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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.
doi:10.1091/mbc.E13-09-0558
PMCID: PMC3873887  PMID: 24173718
2.  Chromatin remodeling complexes in the assembly of long noncoding RNA-dependent nuclear bodies 
Nucleus  2015;6(6):462-467.
Paraspeckles are subnuclear structures that assemble on nuclear paraspeckle assembly transcript 1 (NEAT1) long noncoding (lnc)RNA. Paraspeckle formation requires appropriate NEAT1 biogenesis and subsequent assembly with multiple prion-like domain (PLD) containing RNA-binding proteins. We found that SWI/SNF chromatin remodeling complexes function as paraspeckle components that interact with paraspeckle proteins (PSPs) and NEAT1. SWI/SNF complexes play an essential role in paraspeckle formation that does not require their ATP-dependent chromatin remodeling activity. Instead, SWI/SNF complexes facilitate organization of the PSP interaction network required for intact paraspeckle assembly. SWI/SNF complexes may collectively bind multiple PSPs to recruit them onto NEAT1. SWI/SNF complexes are also required for Sat III (Satellite III) lncRNA-dependent formation of nuclear stress bodies under heat shock conditions. Organization of the lncRNA-dependent omega speckle in Drosophila also depends on the chromatin remodeling complex. These findings raise the possibility that a common mechanism controls the formation of lncRNA-dependent nuclear body architecture.
doi:10.1080/19491034.2015.1119353
PMCID: PMC4915484  PMID: 26709446
long noncoding RNA; nuclear body; paraspeckle; nuclear stress body; omega speckle; chromatin remodeling complex; RNA-binding protein; prion-like domain (PLD)
3.  Paraspeckles modulate the intranuclear distribution of paraspeckle-associated Ctn RNA 
Scientific Reports  2016;6:34043.
Paraspeckles are sub-nuclear domains that are nucleated by long noncoding RNA Neat1. While interaction of protein components of paraspeckles and Neat1 is understood, there is limited information on the interaction of non-structural RNA components with paraspeckles. Here, by varying paraspeckle number and size, we investigate how paraspeckles influence the nuclear organization of their non-structural RNA component Ctn RNA. Our results show that Ctn RNA remains nuclear-retained in the absence of intact paraspeckles, suggesting that they do not regulate nuclear retention of Ctn RNA. In the absence of Neat1, Ctn RNA continues to interact with paraspeckle protein NonO to form residual nuclear foci. In addition, in the absence of Neat1-nucleated paraspeckles, a subset of Ctn RNA localizes to the perinucleolar regions. Concomitant with increase in number of paraspeckles, transcriptional reactivation resulted in increased number of paraspeckle-localized Ctn RNA foci. Similar to Neat1, proteasome inhibition altered the localization of Ctn RNA, where it formed enlarged paraspeckle-like foci. Super-resolution structured illumination microscopic analyses revealed that in paraspeckles, Ctn RNA partially co-localized with Neat1, and displayed a more heterogeneous intra-paraspeckle localization. Collectively, these results show that while paraspeckles do not influence nuclear retention of Ctn RNA, they modulate its intranuclear compartmentalization.
doi:10.1038/srep34043
PMCID: PMC5036046  PMID: 27665741
4.  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.
doi:10.4161/rna.23547
PMCID: PMC3672290  PMID: 23324609
3′-end processing; RNA-binding protein; RNA-protein interaction; long non-coding RNA; nuclear bodies
5.  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.
doi:10.1093/nar/gku579
PMCID: PMC4117792  PMID: 25013176
6.  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.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1756-6606-6-31
PMCID: PMC3729541  PMID: 23835137
Long non-coding RNA; Paraspeckle; NEAT1_2; TDP-43; FUS/TLS; Amyotrophic lateral sclerosis; Electron microscopy
7.  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.
doi:10.1083/jcb.200906113
PMCID: PMC2742191  PMID: 19720872
8.  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.
doi:10.1083/jcb.201011110
PMCID: PMC3082198  PMID: 21444682
9.  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.
Summary
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.
doi:10.1016/j.molcel.2009.01.026
PMCID: PMC2696186  PMID: 19217333
10.  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.
doi:10.1093/hmg/ddt622
PMCID: PMC3976330  PMID: 24334610
11.  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.
doi:10.1101/cshperspect.a000687
PMCID: PMC2890200  PMID: 20573717
12.  Circadian RNA expression elicited by 3’-UTR IRAlu-paraspeckle associated elements 
eLife  null;5:e14837.
