[Cell Cycle 4:1, 102-105; January 2005]; ©2005 Landes Bioscience
The Nucleolus
Spotlight on Nucleolus
Playing by Different Rules? Peter Shaw John Doonan
Department of Cell and Developmental Biology; John Innes Centre; Colney Norwich, UK Correspondence to: Peeter Shaw/ John Doonan; Department of Cell and Developmental Biology; John Innes Centre; Colney. Norwich NR4 7UH UK; Email:
[email protected]/
[email protected] Received 12/09/04; Accepted 12/13/04
Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=1467
KEY WORDS nucleolus, mitosis, FEAR pathway, nucleolar organization
ABSTRACT The nucleolus, although an integral part of all eukaryotic nuclei, plays by its own rules in many respects. Cytologically, it is the most prominent subnuclear domain; functionally, it is the site of transcription of 3 of the ribosomal RNAs from the tandemly repeated rDNA, and subsequently of ribosome biogenesis; biochemically, it possesses transcriptional and post-transcriptional machinery not shared with the rest of the nucleus. Making the huge number of ribosomes required by the cell represents an enormous investment of metabolic activity, and so nucleolar function can easily become a de facto limit to cell growth: nucleolar activity must also be actively regulated, but the detailed regulatory networks linking the nucleolus with cellular metabolism are still unclear. Several recent reports have now shown that segregation of the rDNA in yeast, along with telomeric DNA, is also controlled differently from the rest of the genome. This short review describes some of the features of the nucleolus and highlights recent progress in understanding this important, but enigmatic, nuclear structure.
WHAT MAKES A NUCLEOLUS? The nucleolus is the specialized sub-nuclear compartment where all known eukaryotes make their ribosomes.1-3 Within the nucleolus, the rDNA—many copies of the gene carrying 3 of the 4 ribosomal RNAs—is transcribed by RNA polymerase I, an enzyme complex that transcribes these and only these genes. In almost all eukaryotes, the rRNA genes are arranged in tandem arrays at one or a few genomic locations. Interestingly, the 4th RNA species in eukaryotic ribosomes, 5S, is transcribed by RNA polymerase III, almost invariably from multiple tandem copies elsewhere in the genome and physically located away from the nucleolus.1 In a very few organisms, including S cerevisiae, the 5S genes are contained within the rDNA tandem repeats along with the other rRNA genes, but are transcribed from the other strand, and again by RNA polIII.1 The reasons for these puzzling differences are completely unknown. What seems to generate the nucleolar structure is transcription of rDNA from multiple tandemly arranged rRNA genes. During mitosis in many organisms the nucleolus breaks down and the rDNA is arranged in a partially condensed form at the Nucleolar Organiser Regions (NORs) of the chromosomes, often visible as secondary constrictions. Some at least of the polI complex proteins and probably other proteins remain attached to the mitotic NORs. In late telophase, polI transcription reinitiates, and other nucleolar components are recruited to the transcription sites in the form of small prenucleolar bodies. This results in a nucleolus forming at the site of each NOR. These smaller nucleoli often then coalesce to a single structure. Transcription of rDNA by polI is, however, not strictly necessary for making ribosomes. A yeast mutant deficient in polI has been rescued by placing the rRNA genes on plasmids under the control of a polII promoter.4 The resulting line, although sick, can survive and make functional ribosomes in the nucleoplasm, rather than within a nucleolus.
