From the Howard Hughes Medical Institute and the Departments of Genetics and Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Received for publication, November 22, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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The KH domain mediates RNA binding in a wide
range of proteins. Here we investigate the RNA-binding properties of
two abundant RNA-binding proteins, Post-transcriptional controls play an important role in the
determination of gene expression. The controls over RNA splicing, transport, localization, translation, and/or stability either contribute to or are the major component(s) of gene modulation during development (1). These controls are often mediated via interactions between specific mRNA sequences and/or structures and
corresponding trans-acting RNA-binding proteins (2, 3). In
several cases such interactions have been described in detail and
emphasize the importance of primary and higher order structural RNA
motifs (4-9). The number and variety of RNA-binding proteins reported
in the literature are rapidly expanding. Some of these RNA-binding
proteins, such as those associated with heterogenous nuclear RNA, show
low sequence specificity, suggesting general packaging functions (3,
10). Others demonstrate high level RNA-binding specificity
suggesting circumscribed functions in gene control. Examples of the
latter group of proteins include cytosolic iron-response
element-binding protein (6, 11), human immunodeficiency virus
Rev response element-binding protein (8), and the sex-lethal
alternative-splicing factor (13). RNA-binding proteins, like their
DNA-binding protein counterparts, tend to be modular in structure with
conserved RNA-binding domains and "auxiliary" domains that function
in the assembly of multiprotein complexes central to specific RNP
functions (14). Four common motifs have been identified in
mRNA-binding proteins as follows: the RNP domain (also called RNA
recognition motif) (15), the RGG box (16), zinc fingers (17), and the
KH (hnRNP1 K
Homology) domain (18, 19).
The KH domain is one of the most commonly identified RNA-binding
motifs. This domain, first identified in hnRNP K (19), has been found
in more than 50 proteins in a wide range of organisms. Although
proteins with single KH domains have been identified (20), these
proteins more commonly contain 2-4 or 15 KH repeats (reviewed in Ref.
21). The KH domain encompasses ~70 amino acids. The conserved
structure of this domain, as defined by NMR and x-ray crystallography,
composes a compact hnRNP K, the founding member of the KH family of RNA-binding proteins,
was initially characterized as a component of the hnRNP complex (19).
This complex is responsible for packaging nuclear heterogeneous
transcripts. Functional motifs in hnRNP K in addition to the three KH
domains have been implicated in nuclear localization, nucleo-cytoplasmic shuttling, binding of protein kinases, and transcriptional control. These data suggest that hnRNP K is involved in
a wide variety of cell functions (26). The only native RNA target
defined for hnRNP K to date is a CU-rich sequence (DICE; differentiation-control element)
repeated multiple times in the 3'-untranslated region (3'-UTR) of
15-lipoxygenase (LOX) mRNA. Binding of hnRNP K to this site
mediates translation silencing of LOX mRNA in erythroid precursors
(27). Due to the ubiquitous distribution and high abundance of hnRNP K,
it is probable that many of its targets and functions remain to be identified.
A major group of KH domain proteins is composed of the Interest in the The Whereas both SELEX--
The SELEX protocol (46) was utilized to obtain high
affinity binding sites for recombinant
RNA (100 µg for the first round) was incubated with recombinant
protein under standard conditions for Generation of Mutant SELEX RNAs--
T7 transcription templates
encoding the mutant variants were created by PCR amplification of
oligonucleotides carrying the corresponding nucleotide changes.
The primers used as template for PCR amplification with primers
A1T7 and A3 were as follows (mutant bases indicated in bold):
R7alpha1A26,34,40 (5'-GCG GAA GCT TCT CTA CAT GCA ATG GAG TGC CTT
GGT GGA AGT GGT AAA GTG AAT ATA AAG
TTG AGA AGG TCA CTC CAC GTG TAG TAT CCT CTC CC-3', Fig. 5A, panel
2); R7alpha1An38 (5'-GCG GAA GCT TCT CTA CAT GCA ATG GAG TGC CTT
TTT TTA AGG GGT AAA GGG AAT ATA AAG TTG AGA AGG TCA CTC CAC
GTG TAG TAT CCT CTC CC-3', Fig. 5A, panel 3); R7alpha1An(3)
(5'-GCG GAA GCT TCT CTA CAT GCA ATG GAG TGC CTT TTT TTA
ATT TTT AAA TTT AAT ATA AAG TTG AGA AGG TCA CTC
CAC GTG TAG TAT CCT CTC CC-3', Fig. 5A, panel 4); R7alpha
Mut 32/38 (5'-GCG GAA GCT TCT CTA CAT GCA ATG GAG TGC CTT TTT
TTA ATT TTT AAA GGG AAT ATA AAG TTG AGA AGG TCA CTC CAC
GTG TAG TAT CCT CTC CC-3', Fig. 5B, panel 2); R7alpha Mut
25/38 (5'-GCG GAA GCT TCT CTA CAT GCA ATG GAG TGC CTT TTT
TTA AGG GGT AAA TTT AAT ATA AAG TTG AGA AGG TCA CTC CAC
GTG TAG TAT CCT CTC CC-3', Fig. 5B, panel 3); R7alpha Mut
25/32 (5'-GCG GAA GCT TCT CTA CAT GCA ATG GAG TGC CTT GGG GGA ATT
TTT AAA TTT AAT ATA AAG TTG AGA AGG TCA CTC CAC GTG TAG
TAT CCT CTC CC-3', Fig. 5B, panel 4); R7K15A34 (5'-GCG GAA
GCT TCT CTA CAT GCA ATG GGT CGG CTT ATT ATT AGT GAT GCA ACC
CAT TGT ACT GGT CCT ATC TAA CTA CAC GTG TAG TAT CCT CTC CC-3', Fig.
7C, center panel); R7K15An33 ((5'-GCG GAA GCT TCT CTA CAT
GCA ATG GGT CGG CTT ATT ATT ATT TAT GCA ACC CAT TGT ACT GGT
CCT ATC TAA CTA CAC GTG TAG TAT CCT CTC CC-3', Fig. 7C, right
panel).
PCR products were cloned as described for the analysis of individual
cDNA clones above, and the primary structure of each mutant
cDNA was confirmed by sequencing. The T7 transcription template was
prepared by PCR amplification with the A1T7 and A3 primers.
