Journal of Cell Science 109, 1241-1251 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
JCS4186
1241
The organization of ribosomal RNA processing correlates with the
distribution of nucleolar snRNAs
A. F. Beven1, R. Lee1,2, M. Razaz2, D. J. Leader3, J. W. S. Brown3 and P. J. Shaw1,*
1Department of Cell Biology, John Innes Centre, Colney, Norwich NR4 7UH, UK
2School of Information Systems, University of East Anglia, Norwich, UK
3Department of Cell and Molecular Genetics, Scottish Crop Research Institute, Invergowrie,
Dundee DD2 5DA, UK
*Author for correspondence
SUMMARY
We have analyzed the organization of pre-rRNA processing by confocal microscopy in pea root cell nucleoli using
a variety of probes for fluorescence in situ hybridization
and immunofluorescence. Our results show that transcript
processing within the nucleolus is spatially highly
organized. Probes to the 5′ external transcribed spacer
(ETS) and first internal transcribed spacer (ITS1) showed
that the excision of the ETS occurred in a sub-region of the
dense fibrillar component (DFC), whereas the excision of
ITS1 occurred in the surrounding region, broadly corresponding to the granular component. In situ labelling with
probes to the snoRNAs U3 and U14, and immunofluorescence labelling with antibodies to fibrillarin and SSB1
showed a high degree of coincidence with the ETS pattern,
confirming that ETS cleavage and 18 S rRNA production
occur in the DFC. ETS, U14, fibrillarin and SSB1 showed
a fine substructure within the DFC comprising closely
packed small foci, whereas U3 appeared more diffuse
throughout the DFC. A third snoRNA, 7-2/MRP, was
localised to the region surrounding the ETS, in agreement
with its suggested role in ITS1 cleavage. All three snoRNAs
were also frequently observed in numerous small foci in the
nucleolar vacuoles, but none was detectable in coiled
bodies. Antibodies to fibrillarin and SSB1 labelled coiled
bodies strongly, though neither protein was detected in the
nucleolar vacuoles. During mitosis, all the components
analyzed, including pre-rRNA, were dispersed through the
cell at metaphase, then became concentrated around the
periphery of all the chromosomes at anaphase, before being
localized to the developing nucleoli at late telophase. PrerRNA (ETS and ITS1 probes), U3 and U14 were also concentrated into small bodies, presumed to be pre-nucleolar
bodies at anaphase.
INTRODUCTION
data is beginning to confirm this interesting idea (Schneiter et
al., 1995).
Since the nucleolus is generally well demarcated cytologically, and since at least part of its function is clearly understood at a molecular level, there have been extensive efforts to
interpret the observed nucleolar ultrastructure in molecular
terms, which, until very recently, has proved remarkably
difficult. In early studies, Miller and Beatty (1969) produced
very clear spread preparations, which showed the axis of the
rDNA, together with many (50 or more per gene) engaged
RNA polymerase I molecules and attached nascent rRNA.
These characteristic ‘Christmas trees’ have still not been
unequivocally demonstrated within the intact nucleolar
structure seen in conventional thin section electron micrographs. The EM appearance of nucleoli is generally described
as being composed of three types of structure: fibrillar centres
(FCs), lightly staining regions ranging from less than 1 µm to
several µm in diameter; dense fibrillar component (DFC), more
densely staining material, often adjacent to FCs; and granular
component (GC), regions where granules, often assumed to be
pre-ribosomal particles, are seen. Many nucleoli, particularly
In most eukaryotic cells the nucleolus is a clearly defined,
prominent, subnuclear domain. It is the site of transcription of
the rDNA (the genes encoding three of the ribosomal RNA
species: 18 S, 5.8 S and 28 S), the subsequent processing of
the precursor rRNA (pre-rRNA), and the biogenesis of preribosomal particles (for reviews see Hadjiolov, 1985;
Hernandez-Verdun, 1991; Risueno and Testillano, 1994;
Scheer and Weisenberger, 1994; Wachtler and Stahl, 1993;
Shaw and Jordan, 1995). Thus a large proportion of the transcriptional and RNA processing activity of most cells takes
place in the nucleolus. It is also involved in much of the cell’s
nuclear and nuclear-cytoplasmic transport, including import of
ribosomal proteins and 5 S RNA (transcribed elsewhere in the
nucleus: see Highett et al., 1993a), and export of pre-ribosomal
particles. Furthermore, several ‘nucleolar’ proteins have been
shown to shuttle between the nucleus and the cytoplasm (e.g.
Borer et al., 1989; Goldfarb, 1991; Meier and Blobel, 1992).
Early studies suggested that the nucleolus is also important in
the export of mRNA (Sidebottom and Harris, 1969), and recent
Key words: Nucleolus, rRNA processing, Fluorescence in situ
hybridization, snoRNA, Confocal microscopy
1242 A. F. Beven and others
in plants, contain a central region, similar to the nucleoplasm
in appearance in EM sections, which is usually called the
nucleolar vacuole, and there is often peripheral, nucleolarassociated chromatin, which in many cases is condensed
rDNA. However, nucleoli vary widely in the organization seen
(see Shaw and Jordan, 1995, for a recent review), so that, for
example, FCs may be small, indistinct or entirely absent, and
DFC may be difficult to distinguish from GC. Recently,
immunolocalization of BrUTP incorporation after short treatments has shown that nascent transcripts are confined to zones
within the DFC, especially at the junctions with the FCs
(Dundr and Raska, 1993; Hozak et al., 1994), and although the
exact interpretation of the FCs is still not completely resolved,
a consensus is emerging that the active genes are associated
with the DFC at the periphery of the FCs.
Most structural and immunocytological analysis of the
nucleolus has been directed at identification of the location of
the rDNA and sites of transcription. In contrast, relatively little
is known about the organization of transcript processing. The
rDNA is transcribed as a 45 S precursor, which is cleaved in
the first stages of ribosome biogenesis. The pre-rRNA includes
a 5′ leader sequence (hereafter referred to simply as the
external transcribed spacer, ETS) preceding the 18 S rRNA,
two internal transcribed spacer sequences (ITS1 and ITS2)
which flank the 5.8 S rRNA, and a short 3′ external transcribed
spacer downstream from the 28 S rRNA (Hadjiolov, 1985).
