One of the most prominent features of a eukaryotic cell is the nucleus, which is a complex and highly dynamic organelle. The nucleus was the first cell compartment to be discovered in 1833 by Robert Brown and is the largest organelle in the human cell. Inside the nuclear membrane is the nucleoplasm, which main function is to store DNA and facilitate an isolated environment where controlled transcription and gene regulation is enabled. The nucleoplasm contains several non-membrane bound substructures. Example images of proteins localized to the nucleus can be seen in Figure 1.
Of all human proteins, 6523 (33%) have been experimentally shown to localize to the nucleus (Figure 2). A Gene Ontology (GO)-based functional enrichment analysis of the nuclear proteins shows highly enriched terms for biological processes related to DNA repair, transcription and RNA processing, chromatin modifications and regulation of gene expression. differentiation and development. Approximately 64% (n=4198) of the nuclear proteins can be detected in additional cellular compartments, and 8% (n=490) are only detected in other nuclear structures. The most common additional localizations except for the nucleoli are the cytosol and vesicles.
Figure 1. Examples of proteins localized to the nucleoplasm and its substructures. PDS5A is thought to keep the sister chromatids in place during mitosis and also plays a role in DNA repair. PDS5A has been localized to the nucleoplasm (detected in A-431 cells). TP53BP1 is involved in DNA damage response and is localized to nuclear bodies (detected in A-431 cells). SRRM2 is known to be involved in pre-mRNA splicing and is localized to nuclear speckles (detected in A-431 cells).
Figure 2. 33% of all human protein-coding genes encode proteins localized to the nucleoplasm. Each bar is clickable and gives a search result of proteins that belong to the selected category.
The structure of the nucleoplasm
The size of the human nucleus varies depending on cell type and cell cycle phase, but is usually around 10 μm in diameter. The nucleus mainly contains DNA and proteins interacting with DNA. To accomodate the long DNA molecules, it is winded around histones in an intricate complex called chromatin. The most densely condensed chromatin, the heterochromatin, is usually organized in the nuclear periphery while the less packed euchromatin is dispersed throughout the whole nucleus (Spector DL. 1993). Many of the nuclear proteins are localized to the entire nucleoplasm where they give rise to a smooth or punctate staining pattern. However, the nucleoplasm is far from homogeneous. It contains several non-membrane bound sub compartments, collectively called nuclear bodies, acting as self-organizing clusters for different nuclear activities. Except for the nucleolus, the most prominent subcompartments are nuclear speckles, Cajal bodies (CBs), Gemini of Cajal bodies (gems) and promyelocytic leukemia bodies (PML bodies) (Lamond AI et al, 1998). A selection of proteins localized to the nucleus that would be suitable as nuclear markers, can be found in Table 1. Highly expressed nuclear proteins are summarized in Table 2. Images showing the different nuclear substructures can be seen in Figure 3.
Table 1. Selection of proteins suitable as markers for the nucleus or its substructures.
Table 2. Highly expressed single localizing nuclear proteins across different cell lines.
Nuclear speckles are formed in interchromatin granule clusters (IGCs) and contain pre-messenger RNA (pre-mRNA) splicing factors such as small nuclear ribonucleoprotein particles (snRNPs) (SWIFT H. 1959; Lamond AI et al, 2003). These granules are connected by fine fibrils, forming clusers that can be seen directly by electron microscopy (Thiry M. 1995). The appearance of nuclear speckles varies between cell lines, but they all share an irregular, mottled, pattern which dynamically may change in both size and shape over time. A selection of the proteins localized to nuclear speckles, appropriate for acting as markers for the structure, can be found in Table 1.
CBs and gems are usually found in close proximity to each other, but CBs mainly contain the protein Coilin and snRNPs, while gems mainly contain the snRNP-interacting complex survival of motor neuron (SMN) (Sleeman JE et al, 1999; Darzacq X et al, 2002; Jády BE et al, 2003; Liu Q et al, 1996; Lefebvre S et al, 1995; Fischer U et al, 1997). PML bodies are characterized by the presence of the PML protein, which act as a hub for assembly of a macromolecular complex that is highly dynamic and can contain a variety of different proteins (Lallemand-Breitenbach V et al, 2010). As both CBs, gems, PML bodies and m other nuclear bodies are all seen as distinct spots scattered throughout the nucleoplasm, they are difficult to differentiate without the use of co-localizing protein markers. Moreover, nuclear bodies vary in size and number dependent on cell line.
Figure 3. Examples showing the different nuclear substructures and staining patterns. LSM2 is a protein that might be involved in pre-mRNA splicing and shows a nucleoplasmic punctate staining pattern (detected in SK-MEL-30 cells). CTBP1 is a corepressor targeting various transcription factors and shows a smooth nucleoplasmic staining pattern (detected in A-431 cells). NOSIP is an E3 ubuquitin-protein regulating several catalytic processes and is localized to the nucleus (detected in U-2 OS cells). RBM25 is involved in pre-mRNA splicing activities and has been shown to localize to nuclear speckles (detected in HaCaT cells). NPAT is a known Cajal body protein and is required for proper G1/S transition. In the Cell Atlas, NPAT localizes to nuclear bodies (detected in CACO-2 cells). DAXX is a transcription corepressor involved in a number of different nuclear activities and is known to localize to several nuclear substructures such as PML bodies and centromeres. In the Cell Atlas, DAXX localizes to nuclear bodies (detected in A-431 cells).
