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.

Integrator subunit 4 is a 'Symplekin-like' scaffold that associates with INTS9/11 to form the Integrator cleavage module.

Integrator (INT) is a transcriptional regulatory complex associated with RNA polymerase II that is required for the 3'-end processing of both UsnRNAs and enhancer RNAs. Integrator subunits 9 (INTS9) and INTS11 constitute the catalytic core of INT and are paralogues of the cleavage and polyadenylation specificity factors CPSF100 and CPSF73. While CPSF73/100 are known to associate with a third protein called Symplekin, there is no paralog of Symplekin within INT raising the question of how INTS9/11 associate with the other INT subunits. Here, we have identified that INTS4 is a specific and conserved interaction partner of INTS9/11 that does not interact with either subunit individually. Although INTS4 has no significant homology with Symplekin, it possesses N-terminal HEAT repeats similar to Symplekin but also contains a ß-sheet rich C-terminal region, both of which are important to bind INTS9/11. We assess three functions of INT including UsnRNA 3'-end processing, maintenance of Cajal body structural integrity, and formation of histone locus bodies to conclude that INTS4/9/11 are the most critical of the INT subunits for UsnRNA biogenesis. Altogether, these results indicate that INTS4/9/11 compose a heterotrimeric complex that likely represents the Integrator 'cleavage module' responsible for its endonucleolytic activity.

Chromatin loops and causality loops: the influence of RNA upon spatial nuclear architecture.

An intrinsic and essential trait exhibited by cells is the properly coordinated and integrated regulation of an astoundingly large number of simultaneous molecular decisions and reactions to maintain biochemical homeostasis. This is especially true inside the cell nucleus, where the recognition of DNA and RNA by a vast range of nucleic acid-interacting proteins organizes gene expression patterns. However, this dynamic system is not regulated by simple "on" or "off" signals. Instead, transcription factor and RNA polymerase recruitment to DNA are influenced by the local chromatin and epigenetic environment, a gene's relative position within the nucleus and the action of noncoding RNAs. In addition, major phase-separated structural features of the nucleus, such as nucleoli and paraspeckles, assemble in direct response to specific transcriptional activities and, in turn, influence global genomic function. Currently, the interpretation of these data is trapped in a causality dilemma reminiscent of the "chicken and the egg" paradox as it is unclear whether changes in nuclear architecture promote RNA function or vice versa. Here, we review recent advances that suggest a complex and interdependent interaction network between gene expression, chromatin topology, and noncoding RNA function. We also discuss the functional links between these essential nuclear processes from the nanoscale (gene looping) to the macroscale (sub-nuclear gene positioning and nuclear body function) and briefly highlight some of the challenges that researchers may encounter when studying these phenomena.

Cajal body function in genome organization and transcriptome diversity.

Nuclear bodies contribute to non-random organization of the human genome and nuclear function. Using a major prototypical nuclear body, the Cajal body, as an example, we suggest that these structures assemble at specific gene loci located across the genome as a result of high transcriptional activity. Subsequently, target genes are physically clustered in close proximity in Cajal body-containing cells. However, Cajal bodies are observed in only a limited number of human cell types, including neuronal and cancer cells. Ultimately, Cajal body depletion perturbs splicing kinetics by reducing target small nuclear RNA (snRNA) transcription and limiting the levels of spliceosomal snRNPs, including their modification and turnover following each round of RNA splicing. As such, Cajal bodies are capable of shaping the chromatin interaction landscape and the transcriptome by influencing spliceosome kinetics. Future studies should concentrate on characterizing the direct influence of Cajal bodies upon snRNA gene transcriptional dynamics. Also see the video abstract here.

Specific genomic cues regulate Cajal body assembly.

The assembly of specialized sub-nuclear microenvironments known as nuclear bodies (NBs) is important for promoting efficient nuclear function. In particular, the Cajal body (CB), a prominent NB that facilitates spliceosomal snRNP biogenesis, assembles in response to genomic cues. Here, we detail the factors that regulate CB assembly and structural maintenance. These include the importance of transcription at nucleating gene loci, the grouping of these genes on human chromosomes 1, 6 and 17, as well as cell cycle and biochemical regulation of CB protein function. We also speculate on the correlation between CB formation and RNA splicing levels in neurons and cancer. The timing and location of these specific molecular events is critical to CB assembly and its contribution to genome function. However, further work is required to explore the emerging biophysical characteristics of CB assembly and the impact upon subsequent genome reorganization.

A 28-kDa splice variant of NADPH oxidase-4 is nuclear-localized and involved in redox signaling in vascular cells.