NUCLEOLAR STRUCTURE
Is the nucleolar structure seen then simply the structural counterpart of the biochemical pathway of rDNA transcription and ribosome biogenesis?5 The many detailed differences in nucleolar structure seen in different organisms and different cell types within a single organism argue that there must be more than this to nucleolar organization. The attempts to find a common structural interpretation of the nucleoli from diverse cell types and organisms have to some extent taken attention away from the diversity seen in nucleolar organization. 102
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The nucleolus is a prominent region of most eukaryotic nuclei. It shows up as a dark region compared with the rest of the nucleus when stained with DNA dyes, indicating that its DNA content is comparatively low. However, it is also the site of a large proportion of the cell’s gene transcription—in many cells ribosomal RNAs account for more than half the mature transcripts produced; the resolution of this apparent paradox is that actively transcribed DNA is the most decondensed. Ultrastructurally, two distinct regions can be distinguished in most nucleoli by thin section TEM: a fibrous region, which is often more densely stained by heavy metal stains, called the dense fibrillar component (DFC); and a region apparently filled with granules about the size of ribosomes—the granular component (see Fig. 1). In many nucleoli there are also other substructures—for example, lightly staining regions or bodies called fibrillar centres (FCs), other cavities or vacuoles, and regions of condensed chromatin. FCs have been at the centre of a great deal of controversy over many years.6 In mammalian nucleoli, at least, they contain RNA polymerase I and rDNA, and have been often proposed as the site of rDNA transcription, although originally FCs were assumed simply to be the interphase counterpart of the mitotic NORs—inactive rDNA. However the sites of transcription can only be unambiguously determined by localization of nascent rRNA, and in two more recent studies in plants7 and human nucleoli,8 detailed analysis of the location of nascent RNA has pointed clearly to the DFC as containing the active transcription units. Current models thus place transcription and the first stages of transcript processing in the DFC, with later stages of ribosome biogenesis in the GC, suggesting that the granules observed are various intermediate complexes on the route to mature ribosomes. In S. cerevisiae, the nucleolus is a crescent-shaped region of the nucleus, abutting the nuclear envelope (Fig. 1). This is different from most higher eukaryotes such as plants and animals, in which the nucleoli are more or less spherical regions, often appearing completely detached from the nuclear envelope, but possibly always connected by an invagination of the nuclear envelope.
THE COMPLEXITY OF NUCLEOLAR COMPOSITION
Immunological based approaches revealed early on that the overall protein composition of the nucleolus was quite distinct from that of the nucleoplasm, and there are many proteins that are considered as nucleolar-specific, such as nucleolin and fibrillarin. In reality most if not all such nucleolar proteins are also found elsewhere in the nucleus. For example, fibrillarin, now known to be the methyl transferase involved in methylating ribosomal RNAs, is also found in high concentrations in Cajal bodies, where the box C snoRNAs that confer specificity to these 100 or so methylations are also found and probably processed. A similar story applies to the pathway responsible for carrying out pseudouridylation of a similar number of uridine residues in the rRNAs. In this case the enzyme is Dyskerin/cbf5p, and each modification has its own cognate box H/ACA snoRNA. Recently, proteomic analyses of the human9 and plant10 nucleolus have been published. In the most recent human study, nearly 700 proteins have been identified. In the published plant study, some 200 proteins were identified, but recent results have increased this to more than 500 identifications (Mckeown P and Shaw P, unpublished). It can be argued that many of the proteins identified are inevitably contaminants—such a large and poorly defined structure as the nucleolus is likely to be impossible to fully separate from other nuclear and cellular components. Nevertheless Pendle et al.10 backed up the proteomic analysis with systematic expression of a subset of www.landesbioscience.com
A
B
C
Figure 1. Diagrammatic representations of nucleolar ultrastructure as seen by thin section electron microscopy. (A) Human (HeLa) culture cell. (B) Plant cell (Pisum sativum root). (C) Budding yeast (S cerevisiae).
