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J. Biol. Chem., Vol. 275, Issue 25, 18845-18850, June 23, 2000
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From the Laboratoire de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 route de Narbonne, 31077 Toulouse Cedex, France
Received for publication, March 21, 2000, and in revised form, April 11, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The first processing event of the precursor
ribosomal RNA (pre-rRNA) takes place within the 5' external transcribed
spacer. This primary processing requires conserved
cis-acting RNA sequence downstream from the cleavage site
and several nucleic acids (small nucleolar RNAs) and proteins
trans-acting factors including nucleolin, a major nucleolar protein.
The specific interaction of nucleolin with the pre-rRNA is required for
processing in vitro. Xenopus laevis and hamster nucleolin
interact with the same pre-rRNA site and stimulate the processing
activity of a mouse cell extract. A highly conserved 11-nucleotide
sequence located 5-6 nucleotides after the processing site is required
for the interaction of nucleolin and processing. In vitro
selection experiments with nucleolin have identified an RNA sequence
that contains the UCGA motif present in the 11-nucleotide conserved
sequence. The interaction of nucleolin with pre-rRNA is required for
the formation of an active processing complex. Our findings demonstrate
that nucleolin is a key factor for the assembly and maturation of
pre-ribosomal ribonucleoparticles.
Ribosome biogenesis is a complex process that involves the
transcription of a large
pre-rRNA1 precursor, its
maturation, and assembly with ribosomal proteins (1, 2). Pre-rRNA
undergoes multiple post-transcriptional nucleotide modification (3) and
nucleolytic processing steps to yield the mature 18, 5.8, and 28 S rRNA
species. The two first endonucleolytic cleavages occur in external
transcribed spacers (ETS) and therefore do not lead to the formation of
mature rRNA ends. The first cleavage also called early or primary
processing occurs within the 5'-ETS (4-6) is conserved from yeast (7) to human (8) and can be found at various positions within the 5'-ETS
(5, 7-11).
Despite its conservation, the role of this cleavage in ribosome
biogenesis is still unknown, and only few factors involved in this
process have been characterized. In yeast, the role of Rnt1p, an RNase
III homologue remains unclear (12, 13). In higher eukaryotes, the major
nucleolar protein nucleolin (15), an endonuclease (14) and several
small nucleolar RNA are also involved (15, 16), but their exact
function remains to be elucidated. In vitro UV cross-linking
has identified a small number of proteins, including nucleolin, that
interact with the RNA substrate (17, 18). The different factors
assemble in a large 20 S complex (18) that could be visualized at the
terminal ends of nascent pre-rRNA (terminal balls) observed on
Miller's Christmas tree by electron microscopy (19, 20). The formation
of this complex seems necessary but not sufficient for
processing (20, 21).
The sequence and RNA structural motif required for the processing have
been extensively studied in vitro (21, 22). In mouse
pre-rRNA, an evolutionary conserved 11-nt motif (+655 to +666) located
just 5-6 nt downstream from the cleavage site is absolutely required
for the processing (21) and for formation of the complex observed at
the 5' end of nascent pre-rRNA (20). A 27-nt RNA (+645 to +672) is
processed very inefficiently in vitro, but it seems to
contain the minimal cis-acting sequence required for
processing (21). The 200 nt that follow this motif are 80% conserved
between mice and humans (5, 8, 23) and stimulate the processing (21).
This RNA sequence can be the targets of several RNA-binding proteins or
small nucleolar RNAs for the recognition of the cleavage site.
Nucleolin, one of the major nucleolar proteins in exponentially growing
cells, is involved in several steps of ribosome biogenesis (24, 25).
Nucleolin binds nascent pre-rRNA close to the transcription initiation
site (26, 27), suggesting that nucleolin plays a role at an early step
of pre-rRNA synthesis. We have previously shown that nucleolin is able
to stimulate the primary processing in vitro (17). Specific
interactions of nucleolin with the pre-RNA substrate and with other
cellular components like the U3snoRNP are required for the processing.
In this study, we show that an 11-nt evolutionary conserved sequence
located just 5-6 nt downstream from the cleavage site is required for
the interaction of nucleolin with the pre-rRNA substrate. This
interaction is required for the assembly of the processing complex.