Recombinant Proteins--
Recombinant proteins (mouse Electromobility Shift Analysis (EMSA)--
EMSA (gel shift)
analysis with recombinant proteins was performed in the above-mentioned
1× binding buffer as described (30) on 5% non-denaturing
polyacrylamide gels run in 0.5% TBE at room temperature. The amount of
recombinant protein used in each incubation is noted above
the respective lanes of each gel. To maximize the accuracy of
inter-experimental comparisons, all EMSA studies were carried out using
a single preparation of recombinant protein (see above). RNase T1
(Roche Molecular Biochemicals) was added to each incubation prior to
gel analysis as described previously (30, 32). RNase was specifically
omitted in the case of the gel shifts shown in Figs. 2, 5, and 7. Only
in Fig. 9 (C-E) was RNase T1 treatment performed. The
binding value for each RNA was determined by plotting log
(complexed/free probe) versus log (protein concentration)
and determining the protein concentration resulting in 50% of the
probe shifted into protein-RNA complexes. These values were then
calculated relative to that found, respectively, for R7 RNase Structural Mapping--
5'-End labeling and RNase mapping
was performed as described (48), except that RNAs were renatured and
probed in 1× binding buffer containing 0.25 µg of tRNA/µl.
Incubations were carried out at 37 °C for 4 min. The reactions were
terminated by addition of formamide loading buffer, and the samples
were electrophoresed on a 10% polyacrylamide sequencing gel. In
lanes C, 1× binding buffer was added in place of diluted
enzyme. RNases T1, T2, and V1 were obtained from Roche Molecular
Biochemicals, Life Technologies, Inc., and Amersham Pharmacia Biotech, respectively.
Analysis of Cellular Extracts--
S-100 extracts from MEL cells
were prepared as described (34). Fractionation of total extract on
Superdex 200 HR 10/30 gel filtration column (Amersham Pharmacia
Biotech) in 1× S100 extract buffer was performed as described with a
flow rate of 0.5 ml/min, collecting 500-µl fractions on an automated
fraction collector. Western and Northwestern analyses were performed as
described previously (30). For Western analyses, the antibody specific to Generation of RNA Pools Selected for High Affinity Binding to
RNAs Selected for High Affinity Binding to
Binding of each of the
Predicted secondary structures of the
The relationship of target structure to binding affinity was
further tested by relating the structure of the SELEX RNAs to that of
native
The importance of the conserved C-patches for
In contrast to the modest increase in the relative binding affinity
caused by loss of a single C-patch, loss of two of the three patches
resulted in a more substantial loss of binding activity (Fig.
5B). A single preserved 5' or central C-patch mediated
binding at an affinity that was more than 10-fold lower than the native SELEX RNA (compare 1st panel with 2nd and
3rd panels). A single preserved 3' C-patch mediated an even
more dramatic loss of binding activity (4th panel). In
addition to resulting in a marked decrease in binding affinity, the
elimination of two of the three C-patches also altered the migration of
the resultant RNP complex. As seen most clearly with the first two sets
of mutations (2nd and 3rd panels), the major
complex migrated more rapidly than that formed by the intact SELEX RNA
(1st panel) or that formed on a mutant RNA with 2 intact
C-patches (Fig. 5A, 3rd panel). The structure of this low
affinity complex was not further defined (see "Discussion"). Taken
together, these data demonstrated that the SELEX RNA target site
comprising three C-rich patches in a single-stranded configuration maximized the affinity of The Consensus Sequence for High Affinity Binding to hnRNP K
Composed a Single C-patch--
SELEX enrichment for RNAs binding to
hnRNP K was carried out in parallel with the studies on
Secondary structure predictions of the hnRNP K SELEX RNAs revealed a
common configuration. The primary consensus sequence (single C-patch)
was presented on top of a stable stem structure or single-stranded
structure bridging two adjacent stems. Six examples of computer folding
of hnRNP K SELEX RNAs are shown in Fig.
7A. RNase mapping of two
selected RNAs (R7K6 and R7K15) confirmed the predicted secondary
structures, specifically emphasizing the single-stranded configuration
of the C-patch (Fig. 7B).
R7K15 was studied in further detail to define the relationship of the
C-patch to hnRNP K binding (Fig. 7C). The dependence of
binding on the identified C-patch was supported by the dramatic loss of
hnRNP K binding upon introducing a single C Specificity of Specific Binding of SELEX RNAs to Native
The
The identity of the cluster of complexes formed with the R7 The optimized RNA-binding sequences for two closely related
RNA-binding proteins, SELEX studies have been previously reported on three additional
KH-proteins as follows: Nova-1, Nova-2, and Vigilin. The neuronal RNA-binding Nova proteins each contain 3 KH domains. SELEX-determined consensus for Nova-1 is ((UCAU(N)0-2)3 (51).
Computer formulations predict that the three short pyrimidine-rich
patches in this target are in single-stranded configuration. In
contrast, SELEX studies of the closely related Nova-2 protein revealed
a consensus binding sequence of a single short binding site,
5'-GAGUCAU-3' (12). The difference in structures of Nova-1 and Nova-2
consensus sequences would appear to parallel the differences between
The The hnRNP K consensus binding motif was
5'-UC3-4 (U/A)2-3' (Fig. 6). The
cytosines in this motif were critical to protein binding (Fig. 7). The
size and structure of the hnRNP K-binding sites were of note for
several reasons. First, their sizes conformed to the 4-5-base patch
that can be recognized by a single KH domain (26). Second, they
conformed to the short DICE motif (5'-UCCCCAA-3') present in 11 copies
within the 192-nucleotide repeat region of the LOX 3'-UTR and described
as a native hnRNP K-binding site (27). Third, the structure was
remarkably similar to the individual C-patches within the The isolation of Interaction of The secondary structures of both sets of SELEX targets appeared to be
crucial to high affinity interactions. The M-fold generated secondary
structures, and RNase mapping demonstrated that the consensus sequences
were encompassed within a single-stranded loop and that these loops
were substantially longer than would have been necessary to accommodate
the consensus binding sites (Figs. 4 and 7). The relevance of RNA
secondary structure to The specificities of hnRNP K and In conclusion, the generation and analysis of SELEX targets to
CP-2KL and heterogeneous nuclear
ribonucleoprotein (hnRNP) K. These proteins constitute the major
poly(C) binding activity in mammalian cells, are closely related on the
basis of the structures and positioning of their respective triplicated KH domains, and have been implicated in a variety of
post-transcriptional controls. By using SELEX, we have obtained sets of
high affinity RNA targets for both proteins. The primary and secondary
structures necessary for optimal protein binding were inferred in each
case from SELEX RNA sequence comparisons and confirmed by mutagenesis and structural mapping. The target sites for
CP-2KL and hnRNP K were
both enriched for cytosine bases and were presented in a
single-stranded conformation. In contrast to these shared
characteristics, the optimal target sequence for hnRNP K is composed of
a single short "C-patch" compatible with recognition by a single KH
domain whereas that for
CP-2KL encompassed three such C-patches
suggesting more extensive interactions. The binding specificities of
the respective SELEX RNAs were confirmed by testing their interactions with native proteins in cell extracts, and the importance of the secondary structure in establishing an optimized
CP-2KL-binding site
was supported by comparison of SELEX target structure with that of the
native human
-globin 3'-untranslated region. These data indicate
that modes of macromolecular interactions of arrayed KH domains can
differ even among closely related KH proteins and that binding
affinities are substantially dependent on the presentation of the
target site within the RNA secondary structure.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
configuration (18, 22-24). This
structure projects an invariant Gly-X-X-Gly loop
between the first and second
-helices and a variable loop between
the second and third
-sheets. These two loops directly participate
in RNA binding by forming a "molecular vise" that makes multiple
sequence-specific contacts with 4-5 contiguous core bases within the
target RNA (24). How such limited contacts can result in high
specificity and high affinity interactions is not clearly understood.
