Phase separated microenvironments inside the cell nucleus are linked to disease and regulate epigenetic state, transcription and RNA processing.

Proteins and RNAs inside the cell nucleus are organized into distinct phases, also known as liquid-liquid phase separated (LLPS) droplet organelles or nuclear bodies. These regions exist within the spaces between chromatin-rich regions but their function is tightly linked to gene activity. They include major microscopically-observable structures such as the nucleolus, paraspeckle and Cajal body. The biochemical and assembly factors enriched inside these microenvironments regulate chromatin structure, transcription, and RNA processing, and other important cellular functions. Here, we describe published evidence that suggests nuclear bodies are bona fide LLPS droplet organelles and major regulators of the processes listed above. We also outline an updated "Supply or Sequester" model to describe nuclear body function, in which proteins or RNAs are supplied to surrounding genomic regions or sequestered away from their sites of activity. Finally, we describe recent evidence that suggests these microenvironments are both reflective and drivers of diverse pathophysiological states.

The SP100 component of ND10 enhances accumulation of PML and suppresses replication and the assembly of HSV replication compartments.

Nuclear domain 10 (ND10) bodies are small (0.1-1 µM) nuclear structures containing both constant [e.g., promyelocytic leukemia protein (PML), SP100, death domain-associated protein (Daxx)] and variable proteins, depending on the function of the cells or the stress to which they are exposed. In herpes simplex virus (HSV)-infected cells, ND10 bodies assemble at the sites of DNA entering the nucleus after infection. In sequence, the ND10 bodies become viral replication compartments, and ICP0, a viral E3 ligase, degrades both PML and SP100. The amounts of PML and SP100 and the number of ND10 structures increase in cells exposed to IFN-ß. Earlier studies have shown that PML has three key functions. Thus, (i) the interaction of PML with viral components facilitates the initiation of replication compartments, (ii) viral replication is significantly less affected by IFN-ß in PML-/- cells than in parental PML+/+ cells, and (iii) viral yields are significantly lower in PML-/- cells exposed to low ratios of virus per cell compared with parental PML+/+ cells. This report focuses on the function of SP100. In contrast to PML-/- cells, SP100-/- cells retain the sensitivity of parental SP100+/+ cells to IFN-ß and support replication of the ΔICP0 virus. At low multiplicities of infection, wild-type virus yields are higher in SP100-/- cells than in parental HEp-2 cells. In addition, the number of viral replication compartments is significantly higher in SP100-/- cells than in parental SP100+/+ cells or in PML-/- cells.

Heterochromatin restricts the mobility of nuclear bodies.

Nuclear bodies are relatively immobile organelles. Here, we investigated the mechanisms underlying their movement using experimentally induced interphase prenucleolar bodies (iPNBs). Most iPNBs demonstrated constrained diffusion, exhibiting infrequent fusions with other iPNBs and nucleoli. Fusion events were actin-independent and appeared to be the consequence of stochastic collisions between iPNBs. Most iPNBs were surrounded by condensed chromatin, while fusing iPNBs were usually found in a single heterochromatin-delimited compartment ("cage"). The experimentally induced over-condensation of chromatin significantly decreased the frequency of iPNB fusion. Thus, the data obtained indicate that the mobility of nuclear bodies is restricted by heterochromatin.

Nuclear speckles are detention centers for transcripts containing expanded CAG repeats.

The human genetic disorders caused by CAG repeat expansions in the translated sequences of various genes are called polyglutamine (polyQ) diseases because of the cellular "toxicity" of the mutant proteins. The contribution of mutant transcripts to the pathogenesis of these diseases is supported by several observations obtained from cellular models of these disorders. Here, we show that the common feature of cell lines modeling polyQ diseases is the formation of nuclear CAG RNA foci. We performed qualitative and quantitative analyses of these foci in numerous cellular models endogenously and exogenously expressing mutant transcripts by fluorescence in situ hybridization (FISH). We compared the CAG RNA foci of polyQ diseases with the CUG foci of myotonic dystrophy type 1 and found substantial differences in their number and morphology. Smaller differences within the polyQ disease group were also revealed and included a positive correlation between the foci number and the CAG repeat length. We show that expanded CAA repeats, also encoding glutamine, did not trigger RNA foci formation and foci formation is independent of the presence of mutant polyglutamine protein. Using FISH combined with immunofluorescence, we demonstrated partial co-localization of CAG repeat foci with MBNL1 alternative splicing factor, which explains the mild deregulation of MBNL1-dependent genes. We also showed that foci reside within nuclear speckles in diverse cell types: fibroblasts, lymphoblasts, iPS cells and neuronal progenitors and remain dependent on integrity of these nuclear structures.

