Poly(A) RNA a new component of Cajal bodies
In European larch microsporocytes, spherical structures 0.5 to 6 μm in diameter are present in which poly(A) RNA accumulates. There were one to several bodies per cell and they were often present in the vicinity of the nucleolus. No nascent transcripts were observed within them. Splicing factors of the SR family, including protein SC35, which participates in bringing the 3′ and 5′ sites closer in the splicing reaction, were also not observed. The absence of the above-mentioned elements within bodies containing poly(A) RNA disqualifies them as sites of synthesis and preliminary stages of primary transcript maturation. However, they contained abundant elements of the splicing machinery commonly occurring in Cajal bodies, i.e., Sm proteins or small nuclear RNA (snRNA). The molecular composition as well as the characteristic ultrastructure of bodies containing poly(A) RNA proves that these were Cajal bodies. This is the first report of such poly(A) RNA localization.
KeywordsLarix decidua Meiotic prophase I Nuclear bodies Coiled body pre-mRNA snRNP
In the nuclei of eukaryotic cells, structures called nuclear bodies are present. Many of them have been shown to be involved in RNA metabolism. The nuclear bodies in mammalian nerve tissue discovered by the Spanish scientist Ramón y Cajal belong to this category (Cajal 1903). Investigations using electron microscopy have shown that these bodies consist of coiled fibrils, resembling a coil of twisted thread, and, therefore, they have been called coiled bodies (CB; Monneron and Bernhard 1969). One hundred years after their discovery, they were named Cajal bodies (CB), in honor of their discoverer (Gall et al. 1999). Progresses in investigations of CBs were made possible by the discovery and characterization of a marker of these bodies, coilin, in animal cells (Raška et al. 1991). Using a double-labeling technique, the characteristic molecular composition of these structures was gradually identified. CBs were found to be rich in, among others, elements of the splicing system. All kinds of U RNAs linked to splicing are present here, such as U1–U5 in the mature form containing a trimethylguanosine cap −m3G at the 5′ end (Raška et al. 1991), U6, and immature forms of these small nuclear RNAs (snRNAs; Smith and Lawrence 2000). In these bodies, the presence of Sm proteins (Raška et al. 1991), which together with U snRNA make up the spliceosome (Lorković and Barta 2004), has been confirmed. It has also been demonstrated that active U1 and U2 snRNA genes are associated with Cajal bodies (Frey and Matera 2001). Unlike coilin in animal cell CBs, no CB marker common for all species has been found in plant cells. Therefore, the Sm proteins or U2 small nuclear ribonucleoproteins (snRNPs) are considered plant cell CB markers (Boudonck et al. 1999; Acevedo et al. 2002). Even though a gene has been found which could be a distant homolog of the animal coilin in Arabidopsis (Collier et al. 2006), it still cannot be considered a Cajal body marker, as the presence of this gene product in CB has not been demonstrated.
Despite the presence of splicing machinery elements and many transcription factors (Schul et al. 1998) in CBs, they are not believed to be a site of transcription and splicing, as nascent transcripts have not been found there and some important splicing factors such as SC35 and U2AF are also absent (Gama-Carvalho et al. 1997). Attempts to show the presence of heterogeneous ribonucleoprotein particles in CBs have also not been successful (Gall 2000). There are publications in which an attempt was made to evaluate whether poly(A) RNA is also present in nuclear domains that accumulate many splicing and transcription factors (Visa et al. 1993; Huang et al. 1994). These investigations performed using electron microscopy in somatic cells did not show the presence of poly(A) RNA in CBs.
Microsporocytes of larch are the cells characterized by a high degree of synchronization during anther development and a long meiosis lasting about 6 months, in which periodical increases and decreases in transcriptional activity occur (Smoliński et al. 2007). During prophase of the first meiotic division in microsporocytes of larch, poly(A) RNA accumulates within structures 0.5–6 µm in diameter. Because of the morphological characteristics of these bodies, including shape, number, and spatial relationship with the nucleolus, they resemble CBs. In this work, an attempt was made to determine whether poly(A) RNA accumulates within Cajal bodies.
