Control of Cajal body number is mediated by the coilin C-terminus

Karl B. Shpargel, Jason K. Ospina, Karen E. Tucker, A. Gregory Matera* and Michael D. Hebert{ddagger}

Department of Genetics, Center for Human Genetics and Program in Cell Biology, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH 44106-4955, USA
{ddagger} Present address: Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS 39216-4505, USA

* Author for correspondence (e-mail: gxm26{at}po.cwru.edu)

Accepted 6 October 2002


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Cajal bodies (CBs) are nuclear suborganelles implicated in the post-transcriptional maturation of small nuclear and small nucleolar RNAs. The number of CBs displayed by various cell lines and tissues varies, and factors that control CB numbers within a given cell have yet to be described. In this report, we show that specific regions within the C-terminus of coilin, the CB marker protein, are responsible for regulating the number of nuclear foci. Despite the fact that the coilin N-terminal domain is responsible for its self-oligomerization activity, truncation or mutation of predicted sites of phosphorylation in the conserved C-terminal region leads to a striking alteration in the number of nuclear bodies. Similarly, coilin constructs from various species display differential propensities to form nuclear foci when expressed in heterologous backgrounds. We mapped the domain responsible for this variability to the coilin C-terminus utilizing chimeric proteins. Furthermore, the activities responsible for regulating coilin self-association must reside in the nucleus, as constructs lacking critical nuclear localization sequences fail to form foci in the cytoplasm. Factors controlling the putative signal transduction cascade that phosphorylates coilin are also discussed. The results point to a model whereby phosphorylation of the coilin C-terminus regulates the availability of the N-terminal self-interaction domain.

Key words: RNA processing, Nuclear bodies, Phosphorylation, SMN, snRNA, snoRNA


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It is increasingly evident that the nucleus, like the cytoplasm, contains a wide variety of subdomains that perform discrete functions. These nuclear substructures are dynamic and their presence probably reflects underlying cellular processes (Dundr and Misteli, 2001Go; Matera, 1999Go; Spector, 2001Go). For example, the most easily recognizable nuclear domain, the nucleolus, marks the site of ribosome synthesis and possibly other cellular events (Andersen et al., 2002Go; Olson et al., 2000Go). The functions of other nuclear domains, such as Cajal bodies (CBs) and Gemini bodies (gems) are less clear, although these domains have been implicated in aspects of small nuclear ribonucleoprotein biogenesis (Gall, 2000Go; Matera, 1999Go). Recent work (Darzacq et al., 2002Go; Jady and Kiss, 2001Go) has revealed the existence of small RNAs (scaRNAs) that are localized specifically in the Cajal body and are important for the post-transcriptional modification of uridine-rich (U) snRNAs, thus strengthening the role for CBs in snRNP biogenesis. Similarly, nuclear bodies in yeast and mammals are thought to be important for maturation of U snRNAs (Mouaikel et al., 2002Go; Verheggen et al., 2002Go). Additionally, gems contain the survival of motor neurons protein, SMN, which is the protein mutated in patients with spinal muscular atrophy (Lefebvre et al., 1995Go; Liu and Dreyfuss, 1996Go). SMN is thought to play a crucial role in cytoplasmic Sm protein assembly onto snRNAs (Meister et al., 2001Go; Meister et al., 2000Go; Fischer et al., 1997Go). A nuclear role for the SMN complex is less well defined, but the available evidence points to snRNP recycling (Pellizzoni et al., 1998Go).

In addition to their ability to move throughout the nucleus, another fascinating feature of nuclear bodies is their capacity to form within the nuclear milieu without apparent support structures (Platani et al., 2002Go). Several nuclear domains, such as Sam68 bodies, PML bodies, Cajal bodies and gems contain `signature' proteins (Sam68, PML, coilin and SMN, respectively) that are used as markers for each domain. Characterization of these marker proteins has revealed that they each share the ability to self-associate (Chen et al., 1997Go; Hebert and Matera, 2000Go; Lorson et al., 1998Go; Perez et al., 1993Go). Indeed, self-association may be a common theme utilized by nuclear body marker proteins to provide a scaffold upon which other components of the respective domain can then coalesce (Hebert and Matera, 2000Go; Misteli, 2001Go). Despite elucidation of marker proteins and characterization of a myriad of other protein and RNA constituents in nuclear bodies, understanding of mechanisms that regulate the size and number of these nuclear domains is lacking. Such mechanisms must exist and are probably propagated either through intrinsic properties of the marker proteins (e.g. structure or expression level) or extrinsic factors that affect protein-protein interaction. Interestingly, Lamond and co-workers recently showed that transient upregulation of SmB protein in cells that normally do not display Cajal bodies promotes the nucleation of these structures (Sleeman et al., 2001Go). This finding supports the idea that Cajal bodies play a role in some aspect of snRNP metabolism and suggests that snRNP expression levels can affect the localization of coilin.

