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1.  NEAT1 long noncoding RNA regulates transcription via protein sequestration within subnuclear bodies 
Molecular Biology of the Cell  2014;25(1):169-183.
Paraspeckles are subnuclear structures formed around NEAT1 lncRNA. Paraspeckles became enlarged after proteasome inhibition caused by NEAT1 transcriptional activation, leading to protein sequestration into paraspeckles. The NEAT1-dependent sequestration affects the transcription of several genes, arguing for a novel role for lncRNA in gene regulation.
Paraspeckles are subnuclear structures formed around nuclear paraspeckle assembly transcript 1 (NEAT1)/MENε/β long noncoding RNA (lncRNA). Here we show that paraspeckles become dramatically enlarged after proteasome inhibition. This enlargement is mainly caused by NEAT1 transcriptional up-regulation rather than accumulation of undegraded paraspeckle proteins. Of interest, however, using immuno–electron microscopy, we find that key paraspeckle proteins become effectively depleted from the nucleoplasm by 50% when paraspeckle assembly is enhanced, suggesting a sequestration mechanism. We also perform microarrays from NEAT1-knockdown cells and find that NEAT1 represses transcription of several genes, including the RNA-specific adenosine deaminase B2 (ADARB2) gene. In contrast, the NEAT1-binding paraspeckle protein splicing factor proline/glutamine-rich (SFPQ) is required for ADARB2 transcription. This leads us to hypothesize that ADARB2 expression is controlled by NEAT1-dependent sequestration of SFPQ. Accordingly, we find that ADARB2 expression is strongly reduced upon enhanced SFPQ sequestration by proteasome inhibition, with concomitant reduction in SFPQ binding to the ADARB2 promoter. Finally, NEAT1−/− fibroblasts are more sensitive to proteasome inhibition, which triggers cell death, suggesting that paraspeckles/NEAT1 attenuates the cell death pathway. These data further confirm that paraspeckles are stress-responsive nuclear bodies and provide a model in which induced NEAT1 controls target gene transcription by protein sequestration into paraspeckles.
PMCID: PMC3873887  PMID: 24173718
2.  Paraspeckle formation during the biogenesis of long non-coding RNAs 
RNA Biology  2013;10(3):456-461.
Paraspeckles are unique subnuclear structures that are built around a specific long non-coding RNA (lncRNA), NEAT1, which is comprised of two isoforms (NEAT1_1 and NEAT1_2) that are produced by alternative 3′-end processing. NEAT1 lncRNAs are unusual RNA polymerase II transcripts that lack introns. The non-polyadenylated 3′-end of NEAT1_2 is non-canonically processed by RNase P. NEAT1_2 is an essential component for paraspeckle formation. Paraspeckles form during the NEAT1_2 lncRNA biogenesis process, which encompasses transcription from its own chromosome locus through lncRNA processing and accumulation. Recent RNAi analyses of 40 paraspeckle proteins (PSPs) identified four PSPs that are required for paraspeckle formation by mediating NEAT1 processing and accumulation. In particular, HNRNPK was shown to arrest CFIm-dependent NEAT1_1 polyadenylation, leading to NEAT1_2 synthesis. The other three PSPs were required for paraspeckle formation, but did not affect NEAT1_2 expression. This observation suggests that NEAT1_2 accumulation is necessary but not sufficient for paraspeckle formation. An additional step, presumably the bundling of NEAT1 ribonucleoprotein sub-complexes, may be required for construction of the intact paraspeckle structure. NEAT1 expression is likely regulated at transcriptional and post-transcriptional steps under certain stress conditions, suggesting roles for paraspeckles in the lncRNA-mediated regulation of gene expression, such as the nucleocytoplasmic transport of mRNA in response to certain stimuli.
PMCID: PMC3672290  PMID: 23324609
3′-end processing; RNA-binding protein; RNA-protein interaction; long non-coding RNA; nuclear bodies
3.  Phosphorothioate oligonucleotides can displace NEAT1 RNA and form nuclear paraspeckle-like structures 
Nucleic Acids Research  2014;42(13):8648-8662.
