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STRUCTURE-FUNCTIONSUBCOMPARTIVlENTSOF THE MAMMALIANCELL NUCLEUS AS REVEALED BY THE ELECTRON MICROSCOPICAFFINITY CYTOCHEMISTRY I. Ragka, M. Dundr and K. Koberna

Inst. Experimental Medicine, Czechoslovak Academy of Sciences, Albertov 4, CS-12800 Prague 2, Czechoslovakia

ABSTRACT This electron microscopy review documents the in situ cytochernical localization of important nuclear structures and relates this to the important nuclear functions of RNA transcription and processing. With the help of specific probes (antibodies, nucleic acid probes), a comprehensive picture of nuclear subcompartrnentalization is beginning to emerge. INTRODUCTION In the last few years we have witnessed a large accumulation of information about the cell nucleus. This is primarily due to recent advances in molecular biology and biochemistry that have enabled a detailed characterization of the main nuclear processes of DNA replication, RNA transcription and processing, the transport of macromolecular complexes as well as the nuclear breakdown and reorganization accompanying mitosis. Knowledge of these processes has almost entirely been obtained from in vitro systems: it is important to emphasize that the efficiency of such cell free systems is just a small fraction of in vivo processes. The challenge that arises is how to transpose the knowledge of the molecular speclficities, as gained by molecular biology, to the context of an intact cell. Affinity cytochemistry methods, including immunocytochemistry and in situ hybridization, represent the most convenient approach to try and bridge the gap between molecular biology and cytology. Nevertheless, there is still a gap between molecular biology and cytology at this moment. One reason is the limited resolution of in situ affinity cytochemistry. The second, more important one results from the nature of morphological approaches which usually depict "static" information. They do not give information about the process itself, but they show structures related to it. We would like to emphasize, at this point, that the cell nucleus is a single compartment. Because of this, we expect that structure-function subcompatlmentalization cannot be governed by the complicated rules that are encountered in the compartmentalizedcytoplasm. In addition, the attribution of a specific function to a specific nuclear structure may not be as equivocal as in the case of true compatiments. There are no definite barriers (such as membranes) in between the various structural nuclear components which, with ~ few exceptions like some metabolically inert heterochromatin (chromosome) areas (Manuelidis, 1990), are dynamic structures in themselves. This review shows that in situ electron microscopic (EM) affinity cytochemistry 0309-1651/92/080771-19/$03.00/0

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(EMAC) used in conjunction with colloidal gold adducts can be used to subcompartmentalize the mammalian somatic cell nucleus into domains which apparently play a role in various aspects of the major nuclear functions. In this review we document results concerning the nucleolus and the nucleoplasmic ribonucleoprotein (RNP) regions, i.e. nuclear regions which are involved in the RNA synthesis, and maturation and transport of ribonucleoproteins (RNPs). T I E NUCLEOLUS The nucleolus is the most prominent nuclear organelle. Here ribosomal RNA (rRNA) is transcribed, processed and assembled into preribosomal particles (Reeder, 1990; Warner, 1990). In fact, nucleolar rRNA synthesis accounts for nearly half of the cellular transcriptional activity. For nucleolar function, the presence of more than 100 ribosomal proteins, nucleolar proteins and RNPs is required, including RNA polymerase I, DNA topoisomerase I, transcription factors, processing enzymes, 5S rRNA and U3, U8, U13 and U14 small nuclear RNAs (snRNAs) (Reeder, 1990; Warner, 1990). Such a large metabolic factory needs a massive import and export of (macro)molecules. Nucleolar morphology has to reflect this active steady state metabolic level (Busch and Smetana, 1970; Smetana and Busch, 1974). Three basic nucleolar components can be distinguished according to their EM appearance (Jordan, 1991; Fig. 1): fibrillar centers (FC) representing an interphasic correlate of nucleolar organizer regions (NOR) of mitotic chromosomes, dense fibfillar components (DFC) and granular components (GC). The nucleolus also contains condensed perinucleolar chromatin which penetrates the nucleolar body and gives rise to intranucleolar condensed chromatin clumps interspaced in between GC, DFC and FC. There is a general agreement that these three nucleolar components correspond in some way to the different events in the transcription of rRNA and the formation of preribosomes. The detailed picture is still unclear, stemming from the fact that the active rRNA genes have been localized by different research groups in both the FC and DFC (see also Jordan, 1991 for a thorough review of this problem). These opinions are based on results obtained by cytochemistry such as osmium amine staining of DNA (Derenzini et al., 1990), electron microscopic autoradiography (EMARG) of incorporated tritiated uridine (which is the only method depicting the active process of rRNA synthesis; Fakan, 1986; Wachtler et al., 1990; Thiry and Goesens, 1991), and affinity cytochemistry which includes various immunocytochemical approaches and in situ hybridization of both rRNA and rDNA (Scheer and Rose, 1984; Thiry and Thiry-Blaise, 1989; Wachtler et al., 1990; Puvion-Dutilleul et al., 1991). However, much of this controversy can be reconciled. The various groups have all used different cell types which exhibit very different transcription activities and contain various kinds of nucleoli. These studies include small ring-shaped nucleoli in resting peripheral lymphocytes, nucleoli with spatially well separated FC and DFC in human Sertoli cells, compact nucleoli with one or a few prominent FCs in Ehrlich ascites cells and nucleoli with multiple FCs in various established cell lines. The biochemical composition with respect to nucleolar transcription enzymes and factors as well as the number of active rRNA genes, confined to individual FC and/or DFC, clearly differ among the various cell types (e.g. Scheer and Rose, 1984; Ochs et al.,

