- Open Access
How to build a paraspeckle
© BioMed Central Ltd 2009
- Published: 16 July 2009
Noncoding RNAs have recently been identified as essential components of the nuclear suborganelles called paraspeckles. This finding will facilitate our understanding of the molecular dynamics and physiological role of these enigmatic macromolecular structures.
- Nuclear Body
- Nucleolar Protein
- Cajal Body
- Target Gene Locus
- Splice Speckle
Paraspeckles are large ribonucleoprotein structures around 0.5 μm in diameter that can be detected in nuclei with a light microscope and appropriate antibody staining, and are currently of unknown function. They were discovered quite unexpectedly as recently as 2002 [1, 2]. Lamond and colleagues conducted a large-scale mass-spectrometric analysis of nucleoli isolated from HeLa cells, which identified 271 nucleolar proteins. Of these proteins, more than 30% were novel or uncharacterized . The localization of a subset of the novel proteins fused with yellow fluorescent protein (YFP) for visual detection was then determined . Surprisingly, one of those fusion proteins was found to co-localize not to the nucleolus itself, but to a novel nuclear compartment or suborganelle.
The protein was found to be ubiquitously expressed in all human cell lines examined , and is localized in granular foci often adjacent to 'splicing-speckles', which are implicated as the reservoir of various splicing factors. Hence, the newly discovered foci were dubbed 'paraspeckles' and the newly characterized protein was named paraspeckle protein 1 (PSP1) . Mass spectrometric analysis of nucleolar proteins demonstrated that a small fraction of this protein, undetectable by fluorescence microscopy, transiently associated with the nucleolus, which explained its original detection as a nucleolar protein .
The number of paraspeckles per interphase nuclei in human cell lines varies between 10 and 20, and their typical size is 0.5 μm in diameter. In addition to PSP1, three proteins, p54nrb (also known as NONO, non-POU domain containing octamer-binding protein), polypyrimidine tract-binding protein-associated splicing factor (PSF), and para speckle protein 2 (PSP2), exhibit a punctate nucleoplasmic distribution, co-localizing to paraspeckles as seen by immunno staining using anti bodies against corresponding proteins [2, 3].
These paraspeckle proteins each contain two RNA-recognition motifs (RRMs). The properties and interaction behavior of PSF, p54nrb, and their homologs in species ranging from Drosophila to mouse have been extensively characterized. PSF and p54nrb interact with a nuclear receptor and with RNA, and also with both single- and double-stranded DNA [4–9]. Both p54nrb and PSF are multifunctional proteins that are implicated in nuclear processes such as transcriptional control, splicing regulation, mRNA 3'-end formation, DNA repair and recombination, and nuclear retention of hyperedited RNAs in various human and mouse cell lines [4–9]. Chromosomal translocations involving the genes encoding PSF or p54nrb can produce chimeric proteins that cause tumorigenesis (see  and references therein). Furthermore, if transcription is inhibited by actinomycin D, all the paraspeckle proteins relocate to a perinucleolar cap . There are several more proteins that meet some of the above criteria, and the list of paraspeckle proteins is therefore expected to expand in the near future. Indeed, Cardinale et al.  recently reported that a pre-mRNA 3'-end processing factor, mammalian cleavage factor I (CF Im68), localizes to paraspeckles. The protein contains one RRM instead of two and moves to the perinucleolar cap when transcription is inhibited .
The identification of paraspeckle proteins immediately prompted investigations of the molecular mechanism by which this membraneless suborganelle is assembled. Fox et al.  reported that PSP1 heterodimerizes with p54nrb both in vivo and in vitro, and that the functioning RRM domains are critical for targeting PSP1 to the paraspeckle. Furthermore, the paraspeckle structure is sensitive to RNase, indicating that RNA is also an essential structural component .
