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Vol. 20, Issue 6, 1661-1670, March 15, 2009
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*Carnegie Institution, Department of Embryology, Baltimore, MD 21218; Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom; Department of Biology, University of North Carolina, Chapel Hill, NC 27599; and Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112
Submitted May 27, 2008;
Revised December 23, 2008;
Accepted January 9, 2009
Monitoring Editor: Marvin Wickens
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Because a CB cannot be identified by morphology alone, it has only recently become clear that CBs exist in organisms as diverse as insects, higher plants, amphibians, mammals, and even budding yeast (reviewed in Gall, 2000; Gall, 2003; Cioce and Lamond, 2005; Matera and Shpargel, 2006). The key to this realization was the discovery of the protein coilin in HeLa cells (Andrade et al., 1991; Raska et al., 1991). Human coilin is a 576-amino acid protein of poorly understood function, which is highly enriched in CBs but also occurs at low concentration throughout the nucleoplasm (Bellini, 2000). Orthologous proteins have been identified by sequence comparison and cloning in Xenopus (Tuma et al., 1993), the mouse (Tucker et al., 2000), and Arabidopsis (Collier et al., 2006), and shown to occur in prominent nuclear bodies. Several attempts to identify Drosophila coilin, either by homology search or by use of heterologous antibodies, met with little success, leading to the suggestion that Drosophila might lack coilin. Nevertheless, it was possible to identify CBs in Drosophila using other molecular markers, particularly splicing small nuclear RNAs (snRNAs), fibrillarin, the survival motor neuron (SMN) protein, and the small CB-specific (sca) RNA U85 (Darzacq et al., 2002; Liu et al., 2006b). We report here the characterization of Drosophila coilin protein and the coil gene. We also analyzed the phenotype of three mutants in the coil gene. Two are protein nulls that lack coilin in all tissues. Typical CBs are missing from the cells of the null mutants, as judged by the absence of discrete foci of several common CB components. The third mutant has no obvious effect on coilin in somatic tissues, but lacks coilin and CBs in germline cells of the ovary and testis. We conclude that coilin is essential for maintenance of CB composition, but it is not required for viability or production of functional germ cells in Drosophila.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Transgenic flies that express GFP-labeled polo kinase (CG12306) were derived from a P-element protein trap screen (Buszczak et al., 2007). Several transgenic lines were made that expressed the mouse coilin gene. A Drosophila Kozak sequence was introduced into enhanced green fluorescent protein (EGFP)-mouse coilin by quick change and subcloned into pCaSpeRhs. We made two P-element constructs of full-length D. melanogaster coilin (CG8710), with EYFP (Venus) at either the amino terminus or carboxy terminus of the protein. The P-element was pUASp, in which the cloned protein is under control of the yeast upstream activating sequence (UAS) (Rorth, 1998). pUASp is a modified version of pUASt (Brand and Perrimon, 1993) and was used because it shows enhanced expression in the ovary. P-element transformation was carried out by standard procedures. Twenty different lines were obtained, 10 with EYFP at the amino terminus and 10 at the carboxy terminus. We made similar P-element constructs of the putative short isoform of CG8710, nine with EYFP at the amino terminus and 7 at the carboxy terminus. Two coilin null lines, coil199 and coil203, were generated by site-specific mutation using zinc-finger nucleases (Beumer et al., 2008). A mutant carrying a piggyBac transposon insertion in the coilin gene, PBac{5HPw+}coilinB220, was obtained from the Bloomington Stock Center (Department of Biology, Indiana University, Bloomington, IN; http://fly.bio.indiana.edu/).
Tissue Preparation
Various tissues from D. melanogaster third instars and adult flies were examined as whole mounts. Fresh tissues were isolated in Grace's insect medium (Grace, 1962) and fixed at room temperature for 10 min in 4% paraformaldehyde in phosphate-buffered saline (PBS: 135 mM NaCl, 2.5 mM KCl, 4.3 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.2). After washing in PBS, samples were used for immunofluorescence or fluorescence in situ hybridization (FISH) immediately. Alternatively, fixed samples were stored at 4°C in 0.5% horse serum and 0.3% Triton X-100 in PBS for immunostaining, or at –20°C in hybridization mix for FISH.
