Nuclear bodies

Many nuclear bodies behave like a drop of . For example, in the Xenopus frog oocytes, the nucleoli are almost perfectly spherical in shape. When two nucleoli meet, they merge with each other, forming a larger nucleolus. A similar fusion is described for Cahal bodies, bodies of histone loci , nuclear speckles, and other bodies. However, some nuclear bodies, such as the nucleolus, consist of several structural components, as evidenced by electron microscopy data. At first glance, this contradicts the notion of nuclear bodies as droplets of a viscous liquid. In Xenopus oocytes, both the granular component and the dense fibrillar component of the nucleoli can undergo fusion and exchange of proteins, but the granular component does this faster. The key proteins of the granular and dense fibrillar components — nucleophosmin and fibrillarin, respectively — can form droplets in the purified form in the presence of RNA, however nucleophosmin droplets fuse and exchange proteins faster than fibrillarin proteins. Physically, nucleophosmin drops are a viscous liquid, and fibrillarin drops are viscoelastic , which explains their slow dynamics. When the purified nucleofozmin and fibrillarin are combined in one drop, they form immiscible phases similar to nucleoli: small droplets of fibrillarin are inside larger drops of nucleofozmin. The immiscibility of the phases is ensured by the difference in surface tension , since drops of fibrillarin in an aqueous solution are more hydrophobic than drops of nucleofosmin. Perhaps in a similar way the inability of different nuclear bodies to merge with each other is explained. For example, the Kakhal nucleoli and bodies are often in close contact, but never merge, possibly due to the high interphase energy barrier ^[3] .

A common property of all nuclear bodies is their structural stability. Individual nuclear bodies are distinguishable during the interphase - from the beginning of the G1 phase to the exit from the G2 phase . During the interphase, the nuclear bodies undergo dynamic movements within the nucleus, and the larger the body, the less it moves. Large bodies, such as nucleoli and speckles, reaching 2-3 microns in diameter, are practically motionless and are capable of only limited local movement. Smaller bodies, such as Cahal bodies and PML bodies , ranging in size from 500 nm to 1 μm , intensively move around the core and undergo frequent fusion and separation ^[4] .

Despite the general structural stability, nuclear bodies are characterized by significant internal dynamism. The main component of nuclear bodies is special proteins, which are also present in the nucleoplasm, although at a significantly lower concentration. Photobleaching experiments have shown that nuclear bodies exchange their main components intensively with the nucleoplasm. Within a few minutes, the molecular composition of nuclear bodies is completely exchanged for previously nucleoplasmic molecules ^[4] .

Due to the lack of surrounding membranes, the shape and size of nuclear bodies is determined by the sum of the interactions of the molecules that make up their composition. Among such interactions, no covalent interactions have been revealed; therefore, the molecules inside the bodies interact with each other through non-covalent weak bonds. The key determining factor is the balance of incoming and outgoing molecules: with an increase in the flow of incoming molecules, the size of the body increases, and its decrease or increase in the flow of leaving molecules leads to a decrease in the body. The molecular mechanisms that determine this balance are poorly understood, but they include post-translational modifications of the proteins that make up nuclear cells. Control over the number of nuclear bodies is also poorly understood. Even the number of nucleoli, which are formed only around a fixed number of chromosome sites - nucleolar organizers , varies between different tissues and cell types. It is known that the number of Cahal bodies is regulated by the marker protein coilin : if several key phosphorylation sites of this protein mutate , the number of Cahal bodies is reduced. Moreover, the size and number of nuclear bodies depend on physiological conditions. So, the number of nucleoli is increased in actively proliferating cells. In lymphocytes , which actively synthesize proteins and therefore need large amounts of rRNA , the nucleoli increase in size. The number of PML bodies is positively associated with stressful conditions ^[5] .

Large nuclear bodies, as a rule, are largely immobile, although they are capable of small movements and fusion with each other. As experiments with experimentally induced interphase nuclei have shown, heterochromatin plays a leading role in limiting the mobility of nuclear bodies. The movement of the nucleoli was independent of actin , and their merger occurred in random collisions. Moreover, each body occupied a separate compartment, limited to heterochromatin. Artificial chromatin overcondensation led to a significant decrease in the frequency of fusion of bodies and, therefore, limited their mobility ^[6] . The mobility of nuclear bodies also has functional significance, influencing various aspects of the functioning of the genome ^[7] .