Nuclear paraspeckles are built co-transcriptionally around a long non-coding RNA, NEAT1. Here we report that transfected 20-mer phosphorothioate-modified (PS) antisense oligonucleotides (ASOs) can recruit paraspeckle proteins to form morphologically normal and apparently functional paraspeckle-like structures containing no NEAT1 RNA. PS-ASOs can associate with paraspeckle proteins, including P54nrb, PSF, PSPC1 and hnRNPK. NEAT1 RNA can be displaced by transfected PS-ASO from paraspeckles and rapidly degraded. Co-localization of PS-ASOs with P54nrb was observed in canonical NEAT1-containing paraspeckles, in perinucleolar caps upon transcriptional inhibition, and importantly, in paraspeckle-like or filament structures lacking NEAT1 RNA. The induced formation of paraspeckle-like and filament structures occurred in mouse embryonic stem cells expressing little or no NEAT1 RNA, suggesting that PS-ASOs can serve as seeding molecules to assemble paraspeckle-like foci in the absence of NEAT1 RNA. Moreover, CTN, an RNA reported to be functionally retained in paraspeckles, was also observed to localize to paraspeckle-like structures, implying that paraspeckle-like structures assembled on PS-ASOs are functional. Together, our results indicate that functional paraspeckles can form with short nucleic acids other than NEAT1 RNA.
PMCID: PMC4117792  PMID: 25013176
4.  The long non-coding RNA nuclear-enriched abundant transcript 1_2 induces paraspeckle formation in the motor neuron during the early phase of amyotrophic lateral sclerosis 
Molecular Brain  2013;6:31.
A long non-coding RNA (lncRNA), nuclear-enriched abundant transcript 1_2 (NEAT1_2), constitutes nuclear bodies known as “paraspeckles”. Mutations of RNA binding proteins, including TAR DNA-binding protein-43 (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS), have been described in amyotrophic lateral sclerosis (ALS). ALS is a devastating motor neuron disease, which progresses rapidly to a total loss of upper and lower motor neurons, with consciousness sustained. The aim of this study was to clarify the interaction of paraspeckles with ALS-associated RNA-binding proteins, and to identify increased occurrence of paraspeckles in the nucleus of ALS spinal motor neurons.
In situ hybridization (ISH) and ultraviolet cross-linking and immunoprecipitation were carried out to investigate interactions of NEAT1_2 lncRNA with ALS-associated RNA-binding proteins, and to test if paraspeckles form in ALS spinal motor neurons. As the results, TDP-43 and FUS/TLS were enriched in paraspeckles and bound to NEAT1_2 lncRNA directly. The paraspeckles were localized apart from the Cajal bodies, which were also known to be related to RNA metabolism. Analyses of 633 human spinal motor neurons in six ALS cases showed NEAT1_2 lncRNA was upregulated during the early stage of ALS pathogenesis. In addition, localization of NEAT1_2 lncRNA was identified in detail by electron microscopic analysis combined with ISH for NEAT1_2 lncRNA. The observation indicating specific assembly of NEAT1_2 lncRNA around the interchromatin granule-associated zone in the nucleus of ALS spinal motor neurons verified characteristic paraspeckle formation.
NEAT1_2 lncRNA may act as a scaffold of RNAs and RNA binding proteins in the nuclei of ALS motor neurons, thereby modulating the functions of ALS-associated RNA-binding proteins during the early phase of ALS. These findings provide the first evidence of a direct association between paraspeckle formation and a neurodegenerative disease, and may shed light on the development of novel therapeutic targets for the treatment of ALS.
PMCID: PMC3729541  PMID: 23835137
Long non-coding RNA; Paraspeckle; NEAT1_2; TDP-43; FUS/TLS; Amyotrophic lateral sclerosis; Electron microscopy
5.  Paraspeckles: nuclear bodies built on long noncoding RNA 
The Journal of Cell Biology  2009;186(5):637-644.
Paraspeckles are ribonucleoprotein bodies found in the interchromatin space of mammalian cell nuclei. These structures play a role in regulating the expression of certain genes in differentiated cells by nuclear retention of RNA. The core paraspeckle proteins (PSF/SFPQ, P54NRB/NONO, and PSPC1 [paraspeckle protein 1]) are members of the DBHS (Drosophila melanogaster behavior, human splicing) family. These proteins, together with the long nonprotein-coding RNA NEAT1 (MEN-ϵ/β), associate to form paraspeckles and maintain their integrity. Given the large numbers of long noncoding transcripts currently being discovered through whole transcriptome analysis, paraspeckles may be a paradigm for a class of subnuclear bodies formed around long noncoding RNA.
PMCID: PMC2742191  PMID: 19720872
6.  Paraspeckles are subpopulation-specific nuclear bodies that are not essential in mice 
The Journal of Cell Biology  2011;193(1):31-39.
Mouse NEAT1 is required for paraspeckle formation in a subset of cells but is not essential for animal health and viability.
