Interaction of two multifunctional proteins. Heterogeneous nuclear ribonucleoprotein K and Y-box-binding protein.

The heterogeneous nuclear ribonucleoprotein (hnRNP) K, a component of the hnRNP particles, appears to be involved in several steps of regulation of gene expression. To gain insight into mechanisms of K protein action, we performed two-hybrid screens using full-length hnRNP K as a bait. Several novel protein partners were identified, including Y-box-binding protein (YB-1), splicing factors 9G8 and SRp20, DNA-methyltransferase, hnRNP L, and hnRNP U. In vitro binding studies and co-immunoprecipitation from cellular extracts provided evidence for direct interaction between hnRNP K and YB-1. Two distinct domains in YB-1 were responsible for binding to K protein. Each protein was able to transactivate transcription from a polypyrimidine-rich promoter; however, this effect was reduced when K and YB-1 proteins were coexpressed suggesting a functional interaction between these two proteins.

Activation of gene expression in eukaryotes encompasses multiple processes including chromatin reorganization, followed by the assembly of transcription factors onto promoter elements, processing of pre-mRNA transcripts, and export of the mRNA into the cytoplasm and translation. Although each of these steps has been intensively explored, little is known of how these processes are linked and what factors are involved. RNA-binding proteins, components of the hnRNP 1 particles, may play such a role (1). The formation of these particles is closely coupled to transcription. There are over 20 proteins that bind to pre-mRNA, which are collectively known as the heterogenous nuclear ribonucleoproteins. These proteins are thought to promote formation of pre-mRNA secondary structure and can influence splice site selection. Some hnRNP proteins shuttle between the nucleus and cytoplasm and potentially can influence mRNA export and translation. RNAbinding proteins not only associate with nascent pre-mRNA but also have been found to contact the basal transcriptional machinery and can directly bind to DNA regulatory elements within the promoter itself (reviewed in Ref. 2). Moreover, recent data suggest that components of hnRNPs may serve as a structural and/or functional link between RNA metabolism and nuclear architecture (3).
The hnRNP K protein, a component of the hnRNP particle, exhibits many of the functions described above. It has a wide tissue distribution and is detected in the nucleus and cytoplasm (4). K protein contains both a classical bipartite-basic nuclear localization signal and the novel K nuclear shuttling domain that allow it to shuttle between nucleus and cytoplasm by utilization of one of the two pathways for nuclear entry (5).
K protein has a strong affinity for polypyrimidine-rich RNA and for single-stranded DNA. K protein is also known to interact with many proteins involved in several cellular processes. Because hnRNP K interacts with many protein kinases such as Src, protein kinases C, and the proto-oncogene vav (6 -8), it has been proposed that hnRNP K is involved in signal transduction and may act as a "docking platform" mediating cross-talk between these molecules (9). K protein may facilitate the ability of other factors to stimulate or repress transcription by recruiting gene-specific or general components of the transcription machinery to promoters, such as TBP, SP1, and the transcriptional repressor, Zik1 (10 -12).
In addition to its involvement in signal transduction and transcription, K protein may participate in the regulation of other nucleic acid-dependent processes. For example, binding of K protein to Eed (13), a putative silencer of homeotic genes, may reflect participation of hnRNP K in chromatin reorganization. K protein-mediated silencing of the lipoxygenase mRNA through the binding to the CU-rich 3Ј-untranslated region (14) represents an example of hnRNP K involvement in translation.
Taken together, these studies suggest that K protein is involved in multiple processes that comprise gene expression and may serve as a link between signal transduction pathways and nucleic acid-dependent processes (7,15). To gain more insight into the mechanisms of K protein action, we performed twohybrid screens using full-length hnRNP K as a bait. We identified several novel protein partners, including YB-1 transcription factor, splicing factors 9G8 and SRp20, DNA-methyltransferase, hnRNP L, and hnRNP U. In this work we describe the interaction between K protein and YB-1, another multifunctional protein.

MATERIALS AND METHODS
Plasmids-For two-hybrid analyses, cDNA encoding full-length K protein was inserted into multiple cloning sites of plasmid pGBT9, which expresses the Gal4 DNA binding domain (CLONTECH). In frame fusion was verified by sequencing using the DyeDeoxy terminator cycle sequencing kit (PE Applied Biosystems) and by in vitro transcription/ translation analysis. Two cDNA libraries were used in the two-hybrid screen. The mouse cDNA library was generated by random-primed cDNA synthesis from a 9 -10-day-old mouse embryo (Dr. S. Hollenberg) and size-selected for inserts in the range of 350 -700 nucleotides. This library was inserted into the transcription activation domain vector, pVP16, to generate VP16-cDNA hybrid proteins. The Jurkat cell cDNA library of Gal4AD-cDNA fusion for the two-hybrid screen was obtained from CLONTECH.
