COMMENTARY
The nucleolus today
DANIELE HERNANDEZ-VERDUN
Institut Jacques Monod, 2 place Jussieu, 75251 Paris Cedex 05, France
Introduction
The nucleolus, the most prominent feature of the interphase nucleus, is involved in ribosome biogenesis (Warner,
1990). During evolution, the nucleolus first appeared at
the time when nuclear envelope compartmentation of the
cells was taking place. I would like in this review to
provide information showing that the nucleolus is a
particular nuclear territory in respect of the compartmentation of nuclear functions.
In eukaryotic cells, the nucleolus is the site at which
ribosomal gene transcription takes place and the machinery necessary for the production of the ribosomal subunits
is assembled (Hadjiolov, 1985). At this site, the 18 S, 28 S
and 5.8 S RNAs are synthesized and assembled with
proteins such as ribosomal protein SI (Htigle et al. 1985a)
and 5 S RNA.
The nucleolus is not a stable organelle. Its structure,
size and organization depend on ribosome biogenesis. The
different steps in this biogenesis correspond to nucleolar
domains that can be identified by their morphology. There
are three basic nucleolar domains, the fibrillar centers
(FCs), the dense fibrillar component (DFC) and the
granular component (GC) (see nucleolar nomenclature
reviewed by Jordan, 1984). They are found in all except a
very few nucleoli. Traditionally, the FCs are considered to
be the storage sites of non-transcribed ribosomal genes,
the DFC is the site of transcription of these genes and the
GC is the site of maturation and storage of the ribosomal
subunits (Goessens, 1984; Hernandez-Verdun, 1986; Sommerville, 1986). However, the actual sites of transcription
are still extremely controversial. Some authors believe
they are located in the FCs (Scheer and Benavente, 1990);
others at the border between the FCs and DFC (Derenzini
et al. 1990; Thiry et al. 1991); and others again in the DFC
only (Hartung et al. 1990; Wachtler et al. 1989). At present,
if we take into account all the results obtained so far, the
most reasonable interpretation is to propose that the
ribosomal transcription units are compacted in the DFC,
as recently concluded by Jordan in an extensive review
(Jordan, 1991). The main results that argue for the
localization of this transcription in the FCs are based on
the labelling of the FCs by anti-polymerase I antibodies
(Scheer and Benavente, 1990). However, the presence of
RNA polymerase I in the FCs does not prove that they
constitute the site of RNA transcription. The polymerases
detected can only be the free form, and the engaged form
cannot be accessible during transcription. As there is an
equilibrium between these two forms of RNA polymerase I
(Marilley and Gassend-Bonnet, 1989; Sentenac, 1985),
Journal of Cell Science 99, 465-471 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
antibodies that reduce the amount of free polymerase
should also stop transcription.
In addition to the studies devoted to the interpretation of
nucleolar structures, others have attempted to draw up an
inventory of the nucleolar proteins, in order to investigate
nucleolar complexity and to understand nucleolar functions. These investigations recently attracted much attention (Sollner-Webb and Mougey, 1991; Warner, 1990) and
in this commentary, I would like to focus particularly on
the information available to date about nucleolar polarity,
nucleolar-specific proteins, nucleolar cell cycle, nucleolar
targeting and small nucleolar RNAs.
Nucleolar polarity
The finding that chromosomes occupy a specific territory
in the nuclear volume, defined as the chromosome domain
(Hilliker and Appels, 1989), was recently demonstrated.
This demonstration was done by chromosome 'painting1,
using specific probes corresponding to a single chromosome, and in situ hybridization to locate specific chromosomes during interphase (Pinkel et al. 1989). It is therefore
not surprising that ribosomal gene distribution follows the
general rule of higher-order nuclear structure. However,
the nucleolus organizer regions (NORs) are greatly
involved in nuclear polarity (Manuelidis and Borden,
1988). Besides constituting a traditional cytological landmark, there is also evidence for polarity between different
NORs, and between NORs and the nuclear envelope. To
underline the importance of nucleolar polarity, only these
two points will be discussed. Nevertheless, there is also
evidence for a specific arrangement of the centromeres in
Sertoli cells that reflects the activation or inactivation of
the ribosomal genes (Haaf et al. 1990) as well as a specific
association between the nucleolus and the centromere of
chromosomes 1 and 9 in neurons (Manuelidis and Borden,
1988).