CP proteins
(
-globin mRNA poly(C)-rich
segment-binding protein; see below).
CPs are also
referred to as PCBPs (poly(C)-
binding protein) and hnRNP E (27, 28).
CP
proteins exist in the cell in multiple isoforms encoded by four
dispersed loci in both mouse and man (21, 29). The three best described
isoforms are
CP-1,
CP-2, and
CP-2KL. All three bind tightly to
poly(C).
CP-2KL is encoded by an alternatively spliced
CP-2 transcript that lacks a single exon (exon 8a) corresponding to
an internal 31-amino acid segment (29) (Fig. 1).
CP-1 and the two
CP-2 isoforms can each independently bind to the h
-globin
mRNA 3'-UTR to form the
-complex that is functionally linked to
-globin mRNA stabilization (30). Tissue surveys reveal that each
of these isoforms is found in a wide range of tissues and cell lines.
RNA and protein analyses suggest that
CP-2KL is the most abundantly
expressed isoform (21, 29).
CP proteins emerged from studies of
post-transcriptional control of gene expression. Stabilization of the human (h)
-globin mRNA in erythroid cell lines is tightly linked to the formation of a binary complex ("
-complex") between a
single
CP molecule and a pyrimidine-rich binding site in the 3'-UTR (30-33). The identification of functionally important
CP binding sites in the 3'-UTRs of additional long lived mRNAs including collagen (35) and tyrosine hydroxylase (36) has further suggested that
the
-complex may serve as a general determinant for high level
mRNA stabilization (34).
CP proteins have also been implicated in translational control. They appear to maintain LOX mRNA in a
translationally silent state until the terminal stages of erythroid differentiation by binding to the 3'-UTR DICE motif in conjunction with
hnRNP K (27, 37). Remarkably,
CP can also mediate translational enhancement. In this case
CP increases the efficiency of
cap-independent translation of picornavirus RNA by binding to two
specific sites within the 5'-UTR internal ribosome entry site as
follows: the 5'-terminal cloverleaf structure of the 5'-UTR (38, 39)
and stem-loop IV (38, 40). The interaction with the 5' cloverleaf, which is dependent on the co-binding of the viral protein 3CD, also
controls the switch from translation to replication of the polio RNA
viral genome (41). Additional
CP-mediated translational controls
have been reported in a variety of unrelated viral systems (42-44).
Finally,
CPs may play a role in translational recruitment of dormant
mRNAs during early development of the Xenopus embryo via
controlled cytoplasmic poly(A) elongation (45). Thus,
CP proteins
appear to be involved in a wide range of post-transcriptional controls
involved in mRNA stability, modification, and expression. These
controls target a specific subset of mRNAs and are mediated in an
apparently sequence-specific and selective manner. Despite this wealth
of descriptive data, the underlying mechanisms involved in these
controls remain undefined.
CP and hnRNP K proteins are closely related in structure and
binding properties. Together they constitute the major poly(C) binding
activity in the cell. Both proteins contain three copies of the KH
domain arranged in a similar manner as follows: KH1 and KH2 at the N
terminus separated from the more C-terminal KH3 domain by a central
region of variable length and sequence (Fig. 1; also see Ref. 21).
Close structural and evolutionary relationships between hnRNP K and the
CPs are further supported by the observation that the primary
sequences of their three corresponding KH domains are more closely
related to each other than are KH domains within the same protein (28,
29). This conservation of KH domain number, sequence, and positioning,
and the shared binding to the DICE element in the LOX 3'-UTR (27)
suggest commonalities in their modes and specificities of RNA binding.
However, the unique ability of
CPs to mediate translational
enhancement, modification, and stabilization of specific mRNAs
suggests that these proteins may be distinct in their relative RNA
binding specificities.
CP and hnRNP K are categorized as poly(C)-binding
proteins, their optimal binding sites appear to be more complex and
distinct from each other than is suggested by their common homopolymer
recognition profiles. For example, hnRNP K cannot bind effectively to
the
-globin 3'-UTR nor can it form the
-complex that is linked to
stabilization of a number of additional mRNAs (30). The structural
basis for
CP binding also appears to reflect more than a simple
recognition of poly(C) as the major isoform of
CP,
CP-2KL, has a
6-fold higher affinity for its
-globin 3'-UTR target sequence than
it does for poly(C)
homoribopolymers.2 The
observation that mutations outside of the defined minimal
CP-binding
site within the
-globin mRNA 3'-UTR can severely decrease
-complex formation (32, 33) further suggests that higher order RNA
structures might be of considerable importance in determining RNA
target preference and affinity. In the present study we define the
optimal sequences and structures of the RNA binding sites for hnRNP K
and
CP-2KL. Parallel analyses of these two sets of protein-RNA
interactions revealed well defined differences in how these two closely
related proteins interface with their respective RNA targets.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CP-2KL or hnRNP K. 600 pmol (corresponding to ~4 × 1014 different molecules) of
the polyacrylamide gel-purified oligonucleotide A2N50, 5'-GCG GAA GCT
TCT CTA CAT GCA ATG GNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN
NNN NNN NNN NNN CAC GTG TAG TAT CCT CTC CC-3', was used as template for
PCR with the two primers A1T7, 5'-GCG AAT TCT AAT ACG ACT
CAC TAT AGG GAG AGG ATA CTA CAC GTG-3', and A3, 5'-GCG GAA GCT
TCT CTA CAT GCA ATG G-3', for 6 cycles in a total volume of 10 ml.
A1T7 encodes the T7 RNA polymerase promoter, and A3 was used as the
downstream primer for PCR amplification as well as serving as primer
for cDNA synthesis by reverse transcriptase. These primers also
carry the recognition sequences for the EcoRI and
HindIII restriction enzymes (shown in italics),
respectively, to facilitate cloning of reverse transcriptase-PCR
products for sequencing. The DNA was phenol/chloroform-extracted,
precipitated, and resuspended in 400 µl of TE. After purification on
a NAP-5 gel filtration column (Amersham Pharmacia Biotech), the DNA was precipitated and resuspended in TE, and 300 µg of template was utilized as template for T7 transcription in the presence of trace amounts of [32P]CTP to generate RNA for the first round
of selection. Upon removal of the transcription template with RQ1 DNase
(Promega), the RNA was phenol/chloroform-extracted, precipitated, and
finally purified on preparative 10% denaturing polyacrylamide gels
containing 8 M urea. The band corresponding to the 95-nt
run-off transcript was identified by exposing on film. The band was cut
out and crushed in a 15-ml plastic tube, and the RNA was eluted in
diethyl pyrocarbonate/H2O by incubation on a shaker at room
temperature overnight. After phenol/chloroform extraction the RNA was
precipitated with ethanol and resuspended in a suitable volume of
diethyl pyrocarbonate/H2O. After gel purification, a total
RNA pool of 6.4 nmol (205 µg) was obtained. This pool was split in
two for parallel SELEX experiments against
CP-2KL and hnRNP K, respectively.
