* Department of Biological Chemistry, University of California, Irvine, California 92697-1700; and Department of Anatomy and
Cell Biology, Health Science Center, University of Florida, Gainesville, Florida 32610-0235
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Abstract |
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The nucleolus in Saccharomyces cerevisiae is a crescent-shaped structure that makes extensive contact with the nuclear envelope. In different chromosomal rDNA deletion mutants that we have analyzed, the nucleolus is not organized into a crescent structure, as determined by immunofluorescence microscopy, fluorescence in situ hybridization, and electron microscopy. A strain carrying a plasmid with a single rDNA repeat transcribed by RNA polymerase I (Pol I) contained a fragmented nucleolus distributed throughout the nucleus, primarily localized at the nuclear periphery. A strain carrying a plasmid with the 35S rRNA coding region fused to the GAL7 promoter and transcribed by Pol II contained a rounded nucleolus that often lacked extensive contact with the nuclear envelope. Ultrastructurally distinct domains were observed within the round nucleolus. A similar rounded nucleolar morphology was also observed in strains carrying the Pol I plasmid in combination with mutations that affect Pol I function. In a Pol I-defective mutant strain that carried copies of the GAL7-35S rDNA fusion gene integrated into the chromosomal rDNA locus, the nucleolus exhibited a round morphology, but was more closely associated with the nuclear envelope in the form of a bulge. Thus, both the organization of the rDNA genes and the type of polymerase involved in rDNA expression strongly influence the organization and localization of the nucleolus.
Key words: nucleus; nucleolus; nuclear envelope; ribosomal DNA (rDNA); RNA polymerases I and II ![]() |
Introduction |
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THE nucleolus is the site of ribosomal DNA (rDNA)1
transcription by RNA polymerase I (Pol I), processing of rRNA transcripts and assembly of ribosomes
(for reviews see Scheer and Weisenberger, 1994; Xue and
Mélèse, 1994
; Shaw and Jordan, 1995
). Both Pol I and
rDNA are present in the nucleolus together with other nucleolar proteins and small nucleolar RNAs (snoRNAs) required for these processes. The nucleolus occupies a discrete subnuclear region and has been the subject of
intensive studies by cell biologists, using a variety of higher
eukaryotic cell systems. In the yeast Saccharomyces cerevisiae, the nucleolus can be seen by immunofluorescence microscopy (IFM) using antibodies against suitable nucleolar
proteins or by EM, as a crescent-shaped region, occupying
a substantial fraction of the nucleus along the nuclear envelope. In contrast to yeast cells, the nucleolus in higher
eukaryotes does not have extensive direct contact with the
nuclear envelope in most systems analyzed; the nucleolus
appears to contain an intranucleolar skeleton that is contiguous (and possibly identical) with the nuclear skeleton connecting to the nuclear envelope (for reviews see Bourgeois and Hubert, 1988
; Hozak, 1996
). It has been proposed that the fibrillar center of the nucleolus, which contains the rDNA transcriptional machinery such as Pol I, as
well as rDNA, is bound to the nucleolar skeleton (Hozak,
1996
; Weipoltshammer et al., 1996
). However, the morphologically defined nucleolar skeleton has not been well characterized biochemically.
It has now been established, at least for salivary gland
polytene nuclei in Drosophila, that a single rRNA gene
copy is sufficient to organize a (mini-) nucleolus (Karpen
et al., 1988). However, it is not known how the nucleolus is
localized to certain locations within the nucleus, that is, to
the nuclear periphery in the case of S. cerevisiae and to the
interior, presumably, by virtue of the overall organization
of the nuclear matrix in higher eukaryotes. It is not known
whether rDNA or a nucleolar protein(s) or the transcribed
rRNA plays the primary role in determining the localization of the nucleolus. However, it is clear that the nucleolus can function normally in yeast without its normal connection to the nuclear envelope (de Beus et al., 1994
).
We have previously studied the nucleolar structures of a
yeast mutant in which the gene (RPA135) for the second
largest subunit of Pol I is deleted and rRNA is synthesized
by RNA polymerase II (Pol II) from a hybrid gene consisting of the 35S rRNA coding region fused to the GAL7
promoter ("GAL7-35S rDNA") on a plasmid. Using IFM
and antibodies against known nucleolar proteins, we found that the intact crescent-shaped nucleolar structure is
absent in this mutant; instead several granules (termed
mininucleolar bodies) that stained with these antibodies
were seen in the nucleus (Oakes et al., 1993). Since the
rDNA template transcribed by Pol II is carried by a plasmid, the possibility has not been excluded that the absence
of the intact nucleolar structure in this particular case is
due to the use of the plasmid template rather than the chromosomal rDNA repeats. Nevertheless, these observations combined with other observations on different (temperature-sensitive) Pol I mutants, which do not carry
rDNA plasmids, have suggested that Pol I is important in
the maintenance of the intact nucleolar structure as a
structural element in addition to its functional role to produce rRNA transcripts (Oakes et al., 1993
).
Significant progress has been made recently in identifying molecular components of the nucleolus and characterizing their roles in relation to nucleolar functions. With respect to the initiation of rDNA transcription in the yeast S.
cerevisiae, at least four transcription factors have been
identified in addition to Pol I: upstream activation factor
(UAF; Keys et al., 1996), core factor (CF; Keys et al., 1994
;
Lalo et al., 1996
; Lin et al., 1996
), Rrn3p (Yamamoto et al.,
1996
), and TATA box-binding protein (TBP; Cormach
and Struhl, 1992; Schultz et al., 1992
; Steffan et al., 1996
).