Plasmids Constructs and in Vitro Transcription--
Mouse rDNA
fragments (+541 to +1250 and +645 to +1250) were amplified by PCR using
the following oligonucleotides: 5'-ETS-541 (5'-ggaagatcttcgctcgttgttctcttg-3') or 5'-ETS-645
(5'-ggaagatctgcgcgtcgtttgctcactc-3') and 5'-ETS-1250
(5'-ggaattcaaactttccaaccccagccgcg-3'). Underlined are the
EcoRI and BglII sites used for the ligation in
pSP72 (Promega) to give pSPETS541-1250 and
pSPETS645-1250. The conserved 11-nucleotide motif (ECM)
was deleted by PCR. Two PCR reactions were performed with
oligonucleotides 5'-ETS-541 and
For the construction of the GST-tagged CHO nucleolin a PCR reaction was
performed with oligonucleotides KTNUC1
(5'-cgcggatccgtgaagctcgcaaaggctggcaaaacc-3') and KTNUC2
(5'-gctctagatcattattcaaacttcgtcttctttccttgtgg-3';
BamHI and XbaI sites are underlined) and the
cDNA of CHO nucleolin as template. The PCR product was subcloned in
pEG(KG) plasmid (gift from Dr. Deschenes, Iowa City, Iowa) and give the
pKTNUCFL plasmid.
Preparation of S100 Extracts, Production and Purification of
Proteins, in Vitro Processing, and UV Cross-linking--
Extract,
processing reaction, and UV cross-linking are performed as described
previously (17). Hamster and Xenopus nucleolin were purified
from exponentially CHO (Computer Cell Belgium), and A6 growing cells
respectively. Recombinant p50 was expressed in Escherichia
coli and purified as described previously (31, 32). The GST-tagged
CHO nucleolin was expressed in Saccharomyces cerevisiae
(JCW25 strain) (gift from Dr. Gartenberg, Piscataway, NJ) and purified
using a glutathione-Sepharose column (Amersham Pharmacia Biotech) as
described previously (39).
Filter Binding Assay--
Filter binding assay using purified
nucleolin protein and in vitro labeled RNA were performed as
described previously (27, 31). Briefly, 10 fmol of labeled RNA were
incubated with different concentration of purified nucleolin in a final
volume of 50 µl of binding buffer (200 mM KCl, 25 mM Tris, pH 7.5, 5 mM MgCl2, 20%
glycerol, 50 µg/ml tRNA, 10 µg/ml bovine serum albumin) for 30 min
at room temperature. Then the reaction mixtures were filtered on a
nitrocellulose membrane and washed three times with binding buffer. The
percentage of bound RNA was determined using a PhosphorImager. Native
gel shift assay (see Fig. 5) was performed as described (16, 18).
Briefly, after the processing reaction, half of the incubated reaction
was directly loaded on a 0.8% TBE agarose gel. After a 5-h migration
at 100 V, the gel was dried and autoradiographied.
Xenopus and CHO Nucleolin Stimulates Processing in Mouse Cell
Extract--
Although the 200 nt surrounding the cleavage site, known
as the M3 box (28), are quite conserved between humans and mice, they
have considerably diverged in Xenopus. To determine whether nucleolin from Xenopus and hamster cells were
interchangeable, we tested and compared the interaction of
Xenopus laevis and CHO nucleolin with the mouse 5'-ETS (Fig.
1A). A labeled RNA
corresponding to nucleotides +541 to +1250 (RNA541/1250)
from the mouse pre-rRNA was incubated in mouse S100 extract in presence
of increasing amount of X. laevis (lanes 2-4) or
CHO (lanes 5-7) nucleolin and subjected to an UV
cross-linking experiment. These two proteins interacted with the same
efficiency with the pre-rRNA substrate. To demonstrate that the
exogenous nucleolin was actually binding to the pre-rRNA substrate and
not competing away inhibitory proteins in the extract that would allow
more endogenous nucleolin protein binding to the RNA, we used a
recombinant GST-tagged protein (GST-CHO nucleolin) in this assay (Fig.
1A, lanes 8-12). This experiment clearly shows
that exogenous GST-CHO nucleolin interacts with the mouse pre-rRNA and
competes for binding with endogenous nucleolin.