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1989). It may therefore be not always feasible to ascribe a given function to a single structural domain. The question then arises as to whether it is at all possible to establish an equivocal structure-function relationship with the approaches that are available? It is necessary to document the most important affinity cytochemistry results which can then be taken into account for further discussion. In some recent reviews FC was proposed as being the sole site of rRNA transcription (Scheer and Benavente, 1990; Thiry et al., 1991) and this view has quickly been accepted by others in the field. Even though such an interpretation may be genuine, the accumulated amount of affinity cytochemical data does not point to such an equivocal interpretation. We shall therefore also discuss results concerning the localization of nucleolar and ribosomal chromatin as well as the localization of most important protein and RNP factors which play an established role during the rRNA synthesis and the rRNP maturation. Nucleolar DNA labelling can be obtained by means of various immunocytochemical approaches. The presence of gold particles in FCs has been interpreted as evidence for the presence of rDNA within this nucleolar component (Scbeer and Benavente, 1990; Thiry et al., 1991). This opinion has been substantiated by the finding of DNAse I sensitive DNA domains, such as one would expect for active rRNA genes, in FCs, but not in DFCs, by means of nick-translation experiments (Thiry, 1991). This system employs the exonuclease activity of an exogenous DNA polymerase at DNAse I nicks for the incorporation of biotinylated bases into DNA. However, in our hands, DNA label can also be observed in DFC of several mammalian cell types as well using this experimental system (Ra~ka et al., 1990; Dundr et al., in preparation; Fig. 1, 2). Furthermore, we were not able to reconcile the label in nick-translation experiments with sensitive DNAse I sites in HeLa cells as we observed a uniform label on mitotic chromosomes under a mild DNase I treatment (Dundr et al., in preparation). It is important to mention two facts conceming the localization of ribosomal genes. Firstly, we should take into account that the picture of transcribing rRNA genes seen in Miller's spreads (Miller, 1981) makes it difficult to reconcile their presence within the nucleolar condensed chromatin clumps. Secondly, rRNA genes represent only about 1% of the total nucleolar DNA (Bachellerie et al., 1977) and only some of the rRNA genes are active (Conconi et al., 1989). Various rDNA sequences have been detected by in situ hybridization method either in FC (Thiry and Thiry-Blaise, 1989; Puvion-Dutillieul et al., 1991) or in DFC (Wachtler et al., 1991)). Our preliminary observations indicate that probes to 18S rDNA hybridize to both FC and to DFC in HeLa cells (Fig. 3). The localization of rRNA transcripts using in situ hybridization has shown their high incidence in DFC and GC. A few transcripts were also localized to the peripheral region of FC (Thiry and Thiry-Blaise, 1989; Puvion-Dutilleul et al., 1991). One of the major arguments supporting FC as the site of transcription is the exclusive presence of RNA polymerase I and DNA topoisomerase I in this nucleolar component (Scheer and Benavente, 1990; Thiry et al., 1991). These enzymes respectively transcribe rRNA genes and activate ribosomal chromatin. However, even though the majority of the gold particles are observed in FC in the postembedding