In the metaphasic plate, the NOR-bearing chromosomes
are closer to each other than would be expected from a
random chromosomal distribution. This observation indicates that there is certainly a polarized arrangement of the
NOR-bearing chromosomes in the interphasic nuclei.
Clearly, the NOR association seems to reveal the nucleolar
fusion that takes place in species that generally possess
several pairs of NORs. After this fusion, or in species
without nucleolar fusion, the positions of the nucleoli
Key words: nucleolus, interphase, nucleus.
465
remain fairly stable, even in rotating nuclei (Bard et al.
1985). The effect of the respective positions of the different
nucleoli might explain the nucleolar dominance in
competition for activation molecules, as several authors
have recently proposed (Appels, 1989; Hilliker and Appels,
1989). As revealed by in situ hybridization of rDNA
sequences, certain ribosomal sites are not associated with
a nucleolus (Manuelidis, 1985; Wachtler et al. 1986). It is
therefore likely that some non-transcribed rDNA regions
are located separately or segregated from the rest of the
NORs. Thus, in non-stimulated lymphocytes, the only
remaining nucleolar structure possesses a nuclear marker
of transcriptional activity (see below), although in a small
amount, but the other NOR sites lack the special proteins
associated with transcription (Manuelidis, 1985; Wachtler
et al. 1986). This specific distribution implies precise
targeting of the proteins associated with the NORs that
are or have been expressed.
Very frequently, in higher eukaryotic cells, the nucleoli
are located at or near the nuclear envelope (reviewed by
Bourgeois and Hubert, 1988), or in yeast nuclei they are
juxtaposed to the inner nuclear membrane (reviewed by
Clark et al. 1990). This position seems related to the
presence of specific skeletal structures at the site of the
nucleolar attachment to the envelope. This nucleolar
skeleton either adheres directly to the nuclear lamina or is
attached to it by a pedicle as visualized in spread lamina
preparations (Bureau et al. 1986). At present, little
information is available about the characterization of the
nucleolar skeleton, especially its specificity compared to
that of the nuclear matrix (reviewed by Bouteille et al.
1983) and its action on the structure of the nucleolar
domain. A filamentous complex enriched in a protein of
145 x 103 Mr, was identified as a specific nucleolar skeleton
in amplified Xenopus laevis nucleoli (Franke et al. 1981).
Moreover, the 180 x 103 MT nucleolar protein that contributes to the general structure of the DFC (SchmidtZachmann et al. 1984) might be one of the nucleolar
skeletal proteins playing a role in nucleolar architecture.
In nuclei with centrally located nucleoli, there is a
folding of the nuclear envelope called the nucleolar canal,
which is in direct contact with the nucleoli (Bourgeois et
al. 1982). This canal was shown to be a nuclear envelope
specialization that depends on the presence of active NORs
(G^raud et al. 1989). We observed PtKl micronucleated
cells in which the chromosomes are segregated in different
micronuclei. The cells were serially sectioned and threedimensional reconstitutions of electron micrographs were
made. The micronuclei possessing ribosomal genes but not
the other micronuclei have a nucleolar canal. Therefore,
the nucleolar canal is controlled by the NOR-bearing
chromosomes, since each of these micronuclei only
contains one chromosome (Ge'raud et al. 1989).
It is possible that the nuclear canal, and the close
relationship between the nucleoli and the nuclear envelope, constitute nuclear envelope specializations that favor
nuclear—cytoplasmic exchanges.
Nucleolar-specific proteins
A nucleolar-specific protein can be defined as a protein
that is specifically located in nucleoli and is involved in
ribosomal biogenesis. A hypothesis was recently formulated that such proteins may reside or be engaged in a
shuttle process between the nucleoli and the cytoplasm
(Borer et al. 1989). The nucleolar-specific proteins do not
466
D. Hernandez-Verdun
end up in the mature ribosome and their role in ribosomal
biogenesis is only conjectural for the great majority. They
have been suggested as having roles in the transcription,
maturation, packaging and transport of ribosomal particles (Reeder, 1990).
