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.

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

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. Materials and MethodsMaterials and Methods are available in the online-only Supplement. Results Nuclear-Localized Nox4 ImmunoreactivityWe used a well-validated polyclonal antibody directed against the C-terminal region of Nox4 11,20,21 to investigate the localization of endogenous Nox4 by immunofluorescence in several cell types, including VSMC, endothelial cells, fibroblasts, and cardiomyocytes. A diffuse reticular staining pattern through the cell that colocalized with an ER marker, protein disulfide isomerase, was evident in most cell types ( Figures 1A and 2) as reported previously. 15,16 In addition, however, there was strong focal and granular Nox4 immunostaining within the nucleus in all cell types. Electron microscopy indicated that the focal Nox4 staining was in the nucleolus ( Figure 1C). We confirmed that the focal intranuclear staining was nucleolar by costaining with 2 different nucleolar markers, nucleophosmin and fibrillarin ( Figure 1B). Preincubation of the Nox4 antibody with the antigenic peptide against which it was raised completely abolished both the perinuclear and nucleolar staining, verifying the specificity of antigen-antibody interaction ( Figure 1D). Furthermore, treatment with a prevalidated Nox4 siRNA to knock down Nox4 protein expression also abolished staining as compared with a scrambled siRNA ( Figure 3A). Nuclear-Localized Nox4 Is a 28-kDa Nox4 Splice VariantTo confirm nuclear and nucleolar localization, VSMCs were fractionated into membrane and nuclear fractions. Immunoblotting for Nox4 demonstrated an ≈65-kDa band corresponding to the expected molecular size of Nox4 but ...

Nuclear bodies contribute to nonrandom 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 small nuclear RNA gene transcriptional dynamics.

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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 β-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.

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 β-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 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.

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 (0.3–10nmol/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 H1 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 99mtechnetium. 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.