Post-translational modification of marker proteins may trigger conformational changes that facilitate self- or other protein interactions crucial for nuclear body assembly. PML bodies, for example, require the SUMO modification of the PML protein in order to properly form (Ishov et al., 1999Go; Kamitani et al., 1998Go; Muller et al., 1998Go). Additionally, the phosphorylation state of Sam68 affects its ability to selfoligomerize (Chen et al., 1997Go) and thus may affect its capacity to form nuclear bodies. Likewise, coilin hyperphosphorylation, which occurs during mitosis (Carmo-Fonseca et al., 1993Go), results in a reduction in self-association (Hebert and Matera, 2000Go). Cajal bodies disassemble during mitosis and reform at early- to mid-G1 phase (Andrade et al., 1993Go; Carmo-Fonseca et al., 1993Go). Thus, the phosphorylation status of coilin might influence Cajal body formation. We have also shown that phosphorylation can control localization by exposing or sequestering a putative nucleolar localization signal (Hebert and Matera, 2000Go). This idea has precedence, as the function of the retinoblastoma protein is thought to be controlled by a similar process of sequential phosphorylation events (Harbour et al., 1999Go).

We have recently shown that symmetrical dimethylation of arginines within the coilin RG box motif is vital for the in vivo incorporation of the SMN complex into CBs (Hebert et al., 2002Go). Without this modification, the SMN complex fails to localize in CBs and forms gems. Therefore, coilin methylation, like phosphorylation, can affect nuclear body formation and composition.

We are interested in understanding how nuclear body size and number are regulated. In this report, we set out to identify regions of coilin, if any, that are responsible for controlling these processes. Surprisingly, we found that coilin proteins from human, mouse and frog displayed different localization patterns when expressed in both HeLa cells and mouse embryonic fibroblasts (MEFs). Additional experiments using MEFs derived from coilin-knockout embryos showed that specific residues within the C-terminus of coilin are important for the proper regulation of CB numbers. Finally, although the coilin self-association domain resides in the N-terminus of the protein, point mutations and small truncations in the distal C-terminus affect its ability to form or target to CBs. We conclude that coilin contains an intrinsic nuclear body formation potential, shared among other nuclear body marker proteins, but is subject to increasing layers of regulation from frog, to mouse, to human.


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Constructs and mutagenesis
GFP-tagged full-length human and mouse coilin constructs were cloned into pEGFP vectors (Clontech) as described previously (Hebert and Matera, 2000Go; Tucker et al., 2001Go). Chimeric constructs were generated by cloning the human coilin N-terminus or C-terminus into the corresponding portion of the GFP-mouse coilin construct utilizing a conserved HindIII site at 1434 bp in the mouse coilin mRNA sequence (GenBank Accession: 7710007). All truncation constructs, deletions and point mutations were generated by QuickChange mutagenesis (Stratagene). Constructs were verified by sequencing. Primer sequences can be provided upon request. SMN, fused to a myc-tag, was a kind gift from G. Dreyfuss (University of Pennsylvania).

Cell culture, transfection and immunofluorescence
The mouse embryonic fibroblast cell line was established from coilinknockout (-/-) mice as described previously (Tucker et al., 2001Go). The coilin-knockout MEF or HeLa cells were grown in DMEM (GIBCO BRL), supplemented with 10% FBS (GIBCO BRL). Cells were grown to subconfluency on chambered slides (Nunc) and transfected for 24 hours. MEF cells were transfected using LippofectAMINE (GIBCO BRL); HeLa cells were transfected using SuperFect (Qiagen) as directed. Cells were fixed in 4% paraformaldehyde, extracted in 0.5% Triton X-100 and processed for microscopy as previously described (Frey and Matera, 1995Go). Immunofluorescence was carried out with myc (1:40; Santa Cruz Biotechnology), R288 [1:100 (Andrade et al., 1993Go)] and R508 [1:200 (Chan et al., 1994Go)] antibodies.