the identified proteins as Green Fluorescent Protein (GFP) fusions, and showed that the vast majority (87%) were nucleolar-located by structural criteria as well. Thus it seems clear that the nucleolus does indeed contain a large number of different proteins, and that many of the proteins in the nucleolus are quite unexpected in the context of ribosome biogenesis. For example, although the expected snoRNA-associated proteins were found, many proteins involved in splicing were also found in both human and plant nucleolar proteomes. Proteins that are thought to be specific to mRNAs were also found. Most strikingly in the plant analysis, Pendle et al10 found at least half of the known components of the post-splicing exon-junction complex (EJC). This complex is thought to couple mRNA splicing, export through the nuclear pores and subsequent cytoplasmic location. It is also involved in the recognition of aberrant transcripts containing premature stop codons, marking such transcripts for nonsense-mediated degradation. All the identified EJC components were shown by GFP fusions to be at least partly localised in the nucleolus, raising the possibility that the nucleolus has a role in mRNA surveillance or export. Similarly, many translation factors were identified in both human and plant nucleolar proteomes. In the past few years, evidence has been presented for mRNA translation occurring in the nucleus.11 Is it possible that such translation occurs as a checking mechanism before mRNA travels to the cytoplasm? Also detected in the proteomic analyses were metabolic enzymes such as glyceraldehyde 3-phosphate dehydogenase (GAPDH). Although at first sight, this would be dismissed as a cytoplasmic contaminant, the GAPDH-GFP fusion was indeed present at high levels in the nucleus and to a lesser extent in the nucleolus. Recently GAPDH has been shown to be an essential component of a transcriptional activator complex regulating mammalian histone H2B expression.12,13 Thus this protein, and other metabolic enzymes, may be involved in coupling nucleolar activity with cellular metabolism. At the level of DNA-interacting proteins, the nucleolus, in plants at least, contains histone variants that appear to be nucleolar specific, and there is a small group of plant nucleolar-specific histone deacetylases. Thus, taken with the transcription of rDNA by a specific RNA polymerase, RNA polI, the genome organization of rDNA, its interacting proteins, and its transcriptional machinery are all distinct from that of the rest of the genome. A second source of nucleolar proteins has been screens where cDNAs have been randomly fused to GFP and the subcellular localization of the fusion proteins determined. This approach has turned up an eclectic mix of proteins localized in the nucleolus, some expected as nucleolar constituents, others not. The former class
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includes many ribosomal proteins. For example, RS11 and S41 were found in the nucleolus of plant cells14 and a NAP57 homolog in fission yeast.15 In pilot screens aimed at systematically localizing proteins in mammalian16 and plant cells,17 2–3% of GFP fusions were highly enriched within the nucleolus. As expected, these include many RNA processing functions (http://gfp-cdna.embl.de/) as well as protein kinases and phosphatases that may regulate various aspects of rRNA metabolism.17 Perhaps more surprising was the finding that certain transcription factor-like proteins were preferentially located in the nucleolus, whereas related family members were found mainly in the nucleoplasm. The serendipitous discovery that many cell cycle regulators reside in the nucleolus, at least for part of the cell cycle, provides another set of functionally important ‘nucleolar’ proteins. They are involved in both the mitotic progression and exit pathways. For example, the BIMG protein phosphatase and the CDC14 phosphatase, involved in metaphase/anaphase and mitotic exit respectively, are both enriched in the nucleolus. Although the nucleolus has been proposed as an “inert” parking space for holding such regulatory proteins, an alternative view is that there is an active protein localisation mechanism that restricts active proteins to where they are required.
