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New type of snRNP containing nuclear bodies in plant cells
Biology of the Cell 95 (2003) 303–310 www.elsevier.com/locate/bicell
New type of snRNP containing nuclear bodies in plant cells Janusz Niedojadło *, Alicja Górska-Brylass Department of Cell Biology, Institute of General and Molecular Biology, Nicolaus Copernicus University, 87-100 Torun´, Poland Received 20 February 2003; accepted 7 May 2003
Abstract 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. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Bizonal body; Coiled body; Splicing; Microspore
1. Introduction The introduction of immunodetection and in situ hybridization techniques has contributed to a better understanding of intracellular distribution of macromolecules involved in pre-mRNA splicing. In animal cell nuclei the splicing system machinery has been found to be linked to at least several structurally well distinguished nuclear subdomains. Among them are perichromatin fibrils (PGs), which are cotranscriptionally enriched in snRNPs (Fakan, 1994; Misteli et al., 1997), interchromatin granules (IGs) also known as “speckles” containing besides snRNP also the splicing factor SC-35 (Spector et al., 1991; Spector, 1996, Cmarko et al., 1999) and coiled bodies (CBs) (for review see: Matera, 1999; Gall, 2000). CBs are increasingly being called “Cajal body” because of their discover Rajmon’y Cajal (Gall et al, 1999). CBs besides macromolecules of the splicing system such as snRNPs also contain a number of factors linked to the transcriptional machinery (RNA polymerase II, PTF-proximal element sequence-binding transcription factor protein) (Grande et al., 1997; Schul et al., 1998) and also even though they are devoid of rRNA are a site of nucleolar protein localization (fibrillarin) (Raška et al., 1991; Bauer et al.,
* Corresponding author. E-mail address: [email protected] (J. Niedojadło). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/S0248-4900(03)00061-3
1994; Jimenez-Garcia et al., 1994; Malatesta et al., 1994). In CBs proteins involved in histone gene transcript maturation (Wu and Gall, 1993; Abbott et al., 1999) and cell cycle regulation factors (cyclin E/cdk2) (Liu et al., 2000) have also been detected. CBs contain protein p80 coilin which is considered to be a marker of these structures (Andrade et al., 1991; Raška et al., 1991). Such a surprising concentration of macromolecules linked with very different nuclear metabolic pathways in CBs has aroused considerable interest and is currently the subject of intensive investigations. However, there is still no full understanding of CB function in the cell. The association of CB with genes of small nuclear RNAs (Smith et al., 1995; Frey and Matera, 1995) and the presence in these structures of RNA polymerase II RNA and PTF protein (Grande et al., 1997; Schul et al., 1998) participating in the activation of the snRNA gene promoter appear to indicate that CBs participate in the first steps of snRNA biogenesis. Post-transcriptional snRNA modifications are also believed to take place in CBs (Sleeman and Lamond, 1999; Smith and Lawrence, 2000). The investigations of Darzacq et al. (2002) have revealed “Cajal bodyspecific snRNAs” (scaRNAs) in these structures which take part in snRNA methylation and pseudouridylation. Though a decisive majority of the results of the investigations concern animal cells there is a fairly general belief about the universal nature and function of coiled bodies in eukaryotic cells.
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In plant cells nuclear structures which morphologically resemble “coiled bodies” of animal cells though they are called different names have been known for a long time (for review see Risueño and Medina, 1986). The term “coiled body” in respect to plant cells was used for the first time by Moreno Diaz de la Espina et al. (1980) based mainly on the ultrastructural similarity of nuclear bodies of onion cells to animal cell CBs. Only in the 90-ies using immunocytochemical methods was it demonstrated that nuclear bodies in meristem cells of peas and in larch meiocytes which ultrastructurally resemble CBs from mammalian cells also contain snRNAs and coilin (Beven et al., 1995; Smolin´ ski and Górska-Brylass, 1996). In plant cells both free CBs and perinucleolar nuclear bodies known in the literature concerning plant cells as “nucleolus associated bodies” (NABs) were found to be the site of accumulation of such elements of the splicing system as snRNA and Sm proteins (Chamberland and Lafontaine, 1993; Gulemetova et al., 1998; Jennane et al., 1999; Wróbel and Smolin´ ski, 2003). The investigations of Boundock et al. (1999) using green fluorescent protein (GFP) showing CB movement within the cell nucleus demonstrated that CB and NAB present different locations of the same structure. This work presents a different, hitherto unknown type of nuclear bodies containing elements of the splicing system in a plant cell. These bodies were described for the first time in larch microspores by Górska-Brylass and Wróbel (1978) and on the basis of simple cytochemical tests were defined by these authors as ribonucleoprotein structures. At present it appears that ultrastructurally bizonal nuclear bodies of the microspore show a remarkable spatial separation of different macromolecules associated with the splicing system (snRNA and Sm proteins). This trait of the microspore nuclear bodies makes it possible to consider them as not only a convenient model for investigations on the assembly and disassembly of snRNP but at the same time proves that the spatial organization of the splicing system in plant cell nuclei, similarly as in animal cells, is differentiated. 2. Results 2.1. Morphology of nuclear bizonal bodies The used silver staining Ag-NOR technique made it possible to establish that in the larch microspore nucleus in addition to the nucleolus there are also two other categories of nuclear bodies containing agyrophylic proteins. These are dense bodies and bizonal bodies (Fig. 1a). Bizonal bodies 1 to 1.8 µm in size are characterized by differentiated degrees of silver integration. At the level of the light microscope within these bodies from one to several areas with much higher silver integration are visible (Fig. 1a,b). Zones with increased silver impregnation by their size and degree of affinity for silver ions correspond to nuclear bodies which occur freely in the nucleoplasm and which we define as “dense bodies”. At the level of the light microscope in squash
Fig. 1a,b. In the nucleus of the microspore there are well visible bizonal (BB) and dense bodies (DB). Bizonal bodies contain two zones with a differentiated degree of silver impregnation indicating a characteristic acentric pattern (a). Bar represent 10µm. The bizonal body containing five areas with a higer silver impregnation (b). Bar represent 1µm; Nu-nucleolus.
