Nucleolus

Nucleolus: The Consummate Nuclear Body Laura Trinkle-Mulcahy University of Ottawa, Ottawa, ON, Canada

11.1 A Brief History The nucleolus is the most prominent structure in the eukaryotic cell nucleus, with its high density and greater refractive index relative to the surrounding nucleoplasm rendering it readily detectable in cytological specimens by both light and electron microscopy (Fig. 11.1). This structure attracted substantial interest in the early days of light microscopy due to its prominence within the cell. It was first described in the early 1830s as a “nucleus within the nucleus,” with the name “nucleolus” coined by the German physiologist Gabriel Gustav Valentin (Harris, 2009; Valentin, 1836). By the late 1800s, the nucleolus had been described in great detail with regard to size, number per cell, and appearance/disappearance during mitosis (Montgomery, 1898). In 1896, the Italian pathologist Giuseppe Pianese noted its increased size within the nuclei of malignant tumor cells (Pianese, 1896), which has since been shown to reflect the high energy demands of hyperproliferative cells and remains a useful prognostic indicator for aggressive tumors (for review, see Montanaro et al., 2008). Advances in microscopy and cytological dye techniques in the late 1800s led to the first description of a fibrous network within the nucleus that Walther Flemming termed “chromatin” (for “stainable material”), although it was later renamed “chromosome” by Heinrich Waldeyer. Flemming also described the sequence of chromosome movements during mitosis as they partition equally into two daughter cells (for review, see O’Connor and Miko, 2008). As the chromosome theory of heredity continued to develop throughout the early 1900s, a series of studies established the nucleolus as a genetically determined structure. Specifically, it was observed in mitotic chromosome spreads that the number and lengths of secondary constrictions (defined as thin regions with little or no DNA detected using the acid-based Feulgen staining method) correlated with the number and size of

Nuclear Architecture and Dynamics. DOI: http://dx.doi.org/10.1016/B978-0-12-803480-4.00011-9 © 2018 Elsevier Inc. All rights reserved.

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Figure 11.1 Nucleolar structure. (A) Nucleoli are detected by differential interference contract (DIC) imaging of live HeLa cells as prominent, ovoid subnuclear structures (arrow) and are readily purified by sucrose gradient fractionation methods (inset). (B) Scanning electron microscopy imaging of purified nucleoli reveals the shell of heterochromatin that surrounds them. (C) Transmission electron microscopy imaging of nucleoli in situ identifies a distinct substructure comprising a fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC). (D) Nucleolar structure and dynamics can be studied via light microscopy, using a range of fluorescent markers. This image shows the distinct localization patterns of transiently expressed GFP-tagged nucleophosmin (GC, blue), endogenous fibrillarin stained using fluorophore-tagged antibodies (DFC, red), and neighboring proteins biotinylated by stably expressed BirA (biotin ligase)-tagged UBF (FC, green).

nucleoli in interphase cells (Heitz, 1931). In 1934, Barbara McClintock demonstrated that chromatin at these regions acts as a nucleolar organizing element (McClintock, 1934), since termed NOR for “nucleolar organizing region.” In the 1940s, nucleic acid staining revealed that nucleoli are enriched in RNA (Brachet, 1940; Caspersson and Schultz, 1940). This observation was eventually followed by the demonstration of ribosomal DNA in NORs (Ritossa and Spiegelman, 1965; Scherrer et al., 1963) and a series of studies identified the nucleolus as the site of ribosome synthesis (Birnstiel et al., 1963; McConkey and Hopkins, 1964; Perry, 1965). These included the striking demonstration of growth arrest and the absence of rRNA synthesis in an anucleolate Xenopus laevis embryo (Brown and Gurdon, 1964). 258

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The invention of the electron microscope (EM) by Knoll and Ruska (1932) enabled the ultrastructural analysis of nucleoli at the nanometer scale, which confirmed that this organelle lacks a membrane and revealed the existence of a perinucleolar shell of condensed chromatin (Fig. 11.1B). It also delineated a tripartite substructure (Fig. 11.1C) comprising a concentric arrangement of three distinct components: an innermost lightly stained fine fibrillar structure (fibrillar enter; FC) mostly surrounded by densely packed fibrils (dense fibrillar component; DFC) and embedded in a grainy peripheral region (granular component; GC) comprising RNP particles of 15
20 nm in size (Bernhard and Granboulan, 1963; Swift, 1963). When coupled with autoradiography to assess the distribution of nucleic acids, the intranucleolar fibrillar regions were shown to be enriched in RNA (Bernhard and Granboulan, 1963), with labeled RNA moving outward from them toward the granular region (von Gaudecker, 1967; Granboulan and Granboulan, 1965; Unuma et al., 1968). These early EM studies culminated in the direct visualization of transcriptionally active ribosomal genes, in preparations of nucleoli dissociated and spread on a liquid surface, as a “Christmas tree” (CT) structure (Miller and Beatty, 1969) (Fig. 11.2B). In these trees, branches of nascent transcripts extend off a central