OBJECTIVE: Reactive oxygen species-generating nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase proteins (Noxs) are involved in cell differentiation, migration, and apoptosis. Nox4 is unique among Noxs in being constitutively active, and its subcellular localization may therefore be particularly important. In this study, we identified and characterized a novel nuclear-localized 28-kDa splice variant of Nox4 in vascular cells. APPROACH AND RESULTS: Nox4 immunoreactivity was noted in the nucleus and nucleolus of vascular smooth muscle cells and multiple other cell types by confocal microscopy. Cell fractionation, sequence analyses, and siRNA studies indicated that the nuclear-localized Nox4 is a 28-kDa splice variant, Nox4D, which lacks putative transmembrane domains. Nox4D overexpression resulted in significant NADPH-dependent reactive oxygen species production as detected by several different methods and caused increased phosphorylation of extracellular-signal-regulated kinase1/2 and the nuclear transcription factor Elk-1. Overexpression of Nox4D could also induce DNA damage as assessed by γ-H2AX phosphorylation. These effects were inhibited by a single amino acid substitution in the Nox4D NADPH-binding region. CONCLUSIONS: Nox4D is a nuclear-localized and functionally active splice variant of Nox4 that may have important pathophysiologic effects through modulation of nuclear signaling and DNA damage.

NADPH oxidase-4 promotes eccentric cardiac hypertrophy in response to volume overload.

AIMS: Chronic pressure or volume overload induce concentric vs. eccentric left ventricular (LV) remodelling, respectively. Previous studies suggest that distinct signalling pathways are involved in these responses. NADPH oxidase-4 (Nox4) is a reactive oxygen species-generating enzyme that can limit detrimental cardiac remodelling in response to pressure overload. This study aimed to assess its role in volume overload-induced remodelling. METHODS AND RESULTS: We compared the responses to creation of an aortocaval fistula (Shunt) to induce volume overload in Nox4-null mice (Nox4-/-) vs. wild-type (WT) littermates. Induction of Shunt resulted in a significant increase in cardiac Nox4 mRNA and protein levels in WT mice as compared to Sham controls. Nox4-/- mice developed less eccentric LV remodelling than WT mice (echocardiographic relative wall thickness: 0.30 vs. 0.27, P < 0.05), with less LV hypertrophy at organ level (increase in LV weight/tibia length ratio of 25% vs. 43%, P < 0.01) and cellular level (cardiomyocyte cross-sectional area: 323 µm2 vs. 379 µm2, P < 0.01). LV ejection fraction, foetal gene expression, interstitial fibrosis, myocardial capillary density, and levels of myocyte apoptosis after Shunt were similar in the two genotypes. Myocardial phospho-Akt levels were increased after induction of Shunt in WT mice, whereas levels decreased in Nox4-/- mice (+29% vs. -21%, P < 0.05), associated with a higher level of phosphorylation of the S6 ribosomal protein (S6) and the eIF4E-binding protein 1 (4E-BP1) in WT compared to Nox4-/- mice. We identified that Akt activation in cardiac cells is augmented by Nox4 via a Src kinase-dependent inactivation of protein phosphatase 2A. CONCLUSION: Endogenous Nox4 is required for the full development of eccentric cardiac hypertrophy and remodelling during chronic volume overload. Nox4-dependent activation of Akt and its downstream targets S6 and 4E-BP1 may be involved in this effect.

Membraneless nuclear organelles and the search for phases within phases.

Cells are segregated into two distinct compartment groups to optimize cellular function. The first is characterized by lipid membranes that encapsulate specific regions and regulate macromolecular flux. The second, known collectively as membraneless organelles (MLOs), lacks defining lipid membranes and exhibits self-organizing properties. MLOs are enriched with specific RNAs and proteins that catalyze essential cellular processes. A prominent sub-class of MLOs are known as nuclear bodies, which includes nucleoli, paraspeckles, and other droplets. These microenvironments contain specific RNAs, exhibit archetypal liquid-liquid phase separation characteristics, and harbor intrinsically disordered, multivalent hub proteins. We present an analysis of nuclear body protein disorder that suggests MLO proteomes are significantly more disordered than structured cellular features. We also outline common MLO ultrastructural features, exemplified by the three sub-compartments present inside the nucleolus. A core-shell configuration, or phase within a phase, is displayed by several nuclear bodies and may be functionally important. Finally, we summarize evidence indicating extensive RNA and protein sharing between distinct nuclear bodies, suggesting functional cooperation and similar nucleation principles. Considering the substantial accumulation of specific coding and noncoding RNA classes inside MLOs, evidence that RNA buffers specific phase transition events, and the absence of a clear correlation between total intrinsic protein disorder and MLO accumulation, we conclude that RNA biogenesis may play a key role in MLO formation, internal organization, and function. This article is categorized under: RNA Export and Localization > RNA Localization RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Nuclear bodies: Built to boost.