preparations structures freely localized in the nucleoplasm have never been observed, whose silver staining would correspond to a zone of low silver integration in a bizonal body. In the bizonal body the higher silver impregnatin zone always shows an acentric location in respect to a zone with a lower affinity for silver ions (Fig. 1a,b). Bizonal nuclear bodies were observed within the whole nucleoplasm. Different silver affinity of two zones of the microspore nuclear bodies is correlated with a considerable ultrastructural differentiation of these zones. The highly silver impregnating zone turns out to be an electron dense area formed of highly packed fibers 5 nm thick. This zone of the nuclear body is subsequently referred to as the “dense zone” (Fig. 2a) A zone with a lower silver impregnation is formed by coiled threads 25 to 40 nm in diameter. The ultrastructure of this part makes it similar to the structure of coiled bodies widely described in animal cells. In order to stress this similarity we call it the “coiled zone” (Fig. 2a). Both parts of bizonal nuclear bodies always form an acentric system. A full submergence of dense zones in the coiled zone has never been observed. 2.2. rRNA and fibrillarin distribution Bizonal nuclear bodies of the microspore containing nucleolar proteins of the Ag-NOR type were found to be structures devoid of the rRNA. The used of in situ hybridization has shown a highly specific localization of rRNA which is best observed by the presence of gold particles corresponding to 18S rRNA in the nucleolus (Fig. 2b) and cytoplasm
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Fig. 2a-d. Ultrastructure of the larch microspore bizonal body. The bizonal body of larch microspores is composed of a dense (DZ) and coiled (CZ) zone (a). rRNA localization by in situ hybridization using an 18S rRNA probe. The greatest number of gold grains occurs over nucleolus (Nu) (arrows) (b) and cytoplasm (C) (c). The bizonal body (BB) is free from gold grains (N-nucleus) (c). Fibrillarin localization. Gold grains occur over the nucleolus (Nu) and also over the coiled zone (CZ) of bizonal nuclear bodies (d). Bar represent 1µm.
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Fig. 3. DNA localization using TdT enzyme. The central area in both bizonal nuclear bodies lacks gold grains. The strikingly labelled chromatin fiber (arrow) associated with the bizonal body. A greater accumulation of colloid gold particles occurs over condensed chromatin. Over the nucleolus (Nu) occur only single gold grains. Bar represent 1µm.
(Fig. 2c). The number of the colloidal gold grain is clearly higher in the cytoplasm then in the nucleoplasm (Fig. 2c). However, in the dense and coil zone of bizonal nuclear bodies gold grains indicating the presence of rRNA were never observed (Fig. 2b,c). In contrast to Ag-NOR proteins which were localized in both zones of the bizonal body the nucleolar protein fibrillarin in addition to the nucleolus occurs in the coiled zone of these bodies (Fig. 2d).
in the coiled zone of bizonal bodies (Fig. 4a). The U2 snRNA hybridization performed in situ indicated that the occurrence of this splicing snRNA is also limited to the coil zone of the bizonal body (Fig. 4c). In contrast to snRNA, Sm proteins which are an element of the snRNP molecule occur in both zones of the bizonal body. The amount and type of distribution of colloid gold grains indicating the presence of Sm proteins turned out to be similar in both zones of the bizonal body (Fig. 4b).
2.3. DNA distribution 3. Discussion The used method of in situ DNA localization using the TdT enzyme proved to be very sensitive and specific for larch microspores. Colloidal gold grains indicating the sites of DNA occurrence in the nucleus are preferentially localized above the chromatin. Interchromatin areas are free from gold grains (Fig. 3). Bizonal bodies proved to be structures devoid of DNA. However, accumulation of gold grains in the direct vicinity of bizonal bodies has been observed and occasionally also single grains of colloid gold on the periphery of these bodies bordering on the nucleoplasm (Fig. 3). 2.4. Distribution of macromolecules linked to splicing: 3mG cap of snRNA, U2 snRNA and Sm proteins Using the immunogold technique with K121 antibodies the presence of 3mG cap of snRNA, was only demonstrated
3.1. Bizonal bodies of the microspore are an element of cellular organization of the splicing system The results obtained in the present work have shown that bizonal bodies occurring in larch microspores contain: snRNA including U2 snRNA and Sm proteins which indicates that these structures are the nuclear domain linked to the splicing process. Elements of the splicing system in various types of animal cells are localized in a number of structurally well defined nuclear domains such as: PF, IG, CB (Spector, 1993; Spector, 2001). However in some more specialized cells the pattern of distribution of elements of the splicing system is more differentiated and also encompasses such structures as: the interchromatin granule-associated zone (IGAZ) containing only U1 snRNA among five splicing snRNAs (Visa et al., 1993),
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Fig. 4a-c. Immunolocalization of 3mG cap of snRNA. Colloidal gold grains occur only over the coiled zone (CZ) of the bizonal body (a). Immunolocalization of Sm proteins. Gold grains indicating the sites of Sm protein localization occur both over the coiled (CZ) and dense zones (DZ) of the bizonal nuclear body (b). Localization of U2 snRNA by in situ hybridization using an oligonucleotide probe. Colloidal gold particles indicative of U2 snRNA presence occur over the coiled zone (CZ) of the bizonal body and selectively in the chromatin area (arrow) (c). Bar represent 1µm.
