Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines

Department of Biochemistry, The University of Mississippi Medical Center, Jackson, MS 39216, USA.
Journal of Cell Science (Impact Factor: 5.43). 07/2009; 122(Pt 11):1872-81. DOI: 10.1242/jcs.044040
Source: PubMed


Cajal bodies (CBs) are nuclear structures that are thought to have diverse functions, including small nuclear ribonucleoprotein (snRNP) biogenesis. The phosphorylation status of coilin, the CB marker protein, might impact CB formation. We hypothesize that primary cells, which lack CBs, contain different phosphoisoforms of coilin compared with that found in transformed cells, which have CBs. Localization, self-association and fluorescence recovery after photobleaching (FRAP) studies on coilin phosphomutants all suggest this modification impacts the function of coilin and may thus contribute towards CB formation. Two-dimensional gel electrophoresis demonstrates that coilin is hyperphosphorylated in primary cells compared with transformed cells. mRNA levels of the nuclear phosphatase PPM1G are significantly reduced in primary cells and expression of PPM1G in primary cells induces CBs. Additionally, PPM1G can dephosphorylate coilin in vitro. Surprisingly, however, expression of green fluorescent protein alone is sufficient to form CBs in primary cells. Taken together, our data support a model whereby coilin is the target of an uncharacterized signal transduction cascade that responds to the increased transcription and snRNP demands found in transformed cells.


Available from: Eric M George
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    • "Lines were cultured as described previously (Sun et al., 2005). GST-coilin WT, GST-coilin N362, GST-coilin C214 and GST-coilin Δ121–291 constructs were previously described (Broome and Hebert, 2013; Velma et al., 2010; Toyota et al., 2010; Hearst et al., 2009). His-tagged WRAP53, GST-coilin 93–291, GST-coilin 93–244, GST-coilin 93–147, GST-coilin 142–199, GST-coilin 194–244 and GST-coilin 239–291 constructs were made by PCR amplification using standard molecular biological techniques. "
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    ABSTRACT: Spliceosomal small nuclear ribonucleoproteins (snRNPs) are enriched in the Cajal body (CB). Guide RNAs, known as small Cajal body-specific RNAs (scaRNAs), direct modification of the small nuclear RNA (snRNA) component of the snRNP. The protein WRAP53 binds a sequence motif (the CAB box) found in many scaRNAs and the RNA component of telomerase (hTR) and targets these RNAs to the CB. We have previously reported that coilin, the CB marker protein, associates with certain non-coding RNAs. For a more comprehensive examination of the RNAs associated with coilin, we have sequenced the RNA isolated from coilin immunocomplexes. A striking preferential association of coilin with the box C/D scaRNAs 2 and 9, which lack a CAB box, was observed. This association varied by treatment condition and WRAP53 knockdown. In contrast, reduction of WRAP53 did not alter the level of coilin association with hTR. Additional studies showed that coilin degrades/processes scaRNA 2 and 9, associates with active telomerase and can influence telomerase activity. These findings suggest that coilin plays a novel role in the biogenesis of box C/D scaRNPs and telomerase.
    Biology Open 03/2014; 3(4). DOI:10.1242/bio.20147443 · 2.42 Impact Factor
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    • "Brilliant II SYBR Green qRT-PCR Master Mix Kit from Agilent (Santa Clara, CA, USA) was used for analysis of RNA from RNA IP experiments, transfected cells and U2 and hTR RNase assays and the manufacturer's suggested thermal cycling parameters, primer concentrations and RNA amounts were used. Primers for GAPDH, 47/45S pre-rRNA, U1, pre-U1, U2 and pre-U2 have been described (Broome and Hebert, 2012; Gilder et al., 2011; Hearst et al., 2009). Primer sequences are: 5.8S rRNA: 59-CGGCTCGTGCGTCGAT-39 forward and 59-CCGCAAGT- GCGTTCGAA-39 reverse; telomerase RNA (hTR): 59-AAATGTCAGCTGCTGG- CCCGTTCG-39 forward and 59-ACCCGCGGCTGACAGAGCCCAAC-39 reverse; pre-processed telomerase RNA (pre-hTR): 59-AGGTTCAGGCCTTTCAGG- CCGCAG-39 forward and 59-GACGGATGCGCACGATCGGCGTTC-39 reverse. "
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    ABSTRACT: The Cajal body (CB) is a domain of concentrated components found within the nucleus of cells in an array of species that is functionally important for the biogenesis of telomerase and small nuclear ribonucleoproteins. The CB is a dynamic structure whose number and size change during the cell cycle and is associated with other nuclear structures and gene loci. Coilin, also known as the marker protein for the CB, is a phosphoprotein widely accepted for its role in maintaining CB integrity. Recent studies have been done to further elucidate functional activities of coilin apart from its structural role in the CB in an attempt to explore the rationale for coilin expression in cells that have few CBs or lack them altogether. Here we show that the RNA association profile of coilin changes in mitosis with respect to that during interphase. We provide evidence of transcriptional and/or processing dysregulation of several CB-related RNA transcripts as a result of ectopic expression of both wild-type and phosphomutant coilin proteins. We also show apparent changes in transcription and/or processing of these transcripts upon coilin knockdown in both transformed and primary cell lines. Additionally, we provide evidence of specific coilin RNase activity regulation, on both U2 and hTR transcripts, by phosphorylation of a single residue, serine 489. Collectively, these results point to additional functions for coilin that are regulated by phosphorylation.
    Biology Open 04/2013; 2(4):407-15. DOI:10.1242/bio.20133863 · 2.42 Impact Factor
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    • "ization . Several of these proteins are implicated in multiple cellular processes . For example , the Ser / Thr phosphatase PPM1G has been shown to influence the subcellular localisation of SMN , to act in pre - mRNA splicing , in dephosphorylation of coilin , histone H2B and γH2AX as well as in histone H2A / H2B exchange ( Allemand et al . 2007 ; Hearst et al . 2009 ; Kimura et al . 2006 ; Murray et al . 1999 ; Petri et al . 2007 ) . The CDC5L complex and its constituents CDC5L , PLRG1 and Prp19 have additionally been linked to the DNA damage response ( Legerski 2009 ; Lu and Legerski 2007 ; Zhang et al . 2005 , 2009 ) . Post - translational protein modifications with SUMO1 and SUMO2 / 3 are well k"
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    ABSTRACT: The nucleolus is the subnuclear organelle responsible for ribosome subunit biogenesis and can also act as a stress sensor. It forms around clusters of ribosomal DNA (rDNA) and is mainly organised in three subcompartments, i.e. fibrillar centre, dense fibrillar component and granular component. Here, we describe the localisation of 21 protein factors to an intranucleolar region different to these main subcompartments, called the intranucleolar body (INB). These factors include proteins involved in DNA maintenance, protein turnover, RNA metabolism, chromatin organisation and the post-translational modifiers SUMO1 and SUMO2/3. Increase in the size and number of INBs is promoted by specific types of DNA damage and depends on the functional integrity of the nucleolus. INBs are abundant in nucleoli of unstressed cells during S phase and localise in close proximity to rDNA with heterochromatic features. The data suggest the INB is linked with regulation of rDNA transcription and/or maintenance of rDNA. Electronic supplementary material The online version of this article (doi:10.1007/s00412-011-0327-8) contains supplementary material, which is available to authorized users.
    Chromosoma 06/2011; 120(5):481-99. DOI:10.1007/s00412-011-0327-8 · 4.60 Impact Factor
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