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J. Biol. Chem., Vol. 277, Issue 33, 29617-29625, August 16, 2002
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From the Department of Molecular Genetics, University of Illinois
College of Medicine, Chicago, Illinois 60607-7170
Received for publication, May 6, 2002, and in revised form, June 3, 2002
Bop1 is a conserved nucleolar protein involved in
rRNA processing and ribosome assembly in eukaryotes. Expression of its
dominant-negative mutant Bop1 The biosynthesis of ribosomes is a major expenditure of cellular
resources, consuming more than half of the total cellular transcriptional and translational productivity in unicellular organisms
such as yeast (1). Accordingly, the rate of ribosome biogenesis is
tightly coordinated with cell growth and proliferation. Although the
precise mechanisms by which cell growth and cell cycle progression are
coordinated are still poorly understood, inroads are being made in
understanding the connection between ribosome biosynthesis and
proliferative signals. For example, growth-promoting signals leading to
activation of phosphatidylinositol 3-kinase or target of rapamycin
result in the preferential translation of ribosomal proteins, at least
in part through phosphorylation of the ribosomal protein S6 (2, 3).
One of the major impediments in understanding the role of ribosome
biogenesis in proliferation control lies in the limited knowledge of
the ribosome biosynthetic machinery in higher eukaryotes. The synthesis
of rRNA and ribosome assembly is a highly complex process that takes
place largely in the nucleolus (4). The mammalian 18 S, 5.8 S, and
28 S ribosomal RNAs are derived from a single 47 S precursor
(pre-rRNA), which is processed to the mature species through a series
of endonucleolytic, exonucleolytic, and modification steps (5).
Targeted inhibition of specific components of ribosome biosynthetic
machinery, a fruitful approach for elucidating pre-rRNA processing in
yeast (6, 7), has not been feasible in mammalian cells until recently.
To address questions relating ribosome biogenesis and cell
proliferation, we have devised a system to block specific steps of
pre-rRNA processing in cultured mouse cells by using a
dominant-negative mutant of the nucleolar protein Bop1 (8, 9).
Bop1 is a component of large nuclear ribonucleoprotein complexes that
represent ribosome precursors. Expression of a Bop1 deletion mutant
that lacks 231 amino acids at the N terminus, Bop1 Remarkably, perturbation of Bop1 activities in murine 3T3 cells by
Bop1 In this study, we have undertaken a deletion analysis of Bop1 to
dissect its structure and function relationships. Deletions of either
the N terminus or C terminus of Bop1 create two different dominant-negative mutants (Bop1 Plasmids--
Expression constructs driving expression of Bop1,
Bop1 Cell Culture--
Cells were cultured as described previously
(8). LAP3 is a clonal cell line derived from NIH 3T3 cells that
constitutively expresses the IPTG-inducible transactivator protein
LAP267 (14) to support expression from pX vectors (13). For stable
transfections and isolation of clonal lines, LAP3 cells were
cotransfected with a puromycin resistance marker pPGK-puro and either
the empty pX11 vector or HA-tagged deletion constructs using calcium
phosphate method (15), followed by selection in medium containing 1 µg/ml puromycin (Sigma). Clonal lines LAP3/1 and Bop1 RNA Blot Analysis--
Total RNA was isolated using Trizol
(Invitrogen) following the manufacturer's protocol. RNA was separated
on 1% agarose-formaldehyde gels and analyzed by Northern blot
hybridization using standard techniques (16). The following
oligonucleotides were used as probes for hybridization: 5.8 S probe
(5'-GCGTTCGAAGTGTCGATGATCAATGTGTCCTGCAATTCAC) complementary to nt
68-108 of mouse 5.8 S rRNA; ITS2-2
(5'-ACTGGTGAGGCAGCGGTCCGGGAGGCGCCGACG) complementary to nt 239-271 of
the ITS2 region; ITS1-4 (5'-GTATCGGTATTTCGGGTGTGAGCGAACTCA) complementary to nt 972-1001 of ITS1; 3'-ETS
(5'-AGAGCGACGGAAGGGGAAAGAGAAACGAAC) complementary to nt 158-187
of 3'-ETS. Probes were 5'-labeled using [ Western Blot Analysis--
Cells were lysed in RIPA buffer (16)
and lysates containing equal amounts of protein, determined by the
DC protein assay (Bio-Rad), were resolved by SDS-PAGE.
Western blot analysis was carried out following a standard protocol
(16) using affinity-purified anti-Bop1 antibodies (9), anti-HA
monoclonal antibodies HA11.1 (Babco), or 12CA5 (Roche Molecular
Biochemicals) and anti-Cdk4 antibodies C-22 (Santa Cruz Biochemicals).
Indirect Immunofluorescence--
Cells were grown on slides,
incubated with IPTG for 12 h, and fixed with paraformaldehyde,
followed by permeabilization with 0.5% Triton X-100 and incubation
with monoclonal anti-HA antibodies in phosphate-buffered saline
containing 0.5% bovine serum albumin for 1 h. After washing,
slides were incubated with anti-mouse Alexa 488-conjugated antibodies
(Molecular Probes) and photographed using MicroMAX digital camera
mounted on an Axioplan II Zeiss microscope. Pictures were processed
using Canvas software (Deneba).