the gems in which SMN proteins were located are most probably linked to snRNA recycling (Liu and Dreyfuss, 1996), the cleavage bodies possessing proteins engaged in the maturation of histone gene transcripts, either characteristic for amphibian oocytes, snurposomes (considered as coiled body counterpart in somatic cells) (Wu et al., 1991; Gall, 2000). These structures may occur as an additional structural element of the splicing system (gems, cleavage bodies) or else they constitute modified morphological counterparts of CB (snurposomes). The presented results indicate similarly as in animals the presence of other structures in addition to PF, IG, CB linker
to the splicing process. The occurrence of bodies other than the coiled body with a differentiated ultrastructure containing snRNP indicates the occurrence of differentiation also in plants among nuclear domains which are components of the splicing system. 3.2. The coiled part of bizonal nuclear bodies is the counterpart of a “coiled body” The coiled zone of bizonal bodies of larch microspores not only ultrastructurally resembles mammalian coiled bodies but also contains macromolecules which participate in intron
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removal and was described in CB of mammalian cells (Carmo-Fonseca et al., 1992; Lamond and Carmo-Fonseca, 1993). In ultrastructurally bizonal nuclear bodies of the microspore, snRNP occurs exclusively in the coiled zone. The performed in situ hybridization using a probe complementary to U2 snRNA has demonstrated that the coiled zone containing splicing snRNAs. In contrast to numerous examples of the spatial relation of CB with the nucleolus which is described in both mammalian cells (Raška et al., 1990; Ochs et al., 1994; Malatesta et al., 1994a) and in plant cells (Beven et al., 1995; Wróbel and Smolin´ ski, 2003) the bizonal bodies of larch microspores rather are not perinucleolar bodies. The coiled zone of bizonal bodies, even though they are devoid of rRNA contain nucleolar proteins Ag-NOR and fibrillarin. Even though similarly to mammalian CB bodies the coiled zone does not contain DNA (Thiry, 1994), fibrils and chromatin clumps directly adhering to it have been observed. Similarly to other researchers attempting to immunolocalize coilin in plant cells using antibodies against animal p80coilin, we did not obtain reproducible results (Straatman and Schel, 2001; Acevedo et al., 2002). Nevertheless, the occurrence of snRNP components including U2 snRNP and fibrillarin in the coiled zone, with a concomitant lack of DNA and rRNA allows us suggest that the coiled zone of the bizonal body is “coiled body-like” that commonly occurs in plant and animal cells. 3.3. Bizonal bodies – a new type of nuclear bodies in the plant cell related to the splicing system A permanent association of the coiled zone which represents coiled body-like structure in the microspore with another electron dense zone forms a hitherto unknown structure of the splicing system in the plant cell. The bizonality of this structure is not a characteristic trait of the Larix decidua Mill. species. In meiocytes of this species structurally homogeneous coiled bodies occur containing a number of antigens from the list characteristic for CB (Smolin´ ski and Górska-Brylass, 1996) In the merystematic cells of the larch roots we also found only structurally homogenous coiled body-like struktures (data unpublished). The occurrence of bizonal nuclear bodies is also not characteristic for the stage of microspore development in all plants. The only CBs described so far for microspores other than the larch in Brassica napus microspores turned out to be homogeneous structures containing SmD proteins and fibrillarin (Straatman and Schel, 2001). It seems that the occurence in the larch microspore of a bizonal body may be linked to the high metabolic activity of these cells. In a developing larch microspore compare to meiotic stages an over sixfold increase of total protein takes place (Chwirot and Górska-Brylass, 1981) with an extremely high level of transcriptional activity. The measurement of H3-uridine incorporation in isolated microspores using a scintillation counter demonstrated an intense RNA synthesis
in the postmeiotic interphase of larch microspores (our unpublished data). In comparison to the meiotic stages, in a young larch microspore a tenfold increase and in later stages of the microspore an over thirtyfold increase of H3-uridine incorporation takes place. A similar situation has been described in amphibian and insect oocytes (Gall, 1991; Gall et al., 1995; Bilin´ ski and Kloc, 2002). In amphibian oocytes which in certain developmental stages are also cells with an extremely high metabolic activity numerous extranucleolar structures with a similar morphology to bizonal bodies called snurposomes (Cajal body) have been observed (Gall, 1991; Gall et al., 1995; Roth, 1995). CBs occurring in amphibian oocytes similarly as bizonal bodies of the larch microspore are characterized by a differentiated ultrastructure and molecular composition (Wu et al., 1991; Gall et al., 1999). The molecular differentiation of bizonal nuclear bodies in larch microspore is expressed by the formation of a zone rich in snRNA and a protein zone containing Sm proteins. In amphibian oocytes both types of snurposomes also contain Sm proteins but only in one (snurposome B) is a large accumulation of splicing snRNA present. Spatial separation of macromolecules involved in splicing in distinct zones of nuclear bodies which occur in cells with exceptionally high transcriptional activity suggests that amphibian oocyte snurposomes and larch microspore bizonal bodies are homologous. The morphology of the larch microspore bizonal body should also be compared to the cleavage body and gems. However, at present the data for performing such comparisons are insufficient. No reliable results have been obtained using polyclonal antibodies to SMN proteins present in gems. There are also no antibodies for the plant counterparts of proteins CPSF-100kDa and CstF 64-kDa, which so far are the only macromolecules identified in the cleavage body. However, the lack of data concerning agrophilic proteins in the cleavage body and gems decreases the possibility of homology between these structures and the dense region of bizonal nuclear bodies characterized by agryrophily. 4. Material and methods Male strobili of Larix decidua Mill. were taken from the same tree, in successive postmeiotic interphase stages in order to ensure constant experimental conditions. For immunocytochemical and in situ hybrydization methods freshly collected male strobili of Larix decidua Mill. were immediately fixed in 4% paraformaldehyde in PBS buffer pH 7.2 overnight at 4 °C. Samples where than rinsed in PBS. For Standard Transmission Electron Microscopy (TEM) after washing in PBS strobili were postfixed in 1% OsO4 in PBS buffer overnight at 4 °C, and then rinsed in PBS several times. Then the strobili were dehydrated in alcohol and embedded in Spurr resin (Sigma) for ultrastructures observation and in LR Gold (Sigma) for immunocytochemistry. Sections were placed on copper or nickel grids, contrasted in 2.5 % lead citrate and 2.5 % uranyl acetate (20 min.
