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© The Rockefeller University Press,
0021-9525/2000//293 $5.00
The Journal of Cell Biology, Volume 149, Number 2,
, 2000 293-306
Original Article |
The Ribosomal RNA Processing Machinery Is Recruited to the Nucleolar Domain before RNA Polymerase I during Xenopus laevis Development
dhernand{at}ccr.jussieu.fr
Transcription and splicing of messenger RNAs are temporally and spatially coordinated through the recruitment by RNA polymerase II of processing factors. We questioned whether RNA polymerase I plays a role in the recruitment of the ribosomal RNA (rRNA) processing machinery. During Xenopus laevis embryogenesis, recruitment of the rRNA processing machinery to the nucleolar domain occurs in two steps: two types of precursor structures called prenucleolar bodies (PNBs) form independently throughout the nucleoplasm; and components of PNBs I (fibrillarin, nucleolin, and the U3 and U8 small nucleolar RNAs) fuse to the nucleolar domain before components of PNBs II (B23/NO38). This fusion process is independent of RNA polymerase I activity, as shown by actinomycin D treatment of embryos and by the lack of detectable RNA polymerase I at ribosomal gene loci during fusion. Instead, this process is concomitant with the targeting of maternally derived pre-rRNAs to the nucleolar domain. Absence of fusion was correlated with absence of these pre-rRNAs in nuclei where RNA polymerase II and III are inhibited. Therefore, during X. laevis embryogenesis, the recruitment of the rRNA processing machinery to the nucleolar domain could be dependent on the presence of pre-rRNAs, but is independent of either zygotic RNA polymerase I transcription or the presence of RNA polymerase I itself.
Key Words: prenucleolar body • nucleologenesis • pre-rRNA • RNA polymerase I transcription • Xenopus laevis development
© 2000 The Rockefeller University Press
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Introduction |
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Processing of rRNAs involves cleavage, methylation, and pseudouridylation of the primary rRNAs (Hadjiolov 1985; Smith and Steitz 1997). Cleavage is controlled by several ribonucleoprotein (RNP) complexes that act in an ordered manner to remove the external transcribed spacers (5'ETS and 3'ETS) and the internal transcribed spacers (ITS1 and ITS2). Fibrillarin (Ochs et al. 1985b) and nucleolin (Ginisty et al. 1998) associated with several small nucleolar RNAs (snoRNAs), including U3, could play a role during the first steps of rRNA processing (for a review see Tollervey 1996). Subsequent cleavages involve endoribonuclease activities such as the MRP RNase complex (Lygerou et al. 1996a,Lygerou et al. 1996b; Dichtl and Tollervey 1997; Pluk et al. 1999; Van Eenennaam et al. 1999) for the ITS1, and protein B23 (Savkur and Olson 1998) and U8 (Michot et al. 1999) for the ITS2. In Xenopus laevis, U8 was shown to be involved both in early 3'ETS and late ITS2 cleavages (Peculis and Steitz 1993). Exoribonuclease activity is required for complete processing of 5.8 S rRNAs by the exosome complex (Mitchell et al. 1997; Briggs et al. 1998; Allmang et al. 1999).
Several proteins of the rRNA processing machinery are detected in prenucleolar bodies (PNBs; Ochs et al. 1985a), which are scattered throughout the nucleus in early G1 (Benavente et al. 1987; Jiménez-Garcia et al. 1989; Ochs and Smetana 1991; Azum-Gélade et al. 1994; Scheer and Weisenberger 1994; Beven et al. 1996; Dundr et al. 1997; Zatsepina et al. 1997). Fibrillarin, nucleolin, Nop52, PM-Scl 100/exosome, and protein B23 are found within these PNBs, as are the U3 and U14 snoRNAs (Azum-Gélade et al. 1994; Gautier et al. 1994; Jiménez-Garcia et al. 1994; Beven et al. 1996; Mitchell et al. 1997; Fomproix and Hernandez-Verdun 1999; Savino et al. 1999). Therefore, the PNBs appear to contain preassembled nucleolar complexes mainly involved in processing steps of the pre-rRNAs (Scheer and Weisenberger 1994). Distinct PNBs are involved in the delivery of specific processing complexes to the nucleolar domain (Fomproix and Hernandez-Verdun 1999; Savino et al. 1999). Since the delivery of the different PNBs follows a temporal order, it has been proposed that reassembly into nucleoli could proceed by a stepwise mechanism that reflects the role of these complexes (Savino et al. 1999). The temporally regulated targeting of PNBs during the cell cycle thus appears dependent on the constituents of these PNBs. However, the involvement of the transcription and/or the transcripts in this process is unknown.
The PNBs fuse at the nucleolar organizer regions (NORs) at the end of mitosis, when rRNA synthesis resumes (Jiménez-Garcia et al. 1994; Fomproix et al. 1998). This fusion is inhibited by microinjection into mitotic cells of antibodies directed against RNA polymerase I (RNA pol I; Benavente et al. 1987) and against DNA topoisomerase I (Weisenberger et al. 1993). Therefore, it has been proposed that fusion of PNBs into nucleoli is dependent on the resumption of rDNA transcription (Scheer et al. 1993).