The 5′ ETS sequence is very long in mammals (3,627
nucleotides in human; Renalier et al., 1989), and is cleaved in
at least 2 stages, whereas it is much shorter in plants and yeast
(approximately 800 nucleotides in pea; Shaw et al., 1995).
Similarly the internal spacers are longer in mammals than in
plants and yeast (Hadjiolov, 1985). Although the coding
regions are well conserved between species, the spacer regions
show much less conservation, and even diverge widely
between closely related species.
Pre-rRNA processing and ribosome assembly require a
number of proteins and small nuclear RNA components, and
thus, by analogy to precursor messenger RNA splicing, it is
likely that the cleavage reactions of pre-rRNA processing
occur in multicomponent ribonucleoprotein complexes (for
recent reviews see Filipowicz and Kiss, 1993; Fournier and
Maxwell, 1993; Mattaj et al., 1993; Morrissey and Tollervey,
1995; Maxwell and Fournier, 1995). A number of small
nucleolar RNAs (snoRNAs) has been isolated from animal and
yeast systems and their functions are the subject of intense
investigation. The nucleolar localisation, association with
rRNP particles and nucleolar proteins (particularly, fibrillarin
in animals and the S. cerevisiae equivalent, Nop1p) and complementarity with short sequences in rRNAs suggest that
snoRNAs are involved in a number of different roles in prerRNA processing or ribosome assembly (Filipowicz and Kiss,
1993; Fournier and Maxwell, 1993; Mattaj et al., 1993;
Morrissey and Tollervey, 1995; Bachellerie et al., 1995;
Maxwell and Fournier, 1995). Although precise functions for
snoRNAs have yet to be determined, some snoRNAs and
nucleolar proteins have already been shown to be required for
normal processing of 18 S rRNA (U3, U14, U22, snR10,
snR30, Nop1p, Gar1p and Sop1p), ITS1 cleavage (7-2/MRP,
Pop1p and Smn1p) and processing of 25 S rRNA (U8)
(Tollervey, 1987; Kass et al., 1990; Savino and Gerbi, 1990;
Li et al., 1990; Tollervey et al., 1991; Hughes and Ares, 1991;
Beltrame and Tollervey, 1992, 1995; Girard et al., 1992;
Jansen et al., 1993; Mougey et al., 1993b; Schmitt and Clayton,
1993; Peculis and Steitz, 1993; Chu et al., 1994; Beltrame et
al., 1994; Lygerou et al., 1994; Tycowski et al., 1994; Liang
and Fournier, 1995). In contrast to animal and yeast systems,
very few snoRNA genes or nucleolar protein genes have been
isolated and characterized from plants. Plant genes encoding
U3 snoRNAs have been obtained from tomato, Arabidopsis,
wheat and maize (Kiss and Solymosy, 1990; Marshallsay et al.,
1990, 1992; Kiss et al., 1991; Leader et al., 1994a), 7-2/MRP
from Arabidopsis and tobacco (Kiss et al., 1992), and U14
from potato and maize (Leader et al., 1994b).
We have previously used fluorescence in situ hybridization
and confocal microscopy to show the relation between the sites
of the active rDNA and the nascent and newly complete transcripts in pea nucleoli (Shaw et al., 1995). The nucleoli in these
plant cells are much larger than most animal nucleoli and thus
show considerable detail with fluorescence microscopy, and
have a characteristic, regular structure, which in our previous
work and in the present study has made it possible to draw clear
conclusions about the organization of their function. We
showed that a probe to the ETS region of the transcript surrounded the small, widely dispersed regions of rDNA,
occupying a distinct sub-region of the nucleolus, which was
demonstrated by parallel electron microscopy to correspond to
the DFC. Later stages of ribosome biogenesis occurred in the
surrounding GC, as monitored by a probe to the entire rRNA
transcript (Shaw et al., 1995). In this paper we refine the
resulting model of nucleolar organization by localization
studies using in situ probes to the first internal transcribed
spacer (ITS1), to the three available plant snoRNAs (U3, U14
and 7-2/MRP) and antibodies to the nucleolar proteins fibrillarin and SSB1.
U3 is the most abundant and best studied snoRNA in animal
and yeast systems and is essential in the first cleavage step of
the 5′ ETS sequence in the processing of pre-rRNA and the
production of 18 S rRNA (Savino and Gerbi, 1990; Kass et al.,
1990; Hughes and Ares, 1991; Mougey et al., 1993b). Sites of
interaction between U3 and pre-rRNA have been mapped to
the ETS regions in several species (Maser and Calvet, 1989;
Stroke and Weiner, 1989; Beltrame and Tollervey, 1992; Tyc
and Steitz, 1992; Beltrame et al., 1994) and an essential U3ETS base pairing interaction has recently been demonstrated
in yeast (Beltrame and Tollervey, 1995; Beltrame et al., 1994).
U14 is also a highly conserved snoRNA which is required in
the processing steps needed to generate 18 S rRNA in yeast
(Li et al., 1990; Fournier et al., 1994) and as for U3, base
pairing interactions have been demonstrated to be essential for
18 S rRNA production (Liang and Fournier, 1995). A third
snoRNA considered in this paper, 7-2/MRP, the RNA
component of RNAse MRP, has been implicated in the A3
cleavage within ITS1 in yeast (Schmitt and Clayton, 1993; Chu
et al., 1994). SnoRNAs normally associate with proteins in the
form of ribonucleoprotein particles (snoRNPs) and are
immunoprecipitable with antibodies against snoRNP proteins.
In particular, many snoRNAs, including U3 and U14 (box C
and D containing snoRNAs) are associated with the highly
conserved nucleolar protein, fibrillarin/Nop1p. Indeed, box C
has been implicated in fibrillarin binding (Baserga et al., 1991).
Finally, in yeast, antibodies against the nucleolar protein SSB1
immunoprecipitate a set of snoRNAs including snR10 and
Organization of rRNA processing 1243
snR11 (Clark et al., 1990). Although these snoRNAs are nonessential snR10 is associated with early events of processing
by virtue of inefficient processing of the largest precursor in
defective mutants.