Metabolic Labeling and Analysis of RNA Transcripts--
Various
cell lines were plated in 6-well plates at 105 cells per
well. One day after plating, cells were either left untreated or
treated with 1 mM IPTG for a further 24 h to induce
expression. Pulse-chase experiments were carried out using
L-[methyl-3H]methionine due to a
rapid turnover of the cellular methionine pool. Cells were preincubated
in methionine-free medium for 15 min and then incubated in medium
containing 50 µCi/ml
L-[methyl-3H]methionine
(PerkinElmer Life Sciences) for 30 min. Cells were then chased in
medium containing 10-fold excess of nonradioactive methionine, after
which RNA was isolated using Trizol, and equal amounts of total RNA
were analyzed by Northern blotting and fluorography (9).
Cells to be labeled with radioactive phosphate (for improved detection
of small RNAs) were pretreated in phosphate-free medium for 1 h
and labeled with 20 µCi/ml [32P]orthophosphate
(PerkinElmer Life Sciences) for 30 min followed by a chase in regular
medium for 2 h. Equivalent RNA amounts were separated on a 8%
polyacrylamide, 7 M urea gel. The gel was stained with
ethidium bromide for photography and then dried for autoradiography.
Antisense Treatment--
Phosphorothioate
oligonucleotides 2-1 (5'-GCTCCTCAGGTGGAGTGAAG-3'), 3-1 (5'-GCAGCAGGTGGTAAATGCGG-3'), and 3-3 (5'-GGCGCAGCAGGTGGTAAATG-3') were obtained from Gemini Biotech. LAP3 cells were transfected with oligonucleotides at 400 nM mixed with Cytofectin (Glen
Research) for 4 h in serum-free medium, after which medium
containing 10% calf serum was added. Cells were incubated for
additional 20 h and harvested for analysis.
Deletion Analysis of Bop1--
In previous studies (13), a genetic
selection was established to identify cDNAs that inhibit cell
proliferation when expressed. By using this selection procedure, a
cDNA that confers a powerful and reversible cell cycle block was
identified, and this cDNA (Bop1
We first determined the subcellular localization of the deletion
mutants by indirect immunofluorescence staining with anti-HA antibodies. All N-terminal deletion mutants were present in the nucleus
in substantial amounts, but their subnuclear distribution was different
(Fig. 2). Bop1C1 (aa 153-732) was
primarily localized to the nucleolus, although it could also be
detected in the cytoplasm. Bop1
Among the C-terminal deletion mutants, only Bop1N2 (aa 1-323) was
primarily localized to the nucleolus. Two other mutants, Bop1N4 (aa
1-489) and Bop1N5 (aa 1-704), were excluded from the nucleus in the
majority of transfected cells and accumulated in the cytoplasm,
indicating that either their nuclear import or retention was impaired.
In a fraction of cells, however, these proteins also exhibited some
degree of nucleolar staining, possibly because they still contained a
nucleolar targeting signal that was unaffected by the deletions that
prevented their efficient nuclear accumulation. A short internal
fragment of Bop1, Bop1
From these observations (summarized in Fig. 1B), we conclude
that efficient nuclear import and/or retention of Bop1 is largely dependent on the C-terminal portion, whereas the nucleolar localization requires the region of Bop1 located between aa 251 and 323. Based on
these data, three large functional regions in Bop1 can be defined as
follows: the N-terminal domain (aa 1-250), the central domain (aa
251-323) required for nucleolar localization, and the C-terminal domain containing WD repeats (aa 324-732), which plays a role in
nuclear localization. Both the N-terminal and central regions of Bop1
contain stretches of polar amino acids termed PEST motifs (Fig.
1A) (9).
Effects of Bop1 Deletion Mutants on Cell Proliferation--
To
determine whether the Bop1 mutants affect cell proliferation, we
induced their expression in stable pools of transfected cells and
subjected these cells to BrdUrd light selection that kills
proliferating cells (13). When the N-terminally truncated mutant
Bop1
A modest increase in cell survival in the BrdUrd light assay was also
observed with the nucleolar mutant Bop1C1 (Fig. 3), indicating that
this mutant protein can also inhibit cell cycle progression in a
fraction of transfected cells. The inhibitory effects of Bop1C1,
however, were much less pronounced than those of Bop1 Both Bop1N2 and Bop1
To investigate whether Bop1N2 might also inhibit pre-rRNA maturation,
we isolated single cell clonal lines that inducibly express Bop1N2.
Indeed, expression of Bop1N2 resulted in a blockade of 28 S rRNA
synthesis as judged by [3H]uridine labeling (data not
shown). To examine the specific pre-rRNA processing steps affected by
Bop1N2, we performed pulse-chase labeling with
[methyl-3H]methionine. A
Bop1N2-inducible clonal line (Bop1N2/7) was either left untreated or
treated with IPTG, pulse-labeled for 30 min, followed by chase with
nonradioactive methionine for various times (Fig.