CP binding (30) in 1× binding
buffer (10 mM Tris-HCl, pH 7.4, 150 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol) with
the following modifications: no carrier RNA or heparin was used in the
first round of selection. In subsequent rounds tRNA was added to 0.5 µg/µl and heparin to 5 µg/µl. Partitioning for the first 5 rounds was performed by nitrocellulose filtration (47). Briefly, after
incubation of RNA and protein at room temperature for 20 min, the
mixture was passed through a pre-wetted nitrocellulose filter
(Millipore). Unbound RNAs were washed through using 5 ml of 1× binding
buffer containing 0.5 mg/ml tRNA. The filter was cut in pieces, and
RNAs binding to protein were eluted by shaking for 30 min at room
temperature in a 7 M freshly prepared urea solution with 2 volumes of added phenol (46). Eluted RNA was subsequently
phenol/chloroform-extracted, precipitated in the presence of glycogen
(Roche Molecular Biochemicals), and reverse-transcribed with
avian myeloblastosis virus reverse transcriptase (Promega) at
50 °C for 20 min after annealing of the A3 oligonucleotide. PCR was
performed with the A1T7 and A3 oligonucleotides for a suitable
(typically 6 to 10) number of cycles, as the yield of the PCR product
was monitored by 7% polyacrylamide gel electrophoresis. We found that
an excessive number of cycles would lead to formation of higher
molecular weight PCR products. It should be noted that for each round
the input RNA was subjected to sizing by gel purification to avoid the
accumulation of less than full-length transcripts and that all RNA was
subjected to a renaturation step by heating to 75 °C for 3 min in
1× binding buffer and slowly cooling to room temperature. The molar
ratio of protein to RNA was decreased gradually from 1:10 for round 1 to 1:250 for rounds 6 and 7. To prevent selection of RNAs toward unwanted targets, the partitioning of binders from non-binders was
sequentially performed in 3 different ways. Rounds 1-5 were performed
by nitrocellulose filtration and subsequent elution (46); round 6 was
performed as preparative gel-shift, cutting out and extracting all
shifted complexes; and round 7 was performed by co-immunoprecipitation
of RNA-protein complexes with FF3 (for
CP2-KL) or anti-T7 tag
antibody (the recombinant hnRNP K used in this study carries an
N-terminal T7 tag) and protein A-Sepharose (Amersham Pharmacia
Biotech). At the ends of the 6th and 7th round, individual cDNA
clones were generated from selected RNAs by digesting the reverse
transcriptase-PCRs with EcoRI and HindIII and
ligating the pool of fragments into pUC19
EcoRI-HindIII. The ligation was transformed into
DH5
, and individual clones were picked in a random and nonexclusive
manner for plasmid preparation and sequencing.
CP-2KL
and human hnRNP K) were expressed as His-tagged variants in
Escherichia coli and purified by standard procedures as
described (30). To avoid differences in values due to unanticipated
variation in recombinant protein activity, all studies in this report
were carried out with a single constant preparation of each of the two
recombinant proteins.
1 binding to
CP-2KL or R7K15 binding to hnRNP K as determined by EMSA. This
"relative dissociation value" is indicated for each of the RNAs in
the figures and throughout the text.
CP-2 and
CP-2KL (lab antibody identifier, FF3) was used at a
1:5,000 dilution. The signals were developed by incubation with horseradish peroxidase-conjugated goat anti-mouse I8G as a
secondary antibody. The complexes were detected using the ECL system
from Amersham Pharmacia Biotech. For Northwestern analyses, poly(C) was
end-labeled with [
-32P]ATP. Gels were run and
transferred as for Western studies. Nitrocellulose membranes were
incubated in Northwestern buffer (Tris-HCl, pH 7.4, 50 mM
NaCl, 1 mM EDTA, 1× Denhardt's solution) for 2 h
with added dithiothreitol (1 mM) and heparin (50 µg/ml).
This was followed by incubation in Northwestern buffer in the presence
of 10 µg/ml tRNA and 100,000 cpm/ml of probe for another 2 h.
The membranes were then washed in Northwestern buffer (3 times for 5 min each), partially dried, and exposed to film.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CP-2KL and hnRNP K--
The SELEX protocol (46) was used to
investigate the RNA binding specificities of
CP-2KL and hnRNP K
(Fig. 1).
CP-2KL was chosen as a
representative
CP isoform based on its high abundance in cells
surveyed (29). High affinity RNA targets were isolated for these two
proteins from a pool of 95-nucleotide (nt) RNAs containing a fully
randomized 50-base central segment. The two SELEX studies were carried
out in parallel under identical experimental settings. The initial RNA
pool complexity was estimated at 4 × 1014 (see
"Experimental Procedures"). Protein binding activities of successive RNA pools were monitored by EMSA. Incubation of an RNA probe
corresponding to the native h
-globin 3'-UTR (
3'-UTR) with 125 ng
of recombinant
CP-2KL resulted in the formation of a distinct
complex (Fig. 2; compare lanes
1 and 2). On the basis of migration position and
poly(C) sensitivity (lane 3), this complex matched the
previously described
-complex involved in stabilization of the
-globin mRNA (32-34). Incubation of the
3'-UTR with lower levels of
CP-2KL (20 ng) resulted in a corresponding decrease in
-complex formation (lane 4). The starting RNA pool for
the SELEX experiment (termed R0) did not form a visible complex when incubated with 20 ng of
CP-2KL (lane 6). In contrast, the
RNA pool after 7 rounds of SELEX against
CP-2KL (R7
) formed
strong, poly(C)-sensitive complexes (lanes 7 and
8, respectively). Likewise, incubation of the R0 RNA pool
with 20 ng of hnRNP K did not result in formation of detectable
complexes (lane 9), whereas the RNA pool obtained after 7 rounds of SELEX against the hnRNP K protein (R7K) formed a cluster of
poly(C)-sensitive complexes when incubated with this protein
(lanes 10 and 11, respectively). Therefore, seven
rounds of SELEX generated RNA pools highly enriched for RNAs binding to
either
CP-2KL or hnRNP K.
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Fig. 1.
Schematic representation of hnRNP K,
CP-2, and
CP-2KL
proteins. KH domains are indicated as filled boxes
(KH 1, KH 2, and KH 3). The position of
CP-2
exon 8a lacking in the
CP-2KL splice variant is indicated. The sizes
of each protein (AA) are shown. The detailed structures of
these proteins have been reported previously (29).
