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,such as nuclear bodies and nuclear speckles. Example images of proteins localized to the nucleus can be seen in Figure 1.
In the Cell Atlas, 6758 genes (34% of all human protein-coding genes) have been shown to encode proteins that localize to the nucleoplasm and its sub-compartments (Figure 2). A Gene Ontology (GO)-based functional enrichment analysis of these proteins shows highly enriched terms for biological processes related to DNA repair, transcription, RNA processing, chromatin modifications, regulation of gene expression. differentiation and development. Approximately 66% (n=4449) of the proteins that localize to the nucleoplasm can also be detected in additional cellular compartments, with 8% (n=521) being detected in the other major nuclear compartments; nucleoli and nuclear membrane. 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).
34% (6758 proteins) of all human proteins have been experimentally detected in the nucleoplasm by the Human Protein Atlas.
2760 proteins in the nucleoplasm are supported by experimental evidence and out of these 865 proteins are enhanced by the Human Protein Atlas.
4449 proteins in the nucleoplasm have multiple locations.
1293 proteins in the nucleoplasm show a cell to cell variation. Of these 1166 show a variation in intensity and 154 a spatial variation.
Proteins localizing to the nucleoplasm are mainly involved in RNA processing, transcription, chromatin modification and DNA repair, differentiation and development.
Figure 2. 34% 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 nucleus of human cells varies in size 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, in a complex called chromatin. At the first level of chromatin organization, the DNA is wrapped around proteins known as histones, which provides both a way of compacting the long DNA molecules as well as mechanisms to regulate DNA-dependent cellular processes. The chromatin is then further compacted and organized in intricate ways, while yet remaining dynamic. The most densely condensed chromatin, known as 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 and nuclear bodies (Lamond AI et al. (1998)). Nuclear speckles, in the form of splicing speckles and paraspeckles, 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 clusters 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 may change in both size and shape over time. Nuclear bodies vary in size, number and location dependent on the type of nuclear body and the cell line. Cajal bodies (CBs) and gemini of Cajal bodies (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 acts 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 CBs, gems, PML bodies and 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.
In the Cell Atlas, there are also annotations of proteins that localize kinetochores in mitosis. Kinetochores are large protein structure that assemble on centromeric chromatin and act as an attachment point for microtubules of the mitotic spindle. While the inner kinetochore persists through the cell cycle, the outer kinetochore is assembled only during cell division. Components of the kinetochore include structural components, motor proteins and regulatory checkpoint proteins. Upon entry into mitosis, there is also certain proteins and RNP complexes that localize specifically to the surface of the condensed mitotic chromosomes, known as the perichromosomal layer (Booth DG et al. (2017); Stenström L et al. (2020); Ljungberg O et al. (1983)). Many of these proteins, including MKI67 that is considered a major organizer of this region, localize to nucleoli, and in particular the rim of nucleoli, in interphase.
A selection of proteins localized to the nucleus, nuclear speckles and nuclear bodies that would be suitable as 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.
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 ubiquitin-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).
Figure 4. 3D-view of the nucleoplasm in U-2 OS, visualized by immunofluorescent staining of HNRNPC. The morphology of nuclei in human induced stem cells can be seen in the Allen Cell Explorer.
The function of the nucleoplasm
The main function of the nucleus is to store and compact the majority of the human genome, but a major function for proteins that localize to the nucleoplasm is also also to participate in and regulate DNA-dependent functions and cellular processes, such as transcription, RNA splicing, RNP assembly, DNA repair, and replication.
Despite the fact that the nuclear substructures are not membrane bound, highly specific tasks are carried out in these regions.
Splicing speckles are enriched for pre-mRNA splicing factors (Lamond AI et al. (2003); Melcák I et al. (2000)), and are thought to act as a regulatory site for transcription and pre-mRNA processing, with transcription occurring in close proximity (Spector DL et al. (1991); Misteli T et al. (1997); Cmarko D et al. (1999)). Paraspeckles can sequester nuclear proteins and RNA, thus providing a means for regulation of gene expression. Both splicing speckles and paraspeckles have highly dynamic compositions. 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)).
Kinetochores have an essential role in ensuring proper segregation of sister chromatids in mitosis and meiosis. Apart from serving as a physical attachment point for spindle microtubules, kinetochores contain a number of motor proteins and regulatory factors that orchestrate and control the movements of chromosomes during cell division.The function of the peripheral layer of mitotic chromosomes is not fully known, but it has been suggested to be involved in mitotic chromosome structure, to act as a physical barrier protecting mitotic chromatin from cytoplasmic proteins following nuclear envelope breakdown, and to keep mitotic chromosomes from sticking to one another (Van Hooser AA et al. (2005)). In agreement, MKI67 is essential for proper chromosome segregation and has been shown to act as an emulsifying shield around the chromosomes during mitosis Booth DG et al. (2014); Cuylen S et al. (2016). In addition, the peripheral layer may act as a landing pad, concentrating nucleolar proteins to aid in nucleolar reactivation during mitotic exit, and helping to ensure equal distribution of its components to daughter cells.
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 transcription, DNA repair, RNA processing and (Figure 5a). Enrichment analysis of the GO domain Molecular Function, gives enrichment of terms related to DNA binding, RNA binding, binding to factors involved in DNA-dependent functions and transcriptional regulation (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
In the Cell Atlas, approximately 66% (n=4449) of the proteins that localize to the nucleoplasm also localize to other cell compartments (Figure 6). 521, 8% only 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, while proteins 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.
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