5). The short lived, primary 47 S rRNA
transcript in mouse cells is rapidly converted to 46 S and then to the
relatively stable 45 S pre-rRNA (see Fig. 4). In the absence of IPTG,
the major 45 S rRNA precursor, which comigrates with the less abundant 47 S and 46 S pre-rRNAs, was processed to 41 S and 36 S precursors and converted by 30 min to 32 S pre-rRNA and mature 18 S rRNA (Fig.
5). Formation of the mature 28 S rRNA was almost completed by 1 h
after the pulse.
After 15 min of chase, Bop1N2 expression led to the appearance of novel
41 S* and 36 S* precursors that migrated slightly slower than the
41 S and 36 S pre-rRNAs (Fig. 5). Strikingly, no 32 S rRNA was
formed by this time. After 30 min of chase, Bop1N2 expression resulted
in a decreased amount of mature 18 S rRNA and the appearance of small
amounts of 36 S and 32 S pre-rRNAs, in addition to the aberrant
41 S* and 36 S* species. Processing of the 47/45 S precursor was
significantly delayed, and this pre-rRNA could be found even after
1 h of chase, in contrast to uninduced cells. New 28 S rRNA in
Bop1N2/7 cells was virtually absent, whereas the mature 18 S rRNA was
produced with only a slightly reduced efficiency. To determine whether
the synthesis of 5.8 S rRNA was affected, we separated small RNA
species labeled with radioactive orthophosphate on a polyacrylamide
gel. Induction of either Bop1
Comparison of the effects of Bop1 Role of Bop1 in Processing of ITS1, ITS2, and 3'-ETS in
Pre-rRNA--
In addition to the major pre-rRNA processing pathway in
mouse cells (Fig. 4), several alternative processing pathways have been
observed (17) in which an altered sequence of cleavages may give rise
to pre-rRNA species different from the precursors depicted in Fig. 4.
To investigate the nature of the novel RNAs (41 S* and 36 S*)
observed in pulse-chase labeling of Bop1N2-expressing cells (Fig. 5),
we performed hybridizations of RNA isolated from clonal cell lines
Bop1
The ITS1-4 probe hybridizes with the region immediately upstream of
the 5' end of 5.8 S rRNA and reveals the steady-state levels of the
major precursors 47 S to 45 S, 41S, and 36 S (see Fig. 4). Induction
of Bop1 mutants changed the relative abundance of rRNA precursors as
compared with control vector-transfected cells and non-induced cells
(Fig. 7). The most significant effect of Bop1
Probe ITS2-2 hybridizes with the 47-45 S, 41 S, 36 S, 32 S, and
12 S pre-rRNAs in all control cells (Fig. 7). Induction of Bop1
The probe that hybridizes with the 3'-ETS region ~160 nt downstream
of site 6 (Fig. 7) reveals steady-state levels of the 47 S and 46 S
rRNA precursors, both of which are short lived (18, 19). A single band
corresponding to these pre-rRNAs is detected in control cells (vector
line and Bop1 mutant lines in the absence of IPTG) (Fig. 7).
Strikingly, Bop1N2 expression causes an appearance of additional bands
that hybridize with the 3'-ETS probe. These bands comigrate with the
novel 41 S* and 36 S* bands detected by pulse-chase analysis (Fig. 5)
and hybridizations with the ITS probes (Fig. 7). Thus, these novel
bands represent 41 S and 36 S precursors with an unremoved 3'-ETS
tail. (An additional 32 S* species representing a 3'-extended 32 S
precursor is visible on this blot, and it is probably obscured on other
blots by the abundant 32 S pre-rRNA). These data indicate that Bop1N2
inhibits the early processing step at site 6, which removes the 3'-ETS from 46 S pre-rRNA (Fig. 4). This inhibition does not completely block
subsequent cleavages at sites 1-3, although the increased steady-state
levels of 47/46 S pre-rRNA detected with the 3'-ETS probe (Fig. 7) and
a slowed conversion of the 47/45 S precursors in pulse-chase labeling
(Fig. 5) suggest that their efficiency is reduced. Notably, very low
levels of the 41 S* and 36 S* bands are also detectable by
hybridization with the 3'-ETS probe after Bop1
Together, the hybridization data show that expression of Bop1N2 results
in slowed cleavage at site 6, giving rise to novel 3'-extended rRNA
species due to a bypass of this inefficient processing step. In
addition, there is accumulation of the 32 S pre-rRNA and an apparent
stabilization of 12 S pre-rRNA despite inhibition of its formation.
Thus, besides the role of Bop1 in processing of 32 S pre-rRNA
demonstrated previously (9), the Bop1N2 mutant implicates Bop1 in
processing of the 3'-ETS and the processing of 12 S pre-rRNA to 5.8 S
rRNA. These results indicate that Bop1 function is important for both
late processing steps that lead to generation of the large subunit
rRNAs (sites 3, 4b, and 5) and at least one early processing step (site
6) (Fig. 4). The Bop1 Down-regulation of Endogenous Bop1 by Antisense Oligonucleotides
Leads to Accumulation of 32 S and 12 S rRNA Precursors--
To
corroborate the results obtained above using dominant-negative mutants
to inhibit Bop1 functions, we employed ASOs to down-regulate expression
of endogenous Bop1. Ten phosphorothioate antisense oligonucleotides
were designed against three potential loops in the Bop1 mRNA
identified with the program mfold 3.1 (20). Two of these
oligonucleotides efficiently inhibited endogenous Bop1 protein
accumulation after introduction into LAP3 cells (Fig. 8A).