Materials and methods
Plant material, isolation of protoplasts, and preparation of material for electron microscopy studies
Anthers of Larix decidua Mill. were taken from the same tree in the diplotene stage of prophase of larch microsporocytes. Anthers were fixed in 4% paraformaldehyde solution in phosphate-buffered saline (PBS) buffer pH 7.2 for 6 h and then washed in PBS and resuspended in hydrolysing medium 4% cellulase Onozuka R10 (Serva, Heidelberg Germany) and 27 U/ml pectinase (Sigma) in 0.01 M citric buffer pH 4.8 for 45 min at 37°C. Anthers were then washed in PBS, squashed to obtain free meiocytes and suspended protoplast matter was spread on gelatine-coated slides placed on dry ice and air-dried. Then, the protoplasts were rinsed with 0.1% Triton X100 solution in PBS buffer pH 7.2 for 10 min and air-dried. After that, protoplasts were used for immunofluorescence and fluorescent in situ hybridization (FISH).
For transmission electron microscopy studies, the cells were fixed in 4% paraformaldehyde, 0.25% GA in Pipes overnight at room temperature, dehydrated in alcohols, and embedded in LRGold resin (Sigma). The material was cut using a Leica ultramicrotome, and sections were placed on nickel-formvar-coated grids.
Design of double-labeling reactions
Several double-labeling immunofluorescent–FISH or immunogold high-resolution in situ hybridization (HISH) reactions (Sm-poly(A) RNA; SC35-poly(A) RNA; bromouridine (BrU)-poly(A) RNA; m3G snRNA-poly(A) RNA) were performed as described below. In all of them, immunocytochemical methods always preceded in situ hybridization methods. This is because when in situ hybridization was applied first, the levels of immunofluorescent signals were very weak. In double-labeling FISH (U2 snRNA-poly(A) RNA), both probes were applied simultaneously in the hybridization medium.
Immunodetection of bromouridine incorporation before FISH in double-labeling reaction
Bromouridine incorporation was performed according to the method of Smoliński et al. 2007 with a long (90 min) incubation time. In immunolabeling experiments, protoplasts were blocked for 30 min with 1% bovine serum albumin (BSA) in PBS and then incubated with primary mouse anti-BrU antibodies (F. Hoffmann-LaRoche Ltd., Rotkreuz, Switzerland) in 1% BSA in PBS (diluted 1:100), pH 7.2, overnight at 4°C, and a goat antimouse secondary antibody conjugated with Alexa Fluor 488 in 0.2% BSA in PBS (diluted 1:500) for 1 h at 37°C. They were then washed in PBS and the FISH poly(A) detection method was applied. Finally, slides were washed in double-distilled water and mounted in Citifluor glycerol solution (Agar Scientific). The results were registered with an argon-ion laser emitting light with a wavelength of 488 nm (blue excitation and green fluorescence) and He-neon laser emitting light with a wavelength of 543 nm (green excitation and red fluorescence). A midpinhole, long exposure time (75 μs), and a ×60 (numerical aperture, 1.4) Plan Apochromat DIC H oil immersion lens were used. Pairs of images were collected simultaneously in the green (Alexa 488 or FITC fluorescence) and red (Cy3) channels. In order to minimize of bleed through between the fluorescence channels, low power of lasers (3–10% of maximum power) and control using single-channel collecting were applied. For bleed through analysis and control, Lucia G software was used (Laboratory Imaging, Prague, Czech Republic). The three-dimensional optical sections were acquired with a 0.7-μm step interval. For image processing and analysis, the EZ 2000 Viewer software package (Nikon Europe BV, Badhoevedorp, the Netherlands) was used. For 4',6-diamidino-2-phenylindole (DAPI) staining, a fluorescence-inverted Nikon Eclipse TE 300 microscope, equipped with a mercury lamp, UV-2EC UV narrow band filter, and DXM 1200 FX digital camera, was used.