Given that the paraspeckle was predicted to be a large ribonucleoprotein complex , the presumed RNA-protein interactions have become a focus of research into the molecular mechanisms underlying paraspeckle formation. Three groups have now independently identified the long-sought architectural RNAs [12–14]. These groups began working from different research perspectives but eventually found the same noncoding RNAs (ncRNAs) - two isoforms, MENε and MENβ, which are transcribed from the same RNA polymerase II promoter but differ in the location of their 3' ends, and the functions of which are largely uncharacterized . Our laboratory  identified MENε and MENβ from the LeLa cell nuclei as a component of the paraspeckle-enriched fraction by biochemical purification. Sunwoo et al.  identified some 200 ncRNAs that are either up- or downregulated during differentiation of the C2C12 mouse myoblast cell line into myotubes . They narrowed down their target to Menε/β by manual examination and subcellular localization analyses. Looking for nuclear-retained abundant ncRNAs in both humans and mouse cells, Clemson and colleagues [14, 16] identified three: the inactivated X-chromosome transcript XIST, and two ncRNAs they called nuclear-enriched abundant transcripts 1 and 2, NEAT1 and NEAT2. NEAT1 is identical to MENε and NEAT2 to the noncoding ncRNA MENα, which resides downstream of Menε/β in the MEN locus.
The MENε/β depletion phenotype was also examined in both human and mouse cells, using knockdown with chimeric antisense oligonucleotides [12, 13] or small interfering RNA (siRNA) . MENε/β knockdown resulted in disruption of the paraspeckles but not of other intranuclear bodies [12–14] (Figure 1). Importantly, there is no degradation of paraspeckle proteins in these knockdowns and no paraspeckles remained intact without MENε/β. Furthermore, the reassembly of paraspeckles disassembled by treatment with an RNA polymerase II inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), was suppressed in MENε/β-depleted cells [12, 13]. These results strongly support the hypothesis that MENε and MENβ are essential for the integrity of the paraspeckle structure.
The physical associations of MENε/β RNAs with paraspeckle proteins have been investigated using immunoprecipitation and the following RNA-protein interactions have been reported: MENβ and p54nrb and MENβ and PSF , Menε/β and p54nrb , and MENε and p54nrb and MENε and PSP1 . Clemson et al.  demonstrated that deletion of the RRM domains of PSP1 abrogates its association with MENε in paraspeckles. Our group  examined the effect of paraspeckle protein depletion on MENε/β RNA levels and paraspeckle structure. We found that depletion of either p54nrb or PSF preferentially decreases MENβ but not MENε, and disrupts paraspeckle structure. Notably, PSP1 depletion did not affect either MENε/β levels or paraspeckle structure. These results suggest that PSP1 plays a role in paraspeckle organization distinct from p54nrb and PSF. Despite some discrepancies among the reports of the three research groups, the consensus that the ncRNAs MENε/β are essential to paraspeckle formation via interactions with the RRM domains of each paraspeckle protein is clear.
Prasanth et al.  have proposed a role for paraspeckles in the posttranscriptional regulation of expression of cationic amino acid transporter 2 (CAT2) gene mRNAs. An RNA called CTN-RNA is transcribed from the protein-coding mouse cationic amino acid transporter 2 gene through alternative promoter and poly(A) site usage and is retained in the nucleus . Under stress, this RNA can be cleaved to produce the protein-coding CAT2 mRNA. However, CTN-RNA is thought to be retained in the nucleus as a result of A-to-I RNA editing in the 3' untranslated region , whereas MENε/β RNAs do not appear to be edited [12–14].
With the currently available knowledge, what else can we determine regarding the physiological function of paraspeckles? The ubiquity of paraspeckles across different tissues must be taken into consideration. Given that most paraspeckle components have previously been identified as involved in transcriptional regulation and RNA processing, it is tempting to speculate that paraspeckles control gene expression. However, the mechanism of paraspeckle action is open to question, as the 'paraspeckle proteins' in fact seem to function primarily in nuclear compartments other than MENε/β-containing paraspeckles [4–10]. One plausible assumption, as has been hypothesized for other intranuclear compartments such as the nucleolus and splicing speckles, is that paraspeckles serve as a warehouse for a number of regulatory proteins that are sequestered in the paraspeckle until required in response to physiological conditions [18–21]. Thus, the availability of regulatory proteins at a target gene locus can be strictly controlled by the paraspeckle.
