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COMMENTARY the Nucleolus Today COMMENTARY The nucleolus today DANIELE HERNANDEZ-VERDUN Institut Jacques Monod, 2 place Jussieu, 75251 Paris Cedex 05, France Introduction antibodies that reduce the amount of free polymerase should also stop transcription. The nucleolus, the most prominent feature of the inter- In addition to the studies devoted to the interpretation of phase nucleus, is involved in ribosome biogenesis (Warner, nucleolar structures, others have attempted to draw up an 1990). During evolution, the nucleolus first appeared at inventory of the nucleolar proteins, in order to investigate the time when nuclear envelope compartmentation of the nucleolar complexity and to understand nucleolar func- cells was taking place. I would like in this review to tions. These investigations recently attracted much atten- provide information showing that the nucleolus is a tion (Sollner-Webb and Mougey, 1991; Warner, 1990) and particular nuclear territory in respect of the compartmen- in this commentary, I would like to focus particularly on tation of nuclear functions. the information available to date about nucleolar polarity, In eukaryotic cells, the nucleolus is the site at which nucleolar-specific proteins, nucleolar cell cycle, nucleolar ribosomal gene transcription takes place and the machin- targeting and small nucleolar RNAs. ery necessary for the production of the ribosomal subunits is assembled (Hadjiolov, 1985). At this site, the 18 S, 28 S and 5.8 S RNAs are synthesized and assembled with proteins such as ribosomal protein SI (Htigle et al. 1985a) Nucleolar polarity and 5 S RNA. The finding that chromosomes occupy a specific territory The nucleolus is not a stable organelle. Its structure, in the nuclear volume, defined as the chromosome domain size and organization depend on ribosome biogenesis. The (Hilliker and Appels, 1989), was recently demonstrated. different steps in this biogenesis correspond to nucleolar This demonstration was done by chromosome 'painting1, domains that can be identified by their morphology. There using specific probes corresponding to a single chromo- are three basic nucleolar domains, the fibrillar centers some, and in situ hybridization to locate specific chromo- (FCs), the dense fibrillar component (DFC) and the somes during interphase (Pinkel et al. 1989). It is therefore granular component (GC) (see nucleolar nomenclature not surprising that ribosomal gene distribution follows the reviewed by Jordan, 1984). They are found in all except a general rule of higher-order nuclear structure. However, very few nucleoli. Traditionally, the FCs are considered to the nucleolus organizer regions (NORs) are greatly be the storage sites of non-transcribed ribosomal genes, involved in nuclear polarity (Manuelidis and Borden, the DFC is the site of transcription of these genes and the 1988). Besides constituting a traditional cytological land- GC is the site of maturation and storage of the ribosomal mark, there is also evidence for polarity between different subunits (Goessens, 1984; Hernandez-Verdun, 1986; Som- NORs, and between NORs and the nuclear envelope. To merville, 1986). However, the actual sites of transcription underline the importance of nucleolar polarity, only these are still extremely controversial. Some authors believe two points will be discussed. Nevertheless, there is also they are located in the FCs (Scheer and Benavente, 1990); evidence for a specific arrangement of the centromeres in others at the border between the FCs and DFC (Derenzini Sertoli cells that reflects the activation or inactivation of et al. 1990; Thiry et al. 1991); and others again in the DFC the ribosomal genes (Haaf et al. 1990) as well as a specific only (Hartung et al. 1990; Wachtler et al. 1989). At present, association between the nucleolus and the centromere of if we take into account all the results obtained so far, the chromosomes 1 and 9 in neurons (Manuelidis and Borden, most reasonable interpretation is to propose that the 1988). ribosomal transcription units are compacted in the DFC, as recently concluded by Jordan in an extensive review In the metaphasic plate, the NOR-bearing chromosomes (Jordan, 1991). The main results that argue for the are closer to each other than would be expected from a localization of this transcription in the FCs are based on random chromosomal distribution. This observation indi- the labelling of the FCs by anti-polymerase I antibodies cates that there is certainly a polarized arrangement of the (Scheer and Benavente, 1990). However, the presence of NOR-bearing chromosomes in the interphasic nuclei. RNA polymerase I in the FCs does not prove that they Clearly, the NOR association seems to reveal the nucleolar constitute the site of RNA transcription. The polymerases fusion that takes place in species that generally possess detected can only be the free form, and the engaged form several pairs of NORs. After this fusion, or in species cannot be accessible during transcription. As there is an without nucleolar fusion, the positions of the nucleoli equilibrium between these two forms of RNA polymerase I (Marilley and Gassend-Bonnet, 1989; Sentenac, 1985), Key words: nucleolus, interphase, nucleus. Journal of Cell Science 99, 465-471 (1991) Printed in Great Britain © The Company of Biologists Limited 1991 465 remain fairly stable, even in rotating nuclei (Bard et al. end up in the mature ribosome and their role in ribosomal 1985). The effect of the respective positions of the different biogenesis is only conjectural for the great majority. They nucleoli might explain the nucleolar dominance in have been suggested as having roles in the transcription, competition for activation molecules, as several authors maturation, packaging and transport of ribosomal par- have recently proposed (Appels, 1989; Hilliker and Appels, ticles (Reeder, 1990). 