Two Different Combinations of RNA-binding Domains Determine
the RNA Binding Specificity of Nucleolin*
Hervé
Ginisty,
François
Amalric, and
Philippe
Bouvet
From the Laboratoire de Pharmacologie et de Biologie Structurale,
CNRS UMR 5089, 205 route de Narbonne, 31077 Toulouse Cedex,
France and the
Ecole Normale Supérieure de Lyon,
CNRS UMR 5665, 46 Allée d'Italie, 69364 Lyon Cedex 07, France
Received for publication, December 11, 2000, and in revised form, January 9, 2001
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ABSTRACT |
Nucleolin is an abundant nucleolar protein
involved in several steps of ribosome biogenesis. The protein is highly
conserved through evolution and possesses four RNA-binding domains
(RBD), which are likely to determine its RNA binding specificity.
Previous studies have shown that nucleolin interacts with two different RNA targets. The first is a small stem-loop structure, the nucleolin recognition element (NRE), found all along the pre-ribosomal
RNA. The second is a short single-stranded RNA sequence, the
evolutionary conserved motif (ECM), located five nucleotides
downstream of the first processing site in the pre-ribosomal RNA 5'
external transcribed spacer. Biochemical, genetic, and structural
studies have shown that the first two RBD of nucleolin are necessary
and sufficient for the specific interaction of nucleolin with the NRE
motif. In this work, we have studied the interaction of nucleolin with
the ECM sequence. Deletion and mutational analyses showed that all four
RBDs of hamster nucleolin were required for the interaction with the
ECM sequence. This RNA binding specificity is conserved between hamster
and Xenopus laevis, whereas the Xenopus protein
does not interact with the NRE. Nucleolin is the first example of a
protein that requires four RBDs for its interaction with an RNA target,
demonstrating that a single protein can use different combinations of
RBD to interact specifically with several RNA sequences.
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INTRODUCTION |
Specific RNA-protein interactions play an important role in gene
expression. One of the most common protein sequence motifs involved in
these interactions is the RNA-binding domain
(RBD),1 also called the RNA
recognition motif (RRM) (1, 2). A single or multiple RBD, often in
combination with other domains involved in protein-protein interaction,
are found in proteins that have diverse functions such as in
pre-mRNA maturation and splicing (3, 4), hnRNA packaging (5), and
mRNA stability and translation (6, 7).
The RBD domain is characterized by two highly conserved motifs called
RNP-1 and RNP-2 found within a weakly conserved 80-amino acid sequence
(1, 2). The crystal and solution structures of several RBDs have
revealed a common structural organization composed of four antiparallel
-strands packed against two
-helices (Refs. 8-10; 14).
The RNP-1 octapeptide and RNP-2 hexapeptide are located in the
1 and
3 strands, respectively. Structures of RBD found in tandem show that
the two independently folded domains do not interact with each other in
Sex-lethal (11, 12), HuC (13), and nucleolin proteins (14). In
contrast, in hnRNPA1, the two domains interact extensively (15, 16).
Despite the highly conserved structure of the RBD, they show a
remarkable ability to interact with diverse RNA sequences and
structures. The tertiary structures of several RBD·RNA
complexes are now available. These are the single RBDs from the U1A and
U2B'' protein bound to a stem-loop structure (8, 17, 18), the
two RBDs of Sex-lethal, hnRNPA1, and poly(A)-binding protein bound to a
single-stranded RNA (12, 16, 19), and the first two RBD of nucleolin
bound to a stem-loop structure (20). These different RNA-protein
complexes revealed that the RBD uses the highly conserved RNP1 and RNP2 motifs in addition to the more highly divergent loops and linker regions specific to each RBD for the specific binding with their respective RNA target.
The presence of several RBD motifs (up to four domains in
poly(A)-binding protein and nucleolin) might suggest that these proteins could interact with several RNA targets or that all RBD are
required for the interaction with a single RNA or both. All three RBDs
of U2AF65 are required for high affinity binding to the
polypyrimidine tract (4). The first two RBDs of poly(A)-binding protein
and nucleolin bind specifically to polyadenylate RNA (21-23) and a short stem-loop structure (24, 25), respectively, as efficiently as
full-length protein; the function of the last two RBD is not known.
