key: cord-0004023-8942b94u
authors: Matos-Perdomo, Emiliano; Machín, Félix
title: Nucleolar and Ribosomal DNA Structure under Stress: Yeast Lessons for Aging and Cancer
date: 2019-07-26
journal: Cells
DOI: 10.3390/cells8080779
sha: 1de4de4c1b8a4d3c18f20867febc8f4a2758dfd9
doc_id: 4023
cord_uid: 8942b94u

Once thought a mere ribosome factory, the nucleolus has been viewed in recent years as an extremely sensitive gauge of diverse cellular stresses. Emerging concepts in nucleolar biology include the nucleolar stress response (NSR), whereby a series of cell insults have a special impact on the nucleolus. These insults include, among others, ultra-violet radiation (UV), nutrient deprivation, hypoxia and thermal stress. While these stresses might influence nucleolar biology directly or indirectly, other perturbances whose origin resides in the nucleolar biology also trigger nucleolar and systemic stress responses. Among the latter, we find mutations in nucleolar and ribosomal proteins, ribosomal RNA (rRNA) processing inhibitors and ribosomal DNA (rDNA) transcription inhibition. The p53 protein also mediates NSR, leading ultimately to cell cycle arrest, apoptosis, senescence or differentiation. Hence, NSR is gaining importance in cancer biology. The nucleolar size and ribosome biogenesis, and how they connect with the Target of Rapamycin (TOR) signalling pathway, are also becoming important in the biology of aging and cancer. Simple model organisms like the budding yeast Saccharomyces cerevisiae, easy to manipulate genetically, are useful in order to study nucleolar and rDNA structure and their relationship with stress. In this review, we summarize the most important findings related to this topic.

A membraneless compartment inside the nucleus of eukaryotic cells was observed for the first time by Fontana in 1781 "corps oviforme", and then described by Valentin in 1836 [1] . These early microscopists set up the foundations to study the most exciting organelle inside the nucleus: the nucleolus. Later on, Barbara McClintock first proposed, in Zea mays, that nucleoli assemble around the Nucleolar Organizer Regions (NORs), which happened to be the coding sequences for the ribosomal RNAs (rRNA) [2] . These special rRNA genes are in multiple copies per genome since rRNAs are required in large amounts and they cannot be amplified like proteins during translation. Thus, cells rely on having multiple rRNA gene copies that are transcribed very actively to fulfil cell demands. These copies are often grouped in tandem arrays traditionally referred to as the ribosomal DNA array (rDNA). In addition to the rDNA, the nucleolus comprises pre-ribosomal particles and pre-rRNA with assembly factors that will progressively mature in the nucleolus, then in the nucleoplasm and finally into the cytoplasm [3] . For many decades, the nucleolus was considered a mere rRNA and 3. The Structure of the Nucleolus and the rDNA in the Yeast Saccharomyces cerevisiae Nucleolar structure can be dissected in simpler model organisms, like the budding yeast Saccharomyces cerevisiae. An extensive knowledge about nucleolar biology does exist in this organism, ranging from rDNA structure and stability to rRNA transcription, processing and ribosome assembly. Detailed reviews about these aspects of the nucleolar and rDNA biology can be found elsewhere and are out of the scope of this review [36] [37] [38] . However, we will briefly summarize several facets about the nucleolar structure to consider for the understanding of how stress influences yeast nucleolar biology. In budding yeast, the nucleolus is a crescent-shaped structure that occupies roughly one-third of the nuclear volume. The rDNA is enclosed inside it, abutting the nuclear envelope. The rDNA is located on the right arm of chromosome XII, the largest chromosome in this yeast species. The basic 9.1 Kb unit of the rDNA is repeated in a tandem head-to-tail continuous array between 100 and 200 times per locus, half of them being transcriptionally active [39] . Besides the transcription units, two non-transcribed spacers (NTS1 and NTS2), also known as intergenic spacers (IGS1 and IGS2), lie between the 5S transcription unit and the 35S. The 35S is transcribed by the RNA polymerase I (RNApol I) and forms the precursor rRNA for the 25S, 5.8S and 18S rRNAs, while the 5S is transcribed by RNA polymerase III (RNApol III). A bidirectional criptic non-coding promoter (E-pro) in the IGS1 region is transcribed by the RNA polymerase II (RNApol II) and is subject to silencing by the sirtuin Sir2, an NAD + -dependent histone deacetylase. Two other important elements are an origin of replication (ARS) in the IGS2 region and a replication fork barrier (RFB) in the IGS1 region. The protein Fob1 at the RFB avoids collision between the transcription and the replication machineries ( Figure 1 ).