By the method of formation, nuclear bodies can be divided into two classes: activity-dependent and activity-independent. The first class includes bodies that are formed at the sites of certain nuclear processes, such as transcription, and their morphology strictly depends on the intensity of the process. Among these bodies is the nucleolus, which is formed on transcribed clusters of rRNA genes (nucleolar organizers). When rDNA transcription is suppressed, the nucleolus undergoes a rapid structural reorganization, and the delivery of additional rRNA genes to plasmids to the nucleus leads to the appearance of additional nucleoli. The bodies of histone loci form around histone genes upon activation of transcription of these genes at the beginning of DNA replication during the S phase . Stressful nuclear bodies and nuclear speckles belong to the same class. The second class includes bodies, for the formation of which there is no need for any kind of nuclear process. Such nuclear bodies are formed in the nucleoplasm and can subsequently be associated with a specific location in the nucleus. These are Cajal bodies and PML bodies. Sometimes they are located in certain places of the nucleus and are even associated with specific loci, however, they form in the nucleoplasm and acquire such a connection later. For example, when genes of small nuclear RNAs activated, they undergo directed, actin-dependent movement to previously formed Cahal bodies ^[8] .

The formation of a nuclear body begins with a nucleation event. During nucleation, the key components of the body lose their mobility, are grouped together and attract other “building blocks”. In activity-dependent bodies, the nucleation is triggered by the processes necessary for the formation of bodies. In the case of the nucleolus, nucleation occurs during the accumulation of nucleolar proteins on rDNA and pre-rRNA, and in the case of bodies of histone loci, during the accumulation of processing factors of the 3'-end of histone pre-mRNAs. In activity-independent bodies, the nucleators are probably structural proteins or RNAs, however, to date, such nucleators have not been identified ^[9] .

Some nuclear bodies can form de novo (from scratch) under physiological or experimental conditions. For example, nucleoli de novo may be formed when rRNA minigens are introduced into cells as part of plasmids. A similar phenomenon has been described for oogenesis in the Xenopus frog, in the oocytes of which, during this process, amplification of thousands of extrachromosomal rRNA genes and the formation of many small nucleoli occur. Nuclear speckles can also form de novo upon activation of transcription processes in the cell after global suppression. In viral infections , the rapid formation of PML bodies occurs: the key proteins of the PML bodies surround the viral genome to form a complete body. This reaction, apparently, serves as a reaction of innate immunity directed against viruses. However, de novo formation is most clearly shown for Cajal bodies. If in cells that normally do not have Cahal bodies, the overexpression of the components of these bodies is temporarily caused, then Cahal bodies will really form. In addition, if the components of Cajal bodies are artificially immobilized on chromatin at random loci, then they will form in these places ^[10] .

The composition of many nuclear bodies includes RNA molecules, which often play an important role in the assembly of these bodies. RNA can participate in the biogenesis of nuclear bodies in two ways. First, RNAs can serve as templates for assembling bodies, for example, in the case of most activity-dependent bodies that form around sites with active transcription. Such RNAs attract the that are part of nuclear bodies, starting the formation of bodies. Secondly, RNA can act as an architectural element in nuclear bodies. For example, also (also known as MEN-ε / β), a long stable polyadenylated RNA molecule localized in the nucleus, is required to form paraspeckles . Knockdown of this RNA using RNA interference leads to the destruction of paraspeckles. In addition, paraspeckles are not detected in the nuclei of human embryonic stem cells that do not express NEAT1 ^[11] .

Theoretically, two main mechanisms for assembling nuclear bodies are possible:

• assembly may include a series of consecutive tightly controlled steps;
• assembly can occur as a result of random interactions of components of nuclear bodies without a clear order.

The experiment described above for assembling Cahal bodies in the areas of chromatin immobilization of the key components of these bodies indicates the latter. However, the question of what happens during the assembly of activity-dependent bodies remains open ^[12] .