Nucleolin is a major nucleolar protein involved in most steps of
ribosome biogenesis (26-29). The interaction of nucleolin with
pre-ribosomal RNA (30-32) is believed to play an important role in
rRNA maturation and pre-ribosome assembly (33, 34). Studies of the
specificity of the interaction of nucleolin with pre-rRNA have revealed
that it interacts with two different RNA motifs. The nucleolin
recognition element (NRE) motif, identified by SELEX, is a small
stem-loop structure (31). Nucleolin binds tightly to similar stem-loop
structures found all along the pre-rRNA (35). The second motif, the
evolutionary conserved motif (ECM), was identified during studies on
the function of nucleolin in the first processing step of ribosomal RNA
(33, 34). The ECM is a short evolutionary conserved 11-nt sequence
found 5 nt downstream of the cleavage site (36). The interaction of
nucleolin with this sequence is absolutely required for the assembly of
the processing complex and maturation of pre-rRNA at this site (33,
34).
Although the first two RBD of nucleolin are required for the specific
interaction with the NRE motif (24, 25), it was of interest to
determine which domains are involved in the binding of the ECM. In this
work we show that all four RBD of nucleolin are required for
interaction with the ECM. This interaction is conserved between hamster
and Xenopus nucleolin. This is the first example of a
protein that utilizes two different combinations of RBD to interact
with two different RNA targets.
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MATERIALS AND METHODS |
Plasmids Constructs and in Vitro RNA Transcription--
Mouse
rDNA fragment from nt 645-1250 was amplified by PCR using the
following oligonucleotides: 5'-ETS-645
(5'-ggaagatctgcgcgtcgtttgctcactc-3') and 5'-ETS-1250
(5'-ggaattcaaactttccaaccccagccgcg-3'). The EcoRI and BglII sites used for the cloning in pSP72 (Promega) to
give pSPETS645-1250 are underlined. The BglII
site present in the resulting plasmid was removed. The final plasmid
was called pSPETS645-1250
BglII, and was
linearized by enzyme BspEI (+677) for in
vitro transcription with T7 RNA polymerase to give
RNA645/677. The NRE cloned between the XbaI and
HindIII sites of pSP64PA plasmid (Promega) encode a
68-nucleotide-long RNA. Labeled RNA was synthesized using
[
-32 P]CTP in the transcription reaction.
Unincorporated nucleotides were removed by gel filtration (G50,
Amersham Pharmacia Biotech), and then the RNA was ethanol precipitated.
Production of Recombinant Proteins--
The different nucleolin
mutants were produced as described previously (25). Nucleolin
mutants were generated by PCR using VentTM DNA polymerase
(New England Biolabs) and hamster nucleolin cDNA as template. PCR
products contained an NdeI and a BamHI site at their 5'- and 3'-ends, respectively, for subcloning in the
corresponding sites of pET15b plasmid (Novagen). The following
oligonucleotides were used: R1N
(5'-ccccatatgaatctgttcattggaaac-3') and R2C
(5'-cccggatccggtaccagtatagtaaagtgaaac-3') for R12; R1N and
R3C (5'-cccggatccggtaccttgtaactccaacctgat-3') for R123; R2N
(5'-ccccatatgacacttttagcaaaaaat-3') and R4C
(5'-cccggatccggtaccggcccagtccaaagtaac-3') for R234; R1N and
R4C for R1234; and X.NdeM
(5'-ggaattccatatgcctgcaaaacgcaaaaaa-3') and XR4C
(5'-cccggatcctcattgggaatctcctttagg-3'). All proteins were quantified using Bradford reagent (Bio-Rad) and checked on SDS-PAGE. Each RNA-binding domain is defined here from the first amino
acid of the
1 sheet to the last amino acid of the
4 sheet. For
Chinese hamster nucleolin, these amino acid residues are: Asn308-Pro381 for RBD1,
Thr394-Gly465 for RBD2,
Thr486-Gly558 for RBD3,
Pro563-Pro646 for RBD4.