UAF and CF are multiprotein complexes. The former
contains Rrn5p, Rrn9p, and Rrn10p encoded by RRN5, RRN9, and RRN10, respectively (Keys et al., 1996
), and
probably three additional proteins that include histones
H3 and H4 (Keener et al., 1997
). The CF consists of three
proteins, Rrn6p, Rrn7p, and Rrn11p, encoded by RRN6,
RRN7, and RRN11, respectively. It has been demonstrated that UAF interacts directly with the upstream element of the promoter and functions, together with TBP, to
recruit CF, which in turn recruits Pol I with the aid of
Rrn3p (Keys et al., 1996
; Steffan et al., 1996
). Thus, if Pol I
plays a role in organizing (and localizing) the nucleolus by
its interaction with rDNA, the transcription factors which
mediate this interaction might also participate in this role.
Regarding the localization of the nucleolus within the
nucleus, it is possible that the tandemly repeated structure
of rDNA on the chromosome, including perhaps its adjacent chromosomal DNA, might be the primary factor; for
example, interactions of rDNA with the nuclear envelope
or nearby structures (in yeast) or with the nucleolar skeleton (in higher eukaryotes) might determine the localization of the nucleolus. Alternatively, or in addition, some
nucleolar proteins, such as Pol I as suggested from previous work (Oakes et al., 1993), might play an important
role in the maintenance of the nucleolar structure and localization. To study this question, we used yeast mutants
in which the chromosomal rDNA repeats were deleted
mostly (Chernoff et al., 1994
) or completely (Wai, H., L. Vu, and M. Nomura, unpublished experiments). Such mutant strains are able to grow by transcribing rDNA carried
on a plasmid. Two kinds of plasmids were used to examine
the significance of Pol I in localization of the nucleolus:
one carries a single native rDNA repeat that contains the
35S rRNA coding region with the intact rDNA promoter
and the 5S rRNA gene ("Pol I rDNA plasmid"); the other
carries the GAL7-35S rDNA fusion gene and the 5S
rRNA gene ("Pol II rDNA plasmid"). It should be noted
that Pol I and related factors are all maintained functionally intact in both systems. Yet rRNA is synthesized exclusively by Pol II in the second system, since there is no
rDNA carrying the native Pol I promoter in the strains
with the Pol II rDNA plasmids. With the strains carrying
the Pol I rDNA plasmid, which is transcribed by the Pol I
transcriptional machinery, the plasmid template and nucleolar proteins were detected mostly along the nuclear
envelope. That is, the nucleolus (mininucleoli in this case)
is predominantly localized to the nuclear periphery, as in
the case of the wild-type yeast cells. In contrast, in the
strains growing by virtue of the Pol II rDNA plasmid, a
round nucleolus with a limited contact with the nuclear envelope was observed. These results as well as other results
obtained for some other yeast mutants are presented in
this paper, and we discuss factors responsible for the organization and localization of the nucleolus in this organism.
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Materials and Methods |
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Materials
The rabbit anti-A190 antibody used in this work was described previously
(Wittekind et al., 1990). Antibodies against Ssb1p were provided by J. Broach (Princeton University, Princeton, NJ). The goat anti-rabbit IgG-
fluorescein conjugate (FITC), goat anti-mouse IgG-rhodamine conjugate
(TRITC), and horse serum were purchased from Sigma Chemical Co. (St.
Louis, MO). All other chemical reagents were from Fisher Scientific
(Fairlawn, NJ), or J.T. Baker Chemical Co. (Phillipsburg, NJ). BioNick labeling system was purchased from GIBCO BRL (Gaithersburg, MD). Biotinylated anti-avidin D and fluorescein avidin DCS were purchased from
Vector Laboratories (Burlingame, CA).
Media, Strains, and Plasmids
YEPD medium contains 1% yeast extract, 2% bacto peptone (Difco Laboratories, Inc., Detroit, MI) and 2% D-glucose. YEP-galactose medium is
the same, except that 2% D-galactose is substituted for D-glucose. Synthetic glucose (SGlu) medium (2% D-glucose, 0.67% yeast nitrogen base)
(Difco Laboratories, Inc.) was supplemented with L-tryptophan and required bases as described by Sherman et al. (1986). Synthetic galactose
medium (SGal) is the same as SGlu but 2% D-galactose is substituted for
glucose. For making solid medium, 2% agar was added.
The yeast strains and plasmids used in this study are described in Table
I (see also Table II). All genetic and cloning techniques were standard
procedures (Sherman et al., 1986; Guthrie and Fink, 1991
). NOY758 was
constructed based on the method of Chernoff et al. (1994)
in the following
way. Control strain NOY505 was first transformed with pRDN-hyg1 using
URA3 for selection. Plasmid pRDN-hyg1 is a 2µ plasmid carrying, in addition to URA3, an rDNA locus (RDN) with a recessive hygromycin-resistant mutation in the 18S rRNA coding region (Chernoff et al., 1994
), and
was a gift from Drs. Y.O. Chernoff and S.W. Liebman (University of Illinois, Chicago, IL). The transformants were directly plated on YEP-galactose medium containing 300 µg/ml hygromycin (Calbiochem-Novabiochem, La Jolla, CA). Several hygromycin-resistant mutants were isolated, grown in the absence of hygromycin repeatedly, and then tested for their
hygromycin resistance. One of the stably hygromycin-resistant mutants
was kept as NOY758, and deletion of most of the chromosomal rDNA repeats was confirmed by Southern analysis. The number of residual rDNA
copies was estimated to be ~5% or less relative to those of the control
strain (NOY505). It is expected, as was observed, that one or a few residual copies must be present in this strain, since deletion of the chromosomal rDNA copies (hygromycin-sensitive allele) is based on unequal homologous recombination between rDNA repeats.