Figure 11.2 Organization and transcription of rRNA genes. (A) NORs contain multiple repeats of rRNA genes (B43 kb) that encode the 45S pre-rRNA transcript, interspersed with IGS regions (B30 kb). This transcript is further processed by a series of posttranscriptional modifications and cleavage events into the 18S, 5.8S, and 28S rRNAs that are incorporated into preribosomal subunits. (B) Diagram depicting the direct visualization of transcriptionally active ribosomal genes in chromosome spreads as Christmas tree (CT) structures, with “branches” of nascent rRNA transcripts extending off a central “trunk” of rDNA and terminating in rRNA processing complex “balls.”

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DNA trunk and terminate in balls at 50 end that represent rRNA processing complexes (Mougey et al., 1993; Scheer and Benavente, 1990; Sharma and Tollervey, 1999). Although these are striking images that correlate nucleolar morphology with various steps of ribosome biogenesis, actively transcribing rDNA genes in the form of CTs have not yet been directly observed in thinly sectioned intact nucleoli, and it remains difficult to reconcile these structures and their required packaging with in situ nucleolar components (Jordan, 1991; Shaw et al., 1995). That said, these observations all contributed to the growing appreciation of the nucleolus as “an organelle formed by the act of building a ribosome” (M´ele`se and Xue, 1995). Interestingly, although condensed chromatin is visible in nucleoli by EM, the amount has generally been believed to be low relative to the rest of the nucleus. This was based initially on the fact that nucleoli appear as dark holes (although a weak signal is present) when cells are stained with intercalating DNA dyes such as 4’,6-Diamidine-2’-phenylindole dihydrochloride (DAPI)

Figure 11.3 Nucleolar DNA and RNA. (A) In HeLa cells stained with the DNA intercalating dye DAPI, nucleoli are observed as darker, minimally stained regions (arrow) compared to the rest of the nucleoplasm. (B) GFP-tagged Histone H2B, which is incorporated into nucleosomes, shows a similar distribution when stably overexpressed in HeLa cells. (C) Nascent rRNA transcripts can be detected in cells via incorporation and staining of the Uridine-5’-triphosphate (UTP) analogue 5-fluorouridine (5-FU). Nucleoplasmic staining reflects levels of Pol II transcription, while nucleolar staining (arrow) reflects levels of Pol I transcription. Scale bar 5 10 µm.

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(Fig. 11.3A), but the same holds true for the distribution of tagged histones incorporated into nucleosomes (Mu¨ller et al., 2007) (Fig. 11.3B) and the visualization of DNA replication by labeled nucleotide incorporation (O’Keefe et al., 1992). Estimations of nucleolar (and nucleolar associated) versus nucleoplasmic DNA concentrations cannot fully account for these results, nor can any other single model that has been proposed, from the dense structure of the nucleolus affecting permeability to dyes and tagged constructs (Hancock, 2004) to the exiting of rDNA from the nucleolar interior for replication (Dimitrova, 2011) (for review, see Smirnov et al., 2016). Developments in nucleic acid technology over the next two decades enabled the molecular dissection of the pathway of ribosome biogenesis, from initiation of rRNA transcription through assembly and export of ribosome subunits, in a range of model systems. The surprising observation that a nonribosomal RNA, specifically the RNA component of the signal recognition particle, is also processed in the nucleolus heralded the advent of the “plurifunctional nucleolus” hypothesis (Jacobson and Pederson, 1998a; 1998b), with subsequent work identifying nucleolar processing of certain transfer RNAs (Bertrand et al., 1998; Jarrous et al., 1999) and small nuclear RNAs (Ganot et al., 1999). It was also shown to function as a domain for protein sequestration in the regulation of cell cycle progression and p53 stabilization (Cockell and Gasser, 1999; Visintin and Amon, 2000). Advances in genomic and proteomic screening that allowed the DNA, RNA, and protein contents of the nucleolus, which is readily purified in large amounts (Busch et al., 1963; Chamousset et al., 2010; Li and Lam, 2015; Maggio et al., 1963), to be mapped under both steadystate conditions and in response to various perturbations further supported the idea of previously unknown roles beyond that of ribosome biogenesis (Andersen et al., 2005; 2002; Bai et al., 2014; Boisvert et al., 2007; N´emeth et al., 2010; Pendle et al., 2005; Politz et al., 2009; Scherl et al., 2002; van Koningsbruggen et al., 2010). The advent of genetically encoded fluorophores confirmed that the nucleolus is a dynamic structure whose contents are in constant flux (Chen and Huang, 2001; Phair and Misteli, 2000; Politz et al., 2003) and which can respond rapidly to a wide range of cellular signals that coordinate cell growth and proliferation. It also provided the means to follow the process of nucleolar disassembly/assembly throughout the cell cycle (Hernandez-Verdun et al., 2013; Leung et al., 2004), via time-lapse imaging and photokinetic analysis of fluorophore-tagged marker proteins known to localize to the FC, DFC, and GC (Fig. 11.1D). More recent innovations in superresolution imaging (Sydor et al., 2015) now permit analysis of nucleolar structure and function by light microscopy at the nanometer scale, in both fixed and live samples, utilizing the diverse range of fluorophore-based tags, probes, and assays that have been developed over the years.