The introduction of the RNP-1 mutations was achieved by PCR
site-directed mutagenesis with the following oligonucleotides: R1(L/F,
L/F)S and R1(L/F, L/F)NS for R1(LL)234; R2 (L/I, L/F)S and R2(L/I,
L/F)NS for R12(LL)34; R3(LL)S and R3(LL) NS for R123(LL)4 NS; R4(LL)S
and R4(LL)NS for R1234(LL); R1(LL)S and R1(LL)NS for X.l.R1(LL)234; X.l.R2 (LL)S and
X.l.R2(LL)NS for X.l.R12(LL)34; R3(L L)S and
R3(LL) NS for X.l.R123(LL)4 NS; R4(LL)S and R4(LL)NS for
X.l.R1234(LL): (R1(L/F, L/F)S,
5'-ggaaatt(a/t)ggttt(a/t)gtggactttgagtc-3'; R1(L/F, L/F)NS,
5'-ccac(t/a)aaacc(t/a)aatttcctatttgtacc-3'; R2(L/I, L/F)S,
5'-aaaggg(c/a)ttgcttt(t/a)attgaatttaagtc-3'; R2(L/I, L/F)NS, 5'-aat(t/a)aaagcaa(g/t)ccctttactcttccc-3'; R3(LL)S,
5'-gggttagcgttaatagaatttgctt-3'; R3(LL)NS,
5'-tattaacgctaaccctttagatttgcc-3'; R4(LL)S,
5'-gggttaggtttagtagacttcaacagtg-3'; R4(LL)NS,
5'-tactaaacctaaccctttagaggaacc-3'; X.l.R2 (LL)S,
5'-aaagggctggcactggttgagtttagcactgaag-3'; X.l.R2 (LL)NS,
5'-ctcaaccagtgccagccctttatttgatccatcat-3'). All recombinant plasmids
were sequenced to confirm the presence of the mutations.
Expression and Purification of Recombinant
Proteins--
BL21(DE3)plysS was transformed with each recombinant
pET15b plasmid. Cells grown at 37 °C in LB (100 mg/liter ampicillin, 20 mg/liter chloramphenicol) were induced with 1 mM
isopropyl-1-thio-D-galactopyranoside for 4 h.
Harvested cells were resuspended in buffer A (50 mM sodium phosphate, pH 8, 300 mM NaCl) and lysed by sonication.
After centrifugation (30 min at 10,000 × g), the
supernatant was recovered and gently mixed for 1 h at 4 °C
after the addition of 1 µl of
Ni2+-nitrilotriacetic acid resin (Qiagen)/ml of
initial culture. After three washes with buffer A and two with buffer B
(50 mM sodium phosphate, pH 6, 300 mM NaCl,
10% glycerol), tagged peptide was eluted with buffer C (buffer B + 0.5 M imidazole). The supernatant was applied on a G25 column
(NAP 5, Amersham Pharmacia Biotech) equilibrated with 100 mM KCl and 10 mM Tris-HCl, pH 7.5. Concentrations were estimated with Bradford reagent (Bio-Rad protein
assay) and checked by SDS-polyacrylamide gel electrophoresis.
RNA Binding Assay--
Gel retardation assay were performed by
incubating 10 fmol of labeled RNA in TMKC buffer (20 mM
Tris, pH 7.4, 4 mM MgCl2, 200 mM
KCl, 20% glycerol, 1 mM dithiothreitol, 0.5 mg/ml tRNA, 4 µg/ml bovine serum albumin) with the indicated amount of protein for
30 min at room temperature. Then they were directly loaded on 8%
polyacrylamide gel (acrylamide:bisacrylmamide = 60:1) containing 5% glycerol in 0.5× TBE at room temperature. The gel was dried and
submitted to an autoradiography. Quantification of RNA retardation was
performed with a phosphorimager (Fuji X-BAS 1000).
UV Cross-link and Filter Binding Assays--
For the UV
cross-linking assay, 500 fmol of labeled RNA were incubated with the
indicated amount of protein at room temperature for 30 min in 10 µl
of TMKC buffer. Samples were subjected to UV light for 5 min as
follows: 10-µl drops were spotted on a thin layer of parafilm on ice
and subjected to UV (
= 254 nm) (Spectrolinker XL1000,
Spectronics Corp.). Then 0.5 µg of RNase A (Sigma) was added to each
sample and incubated for 30 min at 37 °C. Proteins were resolved on
SDS-PAGE.
Filter binding assays using purified nucleolin protein and in
vitro labeled RNA were performed as described previously
(25). Briefly, 10 fmol of labeled RNA were incubated with different concentrations of purified nucleolin or recombinant proteins in 50 µl
of binding buffer (200 mM KCl, 25 mM Tris, pH
7.5, 5 mM MgCl2, 20% glycerol, 75 µg/ml
poly(A), 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.