NUCLEOLAR DIVISION AND THE FEAR PATHWAY
Nucleolar breakdown and division are closely coordinated with mitosis but do not necessarily occur at the same time. Breakdown and separation of the nucleolus in yeast is a late mitotic event18,19 and the nucleolus persists as a recognizable region until anaphase. In fungi, many of the nucleolar components are retained until late mitosis and are separated by a mechanism that may involve the telophase spindle: for example the BIMG protein phosphatase, a nucleolar resident involved in the metaphase anaphase transition, is retained in the nucleolus throughout mitosis and can be observed streaming from the mother nucleolar mass into the daughters during telophase.20 Two pathways are involved in nucleolar segregation in budding yeast, the FEAR (cdc fourteen early anaphase release) pathway that initiates breakdown and the MEN (mitotic exit) pathway that maintains it. Breakdown is dependent on mitotic progression past the metaphase/anaphase checkpoint and proteins involved in this checkpoint are required for normal breakdown and segregation. In budding yeast meiosis, nucleolar breakdown is dependent on separase, the protease that dissolves the cohesin cross-bridges that hold the chromatin together.21 However, the actual separation of the rDNA, and also the telomeres, is separase-independent but requires cdc14 and condensin.22 Cohesin, normally required to hold sister chromatids together during early mitosis, is not required to hold the regions containing rDNA.22 Cdc14p is a dual specificity protein phosphatase that acts as the effector of the FEAR signaling transduction pathway. Cdc14p is anchored in the nucleolus by the NET1 protein23 and is involved in the inactivation of the mitotic CDK protein kinase to enable mitotic exit. Net1 may anchor other nucleolar proteins including Pol I and it stimulates rRNA synthesis both in vitro and in vivo. Condensin, the protein that reorganizes chromosomes into their highly compact mitotic structure, becomes highly enriched on the rDNA during mitosis.24,25 Cdc14p also targets condensin to rDNA during anaphase, promoting its compaction. Why compaction of the rDNA is temporally separated from that of the rest of the genome is unclear, but may be related to late mitotic retention of the nucleolus. A recent paper by Machin et al.26 shows that resolution of the sister rDNAs is independent of spindle function 104
but occurs prior to cdc14p-mediated compaction of the rDNA. They suggest that rDNA compaction is required for full separation of rDNA. Taken together, these findings highlight the special status of the rDNA in yeast mitosis and suggest that the physical retention of the Cdc14 phosphatase within the nucleolus until after the metaphase/anaphase transition may provide a structural mechanism to impose order on the later mitotic events in yeast. Does FEAR-mediated nucleolar separation occur in higher eukaryotes? While a recent report indicates that Cdc14-like proteins are present in the nucleolus and play an, as yet undefined, role in cell division,27 it seems likely that the mechanism of nucleolar disassembly and rDNA separation is considerably different. Nucleolar disassembly in mammalian cells is a step-wise process that commences just before nuclear envelope breakdown and is complete prior to metaphase. Mitotic disassembly is probably directly under the control of the CDK1 kinase, which also inhibits RNA polymerase 1 activity.28 Additional mechanisms for nucleolar disassembly and size control have been identified. The relationship between nucleolar size/number and growth rate/cell size and a decreased nucleolar size in response to nutrient starvation has long been recognized and it has been shown29 that ribosome biogenesis is intimately linked to cell size through Sfp1, a transcription factor that controls the expression of at least 60 genes implicated in ribosome assembly. Rapamycin, an inhibitor of the TOR kinase, and nutrient restriction both decrease nucleolar size30 leading to a reduction in pol1 localisation and activity. Hypoacetylation mutants of histone H4 reduce the effects of TOR inhibition and starvation, while hyperacetylation mimic it. In Xenopus oocytes two proteins have been identified that can induce nucleolar disassembly independently of transcriptional inhibition.31 Thus, multiple mechanisms control the assembly, size and disassembly of the nucleolus, some of which are closely associated with Pol1 transcriptional activity but others seem to be independent.