Paraspeckles are nuclear bodies form around the long non-coding RNA, Neat1, and RNA-binding proteins. While their role is not fully understood, they are believed to control gene expression at a post-transcriptional level by means of the nuclear retention of mRNA containing in their 3’-UTR inverted repeats of Alu sequences (IRAlu). In this study, we found that, in pituitary cells, all components of paraspeckles including four major proteins and Neat1 displayed a circadian expression pattern. Furthermore the insertion of IRAlu at the 3’-UTR of the EGFP cDNA led to a rhythmic circadian nuclear retention of the egfp mRNA that was lost when paraspeckles were disrupted whereas insertion of a single antisense Alu had only a weak effect. Using real-time video-microscopy, these IRAlu were further shown to drive a circadian expression of EGFP protein. This study shows that paraspeckles, thanks to their circadian expression, control circadian gene expression at a post-transcriptional level.
DOI: http://dx.doi.org/10.7554/eLife.14837.001
eLife digest
Many biological features of animals, including body temperature and hormone levels, follow daily rhythms that repeat every 24 hours. These so-called circadian rhythms are driven by an internal body clock and are essential for the organism to adapt to the daily cycle of light and dark. Circadian rhythms also take place inside individual cells – for example, the amount of a given protein in a cell often rises and falls over each 24-hour period.
To generate these daily fluctuations, the processes used to make proteins based on the instructions encoded within a gene must be carefully controlled. Genes are first copied or ‘transcribed' into intermediate molecules called messenger RNAs (mRNAs). These mRNA molecules must then travel out of the cell’s nucleus before they can be de-coded to produce proteins. This means that daily fluctuations in mRNA and protein levels could occur because the rate at which the DNA is transcribed fluctuates or because controlling the steps that occur after transcription. However it is not clear how much these post-transcriptional steps contribute to circadian rhythms inside cells.
Recently, structures called paraspeckles were seen inside the nucleus. These structures are made from a long RNA molecule that does not code for a protein, and a number of proteins that can bind mRNA molecules. Paraspeckles are thought to prevent certain mRNAs from leaving the nucleus and therefore stop them from being decoded to make proteins. Torres et al. have now investigated whether paraspeckles may play a role in circadian rhythms.
Torres et al. looked at the long non-coding RNA and several proteins that are known to be components of paraspeckles in cells taken from the pituitary glands of rats using a variety of techniques. These cells were chosen because they were known to have a working circadian clock. The analysis showed that the levels of these components, as well as the number of paraspeckles within the nucleus, changed over the course of a daily cycle.
Torres et al. then confirmed that mRNAs containing a sequence that is known to recruit mRNAs to paraspeckes (the IRAlu sequence) could be also retained in the nucleus or released with a circadian rhythm. This pattern was lost when the paraspeckles were disrupted.
These findings suggest that daily fluctuations in protein levels can be post-transcriptionally controlled by paraspeckles rhythmically retaining mRNAs in the nucleus. Future studies could explore whether it may be possible to control circadian rhythms by targeting the paraspeckles, which could help to improve conditions where the internal body clock goes wrong.
DOI: http://dx.doi.org/10.7554/eLife.14837.002
doi:10.7554/eLife.14837
PMCID: PMC4987140  PMID: 27441387
pituitary cells; paraspeckle; circadian oscillators; circadian rhythm; nuclear RNA retention; Other
13.  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.
doi:10.1016/j.molcel.2009.06.027
PMCID: PMC2749223  PMID: 19716791
14.  Long noncoding RNA NEAT1 promotes laryngeal squamous cell cancer through regulating miR-107/CDK6 pathway 
Background
Long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) plays key role in the progression of some human cancers. However, the role of NEAT1 in human laryngeal squamous cell cancer (LSCC) is still unknown. We therefore investigated the expression and function of NEAT1 in LSCC.
Methods
NEAT1 level in LSCC and adjacent non-neoplastic tissues were detected by qRT-PCR. NEAT1 was knockdown in LSCC cells and cell proliferation, apoptosis and cell cycle were examined. The growth of xenografts with NEAT1 knockdown LSCC cells was analyzed.
Results
NEAT1 level was significantly higher in LSCC than in corresponding adjacent non-neoplastic tissues, and patients with neck nodal metastasis or advanced clinical stage had higher NEAT1 expression. Moreover, siRNA mediated NEAT1 knockdown significantly inhibited the proliferation and induced apoptosis and cell cycle arrest at G1 phase in LSCC cells. The growth of LSCC xenografts was significantly suppressed by the injection of NEAT1 siRNA lentivirus. Furthermore, NEAT1 regulated CDK6 expression in LSCC cells which was mediated by miR-107.
