From the Howard Hughes Medical Institute and Department of Biochemistry & Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148
Received for publication, November 9, 2000, and in revised form, December 7, 2000
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ABSTRACT |
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The heterogeneous nuclear ribonucleoprotein
(hn- RNP) C proteins, among the most abundant pre-mRNA-binding
proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding
domain. The RNA-binding domain (RBD) is comprised of ~80-100 amino
acids, and its structure has been determined. However, relatively
little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are
essential for its binding to RNA. The identified mutants were further
tested for binding to poly(U)-Sepharose, a substrate to which wild type
hnRNP C1 binds with high affinity. We found both previously predicted,
highly conserved residues as well as additional residues in the RBD to
be essential for C1 RNA binding. We also identified three mutations in
the leucine-rich C1-C1 interaction domain near the carboxyl terminus of
the protein that both abolished C1 oligomerization and reduced RNA
binding. These results demonstrate that although the RBD is the primary
determinant of C1 RNA binding, residues in the C1-C1 interaction domain
also influence the RNA binding activity of the protein. The
experimental approach we described should be generally applicable for
the screening and identification of amino acids that play a role in the
binding of proteins to nucleic acid substrates.
The heterogeneous nuclear ribonucleoprotein
(hnRNP)1 proteins are a group
of about 20 abundant proteins that bind nascent RNA polymerase II
transcripts and are involved in various aspects of pre-mRNA
processing, mRNA transport, and mRNA metabolism (1-4). Among
them, the hnRNP C1 protein is one of the most avid pre-mRNA-binding proteins, and it has been shown to preferentially bind to uridine-rich RNA sequences (5-9). In vitro selection/amplification from
pools of random sequence RNA (SELEX procedure) demonstrated that C1 binds avidly to sequences containing a stretch of five or more uridines, its high affinity "winner sequence," with an apparent dissociation constant (Kd) of about 170 nM (10). Recent studies have reported that C1 protein also
binds specifically and with high affinity to several U snRNAs (11, 12).
C1 also binds U6 snRNA, which contains an elongated uridylated stretch at the 3' end and induces disruption of U4:U6 snRNA base pairing (13).
The RNA binding activity of hnRNP C1 has been thought to be mainly
conferred by its single RNP motif RNA-binding domain (RBD) comprising
the amino-terminal 94 amino acids (9, 10). The RBD (also referred to as
RRM, which stands for RNA-recognition motif) is the most
prevalent RNA-binding motif in eukaryotes (1, 14-20). It is an
evolutionarily conserved domain present in pre-mRNA-, mRNA-,
pre-rRNA-, and snRNA-binding proteins, including hnRNP proteins,
splicing factors, and polyadenylation factors (1, 15). The RBD is
comprised of ~80-100 amino acids in which two consensus sequences,
an octapeptide termed RNP1 and a hexapeptide termed RNP2, about 30 amino acids apart, and many other hydrophobic amino acids are
particularly highly conserved (15, 17, 18, 20). The RBD is folded into
a compact domain structure of More recent studies, however, have shown that C1 lacking the canonical
RBD retained considerable RNA binding in vitro to U1, U2,
and U6 snRNA as well as to its SELEX winner sequence (12, 29). It was
suggested that instead of the RBD, the major determinant for C1 RNA
binding is a highly basic domain that consists of residues from
Val140 to Asn161 and immediately precedes a
leucine zipper motif. The zipper motif forms a coiled-coil structure
that mediates C1 oligomerization. In general, the basic zipper motif
present in C1 protein is reminiscent of the DNA-binding bZIP motifs
found in many transcription factors. However, compared with the
essential role of the RBD, the importance of bZIP motif in RNA binding
by intact C1 has not been fully established, because site-directed
mutagenesis failed to generate expressible proteins for functional
assays (12, 29). Here we describe a method to systematically identify,
in the context of full-length C1 protein, amino acids that are
essential for RNA binding. It is based on a functional screening of a
randomly mutagenized C1 expression library constructed in phage. This
screen identified many conserved and thus expected residues in the RBD
to be essential for C1 RNA binding. It also implicated previously
unidentified amino acids, particularly, residues in the C1-C1
interaction domain (CID), to influence its binding to RNA.
Additionally, these identified mutations in CID resulted in a defect in
C1-C1 interaction, indicating a connection between the ability of C1 to
form oligomers and the RNA binding activity of this protein. This
functional screening method should be generally applicable to any
protein of interest to identify amino acids that are required for the
binding to its cognate nucleic acid or protein ligands.