The remarkable dynamics of paraspeckle proteins have been noted since the discovery of paraspeckles, as proteomic analyses also identified all these proteins in the perinucleolar compartment [1, 2]. When paraspeckle proteins relocate to the perinucleolar compartment, the MENε/β RNAs have dissociated, and are degraded  or relocate to either splicing speckles  or the nucleolus . Paraspeckle proteins diffuse across the nucleoplasm in the absence of the MENε/β RNAs [6, 12, 13]. It is possible that posttranslational modifications such as phosphorylation and methylation could alter the interaction between the MENε/β RNAs and paraspeckle proteins, and could increase the affinity of paraspeckle proteins for the perinucleolar compartment.
There is an apparent difference in the number and distribution pattern of paraspeckles in the nucleus between the G1 phase and the rest of interphase. In addition, each cell line that has been observed displays a unique paraspeckle distribution pattern, which may represent the physiological status of the cells. These observations inevitably raise questions as to the precise mechanisms of paraspeckle formation and translocation. Is an individual paraspeckle formed on the MEN locus, or is a large paraspeckle precursor formed and then subsequently divided into several daughter paraspeckles? How do paraspeckles depart from the MENε/β loci? Do paraspeckles roam through the nucleus or are they destined for specific target locations? These questions are inextricably intertwined if both the formation and movement of paraspeckles are dependent on the nuclear domains with which paraspeckles associate, that is, the MENε/β loci and putative target gene loci. In addressing these questions, comparisons with the formation of other nuclear bodies may be useful. The nucleolus is formed at the nucleolar organizer region (NOR) containing the rRNA genes, and its formation is dependent on rRNA trans cription. Additional nucleoli can be formed by introducing extrachromosomal NORs . Cajal bodies, involved in small nuclear ribonucleoprotein (snRNP) and small nucleor RNP (snoRNP) biogenesis, also closely interact with particular gene loci such as those for spliceosomal small nuclear RNAs (snRNAs) and histones, and are recruited or formed de novo in a microenvironment in which the local concentration of their substrates, snRNAs, is elevated . Thus, gene loci provide nucleation sites for nuclear body formation and may be a target for transcriptional regulation or modulation by nuclear bodies [18–21]. Interestingly, the RRM protein NonA, the Drosophila counterpart of p54nrb, forms a complex with other RNA-binding proteins in developmentally regulated 'puffs' on polytene chromosomes . It will be of great interest to determine whether paraspeckles also target particular gene loci in specific physiological conditions (Figure 2).
Having ncRNAs as part of their structure gives paraspeckles unique properties; for example, unlike other intranuclear bodies, paraspeckle structure persists during most of mitosis, with the exception of telophase, in the absence of association with condensed chromatin . This observation implies that long ncRNAs can themselves function as a scaffold for nucleation. In contrast, nucleoli and Cajal bodies disassemble when cells enter mitosis because association with their target loci is a prerequisite for nucleation [24, 25]. It should be noted that RNAs associated with these nuclear bodies (for example, pre-rRNA and snRNA) are relatively small compared to MENε/β). The biogenesis of Cajal bodies exhibits the hallmarks of stochastic self-organization . An important focus of future investigations will be to determine to what extent paraspeckle formation is consistent with the self-organization model.
The identification of MENε/β as a component of paraspeckles has raised many more questions, rather than simply answering the question of what a paraspeckle is. The depletion of MENε/β RNA profoundly affects the structural integrity of paraspeckles, which does not necessarily exclude the possibility of the presence of other structural/functional RNAs in paraspeckles. Transcriptome analysis of isolated paraspeckles, for example, may lead to the identification of ancillary RNA components. Through mechanical and functional characterization of paraspeckles, with emphasis on the RNA components, we will gain substantial insights into the dynamic nature of these nuclear bodies - in particular, how they are assembled into large ribonucleoprotein complexes and how they find their targets on chromatin and/or in particular nuclear domains. These insights should be relevant to our understanding of the dynamics of other nuclear bodies as well.
We thank members of the Hirose laboratory, in particular T Naganuma, K Aoki and T Kawaguchi for helpful discussions. We also thank K Watanabe and T Misteli for their continuous support and encouragement.