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RESULTS |
Nucleolin Utilizes Different RNA-binding Domains to Interact with
Two Different RNAs--
We have previously shown that nucleolin
interacts with two different RNA targets, the NRE and the ECM motifs
(31, 34). The NRE motif is a short stem-loop structure found along the
ribosomal RNA, whereas the ECM is an 11-nt motif located 5 nt
downstream of the first processing site (34, 35). The first two
RNA-binding domains (R12) of nucleolin are required for the interaction
with the NRE sequence (24, 25). To determine whether the same
RNA-binding domains were involved in the specific recognition of the
ECM motif, we compared the interaction of the NRE and the ECM sequences
with several recombinant proteins, which contain different numbers of
nucleolin RBD (Fig. 1A). RNAs
that contain the NRE or the ECM motif were in vitro
transcribed in the presence of [
-32P]CTP. The NRE RNA
is 68 nt long and contains the stem-loop structure selected by SELEX
(31). RNA645/677 contains sequences 645-677 from mouse
ribosomal RNA that include the conserved ECM motif. We have previously
shown that interaction of nucleolin with this RNA requires the ECM
sequence (34). These labeled RNAs were used in a UV cross-linking
experiment in the presence of increasing amounts of R12 and R1234
proteins (Fig. 1B). After UV cross-linking and RNase
digestion, proteins were resolved on SDS-PAGE. As previously described
(24, 25), the NRE RNA interacts with R12 and R1234 with the same
efficiency (Fig. 1B, lanes 1-8). In contrast, no cross-linking was observed between RNA645/677 and R12
(lanes 9-11), whereas the R1234 protein interacts with this
RNA (lanes 12-14). This experiment suggests that different
combinations of RBDs are used for the specific interaction with
the NRE and the ECM motifs.

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Fig. 1.
Nucleolin utilizes different RBD combinations
for the interaction with the NRE and ECM RNAs. A,
schematic representation of the nucleolin deletion mutants used in this
study. RBDs are shown in gray boxes. The black
boxes indicate the (his)6 tag added at the N terminus
of each peptide. The Gar domain is indicated by a striped box at the
C-terminus of nucleolin. B, UV cross-linking assay.
Different amounts of recombinant R12 (lanes 1-4 and
9-11), R1234 (lanes 5-8 and 12-14),
R123 (lanes 15-17), or R234 (lanes 18-20) were
incubated with labeled in vitro transcribed NRE (lanes
1-8) or RNA645/677 (lanes 9-20). Protein
concentrations are 125 nM (lanes 1 and
5), 250 nM (lanes 2, 6, 9, 12, 15, and 18), 500 nM (lanes 3, 7, 10, 13, 16, and 19), and 1000 nM (lanes 4, 8, 11, 14, 17, and 20). After RNase A digestion, labeled
proteins were resolved on SDS-PAGE. C, filter binding assay.
Purified nucleolin or recombinant proteins were incubated
with labeled RNA645/677 at room temperature. After
filtration through a nitrocellulose filter, the retained, labeled RNAs
(in complex with the protein) were quantified using a
phosphorimager.
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To determine which RBDs are involved in the interaction with
RNA645/677, several recombinant proteins (see Fig.
1A) were produced in Eschrichia coli, purified,
and used in the UV cross-linking assay (Fig. 1B). Deletions
of the first (R234, lanes 18-20) or the fourth (R123,
lanes 15-17) RBD drastically reduced the cross-linking level
compared with R1234 (lanes 12-14). To further confirm this experiment, we compared the interaction of full-length nucleolin with
several recombinant proteins using a filter binding assay (Fig.
1C). Proteins and labeled RNAs were incubated at room
temperature and filtered through a nitrocellulose filter. Only the RNA
bound to the protein was retained on the filter, and the percentage of
bound RNA could be determined using a phosphorimager. Nucleolin and the
R1234 protein interact with the same affinity with RNA 645/677 (Kd of about 75 nM).
Deletions of the last RBD (R123), the last two RBDs (R12), or the first
two RBDs (R34) drastically reduced the interaction with this RNA. These
experiments show that all RBD domains of nucleolin are required for the
interaction with RNA645/677.
To further characterize the interaction of the four RBDs with this RNA
target, point mutations were performed within each RBD. A
characteristic feature of the RBD is the octamer RNP-1 motif that
contains several aromatic residues involved in RNA binding. The
presence of these aromatic residues is absolutely required for the
binding activity of most RBDs (25, 37). The mutation of these amino
acids to leucine residues in each RBD was performed (Fig.