Antibodies
Rabbits and guinea pigs were injected with fragments of D. melanogaster coilin that had been expressed in Escherichia coli. Amino acids 1-146 with a glutathione transferase (GST) tag produced a soluble product. Amino acids 147-634 with a 6-His tag produced an insoluble product. Altogether, four rabbit antibodies were produced, two against the amino-terminal fragment (R1 and R2) and two against the carboxy-terminal fragment (R3 and R4). Similarly, four guinea pig antibodies were made (GP1-4). Crude sera were diluted 1:1000 or 1:2000 for immunostaining or 1:15,000 for Western blots. Other primary antibodies were as follows: rabbit polyclonal serum against D. melanogaster Lsm10 and Lsm11 (Liu et al., 2006b), affinity-purified chicken polyclonal serum against D. melanogaster CID (Blower and Karpen, 2001), rabbit polyclonal serum against Drosophila SMN (Ilangovan et al., 2003); mouse monoclonal antibody (mAb) 72B9 against fibrillarin (Reimer et al., 1987), mouse mAb Y12 against the "Sm" epitope (Lerner et al., 1981), mouse mAb against chicken tubulin (catalog no. T9026; Sigma-Aldrich, St. Louis, MO), mouse mAb against green fluorescent protein (GFP), and rabbit polyclonal serum against GFP (Torrey Pines BioLabs, Houston, TX). Secondary antibodies were goat anti-mouse, goat anti-rabbit, goat anti-chicken, and goat anti-guinea pig labeled with Alexa 488, 568, or 594 (Invitrogen, Carlsbad, CA).
Immunostaining
Whole mounts or cultured cells were stained with a primary antibody overnight, rinsed in PBS, and stained 4 h or overnight with a secondary antibody plus 1 µg/ml the DNA-specific dye 4',6-diamidino-2-phenylindole (DAPI). To facilitate penetration of reagents into whole tissues, 0.3% Triton X-100 was included in all solutions. Tissues were rinsed in PBS + Triton X-100 and equilibrated for a few minutes in mounting solution (50% glycerol + 1 mg/ml 1,4-diaminobenzene) before mounting under a coverslip on standard 3- x 1-in. glass slides. Coverslips were usually ringed with nail polish. Slides were stored at –20°C for days or weeks without loss of signal.
FISH
Fluorescent RNA probes labeled with Alexa-488-uridine 5'-triphosphate (UTP), Alexa-546-UTP, or Cy5-UTP were prepared by in vitro transcription from DNA clones, polymerase chain reaction (PCR) products, or deoxyoligonucleotides as described previously (Liu et al., 2006a,b). One or more probes were prepared for each specific RNA species (U2, U4, U5, U6, and U7 snRNAs, and U85 scaRNA). Probes were diluted in the following hybridization mix: 50% formamide, 5x SSC (1x SSC is 0.15 M NaCl + 0.15 M Na citrate, pH 7.0), 10 mM citric acid, 50 µg/ml heparin, 500 µg/ml yeast tRNA, and 0.1% Tween 20. Tissues for in situ hybridization were incubated at 42°C for several hours or overnight, depending on the probe size. In many cases, tissues were observed while still in the hybridization mix (with 1 µg/ml DAPI). Otherwise tissues were rinsed in PBS and mounted in 50% glycerol mounting solution.
