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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.

The transcription factors MyoD and Myf-5 control myoblast identity and differentiation. MyoD and Myf-5 manifest opposite cell cycle-specific expression patterns. Here, we provide evidence that MyoD plays a pivotal role at the G[sub 2]M transition by controlling the expression of p21[sup Waf1/Cip1] (p21), which is believed to regulate cyclin B-Cdc2 kinase activity in G[sub 2]. In growing myoblasts, MyoD reaccumulates during Ge concomitantly with p21 before entry into mitosis; MyoD is phosphorylated on Ser5 and Ser200 by cyclin B-Cdc2, resulting in a decrease of its stability and down-regulation of both MyoD and p21. Inducible expression of a nonphosphorylable MyoD A5/A200 enhances the MyoD interaction with the coactivator P/CAF, thereby stimulating the transcriptional activation of a lueiferase reporter gene placed under the control of the p21 promoter. MyoD A5/A200 causes sustained p21 expression, which inhibits cyclin B-Cdc2 kinase activity in G[sub 2] and delays M-phase entry. This Ge arrest is not observed in p21[sup -/-] cells. These results show that in cycling cells MyoD functions as a transcriptional activator of p21 and that MyoD phosphorylation is required for G[sub 2]/M transition. [ABSTRACT FROM AUTHOR]

Two specific inhibitors of cyclin-dependent kinase 2 (Cdk2), roscovitine and olomoucine, have been shown recently to induce nuclear accumulation of wt p53 and nucleolar unravelling in interphase human untransformed IMR-90 and breast tumor-derived MCF-7 cells. Here, we show that the early response of MCF-7 cells to roscovitine is fully reversible since a rapid restoration of nucleolar organization followed by an induction of p21WAF1/CIP1, a downregulation of nuclear wt p53 and normal cell cycle resumption occurs if the compound is removed after 4 h. Interestingly, similar reversible effects are also induced by the casein kinase II (CKII) inhibitor, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole. Upon short-term treatment also, both compounds significantly, but reversibly, reduce the level of 45S precursor ribosomal RNA. Cells exposed to the two types of protein kinase inhibitors for longer times keep exhibiting altered nucleolar and wt p53 features, yet they strikingly differentiate in that most roscovitine-treated cells fail to ever accumulate high levels of p21WAF1/CIP1 in contrast with DRB-treated ones. In both cases, however, the cells eventually fall into an irreversible state and die. Moreover, we found that constitutive overexpression of p21WAF1/CIP1 alters the nucleolar unravelling process in the presence of DRB, but not of roscovitine, suggesting a role for this physiological Cdk inhibitor in the regulation of nucleolar function. Our data also support the notion that both roscovitine- and DRB-sensitive protein kinases, probably including Cdk2 and CKII, via their dual implication in the p53-Rb pathway and in ribosomal biogenesis, would participate in coupling cell growth with cell division. Oncogene (2001) 20, 5951–5963. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We report the characterization of a nucleolar localization sequence (NoLS) that targets the green fluorescent protein (GFP) into the granular component (GC) of nucleoli. This NoLS interacts in vitro specifically and directly with the major nucleolarprotein B23 and more precisely with the region of B23 including the two acidic stretches. The affinity of NoLS for B23 is stronger than that of the HIV-1 Rev protein in vitro. Moreover, B23-NoLS interaction also occurs in vivo. Indeed, (1) NoLS confers on the GFP the behavior of B23 throughout the cell cycle, (2) the GFP-NoLS fusion and B23 remain colocalized after drug treatments, (3) a selective delocalization of B23 from nucleoli to nucleoplasm induces a concomitent delocalization of the GFP-NoLS fusion, and (4) the fusion of NoLS to fibrillarin makes it possible to colocalize fibrillarin and B23. Interestingly, by fusing NoLS to fibrillarin, both fibrillarin and the fibrillarin partner Nop56 are mislocalized in the GC of nucleoli. Similarly, by fusing the NoLS to MafG, part of the nuclear transcription factor NF-E2 composed of both MafG and p45 NF-E2, NF-E2 is redirected from the nucleoplasm to the nucleoli. Thus, we propose that the NoLS may be used as a tool to visualize and prove protein interactions in a cellular context. [ABSTRACT FROM AUTHOR]