Fob1 is also necessary to induce double strand breaks (DSB) and homologous recombination (HR) at this site, a mechanism used for expansion and contraction of the rDNA [40] . Sir2 regulates the recombination between different copies of the rDNA [41], tuning a sister chromatid equal/unequal HR pathway that depends on the E-pro transcriptional status [42] . An alternative non-HR pathway is also involved in the amplification of the rDNA array [43] . Different pathway choice of amplification was proven to be modulated by nutrient availability, and this, in turn, requires TOR signalling over multiple histone deacetylases (HDACs) of the sirtuin family [44] . Very recently, a model has been proposed whereby Sir2 is regulated by the amount of upstream activator factors (UAF), thus affecting the rDNA recombination outcome (amplification or maintenance). This model represents a mechanism to count and adjust the rDNA copy number [45] . One important feature of old yeast cells (and of sgs1 and sir2 mutants), is the formation of extrachromosomal rDNA circles (ERCs); these can cause aging, presumably by their accumulation leading to nucleolar enlargement and fragmentation [46] . The rDNA is subject to perinuclear membrane attachment through the inner nuclear membrane (INM) chromosome linkage INM proteins (CLIP) and mitotic monopolin complex (Cohibin) [47] . CLIP (Heh1 and Nur1 in yeast) and Cohibin (Csm1 and Lrs4) are also involved in rDNA silencing and stability through tethering of the rDNA [48] . The rDNA is tightly associated to this perinuclear membrane [49] in order to keep it aside from the HR machinery [50] ; the rDNA is the most unstable region in the genome due to its repetitive nature and high recombination rate [51] . Interestingly, the nuclear envelope adjacent to the nucleolus was shown to have different properties and abilities during membrane expansion [52] . Separation of the nucleolus from the rest of the genome is thought to emerge through differential physical properties [53, 54] , resulting in different aggregation and phase separation, either as a polymer or as a liquid phase [55, 56] . Although not entirely proven, rDNA size, nuclear envelope metabolism and liquid phase properties of the nucleolus contribute altogether to its actual shape and morphology. In addition, rDNA condensation seems to play a central role in quickly reshaping the nucleolus within a cell cycle, as we describe in the next chapter. Figure 1 . Schematic representation of the ribosomal DNA array; Top lef: A yeast cell with the rDNA portrayed as a two coiled chains (black) inside the nucleolus (Nu) (green), which occupies the upper part of the nucleus (light purple) in this drawing. Top middle: The rDNA (green) is located on the right arm (here left) of chormosome XII. Middle: Representation of the basic 9.1 Kb unit, repeated 100-200 times in tandem. The 35S transcription unit (transcribed by the RNApol I) is depicted (18S, 5.8S and 25S). These are separated by internal transcribed spacers (ITS1 and ITS2) (not shown), besides external transcribed spacers which lie at the 18S and 25S ends (not shown). The 35S and the 5S are separated by two intergenic regions (IGS1 and IGS2). Bottom middle: Specific features in the IGS1 and IGS2 regions. IGS1: E-pro, cryptic bidirectional promoter (RNApol II), silenced by Sir2; RFB, replication fork block. Binding of Fob1 at RFB, creates a unidirectional barrier for oncoming replication to avoid collision with ongoing transcription from the 35S. Direction of arrows represents direction of transcription; IGS2: ARS, origin of rDNA replication.

During a single cell cycle, the copy number of the rDNA array is thought to change little. Nevertheless, its morphology under the microscope goes through astonishing changes. Pioneering works using fluorescence in situ hybridization (FISH) proved that the rDNA in G1 is organized in locally-constrained disseminated clusters i.e., the rDNA units are stained by the FISH probe as scattered foci within the nucleolar space [57] . This structure was referred to as puff and likely represents clusters of condensed rDNA units connected by strings formed by other less-condensed units. When cells reach G2/M, the rDNA is either clustered in a single focus or is forming an arc at the nuclear periphery [58] . Protracted G2/M arrest make the rDNA to protrude out of the nuclear mass and adopt the most spectacular structural reorganization observed for any yeast chromosome region: the rDNA loop [57, 59, 60] . This loop is not only seen by FISH but with other in vivo techniques that used GFP chimeras for proteins that tightly bind to the rDNA units; e.g., Fob1 and Net1 [61] , or LacI-GFP bound to lacOs inserted all along the rDNA [62] . Much more recently, similar observations on the rDNA structure have also been obtained by a modified FISH-DAPI technique [63] and through the novel CRISPR/dCas9-GFP imaging technology [64] . The rDNA loop has been extensively studied as a model for the chromosome condensation process that occurs in metazoan prophase, despite the controversy about whether yeast chromosomes actually condense as their higher eukaryotes counterparts. Regardless, the amenability of yeast genetics was determinant to test how the structural maintenance of chromosome (SMC) complexes shape mitotic chromosomes [58, 59, [65] [66] [67] [68] [69] . In these works, it was firmly established that both condensin and cohesin SMC complexes were essential for the transition from puff to loop. Another common partner with SMC complexes and key player in the condensation of metazoan chromosomes, topoisomerase II (Top2), proved to be dispensable [59] . Not only SMC complexes and Top2 have been assessed for their role in the rDNA structure, several cell cycle regulators play critical roles in rDNA reshaping. This is especially important after anaphase onset, where the loop is lost but rDNA condenses back into short lines or clusters [58, 61] . Among cell cycle regulators that control rDNA morphology at this stage we find the Polo Like Kinase 1 ortholog Cdc5, Aurora Kinase B ortholog Ipl1 and the master cell cycle phosphatase Cdc14 [58, 61, [70] [71] [72] .

The fine structure of the rDNA was established by electron microscopy (Miller spread technique) [73] [74] [75] , and the nucleolar ultrastructure localization, assembly and organization by mutant analysis [76, 77] . The arrangement, in the case for the Miller spread, was similar to that of a 'Christmas tree', the stem or trunk formed by the rDNA, while ongoing transcription (rRNA) forming the branches, with knobs (representing Christmas baubles) being the rRNA processing machinery (SSU processome). Non-transcribed units appeared as 'naked' rDNA without branches [78, 79] . Moreover, transcription of the rDNA by RNApol I and rRNA processing and modification take place temporally and spatially in a concomitant manner [80] . Three striking characteristics of the nucleolus highlight its central role in cell growth and metabolism, as well as the enormous resources it consumes. Firstly, RNA polymerase I (RNApol I) transcription activity (60 nt/sec) accounts for 60% of the total transcription in growing yeast, and 50% of total RNA polymerase II (RNApol II) activity is devoted to ribosomal proteins (RPs) genes. Secondly, the high rate of ribosome production (approximately 2000 per min) accounts for 50% of the total protein mass in the cell. Thirdly, most of the splicing resources (90%) are invested in messenger RNA (mRNA) coding for RPs [81, 82] . This clearly indicates that transcription of this locus and ribosome biogenesis must be tightly coupled and that nutrient status and availability, as well as environmental conditions (internal and external), have a profound effect on the nucleolus [44, [83] [84] [85] . In fact, ribosome biogenesis and cell size are related through Sfp1 transcriptional regulation [86] [87] [88] , as well as to the S6 kinase (Sch9 in budding yeast and Sck2 in fission yeast) [89] ; both regulated, in turn, by the nutrient sensing TORC1 complex [90] [91] [92] . Moreover, cell growth and cell division are coupled through ribosome content, translation initiation rate and the G1 cyclin CLN3 expression [93] .