Fluorescence Microscopy
Images were taken with a 40x (numerical aperture [N.A.] 1.25) or a 63x (N.A. 1.40) plan apochromatic objective on a laser-scanning confocal microscope (SP2 or SP5; Leica, Exton, PA). Images were taken with the laser intensity and photomultiplier gain adjusted so that pixels in the region of interest were not saturated ("glow-over" display). In most cases, contrast and relative intensities of the green (Alexa 488), red (Alexa 546, 568, and 594), far red (Cy5), and blue (DAPI) images were adjusted with Photoshop (Adobe Systems, Mountain View, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). One of these, which we called the CB, contained at least four molecules found in vertebrate CBs—U2 snRNA, U85 scaRNA, SMN, and fibrillarin. The second body contained the U7 small nuclear ribonucleoprotein (snRNP), and because it invariably associated with the histone gene locus, we called it the histone locus body (HLB). CBs and HLBs were frequently close to each other or touching, but they could also be far apart in the nucleus. We were puzzled by the existence of two separate nuclear bodies, each of which contained canonical CB components. Previous studies in mice showed that when coilin is knocked out, the CB is replaced by three separate bodies, each of which contains a subset of CB components (Tucker et al., 2001; Jády et al., 2003). These findings led to the idea that coilin might be the "glue" that holds the CB together and that the presence of two bodies in Drosophila might be due to the lack of coilin (Liu et al., 2006b). To test this hypothesis, we made transgenic flies that expressed EGFP fused to mouse coilin. Despite its expression in a heterologous system, mouse coilin localized to discrete nuclear foci that were often adjacent to HLBs but sometimes were completely separate from them (Figure 1A). Thus, the HLB and the coilin-positive body exhibited the same relationship as in wild-type flies. This observation suggested that mouse coilin interacts with components of Drosophila CBs and/or HLBs. Regardless, CBs and HLBs did not fuse together in the presence of mouse coilin—they retained separate identities. However, one could argue that mouse coilin might not function properly in Drosophila cells (see Supplemental Data). It thus remained imperative to determine whether Drosophila indeed lacked an orthologue of vertebrate coilin.
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; Raska et al., 1991). Since then, orthologous proteins in other vertebrates (Tuma et al., 1993; Tucker et al., 2000) and in Arabidopsis (Collier et al., 2006) have been widely used as specific biochemical markers for the CB. However, attempts to identify coilin in Drosophila, Caenorhabditis, and other invertebrates have been unsuccessful.
Vertebrate coilins show two conserved regions at the ends of the protein with considerable sequence divergence in the middle (Bellini, 2000; Tucker et al., 2000). In earlier BLAST searches of the D. melanogaster genome, neither we nor others could find an orthologue of human, mouse, or Xenopus coilin. However, when we carried out BLAST searches at lower stringency, we found some similarity between the amino- and carboxy termini of vertebrate coilins, especially zebrafish coilin, and the corresponding regions of D. melanogaster gene CG8710 (Figure 2 and Supplemental Data). Encouraged by this finding, we cloned EYFP-tagged versions of CG8710 and made transgenic flies that expressed the protein under control of the GAL4-UAS system. We saw highly specific expression of CG8710 in CBs, leading us to conclude that we had at last found the elusive Drosophila coilin gene.
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To analyze the distribution of coilin at the cellular level, we produced antibodies against two fragments of CG8710, one from the amino terminus (amino acids [aa] 1-146) and one from the carboxy terminus of the molecule (aa 147-634). On Western blots of ovary extract, each antibody recognizes a band at Mr 
90 kDa, which we presume represents the full-length protein (Figure 3A). CG8710 encodes a 634-amino acid protein with predicted molecular weight (MW) of 70.6 kDa. The discrepancy between the Mr and the predicted MW is not surprising, because a similar discrepancy has been seen with human, mouse, and Xenopus coilin, all of which have Mr 80 kDa, although their calculated MWs are 59.6 kDa for Xenopus and 62.6 and 62.3 kDa for human and mouse, respectively. The 90-kDa band is missing from Western blots of ovarian tissue from three coilin mutants, which we describe later (Figure 3A). For this reason, we are confident that the 90-kDa band represents full-length Drosophila coilin.