Involvement of SRSF11 in cell cycle-specific recruitment of telomerase to telomeres at nuclear speckles.

Telomerase, a unique ribonucleoprotein complex that contains the telomerase reverse transcriptase (TERT), the telomerase RNA component (TERC) and the TERC-binding protein dyskerin, is required for continued cell proliferation in stem cells and cancer cells. Here we identify SRSF11 as a novel TERC-binding protein that localizes to nuclear speckles, subnuclear structures that are enriched in pre-messenger RNA splicing factors. SRSF11 associates with active telomerase enzyme through an interaction with TERC and directs it to nuclear speckles specifically during S phase of the cell cycle. On the other hand, a subset of telomeres is shown to be constitutively present at nuclear speckles irrespective of cell cycle phase, suggesting that nuclear speckles could be the nuclear sites for telomerase recruitment to telomeres. SRSF11 also associates with telomeres through an interaction with TRF2, which facilitates translocation of telomerase to telomeres. Depletion of SRSF11 prevents telomerase from associating with nuclear speckles and disrupts telomerase recruitment to telomeres, thereby abrogating telomere elongation by telomerase. These findings suggest that SRSF11 acts as a nuclear speckle-targeting factor that is essential for telomerase association with telomeres through the interactions with TERC and TRF2, and provides a potential target for modulating telomerase activity in cancer.

Identification of a chemical inhibitor for nuclear speckle formation: implications for the function of nuclear speckles in regulation of alternative pre-mRNA splicing.

Nuclear speckles are subnuclear structures enriched with RNA processing factors and poly (A)(+) RNAs comprising mRNAs and poly (A)(+) non-coding RNAs (ncRNAs). Nuclear speckles are thought to be involved in post-transcriptional regulation of gene expression, such as pre-mRNA splicing. By screening 3585 culture extracts of actinomycetes with in situ hybridization using an oligo dT probe, we identified tubercidin, an analogue of adenosine, as an inhibitor of speckle formation, which induces the delocalization of poly (A)(+) RNA and dispersion of splicing factor SRSF1/SF2 from nuclear speckles in HeLa cells. Treatment with tubercidin also decreased steady-state MALAT1 long ncRNA, thought to be involved in the retention of SRSF1/SF2 in nuclear speckles. In addition, we found that tubercidin treatment promoted exon skipping in the alternative splicing of Clk1 pre-mRNA. These results suggest that nuclear speckles play a role in modulating the concentration of splicing factors in the nucleoplasm to regulate alternative pre-mRNA splicing.

In and around the nucleus.

Studies of how the eukaryotic nucleus is functionally organized have led to the realization that nuclei are incredibly dynamic. Many nuclear structures are actually by products of a large steady-state flux of macromolecules through a given domain. A recent conference in the south of France on Nuclear Structure and Dynamics brought together scientists with diverse perspectives on the nucleus to try to provide a more coherent picture of the nucleus's dynamic organization and how this architecture is entwined with epigenetic control of gene expression.

Localization of rRNA transcribed spacer domains in the nucleolinus and maternal procentrosomes of surf clam (Spisula) oocytes.

The nucleolinus is a nuclear subcompartment long ago posited to play a role in cell division. In a recent study using surf clam oocytes, cytoplasmic foci containing a nucleolinar protein were shown to later recruit γ-tubulin, identifying them as centrosomal precursors. (1) We now demonstrate the presence of structural RNAs from the nucleolinus in these procentrosomes. They include the well-known but poorly understood rRNA-transcribed spacer regions. In situ hybridization revealed a specific and dynamic association of these structural RNAs with the cell division apparatus that extends through the early stages of meiosis. In addition to their bearing on the debate over the nature of centrosome- and spindle-associated RNAs, the observations also suggest that rRNA spacer regions are not simply waste products to be discarded immediately, but may be functional byproducts that play a role in formation of the cell division apparatus.

