The Movement of Coiled Bodies Visualized in Living Plant Cells by the Green Fluorescent Protein

Coiled bodies are nuclear organelles that contain components of at least three RNA-processing pathways: pre-mRNA splicing, histone mRNA 3'- maturation, and pre-rRNA processing. Their function remains unknown. However, it has been speculated that coiled bodies may be sites of splicing factor assembly and/or recycling, play a role in histone mRNA 3'-processing, or act as nuclear transport or sorting structures. To study the dynamics of coiled bodies in living cells, we have stably expressed a U2B"-green fluorescent protein fusion in tobacco BY-2 cells and in Arabidopsis plants. Time-lapse confocal microscopy has shown that coiled bodies are mobile organelles in plant cells. We have observed movements of coiled bodies in the nucleolus, in the nucleoplasm, and from the periphery of the nucleus into the nucleolus, which suggests a transport function for coiled bodies. Furthermore, we have observed coalescence of coiled bodies, which suggests a mechanism for the decrease in coiled body number during the cell cycle. Deletion analysis of the U2B" gene construct has shown that the first RNP-80 motif is sufficient for localization to the coiled body.     

The dynamics of coiled bodies in the nucleus of adenovirus-infected cells.

The coiled body is a specific intranuclear structure of unknown function that is enriched in splicing small nuclear ribonucleoproteins (snRNPs). Because adenoviruses make use of the host cell-splicing machinery and subvert the normal subnuclear organization, we initially decided to investigate the effect of adenovirus infection on the coiled body. The results indicate that adenovirus infection induces the disassembly of coiled bodies and that this effect is probably secondary to the block of host protein synthesis induced by the virus. Furthermore, coiled bodies are shown to be very labile structures, with a half-life of approximately 2 h after treatment of HeLa cells with protein synthesis inhibitors. After blocking of protein synthesis, p80 coilin was detected in numerous microfoci that do not concentrate snRNP. These structures may represent precursor forms of the coiled body, which goes through a rapid cycle of assembly/disassembly in the nucleus and requires ongoing protein synthesis to reassemble.     

New type of snRNP containing nuclear bodies in plant cells

In larch (Larix decidua Mill.) microspores a new type of nuclear bodies has been found which are an element of the spatial organization of the splicing system in plant cell. These are bizonal bodies, ultrastructurally differentiated into a coiled part and a dense part. Using immunocytochemistry and in situ hybridization at the EM level, the coiled part of the bizonal body was found to contain snRNA including U2 snRNA, Sm proteins and nucleolar proteins of the agyrophilic type and fibrillarin. The dense part contains Sm proteins but lacks snRNA. Such a separation of macromolecules related to splicing occurring within the bizonal bodies microspore is striking by the similarity of these bodies to amphibian oocyte snurposomes. The occurrence in plant cells, beside widely known coiled bodies (CBs), also of other nuclear bodies related to splicing proves that in plants similarly as for animals the differentiation among domains containing elements of the splicing system occurs.     

The relationship between SMN, the spinal muscular atrophy protein, and nuclear coiled bodies in differentiated tissues and cultured cells

The spinal muscular atrophy protein, SMN, is a cytoplasmic protein that is also found in distinct nuclear structures called "gems." Gems are closely associated with nuclear coiled bodies and both may have a direct role in snRNP maturation and pre-RNA splicing. There has been some controversy over whether gems and coiled bodies colocalize or form adjacent/independent structures in HeLa and other cultured cells. Using a new panel of antibodies against SMN and antibodies against coilin-p80, a systematic and quantitative study of adult differentiated tissues has shown that gems always colocalize with coiled bodies. In some tissues, a small proportion of coiled bodies (<10%) had no SMN, but independent or adjacent gems were not found. The most striking observation, however, was that many cell types appear to have neither gems nor coiled bodies (e.g., cardiac and smooth muscle, blood vessels, stomach, and spleen) and this expression pattern is conserved across human, rabbit, and pig species. This shows that assembly of distinct nuclear bodies is not essential for RNA splicing and supports the view that they may be storage sites for reserves of essential proteins and snRNPs. Overexpression of SMN in COS-7 cells produced supernumerary nuclear bodies, most of which also contained coilin-p80, confirming the close relationship between gems and coiled bodies. However, when SMN is reduced to very low levels in type I SMA fibroblasts, coiled bodies are still formed. Overall, the data suggest that gem/coiled body formation is not determined by high cytoplasmic SMN concentrations or high metabolic activity alone and that a differentiation-specific factor may control their formation.     