Our results suggest that the initial stages of pre-rRNA processing, as well as rRNA transcription, take place in a process
which is spatially highly organized. We also demonstrate that
during cell division, when the nucleolar structure is dispersed
and synthesis and processing of rRNA ceases, pre-rRNA
persists. In parallel with the other components we have
analyzed, it is distributed throughout the cytoplasm during
mitosis, and then relocated to the chromosomal periphery and
to presumed prenucleolar bodies at anaphase.
was cloned directly into the pCRII TA cloning vector (Invitrogen).
That the correct sequence had been cloned and the direction of
insertion was determined by sequencing. The sequence determined for
the ITS1 region of the pea pHA1 clone was:
MATERIALS AND METHODS
U3 probe
No report of a U3 snRNA sequence from Pisum sativum is available,
but examination of the U3 snRNA sequences in the EMBL sequence
database identified a 41 base stretch at the beginning of the coding
region that showed virtually 100% homology among all plant
sequences:
Specimen preparation
Seeds of Pisum sativum L. (cultivar Alaska) were imbibed in aerated
water for 12 hours then germinated at 18°C for 2 days on watersoaked tissue paper. The terminal 3-5 mm of the radicle was excised
under water, and fixed in 4% (w/v) formaldehyde/0.1% (v/v) glutaraldehyde in PEM buffer (PEM: 50 mM PIPES-KOH, pH 6.9, 5 mM
EGTA, 5 mM MgSO4) for 1 hour at room temperature. After washing
3 times in TBS (TBS: 25 mM Tris-HCl, pH 7.4, 140 mM NaCl, 3
mM KCl), 30-40 µm vibratome (Bio-Rad Polaron Microcut H1200)
sections were cut under water and dried down on to glutaraldehydeactivated γ-aminopropyl triethoxy silane-coated multiwell slides.
Slides were then used immediately. Sections of this thickness
typically contained 2-3 layers of cells; nearly all nuclei were entirely
contained within the section, and only nuclei that were clearly
complete were analyzed. Tissue sections were treated four times for
15 minutes at room temperature with freshly prepared sodium borohydride solutions (l mg/ml in PBS) (PBS: 140 mM NaCl, 3 mM KCl,
4 mM Na2HPO4, 2 mM KH2PO4, pH 8.0) to reduce autofluorescence
caused by glutaraldehyde. Sections were permeabilized with 2% (w/v)
cellulase (Onozuka R-10) in TBS for 1 hour at room temperature.
Preparation of probes
ETS probe
The ETS probe was generated as previously described (Shaw et al.,
1995).
ITS1 probe
An ITS1 sequence of 239 bases was subcloned from the pHA2 pea
rDNA clone (the clone was a gift from Professor W. Thompson, North
Carolina State University, Department of Botany, Raleigh, North
Carolina, USA; Jorgensen et al., 1987). There is no sequence data
available for the internal spacer regions of pea rDNA, and although
the coding regions of rDNA are highly conserved, the spacer regions
can be very variable (Hadjiolov, 1985). The nucleotide sequence of
the region of the pHA2 clone containing the first internal transcribed
spacer (ITS1) was therefore determined (using an Applied Biosystems
automatic sequencer). Examination of several of the plant rDNA
sequences available in the EMBL database (L. esculentum, N. rustica,
P. deltoides, V. faba) showed that there were long sequences which
were absolutely conserved within the 18 S and 5.8 S coding regions
across these diverse species, although there was little or no conservation in the intervening ITS regions. Oligonucleotides were synthesised from conserved sequences at the 3′ end of the 18 S and the
5′ of the 5.8 S sequences to enable sequencing of the ITS1 region of
the pHA2 pea clone. From this sequence data, PCR primers were
designed at the exact start and finish of the ITS1 region and were used
to amplify the ITS1 region from the pHA2 clone. The PCR product
1 5′-GTCGATGCCT TATATGCAGT CCAACACGTG AATTAGTTTG
41
AACACATGCG GTGGGCTTGA GGTGTTTCAC ACCCCAGCTT
81
GCCATTGGCA TCGGAGGGGA ACGACAAAAT GCGTTCTCTT
121
CTGTGCCAAA ACTCAAACCC CGACGCTGAA TGCGTCAAGG
161
AAATTTAACT TTGCTCTGAG CACATCTGCA TGGCACCGGA
201
GACGGTTCCC GTGCGGGTTG TGTTTTGACA CATTAATAT-3′
(Sequence deposited with EMBL database: accession number:
X95254).
Anti-sense or sense probes were produced by in vitro transcription
using SP6 after linearizing with EcoRV or T7 after linearizing with
BamHI, respectively.
5′-ACGACCTTAC TTGAACAGGA TCTGTTCTAT AGGCTCGTAC C-3′
Two complementary oligonucleotides were synthesised to include
this sequence and a BamHI and EcoRI site at the 5′ and 3′ end, respectively, when annealed together. The double-stranded oligonucleotide
was then cloned into the BamHI/EcoRI sites of the Bluescript KS+
vector. Transformants were selected and sequenced to ensure that the
sequence had been inserted correctly. The orientation of the insert was
such that, as for the ETS clone, T7 RNA polymerase produced sense
RNA and T3 produced anti-sense (i.e. complementary to the endogenous U3 snRNA). Linear templates for the in vitro transcription were
produced by digestion with BamHI or EcoRI for transcription with T3
or T7 RNA polymerase, respectively.
U14 probe
A probe to maize U14 was made as described previously (Leader et
al., 1994a) (plasmid pgMU14.1d, in pGEM3zf+, transcribed by T7
RNA polymerase to produce anti-sense probe after linearisation with
HindIII).
7-2/MRP probe
A clone of an Arabidopsis 7-2/MRP sequence (plasmid pAMRP1.3)
was a generous gift from Professor W. Filipowitz (Friedrich-MiescherInstitut, Basel, Switzerland) (Kiss et al., 1992), and anti-sense probe
was transcribed by T3 RNA polymerase after linearisation with SacI.