Nuclear architecture--an island no more.

The eukaryotic nucleus has been the neglected child of cell biology. The "International Symposium on Functional Organization of the Nucleus" held in January on Awaji Island, Japan, highlighted recent work on nuclear organization and function. Emerging from this conference was a holistic view in which diverse chemical and physical signals link the nuclear and cytoplasmic compartments of cells.

Interplay of cGAS with micronuclei: Regulation and diseases.

In higher eukaryotes, sophisticate regulation of genome function requires all chromosomes to be packed into a single nucleus. Micronucleus (MN), the dissociative nucleus-like structure frequently observed in aging and multiple disease settings, has critical, yet under-recognized, pathophysiological functions. Micronuclei (MNi) have recently emerged as major sources of cytosolic DNA that can activate the cGAS-STING axis in a cell-intrinsic manner. However, MNi induced from different genotoxic stressors display great heterogeneity in binding or activating cGAS and the signaling responses downstream of the MN-induced cGAS-STING axis have divergent outcomes including autoimmunity, autoinflammation, metastasis, or cell death. Thus, full characterization of molecular network underpinning the interplay of cGAS and MN is important to elucidate the pathophysiological roles of immunogenic MN and design improved drugs that selectively target cancer via boosting the MN-derived cGAS-STING axis. Here, we summarize our current understanding of the mechanisms for self-DNA discrimination by cGAS. We focus on discussing how MN immunogencity is dictated by multiple mechanisms including integrity of micronuclear envelope, state of nucleosome and DNA, competitive factors, damaged mitochondrial DNA and micronucleophagy. We also describe emerging links between immunogenic MN and human diseases including cancer, neurodegenerative diseases and COVID-19. Particularly, we explore the exciting concept of inducing immunogenic MN as a therapeutic approach in treating cancer. We propose a new theoretical framework to describe immunogenic MN as a biological sensor to modulate cellular processes in response to genotoxic stress and provide perspectives on developing novel experimental approaches to unravel the complexity of MN immunogenicity regulation and immunogenic MN pathophysiology.

RAG2 abolishes RAG1 aggregation to facilitate V(D)J recombination.

RAG1 and RAG2 form a tetramer nuclease to initiate V(D)J recombination in developing T and B lymphocytes. The RAG1 protein evolves from a transposon ancestor and possesses nuclease activity that requires interaction with RAG2. Here, we show that the human RAG1 aggregates in the nucleus in the absence of RAG2, exhibiting an extremely low V(D)J recombination activity. In contrast, RAG2 does not aggregate by itself, but it interacts with RAG1 to disrupt RAG1 aggregates and thereby activate robust V(D)J recombination. Moreover, RAG2 from mouse and zebrafish could not disrupt the aggregation of human RAG1 as efficiently as human RAG2 did, indicating a species-specific regulatory mechanism for RAG1 by RAG2. Therefore, we propose that RAG2 coevolves with RAG1 to release inert RAG1 from aggregates and thereby activate V(D)J recombination to generate diverse antigen receptors in lymphocytes.

Superresolution light microscopy of the Drosophila histone locus body reveals a core-shell organization associated with expression of replication-dependent histone genes.

The histone locus body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of replication-dependent (RD) histone mRNAs, which are the only eukaryotic mRNAs lacking a poly-A tail. Many nuclear bodies contain distinct domains, but how internal organization is related to nuclear body function is not fully understood. Here, we demonstrate using structured illumination microscopy that Drosophila HLBs have a "core-shell" organization in which the internal core contains transcriptionally active RD histone genes. The N-terminus of Mxc, which contains a domain required for Mxc oligomerization, HLB assembly, and RD histone gene expression, is enriched in the HLB core. In contrast, the C-terminus of Mxc is enriched in the HLB outer shell as is FLASH, a component of the active U7 snRNP that cotranscriptionally cleaves RD histone pre-mRNA. Consistent with these results, we show biochemically that FLASH binds directly to the Mxc C-terminal region. In the rapid S-M nuclear cycles of syncytial blastoderm Drosophila embryos, the HLB disassembles at mitosis and reassembles the core-shell arrangement as histone gene transcription is activated immediately after mitosis. Thus, the core-shell organization is coupled to zygotic histone gene transcription, revealing a link between HLB internal organization and RD histone gene expression.