Construction of the hnRNP C1 Mutant Library--
To generate
random point mutations in the coding region of the hnRNP C1 protein,
error-prone PCR was performed using the standard protocol according to
Leung et al. (30) with several modifications. The entire
coding region of human hnRNP C1 cDNA (873 base pairs) was amplified
by using the plasmid pHC12 (31) as the template and by using primers
5'-TCGAATTCGATGGCCAGCAACGTT-3' and 5'-CAGGCTCGAGACCCCACTATGTGCTTAA-3', which contain EcoRI or XhoI restriction enzyme
site, respectively. Mutation frequency was estimated to be about
0.25-0.4% when using the following PCR conditions. Reaction mixtures
(100 µl) contained 10 ng of template, 80 pmol of each primer, 1 mM of each dNTP, 16.6 mM
(NH4)2SO4, 67 mM
Tris-HCl, pH 8.8, 6.1 mM MgCl2, 6.7 µM EDTA, pH 8.0, 0.17 mg/ml bovine serum albumin, 10%
dimethyl sulfoxide, 10 mM Screening the Mutant Library and Identifying Mutations--
To
screen the mutant library, Escherichia coli XL-1 Blue
bacteria were infected at ~200 plaque-forming units/plate and plated onto 50 LB plates (100 mm) with top agarose. After incubation at
37 °C for 4 h, each plate was overlaid with a nitrocellulose filter impregnated with 10 mM
isopropylthio- Production of Wild Type and Mutant C1 Proteins--
Wild type
and mutant hnRNP C1 proteins were produced in vitro using
TnT T7/T3 polymerase coupled rabbit reticulocyte lysate system
(Promega) in the presence of [35S]methionine (Amersham
Pharmacia Biotech) according to the manufacturer's protocols. The
constructs used for TnT were either in pBluescript SK( Site-directed Mutagenesis--
The MT2N mutant was constructed
using PCR to generate two partial COOH-terminal fragments. The more 5'
terminal of these fragments spanned the unique BsrGI site in
C1. The 3' primer for this fragment generated the V194N mutation and a
silent mutation that formed an XhoI site at amino acids 198 and 199. The second fragment was amplified by PCR to contain the
same engineered XhoI site, the L201N mutation, and the
remaining COOH-terminal end of C1 followed by an XbaI site.
These fragments were digested with BsrGI-XhoI and
XhoI-XbaI, respectively, and both were
subsequently inserted into BsrGI-XbaI cleaved
pcDNA3-Myc-C1 plasmid (33) that contained the amino-terminal
portion of the C1 cDNA. The mutations were confirmed using the
Sequenase version 2.0 DNA sequencing kit (United States Biochemicals).
Ribonucleotide Homopolymer Bead Binding Assays--
Binding of
in vitro transcribed and translated hnRNP C1 and mutants to
AGpoly(U) type 6 beads (Amersham Pharmacia Biotech) was carried out as
previously described (6). 5 µl of in vitro produced
proteins were used in each binding reaction. The poly(U)-binding buffer
contains 10 mM Tris-HCl, pH 7.5, 2.5 mM
MgCl2, 0.5% Triton X-100, 1 µg/ml pepstatin A, 1 µg/ml
leupeptin, 0.5% aprotinin, and various concentrations of NaCl as indicated.
In Vitro Protein Binding Assays--
In vitro protein
interaction assays were carried out as previously described (34).
Briefly, purified GST or GST-C1 proteins (2 µg) bound to 30 µl of
glutathione-Sepharose 4B resin (Amersham Pharmacia Biotech) were
incubated with 5 µl of in vitro translated protein in 500 µl of binding buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM EDTA, 0.1% Nonidet P-40, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 0.5% aprotinin).
Following incubation at 4 °C for 1 h, the resin was washed five
times with 1 ml of binding buffer. Bound proteins were then eluted in
SDS-PAGE sample buffer, separated by 12.5% SDS-PAGE, and visualized by fluorography.
In Vitro Transcription and Gel Mobility Shift
Assays--
In vitro transcription and purification of U2
snRNA were done as previously described (35). The
32P-labeled and polyacrylamide gel-purified RNA probe
(2 × 104 cpm) was incubated with the indicated
recombinant His-tagged C1 proteins (25-250 nM) in 20 µl
of reaction mixture containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mg/ml yeast RNA (Sigma), and 5% glycerol at
room temperature for 20 min. Reaction mixtures were loaded onto 4%
nondenaturing polyacrylamide gels with 1/4× TAE buffer (40 mM Tris-acetate, pH 7.5, 1 mM EDTA) that
had been prerun at 100 V for 1 h. Gels were run at 100 V until
bromphenol blue (loaded into an empty lane) migrated about 10 cm into
the gel. The gel was then dried for autoradiography.