CONCLUSION
Given the importance of the nucleolus in protein synthesis, growth, cell size control and mitosis, genetic and biochemical dissection of nucleolar structure and function should continue to reveal insights into all these aspects of cellular function. Many questions remain to be answered. For instance, is nucleolar association of the FEAR pathway restricted to fungi, or to lower eukaryotes, or will it turn out to be a general phenomenon? The organism (budding yeast) in which the FEAR pathway has been shown to be associated with nucleolar segregation does not undergo nuclear envelope breakdown at mitosis, whereas higher plant and animals, which so far have not shown any sign of the FEAR pathway in nucleolar segregation, do break down their nuclear envelope. It would be interesting to know what happens in fission yeast—often used as a model for higher eukaryotic mitosis. It should also be remembered what a restricted group of organisms have been analysed in any detail—a handful of species restricted to three groups—ascomycete fungi, higher plants and animals. Is the FEAR pathway the cause or the consequence of late rDNA segregation in budding yeast? This mechanism also applies to segregation of telomeric DNA. Why should nucleolar and telomeric DNA be separated late, and does this apply to any other repetitive sequences? It may turn out that there are specific and immediate mechanistic reasons for the different segregation of these genomic regions. On the other hand, the reasons may be to do with evolutionary origins and pressures. The nucleolus/rDNA must be under very
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intense selective pressure compared to other parts of the genome, because of the cell’s immense requirement for ribosomes. It can also be argued that ribosomes and the machinery for making them had a distinctly different evolutionary origin from the rest of the nucleus.32 According to the ‘RNA world’ hypothesis, RNA evolved first, then learnt how to make proteins—i.e., precursors to ribosomes developed. Only later was DNA adopted as a medium for information storage. Thus ribosomes and ribosome biogenesis would be derived from a very ancient system. This may be an explanation of why the nucleolus has its own rules in so many ways. References
27. Kaiser BK, Nachury MV, Gardner BE, Jackson PK. Xenopus Cdc14 alpha/beta are localized to the nucleolus and centrosome and are required for embryonic cell division. BMC Cell Biol 2004; 5:27. 28. Sirri V, Hernandez-Verdun D, Roussel P. Cyclin-dependent kinases govern formation and maintenance of the nucleolus. J Cell Biol 2002; 156:969-81. 29. Jorgensen P, Nishikawa JL, Breitkreutz BJ, Tyers M. Systematic identification of pathways that couple cell growth and division in yeast. Science 2002; 297:395-400. 30. Tsang CK, Bertram PG, Ai W, Drenan R, Zheng XF. Chromatin-mediated regulation of nucleolar structure and RNA Pol I localization by TOR. EMBO J 2003; 22:6045-56. 31. Gonda K, Fowler J, Katoku-Kikyo N, Haroldson J, Wudel J, Kikyo N. Reversible disassembly of somatic nucleoli by the germ cell proteins FRGY2a and FRGY2b. Nat Cell Biol 2003; 5:205-10. 32. Staub E, Fiziev P, Rosenthal A, Hinzmann B. Insights into the evolution of the nucleolus by an analysis of its protein domain repertoire. Bioessays 2004; 26:567-81.