FISH in double-labeling reactions
For hybridization, the probe was resuspended in hybridization buffer (30% v/v formamide, 4× SSC, 5× Denhardt's buffer, 1 mM ethylenediaminetetraacetic acid, 50-mM phosphate buffer) at a concentration of 50 pmol/ml. Hybridization was performed overnight at 37°C. The following DNA oligonucleotides were used: antisense poly(A) RNA—5′ Cy3 T(T)29 3′; sense poly(A) RNA—5′ Cy3 A(A)29 3′ (IBB PAN, Warsaw, Poland); antisense 5′ Alexa 488 T(T)29 3′; sense 5′ Alexa 488 A(A)29 3′ (Sigma St. Louis, MO, USA); antisense U2 snRNA—5′ Cy3 ATATTAAACTGATAAGAACAGATACTACACTTG 3′; and sense U2 snRNA—5′ Cy3 CAAGTGTAGTATCTGTTCTTATCAGTTTAATAT (IBB PAN, Warsaw, Poland).
Immunofluorescent methods for double-labeling reactions
Immunofluorescent localization: SC35 protein–anti-SC35 antibody (Sigma) assays were performed according to the method of Zienkiewicz et al. 2008a and 3mG cap–anti-3mG antibody (Calbiochem, Bad Soden, Germany) assays were performed according to the method of Zienkiewicz et al. 2006. Sm proteins were detected via incubation with primary mouse anti-Sm Y12, according to the method of Zienkiewicz et al. 2008b or performed using primary human anti-Sm ANA no. 5 antibody (La Jolla Scripps Institute) in 0.2% acetylated BSA in PBS (1:100) in a humidified chamber at 8°C overnight and secondary antibodies: antihuman antibody fluorescein isothiocyanate (Sigma).
HISH immunogold monolabeling of poly(A) RNA and double labeling with Sm proteins
Ultrathin sections were pretreated with prehybridization buffer (hybridization buffer without probe composition the same as for FISH) for 1 h at room temperature and then incubated with antisense poly(A) RNA probe in hybridization buffer (the probe was labeled at the 3′ end with digoxigenin nucleotides and terminal deoxynucleotidyl transferase; Roche). Hybridization was performed in a sealed humidified chamber for 20 h at room temperature. Posthybridization washing was done according to Smoliński et al. 2007. Nonspecific antigens were blocked with high-salt PBS buffer containing 0.05% acetylated BSA for 0.5 h. DNA–RNA hybrids were localized by incubation with sheep antidigoxigenin antibody coupled to 10-nm-diameter colloidal gold particles (Orion Diagnostica, Espoo, Finland; 1: 30 in PBS buffer containing 0.02% acetylated BSA for 1 h at room temperature). Grids were then rinsed and contrasted according to Smoliński et al. 2007. The material was examined and photomicrographs were taken under a JEOL 1010 electron microscope at 80 kV.
In double-labeling reactions, immunogold localization of Sm proteins was performed (which precedes HISH) using primary human anti-Sm ANA no. 5 antibody (La Jolla Scripps Institute) in 0.2% acetylated BSA in PBS (1:100) at 8°C in a humidified chamber overnight and secondary antihuman antibody coupled to 15-nm-diameter colloidal gold particles (BioCell, Cardiff, UK).
For in situ hybridizations (high resolution) and immunofluorescent methods, control treatments consisting of incubations without the first antibody were performed. For the in situ hybridizations, high resolution and fluorescent, sense-labeled probes and ribonuclease-treated samples were used as additional controls. For detection of new transcripts, a control reaction was performed on sections by omitting the incubation with bromouridine. All control reactions produced negative results, or the result of the control reaction was very low compared with that of standard reactions (supplementary data-Fig. S2).
Because of the presence of poly(A) RNA in these bodies, they were examined to see whether they are the site of messenger RNA (mRNA) synthesis and maturation. The localization of the nascent transcript and poly(A) RNA, as identified by double labeling, indicated that BrU-RNA occurs in the form of clusters in the nucleoplasm and is absent in nuclear bodies containing poly(A) RNA. Even after a prolonged time of incubation with BrU up to 1.5 h—when the transcript was already visible in the area of the cytoplasm—the nascent transcript was not localized in nuclear bodies containing poly(A) RNA (Fig. 1). Thus, these bodies are not the site in which the synthesis of new transcripts occurs.