Protein Cross-linking and Western Blot Analysis--
For
cross-linking, gel mobility shift reactions were prepared with cold RNA
probe, but instead of loading onto gels, glutaraldehyde (final
concentration, 0.01%) was added, and the reaction mixtures were
incubated at room temperature for 30 min. Reactions were stopped by
adding Identification of RNA-binding Mutants of hnRNP C1--
To identify
hnRNP C1 mutants that do not bind RNA, a
The phagemids containing mutated hnRNP C1 coding regions were in
vivo excised out of the
To verify that the mutants scored by the filter binding screening of
the library indeed produced RNA binding defective hnRNP C1, the mutant
C1 proteins were produced by in vitro transcription and
translation and tested for binding to poly(U) beads at 0.1 M NaCl. The results of the binding assays for several of
these mutants are shown in Fig. 2.
Compared with wild type C1 protein, mutants F19S and Q56H completely
abolished poly(U) binding, and mutants S16F and G51Y significantly
reduced binding. These results demonstrate that the filter binding
screening successfully identified RNA binding defective mutants of
hnRNP C1.
The various C1 mutants we characterized, and their relative poly(U)
binding avidity is listed in Fig.
3B. The positions of the
mutations on the
Our library screening identified amino acid changes that have not been
previously predicted to be involved in the interaction with RNA. For
example, multiple mutations were observed at Val28
(V28(G/I)) and at Asp71 (D71(V/G)). These residues are
located at the termini of the loop structures connecting the The Effect of Point Mutations in CID on hnRNP C1-C1
Interaction--
In isolated HeLa nuclear hnRNPs, the C1 and C2
proteins are present at a stoichiometry of (C1)3C2 possibly
as tetramers (40). Bacterially expressed C1 proteins spontaneously form
C1 tetramers (41). CD spectra analysis of C1 deletion mutants revealed
that a leucine-rich coiled-coil domain
(Leu180-Glu207) mediates C1-C1 interaction
(29). Using the yeast two-hybrid system, we have also mapped CID to the
same region.4 However, it has
not been previously known that in the context of full-length C1
protein, the coiled-coil motif is a determinant of C1 protein-protein
interaction. Because three point mutations (L187Q, Q192P, and L201P)
found in the search for RNA binding defective mutant C1 were located
within the CID, we tested the effect of these mutations on C1-C1
interaction. CID is predicted to be comprised of four heptad repeats
(Fig. 4A). Hydrophobic residues at positions 1 and 4 likely form the hydrophobic inner core of
the coiled-coil. Mostly charged residues at other positions likely form
the polar outer surface. The L187Q and L201P mutations change the
hydrophobic leucine residue to the polar residue glutamine or to
proline, respectively. The Q192P mutation changes the amino acid from
polar to aliphatic. We also engineered a mutant (MT2N) that changes two
hydrophobic residues (Val194 and Leu201) to
polar residues by site-directed mutagenesis. All these mutations are
predicted to disrupt the The Effect of C1 Oligomerization on RNA Binding Activity--
The
finding of C1 protein-protein interaction defective mutations in the
screening for RNA binding defective C1 prompted us to examine C1
oligomer RNA binding by gel mobility shift analysis. We first
bacterially expressed and purified His-tagged wild type and mutant C1
proteins (Fig. 5A). The Q56H
mutant has a mutation in the RBD that completely abolished
poly(U)-binding in the in vitro bead binding assay (Fig. 3).
The L187Q mutation is located in the CID, and it disrupted C1-C1
protein interaction in vitro (Fig. 4). Previous studies have
shown that hnRNP C protein tetramer binds U1, U2, and U6 snRNAs with
high affinity (11-13). Therefore, U2 snRNA was transcribed in
vitro in the presence of [32P]UTP and used in gel
mobility shift assays. As shown in Fig. 5B, wild type C1
readily bound the U2 snRNA probe. In contrast, the Q56H mutant did not
bind U2 snRNA under the same conditions. Furthermore, the Q56H mutant
was unable to bind Ad2 pre-mRNA (42) under similar gel mobility
shift conditions (data not shown). These results further confirmed that
the RBD is the primary determinant of C1 RNA binding activity, because
a single mutation in the RNP1 consensus motif abolished C1 binding to U
snRNA, pre-mRNA, and poly(U) homopolymers. When L187Q mutant C1 was
used at low concentration, a faster migrating RNA-protein complex than
that of wild type C1 was detected (Fig. 5B). As the amount
of L187Q protein increased, we observed a gradual increase in the size
of the shifted RNA-protein complex. This is likely due to binding of
multiple C1 molecules on one RNA at higher protein concentrations.
Nonetheless, the L187Q-U2 snRNA complex is smaller than that of the
wild type C1, mostly likely because of the inability of L187Q to form
oligomers on RNA.