Mutational analysis of p80 coilin indicates a functional interaction between coiled bodies and the nucleolus

Coiled bodies are conserved subnuclear domains found in both plant and animal cells. They contain a subset of splicing snRNPs and several nucleolar antigens, including Nopp140 and fibrillarin. In addition, autoimmune patient sera have identified a coiled body specific protein, called p80 coilin. In this study we show that p80 coilin is ubiquitously expressed in human tissues. The full-length human p80 coilin protein correctly localizes in coiled bodies when exogenously expressed in HeLa cells using a transient transfection assay. Mutational analysis identifies separate domains in the p80 coilin protein that differentially affect its subnuclear localization. The data show that p80 coilin has a nuclear localization signal, but this is not sufficient to target the protein to coiled bodies. The results indicate that localization in coiled bodies is not determined by a simple motif analogous to the NLS motifs involved in nuclear import. A specific carboxy-terminal deletion in p80 coilin results in the formation of pseudo-coiled bodies that are unable to recruit splicing snRNPs. This causes a loss of endogenous coiled bodies. A separate class of mutant coilin proteins are shown to localize in fibrillar structures that surround nucleoli. These mutants also lead to loss of endogenous coiled bodies, produce a dramatic disruption of nucleolar architecture and cause a specific segregation of nucleolar antigens. The structural change in nucleoli is accompanied by the loss of RNA polymerase I activity. These data indicate that p80 coilin plays an important role in subnuclear organization and suggest that there may be a functional interaction between coiled bodies and nucleoli.     

The behavior of the coiled body in cells infected with adenovirus in vitro

The coiled body is a phylogenetically conserved nuclear organelle whose function is not known. Probes for detection of p80-coilin, an 80 kDa protein enriched in the coiled body, have made possible studies determining the behavior of the coiled body during the cell cycle, in proliferating cells, as well as reports suggesting some relationship of the coiled body to mRNA splicing and to the nucleolus. The objective of this study is to examine the distribution of p80-coilin and nucleolar proteins in cells infected with adenovirus in vitro. HeLa cells grown as monolayers were infected with successive dilutions of type 5 human adenovirus culture and fixed in methanol/acetone at different time points. Single and double indirect immunofluorescence was performed with human autoantibodies to p80-coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM-Scl, and To, as well as rabbit polyclonal serum to p80-coilin (R288) and mouse monoclonal antibody to adenovirus 72-kDa DNA-binding protein. Indirect immunofluorescence (IIF) with anti- p80-coilin antibodies showed that the usual bright dot-like coiled body staining pattern was replaced in infected cells by 1–5 clusters of tiny dots at the periphery of the nucleus. This phenomenon was first detected within 12 h of infection and affected more severely cells with increased length and load of infection. Cells subjected to heat shock presented no such alteration. Double IIF showed that cells with abnormal coiled body appearance expressed the viral 72-kDa DNA-binding protein. Nucleolar proteins RNA polymerase I and NOR-90/hUBF became associated with the p80-coilin-enriched clusters and were no longer detected in the nucleolus. Other nucleolar proteins, like PM-Scl and To, remained associated to the nucleolus and were not detected in the newly formed clusters. Fibrillarin had a heterogeneous behavior, being restricted to the nucleolus in some infected cells while in some others it was associated with the p80-coilin-enriched clusters. Thus our results showed that in vitro adenovirus infection induced radical redistribution of nucleolar and coiled body constituents into newly formed structures characterized by clusters of tiny dots in the periphery of the nucleus. The fact that three major proteins involved in rRNA synthesis and processing colocalized with p80-coilin in these clusters may bring additional support to the idea that the coiled body and p80-coilin may be implicated in functions related to the nucleolus.     