Genome-wide screen of three herpesviruses for protein subcellular localization and alteration of PML nuclear bodies.

Herpesviruses are large, ubiquitous DNA viruses with complex host interactions, yet many of the proteins encoded by these viruses have not been functionally characterized. As a first step in functional characterization, we determined the subcellular localization of 234 epitope-tagged proteins from herpes simplex virus, cytomegalovirus, and Epstein-Barr virus. Twenty-four of the 93 proteins with nuclear localization formed subnuclear structures. Twelve of these localized to the nucleolus, and five at least partially localized with promyelocytic leukemia (PML) bodies, which are known to suppress viral lytic infection. In addition, two proteins disrupted Cajal bodies, and 19 of the nuclear proteins significantly decreased the number of PML bodies per cell, including six that were shown to be SUMO-modified. These results have provided the first functional insights into over 120 previously unstudied proteins and suggest that herpesviruses employ multiple strategies for manipulating nuclear bodies that control key cellular processes.

Nuclear stress granules: the awakening of a sleeping beauty?

Nuclear stress granules are subnuclear compartments that form in response to heat shock and other stress stimuli. Although many components of nuclear stress granules have been identified, including HSF1 and pre-mRNA processing factors, their function remains a mystery. A paper in this issue describes the stress-induced transcriptional activation of one of the nuclear stress granule target sites, a heterochromatic region that has been considered silent (Jolly et al., 2004). These intriguing findings will certainly give the research of these structures a new twist.

Stress-induced transcription of satellite III repeats.

Exposure of mammalian cells to stress induces the activation of heat shock transcription factor 1 (HSF1) and the subsequent transcription of heat shock genes. Activation of the heat shock response also correlates with a rapid relocalization of HSF1 within a few nuclear structures termed nuclear stress granules. These stress-induced structures, which form primarily on the 9q12 region in humans through direct binding of HSF1 to satellite III repeats, do not colocalize with transcription sites of known hsp genes. In this paper, we show that nuclear stress granules correspond to RNA polymerase II transcription factories where satellite III repeats are transcribed into large and stable RNAs that remain associated with the 9q12 region, even throughout mitosis. This work not only reveals the existence of a new major heat-induced transcript in human cells that may play a role in chromatin structure, but also provides evidence for a transcriptional activity within a locus considered so far as heterochromatic and silent.

Conception of an image data base for cell nuclei and geometric algorithms for diagnosis and therapy monitoring.

Parameters of the genome architecture of cell nuclei like copy number changes of genes or numerical and structural aberrations of chromosomes displayed by changes of size, shape, form and geometric arrangement of the related territories and domains play an important role in tumour diagnosis and monitoring of tumour therapy. We have defined data structures for such parameters, accompanied by meta data describing cell biology and microscopy protocols, and developed algorithms to deduce geometric data from microscopic raw images of fluorescently labelled cell nuclei. The statistical evaluation of nucleus geometry and architecture data is a valuable aid for diagnostic decisions and monitoring of cancer development, as indicated by several research case studies. The algorithms and data storage devices are presently administrated by different operating systems. Unification of workflow is being achieved for a local cluster, but gridification is still subject to problems of licensing, monitoring, and administering systems, including data security.

DNA damage induces alternative lengthening of telomeres (ALT) associated promyelocytic leukemia bodies that preferentially associate with linear telomeric DNA.

The linear chromosomes of vertebrates terminate in telomeres that consist of a tandemly repeated hexameric sequence, 5'TTAGGG3'. Telomeres form a protective loop structure (t-loop), which is thought to prevent them from being recognized as a double-strand break. Approximately 10% of human tumors prevent shortening of their telomeres by using a recombination-mediated alternative lengthening of telomeres (ALT) mechanism. ALT-positive human cells contain extrachromosomal telomere repeat (ECTR) DNA that may either be circular or linear. It has been proposed that ECTR may be generated by recombination events involving the t-loop. A proportion of the cells within ALT-positive cell populations contain promyelocytic leukemia (PML) nuclear bodies that contain telomeric DNA and telomere-binding proteins that are called ALT-associated PML bodies (APB). Although the presence of APBs is very useful for determining whether tumors and cell lines use the ALT mechanism, the function of APBs is unknown. It has previously been shown that telomeric DNA is particularly susceptible to damage by hydrogen peroxide and N-methyl-N'-nitro-N-nitrosoguanidine. We report here that these DNA-damaging agents induce both linear and circular ECTR DNA in ALT cells and increase the proportion of cells that contain APBs. We partially purified APBs and showed that the telomeric repeat DNA they contain is predominantly linear. We propose that a function of APBs is to sequester linear telomeric DNA.