Label was incorporated into the probes in each case by including
either digoxygenin-11-UTP (Boehringer), biotin-21-UTP (Clontech)
or fluorescein-12-UTP (Boehringer) in the in vitro transcription
reaction. The probe size for the ETS probe was reduced to approximately 200 bases by a mild carbonate hydrolysis (Cox et al., 1984) to
improve penetration into the specimen. To confirm the specificity of
the ETS and ITS1 probes, they were hybridised to blots of restriction
digests (BamHI and HindIII) of the original pHA2 clone DNA, under
standard conditions (Sambrook et al., 1989). The ETS probe
hybridized only to the 1.31 kb band containing the ETS sequence. The
ITS1 probe hybridized only to the 2.52 kb band containing the ITS
and 5.8 S sequences.
In situ hybridization
In situ hybridization to tissue sections was carried out as described
previously (Highett et al., 1993a,b). In some experiments where fluorescein-labelled probe was used, no further reactions were used for
probe detection. In others, the labelling was amplified by FITCcoupled antifluorescein and FITC-coupled secondary antibodies.
1244 A. F. Beven and others
Digoxygenin-labelled probe was detected by fluorescent antibody
labelling as described previously (Highett et al., 1993a), again in some
cases amplified by subsequent levels of secondary and tertiary
antibody labelling. Biotin-labelled probes were detected using extravidin-cy3 (Sigma). In control experiments the in situ labelling was
preceded by either DNase or RNase digestion as described previously
(Highett et al., 1993a,b). The sections were counterstained for total
DNA with 40 µM 7AAD (7 amino, actinomycin D) for 10 minutes
or with l µg/ml DAPI for 1 minute. In double labelling experiments
the two probes were mixed and added to the tissue together.
Immunofluorescence labelling
Antibodies
An antibody against fibrillarin (72B9) was a generous gift from Dr E.
Tam and Dr Bob Ochs (Scripps Research Institute, La Jolla, California, USA). Antibodies against yeast SSB1 (4A3A2 and 9C4B7) were
generous gifts from Dr Jim Broach (Princeton University, Princeton,
NJ, USA).
Labelling
Root tips for immunofluorescence labelling were fixed for 1 hour in
4% (w/v) freshly prepared formaldehyde in PEM buffer, then washed
in the same buffer. The root tips were then sectioned with a vibratome,
and the sections were treated with 2% cellulase, as for in situ labelling.
Sections were incubated overnight with appropriate dilutions of
primary antibodies in TBS (TBS: 25 mM Tris-HCl, pH 7.4, 140 mM
NaCl, 3 mM KCl). The bound primary antibody was detected with
fluorescein or cy3-conjugated secondary antibody, and in some experiments the signal was amplified by successive layers of tertiary and
quaternary fluorescently-labelled antibodies.
Optical microscopy
Confocal optical sections stacks were collected using a Bio-Rad
MRC-600 or a Bio-Rad MRC-1000UV confocal scanning microscope
as described previously (Rawlins and Shaw, 1990). Image processing
and interpretation was performed on a Titan workstation (Kubota
Pacific Computer Company). In the data sets shown in Fig. 2G,H,I,
image deconvolution with the non-linear, constrained algorithm due
to Jannson was used (Agard et al., 1989; Lee et al., 1993; Shaw, 1994).
Montages of images were produced for display and interpretation of
the various data sets. Alternatively, series of projections were calculated at different angles (Agard et al., 1989) and displayed as stereo
pairs or sequentially to give the impression of rotation. In the calculation of projections, either the mean or the maximum intensity along
each site line was used, usually the latter. Images were photographed
directly from the monitor, or were transferred to a PC or a Macintosh
computer, assembled into composite images using Adobe Photoshop
and printed on a Tektronix Phaser IISDX dye sublimation printer.
RESULTS
Nucleolar distribution of ETS and ITS1
In this paper we have analyzed cells from the highly active pea
root meristematic tissue, where the majority of nuclei have
large, very active nucleoli and prominent central nucleolar
vacuoles. As we showed previously, the ETS probe, which
would be expected to label nascent and newly completed prerRNA transcripts, has a characteristic pattern of labelling,
which in single central optical sections has the appearance of
an annulus, broken by thin gaps into several pieces (Shaw et
al., 1995). In 3-D reconstructions this labelling is seen to
comprise several elongated and meandering regions arrayed
around an empty central region which includes the vacuole.
We have shown that the ETS-labelled region corresponds
broadly to the DFC (Shaw et al., 1995). Fig. 1 shows a series
of confocal images of pea root vibratome sections, in each case
labelled by fluorescence in situ hybridization both with ETS
probe (red) and one of the other probes (ITS1, U3, U14, 72/MRP, green).
The ITS1 sequence from pea (pHA2, isolated from Pisum
sativum, cv Alaska) was determined, and used to generate an
anti-sense probe, which would be expected to label pre-rRNA
up to the point at which ITS1 is excised. Fig. 1A shows a
specimen doubly labelled with ETS and ITS1. The ETS
labelling is not uniform within the DFC region, but comprises
many closely packed small foci (see also Fig. 2A,D,G). These
foci may represent discrete transcription or processing units,
and we estimate there may be several hundred of them in the
largest nucleoli, comparable to estimates of the number of
active genes. The ITS1 labelling (green in Fig. 1A), on the
other hand, is more widely distributed through the nucleolus.
It shows fainter labelling in the ETS region, and brighter
labelling in the surrounding region, which corresponds to the
GC in EM ultrastructure. Thus when the two images are superimposed (Fig. 1A, right-hand image), the ETS label is seen to
be located in ‘shadowed’ regions of the ITS1 labelling. Neither
ETS nor ITS1 labelling was observed in the nucleolar
vacuoles.
Distribution of U3
Fig. 1B shows a single confocal section from pea root tissue
double labelled with anti-sense probe to the ETS (red) and U3
(green). It is clear that U3 is seen throughout the region of ETS
labelling (DFC), but not in the surrounding GC (c.f. Fig. 1A).
This is seen clearly by comparing the superimposed images in
Fig. 1A and B (right-hand panels). However, many nucleoli,
especially those with large vacuoles, also show clear labelling
of clusters of small foci in the vacuole, which in some cases
appear to be arranged in lines (e.g. arrow in Fig. 1B). Although
distinct foci of U3 labelling can be seen in the vacuole, the U3
labelling in the DFC does not show the many small foci as
clearly as in the ETS labelling pattern, but has a more uniform
distribution throughout the DFC. The foci of labelling with U3
in the absence of any transcript (ETS or ITS1) labelling in the
vacuoles is emphasised by the superimposed image in Fig. 1B
(right-hand panel). We have never observed labelling of
nuclear and nucleolar associated bodies (coiled bodies) with a
probe to U3.
