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.     

Self-association of coilin reveals a common theme in nuclear body localization

We have found that coilin, the marker protein for Cajal bodies (coiled bodies, CBs), is a self-interacting protein, and we have mapped the domain responsible for this activity to the amino-terminus. Together with a nuclear localization signal, the self-interaction domain is necessary and sufficient for localization to CBs. Overexpression of various wild-type and mutant coilin constructs in HeLa cells results in disruption of both CBs and survival motor neurons (SMN) gems. Additionally, we have identified a cryptic nucleolar localization signal (NoLS), within the coilin protein, which may be exposed in specific coilin phospho-isoforms. The implications of these findings are discussed in light of the fact that other proteins known to localize within nuclear bodies (e. g., PML, SMN and Sam68) can also self-associate. Thus protein self-interaction appears to be a general feature of nuclear body marker proteins.     

Symmetrical dimethylarginine methylation is required for the localization of SMN in Cajal bodies and pre-mRNA splicing

The nuclear structures that contain symmetrical dimethylated arginine (sDMA)-modified proteins and the role of this posttranslational modification is unknown. Here we report that the Cajal body is a major epitope in HeLa cells for an sDMA-specific antibody and that coilin is an sDMA-containing protein as analyzed by using the sDMA-specific antibody and matrix-assisted laser desorption ionization time of flight mass spectrometry. The methylation inhibitor 5'-deoxy-5'-methylthioadenosine reduces the levels of coilin methylation and causes the appearance of SMN-positive gems. In cells devoid of Cajal bodies, such as primary fibroblasts, sDMA-containing proteins concentrated in speckles. Cells from a patient with spinal muscular atrophy, containing low levels of the methyl-binding protein SMN, localized sDMA-containing proteins in the nucleoplasm as a discrete granular pattern. Splicing reactions are efficiently inhibited by using the sDMA-specific antibody or by using hypomethylated nuclear extracts, showing that active spliceosomes contain sDMA polypeptides and suggesting that arginine methylation is important for efficient pre-mRNA splicing. Our findings support a model in which arginine methylation is important for the localization of coilin and SMN in Cajal bodies.     

p80 coilin,a coiled body-specific protein,interacts with ataxin-1, the SCA1 gene product

Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder characterized by ataxia and progressive motor deterioration. SCA1 is associated with an elongated polyglutamine tract in ataxin-1, the SCA1 gene product. Using the yeast two-hybrid system and co-immunoprecipitation experiments, we have found that p80 coilin, coiled body-specific protein, binds to ataxin-1. In further experiments with deletion mutants, we found that the C-terminal regions of ataxin-1 and p80 coilin were essential for this interaction. In HeLa cells that have been co-transfected with ataxin-1 and p80 coilin, the p80 coilin protein co-localizes with ataxin-1 aggregates in the nucleoplasm. However, immunohistochemical analysis and immunofluorescence assays showed that mutant ataxin-1 aggregates do not redistribute p80 coilin's dot-like structures in the Purkinje cells of SCA1 transgenic mice. This feature of the interaction between ataxin-1 and p80 coilin suggests that p80 coilin might be implicated in altering the function of ataxin-1.     

Coilin, more than a molecular marker of the cajal (coiled) body

The Cajal (coiled) body is a discrete nuclear organelle that was first described in mammalian neurons in 1903. Because the molecular composition, structure, and function of Cajal bodies were unknown, these enigmatic structures were largely ignored for most of the last century. The Cajal body has now regained the interest of biologists, due to the isolation of a protein marker, coilin. Despite current widespread use of coilin to identify Cajal bodies in various cell types, its structure and function are still little understood. Here, I would like to discuss what we have learned about coilin and suggest a possible role for coilin in RNA processing and cellular trafficking, especially in relation to Cajal bodies and nucleoli. Although coilin has been investigated primarily in somatic cells, I will emphasize the advantages of using the amphibian oocyte to study nuclear proteins and organelles.     

Ongoing U snRNP biogenesis is required for the integrity of Cajal bodies

Cajal bodies (CBs) have been implicated in the nuclear phase of the biogenesis of spliceosomal U small nuclear ribonucleoproteins (U snRNPs). Here, we have investigated the distribution of the CB marker protein coilin, U snRNPs, and proteins present in C/D box small nucleolar (sno)RNPs in cells depleted of hTGS1, SMN, or PHAX. Knockdown of any of these three proteins by RNAi interferes with U snRNP maturation before the reentry of U snRNA Sm cores into the nucleus. Strikingly, CBs are lost in the absence of hTGS1, SMN, or PHAX and coilin is dispersed in the nucleoplasm into numerous small foci. This indicates that the integrity of canonical CBs is dependent on ongoing U snRNP biogenesis. Spliceosomal U snRNPs show no detectable concentration in nuclear foci and do not colocalize with coilin in cells lacking hTGS1, SMN, or PHAX. In contrast, C/D box snoRNP components concentrate into nuclear foci that partially colocalize with coilin after inhibition of U snRNP maturation. We demonstrate by siRNA-mediated depletion that coilin is required for the condensation of U snRNPs, but not C/D box snoRNP components, into nucleoplasmic foci, and also for merging these factors into canonical CBs. Altogether, our data suggest that CBs have a modular structure with distinct domains for spliceosomal U snRNPs and snoRNPs.     

