Structure and function of the nucleolus.

The activity of the ribosomal RNA genes generates a distinct subnuclear structure, the nucleolus, which is the site of ribosome biogenesis. The signals that target proteins and snoRNAs (small nucleolar RNAs) to the nucleolus, the nuclear import of ribosomal proteins, the export of the completed ribosomal subunits and the molecular organization of the nucleolus have been the subject of intense research during the past year. Evidence is accumulating that nucleoli functionally interact with coiled bodies and are also involved in the maturation of non-ribosomal RNA species.     

Nucleolar binding sequences of the ribosomal protein S6e family reside in evolutionary highly conserved peptide clusters

Proteomic analyses of the nucleolus have revealed almost 700 functionally diverse proteins implicated in ribosome biogenesis, nucleolar assembly, and regulation of vital cellular processes. However, this nucleolar inventory has not unveiled a specific consensus motif necessary for nucleolar binding. The ribosomal protein family characterized by their basic nature should exhibit distinct binding sequences that enable interactions with the rRNA precursor molecules facilitating subunit assembly. We succeeded in delineating 2 minimal nucleolar binding sequences of human ribosomal protein S6 by fusing S6 cDNA fragments to the 5' end of the LacZ gene and subsequently detecting the intracellular localization of the beta-galactosidase fusion proteins. Nobis1 (nucleolar binding sequence 1), comprising of 4 highly conserved amino acid clusters separated by glycine or proline, functions independently of the 3 authentic nuclear localization signals (NLSs). Nobis2 consists of 2 conserved peptide clusters and requires the authentic NLS2 in its native context. Similarly, we deduced from previous publications that the single Nobis of ribosomal protein S25 is also highly conserved. The functional protein domain organization of the ribosomal protein S6e family consists of 3 modules: NLS, Nobis, and the C-terminal serine cluster of the phosphorylation sites. This modular structure is evolutionary conserved in vertebrates, invertebrates, and fungi. Remarkably, nucleolar binding sequences of small and large ribosomal proteins reside in peptide clusters conserved over millions of years.     

Studies on chromatin organization in a nucleolus without fibrillar centres

In embryonic cell-line derivative KCo of Drosophila melanogaster, the nucleolus, like most nucleoli, contains a small proportion of ribosomal DNA (1-2% of the total nucleolar DNA). The ribosomal DNA is virtually the only active gene set in the nucleolus and is found among long stretches of inactive supercoiled heterochromatic segments. We have demonstrated by use of a Feulgen-like ammine-osmium staining procedure that, depending on the state of growth, more or less fibres of decondensed DNA emanating from the intra-nucleolar chromatin (which is in continuity with the nucleolus-associated chromatin) ramify and unravel within the central nucleolar core to be transcribed. The nucleolus expands or contracts with the variation of activity and could belong to a supramolecular matricial structure such as is shown after extraction of the nuclei. After a long period of exposure to high doses of actinomycin D, the central nucleolar core became an homogeneous fibrous structure that could be interpreted as an aggregate of protein skeletal elements. The mechanism of repression and derepression of the nucleolar chromatin could thus be explained by a mechanism involving in part a sub-nucleolar structure. We propose a schematic organization of the nucleolar chromatin in KCo cells of Drosophila and discuss it in relation with other nucleolar organizations.     

A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability.

In order to study the structural and functional organization of the eukaryotic nucleolus, we have started to isolate and characterize nucleolar components of the yeast Saccharomyces cerevisiae. We have identified a major 38 kd nucleolar protein (NOP1), which is located within nucleolar structures resembling the dense fibrillar region of mammalian nucleoli. This 38 kd protein is conserved in evolution since affinity-purified antibodies against the yeast protein stain the nucleolus of mammalian cells in indirect immunofluorescence microscopy and the yeast protein is decorated by antibodies directed against human fibrillarin. Affinity-purified antibodies against the yeast NOP1 efficiently precipitate at least seven small nuclear RNAs involved in rRNA maturation. We have cloned the gene encoding the yeast NOP1 protein. Haploid cells carrying a disrupted copy of the gene are not viable, showing that NOP1 is essential for cell growth. The gene codes for a 34.5 kd protein which contains glycine/arginine rich sequence repeats at the amino terminus similar to those found in other nucleolar proteins. This suggests that NOP1 is in association with small nucleolar RNAs, required for rRNA processing and likely to be the homologue of the mammalian fibrillarin.     