To examine C1 oligomerization under gel mobility shift conditions, we
used glutaraldehyde as a cross-linking reagent (36) and then analyzed
C1 protein oligomer formation by SDS-polyacrylamide gel electrophoresis
followed by Western blotting with 4F4 (Fig. 5C). At a final
concentration of 125 or 250 nM, both wild type C1 and the
L187Q mutant migrated at about 45 kDa, which corresponds to the size of
the monomeric C1 as observed when no cross-linking reagent was added.
For wild type C1 cross-linked with 0.01% glutaraldehyde, the band
representing the monomer form dramatically diminished, whereas a band
corresponding to the oligomeric form appeared. The size of the
oligomerized C1 indicates the formation of C1 tetramers or even larger
oligomers. Under the same cross-linking conditions, however, L187Q
mutant C1 is almost exclusively monomeric. Taken together with the gel
shift analysis shown in Fig. 5B, these results suggest that
wild type C1 binds RNA as an oligomer, whereas L187Q mutant binds RNA
as a monomer.
The fact that the L187Q mutant is able to bind U2 snRNA with similar
apparent affinity as the wild type C1 protein raised the question of
why we found L187Q as an RNA binding defective mutant in the library
screening. To address this issue, we produced wild type C1 and mutant
C1 (L187Q and Q192P) by in vitro transcription and
translation, and performed poly(U) bead binding assay under more
stringent conditions. The ability to bind poly(U) even at 2 M NaCl is a characteristic property of C1, which
distinguishes C1 and C2 as high affinity specific binders of poly(U)
from the other major hnRNP proteins (6). Consistent with this, we found that wild type C1 bound to poly(U) equally well at 0.1, 1, and 2 M salt concentrations (Fig.
6). For mutants L187Q and Q192P, RNA
binding was similar to that of the wild type at 0.1 M NaCl. However, the RNA binding of these mutants was significantly reduced at
1 M NaCl and completely abolished at 2 M NaCl
(Fig. 6). These findings show that mutations in CID reduce C1 RNA
binding under high salt conditions and indicate a role for CID in the
RNA binding activity of this protein.
Compared with site-directed mutagenesis and deletional mutagenesis
approaches, random PCR mutagenesis coupled with phage display screening
provides an easy and unbiased way to quickly identify essential amino
acid residues that are involved in RNA binding in the context of
full-length C1 protein. Although other mutagenic chemicals can be used
(43), the production of a large pool of random mutations by use of PCR
with Taq DNA polymerase under essentially standard reaction
conditions is extremely simple and efficient (37). It has been reported
that the mutagenesis conditions can be modified to produce the desired
frequency of mutations from 0.25 to 2% within a specific region that
ranges from 200 to 1,200 base pairs (30, 37). The 4F4 epitope, near the
carboxyl terminus of hnRNP C1, provided a convenient marker for the
expression of full-length or near full-length C1 proteins. It is also
possible, however, to construct any other terminal epitope tag to
ensure expression of full-length proteins. This method should be
readily applicable to many other RNA-protein interactions as well as
DNA-protein interactions. Recently, a similar approach was used to
study protein-protein interactions of Ran-binding protein 1 (RanBP1)
and identified mutations that disrupt its association with RanGTPase
and hence affect the function of Ran (44). Finally, this method could also be used to engineer proteins with novel ligand binding specificity or higher substrate binding affinity.
CID was defined as the coiled-coil motif that mediates C1
protein-protein interaction using CD and velocity sedimentation studies
of C1 deletion constructs (29). Efforts to create C1 mutant protein
containing L187A/V194A double mutations failed, because the mutations
likely resulted in incorrectly folded polypeptides that are degraded
rapidly in E. coli cells. (29). Here we identified three C1
mutants containing point mutations in their CID (L187Q, Q192P, and
L201P) that could be displayed by phage. Furthermore, we were able to
produce these mutant proteins by coupled transcription and translation
and by expression and purification from E. coli cells.
Compared with wild type C1, these mutants are incapable of binding to
C1 in vitro as measured by protein binding assays (Fig.
4B). They form a smaller complex with U2 snRNA, most likely because of a defect in oligomer formation (Fig. 5, B and
C), and gel filtration analysis indicates that they form a
smaller complex than wild type C1.3 These results
demonstrated that a single amino acid change in CID in the context of
full-length C1 protein is detrimental to C1 oligomerization, thus
establishing the crucial role of CID in mediating C1-C1 interaction.
