CNRS, Université Paris VI, Université Paris VII, Institut Jacques Monod, 2 place Jussieu, 75251 Paris Cedex 05, France
* Author for correspondence (e-mail: dhernand{at}ccr.jussieu.fr )
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Summary |
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Key words: Nucleolus, Cell cycle control, CDK, pol I transcription, rRNA processing, Nuclear body
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Introduction |
---|
The nucleolus is the factory in which ribosome subunits are synthesized and
assembled before being exported to the cytoplasm (for a review, see
Hadjiolov, 1985;
Mélèse and Xue,
1995
; Shaw and Jordan,
1995
). Ribosome biogenesis is accomplished by specific
transcription and processing machineries. Consequently, the establishment of
nucleolar functions at the end of mitosis depends on their activation,
targeting and/or recruitment (Fig.
1). In cycling cells, nucleolar assembly is generally initiated
during telophase and continues for 1-2 hours into early G1 phase
(Fig. 1). In this case, mitosis
follows an interphase during which nucleoli have been fully active, and
nucleolar assembly benefits from machinery and complexes inherited from the
previous cell cycle. Similarly, during embryogenesis, de novo nucleolar
assembly integrates material of maternal origin, but it is programmed over
several cell cycles, as nuclei become progressively more competent to organize
and control the transcription of specific genes. In cycling cells and during
embryogenesis, the assembly process has common features, although their
duration is variable. In addition to the products of RNA polymerase I (pol I)
transcription and processing of the precursor ribosomal RNAs (pre-rRNAs),
ribosome biogenesis needs pol III transcription of 5S rRNAs as well as
ribosomal protein import. The details of these other pathways involved in
nucleolar assembly at the exit of mitosis are still lacking; they are
therefore not discussed here.
|
Early work suggested that nucleolar assembly depends on the activation of
the pol I transcription machinery
(Benavente, 1991;
Scheer and Hock, 1999
;
Thiry, 1996
). This generates
pre-rRNAs (47S in mammals), which recruit the rRNA-processing machinery.
Noticeably, proteins and small nucleolar RNAs (snoRNAs) involved in rRNA
processing were observed in nuclear bodies, called prenucleolar bodies (PNBs),
before localizing at sites containing newly transcribed rRNAs
(Jiménez-Garcia et al.,
1994
). From these observations, Spector and co-workers concluded
that PNBs are mobile nuclear bodies that participate in the delivery of the
rRNA-processing complexes to sites of ribosomal gene (rDNA) transcription
(Jiménez-Garcia et al.,
1994
).
Here, we discuss recent findings that have illuminated the cell cycle controls on nucleolar assembly, the dynamics of delivery of the processing machinery and the role of pre-rRNAs in stabilizing nucleolar machinery. It is now clear that activation of pol I transcription at exit of mitosis is not sufficient to generate nucleolar assembly (Fig. 2). These new findings provide a more integrated view of the assembly process and its dynamics in which the localization of a component reflects its time of residency and binding affinity.
|
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Background: pol I transcription and rRNA processing |
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Processing of pre-rRNAs is very complex, involving cleavage, methylation
and pseudouridylation (for reviews, see
Smith and Steitz, 1997;
Tollervey, 1996
). Cleavages
that remove the 5' and 3' external transcribed spacers and the
internal transcribed spacers are controlled by several ribonucleoprotein (RNP)
complexes, which act sequentially. There are early and late processing
complexes, which act at early steps and late steps of rRNA processing,
respectively. For example, fibrillarin participates in early rRNA processing,
and Nop52 participates in late rRNA processing
(Savino et al., 1999
).
![]() |
Cell cycle control of nucleolar assembly |
---|
How is pol I transcription repressed during mitosis? Some components of the
pol I transcription machinery, such as SL1
(Heix et al., 1998) and TTF-1
(Sirri et al., 1999
), are
mitotically phosphorylated by CDK1cyclin-B.
