Nucleoplasm

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 enanble DNA-dependent processes such as transcription to occur in a controlled environment. The nucleoplasm contains several non-membrane bound substructures, such as nuclear bodies and nuclear speckles. Example images of proteins that localized to the nucleus can be seen in Figure 1.

In the subcellular resource, 6842 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 genes encoding nuclear proteins shows an enrichment of genes associated with biological processes related to DNA repair, transcription, RNA processing, chromatin organization, regulation of gene expression, differentiation and development. Approximately 70% (n=4761) of the proteins that localize to the nucleoplasm can also be detected in additional cellular compartments, with 8% (n=558) only 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.


PDS5A - A-431

TP53BP1 - A-431

SRRM2 - A-431

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 play 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).

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

  • 34% (6842 proteins) of all human proteins have been experimentally detected in the nucleoplasm by the Human Protein Atlas.
  • 2865 proteins in the nucleoplasm are supported by experimental evidence and out of these 732 proteins are enhanced by the Human Protein Atlas.
  • 4761 proteins in the nucleoplasm have multiple locations.
  • 1342 proteins in the nucleoplasm show single cell variation.

  • Proteins localizing to the nucleoplasm are mainly involved in RNA processing, transcription, chromatin organization, DNA repair, differentiation and development.

Figure 2. 34% of all human protein-coding genes encode proteins that have been shown to localize 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

Substructures

  • Nucleoplasm: 6244
  • Nuclear speckles: 497
  • Nuclear bodies: 611
  • Kinetochore: 7
  • Mitotic chromosome: 75

The nucleus of human cells varies in size depending on cell type and cell cycle stage, 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 a mechanism 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 subcellular resource, there are also annotations of proteins that localize to kinetochores or the perichromosomal layer during mitosis. Kinetochores are large protein structures that assemble on centromeric chromatin and act as an attachment site 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 proteins, motor proteins and regulatory checkpoint proteins. Upon entry into mitosis, there are 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, also 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 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.

Gene
Description
Substructure
TAF15 TATA-box binding protein associated factor 15 Nucleoplasm
SMARCAD1 SWI/SNF-related, matrix-associated actin-dependent regulator of chromatin, subfamily a, containing DEAD/H box 1 Nucleoplasm
SRRM2 Serine/arginine repetitive matrix 2 Nuclear speckles
RBM25 RNA binding motif protein 25 Nuclear speckles
PML PML nuclear body scaffold Nuclear bodies
SMN2 Survival of motor neuron 2, centromeric Cytosol
Nuclear bodies
MKI67 Marker of proliferation Ki-67 Mitotic chromosome
Nucleoli rim
Nucleoplasm
RSL1D1 Ribosomal L1 domain containing 1 Mitotic chromosome
Nucleoli rim
Primary cilium
Vesicles
CENPC Centromere protein C Kinetochore
Midbody
Nuclear bodies
Nucleoplasm

Table 2. Highly expressed single localizing nuclear proteins across different cell lines.

Gene
Description
Average nTPM
RPS19 Ribosomal protein S19 3465
RAN RAN, member RAS oncogene family 1445
HNRNPA1 Heterogeneous nuclear ribonucleoprotein A1 1141
H2AZ1 H2A.Z variant histone 1 1016
HNRNPC Heterogeneous nuclear ribonucleoprotein C 923
HMGB1 High mobility group box 1 787
HNRNPK Heterogeneous nuclear ribonucleoprotein K 713
HNRNPA3 Heterogeneous nuclear ribonucleoprotein A3 586
ATF4 Activating transcription factor 4 504
MORF4L1 Mortality factor 4 like 1 496


LSM2 - SK-MEL-30

CTBP1 - A-431

NOSIP - U2OS


RBM25 - HaCaT

NPAT - CACO-2

DAXX - A-431

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 co-repressor 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 U2OS 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 subcellular resoure, NPAT localizes to nuclear bodies (detected in CACO-2 cells). DAXX is a transcription co-repressor 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 subcellular resource, DAXX localizes to nuclear bodies (detected in A-431 cells).


Figure 4. 3D-view of the nucleoplasm in U2OS, 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 condense the majority of the human genome, but a major function for proteins that localize to the nucleoplasm is 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 muscular 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 genes encoding proteins mainly localized to the nucleus shows functions that are well in-line with known functions for this compartment. The enriched terms for the GO domain Biological Process are mainly related to transcription and DNA repair (Figure 5a). Enrichment analysis of the GO domain Molecular Function, gives enrichment of terms related to DNA binding, RNA binding, chromatin binding, and regulation of transcription as well as replication (Figure 5b).