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RNA polymerase I labelling protocol performed on various ceil types, the label is also present in DFC (Ra~ka et al., 1989, 1990; Fig. 4). Furthermore, the NOR 90 antigen corresponding to the Upstream Binding Factor controlling the activity of RNA polymerase I (Rodriguez-Sancbez et al., 1987; Jantzen et al., 1990), is found both in FC and DFC (Rodrigo et al., 1991; Fig. 5). With regard to the localization of DNA topoisomerase I, observations showed the highest incidence of gold particles over DFC, with fewer particles located over other nucleolar components of several ceil types (Ra~ka et al., 1989, 1990; Fig. 4). In our opinion the RNA polymerase I and DNA topoisomerase I localization results obtained by the preembedding method do not entirely reflect their genuine localization in the ceil (Ra~ka et al., 1990). Fibrillarin is a highly conserved protein associated with U3, U8 and U13 snRNPs (Tyc and Steitz, 1989). These snRNAs contain a trimethylguanosine (m3G) cap and a direct role (roles; Savino and Gerbi, 1990) of U3snRNP in the early processing of precursoric rRNA was recently demonstrated (Kass et al., 1990).

Fig. 1. Nucleolus of a HeLa cell embedded in Lowicryl K4M. The section was heat denatured and immunostained for ssDNA (Thiry and Thiry-Blaise, 1989). 5 nm gold particles are concentrated over condensed chromatin (C), including intranucleolar chromatin clumps. Nucleolar components are designated: FC (F), DFC (D), GC (G). Some nucleolar label is present over FC (small arrowheads) and DFC (large arrow heads). Note that the nucleolus associated coiled body (B) is not labelled. Nucleus: N, cytoplasm: Cy, nucleolus: Nu, interchromatin granules: Ig. This figure and Fig. 3 were obtained from a collaboration with Dr. M. Thiry. The bar in this and all consecutive figures corresponds to 0.1 /am. Fig. 2. Nucleolus of a cryosectioned HeLa cell according to Tokuyasu (1989; Griffiths et al., 1984). Nick translation was performed on this cryosection. 5nm gold particles reveal the presence of incorparated dUTP tagged with biotin. Intranucleolar chromatin clumps axe heavily labelled, gold particles are also found in FC and DFC (small and large arrowheads). We should emphasize that Tokuyasu's method is flexible so that the contrast of various nuclear components can be increased or even changed from negative to positive contrast. For instance, a positive contrast of RNP structures can be obtained if polyvinylalcohol postembedding is used (e.g. Fig. 9) instead of the usual methylcellulose procedure. Fig. 3. Nucleolus of a Lowicryl K4M emdedded HeLa ceil. In this preliminary experirnent, in situ hybridization was performed with 18S rRNA sense probe of mouse origin (Thiry and Thiry-Blaise, 1989; the indentity between mouse and human 18S rDNA exceeds 99%). 5 nm gold particles, which reflect the presence of biotins in the 18S rRNA probe, are found in FC as well as in DFC. One linearly arranged cluster of gold particles is found both in FC and DFC (two arrowheads with an antiparallel orientation). Fig. 4. Cryosection of a HeLa ceil nucleolus immunostained for RNA polymerase I (10 um particles) and for DNA topoisomerase I (5 nm particles). The label due to RNA polymerase I is enriched in FC, that of DNA topoisomerase I (small and large arrowheads) in DFC.