Previous experiments (17) have established a correlation between an
increase in specific interaction of nucleolin with the pre-rRNA
substrate and an activation of the primary processing. We then
determined whether the interaction of X. laevis nucleolin with the pre-rRNA was also able to stimulate the processing. In the
presence of an excess of X. laevis (Fig. 1B,
lanes 3-5), CHO (lanes 7-9), or recombinant
GST-CHO nucleolin (lanes 10-14), the processing reaction is
markedly activated. Altogether these data show that all these proteins
are able to activate the processing reaction and that nucleolin-binding
site on pre-rRNA is conserved between X. laevis and mouse
despite the low sequence similarity within the 5'-ETS of these two species.
The ECM Is Required for Nucleolin Interaction with the
Pre-rRNA--
Interactions of nucleolin with different truncations of
RNA541/1250, in absence of cellular extract, were studied
to map the binding site of nucleolin (Fig.
2). We used a filter-binding assay with
purified CHO nucleolin and labeled in vitro transcribed RNA (27). Deletion of the first 104 nt or of the last 573 nt of RNA541/1250 did not affect nucleolin binding affinity
(Kd of 180 nM ± 40 nM)
(Fig. 2), indicating that nucleolin binding site is contained within
the 32-nt RNA645/677. We used a previously characterized
small 68-nt RNA (NS) which does not interact with nucleolin (15, 27,
28, 29) as a negative control for these interactions. The 32-nt RNA
(RNA645/677) sufficient for the specific interaction of
nucleolin contains an evolutionary conserved 11-nt motif (ECM), which
is always located 5 or 6 nt after the processing site (9, 20, 29) (Fig.
2A). To determine whether this motif was required for
nucleolin interaction, the 11-nt motif was deleted within
RNA541/764 and RNA541/677 to give
RNA541/764
These RNAs were used as competitor in UV cross-linking and in
vitro processing assays (Fig. 3,
B and C). These competition assays have been
previously used successfully to study the sequences and structural
motifs required for the processing (9, 21). Labeled
RNA541/1250 was incubated in the extract and subjected to
UV cross-linking (Fig. 3B, lane 1) and processing
reaction (Fig. 3C, lane 2). Addition of RNA
competitors that contain the ECM (RNA541/677 and
RNA645/677) were able to compete nucleolin interaction with labeled RNA541/1250 (Fig.
3B, lanes 3 and 5) and abolished
processing of the RNA substrate (Fig. 3C, lanes 4 and 8). In contrast, the RNA competitor that does not
contains this 11-nt motif (RNA541/677 In Vitro Selection with Nucleolin Identifies an UCGA Motif
Contained in the ECM--
A SELEX experiment performed with nucleolin
has characterized several RNA binding sequences (27). Half of these
sequences form a small stem-loop structure composed of a short stem (5 base pairs) and a 7-10-nt loop containing the motif (U/G)CCCGA. This motif interacts with high affinity (Kd of 5-20
nM) with nucleolin (27, 30). The other sequences interact
with lower affinity (Kd of 100 nM) and
show much higher sequence diversity (Fig.
4A). Alignment of these SELEX
sequences highlight an UCGA motif contained in the ECM (Fig.
4A), indicating that this motif could be a key determinant
of nucleolin RNA-binding specificity. One of the selected sequences
(N25-358) was used in competition experiments (Fig. 4, B
and C). When unlabeled N25-358 was added to the cell
extract, the interaction of nucleolin with RNA541/1250
decreased significantly (Fig. 4B, compare lane 2 with lanes 1 and 4) and reduced the basal
processing activity of the extract (Fig. 4C, compare
lanes 5 and 2). When excess of nucleolin and RNA
competitor are added at the same time, the interaction of nucleolin
with labeled RNA541/1250 and processing activity of the
extract was restored (Fig. 4, B, lanes 2 and
3, and C, lanes 5 and 6,
respectively). An RNA sequence, NS, which does not interact with
nucleolin (17, 31) was neither able to compete nucleolin cross-linking
with RNA541/1250 nor processing activity (Fig. 4,
B and C, lanes 4 and 5 and
lanes 3 and 4, respectively). This add-back
experiment demonstrated that N25-358 titrated specifically nucleolin
and strongly suggests that the UCGA motif present in the ECM interacts
with nucleolin. It is also interesting that full activity of the
extract was restored when nucleolin is added to N25-358 treated
extracts, whereas this was not possible with RNA541/677 and
RNA645/677 competitors. These two RNAs are competent for
processing complex formation, although this is not the case of N25-358
(data not shown). Therefore, the selected sequence titrates
specifically nucleolin, whereas RNA541/677 and
RNA645/677 might titrate other factors required for the
processing.