0.00.51.01.52.02.53.03.5Fold EnrichmentBeta-catenin-TCF complex ass...Positive regulation of viral...Viral latencyPhosphorylation of RNA polym...DNA ligationNegative regulation of oligo...Proteasome assemblyMRNA transcription by RNA po...Base-excision repairTranscription initiation fro...Dosage compensationNegative regulation of trans...Single strand break repairDNA-templated transcription,...Positive regulation of mitot...Peripheral nervous system ne...Peripheral nervous system ne...Regulation of helicase activityRegulation of mesenchymal ce...Positive regulation of chrom...Mismatch repairTelomere maintenance via tel...Positive regulation of chrom...Pri-miRNA transcription by R...DNA-templated transcription,...Covalent chromatin modificationNegative regulation of chrom...Definitive hemopoiesisInterstrand cross-link repairEndocardium developmentRegulation of skeletal muscl...RNA splicingGenetic imprintingMaintenance of cell numberRegulation of DNA bindingSignal transduction involved...Protein localization to chro...Regulation of development, h...Regulation of transcription ...Signal transduction by p53 c...Positive regulation of trans...Response to ionizing radiationSteroid hormone mediated sig...Spleen developmentHistone mRNA metabolic processAutonomic nervous system dev...Regulation of DNA-templated ...Positive regulation of stem ...Negative regulation of chrom...Regulation of chromosome sep...Regulation of nuclease activityDNA damage response, detecti...Cranial nerve developmentStem cell differentiationMeiosis I cell cycle processPositive regulation of cellu...Positive regulation of trans...Cell agingNuclear transportResponse to arsenic-containi...Cell fate commitmentRegulation of type I interfe...Embryonic organ developmentColumnar/cuboidal epithelial...Meiotic nuclear divisionCellular response to leukemi...Response to leukemia inhibit...Mitotic nuclear divisionNegative regulation of cold-...Animal organ formationRegulation of cellular amino...Protein autoubiquitinationRoof of mouth developmentNotch signaling pathwayRegulation of embryonic deve...Interleukin-1-mediated signa...Regulation of Wnt signaling ...Mesoderm developmentGland developmentRespiratory system developmentNucleus organizationMuscle organ developmentResponse to fibroblast growt...Regulation of morphogenesis ...Fat cell differentiationCold-induced thermogenesisRegulation of cold-induced t...Neuron apoptotic processOsteoblast differentiationCellular response to environ...Cellular response to extrace...Regulation of protein stabilityProtein dephosphorylationPost-translational protein m...Regulation of small molecule...Visual system developmentShow full plot

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.

0.00.51.01.52.02.53.03.5Fold EnrichmentHMG box domain bindingMismatched DNA bindingRNA polymerase II general tr...DNA secondary structure bindingDamaged DNA bindingTransforming growth factor b...RNA polymerase II core promo...Promoter-specific chromatin ...Histone acetyltransferase bi...BHLH transcription factor bi...DNA N-glycosylase activityEnhancer bindingAcetylation-dependent protei...General transcription initia...DNA replication origin bindingMismatch repair complex bindingC2H2 zinc finger domain bindingClass I DNA-(apurinic or apy...E-box bindingNuclear receptor activityTranscription factor activit...Histone acetyltransferase ac...DNA-binding transcription re...DNA-(apurinic or apyrimidini...Histone kinase activityIntronic transcription regul...Intronic transcription regul...RNA polymerase II transcript...Transcription coactivator ac...DNA-binding transcription ac...Peroxisome proliferator acti...Steroid hormone receptor act...Single-stranded DNA bindingHistone deacetylase bindingHistone bindingChromatin DNA bindingHistone methyltransferase ac...CAMP response element bindingCyclin-dependent protein ser...Methylation-dependent protei...Proteasome bindingDemethylase activityCyclin bindingCatalytic activity, acting o...Methyl-CpG bindingP53 bindingHistone deacetylase activityTranscription cofactor bindingHelicase activitySnRNA bindingModification-dependent prote...RNA polymerase activityAU-rich element bindingDNA binding, bendingTBP-class protein bindingTelomerase RNA bindingPolyubiquitin modification-d...Ran GTPase bindingProtein serine/threonine/tyr...Nucleocytoplasmic carrier ac...SMAD bindingProtein tyrosine/serine/thre...Nucleotidyltransferase activityBeta-catenin bindingNuclease activitySingle-stranded RNA bindingUbiquitin-like protein bindingPre-mRNA bindingProtein N-terminus bindingDioxygenase activityCatalytic activity, acting o...Protein C-terminus bindingUbiquitin-like protein trans...Protein serine/threonine kin...Show full plot

