Verheggen C et al. (1998), Presence of pre-rRNAs before activation of poly...

XB-ART-14310

J Cell Biol 1998 Sep 07;1425:1167-80. doi: 10.1083/jcb.142.5.1167.

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Presence of pre-rRNAs before activation of polymerase I transcription in the building process of nucleoli during early development of Xenopus laevis.

Verheggen C, Le Panse S, Almouzni G, Hernandez-Verdun D.

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During the early development of Xenopus laevis, we followed in individual nuclei the formation of a nucleolus by examining simultaneously its structural organization and its transcriptional competence. Three distinct situations were encountered with different frequencies during development. During the first period of general transcriptional quiescence, the transcription factor UBF of maternal origin, was present in most nuclei at the ribosomal gene loci. In contrast, fibrillarin, a major protein of the processing machinery, was found in multiple prenucleolar bodies (PNBs) whereas nucleolin was dispersed largely in the nucleoplasm. During the second period, for most nuclei these PNBs had fused into two domains where nucleolin concentrated, generating a structure with most features expected from a transcriptionally competent nucleolus. However, RNA polymerase I-dependent transcription was not detected using run-on in situ assays whereas unprocessed ribosomal RNAs were observed. These RNAs were found to derive from a maternal pool. Later, during a third period, an increasing fraction of the nuclei presented RNA polymerase I-dependent transcription. Thus, the structural organization of the nucleolus preceded its transcriptional competence. We conclude that during the early development of X. laevis, the organization of a defined nucleolar structure, is not associated with the transcription process per se but rather with the presence of unprocessed ribosomal RNAs.

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Species referenced: Xenopus laevis
Genes referenced: eif3a nts ubtf uqcc6