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Fig. 2.
Analysis of SELEX RNA pools from rounds 0 and
7. Binding of recombinant CP-2KL or recombinant hnRNP K to
human
-globin 3'-UTR (
3'-UTR) (lanes 1-5), or
starting RNA library (R0) (lanes 6 and
9), or the pool present after seven rounds of SELEX against
CP-2KL and hnRNP K (R7
and R7K,
respectively) (lanes 7, 8, 10, and 11). EMSA
assays were performed with the amounts of protein indicated above each
lane. The presence or absence of poly(C) competitor is indicated by + or
, respectively. The smearing of the free probe in lanes
6-11 reflects the structural complexity of the RNAs in the RNA
pools.
CP-2KL Displayed an
Extended Single-stranded Structure Encompassing a Triplicated Poly(C)
Patch--
EMSA of the RNA pools after 7 rounds (Fig. 2) were
indistinguishable from similar analyses of round 6 SELEX RNAs (data not shown). Thus, we inferred that both pools were similarly enriched for
high affinity protein binders. The primary sequences of a randomly
chosen set of 26 cloned RNAs from rounds 6 and 7 (R6 and R7) were
determined and compared (Fig. 3). The
most striking common feature of these RNAs was the presence of three
C-patches. Each of these C-patches was 3-5 nucleotides in length (2 of
the 78 patches had only 2 Cs). No RNAs found at this stage of the
CP-2KL SELEX experiment contained less than three such C-patches. The C-patches were uniformly flanked by A- and U-rich segments with a
strong bias against G bases throughout the conserved region. The
spacing between the C-patches was variable but tended to be quite
short. The consensus sequence was
5'-(A/U)2C3-5(A/U)2-6C3-5(A/U)2-6C3-5(A/U)2-3'.
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Fig. 3.
Sequences of
CP-2KL SELEX RNAs after rounds 6 and 7 of
selection. The larger font sequences correspond to
bases originating from the 50-base randomized segment, and the
smaller font bases represent the fixed sequence flanking
regions used for library amplification (see "Experimental
Procedures"). Alignments of the 50-nt SELEX sequences were made by
eye; gaps are indicated by dashes and were introduced in
each of the SELEX sequences to aid alignments. The poly(C) patches are
highlighted in red, and the consensus sequence derived from
all 26 cloned RNAs is shown at the bottom. The names of the
individual clones are indicated to the left of each
corresponding sequence. R6 and R7 prefixes
indicate that the clones were isolated from the RNA pool after 6 or 7 rounds of selection, respectively. R7
1, shown
in bold, was the representative target used for detailed
analysis.
CP-2KL-selected RNA targets to
CP-2KL was
quantified by EMSA. We have previously reported the
Kd(app) of the native
-complex is
0.5 × 10
9 M (30). This
value was determined by incubating native
-globin 3'-UTR with
unfractionated cell extracts. In the present study we have used
recombinant
CP-2KL protein for the binding studies; the apparent
Kd value for the native
-globin 3'-UTR with the
recombinant
CP-2KL is 20 × 10
9. The
difference between these two values is likely to reflect the fact that
only a fraction of the recombinant protein appears to be biologically
active (27).3 Therefore all
binding studies reported in the present study were carried out with a
single preparation of recombinant
CP-2KL to minimize interassay
variation, and the dissociation value for each RNA is reported relative
to that of the index SELEX R7
1. The "relative dissociation
values" of each of the SELEX RNAs were found to be within 2-fold of
each other and were on average 10-20-fold lower (i.e.
10-20-fold higher binding affinity) than for the native
-complex
(see below for examples). The SELEX procedure thus enriched for a set
of RNAs with a common primary sequence motif and a remarkably high
affinity for
CP-2KL.
CP-2KL selected RNAs were
generated using the M-fold program version 3.0 (49, 50). Each of the
selected RNAs had a high probability of assuming a secondary structure
with a number of consistent properties. Six representative examples are
shown in Fig. 4A. All of these
RNAs contained an extensively base-paired stem topped by a large loop. In all cases the primary consensus sequence was entirely encompassed within the loop. There was no apparent conservation of primary sequence
in the variable part of the stem regions. The predicted secondary
structures of two of the RNAs (R7
1 and R7
2) was verified experimentally by probing in vitro synthesized
32P-5'-end-labeled RNAs with three structure-specific
ribonucleases as follows: RNases T1, T2, and V1. The results were
consistent with the M-fold predictions (Fig. 4B). Of
particular note was the marked sensitivity of the entire consensus
sequence to cleavage by the single strand-specific RNase T2. These data
suggested that all of the RNAs selected for high affinity binding to
CP-2KL conformed to a general structure in which the conserved
primary sequences, consisting of three closely linked C-patches, were presented in an extended and uninterrupted single-stranded
configuration.
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Fig. 4.
Structures of high affinity
CP-2KL binders. A, computer
(M-fold)-generated secondary structures of the
CP-2KL SELEX RNAs.
Nucleotides conforming to the consensus binding sequence are in
red characters and underscored by curved
lines. The borders between the remainder of the randomized 50-nt
region (black) and the constant regions (blue)
are indicated by short horizontal lines. It should be noted
that the constant regions present in the SELEX RNAs are not passive
sequence elements but are shown to contribute to the overall structures
of these RNAs. All binding studies were performed on these full-length
95-nt RNAs. Watson-Crick base pairing is indicated by lines
connecting bases in the stems; U-G base pairing (dots), and
free energies (kcal/mol) are indicated. B, structure mapping
of 32P-5'-end-labeled R7
1 and R7
2. End-labeled RNAs
were mapped by partial cleavage with structure-specific RNases T1
(cleaves just 3' of single-stranded G residues), T2 (cleaves in
single-stranded regions with no sequence preference), and V1 (cuts
predominantly in double-stranded regions or adjacent to stacked
nucleotides). The results of the in vitro structural
analyses are superimposed on the secondary structure models of R7
1
and R7
2. Cleavage sites for RNase T1, T2, and V1 are indicated;
open symbols indicate weak and filled symbols
indicate strong cleavage sites. Loop regions, identified by T2
cleavage, are indicated on the autoradiographs by a bracket.
Nucleotide positions are numbered from the 5'-end. Lane
M contains the ladder resulting from partial alkaline
degradation of the 5'-end-labeled RNA. C, structural map of
32P-5'-end-labeled h
-globin 3'-UTR. The results of the
RNase mapping (data not shown) are superimposed on the M-fold-generated
diagram. The experimentally defined cis-element involved in
-complex assembly (33) is highlighted in red and
underlined.