Because of numerous reports on the presence of poly(A) RNA within speckles, we investigated whether the accumulation of poly(A) RNA observed in larch microsporocytes corresponds to these same compartments. For this purpose, double labeling was performed to detect poly(A) and protein splicing factor SC35; SC35 is considered to be a marker for speckles. The results indicated that the SC35 factor does not occur in the area of spherical bodies containing poly(A) RNA (Fig. 2, a). The splicing factor analyzed accumulated in the nucleoplasm mainly in the form of large irregular areas at the boundary of the nucleus, which would correspond to speckles (Fig. 2, a). Apart from round nuclear bodies, speckles were the second nuclear compartment, an area in which poly(A) RNA was also observed (Fig. 2, b). Using the immunogold technique, the presence of poly(A) RNA in regions corresponding to speckles was confirmed at the ultrastructural level—interchromatin granules (supplementary data—Fig. S1).
The localization of poly(A) RNA using electron microscopy indicated that a specific accumulation of this RNA class occurs in oval Cajal bodies built of coiled fibrils (Fig. 3a, b). In the area of CBs, a high level of labeling with gold particles, which often occurred in clusters, was observed (Fig. 3b). Similar cluster labeling was observed in the surrounding nucleoplasm (Fig. 3c). Mean density of gold particles over CB (14.7 ± 2.8/μm2) was over two times higher in comparison with mean density over nucleoplasm (6.3 ± 1.5/μm2).
The number, characteristic morphology, and frequent spatial relationship of nuclear bodies containing poly(A) RNA with the nucleolus, as observed by confocal microscopy, indicates that they are the same Cajal bodies in which poly(A) RNA was localized at the ultrastructural level. In order to confirm these findings, double labeling to detect poly(A) RNA and different splicing factors considered as plant markers of Cajal bodies was performed. The simultaneous localization of poly(A) RNA (Fig. 4a) and core spliceosomal Sm proteins (Fig. 4b) indicated that Cajal bodies containing Sm proteins are the same bodies in which poly(A) RNA is present (Fig. 4c). However, occasionally, the colocalization of poly(A) RNA and Sm proteins within a single CB was not complete; the analyzed molecules occurred independently of each other (Fig. 4d). Mature forms of snRNA localized using an antibody against the trimethylguanosine cap (m3G) demonstrated an accumulation specifically within Cajal bodies, whereas in the nucleoplasm they were localized in a dispersed form (Fig. 5b). Double labeling to detect m3G snRNA and poly(A) RNA (Fig. 5a) showed the colocalization of Cajal bodies and structures containing poly(A) RNA (Fig. 5c). We obtained a similar result when a pool of only one kind of snRNA—U2 snRNA—was analyzed (Fig. 6a–c). In some cases, it was observed that CB did not contain poly(A) RNA, or, in bodies where poly(A) RNA was present, one of the splicing factors did not occur (data not shown).
The method of double labeling was also used to detect ultrastructural colocalization of poly(A) RNA and one of the CB markers—the Sm proteins. The immunogold technique showed that gold particles indicating the presence of poly(A) RNA were localized within CB individually and in the form of clusters (Fig. 7). Gold particles indicating the presence of Sm proteins were also localized in the same nuclear body. The level of Sm protein labeling in CB was much higher than that of poly(A) RNA (Fig. 7).
The investigations performed herein have demonstrated that in larch diplotene microsporocytes poly(A) RNA accumulates in nuclear bodies which demonstrate a regular spherical shape and range from 0.5 to 6 µm in diameter. Reports on the spatial organization of successive stages of pre-mRNA maturation indicate that poly(A) RNA is concentrated in areas corresponding to nuclear speckles (Shopland et al. 2002). Within speckles, factors necessary for mRNA synthesis, maturation, and export are present, including the SC35 protein (considered a marker of speckles), snRNP, hyperphosphorylated RNA polymerase II, and mRNA export factors (Reed and Magni 2001), among others. This could suggest that the accumulations of poly(A) RNA in the form of bodies in larch microsporocyte nuclei represent nuclear speckles. Data from the literature indicate that speckles do not have such regular shapes (Ali et al. 2003) as the spherical bodies in larch microsporocytes. Thus, the properties of larch nuclear bodies, such as shape and, in particular, the absence of SC35 protein, do not correspond to the characteristics of speckles.
