From the Departments of Molecular Biophysics and
Biochemistry and ¶ Genetics, Yale University School of
Medicine, New Haven, Connecticut 06510-8024
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ABSTRACT |
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Nucleolin is a very abundant eukaryotic protein
that localizes to the nucleolus, where the rDNA undergoes
transcription, replication, and recombination and where rRNA processing
occurs. The top (non-template) strand of the rDNA is very guanine-rich
and has considerable potential to form structures stabilized by G-G
pairing. We have assayed binding of endogenous and recombinant
nucleolin to synthetic oligonucleotides in which G-rich regions have
formed intermolecular G-G pairs to produce either two-stranded G2 or
four-stranded G4 DNA. We report that nucleolin binds G-G-paired DNA
with very high affinity; the dissociation constant for interaction with
G4 DNA is KD = 1 nM. Two separate
domains of nucleolin can interact with G-G-paired DNA, the four RNA
binding domains and the C-terminal Arg-Gly-Gly repeats. Both domains
bind G4 DNA with high specificity and recognize G4 DNA structure
independent of sequence context. The high affinity of the nucleolin/G4
DNA interaction identifies G-G-paired structures as natural binding
targets of nucleolin in the nucleolus. The ability of two
independent domains of nucleolin to bind G-G-paired structures
suggests that nucleolin can function as an architectural factor in
rDNA transcription, replication, or recombination.
Transcription and processing of rRNA occur within a specialized
subnuclear compartment, the nucleolus. In cells that are actively transcribing the rDNA, nucleoli appear to be composed of three compartments: the fibrillar center, which contains DNA that is not
being transcribed; the dense fibrillar component, where rDNA transcription occurs; and the peripheral granular component, where pre-rRNA processing and pre-ribosome assembly take place (1, 2). In
proliferating cells, RNA polymerase I (pol
I)1 and other components of
the transcription complex localize to the dense fibrillar component,
whereas molecules essential for rRNA processing, like fibrillarin and
the small nucleolar RNAs, localize to the peripheral granular component
(for review, see Refs. 3-5). The rate at which the rDNA is transcribed
in actively dividing cells is remarkable. Electron microscopic analysis
shows that during active rDNA transcription in metazoan cells, the
spacing between pol I complexes is only 100 base pairs (6).
One of the most abundant proteins in the nucleoli of vertebrate cells
is the highly conserved protein, nucleolin. Mammalian nucleolin is 709 amino acids in length and consists of an unusual grouping of sequence
and structural motifs (7-14). The N-terminal region of nucleolin
houses several long stretches of acidic residues with the potential to
function as "acid blobs" in activation of transcription (15). The
central region of nucleolin contains four RNA binding domains (RBDs;
also called RNA recognition motifs or RRMs). RBDs are common among
proteins that interact with single-stranded nucleic acids (16, 17), and
the RBDs of nucleolin are believed to mediate interactions of nucleolin
with RNA (18-22). The C terminus of nucleolin contains nine repeats of
the tripeptide motif arginine-glycine-glycine (RGG), in which the
arginine residues are dimethylated (23, 24).
The distribution of nucleolin within the nucleolus is unusual. Whereas
proteins like pol I and fibrillarin appear to be restricted to a single
compartment of the nucleolus, nucleolin is abundant within both the
dense fibrillar component and the granular component (for review, see
Ref. 4). The presence of nucleolin in the peripheral granular component
is consistent with the participation of nucleolin in rRNA processing
and ribosome assembly (19-22). The fact that nucleolin is abundant
within the dense fibrillar component suggests that nucleolin also
functions in other processes, including transcription, replication, or
recombination of the rDNA. Nonetheless, conserved and specific
interactions of nucleolin with the duplex rDNA have not been reported.
The rDNA transcription unit includes the regions that template mature
18, 5.8, and 28 S RNAs and external and internal transcribed spacer
regions (Fig. 1A). In all
eukaryotes, the entire transcribed region of the rDNA is very rich in
the base guanine (34.2% in humans) within the spacers, as well as
within the regions that template the mature rRNAs. The G-richness is
restricted to a single strand, the non-template strand, and most
guanines are within runs that contain three or more consecutive Gs
(Fig. 1B).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The human rDNA is G-rich. A,
the 13.4-kilobase human rRNA transcription unit. Regions that template
18, 5.8, and 28 S rRNAs are shaded; the external transcribed
spacers (ETS) and internal transcribed spacers
(ITS) are unfilled. B, graphical
representation of the occurrence of G-runs within the transcribed
region of the human rDNA. The 13.4-kilobase region shown in
A was searched for runs of three or more G residues using
the program FINDPATTERNS of the GCG suite of programs. The number of
G-runs per 500-base pair (bp) interval is shown. Both the
top (non-template) strand of the rDNA and the pre-rRNA transcript will
contain many runs of Gs, as shown.