The effects of down-regulation of Bop1 by the ASOs were analyzed by
hybridization of total RNA using ITS2-2 and 5.8 S probes (see Fig. 7).
Cells subjected to mock transfection, as well as cells transfected with
an ASO that did not decrease Bop1 protein level (3-3, Fig.
8A), exhibited essentially similar steady-state levels of
the 45 S, 41S, 36 S, 32 S, and 12 S pre-rRNAs (Fig. 8B).
Down-regulation of Bop1 with ASOs 2-1 and 3-1 caused an increase in
36 S, 32 S, and 12 S pre-rRNAs (Fig. 8B), indicating that
insufficient levels of Bop1 in the cell can reduce the efficiency of
processing in both ITS1 and ITS2. Importantly, the ASO-mediated
down-regulation of endogenous Bop1 resulted in pre-rRNA processing
defects overlapping with those caused by deletion mutants: inhibited
processing of 36 S pre-rRNA (an effect shared with Bop1 Much insight into the functions of trans-acting protein
factors in ribosome biogenesis has been made possible through genetic approaches in yeast (6, 7). In this study, we demonstrate that
transient expression of dominantly acting mutants provides a viable
experimental approach to study the pre-ribosome maturation machinery in
genetically less malleable systems such as mammalian cells. Exploration
of Bop1 functions in pre-rRNA processing using this approach revealed
that Bop1 is involved in processing events in the 3'-ETS and intragenic
spacers ITS1 and ITS2. These findings suggest a functional link that
exists between several specific processing steps in pre-rRNA
maturation. Moreover, these results establish a set of useful reagents
for future studies of pre-rRNA processing in mammalian cells.
Furthermore, we show that the effects of Bop1 dominant-negative mutants
on pre-rRNA processing are correlated with those on cell cycle
progression, providing support to the idea that surveillance of
ribosome biogenesis may serve as a mammalian cell cycle checkpoint
(12).
Bop1 Is Involved in Processing of Intragenic Spacers and the
3'-ETS--
Potent dominant mutants interfering with Bop1
functions can be created by deletions of either N- or C-terminal
domains of Bop1 (Fig. 1). Remarkably, the processing steps affected by
the dominant-negative forms of Bop1 are not identical, suggesting that
Bop1 is a multifunctional protein that harbors domains involved in
different aspects of ribosome formation. Expression of the Bop1 mutants
most significantly affects late processing steps in ITS1 and ITS2 and,
unexpectedly, also interferes with the early processing of the 3'-ETS
at site 6 (Fig. 4). Notably, the presence of 32 S* to 41 S* species
in the Bop1N2/7 cell line (Figs. 5 and 7) implies that cleavage at site
6 is not an obligatory step for subsequent processing of mouse pre-rRNA
at sites 1-3. Consistent with effects of dominant-negative mutants, a
reduced efficiency of processing in the ITS1 and ITS2 is also observed
after antisense-mediated down-regulation of endogenous Bop1 (Fig. 8).
Taken together, these findings demonstrate that Bop1 is involved in
processing of three transcribed spacer regions in mouse pre-rRNA as
follows: ITS1, ITS2, and 3'-ETS (Fig.
9).
Interdependence of distant cleavage reactions in eukaryotic
pre-rRNA has been observed in other organisms. In
Schizosaccharomyces pombe, deletions in the 3'-ETS inhibit
both the removal of the 3'-ETS region and processing in the ITS1 (21,
22). Likewise, the integrity of a stem loop within 3'-ETS is required
for internal cleavages within ITS1 in S. cerevisiae (23), in
which efficient production of 25 S rRNA also requires ITS1 sequences
(24, 25). Recent studies (26) show that proper secondary structures in ITS2 are important for 5.8 S and 25/28 S rRNA synthesis. In addition to cis-acting elements, one known trans-acting
factor, U8 small nucleolar RNA, was shown previously (27) to be
involved in processing of both the ITS regions and 3'-ETS in
Xenopus oocytes. The present study demonstrates that linkage
between processing of the ITS regions and 3'-ETS also exists in
mammalian cells and identifies Bop1 as a protein factor required for
the proper execution of these processing events.