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11.2 Ribosome Biogenesis Although now better appreciated as a functionally diverse signaling hub within the cell, the nucleolus is still best known for its key role in the generation of ribosomes, the molecular machines that govern the translation of mRNA into proteins. Ribosomes are complex ribonucleoprotein structures comprised of 80 ribosomal proteins associated with 4 rRNA molecules (5S, 18S, 5.8S, and 28S) and organized into two subunits (see Fig. 11.4 for overview). It is estimated that over half of the cell’s capacity is dedicated to making pre-rRNA (for review, see Granneman and Tollervey, 2007), and a large excess of ribosomal proteins, with constant turnover mediated by the proteasome, is also maintained (Lam et al., 2007; Scharf et al., 2007). Although this provides cells with the flexibility to rapidly adjust to changes in metabolic demand, it is an energetically expensive process, and its coordinated shutdown is thus a key strategy used by the cell to maintain energy homeostasis under conditions of cellular stress, such as nutrient deprivation and heat shock (for review, see Boulon et al., 2010; Grummt, 2013). The process of ribosome biogenesis, from initial transcription of rRNA through export and assembly of mature ribosomes, is a complex pathway that requires

Figure 11.4 Ribosome biogenesis pathway. Overview of the steps involved in ribosome biogenesis, from initial transcription of the 45S precursor rRNA in the nucleolus to the cytoplasmic assembly of the 60S and 40S subunits into a mature ribosome.

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coordination of RNA Pol I, Pol II, and Pol III activity, along with the participation of a class of small nucleolar ribonucleoproteins that guide chemical modifications of the rRNA such as methylation and pseudouridilation (for review, see Lui and Lowe, 2013). More than 250 nonribosomal accessory proteins are also required and include exo and endonucleases, GTPases, AAA-ATPases, RNA helicases, methyltransferases, isomerases, and export factors. In mammalian cells, the 18S, 5.8S, and 28S rRNAs are produced by cleavage and processing of a 45S precursor rRNA that is transcribed by nucleolar RNA Polymerase I from clusters of 43 kb rDNA gene arrays on chromosomes 13, 14, 15, 21, and 22 (Gonzalez and Sylvester, 1995; Henderson et al., 1972), while the 5S rRNA is transcribed by nucleoplasmic RNA Polymerase III from a cluster of 2.2 kb gene repeats on chromosome 1 (Little and Braaten, 1989; Sørensen et al., 1991). Ribosomal proteins (and processing and export factors) are transcribed by nucleoplasmic RNA Polymerase II, translated in the cytoplasm and imported into the nucleolus. Our current knowledge of the sequential steps in rRNA processing and ribosomal subunit assembly is largely based on work carried out in the powerful yeast model system (for comprehensive reviews, see Fatica and Tollervey, 2002; Gerhardy et al., 2014; de la Cruz et al., 2015; Woolford and Baserga, 2013), while ongoing advances in genomic, proteomic, and structural analysis techniques are facilitating a similarly detailed dissection of pre-rRNA processing and ribosomal subunit assembly in mammalian nucleoli. Although the general consensus scheme is upheld in higher organisms, studies have highlighted certain differences in pre-rRNA processing steps and identified unique factors and medically relevant findings that are specific to mammals (Badertscher et al., 2015; Kamath et al., 2005; Maggi and Weber, 2005; Mullineux and Lafontaine, 2012; Tafforeau et al., 2013; Viktorovskaya and Schneider, 2015; Wild et al., 2010; Yao and Yang, 2005; Ebersberger et al., 2014; Henras et al., 2008).