CDK1cyclin-B-mediated phosphorylation of SL1 abrogates its
transcriptional activity in vitro (Heix et
al., 1998
), and CDK1cyclin-B is necessary not only to
establish repression but also to maintain it from prophase to telophase.
Indeed, in vivo inhibition of CDK1cyclin-B leads to dephosphorylation
of the mitotically phosphorylated forms of components of the pol I
transcription machinery and restores pol I transcription in mitotic cells
(Sirri et al., 2000
).
Interestingly, the restoration of pol I transcription by in vivo inhibition
of CDK1cyclin-B in mitotic cells leads to the accumulation of
pre-rRNAs, which are not processed to form mature rRNAs. Therefore, the
activation and/or relocalization of the pre-rRNA-processing machinery that
normally occurs at exit from mitosis (Fig.
1) is not, or not exclusively, dependent on inhibition of
CDK1cyclin-B; pol I transcription and pre-rRNA processing might thus be
regulated by distinct mechanisms. Recent results show that inhibition of
CDK1cyclin-B in mitotic cells induces the formation of PNBs, but that
the presence of CDK inhibitors prevents proper relocalization of the
pre-rRNA-processing machinery from these PNBs to the reforming nucleoli in
early G1 phase (Sirri et al.,
2002). An (or more than one) unidentified CDK therefore seems
indispensable for proper localization of the processing machinery
(Fig. 2), restoration of
pre-rRNA processing and, consequently, formation of a functional
nucleolus.
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Dynamics of the rRNA-processing machinery |
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At exit from mitosis, the processing machinery is delivered to the
nucleolus through PNB formation and by directional flow. However, during
interphase, analysis of the dynamics of fibrillarin by fluorescence recovery
after photobleaching (FRAP) shows that fibrillarin exchanges rapidly between
the nucleoplasm and nucleolus through an ATP-independent diffusion mechanism
(Dundr et al., 2000;
Phair and Misteli, 2000
;
Snaar et al., 2000
). If this
diffusion mechanism is also present at exit from mitosis, PNB formation could
be determined by interactions(s) between the rRNA-processing complexes and
partners that no longer exist after nucleolar assembly (see the discussion of
the role of rRNAs below).
In addition to the PNBs, there are other bodies that contain nucleolar
components. For example, several nucleolar components involved in pre-rRNA
processing, including U3 snoRNA, fibrillarin, nucleolin, B23 and Nop52, can
accumulate in large cytoplasmic particles termed nucleolus-derived foci (NDF)
during anaphase and telophase of various mammalian cell lines
(Dundr et al., 1997;
Dundr et al., 2000
;
Dundr and Olson, 1998
). NDFs
contain partially processed pre-RNAs that persist throughout mitosis
(Dundr and Olson, 1998
). They
move quickly within the cytoplasm, and when they contact the nuclear envelope
they disappear into the nucleus (Dundr et
al., 2000
). The dynamics of NDFs in the cytoplasm could indicate
that they are associated with motors, but there is presently no direct
evidence supporting this hypothesis. To explain why NDFs are not found in all
cells, Dundr and Olson have suggested that, in the case of high levels of
expression of components of the processing machinery, the excess material that
is not retained around the chromosomes forms these cytoplasmic aggregates
(Dundr et al., 1997
).
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The role of rRNAs in nucleolar assembly |
---|
These partly processed rRNAs inherited from mitosis are found in PNBs and
NDFs in which processing complexes are also present
(Dousset et al., 2000;
Dundr and Olson, 1998
). In
addition, pre-RNAs and rRNA processing intermediates can be immunopurified in
mitotic nucleolar processing complexes
(Pinol-Roma, 1999
). This could
indicate that the processing complexes forming PNBs and NDFs are nucleated by
these pre-rRNAs (Dousset et al.,
2000
; Dundr et al.,
2000
; Dundr and Olson,
1998
; Pinol-Roma,
1999
). Therefore, stable mitotic pre-rRNAs are clearly partners of
the nucleolar machinery during nucleolar assembly, but their role still
remains to be characterized.