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The coilin self-interaction domain affects epitope recognition
Previously, we showed that the N-terminus of human coilin encodes a self-association domain that mediates targeting to Cajal bodies (Hebert and Matera, 2000Go). Constructs lacking this domain fail to accumulate in CBs (Hebert and Matera, 2000Go). Subsequently, we analyzed one of these constructs, myc-coilin(94-576), with two different coilin polyclonal antibodies, R508 (Chan et al., 1994Go) and R288 (Andrade et al., 1993Go). Since these antisera were each raised against C-terminal regions of coilin, both of the epitopes should be present in the N-terminal deletion construct (Fig. 1A). However, despite high levels of coilin(94-576) expression in transfected HeLa cells (as assessed by antibodies to the myc-tag), antibody R508 does not detect the mutant protein (Fig. 1B, top panels). Indeed, the signal obtained from R508 was derived almost entirely from the endogenous coilin, whereas the ectopically expressed myccoilin(94-576) was not detected. On the other hand, nucleoplasmic staining of coilin(94-576) was readily observed by staining with R288 (Fig. 1B, bottom panels). Thus, although coilin(94-576) contains the epitopes for both antibodies, only R288 reacts with this mutant protein. Notably, R508 was raised against a peptide corresponding to the extreme C-terminus of coilin, whereas R288 was raised against the entire C-terminal domain (Fig. 1A). We therefore conclude that the presence of the coilin self-association domain, which resides in the N-terminus, can affect C-terminal epitope recognition.



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Fig. 1. Deletion of the coilin self-association domain affects epitope recognition. (A) Schematic of human coilin showing the location of the self-interaction/CB-targeting domain (Hebert and Matera, 2000Go), two nuclear localization signals (NLS), nucleolar localization signal (NoLS) (Hebert and Matera, 2000Go) and the RG box that mediates interaction with SMN (Hebert et al., 2001Go). The regions of coilin that were used to generate anti-coilin antibodies R288 (Andrade et al., 1993Go) and R508 (Chan et al., 1994Go) are also indicated. (B) Coilin lacking the self-association domain is not recognized by anti-coilin Ab R508. HeLa cells transfected with myc-tagged coilin lacking the first 93 amino acids were subject to staining with anti-myc (left panels) or anti-coilin (right panels) antibodies.

 

The C-terminus of coilin regulates Cajal body number
In addition to finding that coilin is a self-associating protein and that this domain affects C-terminal epitope recognition, we also have shown that fusion of GFP to the coilin C-terminus results in formation of numerous foci (Fig. 2) (Hebert and Matera, 2000Go). Normal coilin localization (diffuse nucleoplasmic plus bright foci) is obtained upon fusion of GFP to its N-terminus (Fig. 2). However, fusion of the GFP moiety to the C-terminus results not only in numerous coilin foci but also in a reduction in the nucleoplasmic pool of protein (Fig. 2). Given the results in Fig. 1, it seemed plausible that fusion of GFP to the C-terminus of coilin produced an aberrantly folded protein, culminating in a large increase in the number of coilin foci. In other words, the coilin C-terminus regulates CB number. This hypothesis is supported by a truncation mutant, coilin(1-481), that produces the same phenotype observed for the coilin-GFP fusion (Fig. 2). Furthermore, larger C-terminal truncations of coilin result in varied patterns of localization, from nucleolar accumulations to large nucleoplasmic inclusions, called pseudo-CBs (Bohmann et al., 1995Go; Hebert and Matera, 2000Go). These results suggest that, in vivo, the coilin C-terminus masks the N-terminal region and that fusion of this domain to GFP or outright truncation of C-terminal residues unmasks the self-association domain.



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Fig. 2. Coilin mutants display unregulated nuclear body formation. Upper panels, HeLa cells were transfected with GFP fused to the N-terminus of coilin (left), GFP fused to the C-terminus of coilin (middle) or a GFP-tagged truncation of coilin (1-481, right). The GFP-coilin(1-481) foci colocalized with other CB markers (SMN and fibrillarin) when transfected into HeLa cells (data not shown). Note that several out-of-focus dots appear blurred. In the lower panels, deletion of the nuclear localization signals (residues 106-234) in the GFP-coilin background results in exclusively cytoplasmic localization without foci in HeLa cells (left). Deletion of the NLSs in the coilin-GFP background results in a similar pattern, with the exception that nuclear foci are detected. Although the bulk of the fluorescence was cytoplasmic, two populations of cells were observed, some with faint nuclear foci and others with brighter foci (middle). When transfected into coilin knockout MEFs, this same coilin{Delta}NLS-GFP construct localized solely to the cytoplasm (right).