Identification of an evolutionary conserved SURF-6 domain in a family of nucleolar proteins extending from human to yeast

The mammalian SURF-6 protein is localized in the nucleolus, yet its function remains elusive in the recently characterized nucleolar proteome. We discovered by searching the Protein families database that a unique evolutionary conserved SURF-6 domain is present in the carboxy-terminal of a novel family of eukaryotic proteins extending from human to yeast. By using the enhanced green fluorescent protein as a fusion protein marker in mammalian cells, we show that proteins from distantly related taxonomic groups containing the SURF-6 domain are localized in the nucleolus. Deletion sequence analysis shows that multiple regions of the SURF-6 protein are capable of nucleolar targeting independently of the evolutionary conserved domain. We identified that the Saccharomyces cerevisiae member of the SURF-6 family, named rrp14 or ykl082c, has been categorized in yeast databases to interact with proteins involved in ribosomal biogenesis and cell polarity. These results classify SURF-6 as a new family of nucleolar proteins in the eukaryotic kingdom and point out that SURF-6 has a distinct domain within the known nucleolar proteome that may mediate complex protein-protein interactions for analogous processes between yeast and mammalian cells.     

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.     

Genome organization in and around the nucleolus

The nucleolus is the largest compartment of the cell nucleus and is where ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins. In addition to rRNA gene clusters that build the core of this subnuclear structure, nucleoli are associated with condensed chromatin. Although the higher order structures of rRNA genes and nucleolus-associated chromatin have been studied for decades, detailed molecular insights into the constituents and organization of the nucleolar genome are only beginning to emerge. Here, we summarize current views on the structural organization of nucleolar DNA and on the targeting and anchoring of chromatin domains to this subnuclear compartment.     

Localization of rRNA genes in the nuclear space of Calliphora erythrocephala Mg. nurse cells during polytenization

Multicolor 3D fluorescence in situ hybridization was used to study arrangement of rRNA genes in Calliphora erythrocephala nurse cell nuclei with different levels of polyteny. It has been shown that the rRNA genes are exclusively localized to chromosome 6, suggesting that chromosome 6 is the only C. erythrocephala chromosome responsible for nucleolar formation. We have also described changes in localization of ribosomal genes within the chromosome territory during polytenization, namely, that rDNA signals are detected in the peripheral region of chromosome territory starting from the stage of polytene chromosomes. In addition, it has emerged that large nucleolus associated with chromosome 6 starts to develop in the central nuclear region in the C. erythrocephala nurse cell nuclei at the stage of a primary reticular structure. The central position and nucleolar structure are retained at the stages when chromosome 6 occupies the central position, that is, at the stages of polytene and bloblike chromosomes. When the nucleus restores a reticular structure but at a higher polyteny level, the displacement of chromosome 6 to the nuclear periphery is accompanied by disruption of the large nucleolus into micronucleoli. The micronucleoli are distributed in the nuclear space retaining their association with the nucleolar-organizing regions of chromosome 6. Thus, our data suggest that the large-scale alterations in the organization of chromosome 6 and the nucleolus during polytenization are the correlated processes directly dependent on the rRNA gene activity. The earlier described dynamics of nucleolar-organizing chromosome territory and nucleolus in the nuclear space is likely to be associated with the change in the total expression activity of the nucleus, which complies with the hypothesis on the correlation between spatial nuclear organization and expression regulation of genetic material.