Net Results of Nucleolar Dynamics

discrete chromatin complexes, although there is no eviThe nucleolus was one of the first subnuclear elements dence that it directly contacts DNA. How Sir2 arrives in observed by early microscopists, yet in the centuries the nucleolus and what the broader significance for that since its discovery, ideas about its significance have localization is are open questions that have now been ranged from dismissal as a cytological artifact to intense addressed through the discovery of new mitotic and focus on it as a dedicated site for ribosome biogenesis. meiotic regulatory proteins and the placement of a preA spate of new studies define mitotic and meiotic roles viously defined cell cycle control protein in close nucleofor the nucleolus in cell cycle control and gene regulalar proximity. tion. These emerging views of expanded functions for New Nucleolar Tenants with Roles in Cell the nucleolus come from evidence that it serves as a Cycle Control privileged site for both recruitment and exclusion of Starting from an interest in Sir2-interacting proteins, regulatory complexes. Among the molecules participatStraight and colleagues (1999) identified the protein ing are newly identified proteins that function in controlNet1 by affinity chromatography. By happy coincidence, ling exit from mitosis (Shou et al., 1999; Straight et al., Net1 had also been identified independently in a screen 1999; Visintin et al., 1999) and that regulate the meiotic for silencing factors (Andrulis and Sternglanz, cited at pachytene arrest checkpoint (San-Segundo and Roeder, http://genome-www.stanford.edu/Saccharomyces/) and 1999). as a component of a nucleolar complex that regulates Established Occupants of the Nucleolus exit from mitosis (Shou et al., 1999; named Cfi1 in Visintin Definition of the nucleolus as the site of rDNA transcripet al., 1999). A perhaps surprising part of this coincition and ribosome biogenesis is well established, and dence is that Shou et al. began by looking for mutants many studies support the idea that these two processes that bypassed the requirement for the protein kinase themselves are responsible for nucleolar architecture Cdc15, and Visintin et al. sought proteins that interacted (Mélèse and Xue, 1995). Additional nucleolar functions with Cdc14, a dual specificity protein phosphatase. have been recognized to include other RNA modification These are two of a group of at least seven essential and finishing steps, not only of rRNA, but also proproteins defining a mitotic exit network (summarized cessing of tRNA and the signal recognition particle RNA, in Shou et al., 1999). They function in addition to the and perhaps even processing of telomerase RNA (Smith reasonably well-defined anaphase-promoting complex/ and Steitz, 1997; Pederson, 1998a). Nucleoli are morcyclosome (APC/C) that programs ubiquitin-mediated phologically diverse, varying in appearance, number, degradation of the cyclin-dependent kinase activity of and size depending on cell type as well as position in Cdc28. the cell cycle and status of rDNA transcription (Shaw Using well-loved yeast genetic tricks, net1 mutants and Jordan, 1995). A number of nonribosomal proteins were observed to suppress mutation of CDC15 and sevlocalize to the nucleolus, and although their functions eral other members of the exit network as well. This are not well understood, it is likely that they contribute suppression apparently works by restoring appropriate to features that are clinically important for staging a degradation and accumulation of two regulators (Clb2 variety of neoplasms. and Sic1, respectively) whose levels are reciprocally In budding yeast, a hallmark of the nucleolus is its modulated in the course of mitotic exit (Shou et al., 1999; relative simplicity. As in mammalian cells, ultrastructural Visintin et al., 1999). The proteolytic angle on this story studies reveal the nucleolus to be relatively electron is reviewed elsewhere (Bachant and Elledge, 1999; dense. In normal yeast, it exists as a single crescentKoepp et al., 1999). shaped structure, occupying up to one-third of the nuEpitope-tagged Net1 was used as an immunoaffinity cleus. Unlike nucleoli from multicellular organisms, nuhook that captured Cdc14, Sir2, and Nan1 as interacting cleolar subcompartments have not been obvious in proteins (Shou et al., 1999). Tagged versions of the proyeast and the structure remains intact during mitosis, teins were used to show that Net1 coprecipitates Cdc14 ultimately being separated along the mitotic spindle dur(Shou et al., 1999; Visintin et al., 1999) and Sir2, but ing late stages of division. Recent studies also distinthat Cdc14 and Sir2 interactions are dependent on Net1 guish the yeast nucleolus as a major site of occupancy (Shou et al., 1999). Nan1 interactions are not yet defined, for the Sir2 protein. With its genetic cohorts SIR1, SIR3, but it is essential for viability. Immunofluorescence localand SIR4, SIR2 was first identified by mutations resulting ization places all four proteins within the nucleolus, and in inappropriate transcription from the silent matingchromatin immunoprecipitation assays confirm nucleotype loci. Reporter genes at telomere-proximal locations lar residence of Net1, Cdc14, and Sir2p (Shou et al., are also silenced, but SIR2 is set apart from the other 1999; Straight et al., 1999; Visintin et al., 1999). These