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Initiation of nucleolar assembly is independent of RNA polymerase I transcription - PubMed

Initiation of nucleolar assembly is independent of RNA polymerase I transcription

T Dousset et al. Mol Biol Cell. 2000 Aug.

Free PMC article

Abstract

This report examines the distribution of an RNA polymerase I transcription factor (upstream binding factor; UBF), pre-rRNA processing factors (nucleolin and fibrillarin), and pre-rRNAs throughout mitosis and postmitotic nucleologenesis in HeLa cells. The results demonstrate that nucleolin, fibrillarin, and pre-rRNAs synthesized at G2/M phase of the previous cell cycle are directly recruited to UBF-associated nucleolar organizer regions (NORs) early in telophase before chromosome decondensation. Unlike the fusion of prenucleolar bodies to the nucleoli, this early recruitment of processing factors and pre-rRNAs is independent of RNA polymerase I transcription. In the absence of polymerase I transcription, the initial localization of nucleolin, fibrillarin, and pre-rRNAs to UBF-associated NORs generates segregated mininucleoli that are similar to the larger ones observed in interphase cells grown under the same conditions. Pre-rRNAs are juxtaposed to UBF-nucleolin-fibrillarin caps that may represent the segregated nucleoli observed by electron microscopy. These findings lead to a revised model of nucleologenesis. We propose that nucleolar formation at the end of mitosis results from direct recruitment of processing factors and pre-rRNAs to UBF-associated NORs before or at the onset of rDNA transcription. This is followed by fusion of prepackaged prenucleolar bodies into the nucleolus. Pre-ribosomal ribonucleoproteins synthesized in the previous cell cycle may contribute to postmitotic nucleologenesis.

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Figures

Figure 1
Figure 1

Nucleolin is recruited directly to UBF-associated NORs early in telophase independent of Pol I transcription. Each row represents the same cells immunolabeled for UBF (first column) and nucleolin (second column), UBF and nucleolin overlay (third column), and differential interference contrast images to identify the cell cycle stages (fourth column). Rows A and B represent cells growing in normal medium (A, metaphase; B, telophase). Rows C–E represent cells at different stages through mitosis to G1 in the presence of 0.04 μg/ml ActD. Nucleolin is recruited before the decondensation of chromosomes (B–B′′′, arrows). The recruitment is not interrupted in the absence of Pol I transcription (D–D′′′, arrows). Toward late telophase, UBF and nucleolin colocalize to cap-like structures that juxtapose or surround dense bodies (Row E, arrows). Row F represents interphase cells treated with 0.04 μg/ml ActD. Cap-like structures (arrows) are associated with segregated nucleoli. All images are 0.8-μm optical sections obtained with the use of confocal laser scanning microscopy. Bars, 10 μm.

Figure 2
Figure 2

The recruitment of fibrillarin directly to UBF-associated NORs (arrows) in early telophase is not disrupted in the presence of 0.04 μg/ml ActD (A–A′′). When cells progress to early G1, fibrillarin is colocalized with UBF to cap-like structures (B–B′′). Each row represents the same cells immunolabeled for UBF (first column) and fibrillarin (second column), UBF and fibrillarin overlay (third column), and phase contrast to identify the cell cycle stages (fourth column). Bar, 10 μm.

Figure 3
Figure 3

ActD at a concentration of 0.04 μg/ml selectively inhibits Pol I transcription without significantly affecting other polymerases and general nuclear organization. A and A′ show the in situ incorporation of Br-UTP, and B and B′ show the same cells corresponding to those in A and A′ immunolabeled with anti-fibrillarin antibody. Daughter cells grown in the presence of ActD form mininucleoli that are immunolabeled with anti-fibrillarin antibody (B, arrows). They do not incorporate Br-UTP (A, arrows), whereas untreated nucleoli (B′, arrows) actively incorporate Br-UTP after pulse labeling (A′). Although Br-UTP incorporation in nucleoli declines in treated cells, the nucleoplasmic Br-UTP incorporation from Pol II and Pol III transcription is not altered (A). The speckled distribution of SC35 (C) and the predominantly nuclear localization of hnRNP A1 (D) are also not changed significantly in treated cells. Bar, 10 μm.

Figure 4
Figure 4

Electron microscopic observations demonstrate that cells progressing into G1 phase in the presence of 0.04 μg/ml ActD assemble segregated mininucleoli (B, arrowheads). In addition, unfused PNBs could be visualized in the nucleoplasm of treated cells (B, arrows). (A) A normal G1 cell nucleus. (B) A G1 cell exiting mitosis in the presence of 0.04 μg/ml ActD. (C) An interphase cell treated with the same concentration of ActD. Bars, 1 μm.

Figure 5
Figure 5

Pre-rRNAs and 28S rRNAs synthesized at the previous cell cycle reenter early G1 daughter cell nuclei. 28S rRNA probe (top) and 5′ETS core probe (bottom) both hybridize to rRNAs that are associated with growing nucleoli (arrows) and nucleolin-containing PNBs (arrowheads). Bar, 10 μm.

Figure 6
Figure 6

Pre-rRNAs (45S and 30S) containing 5′ETS core are detected through mitosis and synthesized during G2/M. (A) Northern blot hybridized with 5′ETS core probe showing that 45S and 30S pre-rRNAs are maintained in mitotic cells (M) synchronized with the use of either colchicine (C) or double thymidine block (S). The levels of these RNAs are less than those in interphase (I). (B) Northern blot hybridized with 5′ETS core probe to RNAs from early G1 cells that were treated with 0.04 μg/ml ActD at G2/M (G2) or telophase (Telo). (−) RNA extracted from control cells that were not treated with ActD. Treatment in G2 significantly reduces the detectable 45S rRNAs, whereas inhibition of Pol I transcription from mitosis to G1 still allows the maintenance of sizable amounts of 45S rRNA.

Figure 7
Figure 7

Pre-rRNAs and possibly 28S rRNAs are recruited to newly formed nuclei in the presence of 0.04 μg/ml ActD. They are colocalized with nucleolin (A and B)-associated PNBs (arrowheads) and are adjacent to UBF (C) and fibrillarin (D)-associated NORs (arrows). Bars, 10 μm.

Figure 8
Figure 8

Scheme of the revised model of nucleologenesis.

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