-globin 3'-UTR target. The native
-globin 3'-UTR binding
target has three C-patches and yet binds to
CP-2KL with a 20-fold
lower relative affinity than the SELEX targets (for example compare
Fig. 2, lanes 4 and 7, and data not shown). The structure of the native
-globin 3'-UTR target as predicted by M-fold
and confirmed by RNase mapping is diagrammed in Fig. 4C (primary data not shown). These data revealed that the first of the
three C-patches previously implicated in
-complex formation (32, 33)
was incorporated in a double-stranded structure. The second patch was
present as a small open loop, and the third and most extensive
segment, a 16-base pyrimidine-pure and C-rich region, although
predicted to be in a single-stranded conformation by M-fold, was not
appreciably sensitive to single strand-specific RNases. These mapping
data stand in marked distinction to the
CP-2KL SELEX targets in
which the three C-patches were encompassed in a continuous and
extensive domain with marked RNase T2 sensitivity (Fig. 4, A
and B). Thus, whereas the primary structure of the native
-globin 3'-UTR target was consistent with the SELEX consensus, its
higher order structure suggested a suboptimal presentation of the
poly(C) patches.
CP-2KL binding was
established by mutagenesis of a representative RNA SELEX target
(R7
1; Fig. 5). C
A transversions were introduced at various positions in the target
sequence. The substitutions were to A rather than G in order to
minimize secondary structural changes. Substitution of all cytosines in
the consensus sequence completely abolished
CP-2KL binding (Fig.
5A, compare 1st and 4th panels). Substitution of a single cytosine in each of the three C-patches caused
a 3.5-fold increase in relative dissociation value (2nd panel), and replacement of all C-bases within the 3'-most
C-stretch resulted in a 2.1-fold increase in relative dissociation
value (3rd panel). A similar 2-3-fold increase in the
relative dissociation value was observed after replacing all C-bases in
either the most 5' or central C-patch (data not shown). Thus, optimal
high affinity binding required the presence of three C-rich patches in
the target RNA, but
CP-2KL binding is still reasonably tight when
two of the three patches remain intact.
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Fig. 5.
Mutational analysis of the consensus
sequences necessary for high affinity binding to
CP-2KL. A, effect of C
A
substitutions within the consensus sequence of the R7
1 SELEX RNA on
CP-2KL binding. The consensus sequence is shown (red) on
the partial structure (top of stem/loop) of the
representative
CP2-KL SELEX RNA, R7
1
(wt). C
A substitutions are indicated with
circles on the partial structures above each
panel, and the designation of the mutant RNA is indicated within
the loop. The EMSA analyses are shown below the
respective RNAs. Increasing amounts of recombinant
CP-2KL are added
to each reaction as indicated by the wedge and concentration
at the bottom of each lane. All studies were carried out using a single
preparation of recombinant protein. The dissociation constant of each
mutant is indicated relative to that of R7
1 (wt)
("Rel. dissoc. value"). B, effect of mutation
at two of the three C-patches within the consensus sequence of the
R7
1 RNA on
CP-2KL binding. Details as in A.
CP-2KL binding.
CP-2KL. The
consensus sequence of the hnRNP K SELEX RNAs isolated after 6 and 7 rounds consisted of a single short conserved sequence motif,
5'-UC3-4(U/A)(A/U)-3' (Fig.
6). Although additional short C-stretches
could be found in some of the RNAs (example, R6K16 and R7K10),
eight high affinity binders contained only one sequence conforming to
this motif (R6K6, R6K7, R7K6, R7K7, R7K15, R7K18, R7K21, and R7K23).
These data suggested that a single short C-stretch was sufficient for
maximal high affinity hnRNP K binding.
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Fig. 6.
RNAs selected against hnRNP K. Details
are the same as noted in Fig. 3. The highlighted R7K15
target was used for subsequent detailed study.
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Fig. 7.
Structures of high affinity hnRNP K
binders. A, computer (M-fold)-generated secondary
structures of hnRNP K SELEX RNAs. Details as in Fig. 4A.
B, structural map of high affinity hnRNP K binders. Markings
as detailed in Fig. 4B. C, effect of point
mutations in the R7K15 RNA consensus sequence on hnRNP K binding.
Details as in Fig. 5. All studies were carried out with a single
preparation of recombinant hnRNP K.
A substitution in
the consensus sequence (compare 1st and 2nd
panels). All evidence of interaction was blocked by mutating all 3 Cs in the patch (3rd panel). The consensus sequence for high
affinity binding to hnRNP K therefore consisted of a single short
C-rich patch presented in a single-stranded configuration.
CP-2KL and hnRNP K for Their Corresponding SELEX
RNAs--
The hnRNP K and
CP-2KL SELEX experiments, carried out in
parallel, yielded distinct consensus sequences. The specificity of the
two sets of high affinity interactions was investigated by
cross-binding comparisons (Fig. 8).
R7K15, the hnRNP K SELEX mRNA, was recognized by hnRNP K (1st
panel) but not
CP-2KL (3rd panel). In contrast,
R7
1, the
CP-2KL SELEX target, was bound by both hnRNP K and
CP-2KL (2nd panel). It should be noted, however, that
this binding of R7
1 to hnRNP K was at a 2.8-fold lower affinity than
to
CP-2KL itself (4th panel). These cross-comparisons
reinforced the conclusion that multiple C-patches are necessary for
high affinity binding to
CP-2KL, whereas a single C-patch is
sufficient for hnRNP K binding.
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Fig. 8.
Selective binding of
CP-2KL and hnRNP K to their respective SELEX
RNAs. The indicated RNA was incubated with increasing amounts of
hnRNP K or
CP-2KL protein (indicated below each lane),
and gel shift analysis was performed and the dissociation value
determined relative to the index R7
1.
CP and hnRNP K in Cell
Extracts--
The SELEX protocol was based on the binding of RNAs to
recombinant (His6-tagged) proteins. To extend our findings,
we determined whether the SELEX RNAs demonstrated binding specificity
to native
CP-2KL or hnRNP K in the context of cell extracts. Studies
were carried out with unfractionated and size-fractionated S100 protein extracts from a mouse erythroleukemia cell line (MEL) known to contain
abundant levels of both proteins (45). The column fractions containing
68-kDa hnRNP K and 38-40-kDa
CPs were identified by Northwestern
analysis (Fig. 9A). The
fractions containing
CP-2 and
CP2-KL were specifically identified
by Western analysis (Fig. 9B). EMSA analysis revealed a
single slowly migrating complex when R7K15 RNA was incubated with the
total MEL extract (Fig. 9C, lane 1). This complex could be
fully competed by poly(C) (lane 2) but was resistant to
competition by poly(CT) (lane 3). In the fractionated
extract, the peak of complex-forming activity with the R7K15 probe
coincided with the peak of hnRNP K activity detected by Northwestern
analysis (fraction 23; Fig. 8A, lane 9). Thus, in the
context of a cellular extract, the R7K15 SELEX RNA bound specifically
to
CP-2KL.
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Fig. 9.