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In agreement with the literature (Ochs et al., 1985), we have observed that fibrillarin is almost exclusively confined to DFC (Ra~ka et al., 1989, 1990). Experiments with rabbit anti-m3G antibodies (L'titmnann et al., 1983) or antisense biotinylated oligonucleotides to U3 snRNA (Carmo-Fonseca et al., 1991a) yield a picture in which all nucleolar components are labelled. The incidence of gold particles in DFC is, however, higher than that in FC (Ra~ka et al., 1989, 1990; Fig. 6). The attribution of (early) rRNA processing events to DFC is compatible with these results. During transcription and processing, rRNA molecules become associated with a number of different macromolecules. Some of these are ribosomal proteins, the others being macromolecules which apparently play a role in the maturation, packaging or transport of ribosomal particles. We shall discuss the localization of ribosomal protein S1 and 5S rRNP which belong to the first category of proteins. It is also hnportant to include results with nucleolin and protein B23. There are a great number of other nucleolar components which have already been mapped by immunocytochemistry. Their role is as yet unknown, and we refer the reader to other reviews for further information (e.g. Hemandez-Verdun, 1991). Chooi and Leiby (1981) clearly showed the association of ribosomal proteins L4 and S14 with transcribing rRNA genes in Miller spreads. It is possible that similar Miller's spreads combined with in situ mapping of these and other ribosomal proteins might provide new insights into the structure-function organization of the nucleolus. Although the antibodies against a number of ribosomal proteins are available, as far as we know, only a few ribosomal proteins were mapped (e.g. Hiigle et al., 1984). In the postembedding procedure, the protein S I was mapped to GC, with a low label present in DFC as well (Ra~ka et al., 1990; Fig. 7).

Fig. 5. Cryosection of a HeLa cell nucleolus labelled with human antibodies targetting NOR 90 antigen. 10 nm gold particles are found in both FC and DFC (small and large arrowheads). Note the presence of a few gold particles in GC which "decorate" the external periphery of DFC (arrows). Fig. 6. A cryosectioned HeLa cell nucleolus. In situ hybridization of U3 snRNA was performed with an antisense oligonucleotide tagged with biotin (Carmo-Fonseca et al., 1991a). The biotins were revealed by an ind~r"ect immunocytochemical method. The few 5 nm gold particles (arrowheads) present in the nucleolar profile, some are found in both FC arid in DFC. Fig. 7. Rat hepatocyte embedded in Lowicryl K4M was immunostained with antibodies to ribosomal protein S 1. The nucleolus and cytoplasm are predominantly labelled (arrowheads). Interestingly, several 5 nm gold particles are observed in between the nucleolus and the cytoplasm (arrows). Fig. 8. Cryosections of a mouse 3T3 cell was imrnunostained with antibodies to nucleolin and protein B23 (from a collaboration with Professor M.O.J. Olson and Dr. K. Sipos). A great majority of 10 nm (nucleolin) and 5 nm (protein B23: arrowheads) gold particles are found in DFC and GC. There are just a few gold particles present in FC. It remains to be established whether a low nucleoplasmic label (arrows) reflects the shuttling of nucleolin and protein B23.