Interaction of Nucleolin with the Pre-rRNA Substrate Is Required
for the Formation of the Processing Complex--
Previous studies have
shown that processing activity is correlated with the formation of a
large ribonucleoprotein complex (9, 18, 20, 22). To study the role of
the interaction of nucleolin with the pre-rRNA in complex formation,
the RNA N25-358 was added to the processing competent extract with
labeled RNA541/1250 and directly loaded on a native agarose
gel (Fig. 5A). Addition of
increasing amount N25-358 was able to prevent the formation of the
correct complex (lanes 5 and 6), whereas the NS
RNA had no effect (lanes 8 and 9). When
RNA541/677 Nucleolin Is Required at an Early Step of the Processing Complex
Formation--
To get an insight on the role of nucleolin in complex
formation, the protein was added at different times in the processing reaction (Fig. 5B). In all previous experiments, cell
extracts and exogenous nucleolin were preincubated 30 min before the
addition of 32P-labeled RNA541/1250
(lanes 3 and 4). Under these conditions, addition
of nucleolin stimulates the processing activity (compare lanes
3 and 4 with lane 2). If nucleolin is
preincubated with the RNA substrate before addition of cell extract
(lanes 9 and 10), the same strong processing
activation is observed. In contrast, when cell extract and pre-rRNA are
incubated for 30 min before the addition of nucleolin, only a weak
activation was observed (lanes 6 and 7). These
results suggest that the activation of the processing is more efficient
when nucleolin interacts with the pre-rRNA substrate first. This first
step might be required for an efficient recruitment of the processing complex.
In these experiments we show that the interaction of nucleolin
with an evolutionary conserved RNA sequence (ECM) in the pre-rRNA 5'-ETS is required for processing and formation of a specific complex.
Nucleolin is well conserved from human to Xenopus (24, 25),
and we show that nucleolin from X. laevis and hamster
interact with the same pre-rRNA sequence and activate processing
activity of a mouse cell extract (Fig. 1). The N-terminal domain of
nucleolin that is not required for RNA binding (31, 32) is needed for complex formation (Fig. 5A) and processing activation (17). The interaction of the N-terminal domain of nucleolin with other factors involved in the processing, like the U3snoRNP (17), seems
therefore well conserved between mouse and Xenopus. A SELEX experiment with hamster nucleolin characterized more precisely the RNA
sequence required for nucleolin interaction with the pre-rRNA and
highlighted the UCGA motif contained in ECM (Fig. 4). The SELEX
selected sequences efficiently compete the interaction of nucleolin
with the pre-rRNA and the formation of specific complexes. This
suggests that nucleolin interacts with the UCGA motif present in the
ECM and that this interaction is required for the formation of a
processing complex. However, this small motif cannot explain by itself
the RNA binding specificity of nucleolin toward the pre-rRNA primary
processing binding site. Phylogenetic and mutagenesis studies suggest
that this sequence is needed in a single-stranded conformation for
processing and specific complex (21, 29). No strong secondary structure
could be found in the SELEX sequences, indicating that the UCGA motif
is also probably in a single-stranded conformation. Future work should
determine the importance of the surrounding sequence for the specific
interaction of nucleolin. The ECM is highly conserved, and its deletion
or point mutation completely inhibits processing (21, 22). Furthermore,
this sequence is required for the presence of the terminal balls at the
5' end of nascent pre-rRNA (20). Therefore, the interaction of
nucleolin with this highly conserved sequence implies that nucleolin
plays an important role in the processing and formation of an active
processing complex.
The minimal pre-rRNA sequence requirement (+645 to +672) for processing
and specific complex formation (21) includes the ECM. The formation of
a ribonucleoprotein complex on this RNA motif was reported to be
necessary but not sufficient for processing (21). The U3snoRNP, which
is absolutely required for processing (16) is only present in complex
formed on larger RNA (9, 16, 18). Sequences downstream of the ECM
together with the interaction between the U3snoRNP and the N-terminal
domain of nucleolin (17) might cooperate for the activation of the
processing reaction. The complex formed on the minimal RNA sequence is
very stable and cannot be displaced (18). If nucleolin is added after complex formation on the pre-rRNA substrate, it is not able to stimulate the processing (Fig. 5B). However, when nucleolin
competitors (Fig. 5A; p50, N25-358) are added at the
beginning of the reaction, the formation of the complex is prevented.