Distribution of U14
Fig. 1C shows a single confocal section double labelled with
ETS (red) and U14 (green). The labelling pattern seen with a
probe to U14 is very similar to that seen with U3 (Fig. 1B).
Again, the ETS/DFC region is labelled, and many small foci
are seen in the nucleolar vacuole (e.g. arrow in Fig. 1C) with
no associated pre-rRNA transcript labelling. However, with
the U14 probe, the DFC labelling is more distinct than U3,
and parallels exactly the ETS distribution of many small foci.
Thus, as with ETS, U14 is restricted to subregions of the DFC,
in contrast to U3 which was observed throughout the DFC
region. No labelling of coiled bodies with U14 probe was
observed.
Distribution of 7-2/MRP
A confocal section double labelled with ETS (red) and 7-
Organization of rRNA processing 1245
2/MRP (green) is shown in Fig. 1D. The labelling with this
probe, although clear, is not so strong as with the previous two
probes. This may be due to sequence divergence between
species (the 7-2/MRP used was cloned from Arabidopsis
thaliana and was only 60% similar to partial tobacco 7-2/MRP
RNAs, (Kiss et al., 1992). The 7-2/MRP probe labels the
nucleolar vacuole most clearly (Fig. 1D, centre panel), in a
similar pattern of small foci to that seen there with U3 and U14
(Fig. 1B and 1C). It also labels the nucleolar region surrounding the ETS/DFC, (which corresponds to the GC) in a similar
way to the ITS1 probe. However, the contrast between the
labelling in the different nucleolar regions is significantly
greater with 7-2/MRP than with ITS1, suggesting that 72/MRP is predominantly in the region surrounding the ETS
labelling (apart from the vacuole), and may be completely
excluded from the ETS-labelled region. This difference in distribution is clearly seen by comparing the various superimposed images in Fig. 1. There some indication of a low level
of labelling in the cytoplasm.
Fig. 1. Vibratome sections of
pea root cells double labelled
in situ with ETS and ITS1,
U3, U14, and 7-2/MRP. In
each case the ETS labelling is
shown in the left-hand panel
in red, the other probe in the
centre panel in green, and the
two labels superimposed in the
right-hand panel.
(A) ETS/ITS1; the ITS1
labelling is concentrated in
regions peripheral to the ETS
label. (B) ETS/U3; the U3
label is seen in the same
region as the ETS (DFC), but
also is located in bright foci in
the central vacuoles (e.g.
arrow). The U3 labelling is
more diffuse within the DFC
than the ETS labelling which
comprises a mass of small foci
(see Fig. 2). (C) ETS/U14; the
U14 and ETS colocalise to a
great extent, and both show
the same substructure within
the DFC. U14 is also located
in bright foci in the vacuole as
is U3 (arrow). (D) ETS/72/MRP; the 7-2/MRP is
localized in regions
surrounding the ETS (GC), as
with ITS1, but shows a greater
contrast, suggesting that it is
more nearly excluded from the
ETS regions. As with the
previous two snoRNAs, 72/MRP is located in small
bright foci in the nucleolar
vacuoles (arrow). Bar, 10 µm.
Distribution of fibrillarin and SSB1
Many of the snoRNAs so far identified are found in association with the nucleolar protein fibrillarin, and can be precipitated with anti-fibrillarin antibodies. We therefore carried out
immunofluorescent antibody labelling with antibodies to fibrillarin (72B9), and also to the yeast nucleolar protein SSB1
(4A3A2 and 9C4B7) which has been found associated with
snR10 and snR11. So far it has not been possible to combine
in situ labelling with antibody labelling in pea root tip tissue,
conditions suitable for one preclude the other, and so double
labelling could not be carried out. Fig. 2A-C shows a comparison of the labelling seen with ETS anti-sense probe (Fig. 2A),
fibrillarin (Fig. 2B) and SSB1 (Fig. 2C). In each case a single
confocal section from a root tip vibratome section is shown.
The distribution of fibrillarin and SSB1 is clearly very similar
to the ETS labelling, both being localised to the DFC. In the
case of both proteins, the labelling comprises the mass of small
foci seen with the ETS label. This is shown more clearly in
Fig. 2D-I where 3-D projections of approximately half of a
1246 A. F. Beven and others
Fig. 2. Comparison of ETS in
situ labelling with antibody
labelling with anti-fibrillarin
and anti-SSB1. Vibratome
sections of pea root cells.
(A) A single confocal section
from a 3-D data set, ETS
label. (B) A single confocal
section from a 3-D data set,
anti-fibrillarin (72B9). (C) A
single confocal section from
a data 3-D data set, antiSSB1 (9C4B7). Bar, 10 µm
(A-C). (D-E) Projections of
the upper half of a single
nucleolus (approximately 4
µm) with each label. (D) ETS
probe. (E) Anti-fibrillarin.
(F) Anti-SSB1.
(G-I) Projections of the same
data sets as in (D-E) after 3-D
deconvolution. The fine detail
is much clearer, and shows a
mass of small foci
approximately 0.5 µm in
diameter in all three cases.
Bar, 5 µm (D-I).
single nucleolus are shown for ETS, fibrillarin and SSB1
labelling, respectively (Fig. 2D,E,F) or in Fig. 2G,H,I in which
the data stacks have been deconvoluted before projection
(Agard et al., 1989; Lee et al., 1993; Shaw, 1994). The latter
shows the fine substructure of the labelling patterns much more
distinctly. The antibody labelling also shows that both fibrillarin and SSB1 are found in nucleolar-associated and other
nuclear bodies (arrows in Fig. 2B and C), which are presumed
to be coiled bodies (Beven et al., 1995). As shown before in
plants, there are generally many small coiled bodies in early
G1 (e.g. see bottom cell in 2C), whereas the larger G2 nuclei
generally have fewer, much larger coiled bodies, which often
appear hollow (Beven et al., 1995). There is no significant
labelling of the nucleolar vacuole by either antibody. Thus,
although they colocalise over the DFC to a high degree, there
is a significant difference between the distribution of fibrillarin
(and SSB1) and the associated snoRNAs U3 and U14 within
the nucleolus.