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), although the effect was not so extreme as in Drosophila. The following description of Drosophila coilin distribution is based on both antibody staining and expression of EYFP-coilin, labeled at the amino terminus. For identification of the HLB, we used FISH for U7 snRNA or immunostaining for Lsm10 and Lsm11 (Liu et al., 2006b), which are specific components of the U7 snRNP.
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Testis. In the testis, coilin exhibits striking cell- and stage-specific variation in its intranuclear distribution, particularly well exemplified in testes from third instar larvae. In wild-type larvae, each somatic hub cell at the tip of the testis displays a single focus of coilin stain. In contrast, the germline stem cells immediately around the hub lack such foci (Figure 7A), although coilin is detectable throughout the nucleus. In this respect they resemble the germline stem cells of the ovary. Spermatogonial nuclei also lack discrete foci of coilin stain. Coilin is dramatically up-regulated in early spermatocytes, in which it is widely distributed throughout the nucleus exclusive of the nucleolus (Figures 7B and 9C). A large amount of coilin persists during the spermatocyte divisions, when it decorates the first and second meiotic spindles (Figure 7C). HLBs are evident in the hub cells and early germline cells (Figure 7A), but disappear by the primary spermatocyte stage.
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Coilin in the Cell Cycle
To determine the behavior of coilin during the cell cycle, we performed live cell imaging on early embryos derived from females that expressed EYFP-coilin (Supplemental Movie 1). We began observations at the 10th cell cycle, at which time we saw a clear fluorescent signal in the blastoderm nuclei at the surface of the embryo. During the ensuing nuclear divisions, coilin underwent extensive movements. In interphase, coilin was detectable throughout the nucleoplasm with multiple brighter foci scattered more or less randomly in the nucleus. As the chromosomes condensed during prophase, these foci became more evident, eventually lining up along the metaphase plate. Because of their position and number, these foci seem to be at or near the centromeres of the chromosomes. At the onset of anaphase, the bright foci suddenly disappeared, leaving a faint coilin signal throughout the spindle. For a short period at late telophase, coilin was detectable only in the midbody. Finally, as the nuclei reformed, coilin reappeared in multiple nuclear foci and throughout the nucleoplasm.
We also examined mitosis in cultured S2 cells, in larval brains, and in adult ovarian follicle cells. The pattern of coilin distribution during mitosis was similar to that seen in the blastoderm; namely, coilin occurred as a band of stain or a row of dots lined up along the metaphase plate. To determine more precisely the localization of coilin relative to the chromosomes, we examined mitosis in flies that expressed GFP-labeled polo kinase (Buszczak et al., 2007), a marker for the kinetochores (Figure 8A). We also stained with an antibody against CID, the Drosophila CENP-A protein (Figure 8, B–E). Both polo kinase and CID occurred as double rows of dots external to coilin, which occurred as a single row of dots or band of stain along the center of the metaphase plate. These relationships suggest that coilin lies between the sister chromatids, either at the centromeres themselves or along the entire pericentromeric heterochromatin.
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). The third mutant, coilB220, is an insertion of a piggyBac transposon in the 5' untranslated region of the gene (http://flybase.bio.indiana.edu/). We used PCR to confirm that the insertion site of the transposon is 22 nt upstream of the ATG translation start site. We are unaware of other coil mutants in public databases. All three coil mutants are homozygous viable and fertile with no obvious morphological phenotype.
Because both coil199 and coil203 lead to frame-shifts in translation at the start of the coding region, we expected them to be protein null mutants. None of the tissues that we have tested from larvae or adults show any staining with antibodies against coilin (Figure 9). Western blots of ovary proteins from these two mutants are completely negative for coilin, whereas proteins from wild-type ovaries show an easily detectable band at 90 kDa (Figure 3).
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The coilB220 mutant is quite different from the null mutants. Immunostaining with antibodies against coilin gives normal signals in CBs of all somatic tissues examined from both larvae and adults. By contrast, germline cells in the ovary and testis are completely negative for coilin (Supplemental Figure S3). As in the coilin-null mutants, HLBs are apparently normal in these tissues. Presumably the insertion of the pBac transposon upstream of the coding region affects sequences required for germline specific expression of coilin. In keeping with the staining results, Western blots of ovary proteins from the coilB220 mutant are negative for coilin (Figure 3).