The Target of Rapamycin (TOR), master regulator of the cell, coordinates metabolism, cell growth and proliferation. TOR is composed by the TOR complex 1 (TORC1) and TOR complex 2 (TORC2) in budding yeast, mTORC1 and mTORC2 in humans. The TORC1 complex is rapamycin sensitive and mainly involved in cell growth by promoting ribosome biogenesis, translation initiation, metabolism and cell cycle progression, while inhibiting, on the other hand, stress responses and autophagy [94, 95] . TORC2 complex is rapamycin insensitive and is involved in actin polarization, cell wall integrity, endocytosis and sphingolipid biosynthesis [96, 97] . Two TOR genes are found in S. cerevisiae (TOR1 and TOR2) as opposed to only one TOR gene in humans [98] . Tor1 only binds to TORC1, while Tor2 (present also in TORC2) binds very weakly and transiently to TORC1 in tor1∆ cells and cannot compensate for the absence of Tor1 in this complex [99] [100] [101] .The TORC1 complex controls all three RNA polymerases [102, 103] . In particular transcription of the 35S rDNA by RNApol I, transcription of RP and Ribosomal biogenesis genes (Ribi) by RNApol II [104, 105] , and transcription of the 5S rDNA and transfer RNA (tRNA) genes by RNApol III [106] ; all needed in order to build ribosomes and synthesize proteins [107] [108] [109] . Remarkably, Tor1 binds to the 35S promoter of rDNA in a rapamycin and nutrient starvation manner [110] , although the exact mechanism whereby Tor1 controls the rDNA transcription is still not clear [111] [112] [113] , nor whether a nuclear resident complete TORC1 pool exists [102, 114] . In addition to 35S promoter binding, Tor1 binds to the 5S promoter, upregulating transcription by the RNApol III [115] , which in turn limits lifespan [116] . Besides, TORC1 controls translation initiation and protein synthesis through phosphorylation of Sch9 (S6 Kinase in humans), and through the stability of eIF-4G and phosphorylation of eIF-4EBP1, both of them eukaryotic initiation factors [91, [117] [118] [119] [120] [121] . In other terms, protein translation accuracy has been linked to proteostasis and lifespan modulation in C. elegans [122] . Likewise, translation inhibition and decreased protein metabolism is linked to lifespan extension and improved proteostasis in this nematode, as well as in mammalian cells [123] [124] [125] . Both lifespan extension and improved proteostasis are influenced by dietary restriction and TOR signalling [126] [127] [128] , establishing TORC1 as a sensor of proteostasis [129] [130] [131] [132] . Certainly, the rDNA/nucleolar structure is influenced by inhibitors of the TORC1 complex [133] [134] [135] . A dramatic shrinkage or compaction of the rDNA, both in cycling and mitotic arrested cells, takes place upon exposure to different stressor such as rapamycin, nitrogen starvation, glucose starvation, calorie restriction, heat stress (HS), oxidative stress and UV radiation, as well as by genetic manipulation of the TORC1 function [63, 64, 133, 135, 136] (Figure 2 ). Former stressors, with the exception of UV, are well-known inhibitors of the TORC1 complex [91, 137, 138] , with an outcome (for some of them) of cease in new ribosome production [139] . Nonetheless, depletion of 60S ribosomes subunits leads to yeast life span extension [140] . Although there is no agreement on the terminology used for this phenomenon (condensation, hypercondensation, compaction, contraction or clustering), all these stresses certainly cause a shrinkage and dramatic reorganization of the nucleolus into a smaller and more compact structure; a small nucleoli phenotype. A model summarising the above interpretations is presented (Figure 3 ). One last interesting observation refers to the involvement of the INM proteins (CLIP), monopolin (Cohibin) and the nuclear envelope in rDNA and nucleolar repositioning under TORC1 inhibition. It was found that part of the nucleolar proteins became separated from the rDNA, and subjected to autophagy, while the rDNA was preserved from this [141] . Whether or not this is related to nucleophagy induced by caloric restriction is a question to explore in the future [142] . , which acquires an enlarged nucleolar phenotype (green); as a result, ribosome biogenesis increases, depicted on the right as ribosomes (grey) with ongoing translation (green ribbons). This impinges on the rate and accuracy of translation, increasing the former while decreasing the latter, thus compromising proteostasis. This will lead in turn to an 'unprotected stress' mode and to aging (dashed line with arrow on the right). Other intrinsic mechanisms dowstream of TORC1 (e.g., mitochondrial dysfunction, autophagy inhibition, etc.) can additionally lead to aging (dashed line on the left). (B) Calorie restriction, rapamycin and stress inhibit TOR, leading to rDNA transcription inhibition and to condensation of the nucleolus (small nucleolar phenotype); reducing ribosome biogenesis, while inhibiting translation rate and/or increasing accuracy of translation, with improved proteostasis. This will lead in turn to a 'protected stress' mode and to lifespan extension and longevity (dashed line on the right). Other intrinsic mechanisms can additionally lead to lifespan extension (dashed line on the left). Nevertheless, excess of stress (ROS: Reactive Oxygen Species, UV: Ultra Violet light, etc) can lead to aging (dashed line pointing towards panel A).

