Figure 9. Fibrillarin and nucleolin were maintained in large PNBs throughout development in the presence of actinomycin D or in in vitro reconstituted nuclei. In a double immunolabeling experiment, nucleolin (a) and fibrillarin (b) were colocalized in the nucleolar domain in nuclei isolated 9 h after fertilization from control embryos. This domain appeared dense by phase-contrast microscopy (c). The nucleolar domain was not formed in most nuclei isolated from embryos treated with 2 mg/ml of actinomycin D (inhibition of RNA pol I, II, and III). Instead, nucleolin (d) and fibrillarin (e) were maintained in large PNBs. A similar dis- tribution was also observed in in vitro reconstituted sperm nuclei (g and h). PNB structures, maintained at the MBT stage in treated embryos, were easily distinguished in nuclei by phase- contrast microscopy (f) and similar to structures formed in in vitro reconstituted nuclei (i). Bars, 10 mm. 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 si- lent stage (Verheggen et al., 1998). In the present study, we observe PNBs in early and late stage nuclei from em- bryos treated with high concentrations of actinomycin D, confirming that PNB assembly during X. laevis embryo- genesis 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 em- bryogenesis. 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). How- ever, the significance of the presence of these complexes in nucleoplasmic foci remains to be elucidated. Several examples of domains for accumulation of tran- scription 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 ac- tivation 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 ad- dition, 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 tran- scription 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 de- pendent 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 ac- tivity, nor the catalytic enzyme RNA pol I itself were re- quired for this recruitment process. Formation of the nu- cleolar domain in the absence of RNA pol I has been observed in yeast mutant strains in which rRNA was tran- scribed 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 chro- mosomal loci. RNA pol I was therefore suggested to play a role in the organization of the nucleolar domain. How- ever, 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 pro- cessing machinery is not recruited when the RNA pol II CTD is deleted (Misteli and Spector, 1999). A different re- cruiting mechanism is used for rRNA processing machin- ery as RNA pol I is dispensable for nucleolar domain as- sembly during X. laevis embryogenesis. Protein recruitment to a specific location can be con- trolled by reversible phosphorylation, which could provide an attractive mechanism for recruitment of PNB to the nu- cleolar domain. For example, the nuclear import of nucle- olin in early blastula of X. laevis has been shown to de- pend 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 The Journal of Cell Biology, Volume 149, 2000 302