Vol. 10, No. 7
MOLECULAR AND CELLULAR BIOLOGY, JUlY 1990, p. 3524-3534
Copyright C) 1990, American Society for Microbiology
Differential Distribution of Factors Involved in Pre-mRNA
Processing in the Yeast Cell Nucleus
JUDITH A. POTASHKIN,t ROBERT J. DERBY, AND DAVID L. SPECTOR*
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724
Received 6 March 1990/Accepted 19 April 1990
The yeast cell nucleus has previously been shown to be divided into two regions by a variety of microscopic
approaches. We used antibodies specific for the 2,2,7-trimethylguanosine cap structure of small nuclear
ribonucleic acids (snRNAs) and for a protein component ofsmall nuclear ribonucleoprotein particles to identify
the distribution of small nuclear ribonucleoprotein particles within the yeast cell nucleus. These studies were
performed with the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae.
By using immunofluorescence microscopy and immunoelectron microscopy, most of the abundant snRNAs
were localized to the portion of the nucleus which has heretofore been referred to as the nucleolus. This
distribution ofsnRNAs is different from that found in mammalian cells and suggests that the nucleolar portion
ofthe yeast nucleus contains functional domains in addition to those associated with RNA polymerase I activity.
The nuclei of mammalian cells are organized into defined
structural and functional domains. The domains which are
readily visible by phase-contrast microscopy are the nucle-
oplasm and the nucleolus. Electron microscopic studies
have shown that the nucleoplasm is composed of euchro-
matic and heterochromatic domains, as well as numerous
fibrillar and granular components (for a review, see refer-
ence 4) about which little functional information is available.
The interphase nucleolus is attached to the nuclear envelope
(23) and is organized into three distinct regions: the fibrillar
centers, the dense fibrillar component, and the granular
component (12, 15, 17). DNA emanating from the chromo-
somal nucleolus-organizing region extends into the nucleolus
via the fibrillar center(s); the chromatin in these regions
(fibrillar centers) of the nucleolus is transcriptionally inac-
tive. Transcriptionally active chromatin is found in the
fibrillar regions surrounding the fibrillar centers. The 80S
preribosomal particles accumulate in the granular region of
In contrast to vertebrate nuclei, yeast nuclei are consid-
erably smaller and appear to have less apparent internal
structure. By phase-contrast microscopy, a dark, crescent-
shaped region is apparent on one side ofthe nucleus (30, 46).
Electron microscopy has revealed that there are two readily
distinguishable regions of a yeast nucleus; one electron-
dense region that occupies about one-third to one-half of the
nucleus and a less electron-dense region (16, 30, 31,45, 46,
50, 56). DNase treatment of isolated nuclei, followed by
electron microscopic examination, has suggested that the
region oflow electron density is composed of chromatin (31,
49). The chromatin region is uniform in appearance and
cannot be easily divided into euchromatic and heterochro-
matic regions (16, 30, 31, 45, 46, 50, 56). The electron-dense
region ofthe nucleus has been referred to as the nucleolus on
the basis of ultrastructural studies which have shown that
this region contains morphological components which re-
semble the nucleolonema (fibrillar regions) seen in nucleoli
of higher eucaryotes (16, 31, 50, 56). In addition, studies
t Present address: Department of Pharmacology and Molecular
University of Health Sciences/The Chicago Medical
School, North Chicago, IL 60064.
have shown that this region is RNA and protein rich (31) and
both the 37S and 28S rRNA precursors are located there (49,
50). In conventional transmission electron micrographs of
the budding yeast Saccharomyces cerevisiae (16, 31, 50) and
the fission yeast Schizosaccharomyces pombe (30, 45), the
nucleolar region which is attached to the nuclear envelope
appears to lack any additional substructure. However, nu-
clei of the fission yeast Schizosaccharomycesjaponicus var.
versatilis show patches of low electron density within the
nucleolar region (43). Similar regions oflow electron density
have been observed in transmission electron micrographs of
S. pombe when the cells are fixed by the method of freeze-
substitution (56). No function has been assigned to these
regions of low electron density.