 

In order to test this idea further, we wanted to determine whether formation of coilin foci could take place in the cytoplasm or if it were restricted to the nucleus. If foci formation were limited to the nucleus, it would imply that important interactions or modifications (e.g. phosphorylation) are localized within this cellular compartment. Alternatively, formation of foci in the cytoplasm would indicate either that coilin modifiers exist in the cytoplasm or that foci formation is an intrinsic property of the coilin N-terminus. The nuclear localization signals (NLSs) reside within the loosely conserved internal region that links the more highly conserved N- and C-terminal domains (Bohmann et al., 1995Go). We therefore created internal deletions spanning coilin residues 106-234. In the GFP-coilin background, the {Delta}NLS protein shows exclusively cytoplasmic localization, lacking foci, in HeLa cells (Fig. 2, lower left panel). Surprisingly, endogenous coilin localization was not altered in cells expressing the GFP-coilin{Delta}NLS (data not shown). Thus, it was possible that deletion of the NLS region could affect the self-association activity of coilin. To test this, and to verify that coilin foci formation is limited to the nucleus, we took advantage of the coilin construct that generates a plethora of dots, coilin-GFP. HeLa cells transfected with coilin{Delta}NLS-GFP typically displayed two to five foci in the nucleus, with additional bright staining throughout the cytoplasm (Fig. 2, lower middle panel). Thus despite the lack of an NLS, a small amount of coilin{Delta}NLS-GFP protein was imported into the nucleus, perhaps by binding to endogenous coilin molecules. In order to test this idea, we transfected coilin{Delta}NLS-GFP into coilin-knockout cells (Tucker et al., 2001Go) and found that, indeed, the construct was completely restricted to the cytoplasm in all cells (Fig. 2, lower right panel). It should be noted that HeLa cells expressing coilin{Delta}NLS-GFP displayed a markedly reduced number of foci compared with the parental construct (Fig. 2, compare upper middle and lower middle panels). Nonetheless, the ability to form bodies in the nucleus and not in the cytoplasm verifies the idea that the subcellular localization of coilin is important for their formation.

Coilins from different species demonstrate specific nuclear body regulatory potentials
When transiently expressed in HeLa cells at low to medium levels, human GFP-coilin properly localizes to the nucleoplasm and the CB. However, high levels of expression result in reorganization of ectopic coilin, endogenous coilin, and other constituents of the CB, such as SMN, to the nucleoplasm (Hebert and Matera, 2000Go). High levels of overexpression do not alter the overall nuclear architecture since other nuclear structures, such as PML bodies, are unaffected (Hebert and Matera, 2000Go). Furthermore, the GFP moiety is not responsible for this phenotype because coilin constructs with a myc-tag display the same effect (K.B.S., M.D.H. and A.G.M. unpublished). In contrast to the results using the human coilin constructs in HeLa cells, high levels of GFP-mouse coilin overexpression do not abolish foci formation. Instead, cells expressing high levels of mouse GFP-coilin displayed the unregulated pattern of coilin foci observed for human coilin(1-481) or coilin-GFP (Figs 2 and 3). Similarly, frog YFP-coilin also displayed a multitude of foci when expressed in HeLa cells (Fig. 3). Surprisingly, transfection of these three coilins into a murine embryonic fibroblast (MEF) cell line lacking endogenous coilin (Tucker et al., 2001Go) revealed additional species-specific regulatory differences. For example, human coilin failed to form CBs in most of these MEFs, even in those with low expression levels (Fig. 3). By contrast, mouse coilin readily formed foci in MEF cells when expressed at low levels. MEF cells expressing high levels of mouse coilin showed only nucleoplasmic staining. Thus expression of heterologous coilins in human cells resulted in dysregulation of CB numbers. When mouse coilin, for example, was expressed in murine cells, the number of coilin foci did not increase. However, the number of coilin foci was completely unregulated when frog coilin was expressed at high levels in both cell types (Fig. 3). This was most easily observed upon expression of frog coilin in the knockout MEFs. In this background, large coilin aggregates were observed, even in the cytoplasm. Furthermore, the pool of nucleoplasmic frog coilin in MEF cells was virtually non-existent.