11.3 Ribosomal Genes and NORs Each mammalian rRNA transcription unit is approximately 43 kb and contains a coding region for 45S pre-rRNA (B13 kb) separated by two internal transcribed spacers (ITS1 and ITS2) and flanked by external spacers (50 ETS and 30 ETS; Fig. 11.2A). Within the gene locus, these transcription units are separated from each other by intergenic spacer (IGS) regions of approximately 30 kb, which contain a high density of DNA sequence repeats and transposable elements, plus regulatory elements such as promoters, spacer promoters, repetitive enhancer elements, and transcription terminators (for review, see McStay and Grummt, 2008; Smirnov et al., 2016). Although initially regarded as “junk” sequences, Pol I binding and transcription from the IGS region have been demonstrated (Zentner et al., 2011, 2014) and IGS transcripts have been shown to regulate both the epigenetic state of rRNA genes (Mayer et al., 2006;

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Santoro et al., 2010) and a reversible stress response that involves nucleolar sequestration of proteins involved in energy-intensive tasks (Audas et al., 2012a, 2012b; Jacob et al., 2013; Lam and Trinkle-Mulcahy, 2015). In human cells, NORs are found on the short arms of each of the five acrocentric chromosomes (13, 14, 15, 21, and 22) and cumulatively contain 300
400 copies of rDNA in tandem arrays arranged primarily in a head-to-toe orientation (Caburet et al., 2005; Henderson et al., 1972; N´emeth and La¨ngst, 2011; Schmickel, 1973; Stults et al., 2008). NORs can be readily visualized in cytohistological samples by silver staining, as they contain accumulations of acidic nonhistone proteins (termed “AgNOR proteins”) that bind silver ions and correspond at the ultrastructural level to an FC (for review, see Trere`, 2000). Activation of RNA Polymerase I-mediated transcription of rDNA at NORs has long been known to trigger nucleolar formation (Karpen et al., 1988; Scheer and Hock, 1999), and rDNA repeats from more than one NOR can cluster together to form a single nucleolus. Although all NORs have the potential to nucleate nucleoli, some remain silent while other fuse in early G1, and not all NORs associate with transcription factors in mitosis (Savino et al., 2001). Even in highly proliferative HeLa cells, only 6 of the 10 NORs are transcriptionally active at a time (Roussel et al., 1996), and although numbers vary, most cultured cells contain only one to three nucleoli (Farley et al., 2015). Nucleolar size has been shown to correlate with efficiency of ribosome biogenesis, which in turn is governed by the demand for ribosomes. It ranges from ,0.5 in terminally differentiated cells to 3
9 µm in actively cycling transformed cell lines (Hernandez-Verdun, 2006). Within nucleoli, silent NORs are interspersed among active NORs (Dammann et al., 1995; Sogo et al., 1984; Zillner et al., 2015). Furthermore, not all rDNA repeats within a single NOR are transcriptionally active (French et al., 2003; McKnight and Miller, 1976; Morgan et al., 1983), indicating that they are not regulated as a cluster. Active genes have been shown to be in a more open, euchromatic state and are associated with nascent rRNA transcripts, while inactive genes are in a closed, heterochromatic state (Conconi et al., 1989; Dammann et al., 1995). Although it remains unclear how the distribution of active/inactive genes is regulated, roles have been demonstrated for methylation, nucleosome positioning, and chromatin remodeling complexes including nucleolar remodeling complex, nucleosome remodeling deacetylase, and Cockayne Syndrome Protein B (for review, see McStay and Grummt, 2008; Tucker et al., 2010). Active genes are also enriched in the Pol I activator UBF (upstream binding factor), which is believed to be important for maintaining an open, euchromatic topology (Kermekchiev et al., 1997; N´emeth and La¨ngst, 2011) and a “bookmark” left on active genes during mitosis, when rRNA transcription temporarily shuts down (Mais et al., 2005). Transcription of the 45S pre-rRNA is a multistep process that starts with formation of a preinitiation complex on the rRNA gene