Multiple effects of Bop1 mutants on pre-rRNA maturation may be
explained by a model in which Bop1 functions by coordinating activities
of various processing factors within preribosomal complexes. Deletions
in parts of the Bop1 molecule that affect different subsets of its
interactions in these complexes may thus impair processing of pre-rRNA
at discrete sites (Fig. 9). This model is corroborated by biochemical
evidence showing that Bop1 is tightly associated with a series of
intermediates in the assembly of large ribosome subunits. The yeast
homolog of Bop1, Erb1p, has been identified recently (28, 29) as a
component of 60-66 S preribosomes. Bop1 cofractionates with
preribosomal particles in mouse cells (9), and the nucleolar mutants
Bop1
Depletion of some proteins involved in 5.8 S/25 S rRNA maturation in
yeast leads to blocked processing and accumulation of 27 S pre-rRNA,
whereas depletion of others results in its loss apparently due to
increased degradation (6). Depletion of the yeast homolog of Bop1,
Erb1p, results in a decrease in the steady-state level of 27SB
pre-rRNA and the disappearance of 7 S pre-rRNA (10). An interesting
feature of Bop1 is that deletions in different parts of this protein
can lead to opposite effects on the levels of the mouse 32 S and 12 S
pre-rRNAs, which are structurally related to the yeast 27SB and
7 S pre-rRNAs. These data suggest that two different types of
non-productive complexes may be formed at late stages of ribosome
maturation. Complexes formed as a result of Bop1 Pre-rRNA Processing and Cell Cycle Progression--
Blocking the
function of Bop1 in a fast and reversible manner not only provides a
novel experimental system for the analysis of the mechanisms of
pre-rRNA processing but also has revealed an intriguing connection
between preribosome assembly and cell cycle progression in mammalian
cells. Induction of Bop1
In this study, we show that two dominant-negative mutants, Bop1 *
This work was supported by National Institutes of Health
Grant CA95627.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, June 4, 2002, DOI 10.1074/jbc.M204381200
2
The abbreviations used are:
ITS1 and ITS2, internal transcribed spacers 1 and 2;
ETS, external transcribed
sequences;
IPTG, isopropyl-
Functional Inactivation of the Mouse Nucleolar Protein Bop1
Inhibits Multiple Steps in Pre-rRNA Processing and Blocks Cell
Cycle Progression*
aklina
Strezoska,
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
in mouse cells blocks rRNA maturation
and synthesis of large ribosomal subunits and induces a reversible,
p53-dependent cell cycle arrest. In this study, we have
conducted a deletion analysis of Bop1 and identified a new mutant,
Bop1N2, that also acts as a potent inhibitor of cell cycle progression.
Bop1N2 and Bop1 are C-terminal and N-terminal deletion mutants,
respectively, and share only 72 amino acid residues. Both mutant
proteins are localized to the nucleolus and strongly inhibit rRNA
processing, suggesting that activation of a cell cycle checkpoint by
Bop1 mutants is linked to their inhibitory effects on rRNA and ribosome synthesis. By using these dominant-negative mutants as well as antisense oligonucleotides to interfere with endogenous Bop1, we
identified specific rRNA processing steps that require Bop1 function in
mammalian cells. Our data demonstrate that Bop1 is required for proper
processing at four distinct sites located within the internal
transcribed spacers ITS1 and ITS2 and the 3' external spacer. We
propose a model in which Bop1 serves as an essential factor in ribosome
formation that coordinates processing of the spacer regions in
pre-rRNA.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, leads to a
specific block in the maturation of the 28 S and 5.8 S rRNAs without
affecting maturation of 18 S rRNA (9). Pulse-chase analysis showed
that Bop1 expression resulted in a partial inhibition of conversion
of the 36 S precursor to 32 S pre-rRNA and a complete inhibition of
synthesis of mature 28 S and 5.8 S rRNAs from 32 S pre-rRNA.
Concomitant with these defects in pre-rRNA processing, expression of
Bop1 abolished formation of new 60 S ribosomal subunits (9). Bop1
is conserved throughout eukaryotes and contains five WD motifs (10),
implicated in protein-protein interactions (11). Analysis of the
Saccharomyces cerevisiae homolog of Bop1,
ERB1, has shown that its functions in pre-rRNA and ribosome
maturation are also conserved. Depletion of Erb1p in yeast resulted in
the inhibition of the synthesis of mature 25 S and 5.8 S rRNA,
paralleling effects of interfering with Bop1 in mouse cells (10).
induced a strong and reversible cell cycle arrest in
G1 (8, 12). This cell cycle arrest was associated with down-regulation of Cdk2 and Cdk4 kinase activities and
hypophosphorylation of pRb, indicating an inability of cells to
progress through G1 into S phase (12). This G1
arrest occurred prior to a significant impediment in the global
translation rate that might occur due to depletion of the cytoplasmic
ribosome pool. Most interestingly, inactivation of functional p53
circumvented the Bop1-induced cell cycle block without restoring
normal pre-rRNA processing. These results have led to the proposal that
ribosome biosynthesis serves as cell cycle checkpoint subject to
surveillance by p53 (12).
and Bop1N2, respectively) that inhibit proliferation and cause overlapping but not identical blocks in
pre-rRNA processing. The comparison of processing defects caused by the
two deletion mutants and the effects of antisense inhibition of
endogenous Bop1 demonstrate a role of Bop1 in processing of the
ITS1,1 ITS2, and the 3'-ETS
in mouse pre-rRNA. Moreover, this analysis shows that effects of Bop1
mutants on cell cycle progression are correlated with effects on
pre-rRNA processing.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, HA-tagged Bop1, and HA-Bop1 in the IPTG-inducible
expression vector pX11 (13) have been described previously (9).
Deletion mutants of Bop1 were obtained by cloning appropriate
restriction fragments of Bop1 into pX11 with an in-frame N-terminal HA
tag sequence. Details of the cloning procedures are available upon request.
/6 have been
described previously (8). BrdUrd light treatment was performed
according to a protocol described previously (8).