Remarkably, during early X. laevis embryogenesis, a unique situation was revealed in which regroupment of fibrillarin and nucleolin around the rDNA occurred before the apparent activation of RNA pol I–dependent transcription (Verheggen et al. 1998). The first cell cycles of X. laevis embryogenesis provide an interesting biological situation since transcription is established de novo after 12 synchronized cell cycles devoid of transcription (Brown and Littna 1964; Newport and Kirschner 1982). At the midblastula transition (MBT), RNA pol II– and III–dependent transcription is activated, whereas RNA pol I transcription is initiated later (Shiokawa et al. 1981a,Shiokawa et al. 1981b; Newport and Kirschner 1982). This biological situation makes it possible to study PNB assembly and delivery in the context of active or inactive RNA pol I transcription. Before MBT, scattered PNBs containing fibrillarin exhibit similar ultrastructural features to postmitotic PNBs and at MBT fibrillarin regroups around the rDNA with maternal pre-rRNAs (Verheggen et al. 1998). At MBT, the association of rDNAs with UBF was demonstrated (Bell et al. 1997; Verheggen et al. 1998), but the presence of other partners of the transcription machinery and, in particular, the RNA pol I complex is not yet established. Indeed, at MBT it was reported that RNA pol I accumulated in nucleoplasmic structures different from PNBs (Bell and Scheer 1999), without information on its association with rDNA.
Nuclei assembled in X. laevis egg extracts contain PNB-like structures with fibrillarin, nucleolin, Nopp180, protein B23 (NO38 in X. laevis), U3, and U8 (Bell et al. 1992; Bauer et al. 1994; Bell and Scheer 1997). Since these PNBs assembled in vitro were not observed to fuse into a nucleolus, a reasonable hypothesis was proposed that this lack of nucleolar assembly is due to the absence of transcription in this system (Bell et al. 1992).
In the present study, we demonstrate that during both X. laevis embryogenesis and in nuclei assembled in vitro, two types of PNBs containing components of the rRNA processing machinery exist. During X. laevis embryogenesis, the recruitment of both types of preassembled complexes to the nucleolar domain occurs at a time when the RNA pol I complex is not detected in the nucleolar domain. Furthermore, this recruitment is not dependent on RNA pol I activity, but correlates precisely with the presence of pre-rRNAs of maternal origin. Pre-rRNAs are absent from nuclei in which RNA pol II and III transcription was inactive and, in this case, recruitment of the rRNA processing machinery does not occur.
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Materials and Methods |
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The rDNA probe for rRNA detection corresponds to the entire X. laevis ribosomal transcription unit inserted into pBR322 (clone pXcr7, kindly provided by F. Amaldi, Universito Tor Vergata, Roma, Italy). Double-stranded DNA probes were labeled with the nick-translation kit (GIBCO BRL) according to the manufacturer's instructions using biotin-14-dCTP. Probes for the detection of U3 and U8 are oligonucleotides complementary to three regions (positions 9-31; 63-85; and 101-122 for U3; and 33-60; 72-93; and 107-136 for U8). They were labeled with biotin-14-dCTP using 3' terminal transferase (Boehringer Mannheim).
Assembly of Nuclei in X. laevis Egg Extract
Eggs were obtained from female X. laevis and interphasic low-speed egg extracts were prepared as previously described (Almouzni 1998). This crude extract can be maintained on ice for 4 h without appreciable decay of nuclear assembly activity. Demembraned X. laevis sperm nuclei were prepared and permeabilized with lysolecithin (Almouzni 1998). For nuclear assembly, 105 sperm heads were added to 100 µl of egg extract and maintained at 23°C. After 45 min, when maximal decondensation of the nuclei occurred, nuclei were processed for photonic and electron microscopies.
Preparation of Embryonic Nuclei from X. laevis
Embryos were produced by in vitro fertilization (Almouzni and Wolffe 1995) and allowed to develop at 23°C in 0.1x modified Barth solution (Gurdon and Wickens 1983) for different times after fertilization. At this temperature, embryos were collected at the following stages: early blastula, 6 h after fertilization (stage 8); midblastula, 7 h after fertilization (stage 8.5); late blastula, 9 h after fertilization (stage 9); early gastrula, 10 h after fertilization (stage 10); gastrula, 11 h after fertilization (stage 10.5); and late gastrula, 13 h after fertilization (stage 12); as specified by Nieuwkoop and Faber 1994. In some cases, actinomycin D was added to inhibit the onset of transcription during development. As dissociation of the blastomeres was required for drug accessibility, embryos 3 h 30 min after fertilization were transferred to Ca2+- and Mg2+-free medium containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, and 7.5 mM Tris HCl, pH 7.6, as previously described (Duval et al. 1990). Actinomycin D (Sigma Chemical Co.) was added to the incubation medium at the desired concentration from a stock solution at 5 mg/ml. As a control, embryos were incubated in Ca2+- and Mg2+-free medium without actinomycin D. Other drugs, such as 
-amanitin and 5,6-dichloro-β-D-ribofuranosylbenzimidazole (DRB; Sigma Chemical Co.) could not be used as inhibitors of transcription in X. laevis embryos. When -amanitin was added to the incubation medium, it did not penetrate into the dissociated blastomeres and DRB had a deleterious effect on the division of the embryos. Suspensions of embryonic nuclei were prepared from embryos taken at precise times after fertilization as described (Verheggen et al. 1998).
Immunofluorescence and In Situ Hybridization
In vitro reconstituted nuclei and suspensions of embryonic nuclei were fixed with 1 vol 4% paraformaldehyde in PBS. The suspensions of fixed nuclei could be stored several weeks at 4°C. For immunofluorescence and in situ hybridization studies, nuclei were centrifuged onto a coverslip. After washing, the different coverslips were postfixed in methanol, permeabilized in 0.1% Triton X-100 (IBI) in PBS, and rinsed.