Nuclei of higher organisms are well structured and have multiple, distinct nuclear compartments or nuclear bodies. Paraspeckles are recently identified mammal-specific nuclear bodies ubiquitously found in most cells cultured in vitro. To investigate the physiological role of paraspeckles, we examined the in vivo expression patterns of two long noncoding RNAs, NEAT1_1 and NEAT1_2, which are essential for the architectural integrity of nuclear bodies. Unexpectedly, these genes were only strongly expressed in a particular subpopulation of cells in adult mouse tissues, and prominent paraspeckle formation was observed only in the cells highly expressing NEAT1_2. To further investigate the cellular functions of paraspeckles, we created an animal model lacking NEAT1 by gene targeting. These knockout mice were viable and fertile under laboratory growth conditions, showing no apparent phenotypes except for the disappearance of paraspeckles. We propose that paraspeckles are nonessential, subpopulation-specific nuclear bodies formed secondary to particular environmental triggers.
PMCID: PMC3082198  PMID: 21444682
7.  An Architectural Role for a Nuclear Non-coding RNA: NEAT1 RNA is Essential for the Structure of Paraspeckles 
Molecular cell  2009;33(6):717-726.
NEAT1 RNA, a highly abundant 4 kb ncRNA, is retained in nuclei in ~10–20 large foci that we show is completely coincident with paraspeckles, nuclear domains implicated in mRNA nuclear retention. Depletion of NEAT1 RNA via RNAi eradicates paraspeckles, suggesting it controls sequestration of the paraspeckle proteins, PSP1 and p54, factors linked to A-I editing. Unlike over-expression of PSP1, NEAT1 over-expression increases paraspeckle number, and paraspeckles emanate exclusively from the NEAT1 transcription site. The PSP-1 RNA binding domain is required for its co-localization with NEAT1 RNA in paraspeckles, and biochemical analyses supports that NEAT1 RNA binds with paraspeckle proteins. Unlike other nuclear retained RNAs, NEAT1 RNA is not A-I edited, consistent with a structural role in paraspeckles. Collectively results demonstrate that NEAT1 functions as an essential structural determinant of paraspeckles, providing a precedent for a ncRNA as the foundation of a nuclear domain.
PMCID: PMC2696186  PMID: 19217333
8.  Compromised paraspeckle formation as a pathogenic factor in FUSopathies 
Human Molecular Genetics  2013;23(9):2298-2312.
Paraspeckles are nuclear bodies formed by a set of specialized proteins assembled on the long non-coding RNA NEAT1; they have a role in nuclear retention of hyperedited transcripts and are associated with response to cellular stress. Fused in sarcoma (FUS) protein, linked to a number of neurodegenerative disorders, is an essential paraspeckle component. We have shown that its recruitment to these nuclear structures is mediated by the N-terminal region and requires prion-like activity. FUS interacts with p54nrb/NONO, a major constituent of paraspeckles, in an RNA-dependent manner and responds in the same way as other paraspeckle proteins to alterations in cellular homeostasis such as changes in transcription rates or levels of protein methylation. FUS also regulates NEAT1 levels and paraspeckle formation in cultured cells, and FUS deficiency leads to loss of paraspeckles. Pathological gain-of-function FUS mutations might be expected to affect paraspeckle function in human diseases because mislocalized amyotrophic lateral sclerosis (ALS)-linked FUS variants sequester other paraspeckle proteins into aggregates formed in cultured cells and into neuronal inclusions in a transgenic mouse model of FUSopathy. Furthermore, we detected abundant p54nrb/NONO-positive inclusions in motor neurons of patients with familial forms of ALS caused by FUS mutations, but not in other ALS cases. Our results suggest that both loss and gain of FUS function can trigger disruption of paraspeckle assembly, which may impair protective responses in neurons and thereby contribute to the pathogenesis of FUSopathies.
PMCID: PMC3976330  PMID: 24334610
9.  Paraspeckles 
Paraspeckles are a relatively new class of subnuclear bodies found in the interchromatin space of mammalian cells. They are RNA-protein structures formed by the interaction between a long nonprotein-coding RNA species, NEAT1/Men ε/β, and members of the DBHS (Drosophila Behavior Human Splicing) family of proteins: P54NRB/NONO, PSPC1, and PSF/SFPQ. Paraspeckles are critical to the control of gene expression through the nuclear retention of RNA containing double-stranded RNA regions that have been subject to adenosine-to-inosine editing. Through this mechanism paraspeckles and their components may ultimately have a role in controlling gene expression during many cellular processes including differentiation, viral infection, and stress responses.
Nuclear structures formed by a long noncoding RNA and DBHS proteins are thought to control gene expression by retaining mRNAs that have undergone adenosine-to-inosine editing in the nucleus.
PMCID: PMC2890200  PMID: 20573717
10.  Altered nuclear retention of mRNAs containing inverted repeats in human embryonic stem cells: Functional role of a nuclear noncoding RNA 
Molecular cell  2009;35(4):467-478.