The formation of nuclear bodies can be based not only on protein – protein and protein – RNA interactions, but also on , which are facilitated by aggregation of protein domains of nuclear bodies. Using the phase transition model, one can explain the fluid-like properties of nuclear bodies, such as the ability to merge and separate, as well as their fast intranuclear dynamics. It is possible that heterochromatin itself has the properties of liquid droplets ^[13] . It was experimentally shown that the proteins and , which are part of the cytoplasmic stress granules and paraspeckles, can provide liquid-liquid phase separation ( English liquid – liquid phase separation, LLPS ) in the presence of RNA. It has been shown that some protein domains undergo LLPS only when combined in specific concentrations. Each nuclear body may have its own ratio of proteins that provide LLPS. LLPS are exposed to protein domains associated with aggregation, such as prion- like domains, as well as domains that promote polymerization (for example, the ( eng. Coiled-coil )), and areas with unexpressed structure ( eng. Low complexity regions ) ^{[ 14]} . A variety of nuclear structures formed by phase separation are involved at various stages of gene expression , such as transcription and RNA processing , affect the epigenetic status of genes and play a role in the development of many diseases ^[15] . Due to phase separation, phosphoinositides can be involved in the formation of nuclear bodies. In 2018, cells containing were found in the nuclei of cells of various organisms; they are known as nuclear lipid islets ( Nuclear Lipid Islets, NLIs ). Nuclear lipid islands are likely to play an important role in the regulation of gene expression, acting as platforms for binding various proteins and facilitating the formation of ^[16] .

The assembly and disassembly of nuclear bodies play an important role in their inheritance by daughter cells during fission . Some nuclear bodies, which are present in cells in a large number of copies, do not disassemble during mitosis , but are divided approximately equally between daughter cells due to their random distribution over the cell volume. Other nuclear bodies, in contrast, are disassembled during cell division and reassemble when daughter cells enter the G1 phase ^[17] .

So, the nucleolus is disassembled during mitosis, since rRNA transcription is suspended due to phosphorylation of transcription factors of , as well as rRNA processing factors. At the beginning of prophase, unprocessed or partially processed pre-rRNAs are accumulated at the periphery of condensed chromosomes along with many processing factors. After the destruction of the nuclear membrane, they exit into the cytoplasm and in the anaphase form many very mobile small bodies. At the beginning of the telophase , when the transcription of rRNA genes is restored, these small bodies are disassembled, and then pre-rRNAs and processing factors form the nucleolus bodies ( English prenucleolar bodies ) in the nucleoplasm of the newly formed nuclei of daughter cells. At the end of the telophase, the chromosomes are decondensed, and pre-rRNA and processing factors exit the nucleolar bodies, forming a nucleolus around the nucleolar organizers. The activity of RNA polymerase I and the resumption of processing of pre-rRNA are also necessary for the formation of the nucleolus after mitosis ^[18] .

At the beginning of mitosis, nuclear speckles are disassembled, and their components are randomly distributed over the cytoplasm. Speckle assembly begins in telophase. Paraspeckles remain stable throughout the cell cycle up to anaphase, when they become randomly scattered throughout the cell (cytoplasmic paraspecules). Cytoplasmic para speckles disappear at the beginning of the telophase, and the formation of nuclear para speckles begins at the end of cell division. The bodies of histone loci exist before the early prometaphase and are finally understood in the metaphase and re-formed in the telophase. Cachal bodies at the beginning of mitosis do not disassemble, but exit into the cytoplasm, where they are not in physical contact with condensed chromosomes. The number and size of Cajal bodies does not change from metaphase to telophase. When the nuclear envelope is formed in the telophase, the Kakhal cytoplasmic bodies are disassembled, and their key component, the coilin protein, quickly enters the nucleus, where it is initially randomly localized, but normal nuclear Kahal bodies are formed in daughter cells by the G1 phase. The number of PML bodies at the beginning of mitosis decreases because their main component, the PML protein, forms characteristic mitotic clusters, losing contact with other PML body proteins. The formation of PML bodies in the nucleus begins in the G1 phase; however, even during the G1 phase, large accumulations of PML protein are still found in the cytoplasm, which then decrease slowly ^[19] .

The table below lists the key nuclear bodies, their properties and the functions performed ^[2] .

Nucleolus

The nucleolus is a separate dense structure in the nucleus. It is not surrounded by a membrane and is formed in the region where rDNA is located - tandem repeats of ribosomal RNA (rRNA) genes , called nucleolar organizers . The main functions of the nucleolus are rRNA synthesis and the formation of ribosomes . The structural integrity of the nucleolus depends on its activity, and inactivation of rRNA genes leads to mixing of the nucleolar structures ^[20] .

