 suppl. 1 (1968) 181. 31. Scherrer, K, Marcaud, L, Zajdela, F, London, I M & Gras, F, Proc natl acad sci US 56 (1966) 1571. 32. Shankar Narayan, K, Steele, W J, Smetana, K & Busch, H, Exptl cell res 46 (1967) 65. 33. Smetana, K, Steele, W J & Busch, H, Exptl cell res 31 (1963) 198. 34. Soeiro, R, Vaughan, M H, Warner, J R & Darnell, J E, J cell biol 39 (1962) 112. 35. Stent, G S, Science 144 (1964) 816. 36. Swift, H. Interpretation of ultrastructure. Symp. int sot cell biol (ed R J C Harris) vol. 2, p. 21 (1962). 37. Unuma, T, Arendell, J P & Busch, H, Exptl cell res 52 (1968) 429. 38. Wisse, E & Tates, A D, Proc 4th europ reg conf E M, Tipografica Polyglotta Vaticana, Rome (1968) vol. 2, p. 465.

Received January 11, 1971. Revised version received February 26, 1971

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