For immunofluorescence labeling, the coverslips were incubated with primary antibodies, followed by FITC- or TRITC-conjugated secondary antibodies (anti-human, anti-mouse, or anti-rabbit IgG; Jackson ImmunoResearch Laboratories) or cy3-conjugated anti-mouse antibodies (Sigma Chemical Co.), rinsed, counterstained with DAPI (4'-6-diamidino-2-phenylindole dihydrochloride; Polysciences, Inc.) and mounted with an antifading solution (Citifluor).
In situ hybridization of rRNAs was performed after immunolabeling as previously described (Verheggen et al. 1998). The rRNA hybridization mixture contained 40% formamide (GIBCO BRL), 10% (wt/vol) dextran sulfate (Sigma Chemical Co.), 50 ng/µl sonicated salmon testes DNA (Sigma Chemical Co.), and the biotinylated rDNA probe diluted to a final concentration of 1 ng/µl in 2x SSC. For the detection of U3 and U8, the hybridization mixture contained 30% formamide, 10% (wt/vol) dextran sulfate, 50 ng/µl sonicated salmon testes DNA, and the 3' end biotinylated oligonucleotides complementary to U3 and U8 diluted to a final concentration of 2 ng/µl in 2x SSC. As a control for the detection of RNA, hybridization was preceded by RNase digestion as described (Highett et al. 1993).
Electron Microscopy
In vitro reconstituted nuclei were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, at 4°C. They were washed in cacodylate buffer, postfixed in 1% OsO4 for 1 h at 4°C, dehydrated in alcohol, and embedded in Epon 812. Ultrathin sections were contrasted with uranyl acetate for 1 h and lead citrate for 2 min, and examined in a Philips EM412 electron microscope.
In Situ Transcription Assay
To localize transcription sites, 5-bromouridine-5'-triphosphate (Br-UTP) incorporation into embryonic nuclei was performed as previously described (Verheggen et al. 1998). In brief, nonfixed nuclei were permeabilized and incubated in transcription buffer in the presence of Br-UTP for 20 min at room temperature (Masson et al. 1996). Before immunolabeling, the nuclei were fixed with 2% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100. A monoclonal anti–Br-deoxyuridine antibody which also recognizes Br-UTP (Boehringer Mannheim) was used. Human antifibrillarin antibodies were used as a nucleolar marker. Transcription and fibrillarin signals were obtained using FITC-conjugated goat anti–mouse and TRITC-conjugated goat anti–human antibodies (Jackson ImmunoResearch Laboratories), respectively.
Optical Microscopy
Images were taken with a Leica epifluorescence microscope equipped with a thermoelectronically cooled charge-coupled device (CCD) camera (Leica). Grayscale images were collected separately with filter sets for FITC and rhodamine/TRITC using an oil immersion lens (63x, NA I.4 plan Apochromat). Grayscale images were pseudocolored and merged using the Adobe Photoshop 5.0 software.
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Fusion of PNB components to the nucleolar domain does not occur in in vitro reconstituted nuclei after several hours of incubation in the egg extract. B23/NO38 distribution remained diffuse and in small PNBs, as in embryonic nuclei 6 h after fertilization. Therefore, we attempted to identify the events that occur in embryonic nuclei and not in reconstituted nuclei that could be responsible for targeting of the rRNA processing machinery to the nucleolar domain.
RNA Polymerase I Was Excluded from the Nucleolar Domain during the Recruitment of Maternally Derived Pre-rRNAs and their Processing Machinery
It is generally accepted that the onset of rDNA transcription in cultured cells at the end of mitosis is an event required for relocalization of the rRNA processing machinery to the nucleolar domain (Scheer et al. 1993). RNA pol I could play a role in this recruitment process. This prompted us to investigate the distribution of RNA pol I at the time of nucleolar assembly in X. laevis embryos.
Immunolabeling of nuclei between 7 h and 11 h after fertilization with an mAb that immunoprecipitates the X. laevis RNA pol I complex (M. Schmidt-Zachmann, personal communication) revealed a speckled distribution (Fig. 5), as recently described (Bell and Scheer 1999). rRNAs were previously detected in the nucleolar domain at the time of PNB gathering and were identified as 40S transcripts, probably derived from a maternal pool (Verheggen et al. 1998). We performed in situ hybridization of rRNAs on nuclei previously labeled with the antibody against RNA pol I, to simultaneously visualize the RNA pol I complex and the pre-rRNAs on the nucleolar domain in nuclei isolated between 7 (Fig. 5, a–c) and 9 h after fertilization (Fig. 5, d–f). Speckle-like structures containing RNA pol I were far from nucleolar domains and consequently from rDNAs. This indicated that pre-rRNAs were maternally derived. A fraction of RNA pol I was found associated with the nucleolar domain only in nuclei 11 h after fertilization (Fig. 5, g–i). This corresponded precisely with the time when RNA pol I transcription began to be observed by the in situ transcription assay (see below).
