Nucleoli

Wagner and Valentin were the first to describe the nucleolus in two independent publications in the 1830s. The nucleolus is a nuclear sub-compartment 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 is also involved in cell cycle regulation and cellular stress responses. Example images of proteins localized to the nucleoli can be seen in Figure 1.

In the subcellular resource, 1461 genes (7% of all protein-coding human genes) encode proteins that have been shown to localize to nucleoli (Figure 2). A Gene Ontology (GO)-based functional enrichment analysis of the nucleolar proteins shows enrichment of genes associated with biological processes related to rRNA synthesis and processing. Approximately 89% (n=1299) of the nucleolar proteins localize to other cellular compartments in addition to nucleoli, with 34% (n=501) of these only localizing to other nuclear compartments. The most common additional localizations outside the nuclear meta compartment is the cytosol and mitochondria.


UTP6 - A-431

RPF1 - SK-MEL-30

NIFK - A-431

Figure 1. Examples of proteins localized to the nucleoli. UTP6 is suggested to be involved in processing of pre rRNA (detected in A-431 cells). RPF1 is a protein believed to be required for ribosome biogenesis (detected in SK-MEL-30 cells). NIFK is known to localize to the nucleoli, but its function is still unclear (detected in A-431 cells).

0%10%20%30%40%50%60%70%80%90%100%% genesNot mappedSecretedOther organellesNucleoli

  • 7% (1461 proteins) of all human proteins have been experimentally detected in the nucleoli by the Human Protein Atlas.
  • 489 proteins in the nucleoli are supported by experimental evidence and out of these 110 proteins are enhanced by the Human Protein Atlas.
  • 1299 proteins in the nucleoli have multiple locations.
  • 352 proteins in the nucleoli show single cell variation.

  • Nucleolar proteins are mainly involved in rRNA synthesis and processing.

Figure 2. 7% of all human protein-coding genes encode proteins localized to the nucleoli. Each bar is clickable and gives a search result of genes encoding proteins that belong to the selected category.

The structure of nucleoli

Substructures

  • Nucleoli: 1106
  • Nucleoli fibrillar center: 319
  • Nucleoli rim: 151

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; the fibrillar center (FC), the dense fibrillar component (DFC) and the granular component (GC) (Boisvert FM et al. (2007); Scheer U et al. (1999)). Upon entry into mitosis, rRNA transcription and RNP processing shuts down and the nucleoli are disassembled. In telophase and early G1, the nucleolar organization is re-established. A selection of proteins localized to the nucleoli that are suitable as nucleoli markers can be found in Table 1. A list of highly expressed nucleolar proteins are summarized in Table 2.

Table 1. Selection of proteins suitable as markers for the nucleoli or its substructures.

Gene
Description
Substructure
DDX47 DEAD-box helicase 47 Nucleoli
RPF1 Ribosome production factor 1 homolog Nucleoli
UTP6 UTP6 small subunit processome component Mitotic chromosome
Nucleoli
NOL10 Nucleolar protein 10 Nucleoli
FTSJ3 FtsJ RNA 2'-O-methyltransferase 3 Mitotic chromosome
Nucleoli
Nucleoli rim
UBTF Upstream binding transcription factor Nucleoli fibrillar center

Table 2. Highly expressed single localized nucleolar proteins across different cell lines.

Gene
Description
Average nTPM
NAA50 N-alpha-acetyltransferase 50, NatE catalytic subunit 179
NOP53 NOP53 ribosome biogenesis factor 159
NOP56 NOP56 ribonucleoprotein 156
NOLC1 Nucleolar and coiled-body phosphoprotein 1 155
POLR2K RNA polymerase II, I and III subunit K 109
NOP16 NOP16 nucleolar protein 95
UTP4 UTP4 small subunit processome component 83
DDX47 DEAD-box helicase 47 80
NOP2 NOP2 nucleolar protein 79
UBTF Upstream binding transcription factor 77

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 (Németh A et al. (2011)). A recent study suggests that the protein MKI67, which is localized to the nucleoli rim, functions like a surfactant to create non-membranous barriers in the cell. Therefore, proteins with similar staining patterns could have a similar function (Cuylen S et al. (2016); Stenström L et al. (2020)). MKI67 and other immunofluorescent images of different nucleolar substructures can be seen in Figure 3.


KRI1 - HEK293

KRI1 - MCF-7

KRI1 - U2OS


NOLC1 - HEK293

UBTF - U2OS

MKI67 - U-251MG

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 U2OS cells. NOLC1 might play a role in maintaining the structure of the fibrillar center and the dense fibrillar component in the nucleoli, an 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 U2OS cells). MKI67 has been found to maintain mitotic chromosome integrity and is a well-known cellular proliferation marker (detected in U-251 MG cells).


Figure 4. 3D-view of nucleoli in U2OS cells, visualized by immunofluorescent staining of NOP56. The morphology of nucleoli in human induced stem cells can be seen in the Allen Cell Explorer.

The function of nucleoli

The nucleolus is responsible for the synthesis, processing and assembly of ribosomes; a complex process controlled in the nucleolar sub regions (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 cellular states. The need for translational capacity varies with different cell cycle phases, and transcriptional capacity 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 of proteins involved in cell cycle regulation and cellular stress responses (Boisvert FM et al. (2007); Visintin R et al. (2000)).