At the first stage of ribosome formation, the RNA polymerase I enzyme transcribes rDNA and forms pre-rRNA, which is then cut into 5.8S, 18S and 28S rRNAs ^[21] . Transcription and post-transcriptional processing of rRNA occurs in the nucleolus with the participation of small nucleolar RNAs (snoRNAs), some of which come from spliced introns of mRNA genes encoding proteins associated with ribosome activity. The assembled ribosomal subunits are the largest structures passing through the nuclear pores ^[22] .

When examined under an electron microscope in the nucleolus, three components can be distinguished: fibrillar centers (FCs), their dense fibrillar component (PFC), and granular component (HA), which, in turn, surrounds PFK. Transcription of rRNA occurs in the FC and on the border of the PC and PFC; therefore, upon activation of the formation of ribosomes, the FCs become clearly distinguishable. RRNA cutting and modifications occur in PFC, and subsequent stages of formation of ribosomal subunits, including loading of ribosomal proteins, occur in HA ^[21] .

Taurus Kahal

Cajal corpuscle (TC) is the nuclear corpuscle found in all eukaryotes . It is identified by the presence of the signature protein coilin and specific RNA (scaRNA). TK also contains SMN protein ( survival of motor neurons ). In TK, a high concentration of splicing small nuclear ribonucleoproteins (snRNP) and other RNA processing factors is observed; therefore, TK are believed to serve as assemblies and / or post-transcriptional modifications of splicing factors. TC is present in the nucleus during interphase, but disappears in mitosis. In the biogenesis of TC, the properties of a self-organizing structure are traced ^[23] .

When the intracellular localization of SMN was first studied by immunofluorescence , the protein was found in the entire cytoplasm, as well as in the nucleolar body, similar in size to TC and often located next to it. For this reason, this body was called the "twin TK" ( Eng. Gemini of CB ) or simply gem. However, it turned out that the HeLa cell line in which the new body was discovered was unusual: in other human cell lines, as well as in the fruit fly, Drosophila melanogaster SMN was colocalized with coilin in TC. Therefore, in the general case, SMN can be considered as an important component of TC, and not as a marker of an individual nuclear body ^[24] .

Body of histone loci

The histone locus body (HLB ) body contains factors necessary for the processing of histone pre-mRNA. As the name implies, histone locus bodies are associated with genes encoding histones; therefore, it is assumed that splicing factors are concentrated in the bodies of histone loci. The body of histone loci is present in the cell during interphase and disappears with the onset of mitosis. The body of histone loci is often considered together with the Kakhal body for several reasons. Firstly, in some bodies of histone loci there is a marker of Kakhal's bodies - coilin. Secondly, these little bodies are often physically close by, so there is some interaction between them. Finally, very large Cajal bodies of amphibian oocytes possess the properties of both bodies ^[23] .

PML Taurus

The bodies of promyelocytic leukemia ( English Promyelocytic leukaemia bodies ), or PML bodies, are spherical bodies scattered throughout the nucleoplasm and reaching about 0.1-1.0 microns in diameter. They are also known under such names as nuclear domain 10 ( Eng. Nuclear domain 10 (ND10) ), Kremer bodies ( Eng. Kremer bodies ) and PML oncogenic domains ( Eng. PML oncogenic domains ). PML bodies are named for one of their key components - Promyelocytic Leukemia Protein (PML). They are often observed associated with Cahal bodies and division bodies ( English cleavage body ) ^[25] . PML bodies belong to the nuclear matrix and can be involved in processes such as DNA replication , transcription, and epigenetic gene silencing ^[26] . The key factor in the organization of these bodies is the PML protein, which attracts other proteins; the latter, according to the ideas of the 21st century, are united only by the fact that they are . Mice in which the PML gene has been deleted are devoid of PML bodies, however, they develop and live normally - this means that PML bodies do not perform irreplaceable biological functions ^[26] .