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It was first necessary to check the ability of actinomycin D when applied to whole embryos to selectively block RNA pol I transcription. This prompted us to use embryos 12 h after fertilization and in situ transcription assays in conditions favoring the detection of RNA pol I transcription (see Materials and Methods). Transcription, revealed by Br-UTP incorporation in isolated nuclei from control and actinomycin D–treated embryos, was characterized by its sensitivity to -amanitin. In control embryos, the transcription detected in nuclei isolated 12 h after fertilization (Fig. 6 a) was RNA pol I–dependent (Fig. 6 c). Indeed, transcription was not sensitive to 100 µg/ml of -amanitin and consequently was not RNA pol II– and III–dependent. Accordingly, with RNA pol I transcription the transcripts were colocalized with fibrillarin (Fig. 6 b and d). In contrast, RNA pol I transcription was not detectable in 95% of the nuclei isolated from embryos treated with 0.5 µg/ml of actinomycin D and collected 12 h after fertilization (Fig. 6 e). This demonstrates inhibition of RNA pol I in actinomycin D–treated (0.5 µg/ml) whole embryos. Interestingly, the recruitment of fibrillarin occurred even when onset of RNA pol I transcription was abolished (Fig. 6 f).
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-amanitin, indicating that it corresponds to RNA pol II and/or III activities (Fig. 8 d). RNA pol II and/or III activities were also detected in most nuclei isolated from embryos treated with 0.5 µg/ml of actinomycin D (Fig. 8 g). In contrast, the onset of RNA pol II and III activities was inhibited in 86% of nuclei isolated from embryos treated with 2 µg/ml of actinomycin D (Fig. 8 j). In the latter nuclei, fibrillarin was maintained in large PNBs (Fig. 7 h and 8 k) and no pre-rRNA was detected by in situ hybridization (Fig. 7 g). In a few nuclei only, fibrillarin and pre-rRNAs were regrouped in the nucleolar domain (Fig. 7g and Fig. h, arrowheads). These nuclei most likely escaped inhibition of RNA pol II and III activities by actinomycin D, as indicated by the detection of fibrillarin clustered to nucleolar domain in the few nuclei in which RNA pol II and III activities were detected (data not shown). The PNBs that remained in the transcriptionally quiescent nuclei (Fig. 7 h and 8 k) were larger than the PNBs I observed in nuclei 6 h after fertilization (Fig. 1 b). Nucleolin, U3, and U8 were also found in these large structures (Fig. 9d and Fig. e). B23/NO38 was maintained in small diffuse PNBs similar to those observed in nuclei isolated 6 h after fertilization (data not shown). A second type of nuclei were in in vitro reconstituted sperm nuclei. Again, the absence of nucleolar domain formation was observed (Fig. 9, g–i). This is consistent with the transcriptional inactive state of these nuclei, which was previously reported (Bell et al. 1992).
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Discussion |
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To date, the mechanisms that govern PNB assembly are poorly understood. By systematic depletion of the egg extract, it has been shown that fibrillarin, nucleolin, B23/NO38, and U3 are dispensable for PNB assembly in reconstituted nuclei (Bell and Scheer 1997). At the end of mitosis, PNB formation and the onset of transcription occur simultaneously, suggesting a common regulatory event (Fomproix et al. 1998). Nevertheless, PNB assembly occurs in the absence of transcription in both cultured cells (Morcillo et al. 1976; Benavente et al. 1987) and reconstituted nuclei (Bell et al. 1992). Nucleolus-like particles with features of PNBs were assembled in a soluble extract of nucleoli (Trimbur and Walsh 1993). PNBs were also induced in interphasic cells which had been released from hypotonic shock and transferred to normal isotonic medium, even in the presence of actinomycin D (Zatsepina et al. 1997). During X. laevis embryogenesis, PNBs are assembled from maternally derived components at a transcriptionally silent stage (Verheggen et al. 1998). In the present study, we observe PNBs in early and late stage nuclei from embryos treated with high concentrations of actinomycin D, confirming that PNB assembly during X. laevis embryogenesis is totally independent of any transcription.
Transcription and Processing Machineries Assemble in Bodies at Locations Distinct from RNA Synthesis and Processing Sites
Accumulation of machineries in domains distinct from their effector sites is not limited to rRNA processing machinery. We also showed that RNA pol I was not at rDNA loci at the time of nucleolar domain formation during X. laevis embryogenesis. Several extranucleolar foci containing RNA pol I distinct from PNBs were observed in both blastula nuclei (Bell and Scheer 1999) and in vitro reconstituted nuclei (Bell et al. 1997). TBP is known to accumulate in these foci at the gastrula stage (Bell and Scheer 1999). However, the significance of the presence of these complexes in nucleoplasmic foci remains to be elucidated.
Several examples of domains for accumulation of transcription and processing machineries have been reported. Stress granules, containing the transcription factor HSF1, formed in response to heat shock and independent of the sites of active transcription (Jolly et al. 1999a). Speckled distribution has been described for components of the mRNA processing machinery (for reviews see Moen et al. 1995; Huang and Spector 1996a; Lamond and Earnshaw 1998; Misteli and Spector 1998). Speckles are prominent in cells with low transcriptional activity, whereas upon activation of transcription, factors are recruited from speckles to sites of active transcription (Jiménez-Garcia and Spector 1993; Huang and Spector 1996b; Jolly et al. 1999b). In addition, coiled bodies are sites where components of the processing machinery localize, but the precise role of these bodies is not yet defined (Boudonck et al. 1999; Frey et al. 1999). Recently, a model was proposed in which the coiled body (cajal body) is the site of preassembly of RNA transcription and processing complexes before transport to the appropriate genes (Gall et al. 1999).