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Fig. 3. Coilins from different species display variation in nuclear body formation. HeLa cells (left two columns) or mouse embryonic cells lacking endogenous coilin (right two columns) were transfected with FP-tagged human, mouse or frog coilin. Representative cells of both low and high expressers are shown. The nucleus is defined by a dotted line for MEF cells transfected with FP-frog coilin.

 

In summary, human coilin is able to effectively target to pre-existing CBs in HeLa cells when expressed at low levels but is unable to target, and indeed disrupts, CBs at high expression levels. In MEF cells lacking endogenous coilin, the human protein is ineffective at nucleating CBs (Fig. 3). Mouse coilin is able to effectively form CBs in mouse cells at low expression levels but, like human coilin in HeLa cells, becomes diffusely nucleoplasmic at higher levels of expression. In the HeLa background, however, mouse coilin displayed numerous foci. Similar findings were observed for frog coilin in both HeLa and MEF cells. At least for mouse and frog coilin proteins, the expression level was an important determinant for nuclear body formation. Human coilin, however, appears to have additional constraints regarding its ability to form nuclear bodies.

Chimeric coilin constructs delineate the Cajal body regulatory region
We next set out to better define the region within coilin that governs CB formation and number. Given that truncation or fusion of the C-terminus resulted in formation of numerous coilin foci (Fig. 2), we speculated that this region might play an important regulatory role. These findings, coupled with the observation that, compared with the human protein, mouse coilin has an increased potential for nuclear foci formation in HeLa cells (Fig. 3), led us to postulate that the C-terminus of mouse coilin imparts differential regulatory constraints. To test this idea, we generated chimeric constructs of human and mouse coilin by swapping the C-terminal 96 amino acids (Fig. 4). Following expression in coilin-knockout MEFs, we assessed the capacity of the mutants to form nuclear foci (Fig. 4). As previously observed (Tucker et al., 2001Go), cells expressing moderate levels of mouse coilin typically display foci (87%), whereas those expressing human coilin rarely display foci (7%; Fig. 4). Furthermore, the number of foci per cell was greatly reduced (0.09 for human versus 2.84 for mouse). Strikingly, replacement of the mouse C-terminus with the corresponding human portion of coilin reduced both the frequency of cells displaying foci and the numbers of foci per cell (Fig. 4, compare 87% for mouse with 1% for the mouse/human chimera). Conversely, replacement of the human C-terminus with the mouse region increased the number of cells that displayed coilin foci by 10-fold (Fig. 4, 7% for human versus 70% for human/mouse). We conclude that the C-terminal 96 amino acids of human coilin downregulates the capacity for nuclear body formation, whereas the corresponding region in mouse coilin increases this capability.



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Fig. 4. Schematic of human and mouse coilin. Chimeric constructs were generated by use of a conserved HindIII site, essentially swapping the amino acids downstream of this site. Numbers of foci per cell and percentages of cells with foci upon transfection into coilin-knockout MEFs are displayed to the right of the representative construct.

 

Critical serine residues in the C-terminus of coilin control nuclear body formation
Despite the apparent phenotypic differences in nuclear-body-forming potential, an alignment of the various coilin C-termini demonstrates a high level of identity in this region (Fig. 5A). Interestingly, frog coilin shows several residues that are substantially changed compared with the corresponding amino acids in human and mouse (e.g. frog contains DEE at 499-501 compared with NGA in human). These differences, although slight, may account for the observed localization phenotypes. Curiously, the biggest differences between human and mouse are in the very C-terminal, serine-rich, portion. Given that human coilin phosphorylation occurs exclusively on serine residues (Carmo-Fonseca et al., 1993Go) and that several of the serines in its C-terminus are predicted to be phosphorylated (NetPhos, Technical University of Denmark; http://genome.cbs.dtu.dk/services/NetPhos/), we decided to systematically generate mutations in this region and assess their nuclear-body-forming potentials. The mutations were each produced in GFP-coilin backgrounds, and the constructs were subsequently transfected into HeLa cells or coilin knockout MEFs. A summary of the mutations and their proclivities to form nuclear bodies in MEF cells is shown in Fig. 5B. The mutation of arginine 561 in human coilin to a stop codon resulted in a variable phenotype. In HeLa cells, human coilin(1-560) was mostly nucleoplasmic with nuclear aggregates, although a small percentage of cells (<10%) appeared normal (data not shown). In coilin-knockout MEFs, human coilin(1-560) mirrors that observed in HeLa, with the exception that no cells displayed coilin foci.



