Nucleoli: Composition, Function, and Dynamics

The nucleus is a highly organized structure. However, unlike cytoplasmic organelles, nuclear substructures are not bounded by membranes, but rather are held together by interactions between their component proteins and nucleic acids, and are thus probably best regarded as giant, extended multimolecular complexes. The nucleolus is the most prominent of these structures. It is the site of transcription by RNA polymerase I (Pol I) of the rDNA, tandemly repeated copies of the genes for three of the four ribosomal RNAs (rRNAs: s-rRNA, 5.8S-rRNA, and l-rRNA), which are called the nucleolar organizer regions (NORs) of the chromosomes (Raska et al., 2006a, 2006b). The rDNA repeats are transcribed as a single precursor RNA, which is edited into the three rRNAs by excising leading, internal, and trailing transcribed spacer sequences. The rDNA repeats are separated by untranscribed intergenic sequences, which are much shorter in plants (2–3 kb) than in mammals and many other vertebrates (10–30 kb; Hadjiolov, 1985). In many cases the size of the intergenic spacers is heterogeneous, as, for example, in Xenopus laevis (3–9 kb). The fourth rRNA, 5S, is transcribed by RNA Pol III from tandem repeats elsewhere in the nucleus (Highett et al., 1993). The nucleolus is also the location at which most of the steps of ribosome assembly and maturation occur. This means about 90 ribosomal proteins, as well as many processing and accessory factors, need to be imported from the cytoplasm to the nucleolus, and the nearly complete small and large ribosomal subunits separately exported to the cytoplasm. Since a rapidly growing cell may require millions of ribosomes to be synthesized, the nucleolus is by far the major destination and origin of nucleocytoplasmic transport in the most active cells. When nuclei are stained with fluorescent DNA dyes such as 4′,6-diamidino-2-phenylindole, the nucleolus is seen as a dark region within the more brightly stained nuclear chromatin. This is an indication that the active rDNA is highly dispersed within the nucleolus. On the basis of their appearance in electron microscope (EM) images, the nucleoli in many animal cells have been described in terms of a tripartite structure, with small, lightly staining regions called fibrillar centers (FCs), surrounded by densely stained material called the dense fibrillar component (DFC); the remainder of the nucleolus contains what appear to be densely packed granules (the granular component [GC]; Shaw et al., 1995; Fig. 1). Originally these granules were assumed to be entirely preribosomal particles in various stages of processing, but recent studies have indicated other types of complexes may occupy specific parts of the GC, which may be segregated into distinct regions of different compositions (Politz et al., 2005). What appear to be FCs are often seen in plants, but the regions assumed to be related to the DFC are much more extensive in plant nucleoli and often no more densely stained than the surrounding granules (Shaw and Jordan, 1995; Fig. 1). In reality the organization of the nucleolus is likely to be more complex than previously thought. A recent article has shown that within the chordates there are species that display a bipartite organization, in essence lacking FCs (Thiry et al., 2011). These authors suggest that the FCs originated with the emergence of the amniotes. A problem with this hypothesis is that some anamniote nucleoli, such as in Xenopus, do contain FCs, as indeed do plants (Raska et al., 2006b). The controversies about nucleolar organization will probably not be fully resolved until we have a better understanding of what these electron microscopic structures represent in molecular and functional terms. Comparison of the structure of animal (A) and plant (B) nucleolar organization. TS, Transcription sites. In plants (B) a central region is often present and is called the nucleolar cavity. The molecular interpretation of these structures has been the subject of a long controversy. Immunogold EM studies showed concentrations of RNA Pol I in the FCs, which were consequently suggested to be the sites of transcription (Scheer and Rose, 1984). In situ labeling, whether at the optical or EM level, showed most rDNA in condensed heterochromatin at the nucleolar periphery, and smaller foci of rDNA labeling corresponding to FCs (Shaw et al., 1995). With more sensitive in situ techniques, fainter, diffuse rDNA labeling was subsequently seen throughout the DFC. The problem with a simple interpretation of these labeling