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each), than examined in TEM (Tesla 550 or Jeol 1010). For the Ag-NOR technique anthers were fixed in Carnoy’s solution. Subsequently a modified reaction according to Howell and Black (1980) was performed. 4.1. Immunolabeling The following primary antibodies were used: antifibrillarin (ANA-N) (Sigma) diluted 1:4, anti-Sm protein (polyclonal, AF-ANA kindly provided by Dr. Pombo) diluted 1:800, anti-three methyl guanosine cap at the 5’ end of splicing U snRNA (Calbiochem, 1:50) and anti-coilin (polyclonal, 205/5 kindly provided by Dr. Lamond) diluted 1:350. In immunolabeling experiments ultrathin sections were blocked 15’ in 1% BSA in PBS, followed by incubation with primary antibodies in a humid chamber in 4 °C overnight (diluted in 1% BSA in PBS). Grids were then washed in PBS and incubated with secondary antibodies in PBS containing 1% BSA at 37 °C for 1h. The following secondary antibodies were used: anti-mouse, and anti-human, anti-rabbit with 10 and 15 nm gold particles (BioCell) diluted 1:50. Controls with performed by omitting incubation with the primary antibodies. 4.2. In situ hybridization For rRNA localization, an inner 18S rDNA repeat element (1200 bp in length) of Pisum sativum (kindly provided by Dr. G. McFadden) was cloned into pBluescript KS plasmid. 18S rRNA sense and antisense probes labelled with digoxigenin were synthesised using the DIG-RNA labelling kit (Roche). In situ hybridization to ultrathin sections was carried out as described Majewska-Sawka and RodriguezGarcia (1997). Controls performed with sense probe. Synthetic probes, 20mer oligonucleotides to detect U2 snRNA were labeled by tailing reaction using TdT enzyme and digoxigenin-dUTP (Roche). Grids were floated in hybridization mixture containing: probe, 4x SSC, 1mg/ml DNA from herring sperm (Sigma), 10% dextran sulfate, and 30% formamide. Hybridization was performed at 42 °C in a humid chamber overnight. The sections were washed with decreasing concentrations of SSC solution (4xSSC, 2xSSC, 1xSSC) and the signal was visualized by incubation for 1 h with sheep anti-digoxigenin antibody coupled with 15 nm gold particles (BioCell). Controls with performed by omitting probe from hybridization mixture. 4.3. DNA detection The TdT immunogold technique was performed according to Thiry (1992) with modifications. Ultrathin sections were incubated for 30 min at 37 °C on a drop of the following solution: 20 µM Br-dUTP (Sigma): 200 mM potassium cacodylate, 25 mM Tris-HCl, 250 µg/ml bovine serum albumin (BSA), pH 6.6; 5 mM CoCI2, 4µM dCTP, dGTP, dATP, 2.5 U/µl terminal transferase (Roche). Sections were then washed with water and incubated successively with PBS, 5%
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BSA in PBS and again PBS. After incubation with anti-BrdUTP antibodies (Roche) diluted 1:200 in PBS for 1 h the grids were washed with PBS and incubated with anti-mouse antibodies coupled with 15nm (BioCell). Finally grids after the above reaction were stained with 5% uranyl acetate and analyzed with a Joel 1010 electron microscope.
Acknowledgements We wish to thank to dr A. Pombo (University of Oxford) for AF-ANA serum, dr A.I Lamond (University of Dundee) for 205/4 serum, dr G. McFadden (University of Melbourne) for 18S rRNA plasmid.
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Raška, I., Andrade, L.E., Ochs, R.L., Chan, E.K., Chang, C.M., Roos, G., Tan, E.M., 1991. Immunological and ultrastructural studies of the nuclear coiled body with autoimmune antibodies. Exp. Cell Res. 195, 27–37. Raška, I., Ochs, R.L., Andrade, L.E.C., Chan, E.K.L., Burlingame, R., Peebles, C., Gruol, D., Tan, E., 1990. Association between the nucleolus and coiled body. J. Struct. Biol. 104, 120–127. Risueño, M.C., Medina, F.J., 1986. In: Ed Barbera-Guillem, E. (Ed.), The nucleolar structure in plant cells. Leioa-Vizcaya, Spain (Cell Biology Reviews) Servicio Editional Universidad del Pais Vasco. Roth, M.B., 1995. Spheres, coiled bodies and nuclear bodies. Curr. Opin. Cell Biol. 7, 325–328. Schul, W., van Driel, R., de Jong, L., 1998. Coiled bodies and U2 snRNA genes adjacent to coiled bodies are enriched in factors required for snRNA transcription. Mol. Biol. Cell. 9, 1025–1036. Sleeman, J.E., Lamond, A.I., 1999. Newly assembled snRNPs associate with coiled bodies before speckles, suggesting a nuclear snRNP maturation pathway. Curr. Biol. 9, 1065–1074. Smith, K.P., Carter, K.C., Johnson, C.V., Lawrence, J.B., 1995. U2 and U1 snRNA gene loci associate with coiled bodies. J. Cell Biochem. 59, 473–485. Smith, K.P., Lawrence, J.B., 2000. Interactions of U2 gene loci and their nuclear transcripts with Cajal (coiled) bodies: evidence for preU2 within Cajal bodies. Mol. Biol. Cell. 11, 2987–2998. Smolin´ ski, D.J., Górska-Brylass, A., 1996. Plant “coiled body” after in situ hybridization and immunocytochemical investigations. Folia Histochem. Cytobiol. 