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core promoter region (for review, see Goodfellow and Zomerdijk, 2013), which requires the binding of a 300 kDa protein complex termed selectivity factor 1 (SL1; TIF1B in mouse) that confers promoter specificity. SL1 is made up of the general transcription factor TBP (TATA-binding protein) and at least four Pol I-specific TBP-associated factors. Binding of SL1, in addition to being essential for recruitment of Pol I to the transcription start site, also promotes the stable interaction of UBF with the rDNA promoter. Following recruitment of the polymerase, transcription is initiated, and subsequent steps include promoter escape, transcript elongation, transcriptional termination, and then reinitiation. Although there has been some debate over where transcription occurs within the nucleolus (reviewed in Cisterna and Biggiogera, 2010; Huang, 2002; Koberna et al., 2002; Raska et al., 2006), the general consensus is that it takes place at the FC/DFC border. This is followed by a “vectorial maturation” of the pre-rRNA, in which the first processing steps occur within the DFC, followed by subsequent processing and assembly of preribosomal subunits in the GC and then export to the cytoplasm. Consistent with this model, nascent RNA transcripts were detected in the DFC following pulse labeling with tritiated uridine (Fakan and Bernhard, 1971) or 5-bromo-UTP (Cmarko et al., 2000), and shown to form and associate with certain processing proteins in this structure (Brown and Shaw, 2008; Henras et al., 2008). Pol I and associated proteins such as UBF and Nopp140 have been detected in the FC (Casafont et al., 2007; Scheer and Rose, 1984; Schwarzacher and Mosgoeller, 2000), and although a transcriptional role has not been definitively ruled out, this is currently believed to represent a reservoir of inactive complexes. The number of FCs within a single nucleolus can vary, and in general, high ribosome biogenesis activity correlates with a larger number of small FCs, while nucleoli in cells with lower metabolic activity, such as lymphocytes, have a single large FC (Hoz´ak et al., 1989; P´ebusque and Seı¨te, 1981). Pol I activity within nucleoli can be monitored in cells by various labeling techniques, such as incorporation and staining of UTP analogs (Fig. 11.3C). Short-term regulation of ribosome biogenesis, such as shutdown in response to acute stress, is primarily mediated via control of the Pol I transcription cycle (for review, see Grummt, 2013). If the stress is reversible, normal nucleolar structure can reform when transcription resumes. In contrast, long-term regulation, such as the reduction in transcriptional activity during development and differentiation, can involve regulation of the total number of active rRNA genes (Haaf et al., 1991) and is likely mediated, at least in part, by epigenetic changes at rDNA promoters.

11.4 Nucleolar Plasticity Transcriptional inhibition leads to reorganization of nucleolar architecture, highlighting the interdependence of structure and function in this organelle. Direct Pol I inhibition, for example using DNA intercalating drugs such as

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actinomycin D, leads to nucleolar segregation, with specific components either retained in a remnant nucleolar body or segregated into various cap structures that form on this residual body (Shav-Tal et al., 2005) (Fig. 11.5). Interestingly, inhibition of RNA Pol II transcriptional activity also causes nucleolar breakdown, albeit in a “dispersal” phenotype in which subnucleolar domains lose their association with each other (Haaf and Ward, 1996; Hancock, 2004; Scheer et al., 1984). This long-time conundrum may finally have been solved by a recent study that demonstrated the contribution of stable short intron-derived RNAs, containing Alu repeat elements and transcribed by Pol II, to nucleolar integrity (Caudron-Herger et al., 2015). Depletion of AluRNAs led to disrupted nucleolar structure and repressed Pol I transcription, while overexpression increased nucleolar size and levels of pre-rRNA. AluRNAs were shown to interact with the abundant nucleolar proteins nucleolin and nucleophosmin (Amin et al., 2008;

Figure 11.5 Nucleolar segregation in response to transcriptional inhibition. Inhibition of Pol I activity by treatment of MCF7 cells with 0.5 µg/mL actinomycin D results in structural changes that include redistribution of components of the FC and DFC regions into distinct caps at the periphery of a smaller, residual GC region. The GC was monitored by transient overexpression of GFP-tagged nucleophosmin (NPM), the DFC by antibody staining of endogenous fibrillarin (FIB) and the FC was by staining neighboring proteins biotinylated by BirA-tagged UBF with Alexa568-streptavidin.

