(Received for publication, October 31, 1994)
From the
We have previously reported that heterogeneous nuclear ribonucleoprotein K (hnRNP K) binds to the pyrimidine-rich strand of the CT element found in the human c-myc gene and activates CT reporter-driven gene expression in vivo. We now characterize the DNA and protein requirements for the interaction of hnRNP K with the CT element. First, hnRNP K is shown to preferentially bind single-stranded DNA over RNA or native double-stranded DNA. Using specific oligoribonucleotide or deoxyribonucleotide probes with specific or nonspecific RNA or DNA competitors, electrophoretic mobility shift assay revealed hnRNP K to be a DNA-binding protein. Specific binding was not simply a reflection of binding to pyrimidine-rich sequences as the number and arrangement of individual CT elements governed interactions with hnRNP K; at least two CT repeats separated by at least three nucleotides are required for binding, indicating the existence of particular stereochemical constraints regulating CT-hnRNP K complex formation. Deletion analysis showed that hnRNP K possesses several nonoverlapping, DNA binding domains, each capable of specific binding with the CT element and preferring DNA over RNA. Each sequence recognition domain is composed of at least one K homology motif, while a larger portion of hnRNP K may be required for stable RNA binding. Additional experiments indicate that the N-terminal 35 residues of hnRNP K are necessary for transactivating the CT element. These results indicate that hnRNP K is a DNA-binding protein and transcriptional activator.
hnRNP ()K was first identified as a component of
hnRNP complexes and has been presumed to facilitate the processing
and/or transport of nuclear pre-mRNA(1, 2) . However,
several recent reports suggest that this same protein is a DNA-binding
protein. We have previously shown that hnRNP K binds and transactivates
a cis-element found within the human c-myc promoter(3) . hnRNP K, when phosphorylated, has also been
reported to bind the human immunodeficiency virus-long terminal repeat
B site(4) . Most recently, hnRNP K has been shown to
interact directly with the SH3 domain of c-src and to bind the vav proto-oncogene product in
vivo(5, 6) . It would seem that hnRNP K is more
than an architectural component of hnRNP complexes.
A motif, first described as a protein sequence homology repeated three times within the hnRNP K primary sequence (and hence termed the K homology, or KH domain), has been reported in a number of nucleic acid-binding proteins, including yeast MER1, Escherichia coli NusA, human glucopyranosyl adenine-6`-phosphate-associated p62, and FMR1(7, 8, 9, 10) . A recent report demonstrated that the integrity of all KH domains in hnRNP K is required for RNA binding and that the RNA binding activity of the FMR1 protein from a severely retarded fragile X patient is impaired by a single point mutation in one of the KH domains. Thus it appears that the KH domain is an RNA-binding motif (11) .
On the other
hand, the domains of hnRNP K that mediate sequence-specific DNA
recognition have not been determined, although involvement of KH
domains might be predicted. The KH proteins p62 and FMR can bind to DNA
as well as to RNA(9, 12) , and a KH domain is part of
the sequence-specific single-stranded DNA binding domain of the far
upstream binding protein (FBP) that interacts with negatively
supercoiled DNA bearing the far upstream element (FUSE) of the human
c-myc gene and that possesses a potent transactivation
domain(13) . ()These results suggest that KH domain
proteins may also bind DNA and participate in transcriptional
regulation.
Is hnRNP K a bifunctional DNA- and RNA-binding protein? Partitioning of protein between DNA and RNA is a regulatory device exploited in prokaryotes and also reported in vertebrates; for example, Xenopus laevis transcription factor TFIIIA binds to both the 5 S rRNA gene and to 5 S RNA(14, 15) . The interaction of dual specificity proteins, including hnRNP K with DNA and RNA in a sequence-specific or nonspecific manner, must be governed by the relative binding affinity and concentration of target binding sites. To determine the extent to which hnRNP K might interact with DNA, the binding affinity of hnRNP K with DNA and RNA was compared. Surprisingly, single-stranded DNA is the preferred target of hnRNP K. Because hnRNP K can also transactivate the CT element, this protein has all the features of a bona fide transcription factor.
The hnRNP K expression plasmid
(full-length hnRNP K) was constructed by inserting the EcoRI
fragment encoding hnRNP K from pHK5 into pcDNAI/AMP (Invitrogen), and
several mutants were constructed from the hnRNP K expression plasmid by
deleting the following fragments from full-length hnRNP K: NKH1+2
(1-367); deletion of the BspEI-XhoI fragment,
NKH3 (1-35, 368-463); deletion of the 997-bp BglII-HpaII fragment from position 211 to 1207,
N-term (1-35); deletion of the BglII-XbaI
fragment. C-term (368-463) was constructed after PCR of the hnRNP
K expression plasmid using the following two oligonucleotides:
AGCTCGAATTCAGAATATGGACGGATATGATTATTCCTATGCAGGGGG and
CTAGATGCATGCTCGAGCGGC. The PCR product was cleaved with EcoRI
and was cloned into pcDNAI/Amp. The reporter plasmids 56-mut was
described in Takimoto et al.(3) .
