Verheggen C et al. (2000), The ribosomal RNA processing machinery is recru...

XB-ART-11194

J Cell Biol 2000 Apr 17;1492:293-306.

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The ribosomal RNA processing machinery is recruited to the nucleolar domain before RNA polymerase I during Xenopus laevis development.

Verheggen C , Almouzni G , Hernandez-Verdun D .

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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.

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Species referenced: Xenopus laevis
Genes referenced: eif3a fubp1 npm1 uqcc6

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	Figure 1. Components of rRNA processing machinery were regrouped in two types of PNBs both in embryonic and reconstituted nuclei. FISH of U3 (a; FITC) was performed after immunolabeling of fibrillarin (b; Texas red) on embryonic nuclei isolated 6 h after fertilization. U3 was distributed in numerous dots dispersed in the nucleoplasm. A large number of these dots colocalized with fibrillarin (c; yellow dots on the merged image). They were called PNBs I. Some other dots of U3 were also seen in the nucleoplasm (c; green dots on the merged image). In contrast, B23/NO38 (e; cy3) was not colocalized with fibrillarin (f, FITC) in a double immunolabeling experiment on embryonic nuclei isolated 6 h after fertilization. Labeling of B23/NO38 was diffuse and partially clustered in small dotted structures. These structures were called PNBs II. Distribution of the rRNA processing machineries was similar in in vitro reconstituted nuclei. Permeabilized sperm nuclei were incubated in low-speed X. laevis egg extract and further treated for in situ hybridization of U3 (i; FITC) after immunolabeling of fibrillarin (j; Texas red). U3 was completely colocalized with fibrillarin (k; yellow dots on the merged image). PNBs I formed in these nuclei were larger and easily distinguished by phase-contrast microscopy (l) in contrast to PNBs I in embryonic nuclei (d and h). Immunolabeling of B23/NO38 on reconstituted nuclei (m) showed the same distribution as in embryonic nuclei 6 h after fertilization. B23/NO38 (m; cy3) was not accumulated on the large PNBs I containing fibrillarin (n; FITC). Bars, 10 μm.
	Figure 2. In vitro reconstituted nuclei observed by EM. a, General view of the nucleus surrounded by the nuclear envelope. The chromatin is decondensed and two types of nuclear bodies are visible: a dense body (arrow) and several gray bodies (arrow heads). Bar, 1 μm. b, High magnification of a dense nuclear body. This body corresponds to PNB I. Granules are densely packed inside. Bar, 0.1 μm. c, High magnification of two gray bodies exhibiting a fibrillar feature. These bodies correspond to PNB II. Bar, 0.1 μm.
	Figure 4. Recruitment of PNB I and PNB II components to the nucleolar domain follows different kinetics. Double immunolabeling of B23/NO38 (FITC) and fibrillarin (Texas red) was performed on embryonic nuclei isolated 7, 9, and 11 h after fertilization. In a fraction of nuclei 7 h after fertilization, B23/NO38 (a) was found colocalized with fibrillarin (b) in large clusters that began to form at the nucleolar domain (c; yellow dots on the merged image). These clusters were seen by phase-contrast microscopy (d; dense area). Nucleolar delivery of PNBs I (fibrillarin) and PNBs II (B23/NO38) thus appears to be initiated at the same time. In nuclei 9 h after fertilization, B23/NO38 (e) was not completely relocalized to the nucleolar domain in contrast to fibrillarin (f). Large dots of B23/NO38 (g; green dots on the merged image) were seen outside of the nucleolar domain (g; yellow area on the merged image). In nuclei 11 h after fertilization, B23/NO38 (i) and fibrillarin (j) were regrouped and colocalized to the nucleolar domain (k). The nucleolar domain appears dense by phase-contrast microscopy (h and l). Bars, 10 μm.
	Figure 3. Targeting of PNB I components to the nucleolar domain. Embryonic nuclei isolated 7 and 9 h after fertilization were labeled with antibodies to fibrillarin (b and e) before performing in situ hybridization of U8 (a and d). In nuclei 7 h after fertilization, U8 (a) and fibrillarin (b) were colocalized on PNBs I and gathered in two areas of the nucleoplasm as a first step through the recruitment process. In most nuclei isolated 9 h after fertilization, fibrillarin (e) and U8 (d) were colocalized in two nucleolar domains. The corresponding DNA staining with DAPI is shown (c and f). Bars, 10 μm.