The number of proteins located in the nucleoli is very
large. Twenty years ago, 97 different nucleolar protein
spots had already been detected by two-dimensional gel
electrophoresis (Orrick et al. 1973). As this detection was
limited to the major nucleolar proteins below 120 x 103 MT,
we may assume that several hundred proteins are
specifically confined within the nucleolar territory. The
notion of nucleolar territory does not exclude the presence
of nuclear proteins, which have also been found to be
associated with other genes, and can accumulate or be
located in the nucleoli. The best example of accumulation
that is not in itself specific for ribosomal gene function is
that of DNA topoisomerase I, which is found at the site of
ribosomal transcription in the nucleoli. Such accumulation was to be expected in view of the fact that the
nucleoli contain the highest concentration of highly active
genes in the nucleus (Reeder, 1990). There are also
numerous proteins that in the nuclei are targeted to the
nucleoli (see below under Nucleolar targeting) and are not
present in other nuclear territories.
RNA polymerase I
This is localized in the nucleoli and is specific for ribosomal
gene transcription of large ribosomal RNAs. This polymerase is a large molecule with complex subunit structures
composed of at least six polypeptides (Sentenac, 1985). Its
two large components, which are highly conserved
(Rowland and Glass, 1990), are involved in the basic
polymerization reaction, whereas the small subunita seem
to have accessory roles. It has been proposed that one of
these roles could be nuclear localization or enzyme
assembly (Rowland and Glass, 1990). In practice, the
active RNA polymerase I is required for the formation of
the nucleolus as its major component, as demonstrated in
yeast (Hirano et al. 1989). Moreover, re-formation of the
active nucleoli was inhibited by injection of antibodies
directed against RNA polymerase I in mitotic cells
(Benavente et al. 1987). However, RNA polymerase I
activity is dependent on the presence of nucleolar
transcription factors such as UBF and SL1 (Jantzen et al.
1990) and on a growth-dependent transcription initiation
factor (TIF-IA) (Schnapp et al. 1990). So far, nothing is
known about the kinetics of the association of these factors
with rDNA and the initial targeting of the DNA binding
proteins. RNA polymerase I is still present in the NORs
during mitosis but there is no transcription at this stage.
We therefore propose as a working hypothesis that the
starting of the rDNA transcription taking place at the
beginning of telophase is induced either by other exogenous proteins or by modifications of the proteins already in
place.
Nucleolin
Nucleolin, also called C23 or 100k, is a phosphorylated
protein present in large amounts in nucleoli with active
ribosomal biogenesis (Caizergues-Ferrer et al. 1987;
Lapeyre et al. 1987). This protein exists in all eukaryotes
(Srivastava et al. 1990) and its molecular weight varies
between 92 and 105xl0 3 M r , depending on the species.
Nucleolin comprises three distinct domains: an acidic
amino-terminal region, four RNA-binding domains and a
glycine-rich carboxyl terminus (Caizergues-Ferrer et al.
1989). It is thought to participate in the early processes of
ribosome biogenesis, such as the regulation of the
transcription by RNA polymerase I, the modulation of the
chromatin conformation in the nucleolus (Erard et al.
1988), and binding to nascent rRNA (Caizergues-Ferrer et
al. 1989; Srivastava et al. 1989), but its function is not
clear. In the nucleoli, nucleolin is found in the DFC and
GC (Biggiogera et al. 1989; Escande-Ge>aud et al. 1985;
Noaillac-Depeyre et al. 1989). It was recently shown to
shuttle between the nucleoli and the cytoplasm (Borer et
al. 1989). Therefore, this protein seems able to follow the
chain of reactions ensuring ribosome biogenesis, but is not
part of the final product.
Nucleolar protein B23
B23 (Mr, 37X103; pi, 5.1) (Michalik etal. 1981), also called
numatrin (Zhang et al. 1989) or No38 (Schmidt-Zachmann
et al. 1987), is a major RNA-associated phosphoprotein
that is considered to be one of the factors responsible for
preribosomal particle assembly (Schmidt-Zachmann et al.