56CT3 was
constructed by inserting the double-stranded CT3 oligonucleotides into HindIII site of
56. The sequence of the CT3 is as
follows: AATTCTCCTCCCCACCTTCCCCACCCTCCCCA (template strand).
GAL4-Nterm was constructed after PCR of 1-35 amino acids of pHK5 using the following two oligonucleotides: CCGGGGATCCGTATGGAAACTGAACAGCCAGAAGAAACC and TTCAACCATCTCATCAGTGTTTCTAGATCTTTT. PCR product was cleaved with BamHI and BglII and was cloned downstream of GAL4 (1-147)(16) . GAL4-Cterm was created by inserting the HpaII-EcoRV fragment of hnRNP K expression plasmid into the SmaI site downstream of GAL4 (1-147). The reporter plasmid contained adenovirus E1bTATA promoter driven by five GAL4 sites coupled to the chloramphenicol acetyltransferase (CAT) gene as described elsewhere (17) .
EMSA (19) was performed with the indicated
amount of recombinant protein incubated in 25 mM Tris, 200
mM glycine, 1 mM EDTA, 50 mM NaCl, 0.5 mg/ml
bovine serum albumin, 0.1% Tween 20, 10% glycerol, 2 µg/ml
poly[d(IC)], and labeled probe as indicated. Twenty
units of rRNasin (Promega) was used with RNA probe and competitors to
avoid degradation. The DNA and RNA probes were 5`-end-labeled with T4
polynucleotide kinase and [
-
P]ATP. Binding
reactions were incubated for 20 min on ice, and protein-DNA complexes
were resolved by electrophoresis on 4 or 7% acrylamide gel in 50 mM Tris, 45 mM boric acid, 0.5 mM EDTA buffer at 4
°C. CT3 oligonucleotide was described above and riboCT3 is
AAUUCUCCUCCCCACCUUCCCCACCCUCCCCA (Clontech). The nonspecific DNA
competitor is the noncoding strand of the FUSE (-1500 bp upstream
of the c-myc P1 promoter), GATCCTATATTCCCTCGGGATTTTTTATTTTGT,
and nonspecific RNA competitor is the corresponding
ribooligonucleotide, GAUCCUAUAUUCCCUCGGGAUUUUUUAUUUUGU. CT1 is
GATCAAGCCAATTCTCCTCCCCACACAGGAAG, CT2 is
AATTCTCCTCCCCACCTTCCCCACACAGGAAG, CT2-3 is
AGCTAACCCTCCCCAATGCCCTCCCCATAG, CT2-9 is
AGCTAACCCTCCCCAATGCCCTCCCCATAG, and CT2-30 is
AGCTAACCCTCCCCAGATCAAGCCTGCGATGATTTATACTCACAGCCCTCCCCATAG. Human DNA
used as a competitor was isolated from Hela cells by addition of an
equal volume of 10 mM EDTA and 0.2% SDS, treated with RNase
and proteinase K, and purified by two phenol extractions, followed by a
phenol/chloroform extraction and ethanol precipitation. Total RNA was
isolated from Hela cells with RNAzol
B (Tel-test) and
treated with DNase to eliminate DNA contamination.
Figure 1:
hnRNP K
prefers to bind nonspecific ssDNA over native DNA (dsDNA) or RNA. EMSA
was performed using 4 ng of bacterially expressed GST or GST-RNP K and
20 fmol of P-labeled single-stranded CT3 pyrimidine
oligonucleotide (dCT3). Lane 1, probe alone; lane
2, GST; lane 3, GST-RNP K; lanes 4-7,
GST-RNP K with 40 ng, 200 ng, 1 µg, and 5 µg of human
single-stranded DNA as competitors; lanes 8-11, with the
same amount of human total RNA; lanes 12-15, with human
double-stranded DNA. DNA-protein complexes were separated from free
probe on a 4% polyacrylamide gel and visualized by
autoradiography.
Figure 2:
hnRNP K binds preferentially to
sequence-specific ssDNA. EMSA was performed on a 4% polyacrylamide gel
using 8 ng of GST-RNP K with 100 fmol of P-labeled
deoxy-CT3 (dCT3) (A) or riboCT3 (rCT3) (B). Specific activity of dCT3 and rCT3 are 2
10
cpm/pmol and 8
10
cpm/pmol, respectively. Lane 1, probe alone; lane 2, GST-RNP K; lanes
3-12, with the indicated molar excess of competitors. The
sequence of probe and competitors are described under ``Materials
and Methods.'' dNS and rNS indicate DNA and RNA
nonspecific competitors.
