Nucleolus: the fascinating nuclear body

Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs are synthesized, processed and assembled with ribosomal proteins. RNA polymerase I synthesizes the ribosomal RNAs and this activity is cell cycle regulated. The nucleolus reveals the functional organization of the nucleus in which the compartmentation of the different steps of ribosome biogenesis is observed whereas the nucleolar machineries are in permanent exchange with the nucleoplasm and other nuclear bodies. After mitosis, nucleolar assembly is a time and space regulated process controlled by the cell cycle. In addition, by generating a large volume in the nucleus with apparently no RNA polymerase II activity, the nucleolus creates a domain of retention/sequestration of molecules normally active outside the nucleolus. Viruses interact with the nucleolus and recruit nucleolar proteins to facilitate virus replication. The nucleolus is also a sensor of stress due to the redistribution of the ribosomal proteins in the nucleoplasm by nucleolus disruption. The nucleolus plays several crucial functions in the nucleus: in addition to its function as ribosome factory of the cells it is a multifunctional nuclear domain, and nucleolar activity is linked with several pathologies. Perspectives on the evolution of this research area are proposed.     

The nucleolus: a model for the organization of nuclear functions

Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins. The size and organization of the nucleolus are directly related to ribosome production. The organization of the nucleolus reveals the functional compartmentation of the nucleolar machineries that depends on nucleolar activity. When this activity is blocked, disrupted or impossible, the nucleolar proteins have the capacity to interact independently of the processing activity. In addition, nucleoli are dynamic structures in which nucleolar proteins rapidly associate and dissociate with nucleolar components in continuous exchanges with the nucleoplasm. At the time of nucleolar assembly, the processing machineries are recruited in a regulated manner in time and space, controlled by different kinases and form intermediate structures, the prenucleolar bodies. The participation of stable pre-rRNAs in nucleolar assembly was demonstrated after mitosis and during development but this is an intriguing observation since the role of these pre-rRNAs is presently unknown. A brief report on the nucleolus and diseases is proposed as well as of nucleolar functions different from ribosome biogenesis.Robert Feulgen Lecture presented at the 48th Symposium of the Society for Histochemistry in Stresa, Lake Maggiore, Italy, 7–10 September 2006.     

Simultaneous visualization of rDNA clusters and the nucleolus by combining fluorescence in situ hybridization and silver staining.

We designed two procedures to visualize simultaneously clusters of ribosomal RNA genes (rDNA) and the nucleolus in plant cells. The procedures combine fluorescence in situ hybridization (FISH) to visualize the rDNA clusters and silver staining to observe the nucleolus. When FISH is followed by silver staining, many minute FISH signals are localized in the nucleolus, and several large FISH signals are seen on the nucleolar periphery. When FISH was applied to the specimens with silver nitrate staining, large FISH signals were visualized in the nucleoplasm associated with the nucleolar periphery, but no signals were seen in the nucleoli. Thus, the two combinations of FISH and silver staining provided different details regarding the arrangement of rDNA clusters in the nucleolus of plant cells.     

Simultaneous visualization of rDNA clusters and the nucleolus by combining fluorescence in situ hybridization and silver staining

We designed two procedures to visualize simultaneously clusters of ribosomal RNA genes (rDNA) and the nucleolus in plant cells. The procedures combine fluorescence in situ hybridization (FISH) to visualize the rDNA clusters and silver staining to observe the nucleolus. When FISH is followed by silver staining, many minute FISH signals are localized in the nucleolus, and several large FISH signals are seen on the nucleolar periphery. When FISH was applied to the specimens with silver nitrate staining, large FISH signals were visualized in the nucleoplasm associated with the nucleolar periphery, but no signals were seen in the nucleoli. Thus, the two combinations of FISH and silver staining provided different details regarding the arrangement of rDNA clusters in the nucleolus of plant cells.     