1987). This protein, identified as a member of the
nucleoplasmin family (Schmidt-Zachmann et al. 1987), has
been found to be widely distributed in higher eukaryotes
with the same apparent molecular weight of
37-38xlO3Afr. It binds cooperatively, with high affinity
for single-stranded nucleic acids, and exhibits RNA helixdestabilizing activity. These features may be related to its
role in ribosome assembly. Protein B23 is mostly located in
the GC (Schmidt-Zachmann et al. 1987) and is associated
with the most mature nucleolar preribosomal RNP
(Dumbar et al. 1989). It was found to migrate out of the
nucleoli when RNA synthesis decreased during serum
starvation (Chan et al. 1985). This protein, which appears
to be involved in the later stages of ribosome assembly,
also seems, like nucleolin, to shuttle between the nucleoli
and the cytoplasm (Borer et al. 1989). The protein
originally described as ribocharin (Hiigle et al. 1985)
turned out to be an isoelectric variant of B23 as mentioned
in the review by Warner (1990). Ribocharin was described
as a protein involved in the transport of large ribosomal
subunits (Hiigle et al. 19856). Therefore, protein B23
appears to be a shuttle protein playing a role in transport
from the nucleolus to the cytoplasm.
Fibrillarin
Fibrillarin was first described in Physarum polycephalum;
it is a nucleolar protein located in the DFC (Ochs et al.
1985). It is a basic protein (pi 8.5) with a molecular weight
between 34 and 36xlO 3 M r . Autoantibodies against fibrillarin have been found in human patients with scleroderma
and can also be induced in mice by mercuric chloride
treatment (Reuter et al. 1989). Fibrillarin has been
conserved from yeast (designated NOP1) (Henriquez et al.
1990; Schimmang et al. 1989) to the human nucleolus and
appears essential for cell growth, since the cells are not
viable in the absence of the corresponding genes (Schimmang et al. 1989). It became one of the 'favourite' nucleolar
proteins when it was demonstrated to be associated with
U3 small nucleolar RNA (snRNA) (Lapeyre et al. 1990;
Lischwe et al. 1985) and subsequently with U8 and U13
snRNAs (Tyc and Steitz, 1989; for review, see Tollervey
and Hurt, 1990).
The Ag-NOR proteins
These are a set of proteins specifically located in the NORs
during mitosis, and have been identified by their ability to
reduce silver under acidic conditions in which most other
cellular proteins remain unstained (Goodpasture and
Bloom, 1975). These proteins are also found during
interphase in the nucleoli and their presence is necessary
for ribosomal gene transcription (Miller et al. 1976). They
have also been used as markers of 'active' NORs. In
electron microscopy, they have been found in the FC and
DFC but never in the GC (Hernandez-Verdun et al. 1980).
On ribosomal transcriptional units spread on grids, the
Ag-NOR proteins have been localized in the transcribed
part of the units and displayed a linear distribution,
indicating preferential localization on the DNP axis
(Angelier et al. 1982). Initially, interest in this set of
proteins was aroused by their ability to localize particular
chromosomal sites via an easy reaction. Then it was
reported that the variability of their staining intensity
revealed the degree of nucleolar activity, which was
presumably transcriptional activity. This explains the
recent rush on the detection of Ag-NOR proteins in cancer
cells (362 papers in the last 3 years), since their amounts
might indicate the level of cell activity. However, although
Ag-NOR protein detection is selective, widely used and
probably a good marker of nucleolar activity, there is at
present no clear way of identifying these proteins. On the
basis of the molecular weights of the bands revealed on
gels by Ag-NOR staining (chiefly 104 and 3'7xlO3Mr, and
also 190, 135, 78 and 29xlO 3 M r ) (Buys and Osinga, 1984;
Lischwe et al. 1979; Pfeifle et al. 19866; Williams et al.
1982), it has been proposed that nucleolin (100xl0 3 M r )
and protein B23 (37xlO3Afr) are the major Ag-NOR
proteins. However, neither has been detected in FC, which
is stained by silver, but both are present in the GC,
although it is never silver stained. The 190xl03Afr band
has been proposed to be the large subunit of polymerase I.
We have no indication of the identities of the other bands
(Masson et al. 1990). A yeast nucleolar protein, SSB-1, was
recently found to be one of the silver-binding nucleolar
proteins, and is strongly associated with snRlO, an snRNA
involved in pre-rRNA processing in yeast (Clark et al.
