Nucleolar Assembly of the Rrna Processing Machinery in Living Cells (original) (raw)
Related papers
Post-mitotic dynamics of pre-nucleolar bodies is driven by pre-rRNA processing
Journal of Cell Science, 2012
Understanding the relationship between the topological dynamics of nuclear subdomains and their molecular function is a central issue in nucleus biology. Pre-nucleolar bodies (PNBs) are transient nuclear subdomains, which form at telophase and contain nucleolar proteins, snoRNPs and pre-ribosomal RNAs (pre-rRNAs). These structures gradually disappear in early G1 phase and are currently regarded as reservoirs of nucleolar factors that participate to post-mitotic reassembly of the nucleolus. Here, we provide evidence from fluorescence in situ hybridization and loss-of-function experiments in HeLa cells that PNBs are in fact active ribosome factories in which maturation of the pre-rRNAs transiting through mitosis resumes at telophase. We show that the pre-rRNA spacers are sequentially removed in PNBs when cells enter G1 phase, indicating regular pre-rRNA processing as in the nucleolus. Accordingly, blocking pre-rRNA maturation induces accumulation in PNBs of stalled pre-ribosomes characterised by specific pre-rRNAs and pre-ribosomal factors. The presence of pre-ribosomal particles in PNBs is corroborated by observation of these domains by correlative electron tomography. Most importantly, blocking pre-rRNA maturation also prevents the gradual disappearance of PNBs, which persist for several hours in the nucleoplasm. In a revised model, we propose that PNBs are autonomous extra-nucleolar ribosome maturation sites, whose orderly disassembly in G1 phase is driven by the maturation and release of their pre-ribosome content.
2010
. Knock-down of CRM1 reproduces the LMB effect. (A) In early G 1 cells, LMB induces the localization of coilin in the cap (arrow) at the periphery of the incipient nucleoli. In control (CTR) cells, coilin (arrowhead) is localized in small foci. Bar: 5 µm. (B) Immunodetection of CRM1 as small nuclear foci already visible in early G1a nuclei. (C) Early G 1 cells in which siRNAs against CRM1 were efficient (∆ CRM1): low CRM1 signal with antibodies compared to CRM1 positive cell in upper right corner (yellow arrow). Fibrillarin is in the cap (enlargment) at the periphery of the incipient nucleoli (white arrow); Nop52 is found both in incipient nucleoli and in PNBs (white arrow and enlargment).
Initiation of Nucleolar Assembly Is Independent of RNA Polymerase I Transcription
Molecular Biology of the Cell, 2000
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. Figure 8. Scheme of the revised model of nucleologenesis.
Experimental Cell Research, 2000
The DEAD box protein, p68, is an established RNA-dependent ATPase and RNA helicase in vitro, but neither the physiological function of this protein nor the macromolecules with which it interacts are known. Using a yeast two-hybrid screen, we identified the nucleolar protein, fibrillarin, as a protein that interacts with p68. Coimmunoprecipitation experiments confirmed that p68 and fibrillarin can form complexes in cellular extracts, and deletion analysis identified regions in each protein responsible for mediating the interaction. Immunofluorescence studies using confocal microscopy revealed that, in interphase cells, while fibrillarin is predominantly nucleolar, p68 shows a diffuse granular nuclear staining but is largely excluded from the nucleoli. Strikingly, both proteins colocalize in nascent nucleoli during late telophase. These data are consistent with a role for p68 either in postmitotic nucleolar reassembly or in the activation of ribosomal DNA transcription/preribosomal RNA processing during telophase and suggest that differential subnuclear compartmentalization may be a mechanism by which interaction of p68 with fibrillarin is regulated in the cell.
Molecular Biology of the Cell, 1994
We have investigated the distribution of U3 snRNA and rRNA in HeLa cells and normal rat kidney cells during interphase and mitosis. U3 snRNA, known to be involved in pre-rRNA processing, was detected in nucleoli and coiled bodies during interphase, whereas rRNA was distributed in the nucleoli and throughout the cytoplasm. By comparison, ribosomal protein S6 was detected in nucleoli, coiled bodies, and in the cytoplasm. During nucleologenesis, pre-rRNA was observed in newly forming nucleoli during late telophase but not in prenucleolar bodies (PNBs), whereas U3 snRNA was detected in forming nucleoli and PNBs. Similar findings to those reported here for the localization of U3 snRNA have been reported previously for the U3 small nuclear ribonucleoprotein fibrillarin. These results suggest that components involved in pre-rRNA processing localize to discrete PNBs at the end of mitosis. The nucleolus is formed at specific telophase domains (nucleolar organizing regions) and the PNBs, cont...
Chromosoma, 1997
The subcellular location of several nonribosomal nucleolar proteins was examined at various stages of mitosis in synchronized mammalian cell lines including HeLa, 3T3, COS-7 and HIV-1 Rev-expressing CMT3 cells. Nucleolar proteins B23, fibrillarin, nucleolin and p52 as well as U3 snoRNA were located partially in the peripheral regions of chromosomes from prometaphase to early telophase. However, these proteins were also found in large cytoplasmic particles, 1-2 µm in diameter, termed nucleolus-derived foci (NDF). The NDF reached maximum numbers (as many as 100 per cell) during mid-to late anaphase, after which their number declined to a few or none during late telophase. The decline in the number of NDF approximately coincided with the appearance of prenucleolar bodies and reforming nucleoli. The HIV-1 Rev protein and a mutant Rev protein defective in its nuclear export signal were also found in the NDF. The mutant Rev protein precisely followed the pattern of localization of the above nucleolar proteins, whereas the wild-type Rev did not enter nuclei until G1 phase. The nucleolar shuttling phosphoprotein Nopp140 did not follow the above pattern of localization during mitosis: it dispersed in the cytoplasm from prometaphase through early telophase and was not found in the NDF. Although the NDF and mitotic coiled bodies disappeared from the cytoplasm at approximately the same time during mitosis, protein B23 was not found in mitotic coiled bodies, nor was p80 coilin present in the NDF. These results suggest that a class of proteins involved in preribosomal RNA processing associate with chromosome periphery and with NDF as part of a system to conserve and deliver preexisting components to reforming nucleoli during mitosis.