The TOR pathway is involved in the rDNA chromatin structure in different ways; epigenetic regulation and condensin activity appear as potential mechanisms. For example, one mechanism might operate through the regulation of the localization of RNA pol I and the Rpd3 histone deacetylase to rDNA [133] , which leads to histone H4 deacetylation and, in turn, to condensin loading onto the array [143] . Upon nutrient availability, TOR inhibits Rpd3 [144] , but this is relieved under starvation or rapamycin treatment, leading to condensin accumulation at the array. In this scenario, condensin acts on the stability of the rDNA [145, 146] ; hence, condensin-mediated nucleolar shrinkage could prevent genetic instability associated with repetitive sequences. On the other hand, Hmo1, a High Mobility Group protein (HMG) involved in the specialized chromatin state at the rDNA [147, 148] , is regulated by TORC1 [105, 149, 150] ; and Hmo1 was required alongside with condensin to mediate both a starvation-induced transcriptional position effect within the rDNA and nucleolar contraction [151] . Mitotic rDNA condensation is also regulated by the Jhd2 demethylase, which acts on histone H3, the maintenance of Csm1/Lrs4 (Cohibin) and condensin association with the rDNA [152] . How is condensin activated outside anaphase under stress conditions? It was shown that the condensation (prior to anaphase) of this locus under stress conditions was independent from Cdc14 activation [135, 153] , the phosphatase required for rRNA transcription inhibition and condensin loading onto the rDNA during anaphase [154] [155] [156] . One possible explanation is through the atypical Rio1 kinase, involved in both ribosome biogenesis and nutrient sensing parallel to TORC1 [157, 158] . Noteworthy, segregation of the rDNA during anaphase is also supported by Rio1, leading to downregulation of RNApol I, stimulation of rRNA processing, rDNA condensation and Sir2 recruitment to the rDNA [159] . Moreover, it was suggested that condensation could occur differentially between active and inactive rDNA repeats by partial downregulation of RNA pol I [160] . Possible mechanisms include condensin overloading, post-translation modifications of condensin and/or epigenetic mechanisms. Regarding the latter, histone modifications (e.g., acetylation), sirtuins and other epigenetic regulators are possible candidates [161] . The sirtuin proteins Hst3 and Hst4, have been shown to be modulated by the TORC1 complex, affecting the acetylation status of histones H3 and H4, independently of the nicotinamidase PNC1 gene expression [162] . It is important to stress that sirtuins are regulated by NAD + levels, which depend on two pathways: de novo pathway (from tryptophan) and the NAD + salvage pathway, the latter governed through the Pnc1 nicotinamidase [163, 164] . Moreover, other members of the sirtuin family such as Sir2 and Hst2 are part of the same longevity pathway involving TOR and calorie restriction. This pathway operates through the upregulation of the PNC1 gene, leading to higher NAD + levels, and hence, increasing sirtuin activity and promoting longevity through the stabilization of the rDNA [164] . In this respect, it was shown that TORC1 inhibition leads to histone deacetylation, and Sir2 enhanced association to the rDNA, through Pnc1 and Net1 [134] . In addition, silencing of the rDNA array by Sir2 is regulated through condensin action, and rapamycin treatment increases condensin binding to the rDNA [165, 166] . Axial contraction and short-range chromatin compaction were shown to rely partially on the deacetylase Hst2 (possibly acting on condensin) in order to promote chromosome condensation in anaphase by removal of acetyl groups from histone H4 [167, 168] . Thus, it is indeed feasible that sirtuins may directly control rDNA condensation. Post-translational modifications of histones are considered to promote chromatin compaction [169] . Remarkably, H4K16 deacetylation is important for chromatin compaction by promoting H2A and H4 interaction [170, 171] , and this epigenetic mark at subtelomeric regions also regulates yeast lifespan [172] . In addition, modifications mediated by acetyl transferases like Nat4, and the loss of histone H4 acetylation, were linked to calorie restriction-mediated longevity and to rDNA silencing [173, 174] . Besides, chromatin remodelers like INO80, histone deacetylases like Rpd3 (mentioned above), and histone chaperones like the FACT complex are needed in order to modulate TORC1 signalling onto chromatin and to facilitate ribosomal DNA nucleosome assembly and transcription [175] [176] [177] . As an example, Histone H3 acetylation at lysine 56 is regulated by TORC1, enabling rDNA transcription and nascent rRNA processing [178] . Finally, in the case of the FACT complex, a small nucleolar phenotype (conserved through evolution) was found in mutants for the Spt16 and Pob3 subunits of this complex [179] . Whether all these modifications have an impact on the nucleolar structure is a question for future research. What is clear is that the rDNA is subjected to epigenetic silencing [180] [181] [182] [183] . Sir2-dependent and independent (as well for other members of the sirtuin family: Hst2, Hst3, Hst4) life span extension mechanisms are a topic of hot debate [164, [184] [185] [186] [187] [188] [189] [190] [191] [192] . Even though sirtuins appear as conserved regulators of aging/longevity [193] , their inclusion in a TOR mediated nucleolar compaction and lifespan extension model needs further clarification [194, 195] .

As in Saccharomyces cerevisiae, different nucleolar rearrangements do occur in higher eukaryotes during stress, the cell cycle or as a result of cell aging. These morphological changes take place despite obvious differences in nuclear and nucleolar physiology; namely, a closed mitosis without nucleolar disassembly in the case of the budding yeast versus an open mitosis with transient nucleolar disassembly in higher eukaryotes [10] . Another major difference lays on the different architectural complexity of the nucleolus. In higher eukaryotes a define set of compartments can be easily distinguished under the microscope: the fibrillary center (FC), the dense fibrillary component (DFC) and the granular component (GC), i.e., a tripartite compartmentalization. By contrast, in the budding yeast only a bipartite compartmentalization, comprising fibrillar and granular components, is present. The main differences are summarised in Table 1 . Each compartment undertakes a specific task; traditionally, FC is where rDNA transcription occurs, DFC where rRNA processing takes place and GC is dedicated to late rRNA processing and pre-ribosomal particles assembly [11] . Thus, the alteration and reorganization of these spatial structures are indicative of alteration in nucleolar function [196] . Remarkably, nucleolar morphology, visualised by NORs silver staining (AgNORs), is correlated to tumour DNA content and ploidy; aneuploid tumours have higher AgNORs counts [197, 198] . This could be due to amplification of the five human acrocentric chromosomes (13, 14, 15, 21 and 22) , where the NORs are located, as proposed in [23] . On the other hand, rDNA copy number variation, increasing the copies of 5S genes while decreasing the 45S copies, have been linked to nucleolar activity, proliferation and inactivation of p53 [199] . All these rDNA changes may operate as a tumour adaptive strategy to promote genome instability.