In many cells, mRNAs containing inverted repeats (Alu repeats in humans) in their 3′-untranslated regions (3′-UTRs) are inefficiently exported to the cytoplasm. Nuclear retention correlates with adenosine-to-inosine editing and is in paraspeckle-associated complexes containing the proteins p54nrb, PSF and PSP1α. We report that robust editing activity in human embryonic stem cells (hESCs), does not lead to nuclear retention. p54nrb, PSF and PSP1α are all expressed in hESCs, but paraspeckles are absent and only appear upon differentiation. Paraspeckle assembly and function depends on expression of a long nuclear-retained noncoding RNA, hNEAT1. This RNA is not expressed in hESCs, but is induced upon differentiation. Knockdown of hNEAT1 in HeLa cells results both in loss of paraspeckles and enhanced nucleocytoplasmic export of mRNAs containing inverted Alu repeats. Taken together, these results assign a biological function to a large noncoding nuclear RNA in the regulation of mRNA export.
PMCID: PMC2749223  PMID: 19716791
11.  Inhibition of long non-coding RNA NEAT1 impairs myeloid differentiation in acute promyelocytic leukemia cells 
BMC Cancer  2014;14(1):693.
Acute promyelocytic leukemia (APL) is characterized by the reciprocal translocation t(15;17), which fuses PML with retinoic acid receptor alpha (RARα). Although PML-RARα is crucially important for pathogenesis and responsiveness to treatment, the molecular and cellular mechanisms by which PML-RARα exerts its oncogenic potential have not been fully elucidated. Recent reports have suggested that long non-coding RNAs (lncRNAs) contribute to the precise control of gene expression and are involved in human diseases. Little is known about the role of lncRNA in APL.
We analyzed NEAT1 expression in APL samples and cell lines by real-time quantitative reverse transcription-PCR (qRT-PCR). The expression of PML-RARα was measured by Western blot. Cell differentiation was assessed by measuring the surface CD11b antigen expression by flow cytometry analysis.
We found that nuclear enriched abundant transcript 1 (NEAT1), a lncRNA essential for the formation of nuclear body paraspeckles, is significantly repressed in de novo APL samples compared with those of healthy donors. We further provide evidence that NEAT1 expression was repressed by PML-RARα. Furthermore, significant NEAT1 upregulation was observed during all-trans retinoic acid (ATRA)-induced NB4 cell differentiation. Finally, we demonstrate the importance of NEAT1 in myeloid differentiation. We show that reduction of NEAT1 by small interfering RNA (siRNA) blocks ATRA-induced differentiation.
Our results indicate that reduced expression of the nuclear long noncoding RNA NEAT1 may play a role in the myeloid differentiation of APL cells.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-693) contains supplementary material, which is available to authorized users.
PMCID: PMC4180842  PMID: 25245097
12.  A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains 
BMC Genomics  2007;8:39.
Noncoding RNA species play a diverse set of roles in the eukaryotic cell. While much recent attention has focused on smaller RNA species, larger noncoding transcripts are also thought to be highly abundant in mammalian cells. To search for large noncoding RNAs that might control gene expression or mRNA metabolism, we used Affymetrix expression arrays to identify polyadenylated RNA transcripts displaying nuclear enrichment.
This screen identified no more than three transcripts; XIST, and two unique noncoding nuclear enriched abundant transcripts (NEAT) RNAs strikingly located less than 70 kb apart on human chromosome 11: NEAT1, a noncoding RNA from the locus encoding for TncRNA, and NEAT2 (also known as MALAT-1). While the two NEAT transcripts share no significant homology with each other, each is conserved within the mammalian lineage, suggesting significant function for these noncoding RNAs. NEAT2 is extraordinarily well conserved for a noncoding RNA, more so than even XIST. Bioinformatic analyses of publicly available mouse transcriptome data support our findings from human cells as they confirm that the murine homologs of these noncoding RNAs are also nuclear enriched. RNA FISH analyses suggest that these noncoding RNAs function in mRNA metabolism as they demonstrate an intimate association of these RNA species with SC35 nuclear speckles in both human and mouse cells. These studies show that one of these transcripts, NEAT1 localizes to the periphery of such domains, whereas the neighboring transcript, NEAT2, is part of the long-sought polyadenylated component of nuclear speckles.
Our genome-wide screens in two mammalian species reveal no more than three abundant large non-coding polyadenylated RNAs in the nucleus; the canonical large noncoding RNA XIST and NEAT1 and NEAT2. The function of these noncoding RNAs in mRNA metabolism is suggested by their high levels of conservation and their intimate association with SC35 splicing domains in multiple mammalian species.
PMCID: PMC1800850  PMID: 17270048
13.  NEAT1 Long Noncoding RNA and Paraspeckle Bodies Modulate HIV-1 Posttranscriptional Expression 
mBio  2013;4(1):e00596-12.
