©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Heterogeneous Nuclear Ribonucleoprotein K Is a DNA-binding Transactivator (*)

(Received for publication, October 31, 1994)

Takeshi Tomonaga David Levens (§)

From the Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

hnRNP (^1)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 kappaB 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) . (^2)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.


MATERIALS AND METHODS

Plasmid Construction

The full-length and the deletion mutants of GST-RNP K, N-terminal-KH1+2 (1-265) and C-terminal-KH3 (329-463), were created by PCR of an hnRNP K cDNA (pHK5, a kind gift from G. Dreyfuss) using the following oligonucleotide: TCAGATGAATTCATATGGAAACTGAACAGCCAGAAGAAACCTTC (5` oligo) and TAAAGCGAATTCTAAGAAAAACTTTCCAGAATACTGCTTCAC (3` oligo) for the GST-RNP K, 5` oligo and GATTCACCGGAATTCACGCATTTAGCTAGCTGGTCCTCGACGAGGGCTCATATCATCATA for the N-terminal-KH1+2, and AGATCACCGCCCGGGACGCATTCAGCTAGCGGCGGCCGG-GGTGGTAGCAGAGCTCGGAAT and 3` oligo for the C-terminal-KH3. PCR products for the GST-RNP K and Nterminal-KH1+2 were restricted with EcoRI, and those for the C-terminal-KH3 were restricted with SmaI and EcoRI, which were cloned into pGEX-2TK. N-terminal region was created by inserting the EcoRl and the BglII fragment of the GST-RNP K into pGEX-2TK.

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 Delta56-mut was described in Takimoto et al.(3) . Delta56CT3 was constructed by inserting the double-stranded CT3 oligonucleotides into HindIII site of Delta56. 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) .

Protein Expression and EMSA

Recombinant protein was purified from extracts of E. coli transformed with the pGEX-2TK(18) , pGEX-hnRNP K, or plasmids encoding mutant proteins by glutathioneagarose affinity chromatography (Sigma). Fusion proteins were eluted with 10 mM glutathione and checked for purity, correct size, and concentration with SDS-polyacrylamide gel electrophoresis.

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(IbulletC)], 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.

Cell Culture, Transfection, and CAT Assays

Hela and COS cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells (5 times 10^6) were resuspended in 250 µl of Dulbecco's modified Eagle's medium and incubated on ice for 10 min with plasmid DNA. Electroporation was performed with Cell-porator (Life Technologies, Inc.) at 230-V, 1180-µF setting, and after the electroshock, the cells were incubated on ice for an additional 10 min. Transfected cells were added to 10 ml of medium and incubated for 48 h before harvesting for CAT assays(20) .


RESULTS

hnRNP K Prefers Nonspecific ssDNA to Native dsDNA or RNA

To determine which nucleic acid hnRNP K binds most tightly, the binding activity of hnRNP K to heat-denatured total human ssDNA, dsDNA, and total cellular RNA was compared. As previously reported, hnRNP K forms a specific complex, detected by EMSA, with the pyrimidine-rich strand of the CT element found upstream of the human c-myc promoter, composed of tandem imperfect repeats of a 9-bp sequence (CT repeat: CCCTCCCCA)(3) . Using a pyrimidine strand probe comprised of three CT repeats (CT3) and bacterially expressed GST-hnRNP K, complex formation was challenged with total RNA, ssDNA, or dsDNA (Fig. 1). Whereas 5 µg of ssDNA virtually eliminated formation of the hnRNP K-probe complex formation, the same amount of RNA had little effect, and dsDNA had no effect. This result showed that hnRNP K binds more tightly with ssDNA than to RNA and is virtually oblivious to dsDNA.


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.



hnRNP K Binds Preferentially to Sequence-specific ssDNA

Although it seemed that hnRNP K preferred ssDNA to RNA, the possibility remained that binding of hnRNP K to the deoxy-CT-element was actually a manifestation of an even stronger sequence preference for a cytidine-rich RNA sequence, vastly underrepresented in total RNA. Therefore the identical sequence, either in RNA (rCT3, three repeats of CCCUCCCCA) or DNA (dCT3, three repeats of CCCTCCCCA), was compared for binding with hnRNP K in the presence of either RNA or DNA competitor (Fig. 2). GST-RNP K was incubated with rCT3 or dCT3 probe and challenged with rCT3 or dCT3 competitor. Under all conditions, dCT3 bound more tightly with hnRNP K than rCT3. The relative ratio of the dissociation constants (K(d)) of hnRNP K-dCT3 and hnRNP K-rCT3 complexes, determined from binding curves, revealed 5-fold greater binding of hnRNP K to DNA than to RNA (data not shown). hnRNP K-dCT3 and hnRNP K-rCT3 complexes are both sequence-specific, as vast excesses of irrelevant sequence DNA or RNA did not perturb binding to the CT element. Thus, dCT3 is the preferred ligand of hnRNP K.


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 times 10^6 cpm/pmol and 8 times 10^5 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.