Nucleolar stress presents here four main features, a decrease in the size and volume of the nucleolus, redistribution and/or fragmentation of the nucleolus, inhibition of rRNA production by RNApol I, and relocalization of nucleolar proteins into the nucleoplasm [200, 201] . Different stresses that impact on the nucleolar structure can have different outcomes. On the one hand, there are stresses that produce nucleolar fragmentation/disintegration; whereas, on the other hand, there are other stresses that alter nucleolar size but not its integrity [7, 8] . Among the former, we find classic antineoplastic drugs like mitomycin C, cisplatin and oxaliplatin, mitoxantrone, doxorubicin, camptothecin (topotecan and irinotecan) and actinomycin D; all but the latter being compounds that generate DNA damage. Specifically, nucleolar aggregates or caps are formed upon DNA damage and actinomycin D treatment [202, 203] . In this respect, it has been suggested a specific DNA damage response (DDR) for the nucleolus (n-DDR), whereby distinct repair features promote the integrity of the rDNA [204] . By contrast, inhibitors of the late rRNA processing such as 5-fluorouracil and homoharringtonine, and TOR inhibitors like rapamycin and its derivatives (temsirolimus and everolimus), belong to the second category of stressors [205, 206] . New compounds such as CX-5461 and CX-3543, selective inhibitors of rDNA transcription by the RNA pol I, show nucleolar disintegration and nucleolin redistribution, respectively [207] [208] [209] . Whereas stressors that fragment the nucleolus could be genotoxic, those that suppress rDNA transcription, rRNA processing and ribosome production could be safer antiproliferative therapies. Other potential stress treatments with such profile, alone or in combination with current antitumor chemotherapy and radiotherapy, could be hypethermia and calorie restriction [210] [211] [212] [213] . Finally, a compound with antimetastatic potential described recently, called metaarrestin, acts through the inhibition of transcription by RNA pol I, reducing the nucleolar volume [214] . Interestingly, stresses with a biological origin, like viral infections, also lead to nucleolar alteration (e.g., enlarged FC) [215] [216] [217] . Another interesting phenotype, the formation of nucleolar aggresomes, is related to the wrong nucleolar turnover of p53 in aging and progeria [218] , as well as to proteotoxic stress, serving the nucleolus as a hub for misfolded proteins storage and proteostasis control. This has been recently reviewed in the context of liquid-liquid phase separation and liquid-solid phase transition of the nucleolus and their role in cancer and neurodegenerative diseases [219] . Finally, we will briefly discuss p53 function in the nucleolus, even though other reviews in this special issue are covering different aspects of it. p53 is a protein regulated at different levels, and its activation upon nucleolar stress depends on the p53-Mdm2 axis. The binding of p53-Mdm2 renders p53 inactive under non-stressed conditions. This happens through the ubiquitin ligase activity of Mdm2 and subsequent p53 degradation by the proteasome. We must mention that 60% of tumours have mutant TP53 [220] , yet, regardless of this p53 mutant status, there are several p53 isoforms that have an impact on p53 transcriptional activity and on tumour progression [221] [222] [223] [224] . It would be valuable to see whether there is a connection between these isoforms and the p53-Mdm2 axis. In response to stressful conditions, several ribosomal proteins are released from the nucleolus into the nucleoplasm: RPL11, RPL23, RPL5 and RPL7. There, they bind to Mdm2, which inhibits the destruction of p53. Moreover, another RP protein, RPL26, binds to the 5 -UTR of p53 mRNA, enhancing its transcription under DNA damage [225] . This nucleolar stress mechanism indicates the cell the synthesis and ensemble status for rRNAs and RPs, establishing a quality control surveillance mechanism [226] . One of the proteins involved in this sensing mechanism is PICT1/GLTSCR2, the homologue of the yeast ribosome biogenesis factor Nop53. PICT1 is bound to RLP11, avoiding its release into the nucleoplasm and hence the binding to Mdm2. This makes Mdm2 available for p53 binding as mentioned above [227] . Besides this, PICT1 also stabilises the tumour suppressor PTEN [228] . When PICT1 is absent from the nucleolus (Pict1 -/or low levels of PICT1), RPL11 is released to inhibit Mdm2. Although PICT1 may function differentially, as a tumour suppressor or as an oncogene, depending on the environment and conditions, low levels of PICT1 have been found on ccRCC with an inverse correlation to the Fuhrman grade system, which classifies tumours based on nuclear/nucleolar abnormalities [229] . In breast cancer tumours, low levels of PICT1 are associated to tumour progression [230] , while cytoplasmic expression of this protein are related to a bad prognostic for non-small cell lung cancer [231] . Finally, PICT1 suppression under hypoxic conditions in glioblastoma tumour cells augments the survival and invasiveness of the tumour [232] . It is possible that tumours cells, subjected to endogenous or exogenous stress, could modulate PICT1 levels as a strategy to impinge on p53 function and/or promote their growth by ribosome biogenesis, depending on the needs. This enhances this nucleolar axis as a putative target in antitumour therapy.