PNB Recruitment to the Nucleolar Domain as the Second Step of Nucleologenesis
Fusion of the PNB components to the nucleolar domain constitutes the second event of delivery of the processing machinery to the nucleolus. This stage was shown to be dependent on RNA pol I activity in cultured cells at the end of mitosis (Giménez-Martin et al. 1974; Ochs et al. 1985a; Benavente et al. 1987; Scheer and Benavente 1990). In contrast, we showed that during the de novo assembly of the nucleolus in X. laevis embryos, neither RNA pol I activity, nor the catalytic enzyme RNA pol I itself were required for this recruitment process. Formation of the nucleolar domain in the absence of RNA pol I has been observed in yeast mutant strains in which rRNA was transcribed from plasmids containing rDNA flanked by an RNA pol II–dependent promoter (Oakes et al. 1998). However, the structure and position of the nucleolar domain were abnormal, even when the plasmids were integrated in chromosomal loci. RNA pol I was therefore suggested to play a role in the organization of the nucleolar domain. However, the authors did not exclude the possibility that a transcription factor, rather than the RNA pol I itself, was involved (Oakes et al. 1998).
Recruitment of the mRNA processing machinery has been shown to be mediated by the COOH-terminal domain (CTD) of the RNA pol II (Corden and Patturajan 1997; McCracken et al. 1997; Steinmetz 1997). Even in the presence of pre-mRNA in the transcription sites, the processing machinery is not recruited when the RNA pol II CTD is deleted (Misteli and Spector 1999). A different recruiting mechanism is used for rRNA processing machinery as RNA pol I is dispensable for nucleolar domain assembly during X. laevis embryogenesis.
Protein recruitment to a specific location can be controlled by reversible phosphorylation, which could provide an attractive mechanism for recruitment of PNB to the nucleolar domain. For example, the nuclear import of nucleolin in early blastula of X. laevis has been shown to depend on its phosphorylation (Mebmer and Dreyer 1993). The phosphorylation state also influences the subnuclear distribution of SR (Ser/Arg-rich) proteins (Misteli and Spector 1998) and their activity in splicing of mRNA during development of Ascaris lumbricoides (Sanford and Bruzik 1999). A hypothesis could be that recruitment of the rRNA processing machinery to the nucleolar domain involves changes in phosphorylation of proteins during X. laevis embryogenesis.
The major event for recruitment of the rRNA processing machinery is likely to be the presence of pre-rRNAs in the nucleolar domain. In cultured cells, the onset of rDNA transcription at the end of mitosis could provide pre-rRNAs required for recruitment of the rRNA processing machinery to the nucleolar domain (Scheer et al. 1993). This reassembly of PNBs is prevented if activation of rDNA transcription is inhibited (Benavente et al. 1987; Scheer and Benavente 1990). Remarkably, during X. laevis embryogenesis, inhibition of rDNA transcription does not prevent nucleolar assembly. Maternally derived pre-rRNAs are maintained during X. laevis embryogenesis and targeted to the nucleolar domain at MBT (Verheggen et al. 1998). Accumulation of these pre-rRNAs in the nucleolar domain could be a prerequisite for the recruitment of the processing machinery. The absence of nucleolar accumulation of maternally derived pre-rRNAs in actinomycin D–treated embryos appears to be a consequence of RNA pol II and III transcription inhibition. An event required for pre-rRNA accumulation could be promoted by zygotic transcription.
Besides the predominant role of pre-rRNAs, additional regulatory mechanisms must exist to allow the delayed translocation of PNB II components relative to PNB I components. During X. laevis embryogenesis, we observed that most of B23/NO38 was recruited later than fibrillarin and nucleolin. A comparable situation was observed for nucleolus formation in telophase cell (Fomproix and Hernandez-Verdun 1999; Savino et al. 1999). Considering that steps for nucleolar assembly are common to both embryonic and cultured cells at the end of mitosis, it is tempting to speculate that nucleolar assembly involves common regulatory pathways in both cases. The fact that components of rRNA processing machinery involved in distinct steps of processing are delayed in their recruitment could be in itself a regulatory mechanism for their stepwise involvement in rRNA processing. This is raising the question whether alteration of the timing of the recruitment process might impair processing of rRNA.
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Acknowledgments |
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This work was supported in part by grants from the Centre National de la Recherche Scientifique (programme Biologie Cellulaire no. 96098) and l'Association pour la Recherche sur le Cancer (contracts nos. 9143 and 5304 to D. Hernandez-Verdun, and no. 1030 to G. Almouzni). C. Verheggen was a recipient of a fellowship from l'Association pour la Recherche sur le Cancer.
Submitted: 20 September 1999
Revised: 29 February 2000
Accepted: 7 March 2000
Abbreviations used in this paper: Br-UTP, 5-bromouridine-5'-triphosphate; DAPI, 4'-6-diamidino-2-phenylindole dihydrochloride; ETS, external transcribed spacers; FISH, fluorescent in situ hybridization; ITS, internal transcribed spacers; MBT, midblastula transition; mRNA, messenger RNA; PNB, prenucleolar bodies; rDNA, ribosomal gene; rRNA, ribosomal RNA; RNA pol I, RNA polymerase I; snoRNA, small nucleolar RNA.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Allmang C., Petfalski E., Podtelejnikov A., Mann M., Tollervey D. & Mitchell P.. The yeast exosome and human PM-Scl are related complexes of 3'-5' exonucleases, Genes Dev., 13, 1999, 2148–2158.
Almouzni, G. 1998. Assembly of chromatin and nuclear structures in Xenopus egg extract. In Chromatin, A Practical Approach. 195–218.