Speckles

Speckles are speckles that contain pre-mRNA splicing factors and are located in the interchromatin regions of the mammalian cell nucleoplasm. Under fluorescence microscopy, speckles look like spotted bodies of irregular shape, of various sizes, and under electron microscopy they look like clusters of interchromatin granules. Speckles are dynamic structures, and the proteins and RNA contained in them can move between speckles and other nuclear bodies, including sites of active transcription. Based on studies of the composition, structure and behavior of speckles, a model was created that explains the functional compartmentalization of the nucleus and the organization of the mechanism of gene expression ^[27] splicing small nuclear ribonucleoproteins ^[28] and other proteins necessary for pre-mRNA splicing ^[27] . Due to the changing needs of the cell, the speckle composition and arrangement change according to mRNA transcription and through regulation of specific protein phosphorylation ^[29] . Splicing speckles are also known as nuclear speckles, compartments of splicing factors, clusters of interchromatin granules and B-snurposomes ( English B snurposomes ) ^[30] . B-snurpososomes were found in the amphibian nuclei of amphibians and in the embryos of the fruit fly Drosophila melanogaster ^[31] . In electron micrographs, B-snurposomes appear attached to or separately from Kahal's bodies. Clusters of interchromatin granules serve as clusters of splicing factors ^[32] .

Paraspeckles

Paraspeckles are irregularly shaped nuclear bodies located in the interchromatin space of the nucleus ^[33] . They were first described in HeLa cells, which have 10–30 para speckles per nucleus, but now para speckles are found in all primary human cells, in cells of transformed lines, and on tissue sections ^[34] . They got their name because of their location in the core - near speckles ^[33] .

Paraspeckles are dynamic structures that change in response to changes in the metabolic activity of a cell. They depend on transcription ^[33] , and in the absence of transcription by RNA polymerase II , paraspecules disappear, and all their constituent proteins (PSP1, p54nrb, PSP2, CFI (m) 68 and PSF) form a crescent perinucleolar cap . This phenomenon is observed during the cell cycle: paraspeckles are present in the interphase and all phases of mitosis, with the exception of telophase . During the telophase, daughter nuclei are formed, and RNA polymerase II does not transcribe anything; therefore, paraspeckle proteins form a perinucleolar cap ^[34] . Paraspeckles are involved in the regulation of gene expression, accumulating those RNAs where there are double-stranded regions that undergo editing, namely, the conversion of adenosine to inosine . Due to this mechanism, paraspeckles are involved in the control of gene expression during differentiation , viral infection, and stress ^[35] .

Peripolar nucleus compartment

The perinucleolar compartment (OK) is an irregularly shaped nuclear body that is characterized by being located on the periphery of the nucleolus. Despite the physical connection, the two compartments are structurally different. OK is usually found in malignant tumor cells ^[36] . OK is a dynamic structure and contains a lot of RNA-binding proteins and RNA polymerase III. OK structural stability is provided by transcription by RNA polymerase III and the presence of key proteins. Since the presence of OK is usually associated with malignancy and with the ability to metastasize , they are considered as potential markers of cancer and other malignant tumors. An association of OK with specific DNA loci was shown ^[37] .

Stress Nuclear Calfs

Stressful nuclear bodies are formed in the nucleus during heat shock. They are formed upon the direct interaction of the transcription factor of heat shock 1 ( ) and pericentric tandem repeats in the sequence of satellite III, which corresponds to sites of active transcription of non-coding transcripts of satellite III. It is widely believed that such bodies correspond to very tightly packed forms of ribonucleoprotein complexes. In stressed cells, they are thought to be involved in rapid, temporary and global changes in gene expression through various mechanisms - for example, chromatin remodeling and capture of transcription and splicing factors . In cells under normal (not stressful) conditions, stressful nuclear bodies are rarely detected, but their number increases dramatically under the influence of heat shock. Stressful nuclear bodies were found only in cells of humans and other primates ^[38] .

Nuclear Orphans

Orphan nuclear bodies are non-chromatin nuclear compartments that have been studied much worse than other well-characterized nuclear structures. Some of them act as places where proteins are modified by SUMO proteins and / or proteasome degradation of proteins labeled with ubiquitin occurs ^[39] . The table below shows the characteristics of known nuclear orphan bodies ^[40] .

• The Nucleus / Tom Misteli, David L. Spector. - New York: Cold Spring Harbor Perspectives in Biology, 2011 .-- 463 p. - ISBN 978-0-87969-894-2 .