LeStourgeon and colleagues (12, 29) raised a question concerning the
critical role of the RBD in C1 RNA binding. The studies they performed
used various C1 deletion mutants and led them to conclude that the
basic region preceding CID (Val140-Asn161) is
the primary determinant of the C protein high affinity binding for both
C1 winner sequence and U snRNAs (12, 29). However, individual amino
acid residues comprising the basic region have not been experimentally
examined to see whether substitution of any of them in intact C1
protein indeed result in loss of RNA binding. In our random mutagenesis
and screening experiments for RNA binding defective mutants, no
mutation was found to map within this basic region, which indicates
that this segment of the protein is not essential for the RNA binding
activity of full-length C1 protein. However, we cannot entirely exclude
the possibility that mutations in this region were not present in our
mutagenized C1 library. One unexpected but very interesting finding to
emerge from our random mutagenesis studies is that several mutations outside of the RBD drastically affect RNA binding. Each of these mutations changes a single residue in CID and results in impaired C1
oligomerization (Fig. 4). Compared with wild type C1, these mutations
very strongly reduced or abolished binding to poly(U) at higher salt
concentrations (1 and 2 M NaCl), although binding at lower
salt concentration (0.1 M NaCl) was not affected (Fig. 6).
Similar defects in ribonucleotide homopolymer binding under high salt
conditions were observed for the mutants of the RNA-binding proteins,
hnRNP K and FMR1 (45). One possible explanation in the case of C1 is
that its oligomerization may affect its affinity for RNA. However, two
findings argue against this notion. First, at high salt concentrations,
C1-C1 interactions were not detected by in vitro protein
binding assays (data not shown). Second, McAfee et al. (29)
found that the binding isotherm of a tetramerization-defective deletion
construct was essentially unchanged. Another possibility is that
mutations in CID simply cause a conformational change that leads to
reduced RNA binding affinity, because the coil structure could be
involved in optimally orienting the RNA-binding sites vis à
vis the RNA.
Nevertheless, among a collection of RNA binding defective mutants, we
found that most of the critical residues are located within, or close
to, the consensus RNP motifs, RNP1 and RNP2. This is consistent with
and adds direct evidence to the conclusions drawn from previous
structural studies on hnRNP C1 RBD and proteins of the same family
(e.g. U1A). For example, for the U1A snRNP protein,
structural modeling implicated several residues in its
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ABSTRACT
INTRODUCTION
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DISCUSSION
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(21-23). The
four-stranded antiparallel
sheets are packed against the two
perpendicularly oriented
helices. The highly conserved RNP1 and
RNP2 consensus sequences are juxtaposed on the central
3
and
1 strands, respectively. NMR studies of hnRNP C1 RBD
bound with its high affinity RNA substrate suggest that the
sheets, the loops connecting the strands of the sheets, and the contiguous NH2- and COOH-terminal regions of the RBD together form an
exposed platform for direct and specific RNA binding (9). Crystal
structure studies of U1A RBD complexed with its cognate U1 snRNA
stem-loop II further support the view that the conserved RNP1 and RNP2
and the COOH-terminal extension of the RBD interact with RNA
extensively (24). Additionally, site-directed mutagenesis has been
carried out on RBDs of many RNA-binding proteins, and these studies
have pinpointed several amino acids, particularly in the conserved RNP1
and RNP2, as essential residues for the RNA binding activity of this
domain. For example, residues Asn9, Thr11,
Tyr13, Gln54, Phe56, and
Gln83 of U1A, which corresponds to Asn15,
Arg17, Phe19, Phe52,
Phe54, and Asn83, respectively, in the hnRNP
C1, were identified as such essential amino acids (25-28).
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-mercaptoethanol, and 4 units
of Taq DNA polymerase (PerkinElmer Life Sciences). Four
identical but separate PCR reactions were subjected to 28 cycles of
95 °C for 1 min, 54 °C for 1 min, and 72 °C for 3 min. The
pooled PCR products were extracted with phenol/chloroform/isoamyl alcohol (25:24:1), ethanol precipitated, cut with EcoRI and
XhoI, and purified on a 1% (w/v) agarose gel. The purified
EcoRI-XhoI fragments were then ligated into the
EcoRI-XhoI cleaved Uni-Zap XR vector arms
(Stratagene). The ligation mixture was packaged using the Gigapack II
Packaging Extract kit (Stratagene) according to the manufacturer's
suggested conditions to generate a Uni-Zap
phage library containing
C1 cDNAs with random mutations. The titer of this library is
~1 × 106 plaque-forming units/ml.