Specific binding of SELEX RNAs to native
hnRNP K and CP-2KL in the context of a
cellular extract. (Note, all fractions were treated in parallel
for the analyses shown in A-D.) A, detection of
the major poly(C) binding activity in the extract. Superdex 200 HR
10/30 column fractions of MEL cell S100 extract were electrophoresed on
an SDS-10% polyacrylamide gel electrophoresis, electroblotted to a
nitrocellulose membrane, and incubated with 32P-labeled
poly(C). The fraction numbers are indicated above respective
lanes. The elution positions of ovalbumin (43 kDa), bovine serum
albumin (67 kDa), and catalase (232 kDa) are indicated
above. The positions of protein size markers for the
SDS-polyacrylamide gel electrophoresis analysis (right) and
hnRNP K and
CP are indicated (left). B,
immunodetection of
CP-2 and
CP-2KL. Protein fractions are as in
A. The filter was probed with the rabbit polyclonal antibody
specific for
CP-2 and
CP-2KL (30). The position of the
CP-2
and
CP-2KL immunoreactive proteins (left) and size
markers (right) are indicated. C, gel shift with
R7K15 probe. Total MEL S100 extract (lanes 1-3) or
fractions 17-33 (lanes 4-20) were incubated with
32P-labeled R7K15 probe and electrophoresed on a native 5%
acrylamide gel. Competition experiments with unlabeled excess poly(C)
(lane 2) or poly(CT) (lane 3) are shown.
The position of the hnRNP K complex is indicated (left).
Note that the rapidly migrating band present in all lanes most
likely corresponded to an RNase T1 digestion fragment of the probe
because this band is also present in the absence of S100 extract (data
not shown). Based on the intensity of this "background" band, it
appears that lane 11 (fraction 24) is underloaded
in this gel. D, gel shift with R7
1 probe. Lanes as
described for C. The position of the
-complex is
indicated (left). E, supershift of complexes
forming on R7
1 RNA with total MEL S100 extract in presence of an
antibody specific to
CP-2 and
CP-2KL. Gel shift analysis was
carried out under conditions identical to D.
Affinity-purified antibodies specific to
CP-2 and
CP-2KL
(lanes 5-7), anti-glutathione S-transferase
(GST) (lane 8), anti-c-Myc (lane 9)
were all derived from rabbits and added at similar protein
concentration to the gel shift incubation mixture. The addition of MEL
S100 extract, poly(C), or poly(CT) competitor is indicated with + and
above each lane.
CP2-KL SELEX RNA, R7
1, was similarly analyzed for binding
activity to native proteins. When incubated in MEL extract, a set of
closely migrating complexes was generated (Fig. 9D, lane 1).
Both complexes were competed by the addition of poly(C) and were
resistant to poly(CT) competition (lanes 2 and
3). We noted that under conditions of a high concentration
of poly(C) (and exclusively under these conditions), where all
CP
proteins are saturated with competitor, the R7
1 probe was free to
form a weak complex of lower mobility than the poly(C)-sensitive
complexes. The CT-binding protein responsible for this complex
was not further characterized. The proteins forming the strong
poly(C)-sensitive complexes with the R7
1 RNA peaked at fractions 26 (lane 13, slightly lower mobility complexes) and
29 (lane 18, slightly higher mobility complexes) (Fig.
8D). These fractions coincided with the presence of
CPs
as identified by Northwestern and Western analyses (A and
B, respectively). Thus, in the context of a native extract, the R7
1 SELEX RNA bound specifically to
CP proteins.
1 RNA
(Fig. 9D, lane 1) was further analyzed. These complexes all
coincided with fractions enriched for
CP (see above). To confirm
that these were exclusively
CP-containing complexes, EMSA supershift
studies were carried out (Fig. 9E). The complete set of
complexes forming with R7
1 in the MEL extract (lane 2) could be quantitatively supershifted with the epitope-specific antibody
recognizing both
CP-2 and
CP-2KL (lane 5) but not with unrelated antisera (lanes 8 and 9). These data
confirmed that the R7
1 bound native CP-2KL and CP-2 with high
affinity and specificity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CP-2KL and hnRNP K, were determined
in the present study. These proteins each contain three
structurally conserved copies of the KH domain (Fig. 1) and
constitute the major poly(C) binding activities in murine and human
cells (Fig. 9A). The two sets of SELEX RNAs revealed
distinct primary consensus sequences (Figs. 3 and 6) encompassed in
consistent secondary structures (Figs. 4 and 7). The data demonstrated
that despite parallels in protein structure,
CP-2KL and hnRNP K have
distinct requirements for high affinity RNA-protein complex formation.
CP-2KL and hnRNP K targets. However, in contrast to hnRNP K and
CP-2KL, structural mapping to confirm the structure of the
RNA-binding sites was not carried out, and cross-competition binding
studies between the two Nova proteins failed to confirm a difference in their respective binding specificities (12). A SELEX study has also
been carried out with Vigilin. The Vigilin protein contains 14 KH
domains. This protein binds unstructured, single-stranded RNAs
containing multiple conserved 5'-(A)nCU-3' and
5'-UC(A)n-3' motifs in a largely G-free region. Approximately
75 bases were required for optimal binding (53). The multiple repeats
in structure and the open configuration of the target site for Vigilin
binding to its RNA target is similar to
CP-2KL and Nova-1. These
three studies support the model that KH domain proteins interact with short and sometimes multiply arrayed RNA target sites and that these
sites must be in single-stranded configuration for optimal interaction.
CP-2KL consensus sequence was
5'-(A/U)2C3-5(A/U)2-6
C3-5(A/U)2-6C3-5(A/U)2-3' (Fig. 3). The three C-rich patches that constituted this consensus all
contributed to
CP-2KL binding (Fig. 5). Remarkably, this tripartite
C-rich consensus sequence bore considerable resemblance to the
previously described
CP-binding site in the native human
-globin
3'-UTR (30, 33). Identical or closely related C-rich regions have also
been identified in the 3'-UTRs of 15-lipoxygenase,
(1)-collagen, and
tyrosine hydroxylase mRNAs (34). These RNP complexes
("
-complexes") formed between these regions, and
CPs appear
to serve as critical determinants of mRNA stability and/or translational control (35-37). Thus, the functionally important
CP-binding sites previously identified in four native mRNAs were consistent with the consensus sequence obtained by
CP-2KL-based SELEX from a library with sequence complexity of >4 × 1014. These data suggest that the native-binding sites are
indeed targeted by
CP and that the spectrum of native high affinity
CP-binding sites may be limited to this single motif.
CP-2KL
consensus. These observations suggested that binding of hnRNP K to its
target RNAs was mediated by a single KH domain. The lower complexity of
the hnRNP K optimal binding site, when compared with that of
CP-2KL, would suggest that its interactions are less stringent in their sequence and structural constraints. This would be consistent with the
inclusion of hnRNP K but not
CP in hnRNP complexes involved in
general mRNA packaging.