Single-stranded DNAs that contain runs of three or more consecutive
guanine residues readily self-associate in vitro to form structures stabilized by G-G pairing (25-31). In these structures, guanines interact via Hoogsteen bonding to form planar rings called G
quartets (Fig. 2A), and the G
quartets stack upon each other to stabilize higher order structures
(Fig. 2B). That guanine-guanine interactions could occur
readily in solution was first established nearly 40 years ago (32).
Although G-G-paired DNA has not been directly observed in
vivo, G-G-paired structures form rapidly and spontaneously
in vitro and are very stable once formed. Because of its
sequence, the G-rich strand of the rDNA has considerable potential to
form G-G-paired structures (Fig. 2A). Formation of such
structures may be stimulated by the unwinding and localized denaturation that accompanies rDNA transcription.
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The observations presented above have led us to investigate the
interaction of nucleolin with G-G-paired DNA. Here we report that
mammalian nucleolin binds tightly and specifically to both four-stranded G4 DNA and two-stranded G2 DNA. The dissociation constant
for binding is KD = 1 nM, which
represents a remarkably high affinity for interaction of a eukaryotic
protein with nucleic acid. Mutational analysis shows that two separable
domains of nucleolin can bind G4 DNA, one comprised of the four RBDs
(RBD-1,2,3,4) and the other comprised of the C-terminal Arg-Gly-Gly
repeats (RGG9). These results suggest that G-G-paired DNA
is a natural binding target of nucleolin within the nucleolus.
Nucleolin may, therefore, be an architectural factor that functions to
organize the G-rich non-template strand of the rDNA during
transcription, replication, or recombination.
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EXPERIMENTAL PROCEDURES |
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Plasmid Construction-- The backbone for construction of deletion mutants of nucleolin was the plasmid pNuc-1,2,3,4-RGG9, which carries human nucleolin residues 284-709, including all four RBDs and the nine RGGs, fused at the N terminus to Escherichia coli maltose-binding protein. Construction of pNuc-1,2,3,4-RGG9 has been described previously (Ref. 14, where it was referred to as pMalNuc). Mutants were constructed as follows. For pNuc-1,2-RGG9, RBDs 1 and 2 (nucleolin residues 268-470) were polymerase chain reaction-amplified from the pNuc-1,2,3,4-RGG9 template with deoxyoligonucleotides 26937 (GCGGATCCAAAGCAGCTCCTGAAGCC) and 26938 (GCACCCTTAGGATTTTGACCTTTCTCTCC); the product was digested with BamHI and Bsu36I and ligated to pNuc-1,2,3,4-RGG9 cleaved with these enzymes. For pNuc-3,4-RGG9, the same procedure was used as for pNuc-1,2-RGG9, except that the polymerase chain reaction was carried out with deoxyoligonucleotides 26939 (CGGGATTCAATAGCACTTGGAGTGG) and 26940 (TGGTTCACCCTTAGGTTTGGC) to amplify RBDs 3 and 4 (nucleolin residues 478-647). For pNuc-1,2,3,4, pNuc-1,2, and pNuc-3,4 constructs, pNuc-1,2,3,4-RGG9, pNuc-1,2-RGG9, and pNuc-3,4-RGG9, respectively, were digested with Bsu36I and PstI, which excised the RGGs, and a synthetic linker made by annealing deoxyoligonucleotides TAAGTAAGTAGCTGATGCA and TCAGCTACTTAC was inserted to provide a stop codon. For pNuc-RGG9, pNuc-1,2,3,4-RGG9 was digested with BamHI and Bsu36I, 3'-filled, and relegated to delete residues 284-646. For pNuc-RGG4, pNuc-1,2,3,4-RGG9 was digested with BglI, the 5'-overhang was filled with Klenow, and a 94-base pair fragment was liberated by HindIII digestion and inserted into pNuc-1,2,3,4-RGG9 that had been digested with BamHI and 3'-filled. All clones involving polymerase chain reaction amplification were sequenced throughout the amplified region.