Almouzni G. & Wolffe A.P.. Constraints on transcriptional activator function contribute to transcriptional quiescence during early Xenopus embryogenesis, EMBO (Eur. Mol. Biol. Organ.) J., 14, 1995, 1752–1765.[Medline]
Azum-Gélade M.-C., Noaillac-Depeyre J., Caizergues-Ferrer M. & Gas N.. Cell cycle redistribution of U3 snRNA and fibrillarin. Presence in the cytoplasmic nucleolus remnant and in the prenucleolar bodies at telophase, J. Cell Sci., 107, 1994, 463–475.[Abstract]
Bauer D.W. & Gall J.G.. Coiled bodies without coilin, Mol. Biol. Cell, 8, 1997, 73–82.[Abstract]
Bauer D.W., Murphy C., Zheng'an W., Wu C.H. & Gall J.G.. In vitro assembly of coiled bodies in Xenopus egg extract, Mol. Biol. Cell., 5, 1994, 633–644.[Abstract]
Bell P. & Scheer U.. Prenucleolar bodies contain coilin and are assembled in Xenopus egg extract depleted of specific nucleolar proteins and U3 RNA, J. Cell Sci., 110, 1997, 43–54.[Abstract]
Bell P. & Scheer U.. Developmental changes in RNA polymerase I and TATA box-binding protein during early Xenopus embryogenesis, Exp. Cell Res., 248, 1999, 122–135.[Medline]
Bell P., Dabauvalle M.C. & Scheer U.. In vitro assembly of prenucleolar bodies in Xenopus egg extract, J. Cell Biol., 118, 1992, 1297–1304.
Bell P., Mais C., McStay B. & Scheer U.. Association of the nucleolar transcription factor UBF with the transcriptionally inactive rRNA genes of pronuclei and early Xenopus embryos, J. Cell Sci., 110, 1997, 2053–2063.[Abstract]
Benavente R., Rose K.M., Reimer G., Hügle-Dörr B. & Scheer U.. Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells, J. Cell Biol., 105, 1987, 1483–1491.
Bentley D.. Coupling RNA polymerase II transcription with pre-mRNA processing, Curr. Opin. Cell Biol., 11, 1999, 347–351.[Medline]
Beven A.F., Lee R., Razaz M., Leader D.J., Brown J.W.S. & Shaw P.J.. The organization of ribosomal RNA processing correlates with the distribution of nucleolar snRNAs, J. Cell Sci., 109, 1996, 1241–1251.[Abstract]
Boudonck K., Dolan L. & Shaw P.J.. The movement of coiled bodies visualized in living plant cells by the green fluorescent protein, Mol. Biol. Cell., 10, 1999, 2297–2307.
Briggs M.W., Burkard K.T.D. & Butler J.S.. Rrp6p, the yeast homologue of the human PM-Scl 100-kDa autoantigen, is essential for efficient 5.8 S rRNA 3' end formation, J. Biol. Chem., 273, 1998, 13255–13263.
Brown D.D. & Littna E.. RNA synthesis during development of Xenopus laevis, the South African clawed toad, J. Mol. Biol., 8, 1964, 669–687.[Medline]
Corden J.L. & Patturajan M.. A CTD function linking transcription to splicing, Trends Biochem. Sci., 22, 1997, 413–416.[Medline]
Dichtl B. & Tollervey D.. Pop3p is essential for the activity of the RNase MRP and RNase P ribonucleoproteins in vivo, EMBO (Eur. Mol. Biol. Organ.) J., 16, 1997, 417–429.[Medline]
Dundr M., Meier U.T., Lewis N., Rekosh D., Hammarskjöld M.-L. & Olson M.O.J.. A class of nonribosomal nucleolar components is located in chromosome periphery and in nucleolus-derived foci during anaphase and telophase, Chromosoma., 105, 1997, 407–417.[Medline]
Duval C., Bouvet P., Omilli F., Roghi C., Dorel C., LeGuellec R., Paris J. & Osborne H.B.. Stability of maternal mRNA in Xenopus embryosrole of transcription and translation, Mol. Cell. Biol., 10, 1990, 4123–4129.
Fomproix N. & Hernandez-Verdun. Effects of anti-PM-Scl 100 (Rr6p exonuclease) antibodies on prenucleolar body dynamics at the end of mitosis, Exp. Cell Res., 251, 1999, 452–464.[Medline]
Fomproix N., Gébrane-Younes J. & Hernandez-Verdun D.. Effects of anti-fibrillarin antibodies on building of functional nucleoli at the end of mitosis, J. Cell Sci., 111, 1998, 359–372.[Abstract]
Frey M.R., Bailey A.D., Weiner A.M. & Matera A.G.. Association of snRNA genes with the coiled bodies is mediated by nascent snRNA transcripts, Curr. Biol., 9, 1999, 126–135.[Medline]
Gall J.G., Bellini M., Wu Z. & Murphy C.. Assembly of the nuclear transcription and processing machinerycajal bodies (coiled bodies) and transcriptosomes, Mol. Biol. Cell., 10, 1999, 4385–4402.
Gautier T., Fomproix N., Masson C., Azum-Gélade M.C., Gas N. & Hernandez-Verdun D.. Fate of specific nucleolar perichromosomal proteins during mitosiscellular distribution and association with U3 snoRNA, Biol. Cell., 82, 1994, 81–93.[Medline]
Giménez-Martin G.C., De la Torre C., Fernandez-Gomez M.E. & Gonzalez-Fernandez A.. Experimental analysis of nucleolar reorganization, J. Cell Biol., 60, 1974, 502–507.