Figure 3:
Arrangement of CT element required for DNA
binding by hnRNP K. (A) hnRNP K did not bind to one or two
tandem CT repeats but did bind to the two repeats when separated. EMSA
was performed on a 4% polyacrylamide gel using 8 ng of GST or GST-RNP K
with 20 fmol of indicated P-labeled oligonucleotides. Lanes 1, 4, and 7, probe alone; lanes
2, 5, and 8, GST; lanes 3, 6,
and 9, GST-RNP K. CT1 indicates the probe that
contained one CT repeat and CT2 the probe that contained two
adjacent CT repeats. CT2-9 indicates the two CT repeats
separated by nine nucleotides. (B) The binding affinity of
hnRNP K to the two repeats increased as the distance between the two
repeats increased. EMSA was performed as above. Lanes 1, 3, and 5, probe alone; lanes 2, 4,
and 6, GST-RNP K. CT2-3 and CT2-30 indicate
the two CT repeats separated by 3 or 30 nucleotides,
respectively.
Figure 4:
Characterization of hnRNP K for ssDNA and
RNA binding. (A) Maps of GST-RNP K and deletion mutants are
shown. The open boxes labeled GST indicate the
glutathione S-transferase fragment present in the fusion
protein. The solid boxes indicate KH domains, and the hatched boxes indicate the N-terminal region. (B)
hnRNP K possesses nonoverlapping DNA binding domains. EMSA was
performed on a 7% polyacrylamide gel using 100 fmol of P-labeled CT3 with several mutants of hnRNP K. Lane
1, probe alone; lane 2, 80 ng of GST; lane 3, 2
ng of GST-RNP K; lane 4, 2 ng of N-terminal-KH1+2; lane 5, 80 ng of C-terminal-KH3; lane 6, 80 ng of
N-terminal. (C) The contributions of different portions of
hnRNP K to recognition of DNA over RNA are not equivalent. EMSA was
performed using 100 fmol of
P-labeled CT3 with 80 ng of
C-terminal-KH3 or 8 ng of N-terminal-KH1+2. The DNA-protein
complex was challenged with the indicated moler excess of CT3 or
riboCT3 as in Fig. 2A. A 4% polyacrylamide gel was used
for the binding with N-terminal-KH1+2, and a 7% polyacrylamide gel
was used for the binding with C-terminal-KH3. dNS and rNS indicate DNA
and RNA nonspecific competitors,
respectively.
In contrast to these observations,
Siomi et al.(11) , using an entirely different assay,
concluded that most of hnRNP K was required to bind nucleic acid,
including all three KH domains. Nevertheless, inspection of their data
suggested that deletions involving KH domain 1 were relatively more
impaired for binding to poly(rC) than with oligo(dC). Conversely,
deletions involving KH domain 3 were more impaired for oligo(dC)
binding. Consistent with this observation, in Fig. 4C,
it is apparent that C-terminal-KH3 possesses an enhanced ability to
discriminate dCT3 over rCT3. Furthermore, other workers have reported
that the C-terminal 75 amino acid residues of hnRNP K, including KH
domain 3, are sufficient to bind ssDNA(21) . The differences in
these observations may be explained by different assays, employing
different nucleic acid targets, and varying in the number and
arrangement of their specific cognate sequences such that all three KH
domains are not always required for complex formation. The
contributions of different portions of hnRNP K to recognition of DNA
over RNA were not equivalent. Comparing the relative ratio of the K values for ssDNA and RNA, the C-terminal-KH3
protein prefers DNA to RNA by 30-fold and the N-terminal-KH1+2
protein by only 10-fold. Because full-length hnRNP K bound DNA 5-fold
more than RNA, a larger portion of hnRNP K may be necessary to
stabilize RNA binding.
Figure 5:
Nonoverlapping DNA binding domains
contribute to transactivation. (A) Maps of hnRNP K expression
vector and the deletion mutants are shown. Full-length hnRNP K or the
deletion mutants were cloned behind the powerful cytomegalovirus
immediate early promoter (CMV promoter). Other symbols are as
shown in Fig. 4A. (B) Transactivation study of
the deletion mutants of hnRNP K. Six picomole of reporter plasmids
expressing CAT from the c-fos minimal promoter driven by CT3
(56CT3, top) or mutant CT elements (
56CT-mut,
bottom) were cotransfected into Hela cells with 2 pmol of either
the vector alone or the expression vectors shown in A. The
transfection was performed by electroporation as described under
``Materials and Methods.''