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Fig. 5. Mutations in mouse coilin affect nuclear body formation. (A) Alignment of the C-termini of human, mouse and frog coilin. The HindIII site used in generating the coilin chimeras is shown. (B) Summary of the mutations generated in this study. All constructs are fused to the C-terminus of GFP, with the exception of human coilin-GFP (FL-GFP). The constructs were transfected into MEF cells lacking endogenous coilin (Tucker et al., 2001Go) and scored for their ability to generate foci. Note that human constructs coilin-GFP and (1-481) as well as mouse construct (1-477) produce numerous unregulated foci. (C) HeLa cells were transfected with GFP-tagged wild-type mouse coilin or mutations of mouse coilin. Endogenous coilin (right column) was detected by using an antibody specific to human coilin (R508). Arrows mark the transfected cells, with concurrent disruption of Cajal bodies (right panels). Arrowheads show non-transfected cells, which display normal coilin localization in CBs and the nucleoplasm (right column). Foci formed by GFP-mouse coilin-SST to DDT were able to recruit SMN and fibrillarin, whereas loss of coilin foci upon overexpression of GFP-mouse coilin-SST to AGA also correlated with a loss of SMN foci (data not shown).

 

The corresponding truncation mutation in mouse coilin (arginine 557 to stop) showed a dramatic change in localization when expressed in HeLa cells compared with full-length mouse coilin (Fig. 5C). Most HeLa cells transfected with mouse coilin(1-556) displayed primarily nucleoplasmic staining, even at lower expression levels. Additionally, high expression levels of mouse coilin(1-556) resulted in nucleoplasmic staining and corresponding disassembly of endogenous CBs (Fig. 5C, arrow). The overexpression of mouse coilin(1-556) in HeLa cells is reminiscent of the pattern seen for the high expression of human coilin in HeLa cells, which likewise results in nucleoplasmic staining and subsequent disruption of CBs (Hebert and Matera, 2000Go). Curiously, overexpression of wild-type mouse coilin in HeLa cells also reduced the amount of endogenous coilin in CBs, despite the observed foci present for mouse coilin (Fig. 5C, top panels, arrow). Thus we conclude that removal of the C-terminal 13 amino acids of mouse coilin effectively makes it behave like the human protein. In support of this idea, MEF cells transfected with mouse coilin(1-556) no longer displayed CBs, but showed nucleoplasmic staining (Fig. 5B). Therefore, these 13 amino acids affect not only the ability of coilin to target to preformed CBs, but the capacity to form foci in the absence of endogenous coilin.

Larger C-terminal truncations of both mouse (1-477) and human (1-481) coilin resulted in formation of multiple foci in MEF cells (Fig. 5B). However, smaller C-terminal deletions (human: 1-516 and 1-560; mouse: 1-512, 1-535 and 1-556) lost the ability to form coilin foci when transfected into MEF cells. Curiously, a construct lacking the C-terminal five residues, mouse(1-564), was capable of generating nuclear bodies whereas mouse(1-556) did not, thus narrowing a region that may structurally inhibit nuclear body formation to residues 478-563 in mouse coilin (Fig. 5B).

Given the serine-rich composition of the C-terminal 13 residues in mouse coilin (5 out of 13), we speculated that the phosphorylation states of these residues might play a role in the ability to form foci. In particular, we were interested in the last two serines of mouse coilin (S567 and S568), which are conserved in human coilin (Fig. 5A). Mutation of these serines, along with the C-terminal threonine in mouse coilin SST to AGA, resulted in a remarkable mislocalization both in MEF and HeLa cells (Fig. 5B,C). Indeed, mouse coilin (AGA) and (1-556) showed virtually identical overexpression localization patterns in HeLa (Fig. 5C). These findings led us to speculate that S567 and S568 in the mouse protein need to be phosphorylated to properly form or target CBs. When the corresponding serines were mutated to aspartates (SST to DDT), mimicking a constitutively phosphorylated state, the ability to form foci in the absence of endogenous coilin and to produce numerous coilin foci in HeLa cells was retained (Fig. 5B,C). However, mutation of mouse residues S567 or S568 (to either D or A) individually had little effect on the localization pattern (Fig. 5B, data not shown). Point mutagenesis of other residues suspected to affect the folding of the C-terminus also failed to produce notable changes in coilin sublocalization compared with the parental construct (Fig. 5B). Thus mutation of both serines 567 and 568 together had an effect whereas no effect was observed when mutated singly.