Conclusion
NEAT1 plays an oncogenic role in the tumorigenesis of LSCC and may serve as a potential target for therapeutic intervention.
doi:10.1186/s13046-016-0297-z
PMCID: PMC4731996  PMID: 26822763
Laryngeal squamous cell cancer; Long noncoding RNA; microRNA; NEAT1; CDK6; miR-107
15.  Inhibition of long non-coding RNA NEAT1 impairs myeloid differentiation in acute promyelocytic leukemia cells 
BMC Cancer  2014;14:693.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2407-14-693
PMCID: PMC4180842  PMID: 25245097
16.  Tumor hypoxia induces nuclear paraspeckle formation through HIF-2α dependent transcriptional activation of NEAT1 leading to cancer cell survival 
Oncogene  2014;34(34):4482-4490.
Activation of cellular transcriptional responses, mediated by hypoxia-inducible factor (HIF), is common in many types of cancer, and generally confers a poor prognosis. Known to induce many hundreds of protein-coding genes, HIF has also recently been shown to be a key regulator of the non-coding transcriptional response. Here, we show that NEAT1 long non-coding RNA (lncRNA) is a direct transcriptional target of HIF in many breast cancer cell lines and in solid tumors. Unlike previously described lncRNAs, NEAT1 is regulated principally by HIF-2 rather than by HIF-1. NEAT1 is a nuclear lncRNA that is an essential structural component of paraspeckles and the hypoxic induction of NEAT1 induces paraspeckle formation in a manner that is dependent upon both NEAT1 and on HIF-2. Paraspeckles are multifunction nuclear structures that sequester transcriptionally active proteins as well as RNA transcripts that have been subjected to adenosine-to-inosine (A-to-I) editing. We show that the nuclear retention of one such transcript, F11R (also known as junctional adhesion molecule 1, JAM1), in hypoxia is dependent upon the hypoxic increase in NEAT1, thereby conferring a novel mechanism of HIF-dependent gene regulation. Induction of NEAT1 in hypoxia also leads to accelerated cellular proliferation, improved clonogenic survival and reduced apoptosis, all of which are hallmarks of increased tumorigenesis. Furthermore, in patients with breast cancer, high tumor NEAT1 expression correlates with poor survival. Taken together, these results indicate a new role for HIF transcriptional pathways in the regulation of nuclear structure and that this contributes to the pro-tumorigenic hypoxia-phenotype in breast cancer.
doi:10.1038/onc.2014.378
PMCID: PMC4430310  PMID: 25417700
17.  Enhanced expression of long non-coding RNA NEAT1 is associated with the progression of gastric adenocarcinomas 
Background
Long non-coding RNAs (lncRNAs) are emerging as new players in the cancer. The aim of this study was to examine the abnormalities of NEAT1 (nuclear paraspeckle assembly transcript 1, also known as MENε/β) in gastric adenocarcinomas (GACs).
Methods
One hundred thirty-one GAC tissues and matched adjacent normal tissues (ANTs) were collected from patients who undergone surgery. Differences in of NEAT1 expression were examined via quantitative reverse transcriptase PCR (qRT-PCR). WST-1 assay and transwell assay were carried out in vitro to investigate the proliferation and migration of GAC cells with alteration in NEAT1 long non-coding RNA (lncRNA) expression.
Results
The expression levels of lncRNA NEAT1 were significantly elevated in GAC tissues (P < 0.001) compared with ANTs. There was also a statistical difference in NEAT1 expression between early and advanced GACs (P = 0.0111). GACs with lymph node metastasis (LNM) expressed higher levels of NEAT1 lncRNA compared with those without LNM (P = 0.004). In the in vitro experiments, the proliferation but not migration of GAC cells was attenuated after NEAT1 knockdown by RNA interference.