Protein Purification-- Full-length (106-kDa) murine nucleolin was purified starting with nuclear extract prepared from PD31 pre-B cells and chromatographed on heparin-agarose resin as described (33). Fractions containing nucleolin were identified at this and subsequent steps by blotting with anti-nucleolin antibodies (14). These fractions were dialyzed against Buffer L (10 mM Tris, pH 7.4, and 1 mM EDTA) containing 0.2 M NaCl, 0.1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride, applied to a Hi-Trap Q (Amersham Pharmacia Biotech) column, and eluted with a 0.2-1.0 M NaCl gradient in Buffer L. Fractions containing nucleolin were dialyzed against Buffer B (10 mM Hepes, pH 7.8, 1 mM EDTA) containing 0.2 M NaCl, applied to a Hi-Trap SP (Amersham Pharmacia Biotech) column, and eluted with a 0.2-1.0 M NaCl linear gradient in Buffer B. Nucleolin-containing fractions were dialyzed against Buffer L containing 0.2 M NaCl, applied to polyguanosine-agarose resin (Sigma), and eluted with Buffer L containing 1.0 M NaCl.
All recombinant proteins were produced by overexpression as described previously (14). As the final step in purification, fusion proteins that contained RGG9 domains were applied to Mono S and eluted with a 0.05-1.0 M NaCl linear gradient in Buffer B. Other proteins were fractionated instead by Mono Q chromatography and eluted with a 0.05-1.0 M NaCl linear gradient in Buffer L. All purified fusion proteins chromatographed as single species on SDS-polyacrylamide gel electrophoresis. Concentrations of proteins were determined by Bradford microassay (Bio-Rad).
Formation of G-G-paired DNAs-- Sequences of oligonucleotides used in binding analyses were: ETS-1, TCTCTCGGTGGCCGGGGCTCGTCGGGGTTTTGGGTCCGTCC; OX-1, ACTGTCGTACTTGATATTTTGGGGTTTTGGGG; TP, TGGACCAGACCTAGCAGCTATGGGGGAGCTGGGGAAGGTGGGATGTGA; and TP-S, AGACCTAGCAGCTATGGGGGAGCTGGGGTATA.
Formation of G-G-paired DNAs was carried out as described by Sen and
Gilbert (25, 26, 34) with minor modifications. Briefly, synthetic
oligonucleotides were incubated at 2-3 mg/ml in TE (10 mM
Tris-HCl, pH 7.4, 1 mM EDTA) containing 1 M
NaCl for G4 DNA formation or 1 M KCl for G2 DNA formation
at 60 °C for 48 h. After incubation, samples were diluted 1:5
with 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 12.5 mM KCl, and 2.5% glycerol, and DNAs resolved on an 8%
nondenaturing polyacrylamide gel (29:1, polyacrylamide:bisacrylamide),
run in 0.5× TBE (50 mM Tris borate, pH 8.2, 0.5 mM EDTA) containing 10 mM KCl at 4 °C at
5-8 V/cm. Bands corresponding to G4 DNA, G2 DNA, and single-stranded
DNA were identified according to their relative mobility by
UV-shadowing or autoradiography and excised. DNAs were eluted from the
crushed gel slices by soaking in TE containing 50 mM NaCl
and 20 mM KCl at room temperature for 8-12 h, precipitated
with ethanol, washed, and stored at 20 °C. G4 DNAs were 5'
end-labeled with T4 polynucleotide kinase (New England Biolabs), and
G-G pairing was verified by assaying characteristic protection of the
guanine N-7 from methylation with dimethylsulfate (35).
DNA Mobility Shift Analysis and Measurements of Binding
Affinities--
Binding to G4 DNA and G2 DNA was carried out in
15-µl reactions containing 10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 100 µg/ml bovine serum
albumin, and 1 fmol of 32P-labeled DNA for 30 min at
37 °C, glycerol was added to a final concentration of 5% (w/v), and
complexes were resolved by gel electrophoresis on 6% (29:1,
acrylamide:bisacrylamide) 0.5× TBE gels at 5 V/cm for 10 h at
4 °C. Affinities were estimated by gel mobility shift assays in
which binding to a fixed amount of G4 DNA was assayed in the presence
of increasing amounts of protein. Protein-DNA complex formation was
quantitated by PhosphorImager analysis of the dried gels, and
KD values were calculated by plotting the
fraction of bound DNA at each protein concentration. Reported KD values are averages from at least three
separate experiments. To verify the very low KD
values for G4 DNA interactions, assays were performed at three DNA
concentrations, 330 fM, 3.3 pM, and 33 pM; the apparent KD was the same at
all concentrations.