Coilin Shuttles between the Nucleus and Cytoplasm In Xenopus Oocytes

Coiled bodies are discrete nuclear organelles often identified by the marker protein p80-coilin. Because coilin is not detected in the cytoplasm by immunofluorescence and Western blotting, it has been considered an exclusively nuclear protein. In the Xenopus germinal vesicle (GV), most coilin actually resides in the nucleoplasm, although it is highly concentrated in 50-100 coiled bodies. When affinity-purified anti-coilin antibodies were injected into the cytoplasm of oocytes, they could be detected in coiled bodies within 2-3 h. Coiled bodies were intensely labeled after 18 h, whereas other nuclear organelles remained negative. Because the nuclear envelope does not allow passive diffusion of immunoglobulins, this observation suggests that anti-coilin antibodies are imported into the nucleus as an antigen-antibody complex with coilin. Newly synthesized coilin is not required, because cycloheximide had no effect on nuclear import and subsequent targeting of the antibodies. Additional experiments with myc-tagged coilin and myc-tagged pyruvate kinase confirmed that coilin is a shuttling protein. The shuttling of Nopp140, NO38/B23, and nucleolin was easily demonstrated by the targeting of their respective antibodies to the nucleoli, whereas anti-SC35 did not enter the germinal vesicle. We suggest that coilin, perhaps in association with Nopp140, may function as part of a transport system between the cytoplasm and the coiled bodies.     

Nucleoli and perinuclear bodies in trophocytes of Panorpa communis contain factors of pre-mRNA splicing

The distribution of pre-mRNA splicing factors and protein coilin was examined in trophocyte nuclei (TN) in polytrophic ovarioles of Panorpa communis. In situ hybridization, using antisense U1 and U6 snRNA 3H-riboprobes, showed that TN were labeled evenly. Immunostaining at light and electron microscopic levels revealed in some TN nucleolar structures containing small nuclear RNP (snRNP) and protein coilin characteristic of the Cajal bodies/coiled bodies (CB). No free CBs were found in TN. These data showed that CB in TN are present only in the nucleoli. One of characteristic features of P. communis trophocytes is the presence of several types of perinuclear bodies (PB) in the cytoplasm. We distinguish between three types of PBs. PB-1 consist of spherical bodies (10-20 microns) with vacuoles composed of closely packed fibrils. PB-2 are irregularly shaped bodies (0.3-2.0 microns) consisting of a fibro-granular material. PB-2 are located near the nuclear envelope and contact the nucleoplasm material through nuclear pores. PB-1 and PB-2 join together to form a complex PB of the third type. All types of PB are not surrounded with a membrane and sometimes have mitochondria on their surface. The immunogold technique at the ultrastructural level revealed snRNP in PB-2. These results have enabled us to make a conclusion that PB-2 may be storage sites of snRNPs required for a future development of the embryo.     

Characterization of human myocardial fibroblasts immortalized by HPV16 E6--E7 genes

SMN, the affected protein in spinal muscular atrophy (SMA), is a cytoplasmic protein that also occurs in nuclear structures called "gems" and is involved in snRNP maturation. Coilin-p80 is a marker protein for nuclear Cajal bodies (coiled bodies; CBs) which are also involved in snRNP maturation, storage or transport. We now show that gems and CBs are present in all fetal tissues, even those that lack gems/CBs in the adult. Most gems and CBs occur as separate nuclear structures in fetal tissues, but their colocalization increases with fetal age and is almost complete in the adult. In adult tissues, up to half of all gems/CBs are inside the nucleolus, whereas in cultured cells they are almost exclusively nucleoplasmic. The nucleolar SMN is often more diffusely distributed, compared with nucleoplasmic gems. Up to 30% of cells in fetal tissues have SMN distributed throughout the nucleolus, instead of forming gems in the nucleoplasm. The results suggest a function for gems distinct from Cajal bodies in fetal nuclei and a nucleolar function for SMN. Spinal cord, the affected tissue in SMA, behaves differently in several respects. In both fetal and adult motor neurons, many gems/CBs occur as larger bodies closely associated with the nucleolar perimeter. Uniquely in motor neurons, gems/CBs are more numerous in adult than in fetal stages and colocalization of gems and CBs occurs earlier in development. These unusual features of motor neurons may relate to their special sensitivity to reduced SMN levels in SMA patients.     

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.