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5S rRNA is synthesized outside the nucleolus by RNA polymerase III and is imported into the nucleolus as RNP (Steitz et al., 1987). The RNP complex containing 5S rRNA is localized in HeLa cell nucleoli, but not in the nucleolar FCs (Ra~ka et al., 1990). Nucleolin is believed to be involved in the activation of ribosomal chromatin and the transcription and processing of rRNA (Olson et al., 1990). Like another abundant nucleolar protein, protein B23, nucleolin was shown to shuttle between the nucleolus and cytoplasm and to be a target of cdc2 kinase (Borer et al., 1989; Peter et al., 1990). Their role as transporters as well as their role in nucleolar reorganization during mitosis is therefore conceivable. Within nucleoli, both nucleolin and protein B23 have mainly been found in DFC and GC (Biggiogera et al., 1989; Ra~ka et al., 1990; Fig. 8). In sununary, whatever the actual arrangement of active ribosomal genes is, the affinity cytochemistry results do not allow the localization of active genes solely to either FC or DFC. The hypothesis that FC and DFC form a structure-function domain for the transcription of rRNA genes seems favourable (Ra~ka et al., 1989, 1990; see also Jordan, 1990 for other posibilities). The amount of accumulated data does enable the localization of processes involving rRNA processing and preribosome formation in DFC and GC. However, it remains to be established to what extent these processes are vectorial. NUCLEOPLASMIC RNP SPACE (NRS) Nucleolar 45S rRNA is synthesized in the nucleolus by RNA polymerase I. All other RNAs are synthesized in the nucleoplasm by RNA polymerases ]I and 11I. The primary RNA transcripts are subjected to co- and post- transcriptional processing (splicing, polyadenylation etc.), transport, storage and degradation (Nigg, 1988). The whole process of extranucleolar RNA transcription and maturation involves a great number of macromolecular protein and RNP complexes containing e.g. RNA polymerases, transcription factors, components of heterogeneous nuclear RNPs (hnRNPs) and components of snRNPs. In contrast to the abundance of molecular biology data (e.g. Dreyfuss, 1986; Nigg, 1988; Lamond, 1991; Nigg et al., 1991), very little in situ information is available about these processes. Only recently, Spector (1990) was able to define a large scale structure-function subcompartmentalization of the cell nucleus. By using EMAC data and EMARG of tritiated thymidine and uridine incorporation from serial sections through CHO 400 cells, he showed that Sm components of snRNPs are concentrated in a continuous reticulum that extends from the surface of the nucleolus to the nuclear envelope. The study showed that in situ sites of replication and transcription occur in complementary, rather than coincident, regions with respect to snRNP space. This review substantiates Spector's findings with EMAC results of various structures involved in the metabolism of RNA. What do we understand by the term "nucleoplasmic RNP space"? The term nucleoplasm refers to the internal part of the cell nucleus. Besides nucleolus, the nucleoplasm includes areas of condensed chromatin and two other morphologically defined regions: 1) perichromatin areas consisting of the border regions around the condensed chromatin clumps, and 2) the intranuclear region or interchromatin space.

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The distinction between perichromatin and interchromatin areas tends to be arbitrary, and nucleoplasm may basically be divided into chromatin areas, nucleolus and interchromatin areas which we call nncleoplasmic RNP areas (Ra~ka et al., 1990). We also need to introduce the notion of euchromatin which is said to be the morphological correlate of transcriptionally active chromatin (Fakan, 1978, 1986). Euchromatin is also present in the nucleoplasm. Using ultrastructural cytochemistry combined with EMARG, this form of chromatin has been localized in the perichromatin region of the cell nucleus (Fakan, 1978, 1986). This morphological concept of extranucleolar RNA synthesis is in harmony with Spector's photographs (Spector, 1990). They show autoradiographic grains due to the incorporated tritiated uridine which more or less decorate the border of chromatin (respectively they decorate immunocytochemically stained snRNP areas). We should emphasize that the presence of euchromatin structures (the existence of which is derived from the molecular and cell biology concepts of interphase chromatin scaffolds; Nigg, 1988) in the perichromatin region of the thin sectioned mammalian cell nucleus has been inferred from EMARG data combined with cytochemistry. This is indicated by EMAC of DNA which enables a clear identification of condensed chromatin beneath a heavy gold label (Ra~ka et al., 1990; see also Fig. 2 for the nucleolar chromatin). However, we cannot always identify the structure beneath a label formed by individual gold particles within the nucleoplasmic RNP space as being euchromatin. Nevertheless, bearing these limitations in mind, it is possible to identify the NRS along with the snRNP space of Spector (1990), with the extranudeolar transcription occurring at the boundaries of NRS. The existence of (only) four categories of structures in N-RS can be established by EM. They are perichromatin fibrils (PFs), perichromatin granules (PGs), interchromatin granules (IGs) and nuclear bodies (NBs). Of the NBs, only simple nuclear bodies (SNBs) and coiled bodies (CBs) are routinely observed (Ra~ka et al., 1990). EMA_RG studies with tritiated uridine have provided evidence that PFs, which are structurally ill defined, contain transcribed extranucleolar RNAs (Fakan, 1978, 1986). However, this morphological definition lacks sufficient specificity as any RNP structure present in NRS, besides IGs, PGs and NBs, can be identified as being (composed of) PF(s). A lack of specific criteria may also apply to the definition of remaining identified nucleoplasmic RNP structures, i.e. IGs, PGs and NBs. In our opinion, a few of these structurally identified nucleoplasmic components may represent heterogeneous categories of different entities. These, however, may be basically involved in the same or similar functions (e.g. processing of different precursor mRNAs). In order to gain a deeper insight into structure-function relationships in the cell nucleus, probes which can define specific structural domains within a seemingly homogeneously looking cell nucleus are gaining increasing importance. The impact of autoantibodies (Tan, 1989) in such studies has to be emphasized. The localization of "autoantigens" such as components of snRNPs has proven to be a breakthrough in the study of nuclear subcompartmentalization (e.g. Lemer et al., 1981; Spector et al., 1983; Ringertz et al., 1986). It represents an additional reference base for the mapping of various antigens in most of today's studies, including this review.