Transcriptional repression, apoptosis, human disease and the functional evolution of the nuclear lamina.

The number and complexity of genes encoding nuclear lamina proteins has increased during metazoan evolution. Emerging evidence reveals that transcriptional repressors such as the retinoblastoma protein, and apoptotic regulators such as CED-4, have functional and dynamic interactions with the lamina. The discovery that mutations in nuclear lamina proteins cause heritable tissue-specific diseases, including Emery-Dreifuss muscular dystrophy, is prompting a fresh look at the nuclear lamina to devise models that can account for its diverse functions and dynamics, and to understand its enigmatic structure.

The human I-mfa domain-containing protein, HIC, interacts with cyclin T1 and modulates P-TEFb-dependent transcription.

Positive transcription elongation factor b (P-TEFb) hyperphosphorylates the carboxy-terminal domain of RNA polymerase II, permitting productive transcriptional elongation. The cyclin T1 subunit of P-TEFb engages cellular transcription factors as well as the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. To identify potential P-TEFb regulators, we conducted a yeast two-hybrid screen with cyclin T1 as bait. Among the proteins isolated was the human I-mfa domain-containing protein (HIC). HIC has been reported to modulate expression from both cellular and viral promoters via its C-terminal cysteine-rich domain, which is similar to the inhibitor of MyoD family a (I-mfa) protein. We show that HIC binds cyclin T1 in yeast and mammalian cells and that it interacts with intact P-TEFb in mammalian cell extracts. The interaction involves the I-mfa domain of HIC and the regulatory histidine-rich region of cyclin T1. HIC also binds Tat via its I-mfa domain, although the sequence requirements are different. HIC colocalizes with cyclin T1 in nuclear speckle regions and with Tat in the nucleolus. Expression of the HIC cDNA modulates Tat transactivation of the HIV-1 long terminal repeat (LTR) in a cell type-specific fashion. It is mildly inhibitory in CEM cells but stimulates gene expression in HeLa, COS, and NIH 3T3 cells. The isolated I-mfa domain acts as a dominant negative inhibitor. Activation of the HIV-1 LTR by HIC in NIH 3T3 cells occurs at the RNA level and is mediated by direct interactions with P-TEFb.

Prespliceosomal assembly on microinjected precursor mRNA takes place in nuclear speckles.

Nuclear speckles (speckles) represent a distinct nuclear compartment within the interchromatin space and are enriched in splicing factors. They have been shown to serve neighboring active genes as a reservoir of these factors. In this study, we show that, in HeLa cells, the (pre)spliceosomal assembly on precursor mRNA (pre-mRNA) is associated with the speckles. For this purpose, we used microinjection of splicing competent and mutant adenovirus pre-mRNAs with differential splicing factor binding, which form different (pre)spliceosomal complexes and followed their sites of accumulation. Splicing competent pre-mRNAs are rapidly targeted into the speckles, but the targeting is temperature-dependent. The polypyrimidine tract sequence is required for targeting, but, in itself, is not sufficient. The downstream flanking sequences are particularly important for the targeting of the mutant pre-mRNAs into the speckles. In supportive experiments, the behavior of the speckles was followed after the microinjection of antisense deoxyoligoribonucleotides complementary to the specific domains of snRNAs. Under these latter conditions prespliceosomal complexes are formed on endogenous pre-mRNAs. We conclude that the (pre)spliceosomal complexes on microinjected pre-mRNA are formed inside the speckles. Their targeting into and accumulation in the speckles is a result of the cumulative loading of splicing factors to the pre-mRNA and the complexes formed give rise to the speckled pattern observed.