-32P]ATP and
T4 polynucleotide kinase.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) encodes an N-terminal deletion
of Bop1 (8). Acting in a dominant-negative manner, expression of
Bop1 blocks maturation of the 28 S and 5.8 S rRNA, 60 S ribosome
subunit biogenesis, and cell cycle progression (9, 12). To understand
the structural elements that mediate Bop1 functions, we constructed a
series of N-terminal and C-terminal deletions of Bop1 linked to an HA epitope tag (Fig. 1A).
IPTG-regulated expression constructs encoding these mutant proteins
were transfected into LAP3 cells, and expression of the deletion
mutants in stable transfected pools induced with IPTG was verified by
immunoblotting (Fig. 1C).
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Fig. 1.
Deletion analysis of Bop1. A,
schematic representation of Bop1 and its deletion mutants. PEST
sequences 1 and 2 are shown as hatched boxes and WD repeats
1-5 as solid black boxes. B, summary of the
subcellular localization of Bop1 deletion mutants and their effects on
cell proliferation and pre-rRNA processing. Predominant localization of
HA-tagged proteins in the nucleolus (No) and nucleoplasm
(Np) is indicated when it occurs in the majority of
transfected cells. The asterisk indicates that the
localization is detected only in a fraction of cells. ND,
not determined. The ability of the deletion mutants to evoke a
reversible cell cycle arrest in LAP3 cells was assessed by the BrdUrd
light assay (see text for details). C, expression of
deletion mutant proteins. Total cell lysates were prepared from cell
populations stably transfected with the IPTG-inducible constructs
indicated after induction with IPTG for 20 h. Lysates were
normalized by protein content, separated by SDS-PAGE, and analyzed by
Western blotting using antibodies against the HA tag.
, as shown previously (9), is
predominantly localized to the nucleolus. Bop1C2 and Bop1C3 were found
in both the nucleus and cytoplasm, whereas Bop1C4 was almost
exclusively nuclear. Interestingly, Bop1C3 (aa 392-732) and Bop1C4 (aa
490-732), although capable of efficient translocation into the
nucleus, appeared to be excluded from the nucleolus (Fig. 2).
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Fig. 2.
Subcellular localization of Bop1 deletion
mutants. Pools of LAP3 cells stably transfected with different
HA-tagged Bop1 constructs were grown on coverslips, induced with IPTG
for 20 h, fixed, permeabilized, and analyzed by indirect
immunofluorescence with a monoclonal anti-HA antibody.
f (aa 231-413), which is likely capable of
entering into the nucleus due to small size, was concentrated in the
nucleolus (Fig. 2). These results suggest that the nucleolar
localization signal is located in the center region of Bop1 (see Fig.
1A).
is expressed in cells, activation of a
p53-dependent checkpoint reversibly blocks the cell cycle
in G1 phase (8, 12). Cells that are transiently arrested by
Bop1 survive the BrdUrd light treatment and form numerous
colonies following the transfer to IPTG-free medium in which Bop1
expression is repressed (Fig. 3). By
using the BrdUrd light assay to analyze cell cycle inhibitory
properties of Bop1 mutants, we found that the C-terminally truncated
mutant Bop1N2 induced a reversible cell cycle arrest in a large
fraction of transfected cells as efficiently as Bop1 (Fig. 3). This
result was surprising because Bop1N2 (aa 1-323) and Bop1 (aa
251-732) are C-terminal and N-terminal deletions, respectively, and
overlap in only a short stretch of 72 amino acids (Fig.
1A).
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Fig. 3.
Expression of Bop1
and Bop1N2 leads to a reversible inhibition of cell
proliferation. LAP3 cells were cotransfected with pPGK-puro, which
confers resistance to puromycin, in combination with different deletion
constructs cloned in the IPTG-inducible pX11 vector, full-length
expression construct pX11-HA-Bop1, or empty pX11. Equal numbers of
stably transfected, puromycin-resistant cells were treated in parallel
with IPTG for 24 h to induce expression and then subjected to
BrdUrd light treatment (13) to selectively kill proliferating cells.
Cells that did not replicate during this period and survived the
treatment were rescued by removal of IPTG, grown for 8 days, and
stained with crystal violet. The number of colonies reflects the number
of cells that were reversibly arrested by expression of the transfected
constructs.
and Bop1N2. In
addition, we did not observe cell cycle inhibition in several
individually isolated clonal lines with low to moderate expression of
Bop1C1, suggesting that its effect may require high expression levels
(data not shown). The remaining Bop1 deletion mutants did not promote
cell survival in the BrdUrd light assay, indicating that these mutants
are not capable of inducing a reversible cell cycle arrest (Fig.
3).
Block Synthesis of 28 S and 5.8 S
rRNAs--
Both Bop1 and Bop1N2 are localized predominantly to the
nucleolus, suggesting that these mutants affect nucleolar functions (Fig. 2). We have shown previously that Bop1 inhibits
processing of pre-rRNA into mature 28 S and 5.8 S rRNAs
(9). Maturation of rRNA in mammalian cells is a complex
process that involves a series of endonucleolytic, exonucleolytic, and
modification steps to generate 18 S, 5.8 S, and 28 S rRNAs from a
primary 47 S transcript (Fig. 4).