In addition to the classical morphological and biochemical
studies, immunolabeling studies have been used to identify
structural and functional subregions of the nucleus. Such an
approach was used in mammalian cells to localize small
nuclear ribonucleoprotein particles (snRNPs) (34, 42, 52,
53). In eucaryotes, there are six abundant small nuclear
ribonucleic acids (snRNAs), referred to as Ul to U6, which
are complexed with several polypeptides to form snRNPs
(for a review, see reference 29). Ul, U2, U4, U5, and U6
have been shown to be essential for pre-mRNA splicing (for
a review, see reference 55). U3 is localized in the nucleolus
(5, 38, 41) and is most likely involved with rRNA processing
(11, 38, 41). All of the U snRNAs, except for U6, have a
2,2,7-trimethylguanosine (m3G) cap structure at their 5'
ends. All of the snRNPs with which these snRNAs are
associated are recognized by anti-Sm serum from individuals
with certain autoimmune diseases, such as lupus erythema-
tosus (33), except for U3, which is nucleolar (39). Anti-Sm
antibodies (27, 33, 34, 48, 51-54) and antibodies that recog-
nize the m3G cap (42) have been used to localize snRNPs in
mammalian cells. The particles are concentrated in clusters
commonly referred to as speckles within the nonchromatin-
containing region of the nucleoplasm (53). Recently, Fu and
Maniatis (14) have shown that a splicing factor which is not
a snRNP protein colocalizes to these speckled nuclear
regions. A three-dimensional reconstruction ofa mammalian
cell nucleus by immunoelectron microscopy has revealed
that this speckled pattern of snRNPs forms a reticular
FIG. 1. Standard transmission electron micrograph of a cell section of S. pombe (a) showing the electron-dense and less electron-dense
portions of the cell nucleus (N). Within the electron-dense portion of the nucleus, irregularly shaped electron-lucid regions were visible (b and
c, arrowheads). These regions resemble fibrillar centers of mammalian cell nucleoli. Usually, one to five such regions were seen in each cell
section. O>ften these regions were surrounded by a region of greater electron density (c, arrows) which resembles the fibrillar component of
mammalian cell nucleoli. Silver-stained cell sections revealed a densely staining latticework within one portion of the cell nucleus (d). When
silver-stained sections were poststained with uranyl acetate and lead citrate, this latticework was observed to be localized within the nucleolar
portion of the cell nucleus (e). The silver-stained regions often surrounded the less electron-dense fibrillar centers (e). Bars, 0.5 p.m.
POTASHKIN ET AL.
network within the nucleoplasm, which extends between the
nuclear lamina-membrane and the nucleolus (53).
In this study, we used immunofluorescense microscopy
and immunoelectron microscopy to evaluate the localization
of components involved in pre-mRNA processing within the
nuclei of the fission yeast S. pombe and the budding yeast S.