-D-galactoside. Following incubation
for 6-8 h at 37 °C, the filter was lifted, replaced with another
isopropylthio-
-D-galactoside-treated nitrocellulose filter, and incubated at 37 °C overnight. The first set of filters was immunoblotted with the anti-C1 monoclonal antibody 4F4 and then
with 125I-labeled goat anti-mouse F(ab')2 as
previously described (32). After washing, the immunoblotted filters
were exposed to x-ray film. The second set of filters was incubated in
screening buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 10 mM
dithiothreitol) for 30 min at room temperature with constant agitation,
blocked with 3% nonfat dried milk and 0.02% sodium azide in
phosphate-buffered saline without MgCl2 for 1 h at
room temperature. After a brief wash with screening buffer, the filters
were incubated in 100 ml of screening buffer containing denatured
salmon sperm DNA (0.1 mg/ml) for 30 min at room temperature. Then,
about 10 pmol of 32P-labeled oligo(dT)25
(25-mer) was added to the screening buffer, and the incubation was
continued for 1 h. After washing five times with screening buffer,
the filters were exposed to x-ray films. Plaques scoring positive for
4F4 and negative for oligo(dT)25 were isolated and excised
using ExAssist helper phage and SOLR E. coli strain as the
host cell (Stratagene) according to the manufacturer's
recommendations. The mutated bases within the C1 cDNA from these
clones were identified by DNA sequencing using the Sequenase version
2.0 DNA sequencing kit (United States Biochemicals). Samples
from each ddNTP terminated reaction were run as single base ladders on
polyacrylamide gels to facilitate rapid identification of mutations.
) vector
(Stratagene) and transcribed by T3 RNA polymerase or in pcDNA3
vector (Invitrogen) and transcribed by T7 RNA polymerase. To produce
recombinant proteins from E. coli, wild type and mutant C1
cDNA fragments were subcloned into EcoRI and
XhoI cleaved pET-28b vector to produce His-tagged proteins
(Novagen) or pGEX-5X-3 vector to produce GST-tagged proteins (Amersham
Pharmacia Biotech). The resulting plasmids were transformed into
BL21(DE3) E. coli (Novagen) for expression and purification
of the fusion proteins according to the manufacturer's recommendations.
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phage expression library
constructed from randomly mutated hnRNP C1 cDNA was screened with a
high affinity nucleic acid probe. To generate random point mutations in
C1, error-prone PCR was performed using the entire coding region of C1
as the template. Under the conditions used, this technique has been
shown to produce mutation frequency of about 0.25-0.4% over 30 cycles
of PCR amplification (30, 37), based on ~10
4
errors/nucleotide synthesized by Taq DNA polymerase (38). To assure that library clones expressed full-length or near full-length C1
protein, a set of replica library filters was screened with 4F4 that
binds to an epitope close to the carboxyl terminus of C1.2
Thus, clones not producing full-length C1
will not be detected with 4F4. Another replica set of library filters
was probed with 32P-labeled oligo(dT)25. We
used oligo(dT)25 instead of poly(U), the more typically
used high affinity substrate for C1 (6, 10), because it is more stable
and produces lower background than poly(U) (data not shown).
Furthermore, in in vitro bead binding assay,
oligo(dT)25 binds to C1 as well as poly(U)
does.3 Fig.
1 (A and B) shows
filter duplicates probed with 4F4 and oligo(dT)25,
respectively. Phage plaques that are positive with 4F4 but negative
with oligo(dT)25 represent phage that likely express
full-length C1 proteins but contain mutations that reduce RNA binding.
After screening ~10,000 clones, we obtained 60 clones that lost
oligo(dT)25 binding capacity.
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Fig. 1.
Identification of potential hnRNP C1 mutants
defective in RNA binding. A and B, phage
display screening of a library containing randomly mutagenized C1
cDNAs. A shows a nitrocellulose membrane immunoblotted
with 4F4. B shows a replica of this membrane probed with
32P-labeled oligo(dT)25. Arrows
point to phage plaques that show positive signal with 4F4 but negative
with oligo(dT)25. They indicate candidate C1 mutants
defective in RNA binding. C, a portion of one sequencing gel
illustrating nucleotide changes in C1 cDNA clone 1 to clone 6. To
facilitate identification of mutations, sequencing reactions terminated
by ddATP (lanes A), ddCTP (lanes C), ddGTP
(lanes G), or ddTTP (lanes T) for each sample
were run side by side. Indicated are a G T change observed for
clone 3 (asterisks) and a C
T change found for clone 5 (inverted open triangles).
phage, and the entire coding region of
each clone was sequenced (39). The sequencing gels were run as single
nucleotide ladders to facilitate rapid identification of mutations. A
portion of such a sequencing gel illustrating representative mutations
found is shown in Fig. 1C. For example, in clone 3, a
guanine is mutated to a thymine, which results in Gln to His change at
amino acid 56 (Q56H), and in clone 5, a cytosine is changed to a
thymine resulting in a silent mutation at the amino acid level. The
loss of oligo(dT)25 binding activity of this clone is
attributed to the presence of a second mutation (data not shown). Among
a total of 60 clones identified in the screening and sequenced, we
found mutations at 29 amino acid positions. Mutations in a few
positions were identified multiple times (data not shown).