1990). Identification of the silver-stained proteins might
permit further characterization, either of these proteins or
of the modifications they undergo by association with
ribosomal transcription.
Other nucleolar-specific proteins
Autoimmune sera directed against the nucleoli (reviewed
by Reimer et al. 1987, and Tan, 1989) contain antibodies
that recognize RNA polymerase I, fibrillarin and protein
B23 (Kindas-Miigge, 1989), as well as many other
nucleolar proteins. Recently, these sera were systematically used to identify and locate new nucleolar proteins.
The finding that it was possible to conserve some of these
during evolution indicates that they might participate in
some fundamental function. This function may be restricted to one nucleolar component such as the 116xlO 3 M r
protein specific for the DFC (Masson et al. 1990). Another
protein, the NOR-90xl0 3 M r protein, seemed specific for
the NORs, even during mitosis (Rodriguez-Sanchez et al.
1987). An association with the preribosomal particle
fraction was proposed for the 35, 37, 69 and 92-93x 103Mr
polypeptides recognized by four different sera (Pfeifle et al.
1986a). Such an association was demonstrated for the
'anti-To' antibodies, which recognize a 40xl0 3 M r protein
forming a complex with 7-2 nucleolar RNA (Reddy et al.
1983). In contrast, PM/Scl proteins, a complex of 11
proteins ranging from 110xl0 3 M r to 20xl0 3 M r characteristic of polymyositis/scleroderma, failed to immunoprecipitate RNA (Reimer et al. 1987). Anti-To and PM/Scl
The nucleolus today
467
antibodies were localized by electron microscopy in the GC
only. Another autoantibody found in scleroderma, called
ScBr, recognized the 94xlO3Afr protein that was distributed in both the DFC and the GC (Hernandez-Verdun et al.
1988).
Although this list of nucleolar-specific proteins is not
exhaustive, it indicates the power of immunological
detection to characterize new nucleolar proteins and
possibly to investigate their role. Note that there are still
few monoclonal antibodies against nucleolar proteins
(Kistler et al. 1984; Schmidt-Zachmann et al. 1984), except
for the 'favourite' nucleolar proteins (RNA polymerase I,
nucleolin, fibrillarin and protein B23) and the cell-cyclerelated nucleolar proteins (Chatterjee etal. 1987; Gerdes et
al. 1984; Verheijen et al. 1989; Waseem and Lane, 1990).
The nucleolar cell cycle
Some nucleolar proteins appear to be cell-cycle-dependent
because they are only found in cycling cells or because
they are regulated by the cell cycle. The proteins in the
first category can be considered to act as nucleolar
markers of the cell cycle by their presence or level of
accumulation in the nucleolus. However, other aspects of
the situation are not clear. For example, the presence of
some of the proteins that are found in highly proliferating
cancer cells and are assumed to be proliferation-associated
nuclear antigens might be related to cell transformation,
needed for ribosomal synthesis or for a rapid cell cycle (for
review see Chatterjee et al. 1987). There are also similar
possibilities with regard to the Ag-NOR proteins, since one
group of investigators found evidence indicating that
these proteins affect the timing of the cell cycle (Trer6 et al.
1989).
Nucleolar cell cycle markers
The first nucleolar cell cycle marker was recognized by the
Ki-67 monoclonal mouse antibody (Gerdes et al. 1984).
However, although it is very specific and routinely used,
we do not yet know which epitopes are recognized. It is,
however, the standard marker for the characterization of
cycling cells (Verheijen et al. 1989).
Among these nucleolar markers, we found the nucleolar
form of the proliferating cell nuclear antigen (PCNA)
(Waseem and Lane, 1990), which is different from the
nuclear form. Nuclear PCNA is an auxiliary protein of
polymerase, and is known to be associated with DNA
replication. When the nucleolar DNA replicates, that is,
during a short period of the S phase, the nuclear form of
PCNA is detected in the nucleoli. We do not know why a
nucleolar PCNA variant is present in the nucleoli
throughout the cell cycle (Waseem and Lane, 1990) but it
does underline the particular nature of the functions in the
nucleolus compared to the rest of the nucleus.