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Ugrinova et al., 2007), which have also been linked to structural integrity. They have thus been proposed to function as a nucleolar scaffold or “glue” holding the subnucleolar components together. Coordinated nucleolar breakdown and reformation also occurs during each cell cycle in mammalian cells, with preexisting complexes transiting through mitosis and acting as the building blocks for new nucleoli once transcription resumes (for review, see Hernandez-Verdun, 2011). Although this is not the case in yeast, where the nucleolus remains intact through mitosis, transcription does briefly halt and rDNA condenses to ensure faithful nucleolar segregation in anaphase (D’Amours et al., 2004; Sullivan et al., 2004; Torres-Rosell et al., 2004). At the onset of mammalian mitosis, rDNA transcription at active NORs is arrested, due at least in part to phosphorylation of several Pol I factors by the mitotic kinase Cdk1-cyclin B (Sirri et al., 2002). As previously noted, these regions can be visualized in stained mitotic chromosome spreads as prominent secondary constrictions (primary constrictions are centromeres) or “gaps” that are B10 3 less condensed than the surrounding chromatin (Heliot et al., 1997). UBF and other Pol I-related factors remain associated with this less condensed rDNA (G´ebrane-Youne`s et al., 1997; Roussel et al., 1993; Sirri et al., 1999; Zatsepina et al., 1996), although Pol I itself does not appear to remain there (Leung et al., 2004). In contrast, repeat regions that were silent during interphase lack UBF and other Pol I-related factors and are fully condensed during mitosis (Grob et al., 2014; McStay and Grummt, 2008). Nucleolar disassembly starts with the ordered release of rRNA processing complexes at the onset of mitosis, followed by inhibition of Pol I transcription and subsequent loss of visible nucleoli by the end of prophase (G´ebrane-Youne`s et al., 1997) (Fig. 11.6). As noted above, certain Pol I-related factors remain associated with previously active NORs, while certain DFC and GC factors are relocalized to a perichromosomal compartment that forms at this stage. They include the DFC proteins fibrillarin, nucleolin, and nopp140, the GC proteins nucleophosmin and Ki-67, several snoRNAs, and partially processed 45S rRNA ˜ ol-Roma, 1999). (Gautier et al., 1992a, 1992b; Hernandez-Verdun, 2011; Pin Although this suggests that processing complexes are maintained during mitosis, Fluorescence/Fo¨rster Resonance Energy Transfer experiments did not detect interactions between protein complex members until initiation of nucleolar assembly (Louvet et al., 2008). The GC protein Ki-67 was recently shown to be essential for the formation of the perichromosal compartment, possibly acting as a scaffold for protein recruitment. Although absence of this structure did not preclude nucleolar reassembly at the end of mitosis, the process was less efficient (Booth et al., 2014). Reassembly starts in telophase, with reversal of the mitotic phosphorylations by the mitotic phosphatases PP1 and PP2A (for review, see Wurzenberger and Gerlich, 2011) and reactivation of rDNA transcription at mitotic NORs

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preloaded with Pol I complexes. This is followed by recruitment of RNA processing complexes via the formation of transient foci termed “prenucleolar bodies” or PNBs (Ochs et al., 1985; Sirri et al., 2002) (Fig. 11.6). Traffic between these foci, which represent accumulations of ribosomal proteins, processing proteins,

Figure 11.6 Nucleolar disassembly and reassembly during mitosis. U2OS cells transiently overexpressing the GC marker nucleophosmin as an mCherry fusion protein and stained with the permeable DNA dye Hoechst 33342 were monitored by multiwavelength timelapse imaging as they progressed through the cell cycle. Z-stacks were acquired every 3 minutes. The top panel shows images from early mitosis, as the nucleoli breakdown and nucleophosmin are released. The middle panel shows images from mid-mitosis, with a pool of nucleophosmin remaining associated with the perichromosomal region (arrows) in metaphase and anaphase, before appearing in the prenucleolar bodies (PNBs; hashed arrows) that start to form in telophase. The bottom panel shows images from early G1, as small nucleoli start to fuse into larger, mature nucleoli (arrowheads). Scale bar 5 10 µm.

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snoRNAs and partially processed 45S RNA recruited from the perichromosomal compartment, and active NORs, is important for recruitment of these factors to the DFC and GC during nucleolar assembly (Hernandez-Verdun, 2011). In early G1, individual NORs start to coalesce into multiple small nucleoli, which then fuse into larger mature nucleoli (Savino et al., 2001) (Fig. 11.6).