Are There Residual CBs in the Coilin-Null Mutants?
In cells derived from coilin-null mice there are no coilin-positive CBs. Instead, typical CB components are distributed among three "residual" bodies, each of which contains a subset of CB components (Tucker et al., 2001; Jády et al., 2003). To determine whether a similar situation occurs in Drosophila, we examined the distribution of several typical CB components in Malpighian tubule cells in the null flies. Nuclei in Malpighian tubules are large and they contain prominent CBs and HLBs. Often the CB and HLB are well separated from the nucleolus, making analysis of their composition easier than in cells where these bodies closely associate with the nucleolus. As already mentioned, U85 scaRNA no longer occurs in a defined structure in the ovary in null flies, and the same is true for Malpighian tubule cells (Figure 11, A–F). We also examined the following CB components: fibrillarin (associated with small nucleolar RNAs), SMN, U2 snRNA, U5 snRNA, and symmetric dimethylarginine (a marker for snRNPs). In no case were these components detectable in a distinct body within the nuclei of the nulls (Figure 11, C–J and Supplemental Figure S2, A–F). In Malpighian tubule cells of wild-type flies, there is usually a single prominent HLB, which contains U7 snRNA, Lsm11 and a low level of coilin (Figures 7, F–H, and 11, A, G, and I). Despite the absence of coilin in the nulls, there is still a body with Lsm11 and U7 snRNA (Figure 11, B, H, and J).
We find what can be considered a residual CB in one cell type, the oocyte. As already mentioned, the germinal vesicle (GV) displays a prominent coilin-positive CB up to about stage 8–9 (Figure 6A). In the coilin-null mutants, the GV is negative for coilin and U85 at all stages (Figures 9A and 10C). By contrast, strong FISH signals for U4 and U6 snRNAs are seen in these GVs (Supplemental Figure S4). In this respect, the GV differs from all other cell types that we have examined, where the absence of coilin and U85-positive foci correlates with the absence of snRNP-positive foci (Figure 11, D, F, and H, and Supplemental Figure S2D).
In summary, except for the GV, we find no evidence for residual CBs in the nuclei of larval and adult tissues of coilin-null mutants. At the same time, what seem to be typical HLBs occur in the cells we have examined. Thus, the absence of coilin disrupts the normal distribution of CB components, but leaves HLBs apparently intact.
Are There Two Isoforms of Drosophila Coilin?
FlyBase (http://flybase.bio.indiana.edu/) lists two potential isoforms of coilin, based on cDNA sequences. Sequence AY118690 encodes a 634-amino acid protein, with predicted MW 70,558 ("long isoform"). Sequence AY060878 includes an intron that is excluded from AY118690. Translation of AY060878 from the same ATG codon as AY118690 leads to a stop codon near the beginning of the intron. Translation from the second in-frame ATG at position 482 would give rise to a 488-amino acid protein of MW 53,850 ("short isoform"). By reverse transcription-PCR analysis we confirmed the existence of two RNAs in wild-type flies, one RNA with the intron and one RNA without. Among the eight antibodies that we produced, four recognize a region that occurs only in the translation product of the long isoform; the other four recognize a region that would occur in both the long and short isoforms. As mentioned, all our antibodies recognize a protein with Mr 90 kDa in ovary extracts. If a short isoform exists, it should occur as a band migrating between 50 and 70 kDa. It should be detected by the four antibodies directed against the carboxy terminus of coilin, but it should not be detected by the four antibodies targeted to the amino terminus of the full-length protein. So far, we have not identified such a band. Thus, if a short isoform does exist, it must be of low abundance, unstable, or occur only in certain tissues.
