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author:("Fox, arcba")
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.  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.
doi:10.1107/S1744309111026212
PMCID: PMC3212370  PMID: 22102035
paraspeckles; PSPC1–NONO heterodimer; RNA-recognition motifs; DBHS-family proteins
3.  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.
doi:10.1128/MCB.00334-12
PMCID: PMC3486183  PMID: 22966205
4.  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
5.  Highly Ordered Spatial Organization of the Structural Long Noncoding NEAT1 RNAs within Paraspeckle Nuclear Bodies 
Molecular Biology of the Cell  2010;21(22):4020-4027.
We describe the spatial organization of the two NEAT1 noncoding (nc)RNAs required for the integrity of the paraspeckle nuclear bodies. The central sequences of the long transcript are internal when its extremities and the short isoform are peripheral, indicating how RNA can contribute to the architecture of nuclear bodies.
Paraspeckles (PSPs) are nuclear bodies associated with the retention in the nucleus of specific mRNAs. Two isoforms of a long noncoding RNA (NEAT1_v1/Menε and NEAT1_v2/Menβ) are required for the integrity of PSPs. Here, we analyzed the molecular organization of PSPs by immuno- and in situ hybridization electron microscopy. Detection of the paraspeckle markers PSPC1 and P54NRB/NONO confirm the identity between PSPs and the previously described interchromatin granule-associated zones (IGAZs). High-resolution in situ hybridization of NEAT1 transcripts revealed a highly ordered organization of IGAZ/PSPs. Although the 3.7-kb NEAT1_v1 and the identical 5′ end of the 22.7-kb NEAT1_v2 transcripts are confined to the periphery, central sequences of NEAT1_v2 are found within the electron-dense core of the bodies. Moreover, the 3′ end of NEAT1_v2 also localize to the periphery, indicating possible architectures for IGAZ/PSPs. These results further suggest that the organization of NEAT1 transcripts constrains the geometry of these bodies. Accordingly, we observed in HeLa and NIH 3T3 cells that IGAZ/PSPs are elongated structures with a well-defined diameter. Our results provide new insight on the ability of noncoding RNAs to form subcellular structures.
doi:10.1091/mbc.E10-08-0690
PMCID: PMC2982136  PMID: 20881053
6.  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
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.  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.
doi:10.1091/mbc.E05-06-0587
PMCID: PMC1266428  PMID: 16148043
9.  Nuclear processes controlled by molecular machines 
Genome Biology  2002;3(6):reports4016.1-reports4016.3.
A report on the 'Nuclear Structure and Function' symposium at the joint spring meeting of the British Society for Cell Biology, British Society for Developmental Biology and Genetics Society, York, UK, 20-23 March 2002.
PMCID: PMC139369  PMID: 12093371

Results 1-9 (9)