experiments is that most Pol I, and most rDNA copies are not transcriptionally active. It was not until fluorescence detection of bromo-uridine labeling was introduced that the sites of transcription could be clearly labeled. In plant cells, this showed the transcription sites to consist of many foci within the DFC, with the smallest foci representing individual rDNA gene repeats (Thompson et al., 1997; González-Melendi et al., 2001; Koberna et al., 2002). Additional labeling with probes to transcribed spacer regions in the prerRNA and with various proteins and other small RNAs known to be involved in ribosome maturation led to a radial model, where the newly formed transcripts moved away from the dispersed genes within the DFC and then subsequently moving into the GC for later processing stages (Brown and Shaw, 1998). The number of rDNA repeats varies greatly among eukaryotes; many plants have thousands of copies, of which only a small proportion seems to be transcriptionally active at any one time. There is evidence that some repeats in human rDNA are inverted and may not be functional (Caburet et al., 2005). In rice (Oryza sativa), fiber fluorescence in situ hybridization has suggested that the repeats are regular and do not contain inversions or rearrangements (Mizuno et al., 2008). Furthermore, physical mapping studies in Arabidopsis (Arabidopsis thaliana) using pulsed field gel electrophoresis showed that restriction endonucleases that cut once per rDNA repeat gave only a single length of fragment (Copenhaver and Pikaard, 1996). Nevertheless the rDNA has not been fully sequenced in any higher eukaryote, as with current technology it is very difficult to analyze such highly repetitive sequences. We therefore do not know absolutely whether all the rDNA repeats are intact functional genes or whether other functional sequences are interspersed with them, and it is merely an assumption that all rDNA repeats are potentially transcribable. As with other genomic regions, current models suggest that rDNA chromatin can adopt three different states: inactive and condensed (heterochromatin), which corresponds to the condensed chromatin at the nucleolar periphery as well as to some intranucleolar condensed regions; active genes in an extended conformation within the DFC; and a poised or potentiated state, available for transcription but not currently being transcribed (McKeown and Shaw, 2009). This latter state may correspond to rDNA within the FCs. The balance between these states, and thus Pol I loading and transcription, is determined by DNA methylation, differences in the histone variants associated with the DNA, remodeling of the DNA, particularly the promoter regions, and the presence of histone modifications (Grummt and Pikaard, 2003). Loading of Pol I and some other factors is maintained through mitosis, and thus the chromatin state can be epigenetically inherited. A particularly clear example of epigenetic control of rDNA is provided by nucleolar dominance in hybrids where the NORs of one parental genome are active, whereas those of the other are inactive (Tucker et al., 2010). An RNAi approach has been used with Arabidopsis suecica, a hybrid of Arabidopsis and Arabidopsis arenosa, to determine factors involved in nucleolar dominance. This has shown the involvement of the histone deacetylases Histone Deacetylase1 and Histone Deacetylase6, the de novo DNA methyltransferase Domains Rearranged Methyltransferase2 (DRM2), and the methylcytosine binding domain proteins Methyl CpG Binding Domain6 (MBD6) and MBD10 (Preuss et al., 2008). Further confirmation that nucleolar dominance in plants involves RNA-directed DNA methylation was obtained by knockdown of RNA Dependent RNA Polymerase2, Dicer-like3, and DRM2, which disrupted the silencing of the Arabidopsis-derived rRNA genes in A. suecica (Preuss et al., 2008). In mammals, rRNA genes are silenced by the nucleolar remodeling complex, NoRC, which is recruited to rRNA genes by 200- to 300-nt RNA species, termed pRNA, derived from intergenic regions of the rDNA (Guetg et al., 2010; Santoro et al., 2010). Over the last 10 to 15 years, it has become clear that the nucleolus is involved in numerous other functions than ribosome biogenesis (Pederson, 1998; Rubbi and Milner, 2003; Olson, 2004; Raska et al., 2006a; Boisvert et al., 2007). Many are RNA-related functions such as RNA processing and assembly of ribonucleoproteins (RNPs). For example, the nucleolus (and associated bodies, particularly Cajal bodies [CBs]) is involved in the maturation, assembly, and export of RNP particles such as