11.5 Building a Nucleolus A fundamental question that has been addressed in various ways over the years is whether it is simply the rDNA arrays that are required for nucleolar formation or if the context of NORs within the genome is also important. When rDNA genes were deleted from yeast chromosomes and replaced by plasmid-encoded copies, for example, multiple small nucleoli were formed but cell growth was compromised (Oakes et al., 1998; Wai et al., 2000). Although this may have been due in part to less efficient rRNA transcription and processing, it does suggest the need for additional factors and/or the importance of the nucleolus in a larger context. Insertion of a single rDNA gene into the polytene chromosomes of Drosophila melanogaster also induced formation of mininucleoli, which were shown to transcribe pre-rRNA and recruit the nucleolar-specific antigen Aj1 (Karpen et al., 1988), while in vitro nuclei assembled in Xenopus egg extract from purified chromatin formed PNBs that could not fuse with each other, suggesting the absence of a functional nucleolar organizer (Bell et al., 1992). Nucleolar formation has been studied in mammalian cells using a synthetic biology approach based on chromosome engineering. “Pseudo-NORs,” which can be used to model mitotic competent NORs and nucleolar FCs, are artificial arrays assembled on non-NOR bearing human chromosomes that contain multiple copies of UBF-binding DNA sequence arrays. Although able to recruit endogenous UBF and the rest of the Pol I transcriptional machinery, and adopt key morphological features of active NORs, pseudo-NORs have no promoter sequences and therefore do not recruit pre-rRNA processing factors or form fully assembled nucleoli (Grob and McStay, 2014; Mais et al., 2005). These studies support a central role for UBF in maintaining NOR competency and establishing the mitotic hallmarks of competent NORs. Although present across animal phyla (Grob et al., 2011), the lack of this protein in yeast may reflect the lack of a need for “mitotic bookmarking” of NORs, given that the nucleolus remains intact throughout (Grob and McStay, 2014). There is, however, a related yeast HMG-box protein called Hmo1 that has been reported to play a similar role in rDNA chromatin organization (Wittner et al., 2011). Construction of functional synthetic nucleoli in human cells was achieved through the integration of ectopic arrays of a “neo-NOR” cassette, which comprises an engineered human rDNA promoter, mouse pre-rRNA coding sequences, and a mouse transcription terminator (Grob et al., 2014) (Fig. 11.7). Neo-NORs are transcriptionally active (albeit at a lower level than

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Figure 11.7 Construction of functional synthetic nucleoli by integration of neo-NORs. This diagram shows the neo-NOR cassette that was engineered to study nucleolar formation.

endogenous NORs), and this activity drives the formation of compartmentalized “neonucleoli” that contain endogenous DFC and GC components and produce mature rRNAs and polysome-associated ribosomes. The ability to distinguish neo-NORs and their transcripts from endogenous NORs/transcripts revealed that B40% of neo-NORs associate with endogenous NORs in large nucleoli in a compartmentalized manner, indicating that there may be “NOR territories” comparable to the chromosome territories that contribute to compartmentalized nuclear architecture (for review, see Cremer and Cremer, 2001). Although these studies suggest that context is not absolutely essential, with functional neonucleoli forming despite the lack of rDNA flanking sequences or a perinucleolar heterochromatic shell, it is likely that these factors play key roles in other aspects of nucleolar biology. Sequences proximal and distal to ribosomal gene arrays have been shown to be conserved among the acrocentric chromosomes, with the distal sequence dominated by a large inverted repeat that was observed by 3D immunofluorescence in situ hybridization to localize to the perinucleolar heterochromatin region (Floutsakou et al., 2013). Interestingly, in the nucleolar segregation that occurs following inhibition of Pol I transcription, the perinucleolar caps form adjacent to these distal sequence regions, suggesting that the regions may be involved in anchoring rDNA to perinucleolar heterochromatin. Additional roles that have been suggested for perinucleolar heterochromatin include maintenance of the genomic stability of rDNA arrays (Peng and Karpen, 2007) and exclusion of Pol II activity from nucleoli (Gagnon-Kugler et al., 2009).

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The nucleolus is already known to play a role in the maintenance of the heterochromatic state of the inactive X chromosome (Zhang et al., 2007), with this condensed structure originally observed as a nucleolar satellite body that was termed the Barr body (Barr and Bertram, 1949). In an attempt to understand the relationship of the nucleolus to global chromatin organization, two studies identified nucleolar-associated chromatin domains (N´emeth et al., 2010; van Koningsbruggen et al., 2010). These represented a surprising 4% of the genome (not including the rDNA repeats), and the majority were found to be inactive heterochromatic regions. Although further work is required, it is clear that the nucleolus contributes more to chromatin organization and preservation of genomic stability than is currently appreciated (for review, see N´emeth and La¨ngst, 2011; O’Sullivan et al., 2013).