34 (supll 2), 90. Spector, D.L., 1993. Macromolecular domains within the cell nucleus. Annu. Rev. Cell Biol. 9, 265–315. Spector, D.L., 1996. Nuclear organization and gene expression. Exp. Cell Res. 229, 189–197. Spector, D.L., 2001. Nuclear domains. J. Cell Sci. 114, 2891–2893. Spector, D.L., Fu, X.D., Maniatis, T., 1991. Association between distinct pre-mRNA splicing component and the cell nucleus. EMBO J. 10, 3467–3481. Straatman, K.R., Schel, J.H., 2001. Distribution of splicing proteins and putative coiled bodies during pollen development and androgenesis in Brassica napus L. Protoplasma 216, 191–200. Thiry, M., 1992. Ultrastructural detection of DNA within the nucleolus by sensitive molecular immunocytochemistry. Exp. Cell Res. 200, 135–144. Thiry, M., 1994. Cytochemical and immunocytochemical study of coiled bodies in different cultured cell lines. Chromosoma 103, 268–276. Visa, N., Puvion-Dutilleul, F., Bachellerie, J.P., Puvion, E., 1993. Intranuclear distribution of U1 and U2 snRNAs visualized by high resolution in situ hybridization: revelation of a novel compartment containing U1 but not U2 snRNA in HeLa cells. Eur. J. Cell Biol. 60, 308–321. Wróbel, B., Smolin´ ski, D.J., 2003. Coiled body in the meristematic cells of the roots of Lupinus luteus L. Biol. Plantarum 46, 223–232. Wu, C.-H.H., Gall, J.G., 1993. U7 small nuclear RNA in C snurposomes of the Xenopus germinal vesicle. Proc. Natl. Acad. Sci. USA 90, 811–823. Wu, Z., Murphy, C., Callan, H.G., Gall, J.G., 1991. Small nuclear ribonucleoproteins and heterogeneous nuclear ribonucleoproteins in the amphibian germinal vesicle: loops, spheres, and snurposomes. J. Cell Biol. 113, 465–483.
New type of snRNP containing nuclear bodies in plant cells Janusz Niedojadło *, Alicja Górska-Brylass Department of Cell Biology, Institute of General and Molecular Biology, Nicolaus Copernicus University, 87-100 Torun´, Poland Received 20 February 2003; accepted 7 May 2003
Abstract 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. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Bizonal body; Coiled body; Splicing; Microspore
1. Introduction The introduction of immunodetection and in situ hybridization techniques has contributed to a better understanding of intracellular distribution of macromolecules involved in pre-mRNA splicing. In animal cell nuclei the splicing system machinery has been found to be linked to at least several structurally well distinguished nuclear subdomains. Among them are perichromatin fibrils (PGs), which are cotranscriptionally enriched in snRNPs (Fakan, 1994; Misteli et al., 1997), interchromatin granules (IGs) also known as “speckles” containing besides snRNP also the splicing factor SC-35 (Spector et al., 1991; Spector, 1996, Cmarko et al., 1999) and coiled bodies (CBs) (for review see: Matera, 1999; Gall, 2000). CBs are increasingly being called “Cajal body” because of their discover Rajmon’y Cajal (Gall et al, 1999). CBs besides macromolecules of the splicing system such as snRNPs also contain a number of factors linked to the transcriptional machinery (RNA polymerase II, PTF-proximal element sequence-binding transcription factor protein) (Grande et al., 1997; Schul et al., 1998) and also even though they are devoid of rRNA are a site of nucleolar protein localization (fibrillarin) (Raška et al., 1991; Bauer et al.,
* Corresponding author. E-mail address: [email protected] (J. Niedojadło). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/S0248-4900(03)00061-3
1994; Jimenez-Garcia et al., 1994; Malatesta et al., 1994). In CBs proteins involved in histone gene transcript maturation (Wu and Gall, 1993; Abbott et al., 1999) and cell cycle regulation factors (cyclin E/cdk2) (Liu et al., 2000) have also been detected. CBs contain protein p80 coilin which is considered to be a marker of these structures (Andrade et al., 1991; Raška et al., 1991). Such a surprising concentration of macromolecules linked with very different nuclear metabolic pathways in CBs has aroused considerable interest and is currently the subject of intensive investigations. However, there is still no full understanding of CB function in the cell. The association of CB with genes of small nuclear RNAs (Smith et al., 1995; Frey and Matera, 1995) and the presence in these structures of RNA polymerase II RNA and PTF protein (Grande et al., 1997; Schul et al., 1998) participating in the activation of the snRNA gene promoter appear to indicate that CBs participate in the first steps of snRNA biogenesis. Post-transcriptional snRNA modifications are also believed to take place in CBs (Sleeman and Lamond, 1999; Smith and Lawrence, 2000). The investigations of Darzacq et al. (2002) have revealed “Cajal bodyspecific snRNAs” (scaRNAs) in these structures which take part in snRNA methylation and pseudouridylation. Though a decisive majority of the results of the investigations concern animal cells there is a fairly general belief about the universal nature and function of coiled bodies in eukaryotic cells.