cerevisiae. Our results show that unlike mammalian cell
nuclei, the nucleolar portion of the yeast nucleus contains
most of the snRNAs and suggest that there is a different
compartmentalization of functional domains in yeast cell
MATERIALS AND METHODS
Growth of yeasts and preparation of spheroplasts. S. pombe
972 and S. cerevisiae AY 1045 (MATot ura3-52) were grown
to the mid-log phase at 30°C in YE medium (0.5% yeast
extract, 3% glucose) and YPD medium (1% yeast extract,
2% peptone, 2% glucose), respectively. Cells were harvested
and washed in phosphate-buffered saline (PBS) (10 mM P04,
0.85% NaCl) at pH 7.3 for fluorescence microscopy or 0.1 M
Sorensen phosphate buffer at pH 5.6 for electron micros-
copy. S. pombe cells were fixed for electron microscopy in
Sorensen phosphate buffer at pH 5.6, and S. cerevisiae cells
were fixed in the same buffer at pH 6.0 to keep the cells at
the same pH as the medium in which they were grown for
better preservation of the cellular ultrastructure. Cells were
fixed for 1 h in 2% formaldehyde-0.01% glutaraldehyde in
PBS for fluorescence microscopy or 2% formaldehyde-0.1%
glutaraldehyde in Sorensen phosphate buffer for electron
microscopy. Formaldehyde solutions were made fresh from
paraformaldehyde. S. pombe cells were then washed three
times in Sorensen phosphate buffer containing 0.3 M glycine,
harvested, suspended at a concentration of 107/ml in 1.2 M
sorbitol-50 mM citrate-phosphate (pH 5.6)-40 mM EDTA-1
mM ,-mercaptoethanol-1 mg of Novozyme (Novolab) per
ml, and incubated at 37°C for 1 h to prepare spheroplasts.
Spheroplasts were washed five times in Sorensen phosphate
buffer before further processing for immunofluorescence
microscopy or immunoelectron microscopy. S. cerevisiae
cells were washed three times for 10 min each time in 0.1 M
Sorensen phosphate buffer containing 0.3 M glycine. Cells
were then washed in 0.1 M dibasic potassium phosphate at
pH 6.5 for 10 min and were then incubated in this buffer
containing 0.1 mg of Zymolyase per ml for 45 min at 30°C.
Spheroplasts were washed three times in 0.1 M dibasic
potassium phosphate, followed by three 10-min washes in
Sorensen phosphate buffer before further processing for
Immunofluorescence microscopy. Spheroplasts were per-
meabilized with 0.2% Triton X-100 in PBS (pH 7.3) contain-
ing 0.5% normal goat serum for 1 min and then washed three
times in PBS containing 1% normal goat serum. Sphero-
plasts (100[lI)were applied to a poly-L-lysine-coated glass
cover slip, and the spheroplasts were allowed to attach for 15
min. The cover slip was washed for 10 min with PBS. The
spheroplasts were incubated with a 1:10 dilution of anti-m3G
(24), D77 (1), or anti-DNA (25) primary antibody in PBS for
1 h at room temperature in a humidified chamber. Subse-
quently, the samples were washed three times in PBS and
incubated with an affinity-purified fluorescein isothiocya-
nate-conjugated goat-anti-mouse immunoglobulin G second-
ary antibody (Organon Teknika) at a dilution of 1:20 in PBS
for 1 h at room temperature in a humidified chamber.
Samples were washed four times in PBS and then counter-
stained with 4',6-diamidino-2-phenylindole 2HCl (Serva) by
using a stock made at 1 mg/ml. The cover slips were
mounted in a medium consisting of 90% Kodak glycerol,
10% PBS, and 4% (wt/vol) n-propylgallate (pH 8.5) and
viewed with a Zeiss Photomicroscope II equipped with an
HBO 100-W mercury light source.
pended and pelleted in 0.5 ml of a 2% melted agar (Difco)
solution and centrifuged in an Eppendorf microcentrifuge.