We speculated that the mutants that cannot form nuclear bodies (mouse 1-556 and SST to AGA) were somehow deficient in coilin self-association. Coimmunoprecipitation assays using these constructs, however, failed to show any differences in the abilities of the mutated coilins to interact with coilin or other components of the CB, such as SMN (data not shown). Therefore, it is possible that the mutations in the mouse coilin background specifically affect the ability to target or form CBs. Our inability to identify human coilin constructs, other than coilin-GFP and GFP-coilin (1-481), that form foci in MEF cells (Fig. 5B) suggests the existence of additional regulatory controls for human coilin.

Other nuclear body proteins display unregulated foci formation
To generalize the idea that, apart from coilin, proteins considered markers for different nuclear bodies can induce foci formation upon overexpression, we surveyed the literature. Notably, various mutants of Sam68, the marker protein for Sam68 nuclear bodies, appear to have lost the ability to regulate nuclear body formation (Chen et al., 1999Go). Cells transfected with wild-type Sam68 typically display two to five nuclear bodies, along with a diffuse nucleoplasmic staining. However, mutants such as Sam68{Delta}L1 produce numerous foci (up to 30) along with a concurrent loss of nucleoplasmic staining (Chen et al., 1999Go). Several human coilin mutants also display a similar phenotype (Fig. 2). PML, which is considered to be the marker protein for PML bodies, also generates more PML bodies when overexpressed in HeLa cells and larger (but fewer) PML bodies when overexpressed in knockout MEFs (Ishov et al., 1999Go; Mu et al., 1997Go).

On the other hand, overexpression of wild-type SMN, which is vital for gem formation, reportedly does not change the number of gems in transfected cells (Pellizzoni et al., 1998Go). We have found, however, that high expression of myc-tagged SMN, both in HeLa cells and coilin knockout MEFs, results in formation of numerous foci (Fig. 6). The additional foci are present both in the nucleus and the cytoplasm. The phenotype is similar to that observed upon ectopic expression of the SMN{Delta}N27 mutant protein (Pellizzoni et al., 1998Go). The main difference is that the enlarged nuclear foci seen with SMN{Delta}N27 (Pellizzoni et al., 1998Go) were not observed in cells overexpressing the wild-type protein. The large cytoplasmic blobs (Fig. 6A, bottom) were observed only at the higher expression levels. In HeLa cells, SMN overexpression produces nuclear foci which sometimes contain coilin and can be considered CBs (Fig. 6A, arrow) and other foci that lack coilin and thus can be considered gems (Fig. 6A, arrowheads). In summary, we conclude that the overall level of SMN expression (Fig. 6) and the extent of coilin arginine dimethylation (Hebert et al., 2002Go) are major determinants in gem formation.



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Fig. 6. Ectopically expressed SMN produces numerous SMN foci. (A) HeLa cells expressing high levels of myc-tagged SMN were visualized by anti-myc antibodies (green). Coilin was detected in the same cell with R508 (in red), and the merged image is shown (right panel). Arrows denote Cajal bodies, which contain both SMN and coilin, whereas arrowheads mark some of the SMN gems, which are present both in the cytoplasm and the nucleus. The bottom panels display some of the large SMN foci that are observed upon high expression of SMN. (B) MEF coilin-knockout cells transiently transfected with myc-SMN were detected by anti-myc antibodies. Arrowheads demarcate some of the foci, including large aggregates, formed in high expressing cells (right).

 


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 Materials and Methods
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 Discussion
 References
 
In this report, we demonstrate that the Cajal body marker protein, coilin, contains a domain capable of regulating the number of nuclear bodies per cell. This domain is located in the C-terminus of the protein and contains putative phosphoserine residues. One of the most interesting aspects of this study is the finding that heterologous coilin proteins display differential nuclear body regulatory potentials in different cellular backgrounds. For example, mouse coilin produces numerous foci in HeLa cells when expressed at high levels, but fails to form foci when expressed at high levels in murine cells. However, frog coilin produces numerous foci both in HeLa and murine cells and can even generate cytoplasmic bodies.

Interestingly, frog coilin does not have a serine-rich C-terminus unlike the mouse and human proteins (Fig. 5A). Furthermore, unlike the human protein (Carmo-Fonseca et al., 1993Go), Xenopus coilin is not highly phosphorylated (M. Bellini, personal communication). It is possible, therefore, that frog coilin is `locked' in a conformation that renders it competent for nuclear body formation and thus is insensitive to phosphorylation. Consequently, frog coilin would be expected to readily form nuclear foci (Fig. 3). Human and mouse coilin appear to have greater regulatory constraints with regard to their ability to form nuclear bodies. This idea is supported by findings using the human coilin{Delta}NLS mutants (Fig. 2), which are incapable of forming cytoplasmic foci. These results suggest that factors responsible for phosphorylating coilin are located in the nucleus.