Bop1 interferes with several steps in pre-rRNA maturation, causing a
loss of 12 S pre-rRNA as well as 28 S and 5.8 S rRNAs and partially
inhibiting the conversion of 36 S pre-rRNA to 32 S pre-rRNA, which
results in the accumulation of the 36 S precursor (9).
View larger version (15K):
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Fig. 4.
Major pre-rRNA processing pathway in the
mouse. The structure of the primary 47S precursor and major
processing sites (labeled 0-6) are shown at the
top. The 47 S pre-rRNA is processed through intermediates
of different sizes designated according to their relative sedimentation
coefficients (S) to eventually yield the mature 18 S,
5.8 S, and 28 S rRNAs. Numbers next to
arrows indicate sites processed at the corresponding
conversion steps. The steps affected by Bop1 and Bop1N2 deletion
mutants and down-regulation of endogenous Bop1 through ASO are
indicated.
View larger version (85K):
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Fig. 5.
Expression of the C-terminal deletion mutant
Bop1N2 inhibits formation of 28 S rRNA. Cells of an inducible
clonal line that expresses HA-tagged Bop1N2 mutant (Bop1N2/7) were
either left untreated or treated with 1 mM IPTG for 24 h, pulse labeled with
L-[methyl-3H]methionine for 30 min, and chased in non-radioactive medium containing excess methionine
for the indicated times. Equal amounts of total RNA were resolved on a
formaldehyde-agarose gel, transferred to a nylon membrane, and
visualized by fluorography. Positions of major precursors are indicated
on the left.
or Bop1N2 led to a nearly complete
inhibition of the 5.8 S rRNA synthesis (Fig.
6).
View larger version (106K):
[in a new window]
Fig. 6.
Expression of Bop1N2 inhibits formation of
5.8 S rRNA. LAP3 clonal lines transfected with either empty pX11
vector (line LAP3/1), pX11-Bop1 (line
Bop1/6), or pX11-HA-Bop1N2 (line Bop1N2/7) were
either left untreated or treated with IPTG for 24 h and
metabolically labeled with radioactive orthophosphate. Following a
chase in non-radioactive medium for 2 h, RNA was isolated, and
equal amounts of RNA for each sample were resolved on an 8% denaturing
polyacrylamide gel, stained with ethidium bromide (left
panel), and analyzed by autoradiography (right
panel).
and Bop1N2 showed that both mutant
proteins abolished the synthesis of mature large ribosome subunit
rRNAs, 28 S and 5.8 S rRNAs, without diminishing the generation of
18 S rRNA significantly (9) (Figs. 5 and 6). However, these two
mutants exerted different effects of the levels of rRNA precursors. In
Bop1-expressing cells, the label from the 32 S precursor disappears after ~1 h (9), indicating that either 32 S pre-rRNA or its derivatives are rapidly degraded. In Bop1N2-expressing cells, the 32 S
precursor is stabilized and remains detectable even after 2 h of
chase (Fig. 5). Bop1 also delayed the processing of 36 S pre-rRNA,
leading to its accumulation (9). In contrast, Bop1N2 expression did not
cause significant accumulation of 36 S pre-rRNA but appeared to delay
early processing steps and also resulted in the formation of novel RNA
species, 41 S* and 36 S* (Fig. 5).
/6, Bop1N2/7, and LAP3/1 (vector-transfected) with
oligonucleotide probes complementary to different regions of the
primary rRNA transcript (Fig. 7).
View larger version (46K):
[in a new window]
Fig. 7.
Hybridization analysis of the processing
block caused by Bop1N2 expression. Clonal LAP3 cell lines
transfected with the empty vector (line LAP3/1), Bop1 (line
Bop1/6), or HA-tagged Bop1N2 (line Bop1N2/7) were either left
untreated (
) or treated (+) with IPTG for 24 h. RNA was
isolated, and equal amounts were separated on a 1% agarose gel,
transferred to a nylon membrane, and hybridized to oligonucleotide
probes complementary to different regions of the mouse pre-rRNA as
indicated at the top. The ITS2-2 blot was exposed for a
shorter time than ITS1-4 to show differences in the abundant 32 S
precursor.
expression was a
strong increase in the 36 S pre-rRNA level, in agreement with previous
data (9). After Bop1N2 induction, 36 S pre-rRNA was only modestly
increased, but two additional bands of a slightly larger size than
36 S and 41 S rRNAs were detected, similar to the 36 S* and 41 S*
RNAs observed in pulse-chase labeling (Fig. 5).
resulted in a strong increase of the 36 S pre-rRNA level, consistent
with the ITS1-4 hybridization, and a decrease in 12 S pre-rRNA (Fig.
7). In Bop1N2 lines, the most striking effect is a large increase in
the 32 S pre-rRNA steady-state level, consistent with its
stabilization observed in pulse labeling experiments (see above), and
the appearance of two additional bands, 41S* and 36 S* (in Fig. 7,
41 S* is not clearly visible due to a short exposure but is readily
detectable after longer exposure times). Interestingly, there is no
significant decrease of the steady-state level of 12 S pre-rRNA,
although the strong accumulation of 32 S pre-rRNA implies that the
cleavage at site 4b, which generates 12 S (Fig. 4), is inhibited.