1. Hadjiolov AA. The nucleolus and ribosome biogenesis. New York: Springer Verlag Wien, 1985. 2. Shaw PJ, Jordan EG. The nucleolus. Annu Rev Cell Dev Biol 1995; 11:93-121. 3. Olson MO, Hingorani K, Szebeni A. Conventional and nonconventional roles of the nucleolus. Int Rev Cytol 2002; 219:199-266. 4. Oakes M, Nogi Y, Clark MW, Nomura M. Structural alterations of the nucleolus in mutants of Saccharomyces cerevisiae defective in RNA polymerase-I. Mol Cell Biol 1993; 13:2441-55. 5. Melese T, Xue Z. The nucleolus—an organelle fomed by the act of building a ribosome. Curr Opin Cell Biol 1995; 7:319-24. 6. Raska I. Oldies but goldies: Searching for Christmas trees within the nucleolar architecture. Trends Cell Biol 2003; 13:517-25. 7. Gonzalez-Melendi P, Wells B, Beven AF, Shaw PJ. Single ribosomal transcription units are linear, compacted Christmas trees in plant nucleoli. Plant J 2001; 27:223-33. 8. Koberna K, Malinsky J, Pliss A, Masata M, Vecerova J, Fialova M, Bednar J, Raska I. Ribosomal genes in focus: New transcripts label the dense fibrillar components and form clusters indicative of “Christmas trees” in situ. J Cell Biol 2002; 157:743-8. 9. Andersen JS, Lyon CE, Fox AH, Leung AK, Lam YW, Steen H, Mann M, Lamond AI. Directed proteomic analysis of the human nucleolus. Curr Biol 2002; 12:1-11. 10. Pendle AF, Clark GP, Boon R, Lewandowska D, Lam YW, Andersen J, Mann M, Lamond AI, Brown JW, Shaw PJ. Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions. Mol Biol Cell 2005; 16:260-9. 11. Iborra FJ, Jackson DA, Cook PR. Coupled transcription and translation within nuclei of mammalian cells. Science 2001; 293:1139-42. 12. Mazzola JL, Sirover MA. Alteration of intracellular structure and function of glyceraldehydes-3-phosphate dehydrogenase: A common phenotype of neurodegenerative disorders? Neurotoxicology 2002; 23:603-9. 13. Zheng L, Roeder RG, Luo Y. S-phase activation of the histone promoter OCA-S, a coactivator complex that contains GAPDH as a key component. Cell 2003; 114:255-66. 14. Cutler SR, Ehrhardt DW, Griffitts JS, Somerville CR. Random GFP:cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc Natl Acad Sci USA 2000; 97:3718-23. 15. Sawin KE, Nurse P. Identification of fission yeast nuclear markers using random polypeptide fusions with green fluorescent protein. Proc Natl Acad Sci USA 1996; 94:15146-51. 16. Simpson JC, Wellenreuther R, Poustka A, Pepperkok R, Wiemann S. Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing. EMBO Rep 2000; 1:287-92. 17. Koroleva O, Tomlinson M, Leader D, Shaw P, Doonan J. High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions. Plant J 2005; 41:162-74. 18. Torres-Rosell J, Machin F, Jarmuz A, Aragon L. Nucleolar segregation lags behind the rest of the genome and requires Cdc14p activation by the FEAR network. Cell Cycle 2004; 3:496-502. 19. Granot D, Snyder M. Segregation of the nucleolus during mitosis in budding and fission yeast. Cell Motil Cytoskeleton 1991; 20:47-54. 20. Fox H, Hickey PC, Fernandez-Abalos JM, Lunness P, Read ND, Doonan JH. Dynamic distribution of BIMGPP1 in living hyphae of Aspergillus indicates a novel role in septum formation. Mol Microbiol 2002; 45:1219-30. 21. Buonomo SB, Rabitsch KP, Fuchs J, Gruber S, Sullivan M, Uhlmann F, Petronczki M, Toth A, Nasmyth K. Division of the nucleolus and its release of CDC14 during anaphase of meiosis I depends on separase, SPO12, and SLK19. Dev Cell 2003; 4:727-39. 22. D’Amours D, Stegmeier F, Amon A. Cdc14 and condensin control the dissolution of cohesin-independent chromosome linkages at repeated DNA. Cell 2004; 117:455-69. 23. Shou W, Sakamoto KM, Keener J, Morimoto KW, Traverso EE, Azzam R, Hoppe GJ, Feldman RM, DeModena J, Moazed D, Charbonneau H, Nomura M, Deshaies RJ. Net1 stimulates RNA polymerase I transcription and regulates nucleolar structure independently of controlling mitotic exit. Mol Cell 2001; 8:45-55. 24. Bhalla N, Biggins S, Murray AW. Mutation of YCS4, a budding yeast condensin subunit, affects mitotic and nonmitotic chromosome behavior. Mol Biol Cell 2002; 13:632-45. 25. Freeman L, Aragon-Alcaide L, Strunnikov A. The condensin complex governs chromosome condensation and mitotic transmission of rDNA. J Cell Biol 2000; 149:811-24. 26. Machin F, Torres-Rosell J, Jarmuz A, Aragon L. Spindle independent condensation-mediated segregation of yeast ribosomal DNA in late anaphase. J Cell Biol 2004; In press.
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