This suggests that the early interaction of nucleolin with the ECM is
required for the formation of an active processing complex.
The ECM is required for the formation of the terminal balls at the 5'
ends of nascent pre-rRNA transcript in Xenopus oocyte (20).
However, in Xenopus oocytes, the primary processing is not
detected (9, 20, 33). Because the formation of these structures is
conserved in all tissues and organisms examined, the formation of this
complex rather than the primary processing may serve an important
function. Our results indicate that nucleolin does not activate the
processing when a stable complex is preformed on the pre-rRNA
substrate, indicating that the ordered formation of a stable complex
that could include nucleolin is necessary for the formation of an
active complex. The regulation of nucleolin, like the phosphorylation
of the N-terminal domain of nucleolin during the cell cycle (34-37),
could modulate the formation of this complex and therefore of the processing.
The N- and C-terminal domains of nucleolin are involved in
protein-protein interactions (17, 38), whereas the central domain
mediates specific interaction with the pre-rRNA (17, 27, 31, 32, 38).
The function of nucleolin in this processing is likely the result of
these different properties. The specific interaction of nucleolin with
the ECM on nascent pre-rRNA might represent the first step in the
assembly of a large ribonucleoparticle involved in this processing.
Nucleolin could then recruit, by protein-protein interaction, other
factors involved in the cleavage, like the U3snoRNP. Nucleolin
interaction with the pre-rRNA could also play a role of a chaperone for
the formation of the correct RNA tertiary structure of the nascent
transcript. The evolutionary conservation of (i) nucleolin, (ii) the
interaction of nucleolin with pre-rRNA sequences involved in the
cleavage, and (iii) the formation of a specific complex at the 5' end
of nascent pre-rRNA indicates that this primary processing event plays
an important and still unknown function for ribosome biogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
M3A
(5'-gagaactccggagcataagagtgagcaacgacgcgcaatcgg-3') or 5'-ETS-1250 and
M3B (5'-cgttgctcactcttatgctccggagttctcttcgggccagggcc-3'). The last
PCR used the first two purified PCR products and oligonucleotides 5'-ETS-541 and 5'-ETS-1250. This DNA fragment was ligated in the BglII and EcoRI sites of pSP72 to give
pSPETS541-1250
ECM. The BglII
site present in the resulting plasmids was removed by BglII
digestion followed by T4 DNA polymerase exonuclease
digestion and religation. The final plasmids were called
pSPETS541-1250BglII, pSPETS645-1250BglII, and
pSPETS541-1250ECMbglII. These plasmids were linearized by different enzymes DdeI
(+656), BspEI (+677), Bsp120I (+764), and
EcoRI (+1250) and used for in vitro transcription
using T7 RNA polymerase to give the corresponding RNAs. Labeled RNA was
synthesized using [
-32P]CTP in the transcription
reaction. Unincorporated nucleotides were removed by gel filtration
(G50; Amersham Pharmacia Biotech), and then the RNA was ethanol
precipitated. Unlabeled RNA competitors were synthesized as described
previously (31).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
X. laevis and hamster nucleolin
are interchangeable for the processing reaction in mouse cell
extract. A, increasing amount of purified X. laevis (X.l, lanes 2-4), CHO (lanes
5-7) or recombinant GST-CHO nucleolin (lanes 8-12)
were added to the processing reaction that contained 10 fmol of
radiolabeled RNA541/1250. Lanes 1 and
8, no exogenous nucleolin was added; lanes 2,
5, and 9, 5 pmol; lanes 3,
6, and 10, 10 pmol; lane 4,
7, and 11, 20 pmol; lane 12, 30 pmol.
After incubation, an UV cross-linking was performed, and labeled
proteins were resolved by SDS-polyacrylamide gel electrophoresis and
autoradiography. B, processing of RNA541/1250 in
presence of X. laevis (lanes 3-5), CHO
(lanes 7-9), or recombinant GST-CHO (lanes
10-14) nucleolin. Lanes 1, 2, 6,
and 10, no exogenous nucleolin was added; lanes
3, 7, and 11, 5 pmol; lanes 4,
8, and 12, 10 pmol; lanes 5,
9, and 13, 20 pmol; lane 14, 30 pmol.