- Andersen JS, Lyon CE, Fox AH, Leung AKL, Lam YW, Steen H, Mann M, Lamond AI: Directed proteomic analysis of the human nucleolus. Curr Biol. 2002, 12: 1-11. 10.1016/S0960-9822(01)00650-9.PubMedView ArticleGoogle Scholar
- Fox AH, Lam YW, Leung AKL, Lyon CE, Andersen J, Mann M, Lamond AI: Paraspeckles: A novel nuclear domain. Curr Biol. 2002, 12: 13-25. 10.1016/S0960-9822(01)00632-7.PubMedView ArticleGoogle Scholar
- Fox AH, Bond CS, Lamond AI: P54nrb forms a heterodimer with PSP1 that localizes to paraspeckles in an RNA-dependent manner. Mol Biol Cell. 2005, 16: 5304-5315. 10.1091/mbc.E05-06-0587.PubMedPubMed CentralView ArticleGoogle Scholar
- Shav-Tal Y, Zipori D: PSF and p54(nrb)/NonO - multifunctional nuclear proteins. FEBS Lett. 2002, 531: 109-114. 10.1016/S0014-5793(02)03447-6.PubMedView ArticleGoogle Scholar
- Auboeuf D, Dowhan DH, Li X, Larkin K, Ko L, Berget SM, O'Malley BW: CoAA, a nuclear receptor coactivator protein at the interface of transcriptional coactivation and RNA splicing. Mol Cell Biol. 2004, 24: 442-453. 10.1128/MCB.24.1.442-453.2004.PubMedPubMed CentralView ArticleGoogle Scholar
- Dong X, Sweet J, Challis JRG, Brown T, Lye SJ: Transcriptional activity of androgen receptor is modulated by two RNA splicing factors, PSF and p54nrb. Mol Cell Biol. 2007, 27: 4863-4875. 10.1128/MCB.02144-06.PubMedPubMed CentralView ArticleGoogle Scholar
- Reim I, Stanewsky R, Saumweber H: The puff-specific RRM protein NonA is a single-stranded nucleic acid binding protein. Chromosoma. 1999, 108: 162-172. 10.1007/s004120050365.PubMedView ArticleGoogle Scholar
- Zhang Z, Carmichael GG: The fate of dsRNA in the nucleus: A p54nrb-containing complex mediates the nuclear retention of promiscuously A-to-I edited RNAs. Cell. 2001, 106: 465-475. 10.1016/S0092-8674(01)00466-4.PubMedView ArticleGoogle Scholar
- Bladen CL, Udayakumar D, Takeda Y, Dynan WS: Identification of the polypyrimidine tract binding protein-associated splicing factor p54(nrb) complex as a candidate RNA double-strand break rejoining factor. J Biol Chem. 2005, 280: 5205-5210. 10.1074/jbc.M412758200.PubMedView ArticleGoogle Scholar
- Shav-Tal Y, Blechman J, Darzacq X, Montagna C, Dye BT, Patton JG, Singer RH, Zipori D: Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional inhibition. Mol Biol Cell. 2005, 16: 2395-2413. 10.1091/mbc.E04-11-0992.PubMedPubMed CentralView ArticleGoogle Scholar
- Cardinale S, Cisterna B, Bonetti P, Aringhieri C, Biggiogera M, Barabino SML: Subnuclear localization and dynamics of the - pre-mRNA 3' end processing factor mammalian cleavage factor I 68-kDa subunit. Mol Biol Cell. 2007, 18: 1282-1292. 10.1091/mbc.E06-09-0846.PubMedPubMed CentralView ArticleGoogle Scholar
- Sasaki YT, Ideue T, Sano M, Mituyama T, Hirose T: MEN ε/β noncoding RNAs are essential for structural integrity of nuclear paraspeckles. Proc Natl Acad Sci USA. 2009, 106: 2525-2530. 10.1073/pnas.0807899106.PubMedPubMed CentralView ArticleGoogle Scholar
- Sunwoo H, Dinger ME, Wilusz JE, Amaral PP, Mattick JS, Spector DL: MENε/β nuclear-retained non-coding RNAs are up-regulated upon muscle differentiation and are essential components of paraspeckles. Genome Res. 2009, 19: 347-359. 10.1101/gr.087775.108.PubMedPubMed CentralView ArticleGoogle Scholar