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NOY759 was constructed by transforming NOY758 with pNOY353
and growing on SGal plates containing 5-fluoroorotic acid (5-FOA) (1 mg/
ml) to select cells that had lost pRDN-hyg1. NOY777 was constructed
from NOY759 by disrupting RPA12 with LEU2 as was done previously
(Nogi et al., 1993) followed by introduction of pRDN-hyg1 by transformation and subsequent screening for loss of pNOY353 after growth in the
presence of L-tryptophan. NOY770 was constructed from NOY758 by a
standard gene replacement method, which used the known sequences
flanking the chromosomal rDNA repeats and replaced any rDNA repeats
remaining in NOY758 by HIS3. (This replacement has a deletion of 297 bp of non-rDNA at the centromere-proximal side of the rDNA repeats.
Non-rDNA at the telomere-proximal side of the rDNA repeat has four
3,652-bp repeats, each of which contains ASP3, an uncharacterized open
reading frame and a single copy 5S rRNA gene, immediately adjacent to
the rDNA repeats. The replacement procedure has deleted the first repeat and 2,908 bp of the second repeat. Details of the method and characterization of the strain will be reported elsewhere.) NOY773 was constructed by transforming NOY770 with pNOY353 and plating on SGal
plates containing 5-FOA to select cells that had lost pRDN-hyg1.
NOY780 was constructed by disrupting RPA12 with LEU2 in NOY773,
transforming with pRDN-hyg1 and then screening for loss of pNOY353
after growth in the presence of L-tryptophan. Construction of NOY408-1a
has been previously described (Nogi et al., 1991
). Strain YJV100 (Venema
et al., 1995
) was a gift from Dr. J. Venema (Vrjie University, Amsterdam,
The Netherlands). This strain is a derivative of NOY408-1a and carries the fusion gene, GAL7-35S rDNA, integrated into chromosomal rDNA repeats by a high copy integrative system. The copy number of the integrated fusion gene (and other vector genes) was estimated to be 20-25 and
was expected to be tandemly arrayed from the mode of integration and
amplification. Approximately equal numbers of the rDNA copies were
also shown to be present (Venema et al., 1995
; see also Tables I and II.
pNOY353 carries the 7,547-bp BamHI-XhoI fragment, which contains
GAL7-35S rDNA (the GAL7 promoter fused to the 35S rRNA coding region) inserted between BamHI and SalI sites of pTV3, a TRP1, ARS, 2µ
plasmid vector (Rose and Broach, 1991). This plasmid also contains the
1,085-bp PvuII-EcoRV fragment carrying the 5S rRNA gene inserted in
the SmaI site upstream of the GAL7 promoter. Plasmid pNOY102 has
been described previously (Nogi et al., 1991
).
IFM and Fluorescence In Situ Hybridization (FISH)
Yeast strains were grown in YEP-galactose liquid medium at 25°C to an
A600 of between 0.1 and 0.3. Cells were fixed in 3.7% formaldehyde and
processed for indirect immunofluorescence microscopy as described previously (Oakes et al., 1993). Cells were then stained with a 1:500 dilution
of rabbit IgG solution containing anti-yeast Pol I A190 subunit and a
1:1,000 dilution of mouse YN2Cl serum containing anti-Ssb1p. The anti-A190 staining was revealed by a 1:2,000 dilution of goat anti-rabbit IgG-
FITC conjugate. The anti-Ssb1p staining was revealed by a 1:2,000 dilution of goat anti-mouse-TRITC conjugate. DNA was stained with DAPI
(4'6-diamidino-2-phenylindole). The protocol used for FISH was as described (Guacci et al., 1994
; Castano et al., 1996
). Plasmids pRDN-hyg1
and pNOY353 were used as probes to detect rDNA. The DNA preparations were digested with restriction enzymes followed by biotinylation using the BioNick labeling system. Hybridized probes were detected by
successive incubations in FITC-avidin (5 µg/ml), biotinylated anti-avidin (5 µg/ml), and finally FITC-avidin (5 µg/ml). IFM and FISH were performed with a Zeiss Axioskop or Axioplan (Carl Zeiss Inc., Oberkochen,
Germany) equipped with a SenSys camera (Photometrics, Tucson, AZ),
using filters for fluorescein, rhodamine, and UV detection. Pictures were
taken digitally or with Kodak T-Max ASA 400 black and white film. Black
and white negatives were scanned into Photoshop (Adobe System Corp.,
Mountain View, CA) using a slide scanner (Polaroid Sprintscan 35; Polaroid, Pennfield, NY). Digital images were pseudocolored and superimposed.
Electron Microscopy
Starting with a fresh patch of cells, yeast strains were grown in YEPD or
YEP-galactose medium to an A600 value of ~0.5 and embedded in Spurr's
epoxy resin as described (Byers and Goetsch, 1991) with the following
modifications. After fixation, cells were incubated in pretreatment solution (Byers and Goetsch, 1991
) for 15 min at ~25°C. Removal of cell walls
was done with 0.5 mg of Zymolyase 100T (ICN Biomedicals, Costa Mesa,
CA) per A600 unit of cells for 1-2 h at ~25°C. Sections were post-stained
with 1% uranyl acetate and lead citrate using standard methods. Photomicrographs were taken on a JEOL 100CX electron microscope.
Morphometric analyses was performed using a digital planimeter. EM negatives were selected solely on the basis of exhibiting sufficient contrast to visualize the nucleolus and nuclear envelope. After 5-10-fold enlargement, prints were marked as follows: the perimeter of the nucleolus in contact with the nuclear envelope with red, and the perimeter not in contact with blue. The linear distance of contact with the nuclear envelope (length of red trace) was divided by the area of the nucleolus (determined from red plus blue traces) to arrive at the "nucleolus-nuclear envelope contact ratio." Data were collected on a per nucleus basis. In cells that contained nucleolar granules not associated with the nucleolus, the data for areas not connected and connected were summed, so that for each nucleus the total linear distance of contact was divided by the total nucleolar area. A total of 119 nuclei from four strains were analyzed (see Fig. 4).
