P54nrb Forms a Heterodimer with PSP1 That Localizes to Paraspeckles in an RNA-dependent Manner □ D □ V

Division of Gene Regulation and Expression, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom.
Molecular Biology of the Cell (Impact Factor: 4.47). 12/2005; 16(11):5304-15. DOI: 10.1091/mbc.E05-06-0587
Source: PubMed


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.

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Available from: Charles S Bond, Oct 10, 2015
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    • "In addition, a number of proteins were identified that were unique to this study including paraspeckle protein 1 (PSPC1), symplekin, FIP1, and nucleophosmin. PSPC1 is a component of a novel subnuclear compartment named paraspeckle and was previously shown to interact with NONO via its coiled-coil domain (Fox et al. 2005). PSPC1 and SFPQ and have also been shown to be coexpressed and interact with Sertoli cells (Kuwahara et al. 2006). "
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    ABSTRACT: Splicing factor proline- and glutamine-rich (SFPQ) also commonly known as polypyrimidine tract-binding protein-associated-splicing factor (PSF) and its binding partner non-POU domain-containing octamer-binding protein (NONO/p54nrb), are highly abundant, multifunctional nuclear proteins. However, the exact role of this complex is yet to be determined. Following purification of the endogeneous SFPQ/NONO complex, mass spectrometry analysis identified a wide range of interacting proteins, including those involved in RNA processing, RNA splicing, and transcriptional regulation, consistent with a multifunctional role for SFPQ/NONO. In addition, we have identified several sites of arginine methylation in SFPQ/PSF using mass spectrometry and found that several arginines in the N-terminal domain of SFPQ/PSF are asymmetrically dimethylated. Furthermore, we find that the protein arginine N-methyltransferase, PRMT1, catalyzes this methylation in vitro and that this is antagonized by citrullination of SFPQ. Arginine methylation and citrullination of SFPQ/PSF does not affect complex formation with NONO. However, arginine methylation was shown to increase the association with mRNA in mRNP complexes in mammalian cells. Finally we show that the biochemical properties of the endogenous complex from cell lysates are significantly influenced by the ionic strength during purification. At low ionic strength, the SFPQ/NONO complex forms large heterogeneous protein assemblies or aggregates, preventing the purification of the SFPQ/NONO complex. The ability of the SFPQ/NONO complex to form varying protein assemblies, in conjunction with the effect of post-translational modifications of SFPQ modulating mRNA binding, suggests key roles affecting mRNP dynamics within the cell.
    RNA 02/2015; 21(3):347-359. DOI:10.1261/rna.045138.114 · 4.94 Impact Factor
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    • "In contrast, PSPC1 is more selectively expressed, acting as a coactivator of transcription [43]. It is also known that PSPC1 can dimerize with p54nrb through the coiled-coil domain to regulate pre-RNA processing, but not with PSF [50]. Nevertheless, until now, little is known about its other functions, especially in DDR. "
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    ABSTRACT: Paraspeckle protein 1 (PSPC1) was first identified as a structural protein of the subnuclear structure termed paraspeckle. However, the exact physiological functions of PSPC1 are still largely unknown. Previously, using a proteomic approach, we have shown that exposure to cisplatin can induce PSPC1 expression in HeLa cells, indicating the possible involvement for PSPC1 in the DNA damage response (DDR). In the current study, the role of PSPC1 in DDR was examined. First, it was found that cisplatin treatment could indeed induce the expression of PSPC1 protein. Abolishing PSPC1 expression by siRNA significantly inhibited cell growth, caused spontaneous cell death, and increased DNA damage. However, PSPC1 did not co-localize with γH2AX, 53BP1, or Rad51, indicating no direct involvement in DNA repair pathways mediated by these molecules. Interestingly, knockdown of PSPC1 disrupted the normal cell cycle distribution, with more cells entering the G2/M phase. Furthermore, while cisplatin induced G1/S arrest in HeLa cells, knockdown of PSPC1 caused cells to escape the G1/S checkpoint and enter mitosis, and resulted in more cell death. Taken together, these observations indicate a new role for PSPC1 in maintaining genome integrity during the DDR, particularly in the G1/S checkpoint.
    PLoS ONE 05/2014; 9(5):e97174. DOI:10.1371/journal.pone.0097174 · 3.23 Impact Factor
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    • "The effect of FUS knockdown on paraspeckles is seemed to be rescued by overexpression of another paraspeckle protein as multiple paraspeckle-like and PSP1-positive structures reappear in COS7 cells with dramatically reduced FUS levels that are expressing GFP-p54nrb/NONO (Fig. 5F and G). We cannot completely exclude that these structures represent small p54nrb/NONO aggregates that also contain PSP1 because of high affinity heterodimerisation of these two proteins (19,40). However, these structures were observed in FUS-depleted cells with a low level of GFP-p54nrb/NONO expression that does not induce aggregate formation in naïve cells, making our suggestion that the protein supports formation of physiological structures, i.e. paraspecles, very credible. "
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    ABSTRACT: Paraspeckles are nuclear bodies formed by a set of specialised 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 mislocalised 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.
    Human Molecular Genetics 12/2013; 23(9). DOI:10.1093/hmg/ddt622 · 6.39 Impact Factor
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