Life-span extension can be achieved by calorie restriction (glucose, protein, amino acids deprivation), or by pharmacological interventions, such as rapamycin and other small molecules (e.g., metformin and resveratrol) [233] [234] [235] . Side effects from acute or long-term treatments could be diminished by intermittent and/or very low doses of rapamycin treatment, fasting-mimicking diets, intermittent fasting, among other regimes [236] [237] [238] . The exact extent of stress (quality, quantity and intensity) and how it affects different cell types, developmental stages and disorders, needs to be evaluated in order to poise the cell and the organism into a protective mode. Although we have focused on a TOR centred view in yeast, other signalling routes, interconnected with TOR itself, like Growth Hormone (GH), Insulin/IGF-1-like, and PI3K-AKT are however important players in more complex higher eukaryotes. Other age-related processes such as mitochondrial dysfunction, also influenced by sirtuins and TORC1, should be taken into account as well [193, 239, 240] . Inhibiting TOR signalling could mediate longevity through both sirtuin and ribosome production (amid others) and its effect on (but not only) rDNA and nucleolar structure and stability. Thus, the nucleolus could be serving as both a genome buffering system and a stress sensor for the cell [241] . In fact, a whole rDNA theory of aging in budding yeast has been proposed by Kobayashi [242] . In addition, a role for the rDNA as an evolutionary conserved clock of aging has been postulated as well [243] . Could a central hub or integrator in the cell, such as TOR, be communicating (through its inhibition) the stress signals into a sensor or buffering system like the rDNA/nucleolus, linking growth and stress and, in that way, enhancing stress responses and protecting the rest of the genome from further damage? This is an interesting hypothesis that remains to be further elucidated. In relation to cancer biology, the use of the nucleolus as a marker in a wide range of tumours continues as a valuable prognostic tool for the pathologist. Meanwhile, the specialized nucleolar nature opens new avenues for potential treatments with less genotoxicity. Finally, one clear conclusion is drawn, the nucleolus is dramatically reduced upon TOR inhibition or caloric restriction, and this is related to longevity. By contrast, an enlarged nucleolus is indicative of aging. This should be used as a predictive hallmark (among others) in aging and longevity research with potential translation into the clinic [244] . 

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The nuclear envelope in genome organization, expression and stability

Structure and function in the budding yeast nucleus

The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus

Ribosomal DNA stability is supported by many 'buffer genes'-introduction to the Yeast rDNA Stability Database

Cohen-Fix, O. Yeast Nuclear Envelope Subdomains with Distinct Abilities to Resist Membrane Expansion

The liquid drop nature of nucleoli

Physical principles and functional consequences of nuclear compartmentalization in budding yeast

Coexisting Liquid Phases Underlie Nucleolar Subcompartments

Enrichment of dynamic chromosomal crosslinks drive phase separation of the nucleolus

Chromosome condesation and sister chromatid pairing in budding yeast

In vivo requirements for rDNA chromosome condensation reveal two cell-cycle-regulated pathways for mitotic chromosome folding

In vivo dissection of the chromosome condensation machinery: Reversibility of condensation distinguishes contributions of condensin and cohesin

Behaviour of nucleolus organizing regions (NORs) and nucleoli during mitotic and meiotic divisions in budding yeast

Spindle-independent condensation-mediated segregation of yeast ribosomal DNA in late anaphase

Visualization of the dynamic behavior of ribosomal RNA gene repeats in living yeast cells

Temperature-dependent regulation of rDNA condensation in Saccharomyces cerevisiae

Live-Cell Imaging of Chromatin Condensation Dynamics by CRISPR

The condensin complex governs chromosome condensation and mitotic transmission of rDNA

Mitotic Chromosome Condensation Requires Brn1p, the Yeast Homologue of Barren

Condensin is required for chromosome arm cohesion during mitosis

Cdc14 phosphatase induces rDNA condensation and resolves cohesin-independent cohesion during budding yeast anaphase

Amon, A. Cdc14 and condensin control the dissolution of cohesin-independent chromosome linkages at repeated DNA

Nucleolar segregation lags behind the rest of the genome and requires Cdc14 phosphatase activation by the FEAR network

Cdc14 and MEN-controlled Cdk inactivation in yeast coordinate rDNA decompaction with late telophase progression

Polo Kinase Regulates Mitotic Chromosome Condensation by Hyperactivation of Condensin DNA Supercoiling Activity

Visualization of nucleolar genes

Morphology of transcription units in Drosophila melanogaster

The terminal balls characteristic of eukaryotic rRNA transcription units in chromatin spreads are rRNA processing complexes

Mutational analysis of the structure and localization of the nucleolus in the yeast Saccharomyces cerevisiae

Assembly and Functional Organization of the Nucleolus: Ultrastructural Analysis of Saccharomyces cerevisiae Mutants

Oldies but goldies: Searching for Christmas trees within the nucleolar architecture

Crosstalk in gene expression: Coupling and co-regulation of rDNA transcription, pre-ribosome assembly and pre-rRNA processing

Yeast Pre-rRNA Processing and Modification Occur Cotranscriptionally

The economics of ribosome biosynthesis in yeast

Economics of ribosome biosynthesis

Transcription Factor UAF, Expansion and Contraction of Ribosomal DNA (rDNA) Repeats, and RNA Polymerase Switch in Transcription of Yeast rDNA

Coordinate control of syntheses of ribosomal ribonucleic acid and ribosomal proteins during nutritional shift-up in Saccharomyces cerevisiae

Hierarchy of elements regulating synthesis of ribosomal proteins in Saccharomyces cerevisiae

Systematic identification of pathways that couple cell growth and division in yeast

A dynamic transcriptional network communicates growth potential to ribosome synthesis and critical cell size

Sfp1 regulates transcriptional networks driving cell growth and division through multiple promoter-binding modes

Genome-wide screen for cell growth regulators in fission yeast

Sfp1 is a stressand nutrient-sensitive regulator of ribosomal protein gene expression

Sch9 Is a Major Target of TORC1 in Saccharomyces cerevisiae

Sfp1 Interaction with TORC1 and Mrs6 Reveals Feedback Regulation on TOR Signaling

Coupling of cell division to cell growth by translational control of the G1 cyclin CLN3 in yeast

TOR regulates ribosomal protein gene expression via PKA and the Forkhead Transcription Factor FHL1

Target of rapamycin (TOR) in nutrient signaling and growth control

Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive

Target of rapamycin complex 2 regulates actin polarization and endocytosis via multiple pathways

Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control

TOR Complex 1 Includes a Novel Component, Tco89p (YPL180w), and Cooperates with Ssd1p to Maintain Cellular Integrity in Saccharomyces cerevisiae

Efficient Tor signaling requires a functional class C Vps protein complex in Saccharomyces cerevisiae

Regulation of ribosome biogenesis: Where is TOR?