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Results |
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IFM and FISH Analyses of Nucleolar Structures in Yeast Mutants in Which the Chromosomal rDNA Repeats Are Deleted
We initially constructed yeast mutants ("rdn") in which
most of the chromosomal rDNA repeats were deleted according to the method described by Chernoff et al. (1994;
see Materials and Methods). Two such rdn
mutant
strains were constructed (see Table II): one strain (NOY758) carries a single native rDNA copy on a plasmid (pRDN-hyg1); another strain (NOY759) carries a 35S
rRNA coding region fused to the GAL7 promoter ("GAL7-
35S rDNA") together with the native 5S rRNA gene on a
plasmid (pNOY353). Nucleolar structures in these two
strains were studied by IFM using antibodies against the
largest A190 subunit of Pol I and those against the nucleolar protein Ssb1p (Clark et al., 1990
), and compared with the nucleolar structure of the parent strain (NOY505)
without an rdn deletion. We observed that in NOY758,
which carries the Pol I rDNA plasmid, both Pol I and
Ssb1p were detected as several fluorescent foci mostly
along the nuclear envelope. In contrast, in NOY759, which
carries the Pol II rDNA plasmid, Ssb1p was seen as a single and occasionally two (but rarely more) fluorescent foci
that were present with minimal contact with the nuclear
envelope (data not shown; see Fig. 1 and below). (It
should be noted that the Pol II plasmid used here
[pNOY353] carries the 5S rRNA gene in addition to the
GAL7-35S rDNA fusion gene to complement the chromosomal rdn deletion, and is different from plasmid
pNOY102. The latter plasmid, which carries the GAL7-35S rDNA gene but not the 5S rRNA gene, was used to allow the growth of strains that are defective for Pol I [Nogi
et al., 1991
; Oakes et al., 1993
; see later sections].)
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We carried out similar analyses using a corresponding
pair in which the chromosomal rDNA repeats are completely deleted by the use of a standard gene replacement
technique starting from a rdn strain used in the above
experiments. These complete deletion strains ("rdn
"
strains) use the same plasmid systems (Table II): one
strain (NOY770) carries the Pol I rDNA plasmid (pRDN-hyg1) and the other strain (NOY773) carries the Pol II
rDNA plasmid (pNOY353). Fig. 1 shows the results of
IFM analysis to localize Pol I and the nucleolar protein
Ssb1p in the rdn
strains and the control strain
(NOY505) without the rDNA deletion. It is to be noted
that all the strains were grown in the galactose medium (to
allow for the growth of the strains carrying the Pol II
rDNA plasmid) and at 25°C (to allow the growth of temperature-sensitive mutants, described below). The control
strain (NOY505) showed localization of both A190 and
Ssb1p at the nuclear periphery in the form of a typical
crescent-shaped nucleolar structure. NOY770, which uses
the Pol I rDNA plasmid, showed a punctate pattern often at the nuclear periphery for both A190 and Ssb1p, and the
two proteins appeared to be colocalized. In contrast,
NOY773, which uses the Pol II rDNA plasmid, showed a
single and occasionally two (but rarely more) foci for
Ssb1p. However, anti-A190 antibodies showed a weak
staining of most of the area of the nucleus and did not
colocalize with Ssb1p. (The Pol I localization in this strain
will be discussed further below.)
To examine localization of the plasmids carrying rDNA genes in these strains, FISH analysis was carried out using the corresponding plasmid DNAs as hybridization probes (see Materials and Methods). The results are shown in Fig. 2. Samples of the control strain (NOY505) showed staining of a crescent- or bar-shaped (or sometimes a dot-shaped) region that appeared to be at or near the nuclear periphery. NOY770, which uses the Pol I rDNA plasmid, showed punctate staining mostly at the nuclear periphery, as in the case of IFM analysis of A190 and Ssb1p. NOY773, which uses the Pol II rDNA plasmid, showed one or a few foci without extensive contact with the nuclear envelope, a pattern similar to that observed for Ssb1p by IFM.
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We have not analyzed localization of plasmid DNA (by FISH) and that of nucleolar proteins (by IFM) simultaneously for the same cells. Nevertheless, combining the results shown in Figs. 1 and 2, we conclude that in chromosomal rDNA deletion strain NOY770 (and NOY758), the Pol I rDNA plasmid, Pol I (and presumably other proteins required for transcription), and Ssb1p (and presumably other nucleolar components required for rRNA processing and modifications) are all colocalized mostly at the nuclear periphery, forming many mini-nucleoli. In contrast, in chromosomal rDNA deletion strain NOY773 (and NOY759), the Pol II rDNA plasmid is localized without extensive contact with the nuclear envelope. Ssb1p (and presumably other nucleolar components required for rRNA processing and modifications) is colocalized with this plasmid template, forming nucleoli that must also contain Pol II and other proteins required for transcription. The Pol I rDNA plasmid (in NOY770) and the Pol II rDNA plasmid (in NOY773) are both present at ~90 copies per cell (Wai, H., unpublished experiments). Thus, the results of both IFM and FISH analyses indicate that many mininucleoli coalesce, forming one or a few nucleoli per cell in NOY773.
It should be noted that in strain NOY773 Pol I is present in the nucleus, but it does not localize to the "Pol II nucleolus," nor does it localize to the nuclear periphery. We measured the cellular amount of A135, the second largest subunit of Pol I, in this strain by SDS-PAGE followed by immunoblot analysis. The amount found was comparable to that in the control wild-type strain (data not shown). In addition, extracts prepared from this strain had specific Pol I transcription activity, indicating the presence of an assembled Pol I in cell extracts (data not shown). Thus, the predominant localization of Pol I to the nuclear periphery appears to require the presence of the intact rDNA gene on the Pol I plasmid.