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In plant cells nuclear structures which morphologically resemble “coiled bodies” of animal cells though they are called different names have been known for a long time (for review see Risueño and Medina, 1986). The term “coiled body” in respect to plant cells was used for the first time by Moreno Diaz de la Espina et al. (1980) based mainly on the ultrastructural similarity of nuclear bodies of onion cells to animal cell CBs. Only in the 90-ies using immunocytochemical methods was it demonstrated that nuclear bodies in meristem cells of peas and in larch meiocytes which ultrastructurally resemble CBs from mammalian cells also contain snRNAs and coilin (Beven et al., 1995; Smolin´ ski and Górska-Brylass, 1996). In plant cells both free CBs and perinucleolar nuclear bodies known in the literature concerning plant cells as “nucleolus associated bodies” (NABs) were found to be the site of accumulation of such elements of the splicing system as snRNA and Sm proteins (Chamberland and Lafontaine, 1993; Gulemetova et al., 1998; Jennane et al., 1999; Wróbel and Smolin´ ski, 2003). The investigations of Boundock et al. (1999) using green fluorescent protein (GFP) showing CB movement within the cell nucleus demonstrated that CB and NAB present different locations of the same structure. This work presents a different, hitherto unknown type of nuclear bodies containing elements of the splicing system in a plant cell. These bodies were described for the first time in larch microspores by Górska-Brylass and Wróbel (1978) and on the basis of simple cytochemical tests were defined by these authors as ribonucleoprotein structures. At present it appears that ultrastructurally bizonal nuclear bodies of the microspore show a remarkable spatial separation of different macromolecules associated with the splicing system (snRNA and Sm proteins). This trait of the microspore nuclear bodies makes it possible to consider them as not only a convenient model for investigations on the assembly and disassembly of snRNP but at the same time proves that the spatial organization of the splicing system in plant cell nuclei, similarly as in animal cells, is differentiated. 2. Results 2.1. Morphology of nuclear bizonal bodies The used silver staining Ag-NOR technique made it possible to establish that in the larch microspore nucleus in addition to the nucleolus there are also two other categories of nuclear bodies containing agyrophylic proteins. These are dense bodies and bizonal bodies (Fig. 1a). Bizonal bodies 1 to 1.8 µm in size are characterized by differentiated degrees of silver integration. At the level of the light microscope within these bodies from one to several areas with much higher silver integration are visible (Fig. 1a,b). Zones with increased silver impregnation by their size and degree of affinity for silver ions correspond to nuclear bodies which occur freely in the nucleoplasm and which we define as “dense bodies”. At the level of the light microscope in squash
Fig. 1a,b. In the nucleus of the microspore there are well visible bizonal (BB) and dense bodies (DB). Bizonal bodies contain two zones with a differentiated degree of silver impregnation indicating a characteristic acentric pattern (a). Bar represent 10µm. The bizonal body containing five areas with a higer silver impregnation (b). Bar represent 1µm; Nu-nucleolus.
preparations structures freely localized in the nucleoplasm have never been observed, whose silver staining would correspond to a zone of low silver integration in a bizonal body. In the bizonal body the higher silver impregnatin zone always shows an acentric location in respect to a zone with a lower affinity for silver ions (Fig. 1a,b). Bizonal nuclear bodies were observed within the whole nucleoplasm. Different silver affinity of two zones of the microspore nuclear bodies is correlated with a considerable ultrastructural differentiation of these zones. The highly silver impregnating zone turns out to be an electron dense area formed of highly packed fibers 5 nm thick. This zone of the nuclear body is subsequently referred to as the “dense zone” (Fig. 2a) A zone with a lower silver impregnation is formed by coiled threads 25 to 40 nm in diameter. The ultrastructure of this part makes it similar to the structure of coiled bodies widely described in animal cells. In order to stress this similarity we call it the “coiled zone” (Fig. 2a). Both parts of bizonal nuclear bodies always form an acentric system. A full submergence of dense zones in the coiled zone has never been observed. 2.2. rRNA and fibrillarin distribution Bizonal nuclear bodies of the microspore containing nucleolar proteins of the Ag-NOR type were found to be structures devoid of the rRNA. The used of in situ hybridization has shown a highly specific localization of rRNA which is best observed by the presence of gold particles corresponding to 18S rRNA in the nucleolus (Fig. 2b) and cytoplasm
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Fig. 2a-d. Ultrastructure of the larch microspore bizonal body. The bizonal body of larch microspores is composed of a dense (DZ) and coiled (CZ) zone (a). rRNA localization by in situ hybridization using an 18S rRNA probe. The greatest number of gold grains occurs over nucleolus (Nu) (arrows) (b) and cytoplasm (C) (c). The bizonal body (BB) is free from gold grains (N-nucleus) (c). Fibrillarin localization. Gold grains occur over the nucleolus (Nu) and also over the coiled zone (CZ) of bizonal nuclear bodies (d). Bar represent 1µm.
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Fig. 3. DNA localization using TdT enzyme. The central area in both bizonal nuclear bodies lacks gold grains. The strikingly labelled chromatin fiber (arrow) associated with the bizonal body. A greater accumulation of colloid gold particles occurs over condensed chromatin. Over the nucleolus (Nu) occur only single gold grains. Bar represent 1µm.
(Fig. 2c). The number of the colloidal gold grain is clearly higher in the cytoplasm then in the nucleoplasm (Fig. 2c). However, in the dense and coil zone of bizonal nuclear bodies gold grains indicating the presence of rRNA were never observed (Fig. 2b,c). In contrast to Ag-NOR proteins which were localized in both zones of the bizonal body the nucleolar protein fibrillarin in addition to the nucleolus occurs in the coiled zone of these bodies (Fig. 2d).
in the coiled zone of bizonal bodies (Fig. 4a). The U2 snRNA hybridization performed in situ indicated that the occurrence of this splicing snRNA is also limited to the coil zone of the bizonal body (Fig. 4c). In contrast to snRNA, Sm proteins which are an element of the snRNP molecule occur in both zones of the bizonal body. The amount and type of distribution of colloid gold grains indicating the presence of Sm proteins turned out to be similar in both zones of the bizonal body (Fig. 4b).