Redistribution of nucleolar components during
mitosis
The nucleolus disappears during mitosis, and transcription and
processing of rRNAs cease. Some nucleolar components, such
as RNA polymerase I (Scheer and Rose, 1984) remain associated with the condensed rDNA, while others, including fibrillarin and U3 have been reported to be associated with the chromosome periphery as ‘passengers’ (Hernandez-Verdun and
Gautier, 1994). Since our labelling experiments were carried out
on intact, active, meristematic root tissue, all stages of mitosis
were represented, and it was possible to analyse the behaviour
of all the components described above during the cell cycle. The
right-hand panels of Fig. 3 show examples from specimens
labelled with anti-sense ETS probe (Fig. 3A), anti-sense U3 (Fig.
3B), and anti-fibrillarin (Fig. 3C); the equivalent images of
DAPI staining to visualize the chromatin are shown in the
respective left-hand panels. These three probes and all the other
probes (not shown) gave very similar distributions during cell
division. Labelling disappeared from the nucleolus very late in
prophase concomitant with the disappearance of the nucleolus,
and was redistributed throughout the cytoplasm by metaphase
(cell 2 in Fig. 3A for ETS; cells 1,2,3 in Fig. 3B for U3; cell 1
in Fig. 3C for fibrillarin). At anaphase and into telophase, the
labelling accumulated at the periphery of all the chromosomes
(cell 1 in Fig. 3A for ETS, anaphase; cell 4 in Fig. 3B for U3,
telophase; cell 2 in Fig. 3C for fibrillarin, anaphase). Labelling
of ITS1 during mitosis (not shown) was very similar to the ETS
Organization of rRNA processing 1247
pattern, and U14 (not shown) was very similar to U3. Although
the peripheral accumulation of fibrillarin seemed to take place
at a slightly later stage than the snoRNAs and pre-rRNA probes
in the images shown (c.f. Fig. 3A, cell 1 and Fig. 3C, cell 3), it
would require double in situ/immunofluorescence labelling to be
certain of this difference. In late telophase/early G1, as the
nucleoli begin to form, all the components mentioned moved
from the chromosome periphery into the forming nucleoli (see,
for example, cell 3 in Fig. 3A in early G1). No labelling of ETS,
ITS1 or any of the other species was seen at the NORs on
metaphase chromosomes. At anaphase, a number of small
bodies as well as the chromosome peripheries were labelled by
ETS, U3 and U14 probes. These bodies were a short-lived stage
in the cycle, and so were often hard to detect. An example of
ETS labelling showing such bodies is displayed inset in Fig. 3A.
This shows a projection of 10 confocal slices (10 µm) of an
anaphase cell from a lower portion of the same data set from
which the larger panel is a single section. These probably
represent prenucleolar or nucleolar precursor bodies (PNBs). It
was not possible to determine whether the bodies also contained
fibrillarin or SSB1, since, unlike the other components, these
proteins are clearly present in coiled bodies which can often
persist through mitosis in these cells, and thus could be confused
with prenucleolar bodies.
DISCUSSION
In a previous paper we showed the relation between the active
rDNA, nascent and newly complete pre-rRNA transcripts, and
Fig. 3. Distribution of ETS, U3
and fibrillarin during mitosis. In
each case a single confocal
section from a 3-D data set
collected from a vibratome
section of a pea root is shown.
The left-hand image shows
DAPI staining of the chromatin,
the right-hand image shows the
relevant probe. The contrast in
the right-hand images has been
set to show the labelling in the
mitotic cells, and so the brighter
interphase labelling is overexposed. (A) ETS probe. At
anaphase (cell 1) the ETS label
is seen at the periphery of all
chromosomes, whereas at
metaphase (cell 2) it is
distributed through the
cytoplasm. At telophase/early
G1 (cell 3) the label becomes
concentrated in the developing
nucleoli. Inset: a projection of a
whole cell further down in the
same 3-D data set, showing
that, in addition to the labelling
at the chromosome periphery,
there is labelling in numerous
small bodies, presumed to be
prenucleolar bodies (arrows).
(B) U3 probe. 3 metaphase cells
(cells 1-3) show a distribution
of labelling throughout the
cytoplasm, whereas the late
anaphase/telophase cell (cell 4)
shows a labelling of the
chromosome periphery.
(C) Anti-fibrillarin. At
metaphase (cell 1) the labelling
is distributed through the
cytoplasm as with the other
probes. At late anaphase (the
top of the chromosomes of cell
2 are just visible) the labelling
is seen around the chromosome
periphery as with the other
probes. However, it is possible that fibrillarin moves to the chromosome periphery later than the previous probes, since it has not yet done so in
the anaphase cell (3), cf cell 1 in A at approximately the same stage. Arrows indicate examples of coiled bodies. Bar, 10 µm.
1248 A. F. Beven and others
Fig. 4. Outline of the processing of pre-rRNA. The RNA processing
events are conventionally divided into early events, giving rise to the
precursor to 18 S rRNA, which involve U3, U14 and fibrillarin
amongst other factors, and late events producing 5.8 S and 28 S
rRNA, which involve 7-2/MRP and other factors.
later stages of ribosome biogenesis in pea root cells (Shaw et
al., 1995). This suggested a radial or vectorial model of
nucleolar organization, with small centres of active rDNA,
possibly in some cases single genes, surrounded by a zone of
nascent transcripts, which in turn was surrounded by a zone of
later processing. Thus, the steps of rDNA transcription and
ribosome biogenesis take place in a highly spatially organized
manner. Fig. 4 shows an outline of the pathway of pre-rRNA
processing (see, e.g. Filipowicz and Kiss, 1993), and Fig. 5
shows in a schematic form the distribution we have observed
for the various probes. Three components (fibrillarin, U3 and
U14) which have been shown to be involved in early pre-rRNA
processing events (5′ ETS cleavage and production of 18 S
rRNA) and SSB1 which, in yeast, associates with snR10
(needed for normal 18 S rRNA accumulation), colocalise to a
high degree with the ETS labelling itself (see Fig. 5C,F).
