Presence of small-nuclear-ribonucleoprotein-containing nuclear bodies in quiescent and early germinatingZea mays embryos

Summary Nucleolus-associated bodies (NABs) occur in interphase nuclei of many plant species. The present work shows that, inZea mays, NABs are present in dry seeds as well as in germinating tissues. The frequency of these nuclear bodies remains more or less constant during the first 24 h of imbibition but decreases significantly during the next 24 h. By the time the nucleolus reaches maturation and contains granular zones, these bodies are still found in close association with the surface of this organelle, as is the case in mature root meristematic cells. Immunocytochemical observations on both dry seeds and germinating tissues further revealed that NABs reacted positively with a monoclonal antibody (mAbK121) recognizing the m3G cap of sn(small nuclear)RNAs. It is, therefore, concluded that the NABs present in such tissues already contain components characterizing snRNPs (small nuclear ribonucleoproteins) in mature tissues. The possible function of NABs as storage deposits of snRNPs in dry seeds and early germinating tissues is discussed. In view of their many similarities with the coiled bodies described in both animal and plant cells, it is most likely that NABs correspond to those structures.Abbreviations BSA bovine serum albumin - DABCO 1,4-diazabicyclo (2.2.2)octane - EDTA ethylenediaminetetraacetic acid - IgM immunoglobulin M - NAB nucleolus-associated body - AO acridine orange - NAC nucleolus-associated chromatin - PBS phosphate-buffered saline - snRNA small nuclear ribonucleic acid - snRNP small nuclear ribonucleoprotein     

Localization of snRNP antigens in nucleolus-associated bodies: study of plant interphase nuclei by confocal and electron microscopy

Using two monoclonal antibodies, mAb Y12 and mAbK121, small nuclear ribonucleoproteins (snRNPs) were localized by immunofluorescence and immunogold electron microscopy in Pisum sativum and Cicer arietinum interphase nuclei. We showed that snRNPs were mostly found in nucleolus associated bodies (NABs) characterizing these two plant species. snRNPs were also found threoughout the nucleoplasm, their distribution being diffuse or speckled depending on the nucleus. Examination of optical sectins furnished by confocal laser microscopy allowed us to obtain more precise information on the number and location of NABs. One, two and rarely three (in green pea) of such structures were observed, their size varied from 0.8 to 1.5 m and they were also systematically observed in intimate contact with the nucleolus. Under electron microscopy, labelling was likewise found to be noticeably higher over NABs than over the nucleoplasm. The possible relationship between NABs and other nuclear bodies found in animal cells and known to be involved in splicing of pre-mRNA is discussed.     

Cytochemical characterization of two types of nuclear bodies in Cicer arietinum L.

The present study was carried out to characterize two distinct types of nuclear bodies, the nucleolus-associated bodies (NABs) and the satellites (SATs) using some specific staining, enzyme and immunogold labeling techniques in Cicer arietinum L. These bodies are of interest as the functional components of plant nucleus. DNA-specific staining and labeling with anti-DNA, a monoclonal antibody, were employed to verify the presence of DNA in NABs as well as in SATs. The enzyme-gold labeling technique was used to compare the relative amounts of RNase-gold and protease-gold labeling over the NABs. In NABs, RNase-gold labeling was relatively low compared to the protease-gold labeling. Ag-NOR staining revealed a similar content of NOR-silver proteins in both NABs and granular zone of the nucleolus. The NABs do not contain any DNA as they show negative response to DNA-specific stains and also when incubated with anti-DNA, only few gold particles are found over these structures. The SATs, on the other hand, react positively with DNA-specific stains, and high labeling is recorded with anti-DNA along with the dense chromatin masses.     

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.     

Coiled Bodies in the Meristematic Cells of the Root of Lupinus luteus L.

The nature of nucleolar associate bodies in the meristematic cells of the root of Lupinus luteus L. was investigated using immunocytochemical methods, in situ hybridisation with light, confocal, and electron microscopy. The nuclear bodies of lupin proved to be structures containing fibrillarin and coilin, but devoid of rRNA and DNA, like animal coiled bodies (CBs). In lupin cells we have observed the occurrence of small nuclear ribonucleoprotein (snRNP) in the cytoplasm, in nucleoplasm, CBs and in nucleoli. This type of snRNP localisation pattern is in agreement with recently presented models of the small nuclear RNA cycle. This revised version was published online in July 2006 with corrections to the Cover Date.     