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RESULTS |
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Binding of G4 DNA by Endogenous Nucleolin-- G4 DNA forms spontaneously in solutions of G-rich synthetic oligonucleotides, but to form G4 DNA at very high yield, synthetic oligonucleotides were incubated at high concentrations at 60 °C for 48 h (32). Under these conditions, over 90% of the starting material typically formed G4 DNA. G4 DNA formation was verified in all cases by dimethyl sulfate footprinting (35). Fig. 2C shows a typical footprint obtained by probing G4 DNA formed from the ETS-1 oligonucleotide. This 40-mer derives from a sequence in the 5'-ETS region of the human rDNA and represents one of many regions in the rDNA that will readily form G-G-paired structures in vitro. Structures of other G-G-paired DNAs used in binding assays were similarly verified (data not shown).
Nucleolin was purified from nuclear extracts of murine PD31 pre-B
cells. The protein preparation was shown to be homogeneous by silver
staining, and the identification of the 106-kDa polypeptide as
nucleolin was confirmed by Western blot analysis with anti-nucleolin antibodies (Fig. 3A). The
ability of nucleolin to bind G4 DNA was assayed by gel mobility shift
using G4 DNA formed from the ETS-1 oligonucleotide. Binding analysis
showed that full-length mammalian nucleolin binds very tightly to G4
DNA formed from the ETS-1 oligonucleotide: KD = 1 nM (Fig. 3B). Similar results were obtained
with G4 DNA generated from other oligonucleotides (not shown).
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Binding of G4 DNA by Recombinant Nucleolin--
Full-length
nucleolin cannot be expressed in E. coli, but deletion of
the N terminus permits good expression of recombinant protein (14). The
Nuc-1,2,3,4-RGG9 fusion protein (nucleolin residues
284-709), which carries RBDs 1, 2, 3, and 4 and the RGG9 domain, was assayed for interaction with G4 DNA formed from the ETS-1
oligonucleotide and shown to bind this G4 DNA with
KD = 0.5 nM (Fig.
4A). Binding produced two
shifted complexes of distinct mobilities, which probably represent
interaction of more than one polypeptide with each G4 DNA substrate,
via protein-DNA or protein-protein interactions.
Nuc-1,2,3,4-RGG9 bound comparably with G4 DNA formed from
the ETS-1 oligonucleotide and other oligonucleotides (data not shown).
Incubation of G4 DNA with nucleolin did not permanently alter DNA
structure, because following addition of SDS and proteinase K to the
binding reaction, all DNA migrated as free G4 DNA (not shown). MBP did
not bind G4 DNA (KD 40 nM) (Fig. 4B).
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Both full-length mammalian nucleolin and recombinant nucleolin (residues 284-709) therefore bind G4 DNA with high affinity. Nucleolin undergoes extensive posttranslational modifications, including phosphorylation and dimethylargininylation (36-38). The high affinity binding of recombinant nucleolin shows that these modifications are not essential for interaction with G-G-paired DNA.
The Nucleolin RBD-1,2,3,4 Domain Binds G4 DNA and G2
DNA--
To identify the domains of nucleolin that interact with
G4 DNA, we began by separating the domain comprised of the four RBDs from the C-terminal RGG9 domain. We assayed binding to G4
DNA by recombinant Nuc-1,2,3,4, which carries RBDs 1, 2, 3, and 4. Nuc-1,2,3,4 bound to G4 DNA (Fig. 5). The
dissociation constant for this interaction (KD = 0.5 nM) is comparable to that of
Nuc-1,2,3,4-RGG9 binding to G4 DNA (Fig. 4). G-rich DNAs
can form several different structures, including G4 DNA, in which G-G
pairing stabilizes interactions between four parallel strands, and G2
DNA, in which two strands associate in antiparallel orientation (for
review, see Ref. 39; see also Fig. 1B). Nuc-1,2,3,4 bound to
G2 DNA with affinity similar to, but slightly lower than, that observed in assays of G4 DNA binding (Fig. 5).
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RGG9 Binds G4 DNA--
The
41-amino acid C-terminal region of nucleolin is comprised of nine
repeats of the motif RGG. Nuc-RGG9, which expresses the
RGG9 domain as a chimeric MBP-fusion protein, bound G4 DNA with KD = 3.3 nM (Fig.