Recent evidence shows that the terminal balls seen on spread
Christmas trees may contain all the components, which would
include those we have visualized, for the initial cleavage
reactions in an ‘ETS-processing complex’ (Mougey et al.,
1993a). This may imply that the initial cleavages take place
before the pre-rRNA transcripts are detached. Garcia-Blanco
et al. (1995) have also shown on partially spread nuclei a strong
association between fibrillarin, RNA polymerase I and nascent
transcripts, and suggested that zones of transcription are
closely associated with zones of transcript processing, which
is supported here.
Although we previously showed that our ETS labelling was
broadly equivalent to the DFC seen in electron micrographs, it
is not uniform within this region. In this paper we show that
in situ labelling with probes to ETS and U14, as well as fibrillarin and SSB1 antibody labelling, all show a characteristic
pattern comprising a mass of small foci, less than 0.5 µm in
diameter. U3, on the other hand, shows a less structured, more
diffuse labelling of the DFC region. This suggests that the
former components are somewhat more restricted in location
than U3. Similarly Cerdido and Medina (1995) have shown fibrillarin to be concentrated in a sub-region of the DFC, surrounding the FCs, in onion cells, rather than being evenly distributed throughout the DFC. In animal cells, fibrillarin has
been reported to be localized throughout the DFC (e.g. Ochs
et al., 1985); whether this apparent difference between plants
and animals reflects a real functional difference, or simply that
the much larger plant DFC permits a finer spatial discrimination is not yet clear, but our labelling of fibrillarin is quite con-
sistent with this interpretation. The somewhat wider distribution of U3 suggests that this snoRNA is involved in later stages
of processing (as suggested for Xenopus U3; Savino and Gerbi,
1990) than fibrillarin, U14 and SSB1, or at least that it remains
associated with the pre-rRNA RNP complex for longer and,
indeed, U3 appears to remain associated with rRNA after 5′
ETS cleavage (Kass et al., 1990; Mougey et al., 1993b).
The distribution of the probe to ITS1 shows that the ETS
cleavage and the subsequent ITS1 cleavage must take place in
distinct nucleolar locations; the ETS is restricted to the DFC,
or a part of it, whereas the ITS1 is clearly present (in fact is
concentrated in) the surrounding region, which must correspond to the GC (see Fig. 5E,F). Our previous results using a
probe to the entire pre-rRNA transcript showed a very similar
labelling pattern to the ITS1 labelling, but because the former
probe would be expected to label targets at all stages of
ribosome biogenesis, we were not able to say at which stage
the transition of the pre-rRNA from the ETS-labelled region to
Fig. 5. Schematic diagram of the observed localization of the various
probes in pea root nucleoli. (A) shows the EM thin section
ultrastructure seen: V, nucleolar vacuole; DFC, dense fibrillar
component; GC, granular component; CB, coiled body. FC, fibrillar
centres. These nucleoli contain many small lightly staining regions
throughout the DFC, of which only the larger can generally be
clearly described as fibrillar centres, but which we have previously
shown parallel the distribution of rDNA (Shaw et al., 1995). In each
segment of the diagram an outline corresponding to the ETS/U14
region is shown. (B) Highly stylized diagram of a possible molecular
organization (not to scale), showing the location of transcription
complexes within the zone defined by the ETS (see Shaw and
Jordan, 1995; Shaw et al., 1995). (C) U3 is localized to a region
which includes the DFC and to granules in the nucleolar vacuole.
(D) 7-2/MRP is restricted to the GC and to granules in the nucleolar
vacuole. (E) ITS1 labelling is seen in both DFC and GC, but is
concentrated in the latter, with no labelling of the nucleolar vacuole.
(F) ETS shows a fine structure consisting of many small foci within
the DFC. U14 closely parallels this distribution, but is also located in
the granules in the nucleolar vacuole. Fibrillarin and SSB1 also show
a similar fine structure to ETS within the DFC, strong labelling of
coiled bodies, but no labelling within the nucleolar vacuole.
Organization of rRNA processing 1249
the surrounding region took place. The present results show
that this transition must take place after ETS cleavage and
before ITS1 cleavage. The strong similarity of labelling of U14
and ETS suggests that cleavages to produce 18 S rRNA also
occur in the ETS/DFC region.The subsequent stages of maturation and ribosome biogenesis take place in the surrounding
region.
The MRP RNP complex in yeast has recently been shown
to share a protein subunit, Pop1p, with RNase P which cleaves
tRNA precursors (Lygerou et al., 1994). A Pop1p mutant was
defective in cleavage of pre-rRNA at the A3 site in ITS1,
showing that Pop1p and thus MRP must be involved in this A3
cleavage. It is known that MRP is concentrated in nucleoli
(Kiss et al., 1992), and in this paper we have shown that MRP
is localized to the GC, and is quite likely to be excluded
entirely from the DFC (see Fig. 5D). This is in agreement with
its proposed role in the later ITS1 cleavage, which we suggest
must occur in the GC.
Matera et al. (1994) labelled HeLa cells with probes to U3,
U8, and U13 snoRNAs, and with an antibody to fibrillarin.
They showed a heterogeneous labelling pattern for U3 and fibrillarin, sometimes punctate, extending throughout the
nucleolus. A probe to U8, which is involved in later cleavages
leading to 25 S rRNA, showed a strikingly different labelling
pattern of ringlike structures surrounding regions of polymerase I and UBF (upstream binding factor, an rRNA gene
transcription factor) labelling. Although the authors described
the U8 labelling as being similar to that expected for the DFC,
it is also possible that the U8 labelling corresponded to the
peripheral zone of the DFC, or to the GC immediately surrounding the DFC. This interpretation would then be very
similar to our 7-2/MRP labelling, which clearly surrounds the
ETS labelling within the DFC. In the plant nucleoli we have
examined, the DFC is a much larger proportion of the nucleolar
volume than in HeLa cells, and the GC generally appears as a
thin region surrounding it. Thus at the optical level of resolution we could not distinguish between 7-2/MRP labelling the
entire GC or a thin zone surrounding the ETS-labelled region
of the DFC.