The classic archetypal function of nuclear bodies is to accelerate specific reactions within their crowded space. In this issue, Tatomer et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201504043) provide the first direct evidence that the histone locus body acts to concentrate key factors required for the proper processing of histone pre-mRNAs.

Spectral imaging to visualize higher-order genomic organization.

A concern in the field of genomics is the proper interpretation of large, high-throughput sequencing datasets. The use of DNA FISH followed by high-content microscopy is a valuable tool for validation and contextualization of frequently occurring gene pairing events at the single-cell level identified by deep sequencing. However, these techniques possess certain limitations. Firstly, they do not permit the study of colocalization of many gene loci simultaneously. Secondly, the direct assessment of the relative position of many clustered gene loci within their respective chromosome territories is impossible. Thus, methods are required to advance the study of higher-order nuclear and cellular organization. Here, we describe a multiplexed DNA FISH technique combined with indirect immunofluorescence to study the relative position of 6 distinct genomic or cellular structures. This can be achieved in a single hybridization step using spectral imaging during image acquisition and linear unmixing. Here, we detail the use of this method to quantify gene pairing between highly expressed spliceosomal genes and compare these data to randomly positioned in silico simulated gene clusters. This is a potentially universally applicable approach for the validation of 3C-based technologies, deep imaging of spatial organization within the nucleus and global cellular organization.

Cajal bodies are linked to genome conformation.

The mechanisms underlying nuclear body (NB) formation and their contribution to genome function are unknown. Here we examined the non-random positioning of Cajal bodies (CBs), major NBs involved in spliceosomal snRNP assembly and their role in genome organization. CBs are predominantly located at the periphery of chromosome territories at a multi-chromosome interface. Genome-wide chromosome conformation capture analysis (4C-seq) using CB-interacting loci revealed that CB-associated regions are enriched with highly expressed histone genes and U small nuclear or nucleolar RNA (sn/snoRNA) loci that form intra- and inter-chromosomal clusters. In particular, we observed a number of CB-dependent gene-positioning events on chromosome 1. RNAi-mediated disassembly of CBs disrupts the CB-targeting gene clusters and suppresses the expression of U sn/snoRNA and histone genes. This loss of spliceosomal snRNP production results in increased splicing noise, even in CB-distal regions. Therefore, we conclude that CBs contribute to genome organization with global effects on gene expression and RNA splicing fidelity.

The vasoactive potential of kisspeptin-10 in the peripheral vasculature.

Splice products of the Kiss1 protein (kisspeptins) have been shown to be involved in a diverse range of functions, including puberty, metastasis and vasoconstriction in large human arteries. Circulating Kisspeptin-10 (Kp-10) plasma levels are low in normal individuals but are elevated during various disease states as well as pregnancy. Here, we investigated the potential of Kp-10, the shortest biologically active kisspeptin, to influence microvascular effects, concentrating on the cutaneous vasculature. Kp-10 caused a dose-dependent increase in oedema formation (0.3-10 nmol/injection site), assessed by Evans Blue albumin dye extravasation, in the dorsal skin of CD1 mice. Oedema formation was shown to be inhibited by the histamine H(1) receptor antagonist mepyramine. The response was characterised by a ring of pallor at the injection site in keeping with vasoconstrictor activity. Therefore, changes in dorsal skin blood flow were assessed by clearance of intradermally injected (99m)technetium. Kp-10 was found to significantly reduce clearance, in keeping with decreased blood flow and providing further evidence for vasoconstrictor activity. The decreased clearance was partially inhibited by co-treatment with the cyclo-oxygenase inhibitor indomethacin. Finally evidence for the kisspeptin receptor gene (Kiss1R), but not the kisspeptin peptide gene (Kiss1), mRNA expression was observed in heart, aorta and kidney samples from normal and angiotensin II induced hypertensive mice, with similar mRNA levels observed in each. We have evidence for two peripheral vasoactive roles for kisspeptin-10. Firstly, plasma extravasation indicative of ability to induce oedema formation and secondly decreased peripheral blood flow, indicating microvascular constriction. Thus Kp-10 has vasoactive properties in the peripheral microvasculature.