6A). The RGG9
domain, therefore, comprises a second and independent high affinity G4
DNA binding domain. Competition experiments carried out in the presence
of cold competitor G4 DNA or single-stranded DNA showed that G4 DNA
effectively competed for binding, whereas the single-stranded
oligonucleotide had no effect, even at 1000-fold molar excess (Fig.
6B). Additional binding and competition studies demonstrated
that recombinant Nuc-RGG9 does not bind duplex DNA or
single-stranded DNA (KD > 1 µM;
data not shown) and that deletion of five of the nine RGG repeats
(Nuc-RGG4) abolished G4 DNA interaction (Fig.
7). The RGG9 domain of
nucleolin binds comparably to G4 DNAs formed from other synthetic
oligonucleotides and, thus, appears to recognize G4 DNA structure
independent of sequence context.
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RBD-3,4 Combines with RGG9 to Produce a High Affinity G4 DNA Binding Domain-- Having identified RBD-1,2,3,4 and RGG9 as separable G4 DNA binding domains, additional deletion analysis was carried out in an attempt to define smaller subdomains capable of high affinity interaction with G4 DNA. Binding assays were carried out with eight different deletion mutants, expressed in E. coli as chimeric MBP fusion proteins, and purified to homogeneity. Results of these experiments, summarized in Fig. 7, showed that Nuc-3,4-RGG9, which carried RBDs 3 and 4 and the RGG9 domain, bound G4 DNA with high affinity (KD = 0.5 nM). Binding affinity was decreased 4-fold (KD = 2 nM) when RBDs 1 and 2 were substituted for RBDs 3 and 4 to produce Nuc-1,2-RGG9.
The importance of the RGG9 domain in G4 DNA recognition is
reinforced by the observation that whereas the Nuc-3,4-RGG9
chimera-bound G4 DNA with relatively high affinity, deletion of
RGG9 to produce Nuc-3,4 resulted in a complete loss of
binding (KD > 40 nM). Similarly, Nuc-1,2 was not active in G4 DNA binding (KD > 40 nM). Finally, complete loss of G4 DNA binding occurred
when the RGG9 region was truncated by deletion of the
N-terminal five RGG repeats to create MBP-RGG4 (Fig.
7).
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DISCUSSION |
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We have shown that the abundant nucleolar protein, nucleolin, binds G-G-paired DNA with very high affinity (KD = 1 nM). Nucleolin can bind to both four-stranded G4 DNA and two-stranded G2 DNA, and nucleolin recognizes G-G-paired structures independent of sequence context. The remarkably high binding affinities suggest that G-G-paired structures are binding targets of nucleolin in vivo. The observation that nucleolin binds G-G-paired structures independent of sequence context shows that this protein will be able to bind G-G-paired structures wherever they might form within the G-rich rDNA.
Dynamic Formation of G-G-paired DNA in the Nucleolus-- Most nuclear DNA is double-stranded, and complementary base pairing will normally protect duplex DNA from forming G-G-paired structures. However, duplex DNA becomes transiently single-stranded during three critical and dynamic processes: transcription, replication, and recombination. Cells have developed sophisticated mechanisms to prevent DNA from adopting alternative structures, including a variety of proteins that bind to transiently exposed single-stranded regions. Nonetheless, these mechanisms are not foolproof. For example, there is considerable evidence that triplet repeat expansion results from formation of non-Watson-Crick structures during replication (see Ref. 40 and references therein).
The sequence composition and the strand asymmetry of the rDNA provide it with considerable potential to form G-G-paired structures. The rDNA is G-rich on the top (non-template) strand, not only within the region transcribed into pre-rRNA but also within the spacers (Fig. 1). During active transcription, pol I molecules pack at extremely high density on the rDNA repeats; electron micrographic analysis shows that the spacing between pol I complexes is only 100 base pairs (6). Transcription at this level requires that a considerable fraction of the rDNA duplex be denatured. We hypothesize that G-G-paired structures form within the G-rich top strand of the rDNA during transcription or when the duplex is transiently denatured during replication or recombination. G-G-paired structures are very stable once formed (26) and would not be predicted to dissociate spontaneously in vivo.