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Fig. 2.
Mutations in C1 reduce binding to
poly(U). In vitro translated and
[35S]methionine labeled wild type (WT) and
mutant (S16F, F19S, Q56H, and G51Y) C1 proteins (5 µl) were bound to
poly(U)-Sepharose beads in poly(U)-binding buffer containing 100 mM NaCl. After extensive washing, bound proteins were
eluted in SDS-PAGE sample buffer and analyzed by SDS-PAGE followed by
fluorography. T lanes show 20% of the proteins used in each
binding. B lanes show proteins retained on the beads after
washing. Sizes are indicated in kilodaltons. The multiple bands around
30 kDa are likely due to internal initiation of translation in
vitro.
domain structure of the RBD are
indicated in Fig. 3A. Most of the mutations lie within the
two RNP consensus motifs that form the two central
-sheets. The
mutations within RNP consensus motifs, e.g. F19(S/L), G21V,
F52L, and Q56H, showed no binding or severely impaired binding in the
poly(U) bead binding assay. Mutations located in close proximity to the
RNP consensus motifs, e.g. S16F and H49(Y/R/N), were less
defective in binding to poly(U). A cluster of mutations near the
COOH-terminal end of the RBD identified in the library screening (D81N,
N83(D/Y), and E87G) had the same affinity as wild type C1 for poly(U)
as measured by the in vitro bead binding assay. Amino acid
residues Phe37, Gly41, and Ala66
are well conserved in several RBD-containing proteins, and they are
located in the two
-helices (Fig. 3A). Mutations in these three positions were identified as RNA binding defective in the initial
library screening. However, these mutants displayed wild type binding
activity in the poly(U) bead binding assay (Fig. 3B). For
two reasons, the poly(U) bead binding assay is probably less sensitive
than the library screening with oligo(dT)25 in detecting a
slight decrease in binding affinity. First, the nucleic acid substrate
used for screening, oligo(dT)25, is 25 nucleotides in
length, whereas poly(U) beads present polyribouridylic acid chains of
about 100 nucleotides. Second, the amount of poly(U) used (3 µM) was in excess over in vitro produced
mutant C1 proteins (0.5 nM), whereas a relatively low
concentration of oligo(dT)25 (0.1 nM) was used
in the library screening. Nevertheless, the above mutations were
generally expected in a successful library screening, because most of
these residues are conserved in many RBD containing proteins (Fig.
3A). Furthermore, previous studies have indicated that
residues located in the
-sheet (22, 26, 28) and in the flanking
amino- and carboxyl-terminal regions (9) are involved in RNA
interaction.
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Fig. 3.
A collection of point mutations identified
from the screening for RNA binding defective C1. A,
sequence alignment of the RBDs from hnRNP and snRNP proteins. Consensus
RNP motifs and conserved amino acid residues are in shaded
boxes. Shown below is the corresponding secondary structure of the
RBD, which consists of a four-stranded -sheets flanked by two
-helices. Indicated with asterisks are amino acid
residues identified in the mutated C1 proteins that are defective in
oligo(dT)25 binding. The number on the
left indicates the position of the first amino acid shown on
the line, and the numbers on top indicate the
positions of corresponding amino acid residues in hnRNP C1.
B, mutants contain amino acid substitutions that are located
in RBD and in CID. Their relative poly(U)-binding avidity at 100 mM NaCl compared with wild type C1 protein is indicated.
+++, wild type binding; +, reduced binding;
, no binding; +++/
,
wild type binding at 100 mM NaCl and no binding at high
salt conditions; n/d, not determined.
-sheets
and the
-helices and thus are likely to be critical for the global
folding of the RBD. Of particular interest, we discovered three
mutations affecting C1 binding to RNA that map outside of the RBD in
the CID (Fig. 3B).
-helical structure or the hydrophobic environment of the coiled-coil. We produced C1 proteins harboring such
mutations individually by in vitro transcription and
translation and tested their binding to bacterially expressed and
purified GST fused to C1 (GST-C1). All four CID mutants abolished C1
homotypic interactions, whereas the wild type protein was able to
interact with GST-C1 (Fig. 4B). These results demonstrated
that CID is critical for protein-protein interaction in the context of
intact C1 protein.
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Fig. 4.
Mutations in CID disrupt C1-C1 protein
interaction. A, CID sequence is shown with numbers 180 and 208 to indicate the positions of the start and the end of CID in
hnRNP C1, respectively. Below the CID sequence, numbers 1-7
show the position of each amino acid in the heptad repeat.