Ginisty H., Amalric F. & Bouvet P.. Nucleolin functions in the first step of ribosomal RNA processing, EMBO (Eur. Mol. Biol. Organ.) J., 17, 1998, 1476–1486.[Medline]
Gurdon J.B. & Wickens M.P.. Use of Xenopus oocytes for the expression of cloned genes, Wu R., Grossman L. & Moldave K., Methods in Enzymology, 1983, 101:370–386, Academic Press, New York.
Hadjiolov, A.A. 1985. The Nucleolus and Ribosome Biogenesis. Vol. 12. M. Alfert, W. Beermann, L. Goldstein, K.R. Porter, and P. Sitte, editors. Springer-Verlag, New York. 268 pp.
Highett M.I., Rawlins D.J. & Shaw P.J.. Different patterns of rDNA distribution in Pisum sativum nucleoli correlate with different levels of nucleolar activity, J. Cell Sci., 104, 1993, 843–852.[Abstract]
Huang S. & Spector D.L.. Dynamic organization of pre-mRNA splicing factors, J. Cell Biochem., 62, 1996, 191–197a.[Medline]
Huang S. & Spector D.L.. Intron-dependent recruitment of pre-mRNA splicing factors to sites of transcription, J. Cell Biol., 133, 1996, 719–732b.
Jiménez-Garcia L.F. & Spector D.L.. In vivo evidence that transcription and splicing are coordinated by a recruiting mechanism, Cell., 73, 1993, 47–59.[Medline]
Jiménez-Garcia L.F., Rothblum L.I., Busch H. & Ochs R.L.. Nucleologenesisuse of non-isotopic in situ hybridization and immunocytochemistry to compare the localization of rDNA and nucleolar proteins during mitosis, Biol. Cell., 65, 1989, 239–246.[Medline]
Jiménez-Garcia L.F., Segura-Valdez M.L., Ochs R.L., Rothblum L.I., Hannan R. & Spector D.L.. NucleologenesisU3 snRNA-containing prenucleolar bodies move to sites of active pre-rRNA transcription after mitosis, Mol. Biol. Cell., 5, 1994, 955–966.[Abstract]
Jolly C., Usson Y. & Morimoto R.. Rapid and reversible relocalization of heat shock factor 1 within seconds to nuclear stress granules, Proc. Natl. Acad. Sci. USA., 96, 1999, 6769–6774a.
Jolly C., Vourc'h C., Robert-Nicoud M. & Morimoto R.I.. Intron-independent association of splicing factors with active genes, J. Cell Biol., 145, 1999, 1133–1143b.
Lamond A.I. & Earnshaw W.C.. Structure and function in the nucleus, Science., 280, 1998, 547–553.
Lygerou Z., Allmang C., Tollervey D. & Séraphin B.. Accurate processing of a eukaryotic pre-rRNA by RNase MRP in vitro, Science., 272, 1996, 268–270a.[Abstract]
Lygerou Z., Pluk H., van Venrooij W.J. & Séraphin B.. hPop1an autoantigenic protein subunit shared by the human RNase P and RNase MRP ribonucleoproteins, EMBO (Eur. Mol. Biol. Organ.) J., 15, 1996, 5936–5948b.[Medline]
Masson C., Bouniol C., Fomproix N., Szöllösi M.S., Debey P. & Hernandez-Verdun D.. Conditions favoring RNA polymerase I transcription in permeabilized cells, Exp. Cell Res., 226, 1996, 114–125.[Medline]
McCracken S., Fong N., Yankulov K., Ballantyne S., Pan G., Greenblatt J., Patterson S.D., Wickens M. & Bentley D.L.. The C-terminal domain of RNA polymerase II couples mRNA processing to transcription, Nature., 385, 1997, 357–361.[Medline]
Mebmer B. & Dreyer C.. Requirement for nuclear translocation and nucleolar accumulation of nucleolin of Xenopus laevis, Eur. J. Cell. Biol., 61, 1993, 369–382.[Medline]
Michot B., Joseph N., Mazan S. & Bachellerie J.P.. Evolutionarily conserved structural features in the ITS2 of mammalian pre-rRNAs and potential interactions with the snoRNA U8 detected by comparative analysis of new mouse sequences, Nucleic Acids Res., 27, 1999, 2271–2282.
Misteli T. & Spector D.L.. The cellular organization of gene expression, Curr. Opin. Cell Biol., 10, 1998, 323–331.[Medline]
Misteli T. & Spector D.. RNA polymerase II targets pre-mRNA splicing factors to transcription sites in vivo, Mol. Cell., 3, 1999, 697–705.[Medline]
Misteli T., Caceres J.F. & Spector D.L.. The dynamics of a pre-mRNA splicing factor in living cells, Nature., 387, 1997, 523–527.[Medline]
Mitchell P., Petfalski E., Shevchenko A., Mann M. & Tollervey D.. The exosomea conserved eukaryotic RNA processing complex containing multiple 3'-5' exoribonucleases, Cell., 91, 1997, 457–466.[Medline]
Moen P.T., Smith K.P. & Lawrence J.B.. Compartmentalization of specific pre-mRNA metabolisman emerging view, Hum. Mol. Genet., 4, 1995, 1779–1789.[Abstract]
Morcillo G., De la Torre C. & Giménez-Martin G.. Nucleolar transcription during plant mitosis, Exp. Cell Res., 102, 1976, 311–316.[Medline]
Newport J. & Kirschner M.. A major developmental transition in early Xenopus embryos. I. Characterization and timing of cellular changes at the midblastula stage, Cell., 30, 1982, 675–686.[Medline]
Nieuwkoop P.D. & Faber J., Normal table of Xenopus laevis (Daudin), 1994, Garland Publishing, Inc, New Yorkpp. 252.