Ribosome biogenesis and cell growth: mTOR coordinates transcription by all three classes of nuclear RNA polymerases

Regulation of Ribosome Biogenesis by the Rapamycin-sensitive TOR-signaling Pathway in Saccharomyces cerevisiae

Hmo1 Is Required for TOR-Dependent Regulation of Ribosomal Protein Gene Transcription

Sch9 partially mediates TORC1 signaling to control ribosomal RNA synthesis

The transcriptional activity of RNA polymerase I is a key determinant for the level of all ribosome components

Is ribosome synthesis controlled by Pol I transcription?

Growth control and ribosome biogenesis

Nutrient regulates Tor1 nuclear localization and association with rDNA promoter

Tor Pathway Regulates Rrn3p-dependent Recruitment of Yeast RNA Polymerase I to the Promoter but Does Not Participate in Alteration of the Number of Active Genes

TOR-dependent reduction in the expression level of Rrn3p lowers the activity of the yeast RNA Pol I machinery, but does not account for the strong inhibition of rRNA production

Molecular basis of Rrn3-regulated RNA polymerase I initiation and cell growth

Where is mTOR and what is it doing there?

Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1

RNA polymerase III limits longevity downstream of TORC1

TOR controls translation initiation and early G1 progression in yeast

Regulation of eIF-4E BP1 phosphorylation by mTOR

Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism

The TOR (target of rapamycin) signal transduction pathway regulates the stability of translation initiation factor eIF4G in the yeast Saccharomyces cerevisiae

A unifying model for mTORC1-mediated regulation of mRNA translation

Regulation of the Elongation Phase of Protein Synthesis Enhances Translation Accuracy and Modulates Lifespan

Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans

Insulin/IGF-1-mediated longevity is marked by reduced protein metabolism

Less is more: Improving proteostasis by translation slow down

mTOR signaling in protein homeostasis: Less is more?

Nutrient signaling in protein homeostasis: An increase in quantity at the expense of quality

Proteostasis and aging

Target of rapamycin (TOR): Balancing the opposing forces of protein synthesis and degradation

mTORC1 links protein quality and quantity control by sensing chaperone availability

TORC1-mediated sensing of chaperone activity alters glucose metabolism and extends lifespan

Spatially Distinct Pools of TORC1 Balance Protein Homeostasis

Chromatin-mediated regulation of nucleolar structure and RNA Pol I localization by TOR

Rapamycin increases rDNA stability by enhancing association of Sir2 with rDNA in Saccharomyces cerevisiae

The ribosomal DNA metaphase loop of Saccharomyces cerevisiae gets condensed upon heat stress in a Cdc14-independent TORC1-dependent manner

Ultrastructural changes in yeast following heat shock and recovery

Transient Sequestration of TORC1 into Stress Granules during Heat Stress

Tor1 and CK2 kinases control a switch between alternative ribosome biogenesis pathways in a growth-dependent manner

Yeast Life Span Extension by Depletion of 60S Ribosomal Subunits Is Mediated by Gcn4

CLIP and cohibin separate rDNA from nucleolar proteins destined for degradation by nucleophagy

Nucleophagy: From homeostasis to disease

Nutrient starvation promotes condensin loading to maintain rDNA stability

The Tor Pathway Regulates Gene Expression by Linking Nutrient Sensing to Histone Acetylation

Compacting DNA During the Interphase

Opposing role of condensin and radiation-sensitive gene RAD52 in ribosomal DNA stability regulation

Hmo1, an HMG-box protein, belongs to the yeast ribosomal DNA transcription system

Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules

Histone H3 and TORC1 prevent organelle dysfunction and cell death by promoting nuclear retention of HMGB proteins

Expression of yeast high mobility group protein HMO1 is regulated by TOR signaling

Condensin and Hmo1 Mediate a Starvation-Induced Transcriptional Position Effect within the Ribosomal DNA Array

Yeast histone H3 lysine 4 demethylase Jhd2 regulates mitotic ribosomal DNA condensation

Late rDNA Condensation Ensures Timely Cdc14 Release and Coordination of Mitotic Exit Signaling with Nucleolar Segregation

Cdc14p/FEAR Pathway Controls Segregation of Nucleolus in S. cerevisiae by Facilitating Condensin Targeting to rDNA Chromatin in Anaphase

Transcription of ribosomal genes can cause nondisjunction

Cdc14 inhibits transcription by RNA polymerase I during anaphase

Rio1 mediates ATP-dependent final maturation of 40S ribosomal subunits

Integrating Rio1 activities discloses its nutrient-activated network in Saccharomyces cerevisiae

Rio1 promotes rDNA stability and downregulates RNA polymerase I to ensure rDNA segregation

Compositional reorganization of the nucleolus in budding yeast mitosis

Transcriptional and Epigenetic Regulation by the Mechanistic Target of Rapamycin Complex 1 Pathway

Saccharomyces cerevisiae TORC1 controls histone acetylation by signaling through the sit4/PP6 phosphatase to regulate sirtuin deacetylase nuclear accumulation

Manipulation of a nuclear NAD + salvage pathway delays aging without altering steady-state NAD + levels

MSN2 and MSN4 link calorie restriction and TOR to sirtuin-mediated lifespan extension in Saccharomyces cerevisiae

Condensin Regulates rDNA Silencing by Modulating Nucleolar Sir2p

Condensin function in mitotic nucleolar segregation is regulated by rDNA transcription