Positions 1 and 4 are in bold to
indicate the corresponding hydrophobic amino acid residues in the
coiled-coil motif. The amino acid residues replaced in each of the
mutants are shown. MT2N was created by site-directed mutagenesis.
L187Q, Q192P, and L201P were identified in the phage display screening.
B, in vitro protein binding assay was performed
to test the binding of 35S-labeled full-length wild type C1
(Myc-C1 or WT) and mutants (MT2N, L187Q, Q192P, and L201P) to purified
GST-C1 (2 µg each). 20% of the input proteins are shown in the lanes
designated Translation or T. Proteins bound to
GST-C1 are shown in the lanes designated Binding or
B. Sizes are indicated in kilodaltons.
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Fig. 5.
Demonstration of U2 snRNA-binding and
oligomerization by C1 wild type and mutants. A,
purified recombinant proteins (2 µg each) on a Coomassie Blue-stained
12.5% SDS-polyacrylamide gel. B, gel mobility shift
analysis. U2 snRNA was in vitro transcribed and labeled with
[32P]UTP and incubated with increasing concentrations of
the indicated C1 proteins (25-250 nM) for 20 min at room
temperature. Samples were then analyzed on a 1/4× TAE 4%
nondenaturing polyacrylamide gel followed by autoradiography. Both free
U2 snRNA and slower migrating C1-U2 snRNA complexes are indicated.
C, oligomer formation of C1 proteins under gel mobility
shift conditions. Following incubation of gel shift reaction mixtures,
proteins were cross-linked by 0.01% glutaraldehyde. Samples were then
resolved by 12.5% SDS-PAGE and transferred to nitrocellulose membrane
for Western blotting with 4F4. Positions of the monomeric and
oligomeric C1 proteins are indicated. Sizes are indicated in
kilodaltons.
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Fig. 6.
Mutations in CID affect C1 poly(U) binding
activity at high salt conditions. Wild type and mutant (L187Q and
Q192P) in vitro translated proteins (5 µl each) were
incubated with poly(U)-Sepharose beads in poly(U)-binding buffer
containing 0.1, 1, or 2 M NaCl. After washing, bound
proteins were analyzed on a 12.5% SDS-polyacrylamide gel followed by
fluorography. 20% of the input samples are shown in the TnT
lanes. Bound proteins under the indicated salt concentrations were
shown in the Binding lanes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet to be
involved in RNA binding. These include Thr11,
Tyr13, Asn15, Asn16, and
Gln54 of U1A, which correspond to Arg17,
Phe19, Gly21, Asn22, and
Phe52 of C1, respectively (22, 28). We found mutations at
these positions in our screen and further demonstrated their inability to bind poly(U), a specific high affinity ribonucleotide homopolymer substrate of C1. Additionally, NMR studies on C1 RBD indicated that
Gly51 is topologically located at the tight turns between
the
-sheets and thus is likely to be part of the RNA-binding surface
(9, 21). Indeed, a mutation was found at this position (G51Y), although its effect on poly(U) binding is less severe. Besides finding mutations
within or near the RNP consensus sequences, we also discovered a
cluster of mutations located at the COOH-terminal end of the RBD. This
finding is in agreement with the conclusion drawn from the crystal
structure studies of U1A RBD complexed with RNA hairpin, in which
hydrogen-bonding was observed between RNA and residues
Arg83, Gln85, and Tyr86 of U1A
(correspond to Asp81, Asn83, and
Leu84 of C1, respectively) (24). Our studies strongly
support the conclusion that the RBD is the primary RNA-binding domain
of C1 and further suggest that structures outside of RBD, such as CID, influence the RNA binding activity of hnRNP C1.
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ACKNOWLEDGEMENTS |
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We thank members of our laboratories, especially Drs. Naoyuki Kataoka, Westley Friesen, Zissimos Mourelatos, and Jeongsik Yong for helpful discussion and critical reading of this manuscript. We also thank Susan Kelchner for secretarial assistance with the preparation of this manuscript.
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FOOTNOTES |
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* This work was supported by a grant from the National Institutes of Health (to G. D.).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.
Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed. Tel.: 215-898-0172; Fax: 215-573-2000; E-mail: gdreyfuss@hhmi.upenn.edu.
§ Present address: Dept. of Biology, Korea University, 5-1, Anam-Dong, Seoungbuk-Ku, Seoul, Korea.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M010207200
2 G. Dreyfuss, unpublished data.
3 L. Wan and G. Dreyfuss, unpublished data.
4 V. Pollard and G. Dreyfuss, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: hn, heterogeneous nuclear; sn, small nuclear; RBD, RNA-binding domain; CID, C1-C1 interaction domain; PCR, polymerase chain reaction; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.
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