The pelleted samples were allowed to solidify at 4°C for 5
min. The sample pellets were removed from the microcen-
trifuge tube, diced into 1-mm cubes, dehydrated with 7-min
washes in 70 and 80% ethanol, and infiltrated in 100% LR
White (hard grade; London Resin Co., Ltd.). The samples
were placed in 100% LR White resin at room temperature for
2 h, and then the LR White solution was changed and the
specimens were left overnight in LR White at room temper-
ature and changed to fresh embedding medium twice on the
following day. The samples were then transferred to fresh
LR White in 00 gelatin capsules. The blocks were hardened
and cured at 55°C overnight. Thin sections were cut by using
a diamond knife on a Reichert-Jung Ultracut-E ultramicro-
tome. Thin (80-nm) sections were picked up on 300-mesh
For immunolabeling, grids were floated face down in m3G
antibody (24) or D77 antibody (1) diluted 1:1 in Tris-buffered
saline containing 20 mM Tris (pH 7.6), 150 mM NaCl, 20 mM
sodium azide, and 1.0% Tween 20 at 4°C overnight in a
humidified chamber. Before incubation with anti-ribonucle-
oprotein particle antibody 58 at a dilution of 1:7,500, sections
were blocked in buffer containing 8% bovine serum albumin
for 30 min. On the following morning, the grids were
equilibrated to room temperature and then washed for 15
min in Tris-buffered saline. The grids were then transferred
to colloidal gold-conjugated goat anti-mouse or anti-human
immunoglobulin G (10- or 15-nm-diameter gold particles;
Janssen Life Sciences Products) diluted 1:20 in Tris-buffered
saline for 1 h at room temperature. Before incubation, the
diluted, colloidal gold-labeled antibody was spun in a micro-
centrifuge for 5 min at room temperature. After incubation,
the grids were washed for 15 min in Tris-buffered saline and
10 min in water and counterstained with Reynolds lead
citrate (43) and uranyl acetate. Samples were examined at 75
kV in a Hitachi H-7000 transmission electron microscope.
Silver staining. LR White cell sections collected on gold
grids were floated for 5 min at 55°C in a mixture that
contained 1 volume of 2% gelatin in 1% formic acid and 2
volumes of 50% silver nitrate solution (32). Grids were
rinsed in distilled water, immersed in 5% thiosulfate solution
for 5 min, and thoroughly rinsed in distilled water again.
Some sections were then poststained in 2% aqueous uranyl
acetate and lead citrate.
Standard electron microscopy and nucleolar silver staining.
Thin (80-nm) sections of yeast spheroplasts embedded in LR
White resin were stained with uranyl acetate and lead
citrate, and the overall ultrastructure of 100 cells was exam-
ined. The nuclear envelopes in these preparations appeared
in negative contrast because of the solubility of lipids in LR
White resin (Fig. la). Most of the nuclei contained a light-
staining region and a dark-staining region (Fig. la). The
percentage of the total nucleus occupied by each of these
regions in each section varied depending upon the plane of
section through the nucleus. The light-staining region has
previously been shown to contain chromatin (31, 50). As in
MOL. CELL. BIOL.
DISTRIBUTION OF snRNAs IN YEASTS
FIG. 2. Indirect immunofluorescence localization of snRNAs, nucleoli, and DNA. Immunofluorescence was performed with spheroplasts
prepared from a wild-type strain of S. pombe by using a fluorescein-conjugated anti-mouse secondary antibody. Phase-contrast views (a, d,
g, j, m, p, s, v, y, bb, ee, and hh) and immunofluorescence micrographs of cells labeled with anti-m3G (b, e, h, and k) to localize snRNAs,
D77 (n, q, t, and w) to localize nucleoli, or anti-DNA antibody (z, cc, ff, and ii) and counterstained with 4',6-diamidino-2-phenylindole-2HCl
to show the distribution of chromatin (c, f, i, 1, o, r, u, x, aa, dd, gg, andjj) are shown. Bar, 5 urm.
VOL. 10, 1990
DISTRIBUTION OF snRNAs IN YEASTS
those studies, the chromatin appeared to be fairly uniform in
appearance, with little to no condensed chromatin or hetero-
la). The dark-staining region of the S.
cerevisiae nucleus has been suggested to contain rRNA
precursors (49, 50). In our preparations, there appeared to be
several (one to five) electron-lucid zones within the nucleolar
region (Fig. lb and c). These regions resembled fibrillar
centers of mammalian cell nucleoli and were often sur-
rounded by a region of greater electron density (Fig. lc,
arrows) which resembles the fibrillar component of mamma-
lian cell nucleoli (6, 36). A similar staining pattern has
previously been observed in the fission yeast S. japonicus
var. versatilis (45) but has not been reported before in S.