ECM and RNA541/677ECM respectively, and these RNA
were used in the filter binding assay (Fig. 2B). The precise
deletion of the 11-nt motif completely abolishes nucleolin interaction
to a level comparable to a negative control for binding (NS and
RNA541/656).
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Fig. 2.
Interaction of nucleolin with pre-rRNA.
Top panel, schematic representation of the different RNAs
used. The black square indicates the position of the ECM,
and the arrow indicates the primary processing site.
Interaction of nucleolin in absence of cellular extract with these
different RNAs was studied using a filter binding. These interactions
were compared with interaction with a 68-nt RNA (NS)
previously described (27-29) that does not interact significantly with
nucleolin. A, deletion of nucleotide sequences at the 5' and
3' end of RNA541/1250. B, comparison of the
interaction of nucleolin with wild type and ECM RNAs. Quantification
was performed using a PhosphorImager. The key to the data points is
shown on the figure.
ECM)
did not significantly compete for nucleolin interaction (Fig.
3B, lane 7) nor RNA541/1250
processing (Fig. 3C, lanes 6). To
demonstrate that RNA541/677 and
RNA645/677 and not
RNA541/677ECM titrated nucleolin, a 5-fold
excess of nucleolin was added to the UV cross-linking and processing experiments. An increase of nucleolin cross-linking and a stimulation of RNA541/1250 processing were observed when no competitors
(Fig. 3, B and C, lanes 2 and
3, respectively) or when
RNA541/677ECM was used (Fig. 3, B
and C, lanes 8 and 7, respectively).
In contrast, nucleolin cross-link was only partially restored (Fig.
3B, lanes 4 and 6), and no activation
of RNA processing was observed (Fig. 3C, lanes 5 and 9) when ECM containing RNA competitors were added. Even
in presence of added nucleolin, RNA competitor and nucleolin are
present in about equimolecular amount, and therefore, unlabeled RNA541/677 and RNA645/677 are still efficiently
competing nucleolin. Furthermore, these two RNAs are sufficient for
formation of processing complex (18, 21). Nucleolin could therefore
form a stable complex with these competitors and titrate other factors
involved in the processing reaction. Altogether, these results
demonstrate that the ECM is required for nucleolin interaction with the
RNA.
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Fig. 3.
The 11-nucleotide ECM is required for the
interaction of nucleolin with the pre-rRNA and nucleolin activation of
the primary processing. A, schematic representation of
the different RNAs used. The black box indicates the
position of the ECM, and the arrow indicates the primary
processing site. In RNA645/677 ECM the
conserved 11 nucleotides have been deleted. B, cross-linking
of nucleolin with 32P-labeled RNA541/1250 in
presence of different RNA competitors. Labeled RNA541/1250
was incubated in cell extract in presence of 40 pmol of unlabeled RNA
competitors, RNA541/677 in lanes 3 and
4, RNA645/677 in lanes 5 and
6, RNA645/677ECM in lanes
7 and 8. A 5-fold excess (compared with nucleolin
present in the extract) of exogenous CHO nucleolin (10 pmol) was added
in lanes 2, 4, 6, and 8. In
lanes 1 and 2, no RNA competitor was added in the
extract. After UV cross-linking, labeled proteins were analyzed by
SDS-polyacrylamide gel electrophoresis, and the gel was
autoradiographied. C, in vitro processing
reaction in presence of different competitors. Labeled
RNA541/1250 was incubated with (lanes 2-9) or
without (lane 1) cell extract in presence of different
unlabeled RNA competitors (20 pmol), RNA541/677 in
lanes 4 and 5,
RNA645/677ECM in lanes 6 and
7, and RNA645/677 in lanes 8 and
9. An excess of nucleolin (10 pmol) was added in lanes
3, 5, 7, and 9. After the
processing reaction, RNA was extracted and analyzed on a 6%
polyacrylamide gel.
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Fig. 4.
Nucleolin SELEX selected sequences titrates
nucleolin-processing activity. A, alignment of RNA
sequences identified in a SELEX experiment with CHO purified nucleolin.