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	Figure 2. Subcellular distribution of the maternal pool of UBF during development. Total (T) or nuclear (N) protein extracts were prepared and analyzed by SDS-8% polyacrylamide gel, blotted onto nitrocellulose and probed with anti-human UBF serum. For A6 cells, an equivalent to 66,000 nuclei was loaded (A6, N, lane 1). Lanes 2 and 3 contain respectively total and nuclear proteins from 1.5 embryos 6 h after fertilization. Lane 4 contains nuclear proteins from 15 embryos 6 h after fertilization (an equivalent to 15,000 nuclei since one embryo contains only 1,000 nuclei at this stage). Lanes 5 and 6 contain, respectively, total and nuclear proteins from 1.5 embryos 13 h after fertilization (one embryo contains ∼15,000 nuclei at this stage).
	Figure 3. Localization by confocal microscopy of UBF and fibrillarin in nuclei from embryos at 6 h (A–A″), 9 h (B–B″), and 13 h (C–C″) after fertilization and in A6 cell (D–D″). Alignments of small beads are seen in green (A–D) using anti-human UBF serum. Fibrillarin labeling with anti-mouse fibrillarin serum in red appears on dot-like structures scattered throughout the nuclei at 6 h after fertilization (A′). Regroupment of fibrillarin is observed around UBF 9 h (B′) and 13 h (C′) after fertilization and in A6 cells (D′). A″–D″ are merged images of both signals on one optical section. The white line indicates the nuclear contour (A″–D″).
	Figure 12. Nuclear events described in this work. Percentage of embryonic nuclei are expressed as a function of time after fertilization. Schematic representations of the embryos at the different times are presented below the graph. The first event described was UBF association with rDNAs in nearly all embryonic nuclei at every time after fertilization ( ). Regroupment of fibrillarin and nucleolin to the nucleolar domain ( ) as well as maternal pre-rRNAs ( ) were observed in a growing fraction of embryonic nuclei between 7 and 13 h after fertilization. The curve corresponding to RNA pol II or pol III activities detected by run-on assays in nuclei ( ) was identical to the two latter curves since transcription activities were detected in nuclei in that nucleolar proteins and maternal pre-rRNAs were regrouped. However, onset of RNA pol I activity was not concomitant with this regroupment. The curve expressing the percentage of nuclei with RNA pol I activity ( ) shows a delay of ∼2.5 h. For simplification, the period that comprises the recruitment of nuclei competent for RNA pol II transcription will be referred to as MBT and the periods before or after as pre-MBT or post-MBT, respectively.
	Figure 4. Localization of nucleolin (Nuc) and fibrillarin (Fib) in nuclei from embryos 6 h (A–A″) and 9 h (B–B″) after fertilization. While dot-like structures are seen using anti-human fibrillarin serum at 6 h after fertilization (A′), nucleolin labeling with anti- human nucleolin serum is diffuse in the nucleoplasm (A). Both proteins are colocalized 9 h after fertilization (B and B′). (A″ and B″) are phase contrast microscopy.
	Figure 5. Fibrillarin becomes clustered near the rDNAs during development. Embryonic nuclei and A6 cells were labeled with anti-fibrillarin human serum (red, A′–C′) before performing rDNA hybridization in situ with an rDNA biotinylated probe (green, A–C). Images were directly numerized from a camera on a fluorescence microscope and then both labelings were merged (A″–C″). At 7 h after fertilization, part of the fibrillarin in red was clustered close to rDNAs in green (A″) but isolated dots of fibrillarin were seen in the nucleoplasm (arrows, compare with Fig. 4 for earlier time). At 9 h after fertilization, all the fibrillarin dots were regrouped around rDNA (B″). In the A6 cell (C–C″), most rDNAs (C) were totally embedded in the largest nucleolar structure labeled by anti-fibrillarin serum (C′). Sites of fibrillarin accumulation are dense in phase contrast (A‴–C‴).
	Figure 6. Ultrastructural analysis of nuclei at different times during X. laevis development: 6 h (A–C), 8 h (D and E) and 13 h after fertilization (F and G). At 6 h after fertilization, the nuclei exhibited decondensed chromatin and numerous nuclear pore complexes (A). Round-shaped dense fibrillar structures similar to PNB were observed (A and B). When sections were incubated with anti-fibrillarin antibodies, these dense fibrillar structures were labeled by protein A gold (C). At 8 h after fertilization, the nucleolar domain was characterized by dense fibrillar component forming a network (D) and containing fibrillarin (gold particles in E). At 13 h after fertilization (F), the nucleolus was formed by dense fibrillar component (DFC) surrounded by granular component (GC).The star indicates a structure similar to fibrillar center. Fibrillarin was localized on DFC whereas no label was detected over the granular component (G). Bar: 0.2 μm.
	Figure 7. rDNA transcription appears after regroupment of fibrillarin. In situ run-on incorporation of Br-UTP (green) in isolated embryonic nuclei (A–C) or A6 nuclei (D) was coupled with fibrillarin labeling (red) using the anti-fibrillarin human serum (A′–D′). Images were directly numerized from a camera on a fluorescence microscope and both labelings combined (A″–D″). Superposition showed that nucleolar transcription was not yet detected 9 h after fertilization (A″) while rDNA transcription was the major transcription observed on nuclei at 11 h (B″) and 18 h (C″) after fertilization, and in A6 nuclei (D″). The white line indicates the nuclear contour (A″–D″).
	Figure 8. Representation of the specific probes corresponding to the X. laevis ribosomal gene (rDNA). Map of the X. laevis 40 S pre-rRNA transcription region (boxed region; rRNA coding region are black and transcribed spacer regions are white) and surrounding rDNA regions designed non transcribed spacer (NTS, black bar). The dotted line indicates the position of the hybridization probes. The rDNA probe is complementary to the entire rDNA, and the two 5′ETS and ITS1 probes are complementary to a part of the transcribed spacer regions. The 5′ETS probe extends between positions +176 and +632 relative to the transcription start site. The ITS1 probe extends between positions +2764 and +3077.
	Figure 9. (a) Presence of rRNAs at the time of fibrillarin regroupment. Embryonic nuclei isolated 7 h after fertilization were labeled with anti-fibrillarin serum (red) before performing rRNA FISH with the rDNA probe (green). All the bright and large foci stained by the anti-fibrillarin (A′) contained rRNAs (A). The rRNA signal was abolished by RNase treatment (B). (b) Presence of rRNAs in nucleolar domain 9 h after fertilization. rRNAs (green) were present on the sites of regroupment of fibrillarin (red; A and B, 2). However, no rRNA was detected when fibrillarin was still dispersed (A and B, 1). C is merge image of rRNA and fibrillarin counterstained by DAPI for DNA. D corresponds to phase contrast microscopy.
	Figure 10. (a) Distribution of rRNAs in cycling cells. A6 cells were labeled with anti-fibrillarin serum (red) before performing rRNA FISH (green) with the rDNA probe (A) or the 5′ETS probe (B). Pre-rRNAs hybridized with the 5′ETS probe occupy a limited region of the functional nucleolus (B–B″) whereas the rRNAs hybridized with the rDNA probe distributed all over the nucleolar domain (A–A″). The white line indicates the nuclear contour (A″–B″). (b) Unprocessed rRNAs are associated with the formation of structurally defined nucleoli. At 9 h after fertilization (A–A″), rRNAs (green) were colocalized with fibrillarin (red). Some rRNAs are present in a region that surrounds the fibrillarin-labeled area (A″). The same localization was obtained using the 5′ETS (B–B″) and ITS1 probes (C–C″) indicating that the rRNAs detected 9 h after fertilization are incompletely processed rRNAs. The white line indicates the nuclear contour (A″–C″).
	Figure 11. A maternal pool of 40 S pre-rRNAs is maintained during early development of X. laevis. A Northern blot was performed using sample of RNAs prepared with embryos at various time after fertilization. Each lane was loaded with RNAs from two embryo equivalents excepted the last one which was loaded with RNAs from 105 A6 cells. The blot was first probed with the 32P-labeled 5′ETS probe that hybridized to 40 S pre-rRNAs. An exposure of 24 h was performed after hybridization to 40 S pre-rRNAs. The same blot was further rehybridized with the 32P-labeled rDNA probe to estimate the RNA loading per lane. A short exposure of 1 h was sufficient to see hybridization to 18 S and 28 S rRNAs. Taking into account this difference, the signal obtained for the 18 S or 28 S rRNAs was 70 times stronger than the signal for the 40 S pre-rRNAs.

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Abramova, Migration of newly synthesized RNA during mitosis. 3. Nuclear RNA in the cytoplasm of metaphase cells. 1973, Pubmed