Given that alterations in coilin phosphorylation levels can affect both self-association and nucleolar localization (Hebert and Matera, 2000Go; Lyon et al., 1997Go; Sleeman et al., 1998Go), it is likely that this modification plays an important role in CB targeting as well. We showed that, unlike human coilin, a chimeric human/mouse construct can readily form foci in murine cells lacking endogenous coilin. A reciprocal construct containing mouse coilin with the human tail displayed a phenotype similar to that of the human protein expressed in the mouse cells (Fig. 4). Considering that mutation of the coilin N-terminal self-association domain alters C-terminal epitope recognition by the anti-coilin Ab R508, we speculated that self-association would be regulated via the C-terminal tail. Coimmunoprecipitation experiments, however, failed to show any change in coilin self-association among the various mutants. Furthermore, the observation that mouse coilin(1-556) and coilin(AGA) disrupt the endogenous coilin in HeLa cells supports the notion that self-interaction is not affected (Fig. 5C). Although it is conceivable that self-interaction is required for nuclear body formation, additional cellular factors [e.g. high levels of Sm proteins (Sleeman et al., 2001Go)] and/or modifications of coilin may be necessary. Since mutation of the mouse coilin C-terminal residues to mimic constitutively phosphorylated serines (SST to DDT) results in wild-type levels of foci formation, we conclude that these sites are normally phosphorylated. Since kinase recognition sites are often dependent on previous phosphorylation events, the phosphorylation of these residues could facilitate additional serine phosphorylation elsewhere within the coilin protein. The fact that mutation of individual S567 or S568 serines to mimic an unphosphorylated state had no effect on mouse coilin localization emphasizes the complexity of coilin phosphorylation. Furthermore, the inability of human coilin to form foci at high expression levels in HeLa cells (or to form any foci at all in MEF cells) may be due to titration of the putative coilin kinases. We have shown that coilin is a substrate for the kinases CDK2-cyclinE (Liu et al., 2000Go) and casein kinase II (Hebert and Matera, 2000Go); however, phosphatases that modulate the phosphorylation state of coilin have not been described. Current studies are focused on identification of residues within coilin that are post-translationally modified and thus alter its conformation, localization and function.

As an alternative model, it is possible that mutation of the coilin C-terminus may not affect phosphorylation, but rather, alter a binding site between coilin and other proteins. The C-terminus may be needed to interact with another protein essential for CB formation, and overexpression of coilin may titrate out this integral component thus dispersing CBs. Another such example exists whereby overexpression of the immediate-early protein IE1 leads to a disruption in PML body formation by interacting with the major structural factor PML (Ahn et al., 1998Go). Additionally, coilin itself may encode the structural integrity of the CB, and the mouse coilin C-terminal mutants deficient in CB formation may simply yield an alteration in coilin structure rather than phosphorylation, leading to CB dispersal. Future experiments will need to address whether the implicated residues are indeed phosphorylated in vivo and if differences in phosphorylation levels can account for CB number differences between cell lines.

In conclusion, various nuclear body marker proteins share a common capacity for self-association and nuclear body formation. However, simply making more protein is not always sufficient to generate additional nuclear foci, especially in the case of CBs. Interestingly CBs can be induced to form in cell lines that do not typically possess them by overexpression of Sm proteins (Sleeman et al., 2001Go). Given that post-translational modification of coilin plays a vital role in Cajal body composition (Hebert et al., 2002Go) and formation (this work), we are especially interested in assessing the modification status of coilin in the nucleoplasmic versus CB compartments. Recently developed methods for CB purification (Lam et al., 2002Go) should greatly aid such an endeavor.


    Acknowledgments
 
We thank E. Chan, U. Fischer, G. Dreyfuss and J. Steitz for reagents. This work was supported by NIH grants GM53034 and NS41617, and by a research grant from the Muscular Dystrophy Association (to A.G.M.). K.B.S. and J.K.O. were supported in part by NIH predoctoral traineeships (GM08613). M.D.H. was supported in part by an NIH postdoctoral fellowship (HD07518) and by a Career Development Grant from the MDA.


    References
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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