pombe. As a means to further evaluate the distribution of
nucleolar components within the electron-dense region of
the S. pombe nucleus, we used a nucleolus-specific silver-
staining cytochemical technique on sections of cells embed-
ded in LR White resin. With the silver-staining procedure,
specific reactivity has been shown to occur at the nucleolus-
organizing region of chromosomes during mitosis (2, 21, 47)
and at the fibrillar centers or fibrillar component of a variety
of animal cell nucleoli (2, 8, 9, 19, 20, 36) during interphase.
Silver-staining nucleolus-organizing region proteins have
been described as acidic proteins (21, 22, 35, 59) whose
presence is related to nucleolar transcriptional activity (18,
40). When the silver-staining procedure was applied to S.
pombe cell sections, specific staining was observed within
one part of the yeast cell nucleus (Fig. ld). The staining
formed a branched distribution pattern which coiled as it
extended throughout one portion of the nucleus (Fig. ld).
The silver-stained regions often surrounded the less elec-
tron-dense fibrillar centers (Fig. le). This type ofdistribution
pattern is very characteristic of the arrangement of the
nucleolonema or fibrillar component reported in a variety of
mammalian cell nucleoli. The nucleolonema is considered to
correspond to the typical active rRNA transcription units
observed upon chromosome spreading (13). To determine
whether the silver staining was contained within the elec-
tron-dense or nucleolar portion of the S. pombe nucleus,
sections were poststained with uranyl acetate and lead
citrate after silver staining to impart contrast to the cell
nucleus. When such sections were observed in the electron
microscope, it was apparent that the silver-staining pattern
was contained within the more electron-dense nucleolar
portion of the nucleus (Fig. le).
localization of snRNAs.
spheroplasts were immunostained with an anti-m3G anti-
body (24) which recognizes the fission yeast homologs ofUl
to U5 snRNAs (3; A. Krainer and D. Frendewey, unpub-
lished results). The snRNAs were localized to the nonchro-
matin or nucleolar portion ofthe nucleus (Figs. 2b, e, h, and
k) as determined by double labeling with the fluorochrome
binds to DNA (Fig. 2c, f, i, and 1). To further confirm that the
immunolabeled snRNAs were present in the nucleolar por-
tion of the nucleus, a nucleolus-specific antibody was used
on another series of samples. D77 is an antibody that
recognizes a nucleolar protein of 38 kilodaltons (kDa) in S.
cerevisiae and has been shown to cross-react with S. pombe
(1). D77 is thought to recognize the yeast homolog of
fibrillarin, which is a protein found in the fibrillar region of
the metazoan nucleolus and is associated with the nucleolus-
specific U3 snRNP (1). Immunofluorescence microscopy of
cells stained with the D77 antibody showed immunoreactiv-
ity in the nucleolar portion of the S. pombe nucleus (Fig. 2n,
q, t, and w), confirming its nucleolar composition. To rule
out the possibility that the chromatin-enriched region of the
nucleus is inaccessible to antibodies, we used a DNA-
specific antibody, 2C10 (25), for immunofluorescence stain-
ing. As shown in Fig. 2z, cc, ff, and ii, the DNA antibody
recognized the same region of the nucleus as the fluoro-
chrome 4',6-diamidino-2-phenylindole-2HCl (Fig. 2aa, dd,
gg, and ii), which clearly demonstrates that antibodies can
penetrate the chromatin-enriched portion of the nucleus.