Construction of the GST⅐K mutants was described previously (6). EFI A /YB-1 cDNA was kindly provided by Dr. K. R. Chen. For the in vitro experiments the NotI fragment carrying full-length EFI A /YB-1 cDNA was subcloned into the NotI site of pBluescript II SK (Stratagene), and the resulting plasmid was denoted pSK-YB-1. To create the YB-1 C mutant, pSK-YB-1 was digested with SalI and religated. The NotI/SalI fragment was isolated and inserted into the NotI/SalI sites of pET28a.
The 0.7-kilobase SalI fragment from pSK-YB-1 containing the N terminus of EFI A /YB-1 was subcloned into the SalI site of the pSK vector. This mutant was named YB-1 NI. The NII, cold shock domain (CSD), NIII, CI, and CII fragments were created by polymerase chain reaction and subcloned into pET28a vector (Fig. 5). All constructs were verified by sequencing.
Flag-YB-1 was generated by insertion of YB-1 cDNA into the EcoRI/ XhoI sites of p18-Flag mammalian expression vector.
PSG5-K and pSG 5 was a gift from Dr. A. Ostareck-Lederer. pSG 5-YB-1 was created by inserting the NotI fragment of EFI A /YB-1 into pSG5.
The plasmids pgst-TBP, p⌬56CT3wt, and p⌬56CT3mut were a gift from Dr. D. Levens. CT3wt-c-fos and CT3mut-c-fos promoter elements were cut out from p⌬56CT3wt and p⌬56CT3mut, respectively, and subcloned into the pGL3-basic vector (Promega). All plasmids were purified using the QIAfilter Plasmid Maxi Kit (Qiagen).
Yeast Strains, Transformation, and Growth Conditions-Transformation of yeast cells was carried out by the method of Klebe et al. (16). Yeast transformants were selected and cultivated on SD synthetic medium (2% glucose and 0.67% yeast nitrogen base without amino acids) supplemented with essential amino acids and nucleotides.
␤-galactosidase expression was detected either by qualitative determination using a filter lift assay (17) with 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside as a substrate or quantitatively by o-nitrophenyl-␤-D-galactopyranoside hydrolysis measured photometrically at 420 nm upon permeabilization of cells (17). The enzymatic activity was normalized to cell culture density expressed as arbitrary units.
Synthesis and Purification of GST Fusion Proteins-GST⅐K and GST⅐TBP constructs were described previously (6,7).
In vitro transcription and translation was performed using the TNT T7 Quick coupled transcription/translation system as per the manufacturer's protocol (Promega, Madison, WI).
In Vitro Binding Studies-2.5 l of the 35 S-labeled products were added to a suspension of 20 l of glutathione or 10 l of either poly(A), (C), (G), (U), or (I) beads in 100 l of binding buffer (12). After mixing for 60 min (4°C), the beads were washed three times with 400 l of binding buffer and then boiled with 30 l of SDS loading buffer; proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and detected by autoradiography.
Transient Transfections-HeLa cells were grown in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum in 60-or 100-mm dishes and were transfected using SuperFect Transfection Reagent as per the manufacturer's protocol (Qiagen). Either pRLnull plasmid (Promega) encoding Renilla luciferase or pSVgal plasmid (Promega) encoding ␤-galactosidase was cotransfected to normalize transfection efficiency. The same protocol was used to transfect NIH3T3 cells. Luciferase activity assay was performed according standard protocols (Promega). ␤-Galactosidase activity was measured using a Galacto-light plus chemiluminescent reporter assay system (Tropix).
Cell extracts were prepared by a modified version of the method of Dignam et al. (18) as described previously (7). Protein concentration was measured using the DC method (Pierce).
Immonoprecipitation and Western Blotting-Transfected HeLa cells were grown for 48 h, and cell extracts were prepared as described above. Rabbit polyclonal anti-K protein antibody 54 was added to 500 l of cell extract, and the mixture was incubated overnight at 4°C with low speed rotation. 20 l of protein A/G PLUS-agarose (Santa Cruz Biotechnology Inc.) and ELB buffer (19) (250 mM NaCl, 0.1% Nonidet P-40, 50 mM HEPES (pH 7.0), 5 mM EDTA) were added to a total volume of 1 ml. The mixture was rotated for another hour at 4°C. The protein A/G beads were washed four times with 1 ml of ELB buffer, and the proteins were eluted by boiling in 30 l 1ϫ loading buffer, separated by SDS-PAGE, and transferred to Immobilon-P membrane (Millipore). The blots were probed with monoclonal M2 anti-Flag antibodies (Sigma). The chemiluminescent detection was performed with ECL Western blotting detection reagents according to standard protocol (Amersham Pharmacia Biotech).

