The Ribosomal RNA Processing Machinery Is Recruited to the Nucleolar Domain before RNA Polymerase I during Xenopus laevis Development Céline Verheggen,*‡ Geneviève Almouzni,‡ and Danièle Hernandez-Verdun* *Institut Jacques Monod, UMR 7592, 75251 Paris, France; and ‡Institut Curie, Section de Recherche, UMR 144, 75248 Paris, France Abstract. Transcription and splicing of messenger RNAs are temporally and spatially coordinated through the recruitment by RNA polymerase II of pro- cessing factors. We questioned whether RNA poly- merase I plays a role in the recruitment of the riboso- mal 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 nucle- olar RNAs) fuse to the nucleolar domain before com- ponents 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 Introduction Defining the coordinating events during RNA transcrip- tion and processing is becoming increasingly important in the context of their regulatory role in gene expression and nuclear organization (for reviews see Lamond and Earn- shaw, 1998; Misteli and Spector, 1998). It has been re- ported that messenger RNA (mRNA)1 transcription and processing is coordinated by the recruitment of processing factors to transcription sites by RNA polymerase II (RNA pol II; Jiménez-Garcia and Spector, 1993; Misteli et al., 1997; Bentley, 1999; Misteli and Spector, 1999). Remark- ably, the activation of ribosomal gene (rDNA) transcrip- Address correspondence to D. Hernandez-Verdun, Institut Jacques Monod, 2 place Jussieu, 75251 Paris Cedex 05, France. Tel.: 331 44 27 40 38. Fax: 331 44 27 59 94. E-mail: dhernand@ccr.jussieu.fr 1Abbreviations used in this paper: Br-UTP, 5-bromouridine-59-triphos- phate; DAPI, 49-6-diamidino-2-phenylindole dihydrochloride; ETS, exter- nal transcribed spacers; FISH, fluorescent in situ hybridization; ITS, inter- nal transcribed spacers; MBT, midblastula transition; mRNA, messenger RNA; PNB, prenucleolar bodies; rDNA, ribosomal gene; rRNA, ribo- somal RNA; RNA pol I, RNA polymerase I; snoRNA, small nucleo- lar RNA. 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 inde- pendent of either zygotic RNA polymerase I transcrip- tion or the presence of RNA polymerase I itself. Key words: prenucleolar body • nucleologenesis • pre-rRNA • RNA polymerase I transcription • Xenopus laevis development tion at the end of mitosis is also accompanied by the recruit- ment of processing complexes (Scheer and Benavente, 1990; Thiry and Goessens, 1996). This therefore raises the issue of whether there is a link between active transcription and processing for ribosomal RNA (rRNA). 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 spac- ers (59ETS and 39ETS) and the internal transcribed spacers (ITS1 and ITS2). Fibrillarin (Ochs et al., 1985b) and nucleo- lin (Ginisty et al., 1998) associated with several small nu- cleolar RNAs (snoRNAs), including U3, could play a role during the first steps of rRNA processing (for a review see Tollervey, 1996). Subsequent cleavages involve endoribo- nuclease activities such as the MRP RNase complex (Lygerou et al., 1996a,b; 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 39ETS and late ITS2 cleavages ª The Rockefeller University Press, 0021-9525/2000/04/293/13 $5.00 The Journal of Cell Biology, Volume 149, Number 2, April 17, 2000 293–305 http://www.jcb.org 293