2A), and each recombinant
protein was purified and used in a gel shift assay (Fig.
2B). Quantification of these gel shifts using a
phosphorimager is shown in Fig. 2C. Wild type protein
(R1234) interacts with high binding affinity (Kd 50 nM) with RNA645/677. Mutation of the RNP1 motif
in RBD1 and -4 (protein R1(LL)234 and R1234(LL), respectively,
drastically reduced the interaction with the RNA (Kd > 500 nM). This result is in agreement with data shown in
Fig. 1, where deletions of either RBD1 or RBD2 completely abolished the
interaction. Remarkably, mutations in RBD2 (R12(LL)34) or RBD3
(R123(LL)4) also drastically affect the interaction with the ECM.
Altogether, these data demonstrate that all four RBDs are required for
the specific interaction of nucleolin with RNA645/677.

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Fig. 2.
Mutations in each RNP1 motif abolish
interaction with the ECM RNA. A, schematic
representation of recombinant R1234 protein and of point mutations that
were introduced in each RNP1 domain. The black box at the N
terminus of the protein represents the (His)6 tag used for
the purification to homogeneity of each mutated protein.
B, gel shift analysis with the wild type (R1234)
and each mutated proteins with labeled RNA645/677. Protein
concentrations were 0, 25, 50 100, 250, 500, and 1000 nM
for each protein. C, RNA binding curves for the
different nucleolin mutants with RNA645/677. The legends
for the data points are shown on the figure.
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Interaction of Nucleolin with SELEX-selected Sequences--
A
SELEX experiment performed with nucleolin (31) identified the NRE motif
in 50% of the selected sequences. The remaining sequences show a
significant homology to the ECM motif and are efficient competitors for
the interaction of nucleolin with the ECM motif (34). To determine
whether the interaction of nucleolin with the selected sequences that
show homology to the ECM also required all four RBDs, one selected
sequence (N25-358) was used in a UV cross-linking experiment (Fig.
3) with several recombinant proteins and
was compared with the cross-linking results obtained with
RNA645/677. Deletion of the first (Fig.
3B, R234, lanes 11and 12) or
the last (R123, lanes 9 and 10) RBD drastically
reduced the interaction with RNA N25-358 compared with wild type R1234 protein (lanes 7 and 8). This experiment
shows that the integrity of all four RBDs of nucleolin is
required for interaction with the SELEX-selected sequence that presents
homology with the ECM motif. It further suggests that the 11 nt found
within this motif and partially in the selected sequence are key
determinants for the specific interaction of the four RBD of
nucleolin.

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Fig. 3.
Interaction of hamster nucleolin with
SELEX-selected sequences requires all four RBDs. A,
sequence alignment of RNA645/677 with one of the
SELEX-selected sequences (N25-358) that shows homology to the ECM
motif (see Ref. 34 for the full alignment). The ECM motif is
highlighted. The capital letters in N25-358
represent the 25 random sequences that have been selected by
SELEX. B, UV cross-linking assay between
RNA645/677 or N25-358 and the R1234, R123, or R234
proteins. Increasing amounts of proteins, 250 nM in
lanes 1, 3, 5, 7, 9, and 11 and 500 nM in lanes 2, 4, 6, 8, 10, and
12 were incubated with labeled RNA and subjected to UV
cross-linking as described under "Experimental Procedures." After
RNase A digestion, proteins were resolved on SDS-PAGE. The
arrows indicate the positions of the expected cross-link
with each peptide.
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Interaction of Nucleolin with the ECM Motif Is Conserved between
Hamster and Xenopus laevis--
Previous experiments have shown that
human, mouse, and hamster nucleolin interacted with the same binding
affinity and specificity with the NRE RNA (25, 35). This interaction
requires the joint action of the first two RBD, which are well
conserved in all these species. Although nucleolin RBDs are well
conserved overall between human, mouse, and X. laevis, some
significant differences can be found in RBD1 and -2 between human and
Xenopus (only 53% identity to the hamster protein) (29). To
determine whether the full-length or the first two RBD
(X.l.R12) from Xenopus interact with the NRE
motif, we performed a cross-link experiment with purified protein (data
not shown) or recombinant X.l.R12 protein and compared it
with the interaction of hamster R12 with the NRE (Fig.