Conclusions
Expression of NEAT1 lncRNA was enhanced in GACs; and NEAT1 may influence GAC progression by promoting tumor growth.
doi:10.1186/s12957-016-0799-3
PMCID: PMC4765228  PMID: 26911892
Gastric adenocarcinoma; Long non-coding RNA; NEAT1; Cell proliferation
18.  Clinical implication of long non-coding RNA NEAT1 expression in hepatocellular carcinoma patients 
Hepatocellular carcinoma (HCC), a primary malignancy of the liver, is associated with high mortality rate and poor prognosis. Emerging evidence showed that novel biomarkers are required toward a better understanding of the biological mechanisms of HCC. NEAT1 (nuclear paraspeckle assembly transcript 1, also known as MENε/β), a novel long non-coding RNA (lncRNA), serves as a crucial regulator in several cancers. However, the correlation between NEAT1 expression with tumorigenesis and metastasis in HCC tissues remains out of the question so far. In the current study, the aim was to evaluate the potential role of NEAT1 expression in HCC tissues and its relationship with clinicopathological parameters. Method: The expression of NEAT1 was detected by qRT-PCR, in 95 cases of adjacent non-cancerous liver and their paired HCC tissues, respectively. The associations of NEAT1 with clinicopathological features and other biological factors were further analyzed. Result: Our results revealed that NEAT1 appeared to have higher expression in the HCC tissues, compared with the adjacent non-cancerous liver tissues. High levels of NEAT1 promoted the clinical features of HCC, including the number of tumor nodes, metastasis, clinical TNM stage, the status of portal vein tumor embolus, vaso-invasion and the infi ltration of tumor cells. Additionally, high NEAT1 expression levels were significantly associated with the expression level of MDTH, NM23 and MALAT1. Conclusion: Our study demonstrates that NEAT1 acts as a pivotal player in tumorigenesis and metastasis of hepatocellular carcinoma.
PMCID: PMC4503113  PMID: 26191242
NEAT1; hepatocellular carcinoma; tumorigenesis; metastasis
19.  A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains 
BMC Genomics  2007;8:39.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2164-8-39
PMCID: PMC1800850  PMID: 17270048
20.  NEAT1 Long Noncoding RNA and Paraspeckle Bodies Modulate HIV-1 Posttranscriptional Expression 
mBio  2013;4(1):e00596-12.
ABSTRACT
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.
IMPORTANCE
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.
doi:10.1128/mBio.00596-12
PMCID: PMC3560530  PMID: 23362321
21.  Tumor hypoxia induces nuclear paraspeckle formation through HIF-2α dependent transcriptional activation of NEAT1 leading to cancer cell survival 
Oncogene  2014;34(34):4482-4490.
Activation of cellular transcriptional responses, mediated by hypoxia-inducible factor (HIF), is common in many types of cancer, and generally confers a poor prognosis. Known to induce many hundreds of protein-coding genes, HIF has also recently been shown to be a key regulator of the non-coding transcriptional response. Here we show that NEAT1 lncRNA is a direct transcriptional target of HIF in many breast cancer cell lines and in solid tumors. Unlike previously described lncRNAs, NEAT1 is regulated principally by HIF-2 rather than by HIF-1. NEAT1 is a nuclear lncRNA that is an essential structural component of paraspeckles and the hypoxic induction of NEAT1 induces paraspeckle formation in a manner that is dependent upon both NEAT1 and on HIF-2. Paraspeckles are multifunction nuclear structures that sequester transcriptionally active proteins as well as RNA transcripts that have been subjected to A-to-I editing. We show that the nuclear retention of one such transcript, F11R (also known as junctional adhesion molecule 1 – JAM1), in hypoxia is dependent upon the hypoxic increase in NEAT1, thereby conferring a novel mechanism of HIF-dependent gene regulation. Induction of NEAT1 in hypoxia also leads to accelerated cellular proliferation, improved clonogenic survival and reduced apoptosis, all of which are hallmarks of increased tumorigenesis. Furthermore, in patients with breast cancer, high tumor NEAT1 expression correlates with poor survival. Taken together, these results indicate a new role for HIF transcriptional pathways in the regulation of nuclear structure and that this contributes to the pro-tumorigenic hypoxia-phenotype in breast cancer.
doi:10.1038/onc.2014.378
PMCID: PMC4430310  PMID: 25417700
Hypoxia; HIF; lncRNA; NEAT1; paraspeckles; F11R RNA
22.  HuR‐regulated lncRNA NEAT1 stability in tumorigenesis and progression of ovarian cancer 
Cancer Medicine  2016;5(7):1588-1598.