A Cascade of Histone Modifications Induces Chromatin Condensation in Mitosis

Axial contraction and short-range compaction of chromatin synergistically promote mitotic chromosome condensation

Mitotic post-translational modifications of histones promote chromatin compaction in vitro

Role of direct interactions between the histone H4 tail and the H2A core in long range nucleosome contacts

Histone Acetylation Regulates Chromatin Accessibility: Role of H4K16 in Inter-nucleosome Interaction

Histone H4 lysine 16 acetylation regulates cellular lifespan

Loss of Nat4 and its associated histone H4 N-terminal acetylation mediates calorie restriction-induced longevity

N-alpha-terminal Acetylation of Histone H4 Regulates Arginine Methylation and Ribosomal DNA Silencing

FACT facilitates chromatin transcription by RNA polymerases I and III

Rpd3-and Spt16-Mediated Nucleosome Assembly and Transcriptional Regulation on

The INO80 chromatin remodeler sustains metabolic stability by promoting TOR signaling and regulating histone acetylation

The histone H3 lysine 56 acetylation pathway is regulated by target of rapamycin (TOR) signaling and functions directly in ribosomal RNA biogenesis

Conserved regulators of nucleolar size revealed by global phenotypic analyses

The Epigenetic Pathways to

The nuts and bolts of transcriptionally silent chromatin in Saccharomyces cerevisiae

Histone H3 N-terminal acetylation sites especially K14 are important for rDNA silencing and aging

Histone Modifications as an Intersection Between Diet and Longevity

Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae

Sir2-independent life span extension by calorie restriction in yeast

Sir2 blocks extreme life-span extension

Cell biology: HST2 mediates SIR2-independent life-span extension by calorie restriction

Twists in the tale of the aging yeast

Sirtuin-independent effects of nicotinamide on lifespan extension from calorie restriction in yeast

Calorie restriction extends the chronological lifespan of Saccharomyces cerevisiae independently of the Sirtuins

Sir2 and calorie restriction in yeast: A skeptical perspective

Dietary restriction: Standing up for sirtuins-Response

It takes two to tango: NAD+ and sirtuins in aging/longevity control

Calorie restriction reduces rDNA recombination independently of rDNA silencing

The implication of Sir2 in replicative aging and senescence in Saccharomyces cerevisiae

Ribosomal DNA and the nucleolus in the context of genome organization

DNA ploidy and p53 expression correlate with survival and cell proliferative activity in male breast carcinoma

Prognostic significance of DNA cytometry in combination with AgNOR investigation

Ribosomal DNA copy number amplification and loss in human cancers is linked to tumor genetic context, nucleolus activity, and proliferation

Cellular stress and nucleolar function

Nucleolar stress: Is there a reverse version

Dynamic Sorting of Nuclear Components into Distinct Nucleolar Caps during Transcriptional Inhibition

Nucleolar responses to DNA double-strand breaks

Double-strand breaks in ribosomal RNA genes activate a distinct signaling and chromatin response to facilitate nucleolar restructuring and repair

The nucleolus: An emerging target for cancer therapy

Targeting the nucleolus for cancer intervention

Anticancer activity of CX-3543: A direct inhibitor of rRNA biogenesis

Targeting RNA polymerase I with an oral small molecule CX-5461 inhibits ribosomal RNA synthesis and solid tumor growth

Discovery of CX-5461, the first direct and selective inhibitor of RNA polymerase I, for cancer therapeutics

Hyperthermia in combined treatment of cancer

Protein and amino acid restriction, aging and disease: From yeast to humans

Therapeutic hyperthermia: The old, the new, and the upcoming

Signaling Confers Resistance to Targeted Cancer Drugs

Metarrestin, a perinucleolar compartment inhibitor, effectively suppresses metastasis

Changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus

RNA viruses: Hijacking the dynamic nucleolus

The nucleolar interface of RNA viruses

Proteasome inhibitors induce nuclear ribonucleoprotein inclusions that accumulate several key factors of neurodegenerative diseases and cancer

Phase-to-Phase With Nucleoli-Stress Responses, Protein Aggregation and Novel Roles of RNA. Front

Wild-type p53: Tumors can't stand it

p53 isoforms can regulate p53 transcriptional activity

Key Regulators of the Cell Fate Decision. Cold Spring Harb

p53β: A new prognostic marker for patients with clear-cell renal cell carcinoma from 5.3 years of median follow-up

p53 -Dependent and -Independent Nucleolar Stress Responses

Ribosome biogenesis surveillance: Probing the ribosomal protein-Mdm2-p53 pathway

A new PICTure of nucleolar stress

Critical Role of PICT-1, a Tumor Suppressor Candidate, in Phosphatidylinositol 3,4,5-Trisphosphate Signals and Tumorigenic Transformation

The nucleolus, an ally, and an enemy of cancer cells

Downregulation of GLTSCR2 expression is correlated with breast cancer progression

PICT1 expression is a poor prognostic factor in non-small cell lung cancer

GLTSCR2 contributes to the death resistance and invasiveness of hypoxia-selected cancer cells

Extending healthy life span-from yeast to humans

The search for antiaging interventions: From elixirs to fasting regimens

Promoting health and longevity through diet: From model organisms to humans

Aging and immortality: Quasi-programmed senescence and its pharmacologic inhibition

A time to fast

Programmed longevity, youthspan, and juventology

The TORC1-Sch9 pathway as a crucial mediator of chronological lifespan in the yeast Saccharomyces cerevisiae

SOD1 Phosphorylation by mTORC1 Couples Nutrient Sensing and Redox Regulation

A new role of the rDNA and nucleolus in the nucleus-RDNA instability maintains genome integrity

Ribosomal DNA and cellular senescence: New evidence supporting the connection between rDNA and aging

Ribosomal DNA harbors an evolutionarily conserved clock of biological aging

The hallmarks of aging