11.6 Physical Properties of Nucleoli Although much is known about the molecular steps involved in nucleolar formation, from their initial nucleation by nascent rRNA transcript through the recruitment of various processing complexes into distinct regions, the underlying physical events remain unclear. In contrast to conventional membranebound cytoplasmic organelles, nuclear bodies such as the nucleolus are able to compartmentalize specific protein and RNA factors without a physical membrane and remain stable while these factors are in constant flux with the surrounding nucleoplasm. Furthermore, their assembly is based on selforganization but is not necessarily sequential. Recent attempts to unify this self-organization model of the formation of distinct structures from soluble constituents with the physical principals that underlie phase transitions from liquid to solid have now provided a framework for the formation of membrane less RNA
protein organelles such as the nucleolus (Bergeron-Sandoval, Safaee, and Michnick, 2016; Courchaine et al., 2016; Feric et al., 2016a; Wu and Fuxreiter, 2016; Zhu and Brangwynne, 2015). Key principles include the concentration-dependent ability of protein
protein and protein
RNA interactions to drive phase transitions, mediated by interactions between intrinsically disordered low complexity sequences (LCSs; enriched in many RNA binding proteins), and the subsequent liquid droplet-like behavior of the resulting structures. These are all demonstrated in a recent study that showed temperature- and protein and salt concentration-dependent formation of liquid phase droplets (driven by a LCS region) by the Ddx4 protein in vitro, and liquid droplet-like synthetic nuclear bodies in vivo (Nott et al., 2015). Consistent with this type of model, nucleolar size in Caenorhabditis elegans was shown to be dependent on the nucleoplasmic concentration of its constituents (S. C. Weber and Brangwynne, 2015), and studies in X. laevis oocytes have shown that nucleoli exhibit liquid droplet-like behavior (Brangwynne et al., 2011), freely diffusing and fusing with each other in a manner consistent

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with the G1 fusion events observed in mammalian cells (Fig. 11.6). While such fluidity would be difficult to reconcile with the original concept of the nucleolus as a dense, compact structure, that view has changed in recent years (Pederson, 2010), due in part to interference microscopy measurements that revealed that the viscosity of the nucleolus is only about twice that of the surrounding nucleoplasm (Handwerger et al., 2005). This viscosity has also been shown to be ATP dependent, suggesting that it is based on active processes (Brangwynne et al., 2011). Although phase transition is an attractive model for nucleolar formation, the basic principles must be extended to accommodate the distinct tripartite architecture that is established and maintained in mature nucleoli. A recent study addressed this issue by demonstrating that purified nucleolar proteins from the separate subcompartments (FC, DFC, or GC) can phase transition into liquid droplets with distinct biophysical properties and coexist without coalescing (Feric et al., 2016b). Interestingly, a more detailed analysis of the properties of the DFC marker protein fibrillarin suggested that different regions of the protein mediate phase transition (disordered domain) and immiscibility (functional domain). Further work is needed to continue to delineate the physical principles underlying nucleolar compartmentalization, and to put it in context with the formation and maintenance of its large number of dynamic macromolecular complexes.

11.7 Conclusion The nucleolus has always been a fertile testing ground for new technologies, ranging from the earliest observations of subcellular structure to the mathematical modeling of the physics of organelle formation. This has benefited not only the field of nucleolar research but also a diverse range of scientific disciplines. We now have a much greater understanding of nucleolar formation and maintenance, and a better appreciation of its role as a signaling hub in the maintenance of cellular homeostasis and the pathological implications of its dysfunction. Although certain fundamental questions remain, such as the implications and coordination of its structural and functional relationship with other nuclear structures, including the Cajal body and specific chromatin regions, it is safe to presume that ongoing technological advances will continue to provide the tools to address them.

Acknowledgments The author would like to thank Sarah Ooi and Drs. Angus Lamond, Carol Lyon, and Yun Wah Lam for providing images used in Figs. 11.1 and 11.5 and apologizes for the many interesting articles that were not discussed or acknowledged here due to space limitations. This work was supported by a Natural Sciences and Engineering Research Council Discovery Grant.

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