Immunoelectron microscopy. To determine whether there
are any snRNAs in the chromatin region that are not
abundant enough to be detected by immunofluorescence, we
used immunoelectron microscopy. To visualize the snRNAs
at the electron microscopic level, we used monoclonal
antibody m3G in combination with secondary antibodies
coupled to colloidal gold. Control sections that were labeled
only with the secondary antibody showed no gold staining
(Fig. 3a). Sections of spheroplasts labeled with both the
primary and secondary antibodies showed most of the gold
particles in the nucleus and minimal background labeling in
the cytoplasm (Fig. 3b). The gold particles located in the
nucleus were concentrated in the electron-dense nucleolar
region (Fig. 3c). The gold particles were counted in 50
individual spheroplasts to quantitate the distribution of sn-
RNAs in yeast cells. The results showed that 85% ofthe gold
particles were localized in the nucleolar region, 11% were
present in the chromatin-enriched region, and 4% were
present in the cytoplasm. Similar localization to the electron-
dense region of the nucleus was observed when an antibody
with strong reactivity to a 34-kDa Ul snRNP protein (S.
Hoch, personal communication) designated A was used (Fig.
To determine whether this distribution pattern of snRNAs
was unique to S. pombe or was also found in the budding
yeast, we examined the localization of snRNAs in S. cere-
visiae. As in S. pombe, the snRNAs in S. cerevisiae were
concentrated in the more electron-dense nucleolar region of
the nucleus (Fig. 5). The lower number of antibody-linked
colloidal gold particles found in the S. cerevisiae nucleus
than in the S. pombe nucleus is consistent with the 10-fold
lower abundance ofsnRNAs in the S. cerevisiae nucleus (44,
To further confirm the nucleolar origin of the electron-
dense nuclear region, thin sections were incubated with
nucleolus-specific antibody D77, which has been shown to
cross-react with S. pombe (1). Most of the gold particles
were located within the dark-staining region of the nucleus
(Fig. 6). The gold particles appeared to be dispersed through-
out this region; however, neither the anti-m3G antibody nor
D77 was observed within the electron-lucid regions of the
nucleolus, suggesting that the snRNAs and fibrillarin are not
FIG. 3. Immunoelectron microscopic localization of snRNAs in the S. pombe nucleus. Ultrathin sections of fission yeast embedded in LR
White resin were incubated with anti-m3G primary antibody, followed by a 15-nm-diameter colloidal gold particle-conjugated secondary
antibody and poststained for electron microscopy. Control cells not incubated with the primary antibody showed no gold labeling (a). Yeast
cells stained with both the primary and secondary antibodies showed most of the gold particles in the nuclei (b). An enlarged view of the
nucleus shows that most ofthe gold particles were present in the electron-dense portion (c). Arrowheads point to representative gold particles.
Bars, 0.5 R,m.
VOL. 10, 1990
POTASHKIN ET AL.
FIG. 4. Immunolocalization of snRNPs in the nucleus of the
fission yeast S. pombe with an autoantibody which recognized the A
protein of Ul snRNP. Ul snRNPs were concentrated in the elec-
tron-dense portion of the cell nucleus. Representative colloidal gold
particles are identified by arrowheads. Bar, 0.5 ,um.
located within these areas of the nucleolus. Twenty cells
were counted to quantitate the distribution of D77 within
yeast cells. Of the gold particles, 78% were located in the
electron-dense nucleolar region, 10% were present in the
chromatin-enriched region, and 13% were present in the
cytoplasm. Interestingly, with the glutaraldehyde fixation
conditions used in this study, the D77 epitope was not
destroyed as it had been reported to be in an earlier study
We localized the abundant snRNAs in wild-type strains of
the fission yeast S. pombe by indirect immunofluorescence
microscopy and immunoelectron microscopy. The results of
these experiments showed that most of the snRNAs were
present within the more electron-dense portion of the nu-
cleus which has heretofore been referred to in the literature
as the nucleolus. In addition, we found a similar localization
ofsnRNAs in the nucleus ofthe budding yeast S. cerevisiae.
These findings are in contrast to the results obtained with
mammalian cells, in which most of the abundant snRNAs
were present in a nonnucleolar network within the non-
chromatin-containing regions of the nucleoplasm (53). Our
results suggest that both structurally and functionally, the
yeast nucleus needs to be reexamined, and perhaps classical
designations of nuclear regions need to be modified to
accommodate new data.