Coiled bodies without coilin.

Nuclei assembled in vitro in Xenopus egg extract contain coiled bodies that have components from three different RNA processing pathways: pre-mRNA splicing, pre-rRNA processing, and histone pre-mRNA 3'-end formation. In addition, they contain SPH-1, the Xenopus homologue of p80-coilin, a protein characteristic of coiled bodies. To determine whether coilin is an essential structural component of the coiled body, we removed it from the egg extract by immunoprecipitation. We showed that nuclei with bodies morphologically identical to coiled bodies (at the light microscope level) formed in such coilin-depleted extract. As expected, these bodies did not stain with antibodies against coilin. Moreover, they failed to stain with an antibody against the Sm proteins, although Sm proteins associated with snRNAs were still present in the extract. Staining of the coilin- and Sm-depleted coiled bodies was normal with antibodies against two nucleolar proteins, fibrillarin and nucleolin. Similar results were observed when Sm proteins were depleted from egg extract: staining of the coiled bodies with antibodies against the Sm proteins and coilin was markedly reduced but bright nucleolin and fibrillarin staining remained. These immunodepletion experiments demonstrate an interdependence between coilin and Sm snRNPs and suggest that neither is essential for assembly of nucleolar components in coiled bodies. We propose that coiled bodies are structurally heterogeneous organelles in which the components of the three RNA processing pathways may occur in separate compartments.     

The Cajal body

The Cajal body, originally identified over 100 years ago as a nucleolar accessory body in neurons, has come to be identified with nucleoplasmic structures, often quite tiny, that contain coiled threads of the marker protein, coilin. The interaction of coilin with other proteins appears to increase the efficiency of several nuclear processes by concentrating their components in the Cajal body. The best-known of these processes is the modification and assembly of U snRNPs, some of which eventually form the RNA splicing machinery, or spliceosome. Over the last 10 years, research into the function of Cajal bodies has been greatly stimulated by the discovery that SMN, the protein deficient in the inherited neuromuscular disease, spinal muscular atrophy, is a Cajal body component and has an essential role in the assembly of spliceosomal U snRNPs in the cytoplasm and their delivery to the Cajal body in the nucleus.     

Newly assembled snRNPs associate with coiled bodies before speckles, suggesting a nuclear snRNP maturation pathway.

BACKGROUND: Small nuclear ribonucleoproteins (snRNPs), which are essential components of the mRNA splicing machinery, comprise small nuclear RNAs, each complexed with a set of proteins. An early event in the maturation of snRNPs is the binding of the core proteins - the Sm proteins - to snRNAs in the cytoplasm followed by nuclear import. Immunolabelling with antibodies against Sm proteins shows that splicing snRNPs have a complex steady-state localisation within the nucleus, the result of the association of snRNPs with several distinct subnuclear structures. These include speckles, coiled bodies and nucleoli, in addition to a diffuse nucleoplasmic compartment. The reasons for snRNP accumulation in these different structures are unclear. RESULTS: When mammalian cells were microinjected with plasmids encoding the Sm proteins B, D1 and E, each tagged with either the green fluorescent protein (GFP) or yellow-shifted GFP (YFP), a pulse of expression of the tagged proteins was observed. In each case, the newly synthesised GFP/YFP-labelled snRNPs accumulated first in coiled bodies and nucleoli, and later in nuclear speckles. Mature snRNPs localised immediately to speckles upon entering the nucleus after cell division. CONCLUSIONS: The complex nuclear localisation of splicing snRNPs results, at least in part, from a specific pathway for newly assembled snRNPs. The data demonstrate that the distribution of snRNPs between coiled bodies and speckles is directed and not random.     