EM Analysis of Nucleolar Structures in Yeast Mutants in Which the Chromosomal rDNA Repeats Are Deleted
Nucleolar structures of the rdn and rdn
strains described above were also studied by EM analysis of thin
sections of these yeast cells (Fig. 3). Compared with the
electron-dense crescent structure adjacent to the nuclear
envelope in the control strain (NOY505; Fig. 3 a), the electron-dense structure corresponding to the nucleolus in the
rdn
strain carrying the Pol II rDNA plasmid (NOY759)
clearly has less contact with the nuclear envelope (Fig. 3
c). In addition, the nucleolus in this rdn
strain appears to be differentiated into two regions, one with greater electron density and the other with relatively less electron
density. The rdn
strain (NOY758) carrying the Pol I
rDNA plasmid showed a clearly different pattern. Here
many small electron-dense foci that do not coalesce into a
single nucleolar structure are seen, and some of them appear to be at or near the nuclear periphery (Fig. 3 b). The
electron density of the nucleolar materials in this strain appears to be relatively uniform, and unlike the rdn
strain
with the Pol II rDNA plasmid, no clear indication of two subregions with different electron densities was noted. We
do not know whether there is any correspondence between these two regions in the strain with the Pol II rDNA
plasmid (Fig. 3 c) and subnucleolar regions defined in the
nucleolus of higher eukaryotes, e.g., the fibrillar center
(FC), the dense fibrillar component (DFC), and the granular component (GC).
EM analyses of nucleolar structures were also carried
out using the rdn strains described above. The nucleolar structures and localizations are very similar to those
seen for the rdn
pair. NOY770, which carries the Pol I
rDNA plasmid, showed many small foci localized mostly
at or near the nuclear periphery (Fig. 3 e), as in the case
of the corresponding rdn
strain NOY758 (Fig. 3 b).
NOY773, which carries the Pol II rDNA plasmid, showed
a rounded single nucleolus that consisted of two subnucleolar regions (Fig. 3 f) as in the case of the corresponding
rdn
strain NOY759 (Fig. 3 c).
To establish the differences between the rdn strain
with the Pol I rDNA plasmid and the rdn
strain with the
Pol II rDNA plasmid with regard to the degree of contact
of the nucleolus with the nuclear envelope, we measured
nucleolus-nuclear envelope contact ratios as described in
Materials and Methods. The ratio of the linear distance of
contact of the nucleolus with the nuclear envelope to the
area of the nucleolus seen on electron micrographs is defined as nucleolus-nuclear envelope contact ratio. This ratio, presented in arbitrary units, is an accurate measure of
the localization of the nucleolus within the nucleus. The
results are shown in Fig. 4, a-c. A comparison of the two
rdn
strains reveals that NOY758 carrying the Pol I
rDNA plasmid (Fig. 4 b) has much higher contacts with
the nuclear envelope than NOY759 carrying the Pol II
rDNA plasmid (Fig. 4 c). The control strain (NOY505;
Fig. 4 a), which transcribes the chromosomal rDNA by Pol
I, also shows much higher contact ratio than the Pol II
plasmid strain (NOY759) and resembles the Pol I plasmid
strain (NOY758), though it is somewhat lower than this
latter strain.
After we completed the present work, Nierras et al.
(1997) published a paper in which they described IFM
analysis of a nucleolar protein, Nop1p, in a strain corresponding to our rdn
strain carrying the Pol I rDNA
plasmid and stated that Nop1p is spread throughout the
nucleus. However, inspection of the published picture suggests that Nop1p is perhaps localized predominantly at the
nuclear periphery forming punctate nucleolar structures in
at least some cells. In fact, electron microscopy of this
rdn
/Pol I rDNA plasmid strain (L-1521; Nierras et al.,
1997
) revealed a nucleolus that was not localized to a single electron-dense region and appeared as numerous smaller areas, many of which were associated with the nuclear periphery (Aris, J.P., unpublished results). Thus, the
nucleolar ultrastructure in this rdn
/Pol I rDNA plasmid
strain was indistinguishable from the rdn
/Pol I rDNA
plasmid strain NOY758 (see Fig. 3 b).
Effects of rpa12 Mutation on Nucleolar Localization
In both rdn and rdn
strains, the nucleolar localization
as well as nucleolar structure is different between the
strains carrying the Pol I rDNA plasmid (pRDN-hyg1),
and those carrying the Pol II rDNA plasmid (pNOY353),
as described above. There are two differences between
these systems. First, the machinery to transcribe rDNA is
different; the former using Pol I and Pol I-specific transcription factors, whereas the latter uses Pol II and Pol II-
related transcription factors. Second, the plasmid templates are different in the promoter region; the former
uses the native rDNA promoter and the latter uses the
GAL7 promoter, although both use the same rRNA-coding region, producing the same rRNA transcript. To determine whether the difference in polymerase is responsible
for the observed difference in nucleolar localization (and
structure), we introduced a rpa12
::LEU2 mutation in the RPA12 locus in rdn
(NOY758) and rdn
(NOY770)
strains carrying the Pol I rDNA plasmid, yielding NOY777
and NOY780, respectively (see Table II). It has been demonstrated previously that RPA12 encoding the A12 subunit of Pol I is not an essential gene, but the rpa12
::LEU2
mutation causes a temperature-sensitive phenotype (Nogi
et al., 1993
). We grew these two rpa12
strains at 25°C in
YEP-galactose medium for EM and IFM analyses, the
same growth condition used for all other strains.
