Wagner and Valentin were the first to describe the nucleolus in two independent publications in the 1830s.
The nucleolus is a nuclear sub-organelle that varies in size and number depending on cell type. The main function of the nucleolus is to
synthesize and assemble ribosomes for later transport to the cytoplasm where translation takes place. The nucleolus has also been found to
be involved in cell cycle regulation and cell stress responses. Example images of proteins localized to the nucleoli can be seen in Figure 1.
In a previous study approximately 500 proteins were identified in the nucleoli, even though the complete nucleolar
proteome might be even larger
(Andersen JS et al, 2005). Of all human proteins, 1297 (7%)
have been experimentally shown to localize to nucleoli (Figure 2). A Gene Ontology (GO)-based functional enrichment analysis of the nucleolar
proteins shows enriched terms for biological processes related to rRNA processing. Approximately 86% (n=1109)
of the nucleolar proteins localize to other cellular compartments in addition to nucleoli, of which 39% (n=435) are other nuclear structures. The most common additional localizations except for the nucleoplasm are the cytosol and mitochondria.Nucleoli fibrillar center: 267
The nucleoli are non-membrane enclosed, highly conserved, sub organelles within the nucleus. They are formed around nucleolus organizer regions (NORs) consisting of ribosomal DNA (rDNA) and are structurally organized into three different sub regions; fibrillar center (FC), dense fibrillar component (DFC) and granular component (GC)
([Boisvert FM et al, 2007; Scheer U et al, 1999 ). A selection of proteins localized to the nucleoli suitable as nucleoli markers, can be found in Table 1.
Table 1. Selection of proteins suitable as markers for the nucleoli or its substructures.
||DEAD-box helicase 47
||Ribosome production factor 1 homolog
||UTP6, small subunit processome component
||Nucleolar protein 10
||BRX1, biogenesis of ribosomes
||FtsJ homolog 3
||Upstream binding transcription factor, RNA polymerase I
||Nucleoli fibrillar center
A majority of the nucleolar proteins show staining throughout the whole nucleolar area, while roughly 20% display a more refined staining pattern.
The staining of fibrillar centers and/or dense fibrillar component appears as clusters of spots for most cell lines while for others, for example MCF-7 and U-251, only one
larger spot is seen. Some proteins localize to the rim of the nucleolus, which is visible as a thin circle around the nucleolus and could be associated to either the GC or the
perinucleolar heterochromatin surrounding the nucleolus
(Németh A et al, 2011). A recent study suggests that the protein
MKI67, localized to nucleoli rim, functions like a surfactant to create
non-membranous barriers in the cell. Therefore, proteins with similar staining patterns could have similar function
(Cuylen S et al, 2016).
MKI67 and other immunofluorescent images of different nucleolar substructures can be seen in Figure 3. During mitosis when transcription shuts down, the nucleoli are disassembled as
no ribosome assembly is required. The size of the nucleolus has also been suggested to correlate with the proliferative ability of cells
(Derenzini M et al, 2000).
Figure 3. Examples of the morphology of the nucleoli in different cell lines as well as the nucleolar substructures and staining patterns.
Immunofluorescent staining of KRI1 in HEK 239, MCF-7 and U-2 OS cells.
NOLC1 might play a role in maintaining the structure of the fibrillar center and the dense fibrillar component in the nucleoli. NOLC1 is localized to the fibrillar center (detected in HEK293 cells).
UBTF is involved in the activation of RNA polymerase I and is localized to the fibrillar center (detected in U-2 OS cells).
MKI67 has been found to maintain mitotic chromosome integrity and is a well-known cellular proliferation marker. MKI67 is localized to the nucleoli rim (detected in U-251 cells). Note that nucleoli rim is currently not a location annotated in the Cell Atlas.
See the morphology of nucleoli in human induced stem cells in the Allen Cell Explorer.