The function of the nucleoplasm
The main function of the nucleus is to store the cell's genetic material, but also to regulate gene expression on a transcriptional level in order to control cellular functions such as cell growth and division. Since the nucleus is membrane enclosed and isolated from the rest of the cell, DNA replication and transcription can be controlled without interfering with the translation that occurs in the cytoplasm (SWIFT H. 1959). Despite the fact that the nuclear substructures are not membrane bound, highly specific tasks are carried out in these regions, which are further described in the subsections below.
Nuclear speckles are thought to function as a storage place for pre-mRNA splicing factors (Lamond AI et al, 2003; Melcák I et al, 2000) as well as a regulatory site for transcription and pre-mRNA processing, even though transcription does not occur within the speckles but rather in close proximity (Spector DL et al, 1991; Misteli T et al, 1997; Cmarko D et al, 1999).
CBs probably function as a modification site of snRNPs into fully functional splicing factors before they enter other parts of the cell (Sleeman JE et al, 1999; Darzacq X et al, 2002; Jády BE et al, 2003). The closely related gems play an important role in the synthesis of cytoplasmic snRNP (Liu Q et al, 1996; Lefebvre S et al, 1995; Fischer U et al, 1997). As previously mentioned, gems contain the SMN1 protein which has been found to be responsible for the onset of spinal muscular atrophy (SMA). SMA is one of the most lethal autosomal recessive disorders and genetic defects in the SMN gene could cause progressive muscle and mobility impairments (Lefebvre S et al, 1995). PML bodies have been found to be highly diverse and have been suggested to perform an ever-growing number of tasks in the cell, ranging from apoptosis regulation to anti-viral protection, and much about the function remains to be unraveled (Lallemand-Breitenbach V et al, 2010).
Gene Ontology (GO) analysis of the proteins mainly localized to the nucleus 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 RNA splicing, transcriptional processes and chromatin modification (Figure 5a). Enrichment analysis of the GO domain Molecular Function, gives hits for terms related to DNA binding activities and transcriptional regulations, such as mismatched DNA binding and pre-mRNA binding (Figure 5b).
Figure 5a Gene Ontology-based enrichment analysis for the nucleoplasm 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 5b Gene Ontology-based enrichment analysis for the nucleoplasm 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.
Nucleoplasmic proteins with multiple locations
Of the nuclear proteins identified in the Cell Atlas, approximately 64% (n=4198) also localize to other cell compartments (Figure 6). 490, 8% localize to other nuclear structures. The network plot shows that the most common locations shared with the nucleus are the cytosol, nucleoli and vesicles. Given that the nucleus is involved both in import and export of proteins to the cytoplasm and other compartments of the cell, these dual locations could highlight proteins functioning in nuclear trafficking as well as proteins functioning in various signaling cascades. Interactions between the nucleus and a number of cellular compartments, including nucleoli and the cytosol, are significantly overrepresented, whileproteins localizing to the nucleus and to the plasma membrane or actin filaments are significantly underrepresented. Examples of multilocalizing proteins within the nucleoplasmic proteome can be seen in Figure 7.
Figure 6. Interactive network plot of nuclear proteins with multiple localizations. The numbers in the connecting nodes show the proteins that are localized to the nucleus and to one or more additional locations. Only connecting nodes containing more than one protein and at least 0.5% of proteins in the nuclear 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 7. Examples of multilocalizing proteins in the nuclear proteome. The examples show common or overrepresented combinations for multilocalizing proteins in the nuclear proteome. IPO7 is functioning in the nuclear import of proteins and is known to be located at both the nucleoplasmic and cytoplasmic side of the nuclear pore complex (detected in A-431 cells). RRAGC is shuttling between the nucleus and the cytoplasm. It plays a crucial role in the initiation of the TOR signaling cascade where it is required for the amino acid induced relocalization of mTORC1 into the lysosomes (detected in U-2 OS cells). SENP3 is located in both the nucleoli and the nucleoplasm known to interact with sumoylated proteins regulating the transcriptional capacity in the cell and is also required for rRNA processing (detected in MCF7 cells).
Expression levels of nucleoplasm proteins in tissue
Transcriptome analysis and classification of genes into tissue distribution categories (Figure 8) shows that a larger portion of the genes encoding proteins localizing to the nucleoplasm and its substructures are detected in all tissues, compared to all genes presented in the Cell Atlas. Significantly smaller portions of these genes are detected in many or in some tissues. Thus, the nucleoplasm is a structure that contains a larger portion of ubiquitously expressed proteins.
Figure 8. Bar plot showing the percentage of genes in different tissue distribution categories for nuclear protein-coding genes compared to all genes in the Cell Atlas. 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
Thul PJ et al, 2017. A subcellular map of the human proteome. Science.