Article

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

Division of Gene Regulation and Expression, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom.
Molecular Biology of the Cell (Impact Factor: 4.55). 12/2005; 16(11):5304-15. DOI: 10.1091/mbc.E05-06-0587
Source: PubMed

ABSTRACT 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.

Download full-text

Full-text

Available from: Charles S Bond, Aug 24, 2015
0 Followers
 · 
108 Views
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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 · 4.62 Impact Factor
  • Source
    • "However, drug treatment and fluorescence loss in photobleaching (FLIP) experiments confirmed that this protein interacted dynamically with the nucleoplasm and nucleolus in a transcriptional-dependent manner (Fox et al., 2005). To fully confirm the presence of a protein in the nucleolus, therefore, it is therefore necessary to take this into consideration and perform FLIP experiments on the transfected cells. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The nucleolus is the most prominent organelle in the mammalian nucleus. It is assembled around rDNA genes and is the site of rDNA transcription and ribosome subunit assembly. However, the presence of proteins with no obvious relationship with ribosome subunit synthesis suggests additional functions for the nucleolus, such as regulation of mitosis, cell cycle progression and proliferation, many forms of stress response and biogenesis of multiple RNPs. The high density and structural stability of the nucleolus make it a relatively easy organelle to isolate. Nucleoli can be isolated from cultured cells that are biochemically, morphologically and at least in part functionally intact. Mass spectrometry analyses have shown that thousands of proteins can be identified reproducibly in purified nucleoli. These proteins, which likely represent most of the human nucleolar proteome, show a considerable functional diversity. The many novel factors and separate classes of proteins identified support the view that the nucleolus may perform additional functions beyond its known role in ribosome subunit biogenesis. Future studies will expand our knowledge of the nucleolar proteomes in other model organisms and will provide a more detailed quantitative picture of the levels of each protein and how this changes under a range of cell growth conditions and in response to stress and other perturbations.
    The Nucleolus, 01/2011: pages 29-42; , ISBN: 978-1-4614-0513-9
  • Source
    • "Individual paraspeckles begin to form in early G1 phase until approximately 1 hour after RNA transcription is initiated (Fox et al. 2005). Importantly, blocking the initiation of RNA polymerase II transcription by the inhibitor DRB at the end of mitosis prevents formation of paraspeckles, suggesting that these structures are dependent on ongoing transcription (Fox et al. 2005; Clemson et al. 2009). The earliest paraspeckles are often positioned next to the NEAT1 gene locus and an increase in NEAT1 expression by ectopic plasmids leads to a corresponding increase in the number of paraspeckles, which is essentially unchanged upon PSP1 overexpression (Clemson et al. 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The nucleus is unique amongst cellular organelles in that it contains a myriad of discrete suborganelles. These nuclear bodies are morphologically and molecularly distinct entities, and they host specific nuclear processes. Although the mode of biogenesis appears to differ widely between individual nuclear bodies, several common design principles are emerging, particularly, the ability of nuclear bodies to form de novo, a role of RNA as a structural element and self-organization as a mode of formation. The controlled biogenesis of nuclear bodies is essential for faithful maintenance of nuclear architecture during the cell cycle and is an important part of cellular responses to intra- and extracellular events.
    Cold Spring Harbor perspectives in biology 11/2010; 2(12):a000711. DOI:10.1101/cshperspect.a000711 · 8.23 Impact Factor
Show more