The function of the nucleoli
The nucleolus is responsible for the synthesis, processing and assembly of ribosomes, a complex process controlled in the nucleolar sub regions; fibrillar center, dense fibrillar component and the granular component
(Boisvert FM et al, 2007; Scheer U et al, 1999; Németh A et al, 2011). The border between the FC and the DFC contains proteins from the RNA polymerase I complex and is the region where pre-ribosomal RNA (pre-rRNA) is transcribed from rDNA. The pre-rRNA is later modified by proteins in the DFC followed by assembly of the ribosome subunits in the GC
(Scheer U et al, 1999). As is the case for the majority of organelles, the proteome of the nucleolus is dynamic and has been shown to consist of multiple overlapping sets of proteins that are interchanging dependent on the cell state.
The need for high translational capacity varies with different cell cycle phases, which in turn is heavily dependent on the amount of ribosomes available. In addition to being responsible for ribosome assembly, the nucleolus has also been found to comprise proteins involved in cell cycle regulation and cell stress responses
(Boisvert FM et al, 2007; Visintin R et al, 2000).
Several genetic disorders such as Werner, fragile X and Treacher Collins syndrome have been linked to nucleolar proteins
(Marciniak RA et al, 1998; Tamanini F et al, 2000; Willemsen R et al, 1996; Isaac C et al, 2000). The nucleolar size increases with the cells proliferative ability, suggesting that the nucleoli play an important role in development of cancer and could therefore be a potential target for cancer therapy
(Drygin D et al, 2010).
Gene Ontology (GO) analysis of the proteins mainly localized to the nucleoli shows functions that are well in-line with already known functions for the structure. The enriched terms for the GO domain Biological Process are related to the rRNA processing and ribosome assembly (Figure 4a), while enrichment analysis of the GO domain Molecular Function gave enriched results for RNA binding activities (Figure 4b). A list of highly expressed nucleolar proteins are summarized in Table 2.
Figure 4.a Gene Ontology-based enrichment analysis for the nucleolar proteome showing the significantly enriched terms for the GO domain Biological Process. Each bar is clickable and gives a search result of proteins that belong to the selected category.
Figure 4.b Gene Ontology-based enrichment analysis for the nucleolar proteome showing the significantly enriched terms for the GO domain Molecular Function. Each bar is clickable and gives a search result of proteins that belong to the selected category.
Table 2. Highly expressed single localized nucleolar proteins across different cell lines.
||EBNA1 binding protein 2
||Nucleolar and coiled-body phosphoprotein 1
||Glioma tumor suppressor candidate region gene 2
||Tubulin polyglutamylase complex subunit 2
||Ribosomal L1 domain containing 1
||NOC2 like nucleolar associated transcriptional repressor
||Nucleolar protein interacting with the FHA domain of MKI67
||Upstream binding transcription factor, RNA polymerase I
Nucleolar proteins with multiple locations
Of the nucleolar proteins identified in the Cell Atlas, approximately 86% (n=1109) also localize to other cell compartments (Figure 5). Of these 1109, 39% (n=435) are other nuclear structures. The network plot shows that the most common locations shared with nucleoli are the nucleoplasm, cytosol and the mitochondria. Given that the nucleoli are responsible for synthesis and assembly of ribosomes that later are exported to the cytoplasm, many of the proteins localized to both the nucleoli and the cytoplasmic structures are most likely involved in translation. The number of proteins localized to the nucleoli and the nucleoplasm as well as the nucleoli and mitochondria are seen more often than expected with the current distribution of multilocalizing proteins, while nucleolar proteins additionally localize to vesicles, the Golgi apparatus or the cytosol are significantly underrepresented. Examples of multilocalizing proteins within the nucleolar proteome can be seen in Figure 6.
Figure 5. Interactive network plot of nucleolar proteins with multiple localizations.