Other experiments provide further support for the notion of a dynamic process of formation and unwinding of G-G-paired structures within the active rDNA. We have recently shown that G-G-paired DNA is the preferred substrate of two eukaryotic helicases, the human BLM helicase, which is deficient in Bloom's syndrome (41), and the Saccharomyces cerevisiae Sgs1p helicase (42). Both these helicases are members of the highly conserved RecQ helicase family. Moreover, S. cerevisiae Sgs1p localizes predominantly to the nucleolus (43, 44), where it could function to maintain the structure of the G-rich rDNA. The human functional homolog of Sgs1p in S. cerevisiae appears to be the WRN helicase (deficient in Werner's syndrome). Like Sgs1p, WRN is a RecQ family helicase that is predominantly nucleolar in localization (45, 46). Unwinding activity mapped to the conserved helicase core domain of Sgs1p (42), strongly suggesting that preferential activity on G-G-paired substrates may be a general property of helicases in this family. It is therefore very likely that WRN will also prove to be active on G-G-paired rDNA substrates.
Nucleolin as an Architectural Factor in rDNA Transcription, Replication, or Recombination-- Two separable domains within nucleolin can bind G-G-paired structures, one comprised of the RBDs 1, 2, 3, and 4 and the other comprised of the C-terminal RGG9 domain. The presence of two independent G-G DNA binding domains would contribute to the ability of nucleolin to organize G-G-paired regions. Nucleolin may thus be an architectural factor, in effect forming a scaffolding for the structured G-rich strand. The presence of long acidic runs in the N terminus of nucleolin is consistent with its function in transcription, but nucleolin is a complex molecule with multiple distinct domains, and it may have multiple functions. We have identified nucleolin as one component of a heterodimeric protein, LR1, induced specifically in B cells activated for immunoglobulin heavy chain switch recombination (14, 33, 47). The rDNA repeats must undergo active recombination to maintain homogeneity of this gene family, and one function of nucleolin may be to stimulate or regulate recombination of the rDNA.
Nucleolin in the Nucleolus-- Nucleolin is abundant in the peripheral granular component of the nucleolus, where rRNA processing occurs, and also in the central dense fibrillar component of the nucleolus, where rDNA transcription occurs (for review, see Ref. 4). Reported functions of nucleolin in rRNA processing (21) and ribosome assembly (24) are consistent with its presence in the nucleolar peripheral granular component. Function in rDNA transcription, replication, and/or recombination is consistent with the observed localization of nucleolin within the nucleolar central dense fibrillar component. The N terminus of nucleolin contains long acidic regions of as many as 38 aspartate and glutamate residues in an uninterrupted stretch, which could function as acid blobs (15) to activate transcription by pol I. The N terminus of nucleolin also contains sites for the mitosis-specific cdc2 kinase (38) and casein kinase II (36, 37). Both of these kinases phosphorylate histone H1, and they could analogously regulate nucleolin in response to cell cycle-dependent controls.
Many proteins have been identified which contain RBDs and RGG motifs, but the mutational analysis of nucleolin makes it unlikely that high affinity binding to G-G-paired DNA is a common property of all RBD/RGG proteins. Most RBD-containing proteins contain only two or three RBDs, and deletion of two of the RBDs of nucleolin to produce Nuc-1,2, Nuc-2,3, or Nuc-3,4 greatly diminished binding affinity (Fig. 7). Similarly, whereas many proteins contain RGG motifs, nucleolin is unusual in that it contains nine repeats of the RGG motif, and deletion analysis showed that Nuc-RGG4 does not bind G4 DNA.
The broad nucleolar distribution of nucleolin has led to considerable
interest regarding its mode of localization within the nucleolus. The
two domains of nucleolin that bind G-G-paired DNA (RBD-1,2,3,4 and
RGG9) are also essential for nucleolar localization (48-50), whereas the N-terminal acidic region is dispensable. The ability to interact with G-G-paired nucleic acids may,
therefore, be essential to localization or retention of nucleolin
within the nucleolus.
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ACKNOWLEDGEMENT |
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We are grateful to Dr. W. P. Russ for invaluable discussions.
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FOOTNOTES |
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* This research was supported by National Institutes of Health Grants R01 GM39799 and P01 CA16038 (to N. M.) and a Ford Foundation postdoctoral fellowship (to L. A. H.).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.
§ Present address: Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms Herts. EN63LD, United Kingdom.
To whom correspondence should be addressed: Depts. of
Molecular Biophysics and Biochemistry and Genetics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510-8024. Tel.: 203-432-5641; Fax: 203-432-3047; E-mail: nancy.maizels{at}yale.edu.
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ABBREVIATIONS |
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The abbreviations used are: pol I, polymerase I; RBD, RNA binding domain; MBP, maltose-binding protein; ETS, external transcribed spacer.
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