Using IFM, we found that in strain NOY780, which has
the genotype rpa12::LEU2 rdn
and carries the Pol I
rDNA plasmid, Ssb1p is localized without extensive contact with the nuclear envelope, forming mostly a single
(and occasionally two) nucleolar structure(s) that resembles
that seen for the rdn
strain carrying the Pol II rDNA
plasmid (NOY773). No punctate pattern at the nuclear
periphery was observed for Ssb1p or Pol I (Fig. 1). As for
Pol I in this strain (NOY780), IFM using anti A190 antibodies showed an apparent colocalization with Ssb1p (Fig.
1), which would be expected from transcription of rDNA
by the mutant Pol I. However, the staining was weak and
quite a few cells did not show a clear signal above the
background. It was previously observed that in rpa12
strains growing at permissive temperatures, the cellular
concentration of A190 was lower than in the control
RPA12 strain, and that Pol I activity in extracts was also
much reduced (Nogi et al., 1993
). The Pol I rDNA plasmid
in this strain (NOY780) was also detected by FISH as one
or a few foci as was observed for the Pol II plasmid strain;
no punctate pattern at the nuclear periphery was observed (Fig. 2). The same results were also obtained for the strain
NOY777, which is rdn
rpa12
and carries the Pol I
rDNA plasmid. NOY777 showed a localization pattern
different from the isogenic RPA12 strain, NOY758, and
similar to that for the rdn
RPA12 strain carrying the Pol
II plasmid (NOY759) (data not shown). Thus, the Pol I
rDNA plasmid and Ssb1p are localized together, forming
one or more nucleoli without extensive contact with the
nuclear envelope.
The same conclusion on the effects of the rpa12 mutation on nucleolar localization was also obtained by analyzing the rdn
rpa12
strain carrying the Pol I plasmid
(NOY777) by EM, as shown in Figs. 3 and 4. Even though
this strain (NOY777) was grown at 25°C and transcribed
the Pol I rDNA plasmid using Pol I, a single rounded nucleolus was observed that had two subnucleolar regions with different electron density (Fig. 3 d). Often the nucleolus of this strain (NOY777) had minimal contact with the
nuclear envelope (Fig. 4 d). Thus, the nucleolus in this
strain resembles the nucleolus observed for the rdn
strain carrying the Pol II rDNA plasmid (NOY759; Fig. 3
c). Many mininucleoli that must have been formed as in
the case of the isogenic RPA12 strain (NOY758; Fig. 3 b), appear to have coalesced mostly into a single nucleolus
away from the nuclear periphery.
It should be noted that the two rpa12::LEU2 strains
grew more slowly than the corresponding control strains
under the growth condition used in these experiments. For
the rdn
strains, the decrease in growth rate caused
by the rpa12
mutation was ~20% (the doubling times
on YEP-galactose medium at 25°C for NOY770 and
NOY780 were 7.3 and 8.8 hours, respectively). For the
rdn
strains, the decrease was ~40% (the doubling times
on YEP-galactose medium at 25°C for NOY758 and
NOY777 were 5 and 7 h, respectively). However, the effect of the rpa12
mutation on growth rate does not account for the striking alteration in the structure and localization of the nucleolus. NOY777, which is rdn
and
carries rpa12
, grew at about the same growth rate as
NOY770, which is rdn
and RPA12, and yet the former
showed a nucleolar localization/structure very different
from the latter (and from its control RPA12 strain, NOY758) as described above.
These observations demonstrate that the predominant localization of the (mini-) nucleolus (and Pol I) to the nuclear periphery requires the presence of an intact Pol I. Deletion of the gene for the A12 subunit prevents the occurrence of many separate mininucleolar foci and their predominant localization to the nuclear periphery even under conditions in which Pol I is sufficiently functional to allow cells to grow at a rate close to that of the wild type.
Nucleolar Structures in a Strain Using Pol II to Transcribe rDNA Template in Chromosome XII
The results described in the previous sections demonstrated the essential role of the transcriptional machinery
in the localization and organization of the nucleolus. The
crescent-shaped nucleolus in normal yeast cells is obviously different from the mininucleoli seen in rdn or
rdn
strains carrying the Pol I rDNA plasmid. Mininucleoli are distributed through the nucleoplasm, although they are predominantly localized at the nuclear periphery.
Thus, the chromosomal rDNA repeats must exert an influence on nucleolar morphology that is absent in the Pol I or
Pol II rDNA plasmid systems. For example, clustering of
rRNA genes in a single locus is expected to prevent the
formation of many independent mininucleoli, and the
presence of DNA flanking the rDNA repeats might have a role in the nucleolar localization. To study this question,
we used strain YJV100, which is a derivative of NOY408-1a and carries the GAL7-35S rDNA fusion gene integrated into the chromosomal rDNA repeats (Venema et
al., 1995
). Like NOY408-1a (Nogi et al., 1991
), the strain
carries a deletion (rpa135
::LEU2) in the essential gene
encoding the A135 subunit of Pol I and can grow only in
galactose medium by transcribing the GAL7-35S rDNA
gene using Pol II. The copy number of this hybrid gene integrated into the chromosomal rDNA in this strain was
previously estimated to be 20-25 and about an equal number of the native rDNA repeats was also present (Venema et al., 1995
). As shown in Fig. 5 (bottom panel), the nucleolus revealed by IFM using anti-Ssb1p antibodies was a single dot localized at the nuclear periphery (see for example,
Fig. 5, arrows; see also the results of EM analysis to be described below). It is clearly different from the crescent
structure seen for normal yeast cells (Fig. 1, NOY505).