The characterization of proliferation-associated nucleolar proteins has been in progress for a long time in
Busch's group (reviewed by Busch et al. 1987). In addition
to the 145xl0 3 M r and 125xlO 3 M r proteins and the
86-70xl0 3 M r complex, they recently described pl20
(Jhiang et al. 1990) and p40 proteins (Chatterjee et al.
1987) as nucleolar markers of cell proliferation. However,
the function of these markers is not clear, since when the
epitope region of pl20 was inhibited by antibody injections, both DNA and RNA syntheses were inhibited
(Valdez et al. 1990).
468
D. Hernandez-Verdun
Cell-cycle-regulated nucleolar proteins
The disorganization of the nucleolar territories and
subsequent dispersion of the nucleolar proteins are among
the first events in mitosis. This dispersion is not simply
due to the ribosomal transcription switch-off, because cellcycle-dependent modifications of the nucleolar proteins
also take place at that time. It has been demonstrated that
two major nucleolar proteins, nucleolin and protein B23,
are highly phosphorylated during mitosis. Results indicated that they are both substrates of p34cdc2 kinase (Peter
et al. 1990). In the same way as for histone HI, the
consensus phosphorylation sequence is a repeated motif
(TPXKK) (Belenguer et al. 1990). This phosphorylation
has been suggested to control the mitotic changes in
nucleolar structures and activity. If correct, this would
mean that the nucleolar dispersion would be controlled by
the same mechanisms as chromosome condensation,
spindle formation and nuclear envelope breakdown (Cochrane et al. 1990). Note that during interphase the activity
of nucleolin also seems to be regulated by phosphorylation,
but at a serine not a threonine site, and by a kinase other
than p34cdc2 (Belenguer et al. 1990).
During mitosis, the nucleolar-specific proteins are
located in different places. Some remain in association
with the NORs, whereas others are scattered in the
cytoplasm or distributed around each chromosome (Sommerville, 1986). RNA polymerase I remains bound to the
NORs, as well as nucleolin, Ag-NOR proteins and DNA
topoisomerase 1. This list, which is not exhaustive
(Courvalin et al. 1986), indicates that the basic components of ribosomal transcription remain together during
mitosis, even in the absence of ribosomal transcription.
This argues in favor of fast activation of the ribosomal
genes at the end of telophase, because only the addition of
one transcription factor and/or a dephosphorylation
process can induce the switch-on of these genes.
Nucleolar targeting
The specific compartmentalization of nucleolar processes
in the nucleus demonstrates the existence of some sort of
cellular machinery that generates this nuclear and
nucleolar organization. The localization of the nucleolar
proteins in specific regions of the nucleolus indicates that
these proteins must contain signals that determine their
final nucleolar destination. The first amino acid sequence
that targets a protein to the nucleolus, i.e. a nucleolar
targeting signal (NOS), was found in the rex protein of the
human T-cell leukemia virus type 1 (HTLV1) (as reviewed
by Hatanaka, 1990). This NOS is a highly basic sequence
of 19 amino acids that is able to target non-nuclear
proteins to the nucleoli. In addition, the NOS contains a
nuclear signal that permits crossing of the nuclear
envelope. Similar nucleolar targeting has been found in
other viral proteins, including the HTV-1 tat and rev
proteins (Cochrane et al. 1990; Siomi et al. 1990).
Furthermore, this location in the nucleolus seems important for rev and rex functions (Cochrane et al. 1990), and
some authors suggested that this specific location is
involved in the export of unspliced viral mRNA (Nosaka et
al. 1989).
Another approach recently showed that a nuclear
localization signal, the SV40 T antigen signal, was able to
bind specifically to the nucleolar protein pl40 (Meier and
Blobel, 1990). This probably illustrates the potential of the
nucleolar-specific protein shuttle, which might be involved
in nucleus-cytoplasm exchanges.