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As showed by Spector (1990) using immtmoperoxidase staining, components of snRNPs, such as Sm antigens, can label the entire NRS. The use of the colloidal gold adducts gives an approximate idea about the concentration of Sm molecules present in various domains of NRS (Fig. 9). Their concentration in CBs is much greater than in clusters of IGs which were originally thought to be the primary sites of accumulation as documented by nuclear speckles from light microscopic immunocytochemistry (LMI). A similar picture of labelling of the NRS is obtained with anti-m3G antibodies (Ra~ka et at., 1990, 1991). The recently described SC-35 antibody to a non-snRNP factor, which is essential for the splicing activity (Fu and Maniatis, 1990), yields a slightly different picture of staining (Koberna et al., submitted; Fig. 10). It does not label CBs; the accumulation of gold particles is most conspicuous over clusters of IGs. LMI results indicate that the staining pattern corresponds only to nuclear speckles (Fu and Maniatis, 1990). Our results show that these speckles correspond above all to clusters of IGs. In addition, EMAC demonstrates that the SC-35 antibody labels other domains of NRS (Fig. 11). The difference in the Sm labelling between clusters of IGs and remaining parts of NRS, excluding CBs, is frequently not as large (Fig. 9) as is the difference in labelling with the use of SC-35 antibody (Koberna et at., submitted; Fig. 10). This illustrates an example where LMI cannot give an appropriate result. In this context, SS-B/La antigen, which was shown to be a termination factor of RNA polymerase IIl (Gottlieb and Steitz, 1989), is localized to clusters of IGs by means of a monoclonal antibody (Carmo-Fonseca et al., 1989). Even though we do observe label over IGs with highly monospecific autoantibodies to SS-B/La antigen, we also observe an overall NRS label which differs from that observed with Sm, U1 snRNP or SC-35 antibodies (Fig. 12). What about probes which target structures involved in the snRNP metabolism to more restricted domains of NRS? A major advance has been reached using stable antisense oligonucleotides to snRNAs (Carmo-Fonseca et at., 1991a,b).

Fig. 9. In this cryosection of HeLa cell, chromatin space, NRS and nucleolus are easily discemable. The cryosection was irnmunolabelled with antibodies to Sm antigen (10 nm gold particles, arrowheads). Note the difference of staining among various domains of NR. CB is intensively stained, the remaining part of NRS (including IGs) exhilSits a much lower concentration of gold particles. A few PGs are designated by arrows. Fig. 10. Cryosection of HeLa cell hnmunolabelled with an autoimmune serum targetting predominantly p80-coilin (5 nm particles) and with SC-35 antibodies (10 run particles. CB (arrows) is heavily labelled with 5 run particles. A few nucleoplasmic 5 run gold particles correspond probably to other minor antibody specificities present in the serum (see also Fig. 13). A cluster of IGs is intensively stained with 10 nm particles, but they are absent in CB. Fig. 11. Lowicryl section of a HeLa cell immunolabelled with the SC-35 antibody. The "homogenous" distribution of 10 nm gold particles in NRS cannot be reconciled with a strictly speckled pattern as observed in LMI.