Most of the human genome is transcribed into protein-noncoding RNAs (ncRNAs), including small ncRNAs and long ncRNAs (lncRNAs). Over the past decade, rapidly emerging evidence has increasingly supported the view that lncRNAs serve key regulatory and functional roles in mammal cells. HIV-1 replication relies on various cell functions. To date, while the involvement of host protein factors and microRNAs (miRNAs) in the HIV-1 life cycle has been extensively studied, the relationship between lncRNAs and HIV-1 remains uncharacterized. Here, we have profiled 83 disease-related lncRNAs in HIV-1-infected T cells. We found NEAT1 to be one of several lncRNAs whose expression is changed by HIV-1 infection, and we have characterized its role in HIV-1 replication. We report here that the knockdown of NEAT1 enhances virus production through increased nucleus-to-cytoplasm export of Rev-dependent instability element (INS)-containing HIV-1 mRNAs.
Long protein-noncoding RNAs (lncRNAs) play roles in regulating gene expression and modulating protein activities. There is emerging evidence that lncRNAs are involved in the replication of viruses. To our knowledge, this report is the first to characterize a role contributed by an lncRNA, NEAT1, to HIV-1 replication. NEAT1 is essential for the integrity of the nuclear paraspeckle substructure. Based on our findings from NEAT1 knockdown, we have identified the nuclear paraspeckle body as another important subcellular organelle for HIV-1 replication.
PMCID: PMC3560530  PMID: 23362321
14.  Long Non-Coding RNA NEAT1 Associates with SRp40 to Temporally Regulate PPARγ2 Splicing during Adipogenesis in 3T3-L1 Cells 
Genes  2014;5(4):1050-1063.
Long non-coding (lnc) RNAs serve a multitude of functions in cells. NEAT1 RNA is a highly abundant 4 kb lncRNA in nuclei, and coincides with paraspeckles, nuclear domains that control sequestration of paraspeckle proteins. We examined NEAT1 RNA levels and its function in 3T3-L1 cells during differentiation to adipocytes. Levels of NEAT1 transcript, measured by RT-PCR, fluctuated in a temporal manner over the course of differentiation that suggested its role in alternative splicing of PPARγ mRNA, the major transcription factor driving adipogenesis. When cells were induced to differentiate by a media cocktail of insulin, dexamethasone, and isobutylmethyxanthine (IBMX) on Day 0, NEAT1 levels dropped on Day 4, when the PPARγ2 variant was spliced and when terminal differentiation occurs The appearance of PPARγ2 coordinates with the PPARγ1 variant to drive differentiation of adipocytes. SiRNA used to deplete NEAT1 resulted in the inability of cells to phosphorylate the serine/arginine-rich splicing protein, SRp40. SiRNA treatment for SRp40 resulted in dysregulation of PPARγ1 and, primarily, PPARγ2 mRNA levels. SRp40 associated with NEAT1, as shown by RNA-IP on days 0 and 8, but decreased on day 4, and concentrations increased over that of IgG control. Overexpression of SRp40 increased PPARγ2, but not γ1. Although lncRNA MALAT1 has been investigated in SR protein function, NEAT1 has not been shown to bind SR proteins for phosphorylation such that alternative splicing results. The ability of cells to increase phosphorylated SR proteins for PPARγ2 splicing suggests that fluxes in NEAT1 levels during adipogenesis regulate alternative splicing events.
PMCID: PMC4276926  PMID: 25437750
lncRNA; NEAT1; 3T3-L1 cells; adipogenesis; SR proteins
15.  P54nrb Forms a Heterodimer with PSP1 That Localizes to Paraspeckles in an RNA-dependent Manner D⃞V⃞ 
Molecular Biology of the Cell  2005;16(11):5304-5315.
P54nrb is a protein implicated in multiple nuclear processes whose specific functions may correlate with its presence at different nuclear locations. Here we characterize paraspeckles, a subnuclear domain containing p54nrb and other RNA-binding proteins including PSP1, a protein with sequence similarity to p54nrb that acts as a marker for paraspeckles. We show that PSP1 interacts in vivo with a subset of the total cellular pool of p54nrb. We map the domain within PSP1 that is mediating this interaction and show it is required for the correct localization of PSP1 to paraspeckles. This interaction is necessary but not sufficient for paraspeckle targeting by PSP1, which also requires an RRM capable of RNA binding. Blocking the reinitiation of RNA Pol II transcription at the end of mitosis with DRB prevents paraspeckle formation, which recommences after removal of DRB, indicating that paraspeckle formation is dependent on RNA Polymerase II transcription. Thus paraspeckles are the sites where a subset of the total cellular pool of p54nrb is targeted in a RNA Polymerase II-dependent manner.