2.3. DNA distribution 3. Discussion The used method of in situ DNA localization using the TdT enzyme proved to be very sensitive and specific for larch microspores. Colloidal gold grains indicating the sites of DNA occurrence in the nucleus are preferentially localized above the chromatin. Interchromatin areas are free from gold grains (Fig. 3). Bizonal bodies proved to be structures devoid of DNA. However, accumulation of gold grains in the direct vicinity of bizonal bodies has been observed and occasionally also single grains of colloid gold on the periphery of these bodies bordering on the nucleoplasm (Fig. 3). 2.4. Distribution of macromolecules linked to splicing: 3mG cap of snRNA, U2 snRNA and Sm proteins Using the immunogold technique with K121 antibodies the presence of 3mG cap of snRNA, was only demonstrated
3.1. Bizonal bodies of the microspore are an element of cellular organization of the splicing system The results obtained in the present work have shown that bizonal bodies occurring in larch microspores contain: snRNA including U2 snRNA and Sm proteins which indicates that these structures are the nuclear domain linked to the splicing process. Elements of the splicing system in various types of animal cells are localized in a number of structurally well defined nuclear domains such as: PF, IG, CB (Spector, 1993; Spector, 2001). However in some more specialized cells the pattern of distribution of elements of the splicing system is more differentiated and also encompasses such structures as: the interchromatin granule-associated zone (IGAZ) containing only U1 snRNA among five splicing snRNAs (Visa et al., 1993),
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Fig. 4a-c. Immunolocalization of 3mG cap of snRNA. Colloidal gold grains occur only over the coiled zone (CZ) of the bizonal body (a). Immunolocalization of Sm proteins. Gold grains indicating the sites of Sm protein localization occur both over the coiled (CZ) and dense zones (DZ) of the bizonal nuclear body (b). Localization of U2 snRNA by in situ hybridization using an oligonucleotide probe. Colloidal gold particles indicative of U2 snRNA presence occur over the coiled zone (CZ) of the bizonal body and selectively in the chromatin area (arrow) (c). Bar represent 1µm.
the gems in which SMN proteins were located are most probably linked to snRNA recycling (Liu and Dreyfuss, 1996), the cleavage bodies possessing proteins engaged in the maturation of histone gene transcripts, either characteristic for amphibian oocytes, snurposomes (considered as coiled body counterpart in somatic cells) (Wu et al., 1991; Gall, 2000). These structures may occur as an additional structural element of the splicing system (gems, cleavage bodies) or else they constitute modified morphological counterparts of CB (snurposomes). The presented results indicate similarly as in animals the presence of other structures in addition to PF, IG, CB linker
to the splicing process. The occurrence of bodies other than the coiled body with a differentiated ultrastructure containing snRNP indicates the occurrence of differentiation also in plants among nuclear domains which are components of the splicing system. 3.2. The coiled part of bizonal nuclear bodies is the counterpart of a “coiled body” The coiled zone of bizonal bodies of larch microspores not only ultrastructurally resembles mammalian coiled bodies but also contains macromolecules which participate in intron
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removal and was described in CB of mammalian cells (Carmo-Fonseca et al., 1992; Lamond and Carmo-Fonseca, 1993). In ultrastructurally bizonal nuclear bodies of the microspore, snRNP occurs exclusively in the coiled zone. The performed in situ hybridization using a probe complementary to U2 snRNA has demonstrated that the coiled zone containing splicing snRNAs. In contrast to numerous examples of the spatial relation of CB with the nucleolus which is described in both mammalian cells (Raška et al., 1990; Ochs et al., 1994; Malatesta et al., 1994a) and in plant cells (Beven et al., 1995; Wróbel and Smolin´ ski, 2003) the bizonal bodies of larch microspores rather are not perinucleolar bodies. The coiled zone of bizonal bodies, even though they are devoid of rRNA contain nucleolar proteins Ag-NOR and fibrillarin. Even though similarly to mammalian CB bodies the coiled zone does not contain DNA (Thiry, 1994), fibrils and chromatin clumps directly adhering to it have been observed. Similarly to other researchers attempting to immunolocalize coilin in plant cells using antibodies against animal p80coilin, we did not obtain reproducible results (Straatman and Schel, 2001; Acevedo et al., 2002). Nevertheless, the occurrence of snRNP components including U2 snRNP and fibrillarin in the coiled zone, with a concomitant lack of DNA and rRNA allows us suggest that the coiled zone of the bizonal body is “coiled body-like” that commonly occurs in plant and animal cells. 3.3. Bizonal bodies – a new type of nuclear bodies in the plant cell related to the splicing system A permanent association of the coiled zone which represents coiled body-like structure in the microspore with another electron dense zone forms a hitherto unknown structure of the splicing system in the plant cell. The bizonality of this structure is not a characteristic trait of the Larix decidua Mill. species. In meiocytes of this species structurally homogeneous coiled bodies occur containing a number of antigens from the list characteristic for CB (Smolin´ ski and Górska-Brylass, 1996) In the merystematic cells of the larch roots we also found only structurally homogenous coiled body-like struktures (data unpublished). The occurrence of bizonal nuclear bodies is also not characteristic for the stage of microspore development in all plants. The only CBs described so far for microspores other than the larch in Brassica napus microspores turned out to be homogeneous structures containing SmD proteins and fibrillarin (Straatman and Schel, 2001). It seems that the occurence in the larch microspore of a bizonal body may be linked to the high metabolic activity of these cells. In a developing larch microspore compare to meiotic stages an over sixfold increase of total protein takes place (Chwirot and Górska-Brylass, 1981) with an extremely high level of transcriptional activity. The measurement of H3-uridine incorporation in isolated microspores using a scintillation counter demonstrated an intense RNA synthesis