the signal recognition particle, telomerase RNP, and processing of precursor transfer RNAs and U6 small nuclear RNA. In addition, the nucleolus has roles in cellular functions such as regulation of the cell cycle, stress responses, telomerase activity, and ageing (Pederson, 1998; Tsai and McKay, 2002; Rubbi and Milner, 2003; Olson, 2004; Raska et al., 2006a; Boisvert et al., 2007; Boulon et al., 2010). Sequestration of specific proteins in the nucleolus or their release is one mechanism by which processes such as the cell cycle or cell death are regulated. The multifunctional nature of the nucleolus is therefore reflected in the complexity of the protein and RNA composition of the nucleolus and in the dynamic composition changes in response to cellular conditions. Initial analyses of the proteome of human cells identified around 450 proteins including ribosomal proteins and proteins known to be involved in ribosome biogenesis (fibrillarin, nucleolin, B23, etc.; Andersen et al., 2002; Scherl et al., 2002). Even at this stage, unexpected proteins like splicing factors, spliceosomal proteins, and translation factors were identified. The increasing resolution of mass spectrometry techniques had led to the current characterization of the nucleolar proteome from human cells of circa 4,500 proteins (Ahmad et al., 2009). Quantitative proteomic analyses have demonstrated the dynamic behavior of nucleolar proteins such as ribosomal proteins and of protein complexes such as Pol I (Andersen et al., 2005; Lam et al., 2007). For example, quantitative changes in the relative levels of nucleolar components (reflecting accumulation or loss) were apparent upon transcriptional inhibition. The degree of change varied greatly for different proteins and showed that ribosomal proteins were highly expressed and either incorporated into ribosomal subunits or rapidly degraded (Andersen et al., 2005; Lam et al., 2007). It is also intriguing that ribosomal protein complexes associate with chromosomes and in particular transcription start sites of tRNAs, although the nature of these complexes and their function are as yet unknown (De et al., 2011). In contrast, characterization of the proteome of plant nucleoli lags significantly behind. In an initial proteomic analysis of purified Arabidopsis nucleoli 217 proteins were identified. In addition to the expected ribosomal and nucleolar proteins, a range of nonribosomal and nonnucleolar proteins including plant-specific proteins, proteins with unknown function, and splicing and translation factors was observed (Pendle et al., 2005). In particular, exon junction complex proteins (known in animal systems to associate with mRNAs after splicing) were identified and their nucleolar localization was confirmed by confocal microscopy of GFP fusions (Pendle et al., 2005; Fig. 2). One of the core exon junction complex components, was shown to from the to the nucleolus and to splicing stress et al., 2009). the is that the of of mRNAs to the nucleolus, the exon junction complex has not yet been identified in plants and with mRNAs in the nucleolus has not been A of an protein splicing to the nucleolus has also been demonstrated et al., this is the only protein to such a and whether it mRNAs is not A number of small nuclear RNP proteins were identified in the nucleolar proteome (Pendle et al., and may be involved in of the spliceosomal in plants and 2008). of the nucleolar proteome in plants is and the of this nuclear a analysis is and be likely to evidence for different functions or of of GFP fusions in cells to proteins identified as nucleolar A to RNA binding proteins with known animal and plant-specific The nucleolus is indicated in by an The functions of the nucleolus in processing of various RNAs and assembly of different are reflected in the different species of RNA. animal cell nucleoli contain the extensive of small nucleolar RNAs as well as tRNAs, RNA recognition and telomerase RNA. In plants, analyses of nucleolar suggest that the plant nucleolus is also involved in many the presence of mRNAs and RNAs in the plant nucleolus and bodies is intriguing in terms of potentially functions in plants, the roles of the nucleolus and of RNAs from purified Arabidopsis nucleoli identified tRNAs, and small RNAs with many known and et al., 2010). In particular, in addition to the expected and of human were The function of has not been determined but it contains to the of contain which do not have to rRNAs or In such have been to be involved in RNA and regulation of gene et al., 2005; and et al., and et al., 2010). three in