Identification of New Protein Partners of K Protein Using the
Yeast Two-hybrid System-Although recent data indicate that hnRNP K protein is involved in many cellular processes, the mechanism of its action is largely unexplored. Thus the identification of new protein partners of K protein provides important information about its cellular function. We performed a two-hybrid screen with full-length K as a bait using two cDNA libraries. The Gal4BD⅐K fusion protein did not have transactivation activity by itself when expressed in Saccharomyces cerevisiae. Yeast strain HF7C expressing Gal4BD⅐K protein was transformed with 9 -10-day-old mouse embryo or Jurkat cell cDNA library; transformants were selected as described under "Materials and Methods." Putative positive clones were retransformed into SFY526 yeast strain and tested for the expression of ␤-galactosidase. False positives were excluded by their ability to activate transcription of His and lacZ reporter genes in the absence of the bait. Sequencing of twenty-one true positive clones from the mouse cDNA library identified several new protein partners of hnRNP K ( Table I). Screening of the Jurkat cDNA library yielded two true positive clones encoding hnRNP L, a component of hnRNP, and the pre-mRNA splicing factor SRp 20. The interaction with splicing factors and hnRNP L suggests that K protein participates in the processing of pre-mRNA. Interaction with scaffold factors and DNA-methyltransferase may reflect the role of K protein in chromatin organization. Seven clones represented YB-1 cDNA with different lengths of insert and different strength of interaction with the bait (Fig. 1). We were particularly interested in YB-1 protein because it shares many functional similarities with hnRNP K and appears to be involved in many cellular processes.
K Protein Interacts with YB-1 in Vitro-To confirm the twohybrid screen we examined whether YB-1 could interact with K protein in vitro. In these experiments we used the rat homologue of YB-1, EFI A . In vitro translated 35 S-labeled YB-1 was incubated with agarose beads bearing GST⅐K protein, as described previously (7). After incubation, beads were washed three times with an excess of binding buffer and one time with the same buffer containing 1% Nonidet P-40. Samples were boiled in SDS loading buffer, and proteins were resolved by SDS-PAGE. In agreement with the two-hybrid screen, a strong interaction between YB-1 and GST⅐K was observed in vitro ( Fig. 2A, lane 2). In contrast to the mammalian K protein, the yeast homolog of hnRNP K, PBP2p (20), did not interact with YB-1 ( Fig. 2A, lane 1). Of note, the YB-1 family proteins are not represented in the yeast genome (confirmed by a search of the completed yeast genome sequence). For some RNA-binding proteins it is known that RNA mediates protein-protein interaction. For example, the primary steps of Sam68 dimerization require cellular RNA (21). Because both K and YB-1 are known to be RNA-binding proteins, we next tested whether the interaction between these proteins can be mediated by RNA. We   (Fig. 2B, lanes 1-5). These experiments were carried out in two different ways. In one set of experiments, 35 S-labeled YB-1 and GST⅐K beads were incubated in the presence of polynucleotides; in another set, GST⅐K beads were preincubated with polynucleotides for 30 min before 35 S-labeled YB-1 was added to the reaction mixture. Identical results were obtained in both experiments. As shown in Fig. 2B (lane 1), poly(U) decreased the interaction of YB-1 with K protein, whereas the other polynucleotides did not alter this binding. When incubated under the same binding conditions, but without K protein, YB-1 was able to bind poly(U) and poly(G) beads (data not shown).
Mapping the Regions of K Protein Involved in Interaction with YB-1-Using a panel of GST⅐K deletion mutants, we next mapped the interaction region in hnRNP K responsible for binding to YB-1. As shown in Fig. 3, 35 S-labeled YB-1 strongly interacted with GST⅐K13 (lane 6) and GST⅐K31 (lane 8), but no binding was detected with the other mutants. These data indicate that the interactive region is located between amino acids 240 and 337. This region, denoted KI, was previously found to be responsible for binding of other K protein partners such as protein kinase C ␦, Zik1, and Eed (7,12,13).
Levens and co-workers (10) demonstrated the interaction between TBP and K in vitro and in vivo. In contrast to YB-1, we observed strong interaction of 35 S-labeled TBP not only with GST⅐K31 and GST⅐K13 but also with GST⅐K7 (Fig. 4, lanes 3, 6,  and 8) and moderate binding to GST⅐K10 (Fig. 4, lane 4) indicating that the only amino acids overlapping the strongest binding fragments are 318 -337, but amino acids external to this region also impact TBP binding.