Abstract
Long noncoding RNAs (lncRNAs) have recently emerged as pivotal regulators in governing fundamental biological processes, as well as in tumorigenesis. The nuclear paraspeckle assembly transcript 1 (NEAT1) is one of the most highly regulated lncRNAs in recent genomic datasets, however, its biological role and regulatory mechanism in ovarian cancer (OC) development and progression are poorly defined. In this study, we identified that NEAT1 was up‐regulated in OC patients and cell lines, and its expression was associated with the FIGO stage and lymph node metastasis. Furthermore, the ectopic expression of NEAT1_1 in OVCAR‐3 cell lines promoted cell proliferation and invasion, whereas knockdown of NEAT1_1 did the opposite. Furthermore, NEAT1_1 was stabilized by an RNA‐binding protein HuR, but suppressed by miR‐124‐3p in OC cells. Accordingly, the increased HuR mRNA and decreased miR‐124‐3p levels were observed in OC patients. These results suggested that lncRNA NEAT1, whose expression was collaboratively controlled by HuR and miR‐124‐3p, could regulate ovarian carcinogenesis and may serve as a potential target for antineoplastic therapies.
doi:10.1002/cam4.710
PMCID: PMC4944886  PMID: 27075229
HuR; lncRNA; miR‐124; NEAT1; ovarian cancer
23.  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.
doi:10.3390/genes5041050
PMCID: PMC4276926  PMID: 25437750
lncRNA; NEAT1; 3T3-L1 cells; adipogenesis; SR proteins
24.  Mining the Human Complexome Database Identifies RBM14 as an XPO1-Associated Protein Involved in HIV-1 Rev Function 
Journal of Virology  2015;89(7):3557-3567.
ABSTRACT
By recruiting the host protein XPO1 (CRM1), the HIV-1 Rev protein mediates the nuclear export of incompletely spliced viral transcripts. We mined data from the recently described human nuclear complexome to identify a host protein, RBM14, which associates with XPO1 and Rev and is involved in Rev function. Using a Rev-dependent p24 reporter plasmid, we found that RBM14 depletion decreased Rev activity and Rev-mediated enhancement of the cytoplasmic levels of unspliced viral transcripts. RBM14 depletion also reduced p24 expression during viral infection, indicating that RBM14 is limiting for Rev function. RBM14 has previously been shown to localize to nuclear paraspeckles, a structure implicated in retaining unspliced HIV-1 transcripts for either Rev-mediated nuclear export or degradation. We found that depletion of NEAT1 RNA, a long noncoding RNA required for paraspeckle integrity, abolished the ability of overexpressed RBM14 to enhance Rev function, indicating the dependence of RBM14 function on paraspeckle integrity. Our study extends the known host cell interactome of Rev and XPO1 and further substantiates a critical role for paraspeckles in the mechanism of action of Rev. Our study also validates the nuclear complexome as a database from which viral cofactors can be mined.
IMPORTANCE This study mined a database of nuclear protein complexes to identify a cellular protein named RBM14 that is associated with XPO1 (CRM1), a nuclear protein that binds to the HIV-1 Rev protein and mediates nuclear export of incompletely spliced viral RNAs. Functional assays demonstrated that RBM14, a protein found in paraspeckle structures in the nucleus, is involved in HIV-1 Rev function. This study validates the nuclear complexome database as a reference that can be mined to identify viral cofactors.
doi:10.1128/JVI.03232-14
PMCID: PMC4403413  PMID: 25589658
25.  NEAT1 is Required for Survival of Breast Cancer Cells Through FUS and miR-548 
Gene Regulation and Systems Biology  2016;10(Suppl 1):11-17.
Increasing evidence shows that long noncoding RNAs (lncRNAs) have important roles in the regulation of multiple cellular processes, including cell division, cell growth, and apoptosis, as well as cancer metastasis and neurological disease progression; however, the mechanism of how lncRNAs regulate these processes is not well established. In this study, we demonstrated that downregulating the expression of the lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in breast cancer cells inhibited cell growth and induced cell apoptosis. In addition, the RNA-binding protein fused in sarcoma/translocated in liposarcoma (FUS/TLS) physically interacted with NEAT1, and reducing the expression of FUS/TLS also induced cell apoptosis. Multiple miRNAs were identified as regulators of NEAT1, but only overexpression of miR-548ar was able to decrease NEAT1 expression and promote apoptosis. These results indicate a novel interaction between NEAT1, miR-548ar-3p, and FUS and their role in the regulation of breast cancer cell apoptosis.
doi:10.4137/GRSB.S29414
PMCID: PMC4849421  PMID: 27147820
lncRNA; NEAT1; miR-548ar-3p; FUS; cell apoptosis

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