To further test for the existence of a short isoform, we made transgenic flies that express EYFP-labeled short isoform. In these flies, fluorescent label was detectable only in the cytoplasm. The cytoplasmic localization could be due to the lack of a nuclear localization signal in the short isoform. Because we have antibodies that recognize only the long isoform, it was possible to probe for the endogenous long isoform in tissues that expressed EYFP-labeled short isoform (Figure 12). We found an inverse relationship between the strength of the cytoplasmic signal (transgenic short isoform) and intensity of antibody stain in the CBs (endogenous long isoform). For example, in follicle cells that strongly expressed the short isoform, no long isoform was seen in the CBs, whereas normal CB stain was evident in nearby cells that did not express the short isoform. Thus, the short isoform, when expressed as an EYFP construct, localizes in the cytoplasm and causes mislocalization of the long isoform. Therefore, it seems unlikely that wild-type cells express any significant amount of the short isoform.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Raska et al., 1991) and has been used as the signature marker for CBs since then, orthologues in invertebrate organisms have been difficult to find for two reasons. First, the amino acid sequence is not strongly conserved (Bellini, 2000); and second, the precise molecular function has been difficult to define. In searching for an orthologue in Drosophila, we relied initially on the relatively weak sequence conservation in the amino and carboxy domains of the protein. Having found what we thought was a likely candidate, CG8710, we made transgenic flies that expressed EYFP-tagged versions of CG8710 protein, and we produced antibodies against two regions of the protein. We found that CG8710 is strongly expressed in CBs, which had been identified previously on the basis of other CB markers (Darzacq et al., 2002; Liu et al., 2006b). For these reasons, we feel confident that we have identified the Drosophila orthologue of mammalian coilin. For the gene itself, we suggest the name coil, corresponding to the name of the mammalian gene (Tucker et al., 2000).
Coilin Mutants
We have analyzed three mutants of the Drosophila coil gene. Two of these, coil199 and coil203, are nulls by two criteria: lack of detectable coilin protein on Western blots and lack of coilin stain in any tissue by immunofluorescence. In wild-type flies, coilin is concentrated in prominent CBs, which are also detectable with a variety of other markers, including fibrillarin, SMN, U2 snRNA, U5 snRNA, SMDA, and U85 scaRNA. In the coilin-null mutants, however, none of these markers are concentrated in defined bodies, suggesting that loss of coilin results in disorganization of CBs. Analysis of a third coil mutant, coilB220, leads to essentially the same conclusion. In this case, coilin stain is absent from germline cells of the ovary and testis, but typical coilin-positive CBs are present in somatic tissues. The absence of coilin from germline cells is correlated with the absence of defined foci of other CB markers.
In contrast to the loss of CBs from nuclei that lack coilin, HLBs seem to be unaffected in the mutants. Even the prominent HLBs of late-stage nurse cells are present in the mutants. These HLBs contain a high level of coilin in wild-type flies, but lack coilin completely in the mutants. Thus, in Drosophila, coilin seems to be necessary for proper assembly of CBs but not for assembly of HLBs.
Coilin mutants have been described in the mouse (Tucker et al., 2001) and in Arabidopsis (Collier et al., 2006). The mouse knockout allele is not complete and could, in principle, express the N-terminal 82 amino acids of coilin, although Western blotting or immunofluorescence did not detect the terminal fragment. Mice homozygous for the knockout allele have reduced viability and fertility, and embryonic fibroblasts derived from these animals lack typical CBs. Instead, there are three residual bodies, each of which contains a subset of typical CB components (Tucker et al., 2001; Jády et al., 2003). In Arabidopsis, the no cajal body 1 (ncb-1) mutant involves a single base substitution in the Arabidopsis coilin gene. It is not known whether the mutants are null for coilin. Plants that are homozygous for ncb-1 show no significant growth defects. However, their cells lack detectable CBs on the basis of three criteria: expression of the CB marker U2B", staining with an antibody against fibrillarin, and ultrastructure in the electron microscope. In Drosophila, cells of coilin-null mutants lack typical CBs and, except in the GV, residual bodies like those in the mouse are not evident. Although the nature of the coilin mutations is different in the three organisms that have been studied—Drosophila, mouse, and Arabidopsis—in each case coilin is required for normal CB formation, but neither coilin nor a typical CB is essential for viability.