the of a ribosomal protein were shown to be involved in regulation of stress response et al., 2011). plants have a number of to no function for example, gene has been The of proteins in the plant nucleolus a function in In only a very mRNAs have been identified in the nucleolus (Pederson, 1998; Olson, In contrast, mRNAs from a range of genes were identified in a from purified Arabidopsis particular was that mRNAs were in the nucleolus and the and therefore were likely to be by the et al., 2009). the localization of proteins involved in and the nucleolus suggested a function for the plant nucleolus in and in biogenesis et al., 2009). However, we have shown that transcripts with are not by and appear to the et al., 2011). other transcripts are to the nucleolus as of the transcripts may in the nucleolus for a different function by a different or may be there of being In this an between the nucleolus and export of particular human RNAs is well 2007; et al., and has been suggested to function for cellular In and animal cells the of whether specific have a nucleolar is in and 2008). the of mRNAs and mRNAs in the plant nucleolus, small which are involved in transcriptional are in a region of the nucleolus or in bodies often associated with the nucleolus called and Pikaard, 2008). The localization of precursor and to also a for the nucleolus in the maturation of and Pikaard, 2008). precursor and are in the nucleolus of cells for assembly, or to (Politz et al., et al., 2009). There is also an between and with some being from and some and et al., et al., et al., 2011). In addition, small RNAs were to be associated with proteins of RNA silencing in and Arabidopsis et al., and small RNAs from a human of gene et al., 2008). The complexity of processes the nucleolus that it is a of RNA For plants, the presence of mRNAs and small RNAs in the nucleolus to that the nucleolus is involved in regulation of in response to cellular conditions. the nature of the nucleolus to many RNA processing and RNP assembly it is not that many animal and plant the nucleolus in and transport of The involvement of the nucleolus in of animal and human is well 2007; et al., 2010; et al., 2011). In plants, a growing number of some with the nucleolus (and other nuclear bodies such as and roles for the nucleolus and nucleolar proteins are et al., 2011). For example, plant can nucleolar proteins for assembly of RNP and and to In the to the protein of to the nucleolus of into structures that with the nucleolus. then nucleolar RNA binding known to be an RNA for assembly of cytoplasmic particles et al., et al., 2008). The nucleolar localization of is for proteins from other plant also the nucleolus and the function of this localization and their interactions with nucleolar proteins are being For example, the protein and protein proteins of are to the nucleolus et al., and of is in plants, that is also involved in of of of proteins is to with plant silencing proteins to the silencing by The protein of A has the localization of to the nucleolus and the of whether this protein components or involved in RNA silencing that are in the nucleus or nucleolus and associated bodies and Pikaard, 2008). The domain of with in the nucleolus and and of accumulation of the that is involved in the and 2009). The silencing protein of also to the nucleus and nucleolus where it with and et al., 2010). However, these interactions are not for of RNA silencing and their far et al., 2010). proteins are also in the the protein of and the protein of but the function of the nucleolar localization is unknown et al., 2011). different proteins and the nucleolus, their interactions with proteins like and the of functions nucleolar structure and function are for the that are likely to into nucleolar The nucleolus, like the other parts of the is dynamic at a number of it and the cell the nucleolus structure is and within the the nucleolar with within the nucleolus and in the and cytoplasm. The nucleolus at the of as most transcription and the nuclear and then with the of rDNA transcription at the of the 2011). the GC components are by the DFC proteins, such as Pol I subunits and binding which DNA with the rDNA the presence of binding is to a in the and 2008). nucleolar components diffuse throughout the whereas such as the protein B23, associate with the periphery of the chromosomes as When rDNA transcription is mitosis, transcripts through the that prerRNA processing is also The nucleolus at the of small bodies, called bodies are formed 2011). When transcription of the rDNA is the bodies as nucleoli are