Figure 6:
The first 35 amino acid residues of hnRNP
K contributes to transactivation. The N-terminal region (Nterm) and the C-terminal region (Cterm) of hnRNP K
shown in Fig. 5A were fused to the GAL4 DNA binding
domain, GAL4 (1-147), driven by SV40 early promoter. One picomole
of either the vector expressing only GAL4 or expressing fusion proteins
was cotransfected into COS7 cells with 2 µg of adenovirus E1bTATA
promoter driven by five GAL4 sites coupled to the CAT gene as a
reporter (p(G4)CAT). The
transfection was performed by electroporation as in Fig. 5B.
hnRNP K was first identified as a component of RNP complexes
of hnRNA in mammalian cells(1, 2) . It possesses a
highly conserved motif, the KH domain, which is present in several
other proteins(22) . The KH domain is commonly considered an
RNA binding domain despite the demonstration that at least three
mammalian KH proteins (hnRNP K, p62, and FMR) have been shown to bind
with either DNA or RNA(3, 9, 12) . DNA versus RNA binding has not been previously compared for these
proteins; if DNA binding is merely a vestige of RNA binding, then their
affinity for RNA should be considerably greater than for DNA, as has
been reported for several bona fide RNA-binding proteins (23) . On the other hand, if some of these KH proteins were to
prove to be bifunctional DNA- and RNA-binding proteins, then their
affinities for DNA and RNA should prove comparable. The results
presented here argue that hnRNP K, the prototype mammalian KH protein,
can function as a transcription factor. First, hnRNP K binds to ssDNA
more tightly than to RNA in a sequence-specific manner. Second, hnRNP K
recognizes a reiterated cis-element that occurs in the natural context
of the c-myc promoter. Third, hnRNP K stimulates gene
expression through the CT element. Fourth, insofar as hnRNP K has
separable DNA-binding and transactivation domains, it behaves
structurally like most conventional gene regulatory proteins. A recent
finding that hnRNP K interacts with TATA-binding protein (TBP) supports
this conclusion. ()These observations raise the possibility
that other RNA-binding proteins with KH domains may also function in a
process requiring DNA binding. In fact, KH domains are found in a
transcription factor of the c-myc gene, FBP, and are essential
for DNA binding(13) .
Might this difference in binding affinity to DNA or to RNA have any physiological relevance? A growing number of factors that bind to DNA or RNA have been reported, some preferring to bind to DNA, whereas others prefer RNA. Several prokaryotic examples serve to illustrate the utility of this dual-binding specificity. E. coli single-strand binding protein (SSB) and T4 gene 32 protein each bind to single-stranded DNA as well as their own mRNAs(24, 25, 26) ; thus these single-stranded DNA-binding proteins partition between intracellular single-stranded DNA and mRNA, thereby regulating their own translation. A recent report demonstrated that bacteriophage T4 DNA polymerase has a 100-fold higher affinity for in vitro transcribed RNA as compared to DNA of the same sequence and suggested that the protein represses its own translation by binding to its mRNA(27) . Partitioning of regulatory proteins between DNA and RNA is not a device unique to prokaryotes. TFIIIA, one of the best characterized eukaryotic transcription factors, partitions between 5 S rRNA and the 5 S rRNA gene, binding to each with comparable affinity(14, 15) . The preferential binding of hnRNP K to DNA over RNA may be a requirement for transcription activation through a relatively smaller number of genomic, single-stranded CT elements operating against competing target sites in hnRNA, in hnRNPs, or directly on other proteins through protein-protein interactions.
How many roles does hnRNP K play? Besides RNA metabolism and
transcription, hnRNP K has recently surfaced in several unexpected
contexts. hnRNP K has been shown to bind the SH3 domain of c-src through a segment of hnRNP K encoded by an alternatively spliced
exon(2, 5, 28) . p95 vav also interacts both in vitro and in vivo with hnRNP K(6) . These
results suggest that hnRNP K may participate in signal transduction,
perhaps as a downstream effecter molecule. Additional work has
demonstrated that hnRNP K binds to TBP, consistent with the idea that
hnRNP K might directly modulate the transcription machinery. hnRNP K
also can bind hyperphosphorylated forms of the Rb protein, ()and nuclear staining of hnRNP K has been reported to be
cell cycle-dependent(29) ; these observations evoke the notion
that hnRNP K function might be coupled to the cell cycle. The multiple
complexes within which hnRNP K can be found in vivo and in
vitro indicate that hnRNP K is not simply a structural component
of hnRNP complexes but is an abundant multifunctional protein,
partitioning between competing complexes and potentially linking
several important cellular processes.