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 subcellular section, approximately 70% (n=4761) of the proteins that localize to the nucleoplasm also localize to other cell compartments (Figure 6). 558 of the proteins in the nucleoplasm (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. Multilocalization 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 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 1.0% 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.


IPO7 - A-431

RRAGC - U2OS

SENP3 - MCF-7

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 U2OS 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 subcellular section. 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.

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

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 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

Spector DL., Macromolecular domains within the cell nucleus. Annu Rev Cell Biol. (1993)
PubMed: 8280462 DOI: 10.1146/annurev.cb.09.110193.001405

Lamond AI et al., Structure and function in the nucleus. Science. (1998)
PubMed: 9554838 

SWIFT H., Studies on nuclear fine structure. Brookhaven Symp Biol. (1959)
PubMed: 13836127 

Lamond AI et al., Nuclear speckles: a model for nuclear organelles. Nat Rev Mol Cell Biol. (2003)
PubMed: 12923522 DOI: 10.1038/nrm1172

Thiry M., The interchromatin granules. Histol Histopathol. (1995)
PubMed: 8573995 

Sleeman JE et al., Newly assembled snRNPs associate with coiled bodies before speckles, suggesting a nuclear snRNP maturation pathway. Curr Biol. (1999)
PubMed: 10531003 

Darzacq X et al., Cajal body-specific small nuclear RNAs: a novel class of 2'-O-methylation and pseudouridylation guide RNAs. EMBO J. (2002)
PubMed: 12032087 DOI: 10.1093/emboj/21.11.2746

Jády BE et al., Modification of Sm small nuclear RNAs occurs in the nucleoplasmic Cajal body following import from the cytoplasm. EMBO J. (2003)
PubMed: 12682020 DOI: 10.1093/emboj/cdg187

Liu Q et al., A novel nuclear structure containing the survival of motor neurons protein. EMBO J. (1996)
PubMed: 8670859 

Lefebvre S et al., Identification and characterization of a spinal muscular atrophy-determining gene. Cell. (1995)
PubMed: 7813012 

Fischer U et al., The SMN-SIP1 complex has an essential role in spliceosomal snRNP biogenesis. Cell. (1997)
PubMed: 9323130 

Lallemand-Breitenbach V et al., PML nuclear bodies. Cold Spring Harb Perspect Biol. (2010)
PubMed: 20452955 DOI: 10.1101/cshperspect.a000661

Booth DG et al., Ki-67 and the Chromosome Periphery Compartment in Mitosis. Trends Cell Biol. (2017)
PubMed: 28838621 DOI: 10.1016/j.tcb.2017.08.001

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

Ljungberg O et al., A compound follicular-parafollicular cell carcinoma of the thyroid: a new tumor entity? Cancer. (1983)
PubMed: 6136320 DOI: 10.1002/1097-0142(19830915)52:6<1053::aid-cncr2820520621>3.0.co;2-q

Melcák I et al., Nuclear pre-mRNA compartmentalization: trafficking of released transcripts to splicing factor reservoirs. Mol Biol Cell. (2000)
PubMed: 10679009 

Spector DL et al., Associations between distinct pre-mRNA splicing components and the cell nucleus. EMBO J. (1991)
PubMed: 1833187 

Misteli T et al., Protein phosphorylation and the nuclear organization of pre-mRNA splicing. Trends Cell Biol. (1997)
PubMed: 17708924 DOI: 10.1016/S0962-8924(96)20043-1

Cmarko D et al., Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection. Mol Biol Cell. (1999)
PubMed: 9880337 

Van Hooser AA et al., The perichromosomal layer. Chromosoma. (2005)
PubMed: 16136320 DOI: 10.1007/s00412-005-0021-9

Booth DG et al., Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery. Elife. (2014)
PubMed: 24867636 DOI: 10.7554/eLife.01641

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