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 Drosophila melanogaster Cajal body

Cajal bodies (CBs) are nuclear organelles that are usually identified by the marker protein p80-coilin. Because no orthologue of coilin is known in Drosophila melanogaster, we identified D. melanogaster CBs using probes for other components that are relatively diagnostic for CBs in vertebrate cells. U85 small CB-specific RNA, U2 small nuclear RNA, the survival of motor neurons protein, and fibrillarin occur together in a nuclear body that is closely associated with the nucleolus. Based on its similarity to CBs in other organisms, we refer to this structure as the D. melanogaster CB. Surprisingly, the D. melanogaster U7 small nuclear RNP resides in a separate nuclear body, which we call the histone locus body (HLB). The HLB is invariably colocalized with the histone gene locus. Thus, canonical CB components are distributed into at least two nuclear bodies in D. melanogaster. The identification of these nuclear bodies now permits a broad range of questions to be asked about CB structure and function in a genetically tractable organism.     

Identification and characterization of coiled body-like structures in pea (Pisum sativum L.)

Coiled bodies (CBs) are nuclear organelles which were considered as "universal" nuclear structures in eukaryotic cells, but the formation and function of CBs, especially in plant cells, remained unclear. In this article we reported that CBs in meristematic cells of pea are oval to round obstacles in nucleus and in adjacent to nucleolus, often have the same electron density with nucleolus. We found that CBs could be stained by the rRNP preference staining method, but no rDNA was detected in the structure. Furthermore, our results of immunoelectron microscopy showed that several processing factors, include fibrillarin, U3 snoRNA and ITS1, were present in CB. It seems probable that CBs is derived structurally from nucleolus and act as transport, storage and processing subnucleolar organelles.     

The SMN Protein is a Key Regulator of Nuclear Architecture in Differentiating Neuroblastoma Cells

The cell nucleus contains two closely related structures, Cajal bodies (CBs) and gems. CBs are the first site of accumulation of newly assembled splicing snRNPs (small nuclear ribonucleoproteins) following their import into the nucleus, before they form their steady-state localization in nuclear splicing speckles. Gems are the nuclear site of accumulation of survival motor neurons (SMNs), an insufficiency of which leads to the inherited neurodegenerative condition, spinal muscular atrophy (SMA). SMN is required in the cytoplasm for the addition of core, Sm, proteins to new snRNPs and is believed to accompany snRNPs to the CB. In most cell lines, gems are indistinguishable from CBs, although the structures are often separate in vivo . The relationship between CBs and gems is not fully understood, but there is evidence that symmetrical dimethylation of arginine residues in the CB protein coilin brings them together in HeLa cells. During neuronal differentiation of the human neuroblastoma cell line SH-SY5Y, CBs and gems increase their colocalization, mimicking changes seen during foetal development. This does not result from alterations in the methylation of coilin, but from increased levels of SMN. Expression of exogenous SMN results in an increased efficiency of snRNP transport to nuclear speckles. This suggests different mechanisms are present in different cell types and in vivo that may be significant for the tissue-specific pathology of SMA.     

Composition of nuclear dense bodies and nucleolus-associated bodies in interphase nuclei of the unicellular green alga Chlamydomonas reinhardtii

Summary— The interphase nucleus of the green alga, Chlamydomonas reinhardtii, displayed two types of bodies some of them, the dense bodies, lying apparently free in the nucleoplasm while the others were attached to the nucleolus and were, therefore, referred to as nucleolus-associated bodies (NABs). The presence of DNA, RNA and histones in dense bodies was investigated by means of post-embedding immunocytochemistry and cytochemistry using a monoclonal antibody to single and double stranded DNA, a polyclonal antibody to rye H3 histones and RNase A-gold complexes. The dense bodies were shown to contain significant amounts of RNA but neither DNA nor histones were detected; their composition was thus similar to that of the dense bodies described in higher plant cells. We propose that dense bodies might be implicated in the assembly of the 25 to 45 nm granules observed throughout the nucleoplasm of Chalamydomonas interphase nuclei. The composition of NABs was found to be distinct from that of the dense bodies since they were labeled by the antibody to DNA, specially in cryofixed and cryosubstituted specimens. The presence of DNA in NABs together with their intimate association to the nucleolus suggest that they may correspond to specific segments of chromosomes.     