The numbers in the connecting nodes show the proteins that are localized to the nucleoli and to one or more additional locations. Only connecting nodes containing more than one protein and at least 0.5% of proteins in the nucleolar proteome are shown. The circle sizes are related to the number of proteins. The cyan colored nodes show combinations that are significantly overrepresented, while magenta colored nodes show combinations that are significantly underrepresented as compared to the probability of observing that combination based on the frequency of each annotation and a hypergeometric test (p≤0.05).
Note that this calculation is only done for proteins with dual localizations.
Each node is clickable and results in a list of all proteins that are found in the connected organelles.
Figure 6. Examples of multilocalizing proteins in the nucleolar proteome. The examples show common or overrepresented combinations for multilocalizing proteins in the nucleolar proteome.
EXOSC10 is known to be involved in multiple RNA processing pathways in the nucleolus, nucleus and the cytoplasm (detected in U-2 OS cells).
APTX is known to be involved in DNA repair and is localized to the nucleoplasm and the nucleoli (detected in PC-3 cells).
SPATS2L is known to localize to the nucleoli and into cytoplasmic stress granules during oxidative stress but the function is unknown (detected in U-2 OS cells).
Expression levels of nucleoli proteins in tissue
The transcriptome analysis (Figure 7) shows that nucleolar proteins are more likely to be expressed in all tissues and less likely to be tissue enhanced, compared to all other genes with protein data in the Cell Atlas.
Figure 7. Bar plot showing the distribution of expression categories, based on the gene expression in tissues, for nucleoli-associated protein-coding genes compared to all genes in the Cell Atlas. Asterisk marks statistically significant deviation(s) (p≤0.05) from all other organelles based on a binomial statistical test. Each bar is clickable and gives a search result of proteins that belong to the selected category.
Relevant links and publications
Andersen JS et al, 2005. Nucleolar proteome dynamics. Nature.
PubMed: 15635413 DOI: 10.1038/nature03207
Boisvert FM et al, 2007. The multifunctional nucleolus. Nat Rev Mol Cell Biol.
PubMed: 17519961 DOI: 10.1038/nrm2184
Cuylen S et al, 2016. Ki-67 acts as a biological surfactant to disperse mitotic chromosomes. Nature.
PubMed: 27362226 DOI: 10.1038/nature18610
Derenzini M et al, 2000. Nucleolar size indicates the rapidity of cell proliferation in cancer tissues. J Pathol.
PubMed: 10861579 DOI: 10.1002/(SICI)1096-9896(200006)191:2<181::AID-PATH607>3.0.CO;2-V
Drygin D et al, 2010. The RNA polymerase I transcription machinery: an emerging target for the treatment of cancer. Annu Rev Pharmacol Toxicol.
PubMed: 20055700 DOI: 10.1146/annurev.pharmtox.010909.105844
Isaac C et al, 2000. Characterization of the nucleolar gene product, treacle, in Treacher Collins syndrome. Mol Biol Cell.
Marciniak RA et al, 1998. Nucleolar localization of the Werner syndrome protein in human cells. Proc Natl Acad Sci U S A.
Németh A et al, 2011. Genome organization in and around the nucleolus. Trends Genet.
PubMed: 21295884 DOI: 10.1016/j.tig.2011.01.002
Scheer U et al, 1999. Structure and function of the nucleolus. Curr Opin Cell Biol.
PubMed: 10395554 DOI: 10.1016/S0955-0674(99)80054-4
Tamanini F et al, 2000. The fragile X-related proteins FXR1P and FXR2P contain a functional nucleolar-targeting signal equivalent to the HIV-1 regulatory proteins. Hum Mol Genet.
Visintin R et al, 2000. The nucleolus: the magician's hat for cell cycle tricks. Curr Opin Cell Biol.
Willemsen R et al, 1996. Association of FMRP with ribosomal precursor particles in the nucleolus. Biochem Biophys Res Commun.
PubMed: 8769090 DOI: 10.1006/bbrc.1996.1126