We have previously shown that the coiled bodies (CBs)
present in these plant cells have a very similar composition to
mammalian coiled bodies, containing spliceosomal snRNAs,
U2B′′ and p80-coilin (Beven et al., 1995). In this paper we
have shown that plant CBs also contain fibrillarin as do
mammalian CBs (Ochs et al., 1994), and also the nucleolar
protein SSB1. The available evidence is divided as to whether
mammalian coiled bodies also contain U3 snoRNA. JimenezGarcia et al. (1994) detected U3 in HeLa and rat kidney cell
CBs, and Bauer et al. (1994) found U3 in bodies closely resembling CBs assembled in vitro from Xenopus egg extract. Conversely, Carmo-Fonseca et al. (1993) found no evidence for U3
in mammalian CBs. There was no sign of labelling of coiled
bodies with probes to either U3 or U14 in the plant cells which
we examined; thus if CBs in these cells contain U3 or U14, it
must be at a level below our detection sensitivity.
On the other hand, in our plant material we observed very
clear and intense labelling of small foci in the nucleolar
vacuoles of many nuclei with all three of the snoRNA probes,
whereas the vacuoles were not significantly labelled with either
the ETS or ITS1 probes or by antibodies to fibrillarin or SSB1.
We also previously noted labelling of foci in the nucleolar
vacuole with probes to the spliceosomal snRNAs U1, U2 and
U6 (Beven et al., 1995). It is possible that the nucleolar vacuole
is the site of some general UsnRNA functions, such as
recycling, storage or post-transcriptional processing.
Recycling of processing complexes, however, may be unlikely
due to the fact that fibrillarin is not found in the vacuole. The
high concentration of snoRNAs that is found in the vacuoles
of some nucleoli may argue for a storage function. Another
possibility is post-transcriptional processing of snoRNAs
before assembly into their respective rRNP complexes.
Numerous snoRNAs, including U14, have recently been
shown to be produced by excision from pre-mRNA introns
(reviewed by Maxwell and Fournier, 1995). It is not yet clear
whether some plant snoRNAs are also intron-encoded but if
this were the case, the presence of spliceosomal snRNAs in the
nucleolar vacuoles could be explained. In addition, however,
we have shown that the genomic organization of plant
U14snoRNAs differs from that of animals in that the U14
genes are clustered and transcribed polycistronically, implying
a different processing strategy (Leader et al., 1994b). Thus, it
is certainly possible that the organization of processing of some
snoRNAs is different in plants, and their maturation may
involve the nucleolar vacuole.
The rapidly dividing root tip material we analyzed allowed
us to survey the distribution of these components in different
stages of the cell cycle. We saw no labelling with the ETS or
any of the other probes at the mitotic NORs. This agrees with
recent observations of Weisenberger and Scheer (1995) who
used an ETS probe in mammalian cells. Since it has been
known for many years that RNA polymerase I remains bound
to the NORs (Scheer and Rose, 1984), these results suggest
that transcripts detach from RNA polymerase I at the cessation
of transcription during mitosis. Furthermore, it is clear from
our results that unprocessed pre-rRNA transcripts persist stably
through mitosis, and indeed condense onto the chromosomes
and into bodies, presumed to be prenucleolar bodies, at
anaphase. It would be of considerable interest to know whether
these unprocessed transcripts are full length, or incomplete,
since this would shed light on the question of whether transcription from initiated polymerases is completed at mitosis,
or simply halted, with the incomplete transcripts detaching
from the polymerase complexes.
Various nuclear proteins, including fibrillarin, have been
suggested to be chromosomal ‘passengers’ throughout mitosis
(Gautier et al., 1992). Other nuclear and nucleolar proteins are
dispersed throughout the cytoplasm at mitosis, as we have
observed for fibrillarin and SSB1, and then reassembled into
prenucleolar bodies at the end of mitosis. The PNBs then
associate with rDNA being actively transcribed by RNA polymerase I to form a nucleolus. The behaviour we have observed
in these plant cells is somewhat different from either of these.
After being dispersed at late prophase and through metaphase,
we found all the components examined here to be redistributed
to the periphery of all the chromosomes at anaphase. Some
components (pre-rRNA, U3 and U14) were also found briefly
in small bodies, which may correspond to PNBs. These results
are in agreement with recent data from Medina et al. (1995).
The composition of PNBs is still not entirely clear, and may
vary in different species. Jimenez-Garcia et al. (1994) did not
find pre-rRNA in PNBs, although they did find U3 and fibrillarin. It is not clear whether the bodies in the cells we have
1250 A. F. Beven and others
studied are formed first and then redistributed to the chromosome periphery, or whether both processes coexist. The
perichromosomal labelling has the appearance of being quite
uniform, rather than groups of foci or speckles as might be
expected from concentrations of PNBs around the chromosomes.
The major conclusion of our study is that the temporal series
of events in the pathway of processing pre-rRNA in the
nucleolus has a clear counterpart in spatial organization, the
initial cleavage of the ETS and production of 18 S rRNA
occurring in one distinct region within the DFC, ITS1 cleavage
and subsequent events in another (GC). This immediately
raises the question of what is responsible for this organization.
One hypothesis would be that the observed structure is purely
the result of the biochemical processes taking place and the
interactions between the various RNA/protein complexes, and
thus that the nucleolus is ‘an organelle formed by the act of
building a ribosome’ (Melese and Xue, 1995). An alternative
hypothesis is that the nucleolar functions are organized on a
structural matrix, for which there is certainly some evidence.
For example, spread preparations of GC have shown a skeletal
component of 6-8 nm filaments (Franke et al., 1979), and Olins
and Olins (1980) showed evidence for a structural framework
in the granular parts of Chironomus nucleoli. However, the difference between these hypotheses may turn out to be slight;
what from a biochemical viewpoint are seen as interacting
RNA and protein complexes may from a structural viewpoint
be exactly what constitutes such a matrix.
This work was supported by the UK Biotechnology and Biological
Sciences Research Council via grant-in-aid to the John Innes Centre,
by the Scottish Office Agriculture, Environment and Fisheries
Department, by Gene Shears, Pty, Sydney, Australia, and by Bio-Rad
UK Ltd (R.L.).
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(Received 16 January 1996 - Accepted 12 March 1996)