Simultaneous visualization of rDNA clusters and the nucleolus by combining fluorescence in situ hybridization and silver staining

We designed two procedures to visualize simultaneously clusters of ribosomal RNA genes (rDNA) and the nucleolus in plant cells. The procedures combine fluorescence in situ hybridization (FISH) to visualize the rDNA clusters and silver staining to observe the nucleolus. When FISH is followed by silver staining, many minute FISH signals are localized in the nucleolus, and several large FISH signals are seen on the nucleolar periphery. When FISH was applied to the specimens with silver nitrate staining, large FISH signals were visualized in the nucleoplasm associated with the nucleolar periphery, but no signals were seen in the nucleoli. Thus, the two combinations of FISH and silver staining provided different details regarding the arrangement of rDNA clusters in the nucleolus of plant cells.     

Cytopathological effects of protein synthesis inhibitor emetine on HeLa cells and their nucleoli

Eukaryotic cell nucleolus is a highly dynamic structure, which is sensitive to all changes within or outside cell borders. Numerous data are available on changes of the nucleolar structure and functions under different treatments. However, almost nothing is known about the action of translation inhibitors on the nucleolus, although these substances, together with TNF-alpha, are commonly used for apoptosis induction, both for scientific and therapeutic purposes. Emetine is one of such inhibitors. We have shown that emetine suppresses cell viability, decreases mitotic index, and induces apoptosis in HeLa cells. Emetine action is irreversible, and it sensitizes cells to unfavourable external conditions. The emetine action causes redistribution of UBF, one of RNA-polymerase I factor, from the nucleolus to nucleoplasm even after a short exposure, i.e. when the morphology of the nucleus and chromatin still keeps its native pattern. It is important that other nucleolar proteins, such as fibrillarin and B23, are not recognized in the nucleoplasm until the very late stages of apoptotic process. A suggestion is made that changes in UBF localization may be associated with the onset of ribosomal repeat cleavage and migration of rDNA-"free" fragments from the nucleolus to nucleoplasm. It looks likely that these changes can serve as an initial morphological indication of apoptosis.     

Mutations in nucleolar proteins lead to nucleolar accumulation of polyA+ RNA in Saccharomyces cerevisiae.

Synthesis of mRNA and rRNA occur in the chromatin-rich nucleoplasm and the nucleolus, respectively. Nevertheless, we here report that a Saccharomyces cerevisiae gene, MTR3, previously implicated in mRNA transport, codes for a novel essential 28-kDa nucleolar protein. Moreover, in mtr3-1 the accumulated polyA+ RNA actually colocalizes with nucleolar antigens, the nucleolus becomes somewhat disorganized, and rRNA synthesis and processing are inhibited. A strain with a ts conditional mutation in RNA polymerase I also shows nucleolar accumulation of polyA+ RNA, whereas strains with mutations in the nucleolar protein Nop1p do not. Thus, in several mutant backgrounds, when mRNA cannot be exported i concentrates in the nucleolus. mRNA may normally encounter nucleolar components before export and proteins such as Mtr3p may be critical for export of both mRNA and ribosomal subunits.     

60S pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm

60S ribosomes undergo initial assembly in the nucleolus before export to the cytoplasm and recent analyses have identified several nucleolar pre-60S particles. To unravel the steps in the pathway of ribosome formation, we have purified the pre-60S ribosomes associated with proteins predicted to act at different stages as the pre-ribosomes transit from the nucleolus through the nucleoplasm and are then exported to the cytoplasm for final maturation. About 50 non-ribosomal proteins are associated with the early nucleolar pre-60S ribosomes. During subsequent maturation and transport to the nucleoplasm, many of these factors are removed, while others remain attached and additional factors transiently associate. When the 60S precursor particles are close to exit from the nucleus they associate with at least two export factors, Nmd3 and Mtr2. As the 60S pre-ribosome reaches the cytoplasm, almost all of the factors are dissociated. These data provide an initial biochemical map of 60S ribosomal subunit formation on its path from the nucleolus to the cytoplasm.     