CP-2KL targets with three C-patches reflected the
power of SELEX to discriminate among targets with relatively close
binding affinities. Mutagenesis of a representative
CP-2KL SELEX
target (R7
1) revealed that slightly lower affinity binding could
occur on less extensive targets. For example, elimination of one of the
three patches resulted in a 2.1-fold decrease in relative binding
affinity (Fig. 5A). Despite this limited loss of binding
affinity, there were no SELEX RNAs that contained less than three
C-patches. Equally remarkable was the observation that elimination of
two of the three C-patches did not completely eliminate
CP-2KL
binding. For example, two of the mutant R7
1 RNAs containing only a
single residual C-patch were still able to mediate protein binding,
albeit at a substantially lower affinity (Fig. 5B). In contrast to the interaction of hnRNP K with its single C-patch target,
the interaction of
CP-2KL with a single C-patch was weak, and the
complex appeared to differ in structure from that with the intact
target (Fig. 5B). This weak and qualitatively distinct interaction could not be attributed to suboptimal (i.e.
nonexposed) structure of the mutated R7
1-binding site because
CP-2KL also demonstrated very weak or no binding to a number of the
hnRNP K SELEX targets that present the single C-patch in an optimized single-stranded conformation. Thus, while a single C-patch may be able
to mediate interaction with an individual KH domain, and is sufficient
for hnRNP K binding, the tandem array of three C-patches maximized
CP-2KL binding to its RNA target.
CP-2KL with mutated SELEX targets containing only a
single residual C-patch not only demonstrated lower binding affinity
than to the triple C-patch target but also resulted in an RNP complex
with a more rapid electrophoretic mobility (Fig. 5B). This
faster migration may have reflected an alteration in the overall
geometry of the RNP complex or a change in the stoichiometry of the
RNA-protein interaction. Of note, the optimal R7
1 target assembled a
similar fast complex when
CP-2KL was added at low concentrations
(Fig. 5B, 1st panel). We cannot exclude that the lower
mobility complex might be generated by multimerization of
CP-2KL on
multiple C-patches, whereas such a multimerization would not be
compatible with a single C-patch configuration as seen in Fig.
5B. This model would be consistent with the ability of
CP-2KL to dimerize in cell extracts and in yeast two-hybrid assays
(38).3 Selection for single C-patches on the hnRNP K SELEX
targets might reflect a corresponding inability of hnRNP K to undergo
productive protein-protein interactions.
CP-2KL binding affinity was highlighted by
comparing the structure of the SELEX target to that of the native
-globin 3'-UTR. Although the primary sequence of the
-globin
3'-UTR-binding site was consistent with the SELEX consensus, its
affinity for
CP-2KL was substantially lower than that of the
CP-2KL SELEX RNAs (Fig. 2, lanes 4 verses 7).
Secondary structure mapping of the
-globin 3'-UTR (summarized in
Fig. 4C) demonstrated that the only segment in a clearly
defined single-strand configuration (RNase T2-sensitive) was a tight
loop containing part of the second C-patch (Fig. 4C). The
20-fold lower relative binding affinity of
-globin 3'-UTR compared
with R7
1 SELEX is similar to that for the mutated R7
1 containing
a single C-patch. Thus the lower binding affinity of the native
-globin 3'-UTR may reflect a suboptimal presentation of the binding
site within the secondary structure which effectively exposes only a
single C-patch. This lower strength of RNA-protein interaction may be
more consistent with its in vivo functions. Further studies using appropriately altered binding sites in model target mRNAs can
address this possibility.
CP-2KL for their respective SELEX
RNAs (Fig. 8) were consistent with their optimized SELEX-binding sites;
a single C-patch was sufficient for high affinity binding by hnRNP K
but not for
CP-2KL, whereas a triple C-patch was necessary for high
affinity binding by
CP-2KL. Cross-binding studies with recombinant
proteins demonstrated that hnRNP K could bind to
CP-2KL SELEX
targets although it appeared to interact most strongly with its own
SELEX targets. This higher affinity of hnRNP K for its own SELEX
targets may have reflected the more limited and sterically constrained
single-stranded binding site than that presented on the
CP-2KL SELEX
RNAs containing multiple exposed C-patches. The specificity of
RNA-protein interactions appeared to be even greater when tested in the
context of native cell extracts. SELEX RNAs bound specificity to their
corresponding native proteins; R7K15 RNA bound to hnRNP K and R7
1
bound to
CPs (Fig. 8, C and D). The
specificity of R7
1, the
CP-2KL SELEX target, for
CP-2KL in the
extract was unexpected as this target contains multiple C-patches that
can be bound by recombinant hnRNP K (Fig. 8). This higher apparent
specificity of R7
1 in the context of extracts may reflect the
relatively lower levels of hnRNP K than
CP in the cytosol (hnRNP K
is predominantly a nuclear protein). The selective interaction of
R7K15, the hnRNP K SELEX target, to hnRNP K and its lack of binding to
the
CP-2KL was consistent with the requirement for multiple
C-patches for high affinity interaction with
CP-2KL.
CP-2KL and hnRNP K in the present study has defined optimal RNA
structures for each of these two KH domain proteins. The physical analysis of the SELEX targets and binding studies using both
recombinant proteins and native cytosolic extracts support the
conclusion that
CP-2KL and hnRNP K have distinct binding
specificities. The greater structural complexities of the RNA targets
binding sites for
CP-2KL verses hnRNP K may correspond to
the respective roles that these two proteins play in sequence-specific
post-transcriptional controls and in nuclear RNA packaging. The
structural basis for these distinct binding interactions will be of
interest for subsequent study.
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ACKNOWLEDGEMENTS |
---|
We thank Nancy E. Cooke and Shelly Wagggoner for detailed comments on the manuscript; Henrik Pedersen for advice on the SELEX protocol; Chenglu Liu and Alexander Chkheidze for providing samples of purified recombinant proteins; and Jessie Harper for expert secretarial assistance.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants HL38632 and CA72765 (to S. A. L.).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: Novo Nordisk, Enzyme Screening, 6E2.09,
Smørmosevej 10-12, DK-2880 Bagsværd, Denmark.
§ Present address: Gen-Probe, Inc., 10210 Genetic Center Dr., San Diego, CA.
¶ Investigator at the Howard Hughes Medical Institute. To whom correspondence should be addressed: Rm. 428, Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-7834; Fax: 215-573-5157; E-mail: liebhabe@mail.med.upenn.edu.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M010594200
2 A. Makeyev and S. Liebhaber, unpublished data.
3 T. Thisted, D. L. Lyakhov, and S. A. Liebhaber, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: hnRNP, heterogeneous nuclear ribonucleoprotein; EMSA, electrophoretic mobility shift assay; UTR, untranslated region; PCR, polymerase chain reaction; LOX, 15-lipoxygenase; nt, nucleotide; h, human; MEL, mouse erythroleukemia.
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REFERENCES |
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