In mammalian cells, the nucleolus is a distinct biochemical
and structural entity within which ribosomal genes and their
products are naturally sequestered from the rest of the
genome and nucleoplasm. Within this highly specialized
non-membrane-bound region of the nucleus, ribosomal gene
transcription, rRNA processing, and preribosomal particle
formation occur (6). It is clear from previous studies with S.
pombe that the electron-dense or nucleolar region of the
nucleus contains an abundance ofRNA and protein (31, 49).
In Saccharomyces carlsbergensis, the 37S and 28S rRNA
FIG. 5. Immunoelectron microscopic localization of snRNAs in the nucleus of the budding yeast S. cerevisiae with an m3G antibody.
snRNAs were concentrated in the electron-dense portion of the cell nucleus (arrowheads). Bar, 0.5 ,um.
MOL. CELL. BIOL.
DISTRIBUTION OF snRNAs IN YEASTS
? t -~.*V .&At
< -* J- '
FIG. 6. Immunoelectron microscopic localization of a nucleolar protein in the S. pombe nucleus. Ultrathin sections of fission yeast
embedded in LR White resin were incubated with antibody D77, followed by a colloidal gold-conjugated secondary antibody, and poststained
for electron microscopy. Yeast cells stained with both the primary and secondary antibodies showed most of the gold particles in the
dark-staining portion of the nucleus, identifying it as the nucleolar component of the nucleus. Arrowheads point to representative gold
particles. Bars, 0.5 FLm.
precursors are located within this region (50). Recently,
Dvorkin et al. (10) have used electron microscopic in situ
hybridization techniques to localize 25S rDNA to the nucle-
olar region of the S. cerevisiae cell nucleus. In addition, the
present study showed that nucleolus-organizing region-spe-
cific silver staining was localized to a portion ofthe electron-
dense region of the S. pombe nucleus. Therefore, at least a
portion of the electron-dense region of the cell nucleus
contained nucleolar components.
The localization of snRNAs and/or snRNPs in mammalian
cells has been studied in a variety of laboratories, and it is
generally agreed that these RNA molecules localize in a
speckled immunostaining pattern within the nuclei of a
variety of mammalian cells and tissues (33, 34, 42, 52, 54).
Recently, D.L.S. has used three-dimensional reconstruction
techniques to demonstrate that these speckled areas con-
nected and form a latticework or network within the nucle-
oplasm (53). In direct contrast to the present findings with S.
VOL. 10, 1990
" r U,j;_-
POTASHKIN ET AL.
pombe, anti-m3G antibodies or antibodies specific for one or
more of the snRNP-associated proteins (70-kDa Ul, 34-kDa
Ul, and 33-kDa U2) did not immunoreact with regions of the
mammalian cell nucleolus (42, 52, 58). This is particularly
interesting with regard to m3G-specific antibodies in light of
the fact that a major mammalian nucleolar snRNA, U3, is
located within the nucleolus. It is possible that the U3 m3G
cap structure is blocked by proteins that bind at or around
the cap structure in vivo. Alternatively, it is possible that the
cap is inaccessible to antibody molecules because of steric
hindrance caused by its three-dimensional position within
the nucleolus. However, antibodies to the nucleolar protein
fibrillarin, which is a U3-specific nucleolar protein (28, 37,
57), are able to penetrate the mammalian cell nucleolus and
provide a specific subnucleolar immunolocalization pattern
The immunolocalization of several nuclear constituents
associated with pre-mRNA processing has been reported in
the budding yeast S. cerevisiae. Last and Woolford (26)
produced antibodies against fusion proteins that contain
portions of the PRP2 or PRP3 open reading frames. These
antibodies were used at the light microscopic level to show
that polypeptides expressed from high-copy-number plas-
mids were localized to the cell nucleus in S. cerevisiae.