Lowercase letters represent nucleotides that are part of the
flanking constant sequence of the oligonucleotide used in the SELEX
experiment. These sequences represent 50% of the selected sequences.
The remaining sequences contained a conserved motif previously
described (27). SELEX sequences are compared with the mouse and
X. laevis evolutionary conserved motif found 5-6-nt
downstream from the processing site (respectively, ECM mouse
and ECM X.l.). B, cross-linking of nucleolin with
32P-labeled RNA541/1250 in presence of
different RNA competitors. Labeled RNA541/1250 was
incubated in cell extract in presence of 20 pmol of unlabeled RNA
competitors, RNA NS in lanes 4 and 5 and RNA
N25-358 in lanes 2 and 3. NS RNA has been
previously studied in detail (27, 30, 31) and does not interact with
nucleolin. In lanes 3 and 5, 10 pmol of purified
CHO nucleolin were added to the reaction. C, in
vitro processing reaction in presence of different competitors.
32P-Labeled RNA541/1250 was incubated with
(lanes 2-6) or without (lane 1) cell extract in
presence of unlabeled RNA competitors, RNA NS in lanes 3 and
4 and RNA N25-358 in lanes 5 and 6.
Nucleolin (10 pmol) was added in lanes 4 and 6.
After the processing reaction, RNA was extracted and analyzed on a 6%
polyacrylamide gel.
ECM is used as competitor, the
formation of the complex is not affected (data not shown) as previously
published (9). These results demonstrate that nucleolin interaction
with the pre-rRNA is required for correct complex formation. A
recombinant protein (p50) containing the RNA-binding domains of
nucleolin competes with full-length nucleolin for the interaction with
the pre-rRNA and inhibit the processing reaction (17). Addition of p50
in the cell extract (lane 3) prevents complex formation.
Altogether these results demonstrate that the interaction of nucleolin
with the ECM is required for the correct assembly of the processing
complex on the pre-rRNA. The N-terminal domain of nucleolin (missing in
the recombinant p50 protein) is also required, suggesting that
nucleolin can recruit other components of the processing complex.
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Fig. 5.
RNA binding activity and the N-terminal
domain of nucleolin are required for the formation of a specific
complex. A, labeled RNA541/1250 was
incubated without (lane 1) or with (lanes 2-9)
cell extract and with unlabeled RNA competitors, N25-358 (lanes
5 and 6) and NS (lanes 8 and 9),
40 pmol (lanes 5 and 8), or 80 pmol (lanes
6 and 9). In lane 3, there was a 20-fold
excess (20 pmol) of recombinant p50 (nucleolin without the N-terminal
domain (17)). After incubation of 60 min at 30° C, the reaction
mixture was loaded on a 0.8% agarose gel. B, interaction of
nucleolin with the pre-rRNA is required at an early step of the
processing reaction. The activation of the processing of
RNA541/1250 by nucleolin was studied in three different
experimental conditions in which the order of addition of the three
components (nucleolin, cell extract, and labeled
RNA541/1250) varies. Preincubation, nucleolin + cell
extract (lanes 2-4), cell extract + labeled
RNA541/1250 (lanes 5-7), and nucleolin + labeled RNA541/1250 (lanes 8-10) were performed
during 30 min before addition of the third component. Increasing
amounts of nucleolin were added: 5 pmol (lanes 3,
6, and 9) and 10 pmol (lanes 4,
7, and 10). After 45 min of incubation, RNAs were
extracted and analyzed on a 6% polyacrylamide gel.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by grants from the Université Paul Sabatier (Toulouse), the Center National de la Recherche Scientifique, and the Association pour la Recherche contre le Cancer.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.
To whom correspondence should be addressed: Laboratoire de
Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 route de
Narbonne, 31077 Toulouse Cedex, France. Tel.: 33-5-61-17-59-51; Fax:
33-5-61-17-59-94; E-mail: bouvet@ipbs.fr.
Published, JBC Papers in Press, April 12, 2000, DOI 10.1074/jbc.M002350200
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ABBREVIATIONS |
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The abbreviations used are: pre-rRNA, precursor ribosomal RNA; ETS, external transcribed spacer; ECM, evolutionary conserved motif; nt, nucleotide(s); PCR, polymerase chain reaction; GST, glutathione S-transferase; CHO, Chinese hamster ovary; NS, non-specific.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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