more than one active is present in the nucleoli at active In plants, these small nucleoli then often together to a single nucleolus as (Shaw and Jordan, 1995). For many cell cells and thus had a to the structures seen as and long have a much more dynamic using fluorescence after of nucleolar and nuclear proteins to GFP have shown that all nucleolar and nuclear proteins are in between the nucleolus and cytoplasm. The nucleolar of nucleolar proteins is only a of and and 2001; and 2005). The between nuclear and nucleolar proteins is that nucleolar proteins a proportion of their in the nucleolus. The structure and the of the nucleolus as a structure the rDNA a small of proteins that then a structure which all the other proteins and The nucleolus (and other nuclear bodies such as thus a of proteins in with the surrounding (Raska et al., A number of dynamic nuclear bodies are either associated with the nucleolus or within it or et al., 2011). The most are the These bodies were identified more than by Cajal in cells, were called accessory bodies, and were to have a with the nucleolus were later bodies of their appearance in the but have been in of their of by GFP showed that and within the nucleus of plant et al., and animal et al., cells, often to the nucleolar periphery or being within the nucleolus. The function of is not well but their is in maturation of RNA complexes such as small and RNA complexes involved in silencing and many components with the nucleolus, particularly a number of small nucleolar and RNA processing proteins such as which various RNA species, and which to in A intranucleolar has been of about components et al., 2011). The composition of seems different from in particular contain small a probably to other The to have an involvement in DNA that DNA The are in the nucleolus, and may be in or with a central region of the nucleolus often called the nucleolar a structure that is particularly prominent in many plant cells and that has been shown to and There are of evidence that the nucleolus has a in and to et al., 2010). In mammals there is evidence the DNA to the nucleolus. is held at a by an that and it for can be by which is in the nucleolus. In one model, release of from the nucleolus then it to with levels consequently 2004; Raska et al., In an of experiments it was shown that the is by many different that the nucleolus and Milner, that the nucleolar structure is a of DNA In this the of by DNA as described is very and may to the rDNA has been associated with stress response to DNA and with the studies of this and rDNA copies are by repeated and their is maintained by gene between the tandem repeats 2008). is by which In at the histone also has a in rDNA number in a number whereas in a rDNA repeat was or 2008). In and cellular a shorter and a 2008). It was shown that cells from the rDNA repeats in the cells as and that this accumulation in is a of and It is that there are cell types or stages in some that require many more rDNA copies than are but in the only about the copies are and in a number of including have been with only a of the number of rDNA copies et al., 2003). There is a between genome size and number of rDNA copies et al., and this has led to a hypothesis that the rDNA may be as a for DNA the of the genome by DNA or The copies present in the rDNA repeats such that copies were available for ribosome 2008). rDNA chromatin also seems to be to the of repeats in as of the component to of repeats (Guetg et al., and in the number of rDNA repeats to a release of heterochromatin silencing throughout the nucleus and 2009). of chromatin assembly in Arabidopsis led to of and rDNA repeats in as well as to DNA et al., 2010). As rDNA repeats are the most gene in the these may to disrupted balance between and However, at in there is evidence that rDNA organization can also the genome by the of and 2008). many in which the nucleolus was to have a and function in ribosome many in recent have to a range of being to this region of the As a of species from different has been used for these we as yet whether all these various functions are in the nucleoli of all For the most what these is the involvement of RNA at some level, in the biogenesis or assembly of the nucleolus may be better regarded as an RNA processing rather than as a ribosome There is clearly much to be to clearly all the of the nucleolus, and to these need to be together within a nuclear The may in the concentrations of factors by to the nucleolus. the may in the RNA may have for such as the of factors in different This may have the various processes