Consistent with inhibition of this cleavage, analysis of
32P-labeled RNA on an agarose gel showed decreased label
incorporation into 12 S pre-rRNA after Bop1N2 induction (data not
shown). These results suggest that Bop1N2 expression has an additional
inhibitory effect on the 12 S pre-rRNA turnover, which may lead to
undiminished steady-state levels of this precursor despite reduced
formation rates. As shown below, a similar stabilizing effect on 12 S
pre-rRNA is also observed after down-regulation of endogenous Bop1 expression.
induction (Fig. 7),
although, as compared with Bop1N2, this is only a minor defect in these cells.
and N2 mutants, acting as dominant-inhibitory
forms of Bop1, thus reveal overlapping but not identical subsets of
Bop1 functions.
View larger version (44K):
[in a new window]
Fig. 8.
Antisense inhibition of endogenous Bop1.
A, the effect of antisense oligonucleotide inhibition on the
Bop1 protein level. Cells were transfected with either a no-oligo
mixture (control sample, lane C), or with
oligonucleotides 2-1, 3-1, or 3-3 for
24 h. Bop1 levels were examined by immunoblotting using antibodies
against Bop1 (9) in equal amounts of protein lysates. A lower portion
of the same blot was probed with anti-Cdk4 antibodies as a loading
control. B, effects of down-regulation of endogenous Bop1 on
pre-rRNA processing. RNA was isolated from cells transfected with
oligonucleotides as in A, and equal amounts of total RNA
were separated on 1% agarose gels and subjected to Northern blot
analysis. Hybridizations were performed with oligonucleotides
complementary to 5.8 S rRNA and ITS2 region (see Fig. 7).
) and 32 S
and 12 S rRNA precursors (similar to Bop1N2), validating the effects
of these mutants as a dominant-negative interference with Bop1 function.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (10K):
[in a new window]
Fig. 9.
Bop1 links processing of ITS1 and ITS2 with
3'-ETS processing. Bop1 is envisaged as part of a multiprotein
complex in which processing of ITS1, ITS2, and 3'-ETS takes place.
Processing at sites 3, 4b, 5, and 6 is inhibited by either deletions of
terminal domains of Bop1 or lowering its levels through antisense
oligonucleotides.
and Bop1N2 are present in the same fractions in sucrose
gradients,2 suggesting that
their dominant-negative effects result from the incorporation of these
defective Bop1 forms into preribosomes.
expression appear
to be open to processing and/or degrading enzymes, as reflected by the
rapid 32 S pre-rRNA turnover. In contrast, Bop1N2 may promote
formation of complexes trapped in a closed conformation in which the
access of RNA-processing enzymes to ITS2 is impaired, thereby causing
an accumulation of 32 S pre-rRNA and stabilization of 12 S pre-rRNA.
Some of the nucleases involved in the removal of the ITS1 and ITS2 have
been characterized in yeast (30, 31); however, the mechanisms that
regulate their activities are not yet known. Dominant-negative mutants
of Bop1 may thus present an interesting experimental system in which to dissect the interactions between nucleases and other components of
preribosome complexes.
in LAP3 cells causes a rapid G1
arrest, which occurs sooner than a measurable repression of global
protein synthesis due to depletion of the translating ribosome pool (9,
12). Most interestingly, inactivation of functional p53 alleviated the
Bop1-induced cell cycle arrest (12), leading us to propose the
hypothesis that perturbations in the ribosome assembly machinery in
mammalian cells trigger a nucleolar stress signal that is subject to
surveillance by p53 as a cell cycle checkpoint (12).
and
Bop1N2, which cause strong but non-identical rRNA processing blocks,
arrest proliferation in transfected cell populations in a potent and
reversible fashion (Fig. 3). Like Bop1, Bop1N2 does not
significantly affect cell viability but rather induces a transient cell
cycle arrest (Fig. 3), which also requires functional p53 (data not
shown). Analysis of cell lines expressing various Bop1 deletion mutants
to date has revealed none in which the effects on rRNA synthesis and
proliferation could be dissociated. The association of effects of Bop1
mutants on pre-rRNA processing and cell proliferation lends further
support to the model in which inhibition of pre-rRNA processing can
lead to a reversible cell cycle arrest. Further analysis of
transdominant mutants of Bop1 as well as other proteins that function
in ribosome biogenesis should provide a useful approach to elucidate
the molecular mechanisms that link ribosome formation to cell cycle
regulation in mammalian cells.
FOOTNOTES
To whom correspondence should be addressed: Dept. of Molecular
Genetics (M/C 669), University of Illinois, 900 South Ashland Ave.,
Chicago, IL 60607. Tel.: 312-996-6978; Fax: 312-996-7034; E-mail:
LFLau@uic.edu.
. Strezoska, D. G. Pestov,
and L. F. Lau, unpublished data.
ABBREVIATIONS
-D-thiogalactopyranoside;
BrdUrd, bromodeoxyuridine;
ASO, anti-sense oligonucleotides;
HA, hemagglutinin;
aa, amino acids;
nt, nucleotides.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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