4A). The full-length protein
or the first two RBD of nucleolin did not interact with the NRE motif,
indicating that key residues involved in the specific binding of the
NRE are not conserved in the Xenopus protein. In contrast,
nucleolin purified from X. laevis (34) or recombinant
protein containing all four RBDs of the Xenopus protein
(X.l.R1234) interacted with RNA645/677 (Fig.
4B). This interaction required the ECM sequence, because deletion of the 11-nt motif completely inhibited the interaction with
X.l.R1234 (Fig. 4B). To determine whether this
interaction required all four RBDs as for the hamster protein,
mutations in the RNP-1 motif of each RBD of the Xenopus
protein were performed. These recombinant proteins were purified and
used in the cross-link assay (Fig. 4C). Mutations within the
RNP-1 motif of RBD1 (R1(LL)234, lanes 4-6), RBD2
(R12(LL)34, lanes 7-9) and RBD4 (R1234(LL) lanes 13-15) had a drastic effect on the interaction with
RNA645/677. The mutation in RBD3 (R123(LL)4) had a weaker
effect (lanes 10-12), however, this is well correlated with
the binding affinity of hamster R123(LL)4 protein (see Fig.
2B). The apparent higher interaction of
X.l.R123(LL)4 with the ECM RNA might result from the
cross-linking assay compared with gel shift analysis for the hamster
protein.

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Fig. 4.
RNA binding specificity of
Xenopus nucleolin. A,
Xenopus nucleolin does not interact with the NRE motif.
Hamster RBD12 (R12) and Xenopus nucleolin RBD12
(X.l.R12) were incubated with labeled NRE RNA, subjected to
UV cross-linking, and then resolved on SDS-PAGE.
B, filter binding assay with X.lR1234
protein and labeled RNA541/677 and
RNA541/677 ECM. C, effect of RNP1
mutations in each RBD of Xenopus nucleolin on the
interaction with RNA645/677. Labeled RNA645/677
was incubated with increasing amount of each recombinant protein (250, 500, or 1000 nM) and submitted to the UV-cross-ling assay
as previously described.
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 |
DISCUSSION |
In this work we show that all four RBDs of nucleolin are involved
in the interaction with the evolutionary conserved RNA sequence (ECM)
located 5 nt downstream of the first processing cleavage site. Deletion
of the first or last RBD (Fig. 1) or mutation of residues within each
RNP-1 motif (Fig. 2) drastically reduced interaction with the ECM. The
ECM sequence is conserved between human, mice, and X. laevis
(38, 39). Interestingly, nucleolin purified from Xenopus is
able to activate the first processing step in vitro (34);
the interaction of the Xenopus nucleolin with the ECM also
requires all four RBD (Fig. 4). This conserved interaction of nucleolin
with the ECM sequence through evolution indicates that nucleolin plays
an important role in this first processing step.
Nucleolin Interacts with Several RNA Targets--
A SELEX
experiment performed with hamster nucleolin (31) has characterized
several RNA binding sequences. Half of these sequences form a small
stem-loop structure (NRE) with a short stem (5 base pairs) and a
7-10-nt loop containing the (U/G)CCCGA motif. Similar motifs are found
all along pre-rRNA, and interaction of nucleolin with these sequences
is believed to be involved in pre-ribosomal assembly (20, 35).
Nucleolin of hamster, mouse, and human origin bind to the NRE motif
with high affinity (25, 31, 35) (Kd 5-20
nM). However, nucleolin from X. laevis does not bind with this RNA structure (Fig. 4). The
first two RNA-binding domains of mouse nucleolin are necessary and
sufficient for the high binding affinity and specific interaction with
the NRE (24, 25). Interestingly, RBD1 and -2 of Xenopus
nucleolin are only 53% identical to the hamster sequence, whereas
human and mouse are about 85 and 96% identical, respectively, to the hamster protein (29). This finding suggests that key amino acids involved in the recognition of the NRE are not conserved within RBD1
and -2 of the Xenopus nucleolin. Indeed, structural analysis of the complex between hamster R12 and NRE has indicated that key amino
acids residues involved in the interaction with the NRE RNA are not
conserved in the Xenopus protein (20); this could explain
why the Xenopus R12 protein does not interact with the NRE.
Whether these RBDs are involved in the interaction with another target
remains to be determined.