During the first cell cycles of Xenopus laevis embryogenesis,
transcription is established de novo at the mid-blastula transition (MBT)
after 12 cell cycles devoid of transcription. One can therefore study assembly
of the nucleolar machinery in the context of active or inactive pol I
transcription. Assembly of a functional nucleolus in Xenopus embryos
takes several cell cycles. Before any transcription, there is association of
UBF with rDNA, sequential formation of PNBs and recruitment of different rRNA
processing complexes. We have demonstrated that in the absence of pol I
transcription, components of the rRNA-processing machinery are recruited to
rDNA in association with pre-rRNAs of maternal origin
(Verheggen et al., 2000;
Verheggen et al., 1998
). An
inactive nucleolus is formed while the RNA pol I complexes accumulate in
nucleoplasmic structures that exclude rDNA
(Bell and Scheer, 1999
;
Verheggen et al., 2000
).
Similarly, in mammalian embryos, functional nucleoli do not develop
immediately after fertilization. Active nucleoli are assembled at
species-specific stages of cleavage (Baran
et al., 1996; Baran et al.,
1997
). In all cases, a nucleolar precursor body (NPB) is present
(Fléchon and Kopecny,
1998
). Nucleolar assembly occurs around these NPBs either over
several cell cycles, when pol I transcription is activated early, or during
one cell cycle, when pol I transcription is activated after several
divisions.
![]() |
Nucleolar assembly in relationship to general nuclear organization |
---|
This large-scale reorganization of the nuclear architecture cannot be due
to interaction between rRNAs for several reasons. First, in living cells the
nucleolar foci initially approach each other without fusing. Second, since
their subsequent fusion takes place at a precise timepoint during the cell
cycle, this is probably a regulated event rather than an event that depends on
the amount of nucleolar activity (which varies in the different foci).
Finally, even silent NORs associate with nucleoli
(Sullivan et al., 2001).
MacStay and co-workers have therefore proposed that clustering of NORs depends
on heterochromatin adjacent to rDNA genes
(Sullivan et al., 2001
). An
additional possibility is that heterochromatic domains of rDNA bound with Net1
and Sir2 (in yeast) maintain rDNA clustering and nucleolar integrity (for a
review, see Carmo-Fonseca et al.,
2000
).
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Conclusions and perspectives |
---|
The pre-rRNAs generated by pol I transcription are localized at the sites of active rDNA gene clusters. The binding affinity of the processing proteins for these pre-rRNAs can explain the compartmentalization of the processing machinery in the functional nucleolus. During nucleolar assembly, pre-rRNAs also appear to participate in compartmentalization of the processing machinery. Mitotic pre-rRNAs are involved in the reformation of the nucleolus after mitosis, and maternal pre-rRNAs in Xenopus embryos are involved in the regrouping of PNBs around rDNA. In both situations, the intriguing question is how the mitotic pre-rRNAs or the maternal pre-rRNAs regroup around rDNA genes. The presence of pre-rRNAs in PNBs and NDFs could also explain the formation of temporarily organized bodies. The stability of these rRNAs could determine their lifetime. Clearly, these questions must be addressed if we are to understand the role of stable rRNAs in the formation and/or maintenance of nuclear structures.
Ribosome biogenesis involves the pol-I, pol-II and pol-III-dependent transcription pathways, the intranuclear translocation of 5S RNAs, the ordered assembly of ribosomal proteins and the export of the small and large ribosomal subunits. Presently we do not know how the coordination between these pathways is regulated and controlled. This is therefore an important goal of research in this area.
Another interesting unanswered question is how the presence of a functional nucleolus contributes to general nuclear architecture and function. The fact that a functional nucleolus is a large nuclear domain (a third of the yeast nucleus), represents the highest concentration of RNA in the nucleus, and is a site of silencing for reporter pol II genes suggests that it has a general role in nuclear function. Indeed, the nucleolus can exclude or sequester molecules that play a role outside the nucleolus. Therefore it will be important to determine whether clustering of rDNA genes has a direct effect on the organization of other genes or on the distribution of heterochromatin.
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Acknowledgments |
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