Relationship of nucleolus-associated bodies with the nucleolar organizer tracks in plant interphase nuclei (Pisum sativum)

Nucleolus-associated bodies (NABs) have long been noted in interphase nuclei of a wide variety of plant species. We have recently shown that these bodies consist largely of snRNPs and that they are located on the nucleolar surface in the immediate vicinity of the nucleolar organizer tracks. The present study revealed that, following exposure of roots to KCN, an agent that induces nucleolar segregation, NABs were intimately associated with intranucleolar chromatin. Although immunocytochemical tests with anti-DNA indicated that NABs contained no demonstrable amounts of DNA, our observations nevertheless add further support to the notion that these bodies are somehow related to the nucleolar chromosomes.     

A distant coilin homologue is required for the formation of cajal bodies in Arabidopsis

Cajal bodies (CBs) are subnuclear bodies that are widespread in eukaryotes, being found in mammals, many other vertebrates and in all plant species so far examined. They are mobile structures, moving, fusing, and budding within the nucleus. Here we describe a screen for Arabidopsis mutants with altered CBs and describe mutants that have smaller Cajal bodies (ncb-2, ncb-3), lack them altogether (ncb-1), have increased numbers of CBs (pcb) or have flattened CBs (ccb). We have identified the gene affected in the ncb mutants as a distant homolog of the vertebrate gene that encodes coilin (At1g13030) and have termed the resulting protein Atcoilin. A T-DNA insertional mutant in this gene (ncb-4) also lacks Cajal bodies. Overexpression of Atcoilin cDNA in ncb-1 restores Cajal bodies, which recruit U2B″ as in the wild type, but which are, however, much larger than in the wild type. Thus we have shown that At1g13030 is required for Cajal body formation in Arabidopsis, and we hypothesize that the level of its expression is correlated with Cajal body size. The Atcoilin gene is unaffected in pcb and ccb, suggesting that other genes can also affect CBs.     

Identification of coiled bodies inBrassica napus nuclei during embryogenesis and early germination

Summary Nucleolus-associated bodies characterize interphase nuclei of many plant species. The recent demonstration that such bodies contain small nuclear ribonucleoproteins as well as coilin clearly indicates that they belong to a larger family of nuclear structures, known as coiled bodies, that have been intensively studied in a variety of animal cell types. In a previous work, we have shown that coiled bodies were present in close association with the nucleolus inZea mays dry seeds as well as during subsequent stages of germination. This study reveals that similar nuclear structures were also present duringBrassica napus embryogenesis starting at the torpedo stage and that they were, likewise, generally located on the nucleolar surface. As in the case ofZ. mays, coiled bodies were observed in cells of dry seeds as well as in those of early germinating tissues. These bodies were labelled with monoclonal antibody K121, an antibody reacting with the unique 5-terminal cap structure containing 2,2,7-trimethylguanosine that characterizes small nuclear RNAs. Owing to their intimate association with the nucleolus in all stages studied, the possibility is considered that, in these plant cells, coiled bodies are assembled on an organizer element located within this organelle.Abbreviations BSA bovine serum albumin - IgM immunoglobulin M - NAB nucleolus-associated body - NAC nucleolus-associated chromatin - PBS phosphate-buffered saline - snRNA small nuclear ribonucleic acid - snRNP small nuclear ribonucleoprotein     

Nuclear bodies in Douglas fir (Pseudotsuga menziesii Mirb.) microspores

The identification of nucleolar proteins and immunocytochemical localization of small nuclear ribonucleoprotein (snRNP) elements revealed the presence of three types of nuclear bodies in Douglas fir microspore nuclei. One type consists of structures resembling Cajal bodies (CBs) and contains nucleolar proteins as well as snRNPs and U2 snRNA. The second type is bizonal bodies, which are nuclear bodies also linked with the splicing system. The bizonal body comprises two parts: the first contains Sm proteins and stains strongly with silver stain, and the second resembles CBs in terms of the degree of silver staining and molecular composition. Douglas fir is the second species after larch where the presence of bizonal bodies has been demonstrated. Pseudotsuga menziesii Mirb and Larix decidua Mill are species with one of the longest microsporogenesis processes known in plants. The presence of bizonal bodies in both species may be linked to the intensification of the splicing processes in microspores with an exceptionally long cell cycle. The third type of structure is dense bodies, whose morphology and degree of silver staining strongly indicate their functional and spatial relationship to the dense part of bizonal bodies.