Oakes M., Aris J.P., Brockenbrough J.S., Wai H., Vu L. & Nomura M.. Mutational analysis of the structure and localization of the nucleolus in the yeast Saccharomyces cerevisiae, J. Cell Biol., 143, 1998, 23–34.
Ochs R.L. & Smetana K.. Detection of fibrillarin in nucleolar remnants and the nucleolar matrix, Exp. Cell Res., 197, 1991, 183–190.[Medline]
Ochs R.L., Lischwe M.A., Shen E., Caroll R.E. & Busch H.. Nucleologenesiscomposition and fate of prenucleolar bodies, Chromosoma., 92, 1985, 330–336a.[Medline]
Ochs R.L., Lischwe M.A., Spohn W.H. & Busch H.. Fibrillarina new protein of the nucleolus identified by autoimmune sera, Biol. Cell., 54, 1985, 123–134b.[Medline]
Peculis B.A. & Steitz J.A.. Disruption of U8 nucleolar snRNA inhibits 5.8S and 28S rRNA processing in the Xenopus oocyte, Cell., 73, 1993, 1233–1245.[Medline]
Pluk H., Van Eenennaam H., Rutjes S.A., Pruijn G.J.M. & Van Venrrooij W.J.. RNA–protein interactions in the human RNase MRP ribonucleoprotein complex, RNA., 5, 1999, 512–524.[Abstract]
Sanford J.R. & Bruzik J.P.. Developmental regulation of SR protein phosphorylation and activity, Genes Dev., 13, 1999, 1513–1518.
Savino T.M., Bastos R., Jansen E. & Hernandez-Verdun D.. The nucleolar antigen Nop 52, the human homologue of the yeast ribosomal RNA processing RRP1, is recruited at late stages of nucleologenesis, J. Cell Sci., 112, 1999, 1889–1900.[Abstract]
Savkur R.S. & Olson M.O.J.. Preferential cleavage in pre-ribosomal RNA by protein B23 endoribonuclease, Nucleic Acids Res., 26, 1998, 4508–4515.
Scheer U. & Benavente R.. Functional and dynamic aspects of the mammalian nucleolus, BioEssay., 12, 1990, 14–20.[Medline]
Scheer U., Thiry M. & Goessens G.. Structure, function and assembly of the nucleolus, Trends Cell Biol., 3, 1993, 236–241.[Medline]
Scheer U. & Weisenberger D.. The nucleolus, Curr. Opin. Cell Biol., 6, 1994, 354–359.[Medline]
Schmidt-Zachmann M.S., Hügle-Dörr B. & Franke W.W.. A constitutive nucleolar protein identified as a member of the nucleoplasmin family, EMBO (Eur. Mol. Biol. Organ.) J., 6, 1987, 1881–1890.[Medline]
Shiokawa K., Misumi Y. & Yamana K.. Demonstration of rRNA synthesis in pre-gastrular embryos of Xenopus laevis, Dev. Growth Differ., 23, 1981, 579–587a.
Shiokawa K., Tashiro K., Misumi Y. & Yamana K.. Non-coordinated synthesis of RNA's in pre-gastrular embryos of Xenopus laevis, Dev. Growth Differ., 23, 1981, 589–597b.
Smith C.M. & Steitz J.A.. Sno storm in the nucleolusnew roles for myriad small RNPs, Cell., 89, 1997, 669–672.[Medline]
Steinmetz E.J.. Pre-mRNA processing and the CTD of RNA polymerase IIthe tail that wags the dog?, Cell., 89, 1997, 491–494.[Medline]
Thiry M. & Goessens G., The nucleolus during the cell cycle, 1996, Springer-Verlag, Heidelbergpp. 146.
Tollervey D.. Trans-acting factors in ribosome synthesis, Exp. Cell Res., 229, 1996, 226–232.[Medline]
Trimbur G.M. & Walsh C.J.. Nucleolus-like morphology produced during the in vitro reassociation of nucleolar components, J. Cell Biol., 122, 1993, 753–766.
Van Eenennaam H., Pruijn G.J.M. & Van Venrooij W.J.. hPop4a new protein subunit of the human RNase MRP and RNase P ribonucleoprotein complexes, Nucleic Acids Res., 27, 1999, 2465–2472.
Verheggen C., Le Panse S., Almouzni G. & Hernandez-Verdun D.. Presence of pre-rRNAs before activation of polymerase I transcription in the building process of nucleoli during early development of Xenopus laevis, J. Cell Biol., 142, 1998, 1167–1180.
Weisenberger D., Scheer U. & Benavente R.. The DNA topoisomerase I inhibitor camptothecin blocks postmitotic reformation of nucleoli in mammalian cells, Eur. J. Cell Biol., 61, 1993, 189–192.[Medline]
Xia L., Liu J., Sage C., Trexler E.B., Andrews M.T. & Maxwell E.S.. Intronic U14 snoRNAs of Xenopus laevis are located in two different parent genes and can be processed from their introns during early oogenesis, Nucleic Acids Res., 23, 1995, 4844–4849.
Zatsepina O.V., Dudnic O.A., Todorov I.T., Thiry M., Spring H. & Trendelenburg M.F.. Experimental induction of prenucleolar bodies (PNBs) in interphase cellsinterphase PNBs show similar characteristics as those typically observed at telophase of mitosis in untreated cells, Chromosoma., 105, 1997, 418–430.[Medline]
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