The structure is also different from that seen for NOY408-1a; in this strain several separate granules, previously
called mininucleolar bodies (Oakes et al., 1993
), were observed (Fig. 5, top panel). Both the original plasmid (pGRIM) integrated into the chromosomal rDNA repeats
in YJV100 and the plasmid (pNOY102) carried by
NOY408-1a contain the GAL7-35S rDNA for Pol II transcription, and their copy numbers are similar. Therefore,
the difference in the nucleolar structure between YJV100 and NOY408-1a must be due to the intranuclear state of
the plasmids. The integrated plasmid copies are probably
physically close together and mininucleoli formed from individual hybrid genes may have coalesced into a single nucleolar structure at the nuclear periphery. In contrast, the
non-integrated plasmid copies in NOY408-1a may not
have such topological restrictions and may be able to form several separate mininucleolar bodies away from the nuclear periphery.
|
The difference between pNOY408-1a and YJV100 was
also clearly demonstrated by EM analysis of these strains.
As was seen in previous work (Oakes et al., 1993), one or a
few mininucleolar bodies were seen mostly away from the
nuclear periphery in thin sections of NOY408-1a cells (Fig.
6 a). The rounded nucleolar bodies contained two ultrastructurally distinct subnucleolar regions, similar to the
Pol II rDNA plasmid systems, and lacked extensive contact with the nuclear envelope (Fig. 6 a). In YJV100 cells, the nucleolus was a single rounded body similar to that
seen for the Pol II rDNA plasmid systems, showing in
many instances a segregation of a very electron-dense area
and a less electron-dense area (Fig. 6 b), and was clearly
different from the normal crescent-shaped nucleolus (Fig.
3 a). However, in YJV100 cells, there is a bulge or outpocketing of the nuclear envelope in the area of the nucleolus (Fig. 6 b). This feature could also be seen often in
IFM analysis of YJV100 cells (see Fig. 5, arrows). It appears that although the polymerase system clearly plays
an important role, the tandemly repeated chromosomal
rDNA structure has, presumably through its connection to
flanking chromosomal DNA regions, an influence on association of the nucleolus with the nuclear envelope.
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Discussion |
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Pol I Transcription Machinery Is Important for Localization of the Nucleolus to the Nuclear Periphery
We have used yeast strains with a chromosomal rDNA deletion and carrying rDNA plasmids to study how the nucleolus is spatially organized within the nucleus. The various kinds of nucleolar morphology/localization observed in the present investigation are schematically shown in Fig. 7 and are summarized in Table II. Comparison of the two types of strains, one carrying the Pol I rDNA plasmid and the other carrying the Pol II rDNA plasmid, has demonstrated a clear difference in the nucleolar structures and localization. The former strains contained many mininucleoli distributed throughout the nucleus, predominantly localized at the nuclear periphery, while the latter strains contained a single (and possibly two but rarely more) rounded nucleolus without an extensive contact with the nuclear envelope. This difference in the degree of nucleolar coalescence and localization is not due to differences in plasmid copy numbers, since both types of strains carry a comparable number of plasmids (~90).
There are two main differences between the Pol I rDNA
plasmid and the Pol II rDNA plasmid systems. First, the
transcription machinery is different. Second, the cis elements, specifically the promoters, on rDNA plasmids are
different. However, the rRNA coding region and the
rRNA transcript are identical between the two systems.
Thus, models invoking some specific affinity between the
DNA encoding rRNA or the rRNA transcript and some
structures at the nuclear periphery cannot explain localization of the nucleus to the nuclear periphery. Furthermore,
the results obtained for the strains with the rpa12 mutation and carrying the Pol I plasmid (Fig. 7 c) indicate that
the presence of the intact rDNA with the intact cis elements alone is not sufficient for predominant localization
to the nuclear periphery. The difference in the nucleolar morphology/localization between NOY758 (rdn
, RPA12,
Pol I rDNA plasmid) and NOY777 (the same, but rpa12
)
is striking (Fig. 7, b and c). This demonstrates the importance of the intact structure of Pol I for nucleolar structure
and localization. One possibility to explain differences between these two strains is that mininucleoli formed on individual plasmid molecules have an inherent tendency to
coalesce, and that interactions between the intact Pol I and the nuclear periphery prevent coalescence of mininucleoli
into a single large nucleolar structure.
Pol I plays an essential role in organizing nucleolar
structure and localization. It is known that interaction of
Pol I with rDNA requires specific transcription factors
such as UAF and CF (see Introduction). Thus, it is reasonable to assume that these transcription factors also play an
important role in nucleolar organization and localization.
It should be noted that purified UAF contains histones H3
and H4 (Keener et al., 1997). UAF might be part of a special chromatin structure unique to the rDNA locus. Certain evidence suggests that rDNA chromatin can assume
two (or more) different structures that can be distinguished by their ability to silence Pol II activity in the
rDNA locus (Bryk et al., 1997
; Smith and Boeke, 1997
;
Fritze et al., 1997
). Our recent experiments showed that
these structures are interchangeable by epigenetic events;
the form able to silence Pol II activity is stabilized by
UAF, Pol I, and perhaps other Pol I-specific transcription factors (Vu, L., M. Oakes, J.P. Aris, and M. Nomura, unpublished experiments). Such chromatin structures may be
responsible for localization of the nucleolus to the nuclear
periphery.
Role of the Chromosomal Context of rDNA in the Localization of the Nucleolus
The localization of mininucleoli to the nuclear periphery
observed in rdn deletion strains carrying the Pol I rDNA
plasmid (Fig. 7 b) may reflect the localization of the crescent-shaped nucleolus adjacent to the nuclear envelope in
normal yeast strains (Fig. 7 a). The chromosomal rDNA is
clustered as a tandem repeat of 100-150 genes on chromosome XII (Petes, 1979). This clustering may limit contact
of the nucleolus to a part of the nuclear envelope, thus
forming the crescent structure, which is different from the nucleolar morphology (Fig. 7 b) seen for the Pol I plasmid
system. In addition, the results (Fig. 7 e) obtained with
strain YJV100 in which multi-copies of the GAL7-35S
rDNA fusion gene are integrated into the chromosomal
rDNA repeats, suggest that, in addition to the proposed
interaction between the Pol I-specific rDNA chromatin structure and some structures at the nuclear periphery,
there may be additional interactions perhaps between
DNA elements flanking rDNA repeats and structures at
the nuclear periphery.
