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 diVerent 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.

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The nucleolus is the ribosome factory of the cells. This is the nuclear domain where ribosomal RNAs are synthesized, processed, and assembled with ribosomal proteins. Here we describe the classical tripartite organization of the nucleolus in mammals, reflecting ribosomal gene transcription and pre-ribosomal RNA (pre-rRNA) processing efficiency: fibrillar center, dense fibrillar component, and granular component. We review the nucleolar organization across evolution from the bipartite organization in yeast to the tripartite organization in humans. We discuss the basic principles of nucleolar assembly and nucleolar structure/function relationship in RNA metabolism. The control of nucleolar assembly is presented as well as the role of pre-existing machineries and pre-rRNAs inherited from the previous cell cycle. In addition, nucleoli carry many essential extra ribosomal functions and are closely linked to cellular homeostasis and human health. The last part of this review presents recent advances in nucleolar dysfunctions in human pathology such as cancer and virus infections that modify the nucleolar organization.

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Sirtuins, also designated class III histone deacetylases, are implicated in the regulation of cell division, apoptosis, DNA damage repair, genomic silencing and longevity. The nucleolar Sirtuin7 (SIRT7) was reported to be involved in the regulation of ribosomal gene (rDNA) transcription, but there are no data concerning the regulation of SIRT7 during the cell cycle. Here we have analyzed the behavior of endogenous SIRT7 during mitosis, while rDNA transcription is repressed. SIRT7 remains associated with nucleolar organizer regions, as does the RNA polymerase I machinery. SIRT7 directly interacts with the rDNA transcription factor UBF. Moreover, SIRT7 is phosphorylated via the CDK1-cyclin B pathway during mitosis and dephosphorylated by a phosphatase sensitive to okadaic acid at the exit from mitosis before onset of rDNA transcription. Interestingly, dephosphorylation events induce a conformational modification of the carboxy-terminal region of SIRT7 before the release of mitotic repression of rDNA transcription. As SIRT7 activity is required to resume rDNA transcription in telophase, we propose that this conformational modification regulates onset of rDNA transcription.

Maintenance of specific heterochromatic domains is crucial for genome stability. In eukaryotic cells, a fraction of the tandem-repeated ribosomal RNA (rRNA) genes is organized in the heterochromatic structures. The principal determinant of rDNA silencing is the nucleolar remodelling complex, NoRC, that consists of TIP5 (TTF-1-interacting protein-5) and the ATPase SNF2h. Here we showed that TIP5 not only mediates the establishment of rDNA silencing but also the formation of perinucleolar heterochromatin that contains centric and pericentric repeats. Our data indicated that the TIP5-mediated heterochromatin is indispensable for stability of silent rRNA genes and of major and minor satellite repeats. Moreover, depletion of TIP5 impairs rDNA silencing, upregulates rDNA transcription levels and induces cell transformation. These findings point to a role of TIP5 in protecting genome stability and suggest that it can play a role in the cellular transformation process.

In higher eukaryotic cells, the nucleolus is a nuclear compartment assembled at the beginning of interphase, maintained during interphase, and disorganized during mitosis. Even if its structural organization appears to be undissociable from its function in ribosome biogenesis, the mechanisms that govern the formation and maintenance of the nucleolus are not elucidated. To determine if cell cycle regulators are implicated, we investigated the putative role of the cyclin-dependent kinases (CDKs) on ribosome biogenesis and nucleolar organization. Inhibition of CDK1–cyclin B during mitosis leads to resumption of rDNA transcription, but is not sufficient to induce proper processing of the pre-rRNA and total relocalization of the processing machinery into rDNA transcription sites. Similarly, at the exit from mitosis, both translocation of the late processing machinery and pre-rRNA processing are impaired in a reversible manner by CDK inhibitors. Therefore, CDK activity seems indispensable for the building of functional nucleoli. Furthermore, inhibition of CDKs in interphasic cells also hampered proper pre-rRNA processing and induced a dramatic disorganization of the nucleolus. Thus, we propose that the mechanisms governing both formation and maintenance of functional nucleoli involve CDK activities and couple the cell cycle to ribosome biogenesis.

Nuclear RNA transcription is repressed when eukaryotic cells enter mitosis. Here, we found that the derepression of ribosomal gene (rDNA) transcription that normally takes place in telophase may be induced in prometaphase, metaphase, and anaphase mitotic HeLa cells, and therefore appears not to be dependent on completion of mitosis. We demonstrate for the first time that in vivo inhibition of the cdc2– cyclin B kinase activity is sufficient to give rise to okadaic acid–sensitive dephosphorylation of the mitotically phosphorylated forms of components of the rDNA transcription machinery, and consequently to restore rDNA transcription in mitotic cells. These results, showing that during mitosis the rDNA transcription machinery is maintained repressed by the cdc2–cyclin B kinase activity, provide an in vivo demonstration of the cell cycle–dependent regulation of rDNA transcription. Interestingly in mitotic cells, the newly synthesized 47S precursor ribosomal RNA (pre-rRNA) is not processed into the mature rRNAs, indicating that rDNA transcription and pre-rRNA processing may be uncoupled. Moreover this suggests that inhibition of the cdc2– cyclin B kinase is not sufficient to activate the 47S pre-rRNA processing machinery and/or to induce its relocalization at the level of newly synthesized 47S pre-rRNA. This in vivo approach provides new possibilities to investigate the correlation between pre-rRNA synthesis and pre-rRNA processing when the nucleolus reforms.