Several genetic disorders such as Werner syndrome, Fragile X syndrome 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)). Moreover, the nucleolar size increases with the proliferative ability of cells, 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 genes encoding nucleolar proteins reveal functions that are well in-line with already known functions for the compartment. The enriched terms for the GO domain Biological Process are related to ribosome biogenesis, but also regulation of gene expression, chromosome organization and signal transduction (Figure 5a), while enrichment analysis of the GO domain Molecular Function gives enrichment for RNA polymerase I activity, as well RNA binding and processing activities (Figure 5b).

0246810121416Fold EnrichmentProtein localization to nucl...Ribosome biogenesisPositive regulation of gene ...SRP-dependent cotranslationa...Viral transcriptionViral gene expressionTranslational initiationDNA replicationSignal transduction by p53 c...

Figure 5a. 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.

024681012141618Fold EnrichmentRNA polymerase I activityRibonuclease P RNA bindingSnoRNA bindingCatalytic activity, acting o...RNA helicase activityRRNA bindingRNA polymerase activityMethylation-dependent protei...Catalytic activity, acting o...S-adenosylmethionine-depende...Histone bindingNuclease activityDNA-binding transcription re...

Figure 5b. 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.

Nucleolar proteins with multiple locations

Of the nucleolar proteins identified in the subcellular section, approximately 89% (n=1299) also localize to other subcellular compartments (Figure 6). 34% (n=501) of the nucleolar proteins only localize to other nuclear structures. The network plot shows that the most common locations shared with nucleoli are nucleoplasm, cytosol and 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 ribosome biogenesis, transport and function. Nucleolar protein that additionally localize to the nucleoplasm or mitochondria are significantly overrepresented, while nucleolar proteins that additionally localize to vesicles, the cytosol or plasma membrane, respectively, are significantly underrepresented. Examples of multilocalizing proteins within the nucleolar proteome can be seen in Figure 7.

Figure 6. 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 at least 1.0% 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.


EXOSC10 - U2OS

APTX - PC-3

SPATS2L - U2OS

Figure 7. 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 U2OS 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 within cytoplasmic stress granules during oxidative stress but its function is still unknown (detected in U2OS cells).

Expression levels of nucleoli proteins in tissue

Transcriptome analysis and classification of genes into tissue distribution categories (Figure 8) shows that genes encoding proteins that localize to nucleoli are more likely to either be detected in all tissues or not detected in any, compared to all genes presented in the subcellular section. Significantly lower portions of nucleoli-associated genes are detected in some or many of the tissues. Thus, many genes encoding nucleolar proteins are ubiquitously expressed.

****Detected in singleDetected in someDetected in manyDetected in allNot detected0.0102030405060708090100%NucleoliAll localized genes

Figure 8. Bar plot showing the percentage of genes in different tissue distribution categories for nucleoli-associated protein-coding genes compared to all genes in the subcellular section. Asterisk marks a statistically significant deviation (p≤0.05) in the number of genes in a category 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

Boisvert FM et al., The multifunctional nucleolus. Nat Rev Mol Cell Biol. (2007)
PubMed: 17519961 DOI: 10.1038/nrm2184

Scheer U et al., Structure and function of the nucleolus. Curr Opin Cell Biol. (1999)
PubMed: 10395554 DOI: 10.1016/S0955-0674(99)80054-4

Németh A et al., Genome organization in and around the nucleolus. Trends Genet. (2011)
PubMed: 21295884 DOI: 10.1016/j.tig.2011.01.002

Cuylen S et al., Ki-67 acts as a biological surfactant to disperse mitotic chromosomes. Nature. (2016)
PubMed: 27362226 DOI: 10.1038/nature18610

Stenström L et al., Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic protein disorder. Mol Syst Biol. (2020)
PubMed: 32744794 DOI: 10.15252/msb.20209469

Visintin R et al., The nucleolus: the magician's hat for cell cycle tricks. Curr Opin Cell Biol. (2000)
PubMed: 10801456 

Marciniak RA et al., Nucleolar localization of the Werner syndrome protein in human cells. Proc Natl Acad Sci U S A. (1998)
PubMed: 9618508 

Tamanini F et al., The fragile X-related proteins FXR1P and FXR2P contain a functional nucleolar-targeting signal equivalent to the HIV-1 regulatory proteins. Hum Mol Genet. (2000)
PubMed: 10888599 

Willemsen R et al., Association of FMRP with ribosomal precursor particles in the nucleolus. Biochem Biophys Res Commun. (1996)
PubMed: 8769090 DOI: 10.1006/bbrc.1996.1126

Isaac C et al., Characterization of the nucleolar gene product, treacle, in Treacher Collins syndrome. Mol Biol Cell. (2000)
PubMed: 10982400 

Drygin D et al., The RNA polymerase I transcription machinery: an emerging target for the treatment of cancer. Annu Rev Pharmacol Toxicol. (2010)
PubMed: 20055700 DOI: 10.1146/annurev.pharmtox.010909.105844