Nucleolar Structures in Strains in Which Pol II Transcribes rDNA
In the rdn deletion strains carrying the Pol II rDNA plasmid, in which the GAL7-35S rDNA hybrid is transcribed
by Pol II, both the template plasmid (and Pol II engaged in
rRNA synthesis) and nucleolar protein Ssb1p are colocalized to regions that do not have extensive contact with the
nuclear periphery. Regarding the Pol II rDNA plasmid, it
is now generally accepted that mRNA transcription does
not take place uniformly in the nucleus, but at many specific places ("transcription foci" or "transcription factories") (Lawrence et al., 1989, 1993
; Jackson et al., 1993
; Spector et al., 1993
; Wansink et al., 1993
; Iborra et al.,
1996
; for review see Cook, 1994
). Perhaps, Pol II molecules present in these transcription factories may be responsible for binding and transcribing the Pol II rDNA
plasmid. For nucleolar proteins, many
including Ssb1p
are complexed with snoRNAs, forming small nucleolar
RNPs (snoRNPs), which interact with precursor rRNA
presumably through rRNA-snoRNA base pairing, as
demonstrated by recent studies (Kiss-László et al., 1996
;
Ganot et al., 1997
; Ni et al., 1997
; for review see Smith and
Steitz, 1997
) (Ssb1p is associated with Box H/ACA snoRNAs, snR10 and snR11, as described by Clark et al., 1990
). Thus, in the yeast cells that grow by synthesizing
rRNA by Pol II, nucleolar proteins (and snoRNA) engaged in rRNA modification, processing, and perhaps ribosome assembly, will be localized to Pol II transcription
factories synthesizing rRNA. However, Pol I would not be
expected to be colocalized with these nucleolar components, as observed in the present work. It is evident that some nucleolar proteins, such as those involved in specific
transcription, Pol I, UAF, and CF, play a primary role in
determining the nucleolar localization, whereas others,
such as those involved in rRNA processing, do not.
Possible Significance of the Nucleolar Localization to the Nuclear Periphery
What is the significance of the nucleolar localization to the
nuclear periphery? The synthesis of ribosomes, the major
nucleolar function, requires extensive nuclear-cytoplasmic
transport of macromolecules, such as the nuclear import of
many ribosomal proteins and the export of ribosomes. Localization of the nucleolus adjacent to the nuclear envelope, thus, might be advantageous for efficient nuclear-
cytoplasmic transport. However, the rpa12 deletion that prevented the nucleolar localization to the nuclear periphery in rdn deletion strains growing at permissive temperature, caused only a small decrease (20-40%) in growth
rate, as described in this paper. Similarly, it was observed
previously that overproduction of Nop2p, a nucleolar protein, causes the nucleolus to become detached from the
nuclear envelope without causing any decrease in growth
rate (de Beus et al., 1994). Thus, the significance of the nucleolar localization to the nuclear periphery is not clear at
the moment. Perhaps there are other important nucleolar functions that cannot be assessed by the simple measurement of growth rate. For example, recent studies have
shown a correlation between redistribution of certain silencing proteins from telomeres to the nucleolus and
lengthening of life span (Kennedy et al., 1997
). A correlation between structural alterations of the nucleolus and
aging of yeast cells has also been observed (Sinclair et al., 1997
). These observations suggest a role of the nucleolus
in the maintenance of normal aging (for review see
Guarente, 1997
). In addition, silencing of some Pol II
genes inserted into the chromosomal rDNA repeats and
its dependence on proteins such as Sir2p have been reported recently (Bryk et al., 1997
; Fritze et al., 1997
; Smith
and Boeke, 1997
; our unpublished work described above). Sir2 protein is also known to repress mitotic and meiotic
recombination between the tandem rDNA repeats within
the nucleolus (Gottlieb and Esposito, 1989
). It is possible
that the nucleolar localization at the nuclear periphery
might be important in such less well-explored (or other
unexplored) nucleolar functions. In this connection, it may
be noted that yeast telomeres have been observed to localize to certain regions of the nuclear periphery in clusters.
Silencing of Pol II genes near telomeres (for review see
Loo and Rine, 1995
) might be related to the nuclear location of telomeres (Klein et al., 1992
; Palladino et al., 1993
;
Pillus and Grunstein, 1995
). With several yeast mutant
strains with different nucleolar localizations as characterized in this work, it should now be possible to study the
question of nucleolar localization in connection with nucleolar events such as those related to silencing and cell
aging.
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Footnotes |
---|
Received for publication 29 January 1998 and in revised form 11 June 1998.
Address all correspondence to Masayasu Nomura, University of California, Irvine, Department of Biological Chemistry, Irvine, CA 92697-1700. Tel.: (949) 824-4564. Fax: (949) 824-3201. E-mail: mnomura{at}uci.edu
We thank Drs. S. Liebman, J.R. Warner, and J. Venema for providing plasmid pRDN-hyg1, strains L1521 and YJV100, respectively; and Drs. S.M. Arfin and T. Pederson for critical reading of the manuscript; and D. Semanko for help in preparation of the manuscript.
This work was supported by U.S. Public Health Grants GM35949 (M. Nomura) and GM48586 (J.P. Aris) from the National Institutes of Health.
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Abbreviations used in this paper |
---|
CF, core factor; FISH, fluorescence in situ hybridization; IFM, immunofluorescence microscopy; Pol I and Pol II, polymerase I and II; rDNA, ribosomal DNA; snoRNA, small nucleolar RNA; UAF, upstream activation factor.
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