Coilin in the Centromere and Spindle
Although coilin is well known as a constituent of CBs and the nucleoplasm, we were surprised to find easily detectable amounts of coilin in the centromeric regions of the chromosomes (embryonic blastoderm, larval brain, and ovarian follicle cells), and in the spindle (spermatocytes and embryonic blastoderm). To our knowledge, coilin has not been described in these locations in normal mammalian tissues, despite many studies in which such a distribution should have been evident. In contrast, a striking accumulation of coilin, fibrillarin, and SMN at interphase centromeres of HeLa and mouse chromosomes was recently demonstrated in cells that were infected with herpes simplex virus type 1 (Morency et al., 2007). The accumulation of coilin at centromeres was also induced independently of virus infection by knockdown of centromere protein B. It is, therefore, unclear whether coilin plays a role in normal centromere function in mammals or is recruited only when centromere composition is altered. A closer examination of Drosophila coilin during the cell cycle may shed light on this question.
Relationship between CBs and HLBs
Our earlier study of Drosophila nuclear bodies emphasized the fact that typical CB markers were distributed between two bodies, one of which we called the CB and the other the HLB (Liu et al., 2006b). Now that we have identified Drosophila coilin, two important facts emerge. First, coilin is not limited to the CB, but it is found at a low concentration in many if not all HLBs. Second, in late-stage nurse cell nuclei, not only do the HLBs contain a high concentration of coilin but also the CBs that were prominent in earlier stages break down and largely disappear.
These data suggest that a reinterpretation of other CBs may be in order, especially those in the Xenopus GV. Shortly after the discovery of coilin in HeLa cells, an orthologue of human coilin was identified in Xenopus and shown to be a prominent component of the multiple spheres or sphere organelles in the GV (Tuma et al., 1993). Because they contained coilin, the spheres were renamed coiled bodies and later Cajal bodies (Wu et al., 1994; Gall et al., 1999). Well before the discovery of coilin, it had been shown that a few spheres in the GV were attached to the chromosomes at the histone gene loci (Gall et al., 1981; Callan et al., 1991). The U7 snRNP was demonstrated in these bodies and indeed was the major snRNP there (Wu and Gall, 1993; Bellini and Gall, 1998). Thus, the structures called CBs in the Xenopus GV are HLBs by definition, even though they contain coilin. It is possible that they are hybrid bodies combining the features of both CBs and HLBs. Alternatively, the Xenopus GV may have lost its CBs during oocyte growth, ending up with HLBs that contain coilin, like those in the Drosophila nurse cell nuclei. We are currently investigating the early stages of Xenopus oogenesis to clarify this issue.
An interesting possibility is that CBs and HLBs are distinct nuclear bodies in other organisms as well. Earlier studies suggested that the mammalian U7 snRNP is colocalized with coilin in CBs (Frey and Matera, 1995; Shopland et al., 2001). However, it seems that at least in some cases the histone genes themselves are adjacent to CBs, not overlapping them (e.g., Figure 2D in Frey and Matera, 1995). Recent studies also suggest that histone processing factors are found in bodies separate from CBs, as defined by the presence of coilin (Narita et al., 2007; Bongiorno-Borbone et al., 2008). A careful reexamination of the exact relationship between the U7 snRNP, histone genes, and coilin needs to be carried out in a variety of mammalian and nonmammalian cell types.
Despite intensive study over nearly 20 years, the precise molecular function(s) of coilin remains obscure. It is our hope that the identification of Drosophila coilin will permit a variety of genetic, cell biological, and molecular studies that will help clarify not only the molecular functions of coilin but also its role in organizing the CB.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Joseph G. Gall (gall{at}ciwemb.edu)
Abbreviations used: CB, Cajal body; HLB, histone locus body; SMN, survival motor neuron.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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