in the postmeiotic interphase of larch microspores (our unpublished data). In comparison to the meiotic stages, in a young larch microspore a tenfold increase and in later stages of the microspore an over thirtyfold increase of H3-uridine incorporation takes place. A similar situation has been described in amphibian and insect oocytes (Gall, 1991; Gall et al., 1995; Bilin´ ski and Kloc, 2002). In amphibian oocytes which in certain developmental stages are also cells with an extremely high metabolic activity numerous extranucleolar structures with a similar morphology to bizonal bodies called snurposomes (Cajal body) have been observed (Gall, 1991; Gall et al., 1995; Roth, 1995). CBs occurring in amphibian oocytes similarly as bizonal bodies of the larch microspore are characterized by a differentiated ultrastructure and molecular composition (Wu et al., 1991; Gall et al., 1999). The molecular differentiation of bizonal nuclear bodies in larch microspore is expressed by the formation of a zone rich in snRNA and a protein zone containing Sm proteins. In amphibian oocytes both types of snurposomes also contain Sm proteins but only in one (snurposome B) is a large accumulation of splicing snRNA present. Spatial separation of macromolecules involved in splicing in distinct zones of nuclear bodies which occur in cells with exceptionally high transcriptional activity suggests that amphibian oocyte snurposomes and larch microspore bizonal bodies are homologous. The morphology of the larch microspore bizonal body should also be compared to the cleavage body and gems. However, at present the data for performing such comparisons are insufficient. No reliable results have been obtained using polyclonal antibodies to SMN proteins present in gems. There are also no antibodies for the plant counterparts of proteins CPSF-100kDa and CstF 64-kDa, which so far are the only macromolecules identified in the cleavage body. However, the lack of data concerning agrophilic proteins in the cleavage body and gems decreases the possibility of homology between these structures and the dense region of bizonal nuclear bodies characterized by agryrophily. 4. Material and methods Male strobili of Larix decidua Mill. were taken from the same tree, in successive postmeiotic interphase stages in order to ensure constant experimental conditions. For immunocytochemical and in situ hybrydization methods freshly collected male strobili of Larix decidua Mill. were immediately fixed in 4% paraformaldehyde in PBS buffer pH 7.2 overnight at 4 °C. Samples where than rinsed in PBS. For Standard Transmission Electron Microscopy (TEM) after washing in PBS strobili were postfixed in 1% OsO4 in PBS buffer overnight at 4 °C, and then rinsed in PBS several times. Then the strobili were dehydrated in alcohol and embedded in Spurr resin (Sigma) for ultrastructures observation and in LR Gold (Sigma) for immunocytochemistry. Sections were placed on copper or nickel grids, contrasted in 2.5 % lead citrate and 2.5 % uranyl acetate (20 min.
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each), than examined in TEM (Tesla 550 or Jeol 1010). For the Ag-NOR technique anthers were fixed in Carnoy’s solution. Subsequently a modified reaction according to Howell and Black (1980) was performed. 4.1. Immunolabeling The following primary antibodies were used: antifibrillarin (ANA-N) (Sigma) diluted 1:4, anti-Sm protein (polyclonal, AF-ANA kindly provided by Dr. Pombo) diluted 1:800, anti-three methyl guanosine cap at the 5’ end of splicing U snRNA (Calbiochem, 1:50) and anti-coilin (polyclonal, 205/5 kindly provided by Dr. Lamond) diluted 1:350. In immunolabeling experiments ultrathin sections were blocked 15’ in 1% BSA in PBS, followed by incubation with primary antibodies in a humid chamber in 4 °C overnight (diluted in 1% BSA in PBS). Grids were then washed in PBS and incubated with secondary antibodies in PBS containing 1% BSA at 37 °C for 1h. The following secondary antibodies were used: anti-mouse, and anti-human, anti-rabbit with 10 and 15 nm gold particles (BioCell) diluted 1:50. Controls with performed by omitting incubation with the primary antibodies. 4.2. In situ hybridization For rRNA localization, an inner 18S rDNA repeat element (1200 bp in length) of Pisum sativum (kindly provided by Dr. G. McFadden) was cloned into pBluescript KS plasmid. 18S rRNA sense and antisense probes labelled with digoxigenin were synthesised using the DIG-RNA labelling kit (Roche). In situ hybridization to ultrathin sections was carried out as described Majewska-Sawka and RodriguezGarcia (1997). Controls performed with sense probe. Synthetic probes, 20mer oligonucleotides to detect U2 snRNA were labeled by tailing reaction using TdT enzyme and digoxigenin-dUTP (Roche). Grids were floated in hybridization mixture containing: probe, 4x SSC, 1mg/ml DNA from herring sperm (Sigma), 10% dextran sulfate, and 30% formamide. Hybridization was performed at 42 °C in a humid chamber overnight. The sections were washed with decreasing concentrations of SSC solution (4xSSC, 2xSSC, 1xSSC) and the signal was visualized by incubation for 1 h with sheep anti-digoxigenin antibody coupled with 15 nm gold particles (BioCell). Controls with performed by omitting probe from hybridization mixture. 4.3. DNA detection The TdT immunogold technique was performed according to Thiry (1992) with modifications. Ultrathin sections were incubated for 30 min at 37 °C on a drop of the following solution: 20 µM Br-dUTP (Sigma): 200 mM potassium cacodylate, 25 mM Tris-HCl, 250 µg/ml bovine serum albumin (BSA), pH 6.6; 5 mM CoCI2, 4µM dCTP, dGTP, dATP, 2.5 U/µl terminal transferase (Roche). Sections were then washed with water and incubated successively with PBS, 5%
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BSA in PBS and again PBS. After incubation with anti-BrdUTP antibodies (Roche) diluted 1:200 in PBS for 1 h the grids were washed with PBS and incubated with anti-mouse antibodies coupled with 15nm (BioCell). Finally grids after the above reaction were stained with 5% uranyl acetate and analyzed with a Joel 1010 electron microscope.
Acknowledgements We wish to thank to dr A. Pombo (University of Oxford) for AF-ANA serum, dr A.I Lamond (University of Dundee) for 205/4 serum, dr G. McFadden (University of Melbourne) for 18S rRNA plasmid.
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