Cell Biology lntemational Reports, VoL 16, No. 8, 1992

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U2, U4, U5 and U6 snRNAs are detected in a small number of NRS foci only whereas U1 snRNA is also widely distributed throughout NRS. This result principally comes from in situ hybridizations performed on fixed cells and has been reconfirmed by in vivo experiments involving the microinjection of oligonucleotides tagged with a fluorochrome. In addition, antibodies to another splicing factor U2AF (Zamore and Green, 1991) have been found to label NRS, with an enrichment in these loci. Recently, an autoantibody targetting a protein component, called p80-coilin, of CBs has been identified (Ra]ka et al., 1990a, 1991; Andrade et al., 1991) and the identity of CBs and NRS foci can now be established (Kobema et al., submitted; Fig. 13). However, besides CBs, some NRS label is observed in in situ hybridizations with not only U1 snRNA, but also with the oligonucleotides to other snRNAs (Kobema et al., submitted). This apparent discrepancy could be explained by a different in situ hybridization protocol for the cells and/or by the fact that U2 snRNAs and particularly U4, U5 and U6 snRNAs are present in a lower copy number in the cell nucleus than U1 snRNA. If these findings are genuine, they may remind us of the results with the SC-35 antibody (see above). Where is the active splicing situated? The results obtained with oligonucleotide probes are very suggestive, but there are three obstacles to the splicing process taking place in CBs.

Fig. 12. Cryosection of a HeLa ceil. The cryosection labelled with autoantibodies to SS-B/La antigen exhibits a widespread NRS distribution of 10 nm gold particles. Fig. 13. Cryosection of a HeLa cell. In situ hybridization with a biotinylated antisense oligonucleotide to U1 snRNA (Carmo-Fonseca et al., 1991a) was performed with a help of an indirect immunocytochernical method (5 nm particles reveal the presence of biotins), the cryosection was then immunolabelled for p80-coilin with an autoimmune serum (10 nm particles). 5 nm particles are enriched in CB (arrowheads), there are also several gold particles present in a cluster of IGs (arrows). Fig. 14. Cryosection of a HeLa cell immunolabelled with a monoclonal antibody targetting the L protein of hnRNPs exhibits a widespread NRS staining (10 nm particles). Note the absence of gold particles in a cluster of IGs. Several gold particles are present over RNP clumps of homogenous electron density (arrows). Fig. 15. CryosectiofL of a HeLa cell from the same preparation shown in Fig. 14. In this micrograph, a heavy label (10 nm particles; arrowsheads) is present on RNP clumps of NRS which we found compatible with large speckles due to hnRNP L protein as observed in LMI (Pinol-Roma et al., 1989). Fig. 16. Lowicryl section co-stained with rabbit antibodies to nuclear domains described by Szostecki et al. (1990) (5 nm particles) and with SC-35 antibodies (10 nm particles). The 5 nm gold particles identify a distinct nuclear domain which we include, according to its EM appearance, in a category of SNBs. Note that SC-35 antibody (as well as anti-p80-coilin antibody (Kobema et al., submitted)) does not co-staha this nuclear domain. Interestingly, several PGs are stained by SC-35 antibody (arrows). From a collaborative work with Dr. C. Szostecki and Dr. H. Guldner.

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First, CBs are not observed in every interphasic cell (Ra~ka et al., 1991;/uadrade et al., 1991). Furthermore, the splicing factor of Fu and Maniatis is not found in these nuclear domains. Finally and most importantly, EMARG does not show incorporated tritiated uridine in CBs (this, to some extent, also applies to the clusters of IGs as well (Fakan, 1978, 1986; Fakan, pers. communication), which are large enough nuclear domains to be seen despite the low resolution power of the method. The EMARG data also make it unlikely that CBs serve as sites of RNA synthesis from repeated genes (e.g. tR_NAs, 5S rRNAs, snRNAs) or as degradation subcompartments for excised introns. The transport structure for preribosomes is also not probable as 5S rRNP and ribosomal protein SI are not found in CBs (Ragka et al., 1991; Kobema et al., submitted). The possibility that the CBs play a role in the transport, maturation (assembly), storage, or recycling of snRNPs seems more likely. We consider the CBs to be a sort of metabolic marker, according to their presence in the cell under various experimental conditions. These conditions include the modulation of cell growth (including the cell cycle related changes), RNA synthesis and processing, and protein synthesis (Kobema et al., submitted). Interestingly, an association between nucleoli and CBs is observed (Fig. 1), particulary in neuronal cells (Ra~ka et al., 1990a). Therefore, CBs may also serve the nucleolus (Dundr et al., in preparation). Taking all the cytochemical data together, if the widespread NRS distribution of various snRNAs is corffinned, we can conclude that the m R N A processing apparatus may, at least in part, already be associated already with the nascent transcripts (Fakan et al., 1986) in NRS. However, it would probably be outside of clusters of IGs and outside of CBs. In situ information of the various steps of m R N A processing, transport and the accompanying involvement of snRNPs is very scarce. More research in this field is necessary, nevertheless, the observed linear tracks of hybridized mRNAs are promising (Xing and Lawrence, 1991). For further progress in the localization of splicing sites, probes specific for the active spliceosomes are evidently necessary, together with in situ hybridization experiments (including microinjection approaches) using probes for various forms of precursor mRNAs. In this respect, the localization of hnRNPs is of importance. The L hnRNP protein (Pinol-Roma et al., 1989) gives a widespread NRS localization with an accumulation of the label in domains consisting of clumps of RNP material (Figs. 14, 15). SUMMARY AND PROSPECTS