The remaining sequences selected by SELEX showed significant homology
with the ECM motif found 5 nt downstream of the first processing site
of rRNA (31, 34). We have shown that the specific interaction of
nucleolin with the ECM motif is required for cleavage of the RNA 5 nt
upstream of this motif in vitro (33, 34). The binding
affinity of nucleolin with this RNA sequence (Kd 100 mM) is lower compared with the interaction with the NRE
sequence (Kd of 5-20 nM). Sequence
analysis of the RNA sequence around the ECM predicts that this motif is
in a single-stranded conformation (36, 39). Previous experiments
suggest that the interaction of nucleolin with this sequence is
required for the formation of the processing complex by recruiting the
different factors through interaction with the N- and C-terminal
domains of nucleolin (33, 34).
Multiple RBDs Are Involved in the Interaction with the ECM
Sequence--
Deletion and mutational analyses of nucleolin RBDs
(Figs. 2-4) show that all four nucleolin RBDs are involved in the
interaction with the ECM sequence. Aromatic residues present in the
RNP-1 sequence of the RBD contribute to RNA binding through
ring-stacking interactions with the bases (9, 40). Mutation of these
residues has been shown to drastically reduce the binding affinity of
the RBD (37, 41). It is remarkable that mutations of these residues in
each RBD of the hamster (Fig. 2) and Xenopus nucleolin (Fig. 4) drastically reduced the interaction with RNA645/677.
This suggests that all four RBDs are involved in the interaction with
the RNA and that this binding is conserved between hamster and
Xenopus. Cooperation of several RBDs to achieve specific
recognition of an RNA target seems to be a common feature of proteins
that contains several RBDs. Two RBDs in hnRNPA1, Sxl, ASF/SF2,
HuD, HuC, and poly(A) binding protein are involved in binding with an
RNA target (21, 40, 42-44). For U2AF65, three RBDs are
required for high affinity binding to the polypyrimidine tract.
(4) Nucleolin is the first example of a RNA-binding protein that
requires four RBD for the specific interaction with an RNA sequence.
The structural basis for the interaction of RBD with a single-stranded
RNA or a stem-loop structure has now been studied for a few RBD·RNA
complexes. These structures are from U1A (8, 17, 45) and U2B''
(18), which bind a stem-loop structure, sxl (12); poly(A)-binding
protein (19); and hnRNPA1 (16), which binds single-stranded RNA and the
structure of the first two RBD of nucleolin bound to the NRE motif
(20). These different structures highlight the important role of the
sheet surface and loop regions within or outside the RBD to
determine the specificity of the interaction. They also show that a
common domain, which is highly conserved in numerous proteins, can
adopt numerous conformations for specific recognition of many
different RNA structures. Structural studies of the interaction of the
four RBDs of nucleolin with the ECM RNA will certainly reveal a new mode of RNA protein interaction.
Another unique feature of nucleolin is its ability to use different
combinations of RBD to interact with different RNAs. The interaction of
nucleolin with the NRE and ECM motifs appears to be exclusive, because
RBD1 and 2 are required for both interactions, and we have been unable
to detect an interaction of both RNAs with the same nucleolin molecule
(data not shown). The interaction of nucleolin with the two different
RNA targets present on pre-rRNA suggests that nucleolin might play
different roles upon interaction with pre-rRNA. How nucleolin
interaction with the NRE and ECM motifs is regulated remains to be
determined, which will certainly reveal important aspects of protein
dynamics. Recent studies of RNA·protein complexes show that
interaction with RNA can induce conformational changes in the protein,
which can influence protein function (45-47). The interaction of
nucleolin with the different RNA targets might induce a selective
conformational change of the protein allowing access of the N- and
C-terminal domains, which are involved in protein-protein interactions.
Depending on these interactions, nucleolin will be incorporated in
different RNP complexes, which could explain its different functions.
 |
FOOTNOTES |
*
This work is supported by grants from the Association pour
la Recherche contre le Cancer (ARC), CNRS, and Fondation pour la Recherche Médicale (FRM).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. Tel.: 33-4-72-728016;
Fax: 33-4-72-728080; E-mail: pbouvet@ens-lyon.fr.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M011120200
 |
ABBREVIATIONS |
The abbreviations used are:
RBD, RNA-binding
domain;
RNP, ribonucleoprotein;
hnRNP, heteronuclear RNP;
pre-rRNA, precursor ribosomal RNA;
ETS, external transcribed spacer;
ECM, evolutionary conserved motif;
NRE, nucleolin recognition element;
nt, nucleotide(s);
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel
electrophoresis;
SELEX, systematic evolution of ligands by
exponentional enrichment.
 |
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