Characterization of DNA Binding Site for hnRNP K

Previous studies of hnRNP K binding to DNA and RNA have employed polymeric or oligomeric nucleic acids composed entirely or largely of cytidine residues. If hnRNP K recognizes a defined motif comprised principally of cytidine, then it should be able to recognize its cognate sequence even when embedded among irrelevant sequences. Alternatively, if hnRNP K binding requires only cytidine-rich nucleic acid then the composition, and not the arrangement, of the nucleotides comprising the probe should determine the binding affinity. It has already been demonstrated that hnRNP K binds to three direct CT repeats; to determine the minimum number and arrangement of CT elements required for binding with hnRNP K, several defined sequence single-stranded deoxyoligonucleotide probes were synthesized and tested by EMSA (Fig. 3A). hnRNP K did not bind to one (CT1) or two tandem repeats (CT2). However, by separating the two CT elements from each other with an irrelevant spacer sequence, molecules forming specific complexes with hnRNP K were created; the binding affinity of hnRNP K to the probe increased as the distance between the two CT elements was increased from 3 bp (CT2-3) to 9 bp (CT2-9) and to 30 bp (CT2-30) (Fig. 3B). Thus, hnRNP K recognizes single-stranded CT elements, not simply base composition, and to be recognized the elements must be arranged in a polymer capable of adapting to certain stereochemical requirements. In these respects hnRNP K behaves similarly to most sequence specific DNA-binding proteins.


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.



hnRNP K Possesses Nonoverlapping DNA Binding Domains

To determine what portions of hnRNP K directed specific binding to dCT3 or rCT3, a series of deletion mutants were expressed as GST fusion proteins and assayed by EMSA (Fig. 4, A and B). Surprisingly, nonoverlapping segments of hnRNP K formed complexes with dCT3 and only the solitary N terminus lacking any KH domain failed to bind to dCT3. The binding observed was specific because complex formation could be competed by CT elements but not by unrelated sequences (Fig. 4C). Therefore, redundant sequence recognition domains reside within the hnRNP K backbone, perhaps reflecting the reiterated nature of the CT element as it occurs in the human c-myc gene.


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(d) 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.

Nonoverlapping DNA Binding Domains Contribute to Transactivation

If hnRNP K recognizes the CT element to stimulate expression, then the same domains that separately bind to dCT3 in vitro might independently facilitate CT element activity in vivo. Therefore, a series of hnRNP K deletion mutants, characterized for binding in vitro were cloned downstream of the cytomegalovirus immediate early enhancer/promoter (Fig. 5A) and were cotransfected with the CT3-minimal fos promoter CAT (Delta56CT3) or mutant CT elements-minimal fos promoter CAT (Delta56CT-mut) reporter plasmids (Fig. 5B). Full-length hnRNP K augmented CT-mediated expression approximately 4-fold. The mutant forms of hnRNP K possessing KH domain 1+2 (NKH1+2) or 3 (NKH3), which retained specific dCT3 binding by EMSA, also mediated CT element activation in vivo. In contrast, the N-terminal region alone, which does not have a KH domain (N-term), had only a marginal effect, indicating that at least one KH domain is required for transactivation. The only mutant that bound in vitro, but failed to transactivate the CT element, was composed solely of C-terminal sequences (KH3) and lacked the N-terminal residues shared by all other constructs. These results, when combined with the in vitro binding data, suggest that at least one KH domain is required for the DNA binding and the N-terminal region is likely to be required for transactivation. It is important to note that transactivation is sequence specific because none of the mutants stimulated the reporter gene containing a mutant CT element.


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 (Delta56CT3, top) or mutant CT elements (Delta56CT-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.''



The N-terminal 35 Amino Acid Residues of hnRNP K Contribute to Transactivation

To test the hypothesis that the N terminus is necessary for transactivation, the first 35 amino acids from the N terminus of hnRNP K, including 11 acidic residues, were fused to the GAL4 DNA-binding domain, GAL4 (1-147). The resulting plasmid was cotransfected along with the CAT gene driven by five GAL4 sites from the adenovirus E1bTATA into COS7 cells (Fig. 6). As predicted, the N terminus of hnRNP K (GAL4-Nterm) increased CAT activity compared to the GAL4 DNA binding domain alone. Although the C-terminal region (KH3) of hnRNP K is required to stimulate the CT element, the GAL4 C-terminal fusion protein did not augment expression from GAL4 sites, consistent with the notion that KH3 supplies a nucleic acid recognition motif but not a transactivation domain. Thus, the first 35 amino acid residues of hnRNP K are important for transactivation.


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.




DISCUSSION

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. (^3)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, (^4)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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 301-496-2176; Fax: 301-402-0043.

(^1)
The abbreviations used are: hn, heterogeneous nuclear; RNP, ribo-nucleoprotein; ss, single-stranded; ds, double-stranded; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; bp, base pair; FUSE, far upstream element.

(^2)
L. Bazar, D. Meighen, R. Duncan, D. Levens, M. Avigan, manuscript submitted for publication.

(^3)
E. Michelotti and D. Levens, manuscript submitted for publication.

(^4)
E. Michelotti and D. Levens, unpublished data.


ACKNOWLEDGEMENTS

We thank Emil Michelotti and Robert Duncan for help of this work, Susan Mackem and Greg Michelotti for comments on the manuscript, Suzanne Sanford for preparing the synthetic oligonucleotides, and Charles Robinson for technical assistance.


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