Download New type of snRNP containing nuclear bodies in plant cells

Biology of the Cell 95 (2003) 303–310
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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 Toruń, 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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J. Niedojadło, A. Górska-Brylass / Biology of the Cell 95 (2003) 303–310
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; Smoliński 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 Smoliński, 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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305
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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307
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
Smoliński, 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 (Smoliński 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; Biliński 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.
J. Niedojadło, A. Górska-Brylass / Biology of the Cell 95 (2003) 303–310
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%
309
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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