1989). As revealed by in situ hybridization of rDNA The number of proteins located in the nucleoli is very sequences, certain ribosomal sites are not associated with large. Twenty years ago, 97 different nucleolar protein a nucleolus (Manuelidis, 1985; Wachtler et al. 1986). It is spots had already been detected by two-dimensional gel therefore likely that some non-transcribed rDNA regions electrophoresis (Orrick et al. 1973). As this detection was 3 are located separately or segregated from the rest of the limited to the major nucleolar proteins below 120 x 10 MT, NORs. Thus, in non-stimulated lymphocytes, the only we may assume that several hundred proteins are remaining nucleolar structure possesses a nuclear marker specifically confined within the nucleolar territory. The of transcriptional activity (see below), although in a small notion of nucleolar territory does not exclude the presence amount, but the other NOR sites lack the special proteins of nuclear proteins, which have also been found to be associated with transcription (Manuelidis, 1985; Wachtler associated with other genes, and can accumulate or be et al. 1986). This specific distribution implies precise located in the nucleoli. The best example of accumulation targeting of the proteins associated with the NORs that that is not in itself specific for ribosomal gene function is are or have been expressed. that of DNA topoisomerase I, which is found at the site of Very frequently, in higher eukaryotic cells, the nucleoli ribosomal transcription in the nucleoli. Such accumu- are located at or near the nuclear envelope (reviewed by lation was to be expected in view of the fact that the Bourgeois and Hubert, 1988), or in yeast nuclei they are nucleoli contain the highest concentration of highly active juxtaposed to the inner nuclear membrane (reviewed by genes in the nucleus (Reeder, 1990). There are also Clark et al. 1990). This position seems related to the numerous proteins that in the nuclei are targeted to the presence of specific skeletal structures at the site of the nucleoli (see below under Nucleolar targeting) and are not nucleolar attachment to the envelope. This nucleolar present in other nuclear territories. skeleton either adheres directly to the nuclear lamina or is attached to it by a pedicle as visualized in spread lamina RNA polymerase I preparations (Bureau et al. 1986). At present, little This is localized in the nucleoli and is specific for ribosomal information is available about the characterization of the gene transcription of large ribosomal RNAs. This polym- nucleolar skeleton, especially its specificity compared to erase is a large molecule with complex subunit structures that of the nuclear matrix (reviewed by Bouteille et al. composed of at least six polypeptides (Sentenac, 1985). Its 1983) and its action on the structure of the nucleolar two large components, which are highly conserved domain. A filamentous complex enriched in a protein of (Rowland and Glass, 1990), are involved in the basic 3 145 x 10 Mr, was identified as a specific nucleolar skeleton polymerization reaction, whereas the small subunita seem in amplified Xenopus laevis nucleoli (Franke et al. 1981). to have accessory roles. It has been proposed that one of 3 Moreover, the 180 x 10 MT nucleolar protein that contrib- these roles could be nuclear localization or enzyme utes to the general structure of the DFC (Schmidt- assembly (Rowland and Glass, 1990). In practice, the Zachmann et al. 1984) might be one of the nucleolar active RNA polymerase I is required for the formation of skeletal proteins playing a role in nucleolar architecture. the nucleolus as its major component, as demonstrated in In nuclei with centrally located nucleoli, there is a yeast (Hirano et al. 1989). Moreover, re-formation of the folding of the nuclear envelope called the nucleolar canal, active nucleoli was inhibited by injection of antibodies which is in direct contact with the nucleoli (Bourgeois et directed against RNA polymerase I in mitotic cells al. 1982). This canal was shown to be a nuclear envelope (Benavente et al. 1987). However, RNA polymerase I specialization that depends on the presence of active NORs activity is dependent on the presence of nucleolar (G^raud et al. 1989). We observed PtKl micronucleated transcription factors such as UBF and SL1 (Jantzen et al.
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