PMCID: PMC1266428  PMID: 16148043
16.  The lncRNA Neat1 is required for corpus luteum formation and the establishment of pregnancy in a subpopulation of mice 
Development (Cambridge, England)  2014;141(23):4618-4627.
Neat1 is a non-protein-coding RNA that serves as an architectural component of the nuclear bodies known as paraspeckles. Although cell-based studies indicate that Neat1 is a crucial regulator of gene expression, its physiological relevance remains unclear. Here, we find that Neat1 knockout (KO) mice stochastically fail to become pregnant despite normal ovulation. Unilateral transplantation of wild-type ovaries or the administration of progesterone partially rescued the phenotype, suggesting that corpus luteum dysfunction and concomitant low progesterone were the primary causes of the decreased fertility. In contrast to the faint expression observed in most of the adult tissues, Neat1 was highly expressed in the corpus luteum, and the formation of luteal tissue was severely impaired in nearly half of the Neat1 KO mice. These observations suggest that Neat1 is essential for the formation of the corpus luteum and for the subsequent establishment of pregnancy under a suboptimal condition that has not yet been identified.
PMCID: PMC4302932  PMID: 25359727
Neat1; Paraspeckles; Corpus luteum; Progesterone; Sfpq; Stochastic failure
17.  Paraspeckle nuclear bodies—useful uselessness? 
Cellular and Molecular Life Sciences  2012;69(18):3027-3036.
The nucleus of higher eukaryotes, such as humans and mice, is compartmentalized into multiple nuclear bodies, an organization that allows for the regulation of complex gene expression pathways that are characteristic of these organisms. Paraspeckles are recently discovered, mammalian-specific nuclear bodies built on a long, non-protein-coding RNA, NEAT1 (nuclear-enriched abundant transcript 1), which assembles various protein components including RNA-binding proteins of the DBHS (Drosophila behavior and human splicing) family. Paraspeckles have been proposed to control several biological processes, such as stress responses and cellular differentiation, but their function at the whole animal level remains unclear. In this review, we summarize a series of studies on paraspeckles that have been carried out in the decade since their discovery and discuss their physiological function and molecular mechanism.
PMCID: PMC3428521  PMID: 22476590
Paraspeckles; Nuclear structures; NEAT1; Noncoding RNA
18.  Distinct Roles of DBHS Family Members in the Circadian Transcriptional Feedback Loop 
Molecular and Cellular Biology  2012;32(22):4585-4594.
Factors interacting with core circadian clock components are essential to achieve transcriptional feedback necessary for metazoan clocks. Here, we show that all three members of the Drosophila behavior human splicing (DBHS) family of RNA-binding proteins play a role in the mammalian circadian oscillator, abrogating or altering clock function when overexpressed or depleted in cells. Although these proteins are members of so-called nuclear paraspeckles, depletion of paraspeckles themselves via silencing of the structural noncoding RNA (ncRNA) Neat1 did not affect overall clock function, suggesting that paraspeckles are not required for DBHS-mediated circadian effects. Instead, we show that the proteins bound to circadian promoter DNA in a fashion that required the PERIOD (PER) proteins and potently repressed E-box-mediated transcription but not cytomegalovirus (CMV) promoter-mediated transcription when they were exogenously recruited. Nevertheless, mice with one or both copies of these genes deleted show only small changes in period length or clock gene expression in vivo. Data from transient transfections show that each of these proteins can either repress or activate, depending on the context. Taken together, our data suggest that all of the DBHS family members serve overlapping or redundant roles as transcriptional cofactors at circadian clock-regulated genes.
PMCID: PMC3486183  PMID: 22966205
19.  Crystallization of a paraspeckle protein PSPC1–NONO heterodimer 
A truncated heterodimer of human PSPC1–NONO protein, a paraspeckle-specific complex, has been crystallized and the diffraction data collected to a resolution of 1.9 Å.
The paraspeckle component 1 (PSPC1) and non-POU-domain-containing octamer-binding protein (NONO) heterodimer is an essential structural component of paraspeckles, ribonucleoprotein bodies found in the interchromatin space of mammalian cell nuclei. PSPC1 and NONO both belong to the Drosophila behaviour and human splicing (DBHS) protein family, which has been implicated in many aspects of RNA processing. A heterodimer of the core DBHS conserved region of PSPC1 and NONO comprising two tandemly arranged RNA-recognition motifs (RRMs), a NONA/paraspeckle (NOPS) domain and part of a predicted coiled-coil domain has been crystallized in space group C2, with unit-cell parameters a = 90.90, b = 67.18, c = 94.08 Å, β = 99.96°. The crystal contained one heterodimer in the asymmetric unit and diffracted to 1.9 Å resolution using synchrotron radiation.