Coiled bodies in nuclei from plant cells evolving from dormancy to proliferation

To characterise the coiled bodies in meristematic nuclei of Saccharum officinarum, immunofluorescence labelling with antibodies against components of the splicing (U2B' and Sm core protein B) and pre-rRNA processing (fibrillarin) complexes was used in cells from the dormant root primordia and from roots at different times after activation to the steady state of proliferation. The number, size and distribution of coiled bodies varied in the meristematic tissue depending on cell activity. While G0 cells in the dry primordia and proliferating cells showed a similar number of coiled bodies attached to their nucleoli, the number of nucleoplasmic coiled bodies greatly increased after the primordia were stimulated to proliferate. Their number remained steady from the time the meristematic population reached the steady state of proliferation, as estimated by flow cytometry. Fractionation studies demonstrated that coiled bodies are a part of the underlying nuclear matrix. Comparison of immunocytochemical and cytochemical data from confocal and electron microscopical studies demonstrated that the nucleolar and nucleoplasmic coiled bodies detected by confocal microscopy shared many features, suggesting that they form a family of closely related structures.     

U2 and U1 snRNA gene loci associate with coiled bodies

The coiled bodies are nuclear structures rich in a variety of nuclear and nucleolar components including snRNAs. We have investigated the possibility that coiled bodies may associate with snRNA genes and report here that there is a high degree of association between U2 and U1 genes with a subset of coiled bodies. As investigated in human HeLa cells grown in monolayer culture, about 75% of the nuclei had at least one U2 gene associated with a coiled body, and 45% had at least one U1 locus associated. In another suspension-grown HeLa cell strain, 92% of cells showed association of one or more U2 genes with coiled bodies. In contrast to the U2 and U1 gene associations, a locus closely linked to the U2 gene cluster appeared associated with a coiled body only in 10% of cells. Associated snRNA gene signals were repeatedly positioned at the edge of the coiled body. Thus, this association was highly nonrandom and spatically precise. Our analysis revealed a much higher frequency of association for closely spaced “doublet” U2 gene signals, with over 80% of paired signals associated as opposed to 35% for single U2 signals. This finding, coupled with the fact that not all genes were associated in all cells, suggested the possibility of a cell-cycle-dependent, possibly S-phase, association. However, an analysis of S- and non-S-phase cells using BrdU incorporation or cell synchronization did not indicate an increased level of association in S-phase. These and other results suggested that a substantial fraction of paired U2 signals represented association of U2 genes on homologous chromosomes rather than only replicated DNA. Furthermore, triple lable analysis showed that in a significant fraction of cells U1 and U2 genes were both associated with the same coiled body. U1 and U2 genes were closely paired in approximately 20% of cells, over 60% of which were associated with a readily identifiable coiled body. This finding raises the possibility that multiple genes of a particular class may be in association with each coiled body. Thus, the coiled body may be a dynamic structure which transiently interacts with or is formed by one or more specific genetic loci, possibly carrying out some function related to their expression. © 1995 Wiley-Liss, Inc.     

Assembly of snRNP-containing coiled bodies is regulated in interphase and mitosis--evidence that the coiled body is a kinetic nuclear structure

Coiled bodies (CBs) are nuclear organelles in which splicing snRNPs concentrate. While CBs are sometimes observed in association with the nucleolar periphery, they are shown not to contain 5S or 28S rRNA or the U3 snoRNA. This argues against CBs playing a role in rRNA maturation or transport as previously suggested. We present evidence here that CBs are kinetic structures and demonstrate that the formation of snRNP-containing CBs is regulated in interphase and mitosis. The coiled body antigen, p80 coilin, was present in all cell types studied, even when CBs were not prominent. Striking changes in the formation of CBs could be induced by changes in cellular growth temperature without a concomitant change in the intracellular p80 coilin level. During mitosis, CBs disassemble, coinciding with a mitotic-specific phosphorylation of p80 coilin. Coilin is shown to be a phosphoprotein that is phosphorylated on at least two additional sites during mitosis. CBs reform in daughter nuclei after a lag period during which they are not detected. CBs are thus, dynamic nuclear organelles and we propose that cycling interactions of splicing snRNPs with CBs may be important for their participation in the processing or transport of pre-mRNA in mammalian cells.