Scattering of the silver-stained proteins of nucleolar organizer regions in Ishikawa cells by actinomycin D.

Scattering of the silver-stained proteins of nucleolar organizer regions (Ag-NOR proteins) was produced by actinomycin D in Ishikawa cells. Scattering of Ag-NOR proteins was found only in cells treated with actinomycin D and various other agents had no effect. Scattering was dose-dependent up to 10(-2) micrograms/ml of actinomycin D, but it was not found at higher concentrations that caused marked inhibition of total DNA and RNA synthesis. Actinomycin D (10(-2) micrograms/ml) caused the following changes: (i) nucleolar segregation and (ii) emergence of dense fibrillar bodies in the nucleoplasm. Ag-NOR proteins were observed on the fibrillar centers and surrounding fibrillar components in control nucleoli, on the fibrillar and amorphous zones in segregated nucleoli, and on the dense fibrillar bodies emerging in the nucleoplasm. The scattering of Ag-NOR proteins was due to the argyrophilic nature of the dense fibrillar bodies. Actinomycin D (10(-1) micrograms/ml) also caused similar morphological alterations in the nucleolus and nucleoplasm, but Ag-NOR proteins were observed only on nucleolar remnants.     

Relative stoichiometry in ribosomal proteins in HeLa cell nucleoli.

Total protein was released from isolated HeLa cell nucleoli by guanidine hydrochloride, purified by cesium chloride density gradient centrifugation, and analyzed by two-dimensional polyacrylamide gel electrophoresis. Conditions of electrophoresis restricted attention to proteins that are positively charged at pH 8.6. Most of the major nucleolar protein spots co-electrophoresed with ribosomal proteins; the majority of ribosomal proteins from both the large and small ribosomal subunits were represented. Several proteins found in association with polysomes but not on ribosomal subunits and several proteins unique to the nucleolus were also identified in these nucleolar protein patterns. In order to determine whether the ribosomal proteins found in the nucleolus represented sizable pools of ribosomal proteins, or merely ribosomal proteins contained in the preribosomal particles, [35S]methionine-labeled nucleoli were mixed with [3H]methionine-labeled polysomes. From analysis of isotopic ratios in individual protein spots it was possible to determine the stoidchiometry of individual ribosomal proteins in the nucleolus relative to their complement on cytoplasmic ribosomes. All but a few proteins exhibited relative nucleolar stoichiometry values of approximately one, indicating that there are not significant pools of most ribosomal proteins in isolated nucleoli.     

Control of ribosome biosynthesis in plant cell cultures under heat shock conditions. II. Ribosomal proteins

The nomenclature and synthesis of acidic and basic ribosomal proteins of plant cell cultures are described, with special regard to ribosome biosynthesis under control and heat-shock conditions. Assembly and processing of preribosomes in the nucleolus require a defined set of ribosomal proteins binding to the nascent pre-rRNA chain. Others are added later on the maturation pathway, mostly in the cytoplasm. Although, under appropriate heat-shock conditions, formation of mature ribosomes is completely blocked, most of the typical ribosomal proteins are still detected in the nuclear fraction. They are constituents of heat-shock preribosomes, which can be processed to normal cytoplasmic ribosomes only if the cells are allowed to recover at 25°C shortly after the labeling period at 40°C. However, if hyperthermic conditions are maintained, the labeled pre-rRNP material is evidently partly broken down. It forms the growing amount of RNP granules (ribosomal wastage) characteristic of the dispersed nucleolus of heat-shocked cells. In addition to the ‘nucleolar’ ribosomal proteins, a few newly formed ribosomal proteins can also be detected in cytoplasmic ribosomes under heat-shock conditions. Most of them belong to the group of exchange proteins whose labeling continues even if pre-rRNA synthesis is blocked by actinomycin D.