The nucleolar proteins tend to gather around the
ribosomal genes at the end of mitosis. As these proteins
are bound in a sequential order, we propose that they
reach their respective targets in response to a specific
signal. The acidic groups of major nucleolar proteins, such
as nucleolin, B23 and pl20, might be involved in this
process. Only one ribosomal gene is able to induce
nucleolus formation by protein aggregation, but although
ribosomal transcription is essential for this purpose it is
not necessarily a primary determinant of nucleolus
formation (Karpen et al. 1988). To investigate the
potential of nucleolar protein aggregation, we followed the
distribution and localization of nucleolar proteins in
micronucleated cells. In the micronuclei that only contained one chromosome, active ribosomal genes and
nucleoli were only found in micronuclei containing NORbearing chromosomes (Labidi et al. 1990). In the others,
nucleolar proteins corresponding to the different nucleolar
domains still accumulated, despite the absence of ribosomal genes (Hernandez-Verdun et al. 1991). These
proteins tend to aggregate in fibrillar structures, but these
structures are distributed at random in the nuclear
volume. Therefore, ribosomal gene transcription seems
necessary in this experimental model, as it is during the
cell cycle, in order to coordinate the correct positioning of
prepackaged nucleolar proteins. This raises the question
of what initial events are necessary for the switch-on of
ribosomal transcription.
The small nucleolar RNAs
Small nuclear ribonucleoproteins (snRNPs) are a class of
stable RNA—protein complexes found in the nuclei of all
types of eukaryotic cells (reviewed by Luhrmann, 1990).
snRNP particles contain an RNA component called a U
RNA, and at least nine proteins, some of which are
recognized by anti-Sm antibodies. The Sm-snRNPs are
located in the nucleoplasm and their roles in pre-mRNA
splicing have been established. In the nucleoli, there is
another class of snRNPs. They contain four different
RNAs (U3, U8, U13 and U14) that are not found in the
nucleoplasmic snRNPs (Li et al. 1990; Tollervey and Hurt,
1990; Tyc and Steitz, 1989). U3 snRNAs, which are
precipitated by anti-fibrillarin antibodies (Lapeyre et al.
1990; Reuter et al. 1989), participate in vitro in the first
processing event (Kass et al. 1990). The role of U3 in vivo
might be to fold pre-rRNA into a conformation dictating
correct cleavage at the processing site located between
ITS1 and the 5.8 S. It has been suggested that UScontaining RNP particles are the distinct electron-dense
'terminal knobs' observed at the 5' end of the nascent
RNAs on molecular spread transcriptional units (Kass et
al. 1990). During development, the synthesis and distribution of U3 snRNA and U3 snRNP follow a pattern
different from that of the other nuclear U snRNAs and
snRNPs (Li et al. 1990). Similarly, in the yeast nucleus,
where the number of snRNAs seems to be much larger
than in mammals, several specific nucleolar snRNPs
(snR3-snR6, snR8-snR10 and snRl28) were found to
associate with various ribosomal RNA precursors (Clark et
al. 1990). The role of some of these snRNPs in processing
ribosomal RNA has been demonstrated (Li et al. 1990) and
a nucleolar protein related to mammalian fibrillarin was
found to associate with them (Schimmang et al. 1989).
Although, the precise roles of these nucleolar snRNPs in
ribosome biogenesis are not yet known (Luhrmann, 1990),
it is clear that their functions differ from those of
nucleoplasmic snRNPs, which are involved in the splicing
of the pre-mRNAs.
Conclusions
There was no nucleolus before the appearance of eukaryotes. Its differentiation seems to be linked to the
regulation of ribosomal biogenesis, the transport of the
latter's products and also to other undefined functions,
especially in relation to the viral proteins. The hypothesis
that the nucleolar territory might constitute an alternative route for the traffic of certain non-nucleolar molecules
is attractive and is in line with the hypothesis formulated
by Harris's group (Sidebottom and Harris, 1969), who
proposed that the nucleoli are involved in the transport of
certain messenger RNAs.
The nucleolus is the site of specific functions that
implicate specific RNA polymerase, specific snRNAa and
specific proteins. It is an organelle dependent on the cell
cycle and the site of intense exchanges between the
nucleus and cytoplasm involving specific shuttled proteins
and specific relationship with the nuclear envelope. In
conclusion, the nucleolus constitutes a very original
nuclear territory that reflects the compartmentation of
nuclear functions. This can in part answer the question of
why there is a nucleolus in the eukaryotes.
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