The structures—similar to speckles containing SC35 and poly(A) RNA observed in larch microsporocyte nuclei—are distinct from the spherical regular bodies in which poly(A) RNA accumulates. The difference of these two nuclear structures in the larch microsporocytes was confirmed by ME investigations. The speckles are visible under an electron microscope as clusters of interchromatin granules which are thought to be the sites of storage and recycling of snRNPs and SR proteins (Lamond and Spector 2003). Poly(A) RNA presence in the larch microsporocytes was noted both in the area of clusters of interchromatin granules (additional data) as well as in nuclear bodies whose ultrastructure and molecular composition prove that they are CB. Their frequent spatial association with the nucleolus is an additional property which is characteristic of Cajal bodies (Ochs et al. 1994).
This is the first report on the presence of poly(A) RNA within Cajal bodies. The localization of poly(A) RNA—on the basis of electron microscope studies in mammalian somatic cells—did not indicate the presence of this type of RNA in CBs (Visa et al. 1993; Huang et al. 1994). So far, similar investigations have not been performed on plant specimen. The discrepancy of the results obtained here with data from the literature could indicate: (1) a difference between CBs in plant and animal cells or (2) an undetectable level of poly(A) RNA (particularly at the level of the electron microscope) in somatic cells, in which this type of RNA constitutes only 1–2% of the total cellular RNA pool (Lequarre et al. 2004). The analyzed microsporocytes are cells of the generative cell line in the meiotic prophase. In generative cells, the poly(A) RNA may rise to levels that represent up to 10–20% of the total RNA pool (Lequarre et al. 2004). The investigations performed here indicate that, in the analyzed microsporocytes, the level of this particular RNA is high. Thus, it cannot be excluded that its detection was easier than in somatic cells.
The question arises whether the observed poly(A) RNA accumulation in Cajal bodies is linked to a high level of transcription or whether these bodies are the site of storage and/or modification of transcripts with a poly(A) tail. The investigations performed herein have shown that, even after 1.5 h of incubation of larch microsporocytes with bromouridine, poly(A) RNA is not present in CBs as newly synthesized RNA. In animals, a common phenomenon is the accumulation of “long-lived mRNA” in oocyte cytoplasm. In mouse oocytes, as much as 8% of total RNA occurs in the form of poly(A) RNA (Picton et al. 1998). It seems that, in plants, the situation is different; poly(A) RNA accumulation may occur in cells of the male germ line and not the female one. In the hyacinth, the mature egg cell is almost completely deprived of poly(A) RNA (Pięciński et al. 2008). In contrast, in the mature pollen grain, in which RNA polymerase II is present in an inactive form (Zienkiewicz et al. 2008c), a large pool of poly(A) RNA transcripts is present (Tuppy 1982; Zienkiewicz et al. 2006). During larch microsporocyte development, the volume is increased several times (Smoliński et al. 2007), and, after the end of metaphase, it divides into four cells (tetrads). The long-lived RNA that is synthesized earlier and accumulates in CBs during the prophase period could thus be used during successive stages of meiosis, which are accompanied by an increase in cell volume.
It cannot be excluded that part of the poly(A) RNA detected in this study are noncoding “mRNA-like” transcripts (Inagaki et al. 2005). They are transcribed by RNA polymerase II and undergo polyadenylation and splicing. Their role is not completely understood, but they are believed to participate in gene regulation and regulation of transcription factors and have an important role in organogenesis and cell differentiation.
It is not known whether poly(A) RNA accumulation in the nucleus in CBs represents an atypical developmental strategy in larch or whether this is a more common process. Thus far, there are no reports on similar phenomena during microsporogenesis in other higher plants. The lack of data on this subject may be due to the fact that the localization of poly(A) RNA during microsporogenesis has not been performed to date, and investigations on Cajal bodies in these cells are still fragmentary (Seguí-Simarro et al. 2006; Niedojadło et al. 2008). The role of poly(A)-RNA-rich CBs in RNA metabolism in larch microsporocyte development requires further detailed investigations.
We thank J. Niedojadło for critical reading of the manuscript. This work was supported by a UMK Grant (project 321-B) and by the grant of Polish Ministry of Science and Higher Education no. N303 290434.
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