QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 11 October 2005
doi: 10.1242/jcs.02613

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sun, J. Articles by Hebert, M. D. Search for Related Content PubMed PubMed Citation Articles by Sun, J. Articles by Hebert, M. D. Social Bookmarking                         What's this?

Interactions between Coilin and PIASy partially link Cajal bodies to PML bodies

¹ Department of Neurology, The University of Mississippi Medical Center, Jackson, MS 39216-4505, USA
² Department of Biochemistry, The University of Mississippi Medical Center, Jackson, MS 39216-4505, USA

View larger version (47K): [in a new window]   Fig. 1. Cajal bodies (CBs) can associate with PML bodies. (Left) HeLa cells were transfected with GFP-coilin to mark CBs (green) and PML bodies were detected with anti-PML antibodies (red). (Right) CBs and PML bodies formed by ectopically expressed proteins can associate. HeLa cells were co-transfected with CFP-coilin (red) and YFP-Sp100 (to mark PML bodies, green). In both panels, DAPI staining was used to identify the nucleus and associated CBs and PML bodies are marked with arrows. Scale bar, 2 µm.

View larger version (111K): [in a new window]   Fig. 2. Cajal bodies and PML bodies can associate with the same U2 gene locus. HeLa cells were subjected to antibody staining to mark the location of CBs (red) and PML bodies (blue), followed by DNA FISH using an U2 gene probe (green). The arrow marks an association between CBs, PML bodies and an U2 gene (inset) in four different cells. Scale bar, 2 µm.

View larger version (78K): [in a new window]   Fig. 3. Residual CBs containing fibrillarin do not associate with PML bodies. Wild-type (WT, panel A) and coilin knockout MEF cells (KO, panel B) were stained with antibodies to PML to mark PML bodies (green) and fibrillarin to detect CBs and residual CBs (red). Fibrillarin normally localizes to nucleoli and CBs, but is also enriched in one kind of rCB (Tucker et al., 2001). An arrow marks an association between a CB and a PML body (panel A), while arrowheads mark residual CBs in KO cells that fail to associate with PML bodies (double arrowhead, panel B). DAPI staining (blue) was used to define the nucleus. Wild-type (WT, panel C) and coilin knockout MEF cells (KO, panel D) were also transfected with YFP-Sp100 (green) to mark PML bodies and stained with anti-fibrillarin (red) to detect CBs and residual CBs (rCBs). Arrows mark some of the associations between CBs and PML bodies in wild-type cells (panel C). Double arrowheads mark PML bodies that fail to associate with rCBs (arrowheads) in knockout cells (panel D). Scale bar, 2 µm.

View larger version (26K): [in a new window]   Fig. 4. PIASy interacts with coilin in vitro and in vivo. (A) Yeast cells transformed with PIASy prey and the indicated coilin baits were spotted onto non-selective (–LW) or selective (–LWHA) media and incubated for 3 days. Note that yeast harboring PIASy prey with coilin baits containing the first 161 or 92 amino acids showed minimal growth on the selective medium, as did yeast with PIASy prey and the C-terminal 214 aa of coilin (362-576) as bait. (B) His-T7-tagged full-length (FL, 1-576) or N-terminal fragments (1-161, 1-362) of coilin, detected by anti-coilin or anti-T7 antibodies, bind GST-PIASy but not GST alone. By contrast, a C-terminal fragment of coilin containing the last 214 aa of coilin (C214) does not interact with GST-PIASy. Ponceau S staining verified that equivalent amounts of GST proteins were used in the assay. The input lanes account for 20% of the coilin or coilin truncations used in the reactions. (C) HeLa cells were co-transfected with empty GFP vector, GFP-coilin or GFP-coilin mutants along with myc-tagged PIASy. GFP-C214 corresponds to the C-terminal 214 aa of coilin (from aa 362-576). Lysates were subjected to immunoprecipitation with anti-GFP antibodies, followed by western blotting with anti-myc antibodies (upper rows in all panels). The blots were reprobed with anti-GFP to monitor the level of the GFP tagged proteins (lower rows in all panels). The input lanes account for 2% the amount of lysate used in the immunoprecipitation reactions. (D) HeLa cells were transfected with empty GFP vector or GFP-PIASy, followed by immunoprecipitation with anti-GFP antibodies and western blotting with antibodies against coilin (top panel) or GFP (bottom panel). The input lane accounts for 2% the amount of lysate used in the immunoprecipitation reactions.

View larger version (49K): [in a new window]   Fig. 5. A mutation in coilin that disrupts PIASy interaction reduces the frequency of CB/PML body association. (A) Coilin knockout MEF cells were co-transfected with myc-PIASy and various GFP-tagged control or mouse coilin constructs. After immunoprecipitation with anti-GFP antibodies and SDS-PAGE, a western blot was conducted with antibodies to the myc-tag (upper panels). The blot was reprobed with anti-GFP to verify that GFP, GFP-mouse coilin (coilin), GFP-mouse coilin (100-150) and GFP-mouse coilin (150-200) were immunoprecipitated (middle and lower panel). The position of the immunoglobulin heavy chain, IgG(H), is noted. (B) Coilin knockout MEF cells were transfected with full-length and mutant GFP-tagged mouse coilin constructs (green) followed by detection of PML bodies using anti-PML (red). DAPI staining (blue) was used to define the nucleus. Cells were scored for associations between CBs and PML bodies. In panels A-C, the percent in the bottom right of the image accounts for the majority of scored cells. For example, GFP-mouse coilin (panel A) forms CBs that associate with PML bodies (double arrowheads) in 80% of cells. By contrast, 92% of cells with CBs (arrows) formed by GFP-mouse coilin (100-150) (panel B) do not show association with PML bodies (arrowheads). In panels D-G, coilin knockout MEF cells were transfected with GFP-mouse coilin (100-150) + NLS and stained with anti-PML. The percent in the bottom right of each image accounts for the number of cells expressing this protein with similar localization patterns. For example, 49% of cells expressing GFP-mouse coilin (100-150) + NLS (panel D) have cytoplasmic accumulations and CBs (arrows) which do not associate with PML bodies (arrowheads). By contrast, only 11% of transfected cells display a strictly nuclear coilin signal and CBs that associate with PML bodies (panel F, double arrowhead). Scale bar, 2 µm.

View larger version (9K): [in a new window]   Fig. 6. Schematic summary of coilin and its role in nuclear organization. Human coilin is a protein of 576 aa and contains two nuclear localization signals (NLSa, NLSb) in addition to an arginine/glycine repeat region (RG). Previous work has shown that the N-terminal 92 aa of coilin are necessary for self-interaction and Thus, are important in CB formation (Hebert and Matera, 2000). Additionally, knockout studies demonstrate that full-length coilin is necessary for canonical CB formation (Tucker et al., 2001). The C-terminus of coilin is important in regulating CB number (Shpargel et al., 2003) and for the interaction with some Sm proteins and snRNPs (Hebert et al., 2001) (our unpublished observations). The RG box of coilin mediates direct interaction with SMN and reduction in arginine methylation correlates with Gem formation (Hebert et al., 2001; Hebert et al., 2002; Boisvert et al., 2002). The association of PML bodies with CBs is mediated in part by interactions between coilin and PIASy (this study). Not shown are interactions with Nopp140 (Isaac et al., 1998) and nucleic acids (Bellini and Gall, 1998).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter