From the Department of Molecular Biology, Vanderbilt University,
Nashville, Tennessee 37235
Based on UV cross-linking experiments, it has
been reported that the C protein tetramer of 40 S heterogeneous nuclear
ribonucleoprotein complexes specifically interacts with stem-loop I of
U2 small nuclear RNA (snRNA) (Temsamani, J., and Pederson, T. (1996)
J. Biol. Chem. 271, 24922-24926), that C protein
disrupts U4:U6 snRNA complexes (Forne, T., Rossi, F., Labourier, E.,
Antoine, E., Cathala, G., Brunel, C., and Tazi, J. (1995) J. Biol. Chem. 270, 16476-16481), that U6 snRNA may modulate C
protein phosphorylation (Mayrand, S. H., Fung, P. A., and
Pederson, T. (1996) Mol. Cell. Biol. 16, 1241-1246), and
that hyperphosphorylated C protein lacks pre-mRNA binding activity.
These findings suggest that snRNA-C protein interactions may function
to recruit snRNA to, or displace C protein from, splice junctions. In
this study, both equilibrium and non-equilibrium RNA binding assays
reveal that purified native C protein binds U1, U2, and U6 snRNA with
significant affinity (~7.5-50 nM) although nonspecifically. Competition binding assays reveal that U2 snRNA (the
highest affinity snRNA substrate) is ineffective in C protein displacement from branch-point/splice junctions or as a competitor of C
protein's self-cooperative RNA binding mode. Additionally, C protein
binds snRNA through its high affinity bZLM and mutations in the RNA
recognition motif at suggested RNA binding sites primarily affect
protein oligomerization.
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INTRODUCTION |
The C protein tetramer of 40 S heterogeneous nuclear
ribonucleoprotein (hnRNP)1
particles is one of the three major core particle proteins involved in
the packaging of pre-mRNA in vertebrates (for review, see Ref. 1).
The tetramer is highly stable in solution and, unlike the A- and
B-group proteins, binds RNA in a salt-resistant manner (2, 3).
Hydrodynamic and ultrastructural studies have shown that three C
protein tetramers bind approximately 700 nt of RNA in a cooperative
manner and fold the RNA into a unique 19 S triangular complex that can
be recovered from intact 40 S monoparticles following their
dissociation in low ionic strength buffers (4-6). Interestingly, when
all of the core particle proteins are allowed to bind long pre-mRNAs in vitro, a contiguous array of 20-nm 40 S
hnRNP particles assemble such that each contains approximately 700 nt
of RNA. The same experiment performed with purified native C protein
alone yields one triangular complex per 700 nt of RNA. These in
vitro activities are consistent with electron micrographs of
native hnRNP complexes that mostly reveal contiguous arrays of 20-nm particles. Thus, C protein may function in vivo as a protein
ruler that nucleates monoparticle assembly (4, 7, 8). C protein's intrinsic ability to melt RNA secondary structure further indicates that it may act as an RNA chaperonin to maintain a single-stranded state and to orient the RNA such that trans-acting factors can access
their cognate sequences (9).
The in vitro reconstitution studies described above together
with C protein's cooperative binding mode suggest that C protein may
completely coat elongating transcripts in vivo. In support of this possibility, it has been reported that C protein is present in
the nonspecific pre-spliceosomal H complex but that it is displaced from splice junctions during the early events of spliceosome assembly (10). In the ontogeny of mRNA, the site-specific displacement of C
protein would be a significant biochemical event. In this context,
several recent reports have implicated snRNAs and their associated
factors in a mechanism that could modulate C protein's affinity for
RNA in a site-specific manner. Although not required for C protein-RNA
interactions in vitro, previous studies have indicated that
both U1 and U2 snRNPs are required for C protein binding to
pre-mRNA (11). More recent reports have suggested that C protein
interacts specifically with stem-loop I of U2 snRNA (12) and that C
protein binds U6 snRNAs that contain elongated uridylate stretches at
their 3' end (13). In the latter studies, it was reported that C
protein disrupts U6:U4 snRNA interactions when elongated U6 snRNAs are
a part of the complexes. Finally, it has been reported that U6 snRNA
plays a role in the modulation of C protein phosphorylation (14) and it
is now well established that hyperphosphorylated C protein lacks RNA
binding activity (15, 16). It is therefore possible that the
displacement of C protein from branch-point/splice-junction sites might
be mediated directly by snRNP binding or by an snRNP-directed C protein
phosphorylation event.
To explore these possibilities further, we have characterized the
in vitro interactions of purified native C protein with U1,
U2, and U6 snRNA. C protein contains a consensus RNA recognition motif
(CS-RRM) at its amino-terminal end (residues 8-87) (17, 18).
Initially, the RRM was believed to be the major determinant of RNA
binding. However, we have recently shown that C protein's high
affinity RNA binding domain is located between residues 140 and 180 (19). This 40-residue RNA binding domain is highly basic (28% Arg and
Lys) and immediately precedes a 28-residue leucine zipper motif
(residues 180-207). Highly basic nucleic acid binding domains
preceding leucine zipper motifs have been well characterized in several
DNA-binding bZIP transcription factors (i.e. GCN4, Fos, and
Jun) (20, 21). It is therefore not surprising that this bZIP-like motif
(the bZLM) is responsible for C protein's wild type RNA binding
activity. Deletion constructs of C protein lacking the CS-RRM, but
retaining the bZLM, are able to bind long lengths of RNA with a
slightly higher affinity than full-length C protein (19). This brings
into question the function of C protein's CS-RRM. Because the
interactions of certain CS-RRMs with snRNA are now well characterized,
we were particularly interested in the possibility that C protein may
bind cooperatively to long pre-mRNAs through its bZLM and to snRNA
through its CS-RRM.
Using both equilibrium and non-equilibrium binding assays, we report
here that C protein's CS-RRM is not responsible for binding U2 or U6
snRNA. Rather, C protein binds these RNAs in a nonspecific manner
through its bZLM motif. We also show that 19 S complex formation can
only be slightly attenuated by the presence of U2 snRNA. Finally, we
will present evidence for the involvement of C protein's CS-RRM in the
tetramer-tetramer interactions associated with formation of 19 S
complexes. These studies further the ongoing search for possible
functions of CS-RRMs and provide additional evidence for C protein's
nonspecific affinity for RNA.
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EXPERIMENTAL PROCEDURES |
C Protein Purification--
Typically, nuclei from approximately
9 billion HeLa S3 cells were isolated as described previously (22, 23).
The nuclei were sonicated briefly on ice and exposed to mild digestion
with micrococcal nuclease (Boehringer Mannheim, 150 units/ml sonicate) for 5 min at 37 °C. Particulate material was removed by
centrifugation (5856 × g for 10 min in a Sorvall SS-34
rotor). Pepstatin, leupeptin, and aprotinin were added to the
supernatant prior to loading on a Hi Trap Q column (anion-exchange;
Amersham Pharmacia Biotech). Proteins were eluted with a 200-460
mM NaCl gradient. Fractions containing C protein were
combined, diluted 3-fold, and further purified using the Amersham
Pharmacia Biotech Mono Q HR 5/5 column. Purified C protein was then
equilibrated in 1× STE via dialysis for 12 h at 4 °C. Protein
concentrations were determined using the BCA assay (Pierce).
In Vitro Transcription--
The 709-nt transcript of the human
-globin gene was prepared by enzyme digestion (BamHI) of
the pHBG709 vector, followed by in vitro transcription using
T7 polymerase as described previously (8). This particular RNA
possesses the coding sequence for exon 1, intron 1, and a portion of
exon 2 of human
-globin. The pHU6-1 (DraI), pMRG3U2
(BamHI), and the pHU1A (SalI) vectors were kindly
provided by Dr. Thoru Pederson. U2 and U6 snRNAs were transcribed using
a T7-MEGA shortscript in vitro transcription kit (Ambion, Inc.) in order to produce high yields of RNA. U1 snRNA, transcribed with S6 polymerase under previously described conditions, was used as a
control substrate (8). As an additional control, a 116-nt RNA was
transcribed from a portion of antisense 18 S ribosomal DNA. In each
case where RNA was radiolabeled, [
-32P]CTP (3000 Ci)
was added to the transcription reaction. The r(U)14, the
SELEX-identified winner sequence, and the 20-nt
-globin sequence were commercially prepared by Cruachem. The sequence of the winner oligonucleotide has been previously described by Gorlach et
al. (38). The
-globin 20-mer is a sequence from IVS1 of human
-globin pre-mRNA that is not thought to function in pre-mRNA
processing (see Burd and Dreyfuss (18) for the nucleotide sequence).
All three oligonucleotides were end-labeled with T4 polynucleotide kinase in the presence of [
-32P]ATP (3000 Ci). RNA
concentrations were determined through A260 measurements. The extinction coefficients of U1, U2, and U6 snRNA are
similar due to their similar uridine percentages (i.e. 24%, 29%, and 27%, respectively). Observed differences in C protein's affinity for these substrates is thus not due to differences in substrate concentrations. Although differences in the extinction coefficient between the 20-nt
-globin transcript and the
r(U)14 substrate could lead to lower molar concentrations
of the homoribopolymer this alone can not explain the absence of
observed binding to these RNAs especially inasmuch as, in separate
experiments, they were used at a 10-fold higher concentration than the
snRNA substrates. Optical density measurements at 280 nm were also
obtained to ensure the absence of protein contamination in the RNA
substrates.
Gel Mobility Shift Assays--
RNA-protein interactions were
examined using gel mobility shift assays. Each 20-µl reaction
consisted of 20 mM Tris-HCl, pH 7.5, 100 mM
NaCl, 1 mM dithiothreitol, 10% glycerol, 0.1% Nonidet P-40, C protein (or a deletion construct of C protein), and 100 pM amounts of the appropriate 32P-labeled RNA.
Increasing concentrations of protein were used in each sample. Each
sample was allowed to incubate for 20 min at room temperature. A 4.5%
nondenaturing polyacrylamide gel (acrylamide to bisacrylamide ratio of
30:1) was pre-electrophoresed in 0.5× TBE for 30 min (Bio-Rad
Mini-PROTEAN II electrophoresis system). The samples were first loaded
into the gel while it was running at 100 V and then electrophoresed for
30 min at 200 V. Radioactive bands were detected with the use of a
Molecular Dynamics PhosphorImager. In Figs. 1, 2, and 6, the apparent
mobility retardation of the free RNA probes seen in both panels is a
result of loading consecutive lanes while the gel was running and does
not represent protein-RNA interactions. Control experiments using
Epstein-Barr virus nuclear antigen-1 (EBNA-1) and EBNA-1 DNA (provided
with the bandshift kit from Amersham Pharmacia Biotech) were performed
to confirm that binding occurs under the conditions described above. As
a negative control, U2 and U6 snRNA was titrated with bovine serum albumin. Bovine serum albumin was not observed to bind either substrate
under these conditions, even at high bovine serum albumin concentrations.
In the case of the band shift assay, dissociation constants were
determined by visually identifying the electrophoretic lane representing the protein concentration at which half of the RNA present
is bound (24, 25). The Kd values listed in Table I
are therefore approximate. This qualitative method has been shown to be
valid for Kd estimations providing the nucleic acid
concentration is at least 10-fold lower than the protein concentration
at the midpoint (24). In this context, Kd estimates
from the band shift assay were confirmed through the equilibrium
binding study described below. The band shift assay was also used to
identify the primary snRNA binding domain. In these studies the
approximate Kd values determined for the M1-F115
construct (the RRM) and the Y119-G290 construct (the bZLM) were
sufficiently dissimilar so as to render the results unambiguous.
Fluorescence Titrations: The Equilibrium RNA Binding
Assay--
Equilibrium binding isotherms were obtained by measuring
the change in the fluorescence signal (enhancement) that occurs when C
protein binds the fluorescent probe RNA, poly-r(
A). The probe
substrate was approximately 3000 nt. Fluorescence measurements were
performed with an SLM Aminco Bowman Series 2 luminescence spectrometer.
Excitation and emission slits were fixed at 4 and 8 nm, respectively.
Titrations were conducted by exciting a fixed amount (0.5-2
µM) of the probe RNA at 310 nm, and measuring the change
in its emission at 410 nm, as a function of increasing protein
concentration. The fluorescent probe RNA was prepared as described
previously (26). The concentration of the probe RNA was determined
using an extinction coefficient (
) of 5330 for optical density
measurements at 260 nm (24). Fluorescence measurements were corrected
for dilution due to the addition of protein sample, and for background
fluorescence. In all cases, the dilution of probe RNA did not exceed
5% of the total sample volume. Inner filter corrections were not
necessary as the quantities of RNA used in these titrations have
negligible absorbance at 310 nm. To determine if native C protein binds
the fluorescent probe through a single binding mode, the macromolecular
binding density function analysis (27) was performed at RNA
concentrations of 0.7, 1.4, and 2.8 µM (28).
The competitor RNAs were used in 2-fold molar excess (total
nucleotides) depending on the strength of the competitor as a substrate
for C protein binding (determined through test titrations). An
expression for the apparent equilibrium constant
(Kapp) for the interaction of C protein with
each competitor RNA was derived considering the following equilibria
(29, 30).
P,
A, and comp are the steady state concentrations of
protein, poly[r(
A)], and competitor RNA, respectively. P
A and
Pcomp are the equilibrium concentrations of protein-(
A)
or competitor RNA complex. The mass action expression defining the
Kapp for the interaction of protein with
poly[r(
A)] (K
A) or competitor RNA
(Kcomp) are shown in Equations 1 and 2.
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(Eq. 1)
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(Eq. 2)
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In a competition titration, the free protein concentration for
both mass action expressions is the same. As a result, both expressions
can be equated through [P]free. The solution for
Kcomp is shown in the following
equation.
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(Eq. 3)
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In the above equation, K
A was derived
from a fit of data from a control titration (no competitor present)
using the McGhee von-Hippel non-cooperative binding equation (31).
Velocity Sedimentation--
Velocity sedimentation
centrifugation in 15-30% linear glycerol gradients was used to
characterize the sedimentation properties of various protein-RNA
complexes and the efficiency of 19 S complex assembly in the presence
of competitor RNAs. The gradients were subjected to centrifugation at
134,400 × g for 24 h at 4 °C in the Beckman SW
28 rotor. The protein and RNA present in consecutive fractions was
precipitated with three volumes of cold ethanol and resolved by
SDS-PAGE (32). The gels were first stained with Coomassie Brilliant
Blue R-250 to identify the fractions containing the protein peak.
Following this, the gels were dried (1 h at 80 °C) and exposed to
phosphor screens to visualize radiolabeled RNA. Radioactive bands were
detected with the use of a Molecular Dynamics PhosphorImager.
The efficiency of 19 S complex formation was calculated by densitometry
using NIH Image. The area under each peak (or for each band) was
determined and the values (which were directly proportional to the
band's density) were summed to determine the total integrated area.
The integrated area for the 19 S bands alone was also determined and
divided by the total in order to calculate percent 19 S complex formed.
C Protein Constructs--
The M1-F115 and the Y119-G290
constructs were generated as described previously (19). The His-tagged
8KTD/A and 13SMNS/A constructs were generated by run-around polymerase
chain reaction using the pET-28a vector, containing the full-length C1
cDNA sequence, as the template. In each case, primers were created
such that the codons that were to be mutated were replaced with alanine (GCG). After kinasing and religating, the vector was then purified by
gel extraction (Qiagen, Inc.). The purified plasmid was then transformed into both DH5
and BL21 DE3 pLys S Escherichia
coli strains. Expression and purification of these C protein
constructs was performed as described previously (19).
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RESULTS |
C Protein-snRNA Interactions Can Be Monitored through
Nonequilibrium and Equilibrium Binding Assays--
Gel mobility shift
assays were performed to determine if interactions between purified C
protein and U1, U2, and U6 snRNA are of sufficient affinity to monitor
under nonequilibrium conditions. Although the focus of this study was
designed to evaluate interactions between C protein and U2 and U6
snRNA, U1 snRNA and a 116-nt transcript of ribosomal DNA were used to
further examine the question of sequence-specific binding. In
vitro transcribed 32P-labeled snRNAs were incubated
with increasing concentrations of purified native C protein (1.0-50
nM), and the preparations were resolved in 4.5%
nondenaturing polyacrylamide gels. As shown in Fig.
1A, C protein binds U2 snRNA
with an approximate Kd of 7.5 nM. C
protein also binds U6 snRNA but with lower affinity (Fig.
1B). In the latter titrations, the protein concentration yielding 50% RNA bound complexes (Kd) could not be
determined due to practical limitations associated with the methodology
for obtaining highly purified native C protein. It is apparent,
however, that the Kd for the interaction of native C
protein with U6 snRNA is not significantly above 50 nM. In
Fig. 2 (A and B), it can be seen that C protein binds U1 snRNA with a
Kd near 10 nM. It can also be seen that
C proteins binds with slightly lower affinity to a 116-nt transcript of
ribosomal RNA (about 25 nM) (see Table
I for summary).

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Fig. 1.
Gel mobility shift analysis of C protein
binding to U2 and U6 snRNA. 100 pM amounts of the
respective 32P-labeled RNAs were incubated with increasing
concentrations of C protein (0-50 nM) for 20 min. at room
temperature. Samples were then electrophoresed on a 0.5× TBE
nondenaturing polyacrylamide gel. RNA was then visualized using
phosphorimaging techniques (see "Experimental Procedures"). C
protein concentration is labeled above the gels. A, both
free U2 snRNA and a slower migrating C protein-U2 snRNA complex are as
identified. The estimated Kd value for this
interaction is 7.5 nM. B, a U6 snRNA band shift
is also observed, indicating C protein-U6 snRNA complex formation at 50 nM concentration of C protein. From this experiment, a
Kd value could not be determined. The apparent
retardation in RNA mobility (lanes 2-5) seen in both panels
is a result of loading consecutive lanes while the gel was running and
does not represent protein-RNA interactions. The position of the
protein-RNA complex is indicated in the figure.
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Fig. 2.
Gel mobility shift analysis of C protein
binding to U1 snRNA and a 116-nt RNA. These RNAs were used as
competitors to determine whether the binding of C protein to U2 and U6
snRNA was sequence-specific. C protein concentration ranged from 0 to
50 nM. A, identified are the migrations of free
U1 snRNA and the C protein-U1 snRNA complex. The Kd
value is estimated to be 10 nM. B, the findings
here show that C protein will also bind to the 116-nt RNA; however, the
approximated Kd value is slightly higher than that
of the C protein-U1snRNA interaction (25 nM). As in Fig. 1,
the apparent mobility retardation of the free RNA probe (especially
apparent in the second and third lanes of
panel A) is due to loading consecutive lanes while the gel
was running and does not represent protein-RNA interactions. The
position of the protein-RNA complex is indicated in both panels.
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To determine if these differences in relative affinity occur in
solution under equilibrium conditions, binding to the three snRNAs and
to the 116-nt control was monitored using fluorescence spectroscopy
(27, 33-35). In this assay, the ability of the individual snRNAs to
compete as substrates with fluorescently labeled poly(A) was
determined. RNA preparations containing a 2:1 µM excess
(total nucleotides) of snRNA over the fluorescent probe RNA were
titrated with increasing amounts of C protein (from 0.5-16.5
nM). In these experiments, C protein binding to competitor
RNA is seen as an attenuation of the binding-induced enhancement of
fluorescence from the probe RNA. Consistent with the order of affinity
observed in the band shift assay, calculations from the data shown in
Fig. 3 reveal that C protein binds U2
snRNA under equilibrium conditions with 2-fold higher affinity than U1
snRNA, a 10-fold higher affinity that U6 snRNA, and a 4-fold higher
affinity than to the 116-nt control substrate. Additionally, C protein
binds the 116-nt control substrate with a 2-fold higher affinity than
U6 snRNA. Although the equilibrium binding assay used here can detect
subtle differences in relative binding affinities, it does not yield
absolute dissociation constants because the competitors are present in
molar excess to the probe and because the poly(
A) probe is of
sufficient length (3000 nt) to accommodate C protein's cooperative
binding mode (36). The results from both assays were not unexpected, as
our previous findings have demonstrated that C protein binds RNA
regardless of sequence or the presence of RNA secondary structure (4, 8, 37). The differences in affinity observed here do not seem adequate
to direct in vivo specificity for any of the RNA substrates
tested.

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Fig. 3.
Equilibrium binding isotherms for the
interaction of purified native C protein with various RNAs. These
RNA binding isotherms reveal the relative abilities of U1, U2, and U6
snRNA (and the 116-nt control) to compete with the fluorescently
labeled probe RNA substrate. The magnitude of fluorescence attenuation
from the probe RNA correlates with increased C protein affinity for the
RNAs indicated. More specifically, these equilibrium binding isotherms
reveal that U2 snRNA is a better substrate competitor than U6 snRNA. In
these experiments the concentration of the fluorescent probe RNA was 1 µM. C protein concentrations ranged from 0 to 20 nM and the concentration of the competitors (U1, U2, U6,
and the 116-nt control) was 2 µM (see "Experimental
Procedures"). The upper solid tracing (denoted probe RNA)
is a nonlinear least squares fit of the titrations of poly-r( A) with
C protein using the McGhee and von Hipple non-cooperative model (31,
47). The lines for the competitor RNAs are interpolations of the data
points. The competitor titrations were not carried to plateau
(Emax) due to limitations of C protein
availability and concentration.
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C Protein Binds U2 and U6 snRNA through Its bZLM--
To
characterize the individual interaction of C protein's two RNA binding
domains with U2 and U6 snRNA, two bacterially expressed deletion
constructs were utilized. The amino-terminal construct (M1-F115)
contains the CS-RRM. The second construct contains the bZLM and C
protein's acidic carboxyl terminus (Y119-G290) (Fig. 4). The band shift assay shown in Fig.
5A reveals that the M1-F115 construct binds U2 snRNA with a Kd near 125 nM (see Table I). It is apparent that at higher protein
concentrations a second RNA-protein complex forms. This result could be
due to the binding of a second mole of protein at high protein
concentrations. In Fig. 5B, both the first and second
complexes are again observed, indicating an interaction between U2
snRNA and the bZLM-containing construct (Y119-G290). The
Kd for the CS-RRM-U2 snRNA interaction is at
least 10-fold greater (indicating a lower affinity) than that of the
bZLM-U2 snRNA. Similar results were obtained for the interaction of
these two RNA binding domains with U6 snRNA. In Fig.
6 (A and B), it can
be seen that the affinity of the bZLM is at least 10-fold higher than
the CS-RRM. As observed with U2 and U6 snRNA, it was found that the
Y119-G290 construct binds with higher affinity than the M1-F115
construct to U1 snRNA and to the 116-nt control (results summarized in
Table I). These results are consistent with our previous studies using
various pre-mRNAs as substrates (19, 36), namely that the bZLM is the high affinity determinant of C protein-RNA interactions.

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Fig. 4.
Schematic representation of full-length C
protein and two deletion constructs. Shown in the top diagram is
the full-length C protein with its four known major domains: the
CS-RRM, the highly basic RNA binding domain and the four-heptad leucine
zipper (or bZLM), and the acidic carboxyl-terminal region. Below the
full-length depiction of C protein are the two deletion mutants,
M1-F115 and Y119-G290, respectively. The M1-F115 mutant consists
primarily of the CS-RRM whereas the Y119-G290 possesses the bZLM and
the acidic carboxyl terminus.
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Fig. 5.
Gel mobility shift analysis of the
interactions between the M1-F115 and Y119-G290 constructs and U2
snRNA. A, U2 snRNA was incubated with an increasing
concentration of M1-F115 (0-11 × 102
nM). The free probe and the slowed migration of complex I
are as indicated (Kd 125 nM). A
second, concentration-dependent complex also is observed
and is identified as complex II. B, U2 snRNA was also
incubated with increasing concentrations of Y119-G290 (0-75
nM). The arrows to the right of the
gel designate the two complexes that form when Y119-G290 binds to U2
snRNA. The approximated Kd of complex I is 10 nM.
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Fig. 6.
Gel mobility shift analysis of the
interactions between the M1-F115 and Y119-G290 constructs and U6
snRNA. A, increasing concentrations of M1-F115
(0-11 × 102 nM) were incubated with U6
snRNA. The Kd of this interaction is estimated to be
125 nM. The migration of the free probe and the slowed
migration of the complex formed are as indicated. B,
increasing concentrations of Y119-G290 (0-100 nM) were
incubated with U6 snRNA. Indicated on the gel are free U6 snRNA and the
Y119-G290/U6snRNA complex (Kd 10 nM). The apparent mobility retardation of the free RNA
probes seen in panel B (lanes 2 and 3)
is a result of loading consecutive lanes while the gel was running and
does not represent protein-RNA interactions.
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C Protein Binds snRNA with Higher Affinity than It Binds
Uridine-rich Oligonucleotides--
Several previous publications have
indicated that C protein displays its highest binding affinity for
homoribopolymers of uridine and for uridine rich oligonucleotides
(38-43). Additionally, in previous UV-induced cross-linking studies on
C protein-snRNA interactions, it has been suggested that C protein may
preferentially bind a uridine-rich loop of U2 snRNA (12) and to an
unusually long polyuridine element at the 3' terminus of U6 snRNA (13). However, in salt-dissociation studies, in in vitro
reconstitution studies (4), and in equilibrium binding assays (36), we
have not observed that native C protein preferentially binds
uridine-rich sequences. It therefore seemed important to compare (via
the band shift assay) C protein's affinity for snRNA (U1, U2 and U6
snRNA) with its affinity for two uridine-rich oligonucleotides
previously described as high affinity substrates (38). Specifically, as substrates we used a 14-nucleotide homoribopolymer of uridine (r(U)14) and the uridine-rich "winner" sequence
identified through selection and amplification (SELEX) (38). A
20-nucleotide sequence (from IVS1 of
-globin) that is not
uridine-rich and that has not been characterized as a high affinity
substrate for C protein binding was used as a control substrate. The
results of the band shift assays shown in Fig.
7 reveal that C protein does not form stable associations with any of the three substrates. Additionally, these titrations were performed at molar RNA concentrations 10-fold higher than those used for the snRNA titrations. At the highest concentration of protein (50 nM), a smear of labeled
r(U)14 can be seen (Fig. 7B). This could result
from the dissociation of weakly interacting species. Consistent with
these findings, we have used fluorescence spectroscopy to monitor C
protein binding in solution to various oligonucleotides suggested as
high affinity target
sequences.2 In these
experiments, it was necessary to use target oligonucleotide concentrations at least 500 fold higher than used here to evaluate C
protein-snRNA and C protein-pre-mRNA interactions.

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Fig. 7.
Gel mobility shift analysis of native C
protein interaction with short oligonucleotides. In each case, the
substrate was incubated with an increasing concentration of full-length
C protein (0-50 nM). A, binding of the
SELEX-identified winner sequence (14-mer) to C protein was monitored;
however, a slower migrating band was not observed, indicating no
complex formation. B, r(U)14 was the probe used
in this second experiment. Minimal interaction can be observed at the
highest level of C protein concentration (50 nM).
C, a small portion of the -globin sequence (20-mer) also
does not interact with C protein at the concentrations used.
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Relative Affinities of C Protein for Pre-mRNA and U2 snRNA: U2
snRNA Moderately Attenuates 19 S Formation--
The results described
above demonstrate that native C protein binds U2 snRNA with significant
affinity. Although the binding of C protein to U2 snRNA appears
nonspecific, it could, however, function in vivo to block
the RNA-activated interactions between tetramers that direct 19 S
complex assembly at splice junctions. Additionally, interactions
between snRNA and C protein might induce the site-specific displacement
of C protein from intronic branch points. Regarding this question, a
series of in vitro reconstitution studies were conducted to
determine the relative affinities of C protein for pre-mRNA and U2
snRNA and to determine if U2 snRNA can dissociate or block the assembly
of 19 S C protein-RNA complexes. In these experiments, purified native
C protein was allowed to associate in solution with a
32P-labeled 709-nt human
-globin transcript containing
all of the first exon and intron and a 205-nt portion of exon 2 (44).
In other experiments, C protein was either preincubated with
32P-labeled U2 snRNA or the latter was present with the
-globin transcript prior to C protein addition. Because C protein,
U2 snRNA, and nascent transcripts are all abundant intranuclear
constituents in actively dividing cells, the molar concentration of the
RNA substrates in most of the competitive reconstitution assays was adjusted to near equality. The various C protein-RNA preparations were
resolved in 15-30% linear glycerol gradients. Following
centrifugation, the distribution of RNA was monitored by pumping the
gradients through a 30-µl flow cell of a diode array
spectrophotometer. Additionally, SDS-PAGE was performed on successive
1-ml gradient fractions to more precisely identify the RNA
(radioactivity) and protein components (Coomassie stain) of the various
gradient-resolved species. In several previous studies, we have shown
that each C protein tetramer binds a 230-nt length of RNA and that
three tetramers associate to fold approximately 700-nt RNA substrates (regardless of sequence or source) into a unique triangular complex that sediments at 19 S. Purified C protein alone sediments at approximately 5.8 S while the 709 nt transcript used here sediments in
a well resolved manner slightly faster that C protein (4) (see Fig.
8, A and B).

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Fig. 8.
Velocity sedimentation analysis of the 19 S
complex. A radiolabeled 709-nt transcript was incubated with
native C protein and then loaded onto a 15-30% glycerol gradient. The
gradient was spun at 4 °C for 24 h. at 134,400 × g and then fractionated into 1-ml fractions. The fractions
were ethanol precipitated and then electrophoresed on a 8.75% Laemmli
gel. The gel was stained for protein and then dried. RNA was visualized
by phosphorimaging techniques. A, distribution of RNA
throughout the gradient. Fraction numbers are labeled above the gels.
The free 709-nt RNA is found in fractions 12 and
13. RNA that is part of the 19 S complex can be seen in
fractions 8, 9, and 10. B,
distribution of Coomassie-stained protein throughout the gradient. The
result shown here indicates that C protein sedimented in the
corresponding 19 S fractions (fractions 8,
9, and 10). Because C protein was present in
excess, unbound protein is observed in fractions 14 and
15. The two isoforms of C protein, C1 and C2, are as
indicated.
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In the control experiment shown in Fig. 8, it can be seen that when C
protein is allowed to bind the 709-nt pre-mRNA transcript in the
absence of snRNA and under conditions of slight protein excess, the
great majority of RNA and protein sediment at 19 S. It can be seen that
a very small amount of unbound RNA (fractions 12 and
13, panel A) and unbound protein (fractions
14 and 15, panel B) sediment as free
moieties. It is also apparent that the great majority of RNA sediments
with C protein about a peak at 19 S. In these SDS-PAGE gels, RNA
secondary structures are not denatured and it can be seen that the
709-nt transcript is folded by C protein to form a unique and stable
but protein-free, slow migrating secondary structure (upper
band in fractions 8-10 of Fig. 8A).
To observe the effects of U2 snRNA on 19 S complex formation, C protein
(300 nM) was incubated with both U2 snRNA (46 nM) and the 709-nt transcript (40 nM)
simultaneously. In Fig. 9A, it
can be seen that most of the pre-mRNA moiety was assembled into 19 S complex. Even though the snRNA used in these experiments was labeled
at approximately one-third the specific activity of the 709 nt
transcript, it is clear that the U2 snRNA was not packaged with
pre-mRNA in the 19 S complex. This demonstrates that, in competition with U2 snRNA, C protein preferentially binds long pre-mRNAs. A small amount of C protein is, however, present in the
same fractions as the bulk of U2 snRNA (fractions 12 and
13, panels A and B). From these
results alone, it can not be determined whether the protein in these
fractions is bound to U2 snRNA alone, if it is bound to both U2 snRNA
and the 709-nt transcript, or if it is coincidental sedimentation. To
resolve this question, reconstitution experiments were performed under
conditions of RNA excess (snRNA = 310 nm, 709-nt transcript = 71 nm, C protein = 110 nm). U2 snRNA was again recovered with
protein that was not committed to 19 S complex (data not shown).
Finally, in the absence of U2 snRNA, the distribution of protein and
pre-mRNA typically appears symmetrical about the 19 S peak. These
findings indicate that in vitro U2 snRNA does not
effectively block 19 S complex assembly on a length of RNA containing
the elements of a functional splice junction. The moderate attenuation
of 19 S complex formation seen in the presence of U2 snRNA is not
inconsistent with C protein moderate affinity for U2 snRNA.
Densitometry of stained bands (shown in Figs. 8B and
9B) reveals that when present at approximate equal molar
concentrations with pre-mRNA, U2 snRNA attenuates 19 S complex
formation by approximately 25%.

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Fig. 9.
Velocity sedimentation analysis of the
competition between U2 snRNA and 709-nt RNA for C protein.
A, distribution of RNA throughout the gradient. Again the
709-nt RNA that is part of the 19 S complex peaks in fractions
8, 9, and 10. The U2 snRNA peaks in
fractions 12 and 13 and is not found to be a part
of the 19 S complex. B, distribution of the
Coomassie-stained protein throughout the gradient. Fractions
8, 9, and 10 contain complexed C protein.
Free C protein is found in fractions 14 and 15.
The small amount of C protein found in fractions 12 and
13 is likely to be bound to U2 snRNA.
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The CS-RRM May Function in Tetramer-Tetramer Interactions in the 19 S Complex--
Having observed that C protein's high affinity
interaction with both pre-mRNA and snRNA is mediated through its
bZLM motif, experiments were designed to gain further information on
the role of the amino-terminal CS-RRM in the assembly of the 19 S C
protein-RNA complex. Site-specific mutants of the CS-RRM were
constructed and characterized regarding their ability to form 19 S
structures. In previous experiments where the amino-terminal domain was
mixed with r(U)8, NMR spectra revealed that residues 8-10
(KTD) and 13-16 (SMNS) are involved in associations with RNA (45). In the present study, these residues were changed to alanines creating two
mutants: 8KTD/AAA and 13SMNS/AAAA (Fig.
10B). These constructs were
separately incubated with the 709-nt
-globin transcript and resolved
in 15-30% glycerol density gradients. Like wild type C protein, the
8KTD/AAA was competent in 19 S complex assembly (Fig. 10A).
However, the 13SMNS/AAAA construct was not only defective in 19 S
complex assembly but directed the assembly of a faster sedimenting
anomalous ribonucleoprotein complex. Because the latter construct
functioned well in RNA binding but caused the assembly of an
artifactual ribonucleoprotein complex, it is likely that the CS-RRM may
function in the RNA-activated tetramer-tetramer contacts that direct 19 S complex assembly.

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Fig. 10.
Velocity sedimentation of the site-specific
C protein mutants 8KTD/AAA and 13SMNS/AAAA. Each mutant was
incubated with a 709-nt RNA and then loaded onto a 15-30% gradient.
The gradients were spun at 4 °C for 24 h at 134,000 × g, fractionated, and then monitored by pumping them through
a 30-µl flow cell of a diode array spectrophotometer. A,
shown are the spectrophotometric results of the effects of these
mutations on 19 S complex formation. Although the 8KTD/AAA mutant
behaved as the native C protein tetramer, the 13SMNS/AAAA mutant could
not efficiently form 19 S complexes. Instead a larger anomalous
ribonucleoprotein particle formed. B, schematic
representation of C protein. Important regions within the protein are
identified. The locations of the site-specific mutations are indicated
at the amino terminus of the CS-RRM.
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DISCUSSION |
Both equilibrium and nonequilibrium binding studies reveal here
that purified native C protein interacts with three snRNAs (U1, U2, and
U6) and a 116-nt ribosomal DNA transcript with significant in
vitro affinities (7.5-50 nM). The differences
observed do not, however, appear sufficient to direct in
vivo binding specificity (46). Although one binding assay monitors
the stability of protein-RNA complexes under nonequilibrium conditions
(the band shift assay) and the other monitors binding in solution under
equilibrium conditions (fluorescence spectroscopy), both assays
revealed the same order of binding affinity (U2 > U1 > 116 > U6). It was observed here that C protein binds snRNAs with
much higher affinity than it binds a 14-nt homoribopolymer of uridine
or the SELEX-identified "winner" sequence. More specifically, no
binding was observed in the band shift assay and, in a separate study,
500-fold higher concentrations of these oligonucleotides were required
to monitor binding under equilibrium conditions (47). Although C
protein binds snRNAs with relatively high affinity, in competition with a 709-nt human
-globin transcript, U2 snRNA, at slight molar excess,
did not effectively block C protein from folding the bulk of the
pre-mRNA into the 19 S C protein-RNA complexes that nucleate 40 hnRNP core particle assembly. This finding is consistent with our
previous report that C protein binds pre-mRNA through a cooperative binding mode (36).
In a previous study on C protein-snRNA interactions, Temsamani and
Pederson (12) did not observe UV-mediated cross-linking of C protein to
U1 snRNA. They did, however, report that C protein can be cross-linked
to stem loop I of U2 snRNA. From the results described here (that C
protein binds U1 and U2 snRNA with similar affinities), one might
expect that C protein would cross-link equivalently to U1 and U2 snRNA.
This discrepancy may be explained by the intrinsically high
photoreactivity of uridine and the presence of four peripherally
located U's in the loop of stem loop I of U2 snRNA (48, 49). In
contrast, U1 snRNA does not possess a loop containing four uridines. In
a second study utilizing UV irradiation, it was observed that C protein
is preferentially recovered in small subfractions of U6 snRNA that
contain an unusually long poly(U) sequences at the 3' terminus. Based
on additional findings, it was further concluded that C protein may
function to specifically dissociate U4-U6 complexes possessing this
unique subset of U6 snRNAs. The binding affinities reported here are not inconsistent with an RNA-dissociating activity, but they suggest that the enhanced photoreactivity of uridine may underlie observations implying binding specificity based on UV-induced cross-links.
Because the C protein tetramer possesses four copies each of two
different RNA binding domains (the CS-RRM and the bZLM), it was of
interest to determine if C protein might specifically bind snRNAs
through its CS-RRM motif. Such an activity could function in
vivo to recruit snRNPs to nascent transcripts, to block C protein binding at branch point/splice junctions, or perhaps to displace nonspecifically bound C protein from splice junctions. Previous studies
have shown that the bZLM is the primary determinant of C protein's
high affinity interaction with pre-mRNAs. Two observations described here indicate that the interaction between C protein and
snRNA is also mediated via the bZLM motif. First, in reconstitution studies where both C protein and U2 snRNA were present at equal molar
concentration prior to C protein addition, no U2 snRNA was recovered in
the 19 S C protein-pre-mRNA complexes. Second, the M1-F115
amino-terminal construct possessing the CS-RRM binds U2 and U6 snRNA
with significantly reduced affinity (8-12 fold), whereas the Y119-290
construct, containing the bZLM motif, binds these RNAs with affinities
equal to or higher than wild type C protein. These two findings support
our previous suggestion that the CS-RRM may function as a negative
allosteric modulator of C protein-RNA interactions (19). The absence of
U2 snRNA in 19 S C protein-RNA complexes formed in the presence of U2
snRNA indicate that the CS-RRM does not independently bind RNA. Other proteins in which deletion constructs, containing the primary determinant for binding, bind tighter than the full-length protein include the Drosophila Sex-lethal protein, E. coli
70 subunit of RNA polymerase, and the
mammalian glucocorticoid receptors (50). The finding that the CS-RRM is
not the primary RNA binding domain is not without precedence. It has
been observed previously that the COOH-terminal RNA binding domain of
the human U1A protein does not bind to any of the following RNAs:
snRNAs, an RNA hairpin, rA16, rU16,
rC16, rA3U3GUA4, or
random RNAs (51).
A finding of considerable interest is the appearance of two
concentration-dependent RNA-protein complexes when either
of the deletion constructs bind U2 snRNA. This result is U2
snRNA-dependent, as we did not observe second complex
formation at high protein concentrations with the other RNAs tested.
The binding of a second mole of protein to U2 snRNA may relate to the
size of U2 (188 nt) versus the other RNA substrates (U6 at
107 nt, the 116-nt control, and U1 at 178 nt). In fact, the overall
order of binding affinity reported here correlates strongly with RNA
length (including the absence of stable binding to r(U)14
and the winner oligonucleotide). In this context, we have shown
previously that a single mole of native C protein occludes
approximately 230 nt of RNA; however, slightly longer substrates can
support the binding of a second tetramer (4). Thus, U2 snRNA appears to
be of sufficient length for a second mole of truncated protein to bind
at high protein concentrations. The binding of a second mole of protein
to substrates of sufficient length has been reported by Kanaar et
al. (50). Using gel mobility shift assays, they determined that
two Drosophila Sex-lethal proteins will bind to transcripts
containing two poly(U) sequences in a
concentration-dependent manner.
In our efforts to evaluate the individual roles of the CS-RRM and the
bZLM in snRNA binding, an observation of particular significance
deserves some mention here. Namely, two C protein constructs, each
possessing mutations at sites in the CS-RRM thought to function in RNA
binding (8KTG/AAA and 13SMNS/AAAA), showed no attenuation in RNA
binding activity. However, the second construct revealed clear defects
in the RNA-activated tetramer-tetramer interactions that lead to 19 S
complex formation. These findings, when taken together with the CS-RRMs
low affinity for RNA, indicate that the amino-terminal RRM may play a
major role in protein-protein interactions. A precedent for this
possibility exists in the findings of others. More specifically, the
NH2-terminal RRM of U2 snRNP protein U2B "is responsible
for interacting with snRNP U2A" and the residues involved in
protein-protein interactions apparently function in binding to U2 snRNA
as well (52, 53). Similarly, both RNA binding domains of the
Drosophila Sex-lethal protein are required for
homodimerization (54). Additionally, it has been reported that the
mammalian spliceosome-associated protein, SAP-49 interacts with SAP-145
through its two RRMs, and that the RRM of snRNP U1A is responsible for
the protein's homodimer formation (55, 56).
We thank James McAfee for initial assistance
with the equilibrium binding assay and Thoru Pederson for providing the
plasmids pHU6-1, pMRG3U2, and pHU1A.