Whether overexpressed PRP2 or PRP3 proteins were local-
ized throughout the nucleoplasm or restricted to a particular
portion of the yeast nucleus was not reported. In a more
recent study, Chang et al. (7) have shown that the PRP11
protein in S. cerevisiae is specifically associated with the 40S
spliceosome and a 30S complex. In addition, those investi-
gators used an antibody to localize the PRP11 protein to the
nonnucleolar portion of the S. cerevisiae nucleus. Of the
anti-PRP11 gold particles, 52% were localized in the cyto-
plasm and were regarded as nonspecific background stain-
ing, while 44% were localized in the nucleoplasm and were
considered specific. In the present study, we used an anti-
body against the m3G cap structure of snRNAs, and upon
quantitation, we observed that 85% of the m3G-labeled
colloidal gold particles were localized to the nucleolar por-
tion ofthe nucleoplasm, 11% were in the chromatin-enriched
portion of the nucleoplasm, and 4% were in the cytoplasm.
Brennwald et al. (3) have shown that this same m3G antibody
immunoprecipitates snRNPs Ul to U5 from S. pombe ex-
tracts and Ul and U2 snRNPs are the most predominant
snRNPs in this organism. In addition, we found a similar
localization by using an antibody which recognizes a protein
component of Ul snRNP. One possibility for the differential
localization of snRNAs observed in the present study is that
most of the snRNAs localized to the electron-dense region of
the nucleus may represent potential sites of splicing com-
plexes. Recently, Fu and Maniatis (14) have shown that a
non-snRNP splicing factor in mammalian cells localizes
within nuclear regions enriched in snRNPs. These findings
support our original model (53) that the nuclear regions
concentrated in snRNPs form a framework within which
events involved in pre-mRNA processing take place or from
which they emanate. If these findings are consistent in yeast
nuclei, we predict that pre-mRNA splicing would occur in
the electron-dense region of the yeast nucleus, where snR-
NAs are concentrated. On the basis ofour findings that most
snRNAs localized to the nucleolar portion of the yeast cell
nucleus, we suggest a modification of the current model of
the yeast nucleus. We propose that the classically designated
nucleolar portion of the yeast nucleus is organized differ-
ently from the mammalian cell nucleolus and contains func-
tional domains in addition to those associated with rRNA
transcription and processing. The dark-staining region of the
nucleus may function in nucleolar processes, including tran-
scription of rDNA and processing of rRNA transcripts, and
in pre-mRNA processing. We therefore suggest that the
dark-staining region be referred to as the non-chromatin-
enriched area of the nucleus, rather than the nucleolus.
Further research is needed to define which nuclear processes
occur in the two parts of the yeast nucleus and the relation-
ship between components within each of these nuclear
regions, as well as between the regions themselves. Such
studies will depend upon the localization of other nucleus-
specific probes. In addition, the yeast system provides for
powerful genetic approaches to define associations between
nuclear structure and function.
We thank James D. Watson for support and encouragement
throughout this work. We are grateful to A. Krainer for the anti-m3G
antibody, J. Aris and G. Blobel for the D77 antibody, D. Stollar for
the anti-DNA antibody, and S. Hoch for anti-ribonucleoprotein
particle autoantibody 58. We thank J. Huberman, J. Hyams, B.
McClintock, M. Spector, D. Tollervey, and J. Woolford for helpful
discussions. We are grateful to G. Conway, J. Huberman, A.
Krainer, B. McClintock, and M. Spector for critical review of the
manuscript. We thank A. Sutton for providing S. cerevisiae cells.
The excellent technical assistance of J. Suhan at the start of this
study is gratefully acknowledged.
This work was partially supported by a grant from the American
Cancer Society (NP-619A) and Public Health Service grants from
the National Institutes of Health (5P30 CA45508-03 and GM42694)
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