In this review we have tried to document and discuss some important EMAC data on the manunalian cell nucleus related to RNA synthesis and processing. The idea of structure-function related nuclear domains within a rather homogeneously looking cell nucleus is emerging from the data, but at the same time we must emphasize how little is actually known about the in situ description of nuclear processes. Probably the nucleolus, which serves only one major function, has been best-documented to date. However, the situation may quickly improve in the near future with the specific localization of genes and transcripts (e.g. Manuelidis, 1990; Berman et al., 1990; Xing and Lawrence, 1991; Carmo-Fonseca et al., 1991a,b) and with the availability of antibodies to new distinct nuclear domains (e.g. Szostecki et al., 1990; Cooke et

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al., 1990; Ascoli and Maul, 1991; Fig. 16). The importance of further progress of two approaches should be emphasized. One is the in situ EMAC approach combined with EMAC performed on Miller's spreads of both nucleolar and (characterized) extranucleolar material. The second is more important because it to some extent avoids the "static" information usually gained by morphologic approaches. It is the time-lapse confocal microscope analysis of the fate of microinjected probes (nucleic acids, antibodies, antigens or various ligands which may include competing synthetic peptides) tagged with a fluorochrome as recently documented by Carrno-Fonseca et al. (1991b). ACKNOWLEDGMENTS The authors would like to thank Professor K. Smetana and Dr. R. Jelhaek for critical reading of the manuscript, J. Otcovskb and Mrs. V. Rohlenovei for preparation of the manuscript and photographs, Dr. V. Mandys nad Mrs. H. Ederovei for help with the cell culturing. They thank Professor U. Aebi, Dr. M. Carmo-Fonseca, Professor G. Dreyfuss, Professor J. Dubochet, Dr. M. Fu, Professor W. W. Franke, Dr. G. Griffiths, Dr. A.I. Lamond, Professor R. Liihrmarm, Professor T. Maniatis, Professor B. Mechler, Professor M.O.J. Olson, Dr. S. Pinol-Roma, Dr. G. Reirner, Dr. A. Richter, Professor U. Scheer and Dr. E.M. Tan for generous gifts of antibodies, antisence oligonucleotides and various (immuno)chemicals. M. Dundr expresses his gratitude to Professor G. Goessens and Dr. M. Thiry for a short stay at the University of Liege. The authors apologize that due to a limited amount of space not all the original publications could be quoted. An additional list of literature is given in Ra~ka et al., (1990). Note added in proof: After the submission of the manuscript, several important papers have appeared dealing with the functional subcompaih~ientalization of the NRS (Wang et al., Proc. Natl. Acad. Sci. USA 88, 7391-7395, 1991, Carter et al., J. Ceil Biol. 115, 1191-1202, 1991, Spector et al., EMBO J. 10, 3467-3481, 1991, Huang and Spector, Genes Dev., 5, 2288-2302, 1991). The results of these publications are in agreement with the claims of this review and show that both endogenous and microinjected transcripts are associated with nuclear regions stained with Sm and SC-35 antibodies, and suggest that these regions may be involved in pre-mRNA processing.

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