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.

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).

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.

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

Nucleolar proteins with multiple locations

Of the nucleolar proteins identified in the Cell Atlas, approximately 86% (n=1090) also localize to other cell compartments (Figure 5). Of these 1090, 39% (n=428) 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.

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.

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.
PubMed: 10982400 

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

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.
PubMed: 10888599 

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

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