PMCID: PMC3212370  PMID: 22102035
paraspeckles; PSPC1–NONO heterodimer; RNA-recognition motifs; DBHS-family proteins
20.  ncRNA- and Pc2 Methylation-Dependent Gene Relocation between Nuclear Structures Mediates Gene Activation Programs 
Cell  2011;147(4):773-788.
Although eukaryotic nuclei contain distinct architectural structures associated with noncoding RNAs (ncRNAs), their potential relationship to regulated transcriptional programs remains poorly understood. Here, we report that methylation/demethylation of Polycomb 2 protein (Pc2) controls relocation of growth control genes between Polycomb bodies (PcGs) and interchromatin granules (ICGs) in response to growth signals. This movement is the consequence of binding of methylated and unmethylated Pc2 to the ncRNAs, TUG1 and MALAT1/NEAT2, located in PcGs and ICGs, respectively. These ncRNAs mediate assembly of multiple co-repressors/co-activators, and can alter the histone marks read by Pc2 in vitro. Additionally, binding of NEAT2 to unmethylated Pc2 promotes E2F1 SUMOylation, leading to activation of the growth control gene program. These observations delineate a molecular pathway linking the actions of subnuclear structure-specific ncRNAs and non-histone protein methylation to relocation of transcription units in the three-dimensional space of the nucleus, thus achieving coordinated gene expression programs.
PMCID: PMC3297197  PMID: 22078878
Noncoding RNA; Nuclear Architecture; Subnuclear Structures; Suv39h1; Pc2; Methylation; KDM4C; Polycomb Body; Interchromatin Granules; E2F1; SUMOylation; Ubiquitination
21.  Paraspeckle protein p54nrb links Sox9-mediated transcription with RNA processing during chondrogenesis in mice  
The Journal of Clinical Investigation  2008;118(9):3098-3108.
The Sox9 transcription factor plays an essential role in promoting chondrogenesis and regulating expression of chondrocyte extracellular-matrix genes. To identify genes that interact with Sox9 in promoting chondrocyte differentiation, we screened a cDNA library generated from the murine chondrogenic ATDC5 cell line to identify activators of the collagen, type II, α 1 (Col2a1) promoter. Here we have shown that paraspeckle regulatory protein 54-kDa nuclear RNA-binding protein (p54nrb) is an essential link between Sox9-regulated transcription and maturation of Sox9-target gene mRNA. We found that p54nrb physically interacted with Sox9 and enhanced Sox9-dependent transcriptional activation of the Col2a1 promoter. In ATDC5 cells, p54nrb colocalized with Sox9 protein in nuclear paraspeckle bodies, and knockdown of p54nrb suppressed Sox9-dependent Col2a1 expression and promoter activity. We generated a p54nrb mutant construct lacking RNA recognition motifs, and overexpression of mutant p54nrb in ATDC5 cells markedly altered the appearance of paraspeckle bodies and inhibited the maturation of Col2a1 mRNA. The mutant p54nrb inhibited chondrocyte differentiation of mesenchymal cells and mouse metatarsal explants. Furthermore, transgenic mice expressing the mutant p54nrb in the chondrocyte lineage exhibited dwarfism associated with impairment of chondrogenesis. These data suggest that p54nrb plays an important role in the regulation of Sox9 function and the formation of paraspeckle bodies during chondrogenesis.
PMCID: PMC2491455  PMID: 18677406
22.  The oestrogen receptor alpha-regulated lncRNA NEAT1 is a critical modulator of prostate cancer 
Nature Communications  2014;5:5383.
The androgen receptor (AR) plays a central role in establishing an oncogenic cascade that drives prostate cancer progression. Some prostate cancers escape androgen dependence and are often associated with an aggressive phenotype. The oestrogen receptor alpha (ERα) is expressed in prostate cancers, independent of AR status. However, the role of ERα remains elusive. Using a combination of chromatin immunoprecipitation (ChIP) and RNA-sequencing data, we identified an ERα-specific non-coding transcriptome signature. Among putatively ERα-regulated intergenic long non-coding RNAs (lncRNAs), we identified nuclear enriched abundant transcript 1 (NEAT1) as the most significantly overexpressed lncRNA in prostate cancer. Analysis of two large clinical cohorts also revealed that NEAT1 expression is associated with prostate cancer progression. Prostate cancer cells expressing high levels of NEAT1 were recalcitrant to androgen or AR antagonists. Finally, we provide evidence that NEAT1 drives oncogenic growth by altering the epigenetic landscape of target gene promoters to favour transcription.