	Figure 5. RNA polymerase I is not associated with the newly formed nucleolar domain. Embryonic nuclei isolated 7, 9, and 11 h after fertilization were labeled with an antibody to RNA polymerase I (a, d, and g) before performing in situ hybridization of the rRNAs (b, e, and h). When the nucleolar domain began to assemble in nuclei 7 h after fertilization, maternally derived rRNAs were detected (b). No RNA polymerase I was seen colocalized to the FISH signal at this time (a; arrowheads). Instead, RNA polymerase I appeared as speckles dispersed in the nucleoplasm. This distribution was maintained in nuclei 9 h after fertilization (d). RNA polymerase I was not yet colocalized with rRNA (e). Only after 11 h of development, RNA polymerase I (g) was seen partially colocalized with rRNAs (h). The corresponding DNA staining with DAPI is shown (c, f, and i). Bars, 10 μm.
	Figure 7. Presence of maternally derived pre-rRNAs for the recruitment of fibrillarin to the nucleolar domain. In situ hybridization of rRNA was performed after immunolabeling of fibrillarin on nuclei isolated 9 h after fertilization from control embryos (a–c) and embryos treated with 0.5 μg/ml (inhibition of RNA pol I; d–f) or 2 μg/ml of actinomycin D (inhibition of RNA pol I, II, and III; g–i). The nucleolar domain was formed in control and 0.5 μg/ml actinomycin D–treated nuclei. rRNAs (a and d) were colocalized with fibrillarin (b and e) in this domain. rRNA was not detected in most nuclei from embryos treated with 2 μg/ml of actinomycin D (g). In this case, fibrillarin was not recruited to the nucleolar domain, but was maintained in large PNBs in the nucleoplasm (h). Only a few nuclei formed a nucleolar domain containing rRNA and fibrillarin (arrowheads in g and h, respectively). The corresponding DNA staining with DAPI is shown (c, f, and i). Bars, 10 μm.
	Figure 6. Fibrillarin is targeted to the nucleolar domain even in the absence of zygotic RNA pol I transcription. The fluorescent in situ transcription assay by immunolabeling of incorporated Br-UTP coupled to immunolabeling of fibrillarin was performed on nuclei isolated 12 h after fertilization from control embryos (A) and embryos treated with 0.5 μg/ml of actinomycin D (B). A, Sites of transcription were labeled in nuclei isolated from control embryos (a) and colocalized with fibrillarin in the nucleolar domain (b). This transcription was RNA pol I transcription because it was not inhibited by incubation of nuclei in 100 μg/ml of α-amanitin before and during incorporation of Br-UTP (c). B, When whole embryos were treated with 0.5 μg/ml of actinomycin D during development, no RNA pol I transcription was detected (e). Although RNA pol I activity was inhibited, fibrillarin was clustered in the nucleolar domain (f) as in control nuclei. Bars, 10 μm.
	Figure 8. Fibrillarin was not recruited to the nucleolar domain when the onset of nonribosomal zygotic transcription was inhibited. The fluorescent in situ transcription assay by immunolabeling of incorporated Br-UTP coupled to immunolabeling of fibrillarin was performed on nuclei isolated 9 h after fertilization from control embryos (A) and embryos treated with actinomycin D (B and C). A, At this time, incorporation of Br-UTP was detected in the nucleoplasm as punctuated signals (a) not colocalized with fibrillarin to the nucleolar domain in control nuclei (b). This transcription signal was inhibited by incubation of nuclei in 100 μg/ml of α-amanitin before and during incorporation of Br-UTP (d). B and C, A non-RNA pol I transcription signal was also detected in nuclei isolated 9 h after fertilization when embryos were treated with 0.5 μg/ml of actinomycin D (g), but not when embryos were treated with 2 μg/ml of actinomycin D (j). In this latter case, fibrillarin was maintained in large PNBs dispersed in the nucleoplasm (k). Arrows indicate the position of the nucleolar domains in a and g. DNA staining with DAPI is shown in (c, f, i, and l). Bars, 10 μm.
	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 μg/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 distribution 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 μm.
	Figure 10. Recruitment of rRNA processing machinery related to transcription during X. laevis development. Processing machinery components were first assembled in the nucleoplasm in two types of PNBs: PNBs I (in purple) containing fibrillarin, nucleolin, U3, and U8; and PNBs II (in red) containing B23/NO38. PNB I and PNB II components were recruited to the nucleolar domain with different kinetics. Material contained in PNBs I was regrouped in the nucleolar domain before material contained in PNBs II. At the time of the recruitment process: zygotic transcription by RNA pol II and pol III was observed (+ in orange); RNA polymerase I was found in the nucleoplasm. It was relocalized on rDNA and activated later (+ in blue); pre-rRNAs of maternal origin were detected at the time of nucleolar domain formation (+ in fuchsia). In nuclei isolated from embryos treated with 2 μg/ml of actinomycin D that inhibited RNA pol II and pol III transcriptions, pre-rRNAs were not detected by FISH and the rRNA processing machinery components were maintained in large PNBs in the nucleoplasm. A similar situation was encountered in in vitro reconstituted nuclei.

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