- Clemson CM, Hutchinson JN, Sara SA, Ensminger AW, Fox AH, Chess A, Lawrence JB: An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles. Mol Cell. 2009, 33: 717-726. 10.1016/j.molcel.2009.01.026.PubMedPubMed CentralView ArticleGoogle Scholar
- Guru SC, Agarwal SK, Manickam P, Olufemi SE, Crabtree JS, Weisemann JM, Kester MB, Kim YS, Wang Y, Emmert-Buck MR, Liotta LA, Spiegel AM, Boguski MS, Roe BA, Collins FS, Marx SJ, Burns L, Chandrasekharappa SC: A transcript map for the 2.8-Mb region containing the multiple endocrine neoplasia type 1 locus. Genome Res. 1997, 7: 725-735.PubMedPubMed CentralGoogle Scholar
- Hutchinson JN, Ensminger AW, Clemson CM, Lynch CR, Lawrence JB, Chess A: A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains. BMC Genomics. 2007, 8: 39-10.1186/1471-2164-8-39.PubMedPubMed CentralView ArticleGoogle Scholar
- Prasanth KV, Prasanth SG, Xuan Z, Hearn S, Freier SM, Bennett CF, Zhang MQ, Spector DL: Regulating gene expression through RNA nuclear retention. Cell. 2005, 123: 249-263. 10.1016/j.cell.2005.08.033.PubMedView ArticleGoogle Scholar
- Misteli T: Protein dynamics: Implications for nuclear architecture and gene expression. Science. 2001, 291: 843-847. 10.1126/science.291.5505.843.PubMedView ArticleGoogle Scholar
- Misteli T: Concepts in nuclear architecture. BioEssays. 2005, 27: 477-487. 10.1002/bies.20226.PubMedView ArticleGoogle Scholar
- Shav-Tal Y, Darzacq X, Singer RH: Gene expression within a dynamic nuclear landscape. EMBO J. 2006, 25: 3469-3479. 10.1038/sj.emboj.7601226.PubMedPubMed CentralView ArticleGoogle Scholar
- Misteli T: Physiological importance of RNA and protein mobility in the cell nucleus. Histochem Cell Biol. 2008, 129: 5-11. 10.1007/s00418-007-0355-x.PubMedPubMed CentralView ArticleGoogle Scholar
- Oakes M, Aris JP, Brockenbrough JS, Wai H, Vu L, Nomura M: Mutational analysis of the structure and localization of the nucleolus in the yeast Saccharomyces cerevisiae. J Cell Biol. 1998, 143: 23-34. 10.1083/jcb.143.1.23.PubMedPubMed CentralView ArticleGoogle Scholar
- Dundr M, Ospina JK, Sung M-H, John S, Upender M, Ried T, Hager GL, Matera SG: Actin-dependent intranuclear repositioning of an active gene locus in vivo. J Cell Biol. 2007, 179: 1095-1103. 10.1083/jcb.200710058.PubMedPubMed CentralView ArticleGoogle Scholar
- Leung AK, Gerlich D, Miller G, Lyon C, Lam YW, Lleres D, Daigle N, Zomerdijk J, Ellenberg J, Lamond AI: Quantitative kinetic analysis of nucleolar breakdown and reassembly during mitosis in live human cells. J Cell Biol. 2004, 166: 787-800. 10.1083/jcb.200405013.PubMedPubMed CentralView ArticleGoogle Scholar
- Carmo-Fonseca M, Ferreira J, Lamond AI: Assembly of snRNP-containing coiled bodies is regulated in interphase and mitosis - evidence that the coiled body is a kinetic nuclear structure. J Cell Biol. 1993, 120: 841-852. 10.1083/jcb.120.4.841.PubMedView ArticleGoogle Scholar
- Kaiser TE, Intine RV, Dundr M: De novo formation of a sub-nuclear body. Science. 2008, 322: 1713-1717. 10.1126/science.1165216.PubMedView ArticleGoogle Scholar