While prostate cancer predominantly exhibits androgen dependence, oestrogen receptor (ER) signalling is also involved. Here, Chakravarty et al. show that ERα regulates the expression of the NEAT1 long non-coding RNA, which in turn promotes tumorigenesis by maintaining an oncogenic programme/cascade.
PMCID: PMC4241506  PMID: 25415230
23.  The nuclear ATPase/adenylate kinase hCINAP is recruited to perinucleolar caps generated upon RNA pol.II inhibition 
FEBS letters  2010;584(22):10.1016/j.febslet.2010.10.044.
hCINAP is an atypical nucleoplasmic enzyme, combining structural features of adenylate kinases and ATPases, which exhibits dual enzymatic activity. It interacts with the Cajal Body marker coilin and its level of expression and enzymatic activity influence Cajal Body numbers. Here we show that upon specific transcriptional inhibition of RNA pol.II, hCINAP segregates in perinuclear caps identified as Dark Nucleolar Caps (DNCs). These are distinct from perinucleolar caps where coilin and fibrillarin (both Cajal Body components) accumulate. In DNCs, hCINAP co-localizes with Paraspeckle Protein (PSP1) and also co-segregates with PSP1, and not coilin, in nuclear and nucleolar foci upon UV irradiation.
PMCID: PMC3839081  PMID: 20974138
Nuclear organization; Cajal Body; Coilin; Paraspeckle Protein 1; Paraspeckle
24.  Properties of the phage-shock-protein (Psp) regulatory complex that govern signal transduction and induction of the Psp response in Escherichia coli 
Microbiology  2010;156(Pt 10):2920-2932.
The phage-shock-protein (Psp) response maintains the proton-motive force (pmf) under extracytoplasmic stress conditions that impair the inner membrane (IM) in bacterial cells. In Escherichia coli transcription of the pspABCDE and pspG genes requires activation of σ54-RNA polymerase by the enhancer-binding protein PspF. A regulatory network comprising PspF–A–C–B–ArcB controls psp expression. One key regulatory point is the negative control of PspF imposed by its binding to PspA. It has been proposed that under stress conditions, the IM-bound sensors PspB and PspC receive and transduce the signal(s) to PspA via protein–protein interactions, resulting in the release of the PspA–PspF inhibitory complex and the consequent induction of psp. In this work we demonstrate that PspB self-associates and interacts with PspC via putative IM regions. We present evidence suggesting that PspC has two topologies and that conserved residue G48 and the putative leucine zipper motif are determinants required for PspA interaction and signal transduction upon stress. We also establish that PspC directly interacts with the effector PspG, and show that PspG self-associates. These results are discussed in the context of formation and function of the Psp regulatory complex.
PMCID: PMC3068692  PMID: 20595257
25.  Molecular and Evolutionary Analysis of NEAr-Iron Transporter (NEAT) Domains 
PLoS ONE  2014;9(8):e104794.
Iron is essential for bacterial survival, being required for numerous biological processes. NEAr-iron Transporter (NEAT) domains have been studied in pathogenic Gram-positive bacteria to understand how their proteins obtain heme as an iron source during infection. While a 2002 study initially discovered and annotated the NEAT domain encoded by the genomes of several Gram-positive bacteria, there remains a scarcity of information regarding the conservation and distribution of NEAT domains throughout the bacterial kingdom, and whether these domains are restricted to pathogenic bacteria. This study aims to expand upon initial bioinformatics analysis of predicted NEAT domains, by exploring their evolution and conserved function. This information was used to identify new candidate domains in both pathogenic and nonpathogenic organisms. We also searched metagenomic datasets, specifically sequence from the Human Microbiome Project. Here, we report a comprehensive phylogenetic analysis of 343 NEAT domains, encoded by Gram-positive bacteria, mostly within the phylum Firmicutes, with the exception of Eggerthella sp. (Actinobacteria) and an unclassified Mollicutes bacterium (Tenericutes). No new NEAT sequences were identified in the HMP dataset. We detected specific groups of NEAT domains based on phylogeny of protein sequences, including a cluster of novel clostridial NEAT domains. We also identified environmental and soil organisms that encode putative NEAT proteins. Biochemical analysis of heme binding by a NEAT domain from a protein encoded by the soil-dwelling organism Paenibacillus polymyxa demonstrated that the domain is homologous in function to NEAT domains encoded by pathogenic bacteria. Together, this study provides the first global bioinformatics analysis and phylogenetic evidence that NEAT domains have a strong conservation of function, despite group-specific differences at the amino acid level. These findings will provide information useful for future projects concerning the structure and function of NEAT domains, particularly in pathogens where they have yet to be studied.
PMCID: PMC4143258  PMID: 25153520

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