To more precisely map the region of K protein responsible for binding to YB-1, we used GST⅐K protein mutants with deletions inside the KI region. Deletions of amino acids 322-337 Numbers represent amino acid residues. B, plasmid containing partial cDNA of mouse YB-1 fused to the VP-16 activation domain was rescued from seven true positive clones from the yeast strain HF7c and then retransformed into S. cerevisiae SFY 526. Quantitative ␤-galactosidase activity was monitored as described under "Materials and Methods." The enzymatic activity was normalized to cell culture density and is expressed as arbitrary units.

FIG. 2. K protein interacts with YB-1 in vitro. A, GST⅐K and
GST⅐PBP2p bound to glutathione beads were incubated with 2.5 l of in vitro translated 35 S-labeled YB-1. Beads were washed three times with binding buffer and one time with binding buffer containing 1% Nonidet P-40. Proteins bound to the beads were eluted with SDS buffer as described under "Materials and Methods" and then resolved by SDS-10% PAGE. The gel was stained (Coomassie Blue) and autoradiographed. 2.5 l of the input (load) used in each reaction was run in lane 3. The arrow marks the position of the in vitro translated 35 S-labeled YB-1. B, GST⅐K protein bound to glutathione beads was incubated with in vitro translated 35 S-labeled YB-1 in the absence (lane 6) or presence of 2 g of given homopolymer RNA (lanes 1-5). Beads were washed, and bound proteins were eluted and analyzed as described above. To map the interactive region in YB-1 that binds K protein, we created a series of deletion mutants (Fig. 5C). The in vitro binding experiments were performed as described above. Surprisingly, we observed equally strong binding of both N-terminal and C-terminal parts of YB-1, indicating the presence of multiple interactive regions (Fig. 5, A and B). The binding of NII polypeptide (amino acids 58 -150), which includes the conservative RNA binding CSD followed by 26 amino acids, was as strong as that of the full-length YB-1 (Fig. 5A, lanes 7). As shown in Fig. 5A, the NIII polypeptide, containing 26 amino acids immediately downstream of the CSD, maintained a strong interaction with GST⅐K (Fig. 5A, lane 9). No interaction was found with the "cold shock" domain (Fig. 5A, lane 8). Fig. 5B illustrates mapping of the second interactive region localized in the C terminus of YB-1. The CI polypeptide did not interact with GST⅐K protein (lane 11); however, the binding of the CII polypeptide containing the amino acids 271-320 was strong (lane 12). Thus the second interactive region responsible for binding to GST⅐K is located in the last 50 amino acids of the C terminus of YB-1 (amino acids 271-320).
These results are in agreement with our two-hybrid system data. As shown in Fig. 1, the highest ␤-galactosidase activity was observed with clones 1 and 2, which contained residues located just downstream of the cold shock domain. Clone 5 had the part of the C-terminal interactive region resulting in an increase of ␤-galactosidase activity in comparison to clones 3 and 4, which are missing this C-terminal region. The middle part of YB-1 has been shown to be responsible for its multimerization and for binding to other protein partners of YB-1 (22,23). The presence of two distinct interactive regions for K protein in YB-1 is a novel observation.
Next, we examined whether TATA-binding protein, a known partner of hnRNP K, can interact with YB-1. As in the case of GST⅐K, the same NIII (amino acids 124 -150) (Fig. 5A, lane 6) and CII (amino acids 271-320) polypeptides (Fig. 5B, lane 6) were responsible for the binding of YB-1 to TBP.
Co-immunoprecipitation of K Protein and YB-1 from Cell Extracts-Because the hnRNP K is highly expressed in HeLa cells, we tested whether endogenous K protein can interact with overexpressed Flag-tagged YB-1. HeLa cells were transfected with Flag-YB-1 construct. Cellular extracts were prepared and examined for the presence of K protein⅐YB-1 complex by co-immunoprecipitation with either pre-immune or anti-K serum followed by Western blotting with anti-Flag antibodies (as described under "Materials and Methods"). In cells transfected with Flag-YB-1 only immune anti-K serum precipitated Flag-YB-1 (Fig. 5, lane 4). Although expression levels of both hnRNP K (4) and Flag-YB-1 are high (lanes 7 and 8), the magnitude of their detectable interaction appears to be lower  4 and 7) and NIII polypeptides (lanes 6 and 9) to GST⅐TBP and GST⅐K was strong. No interaction was found with the cold shock domain (lanes 5 and 8). B, the GST⅐K and GST⅐TBP proteins bound to glutathione beads were incubated with 2.5 l of in vitro translated YB-1 35 S-labled C-terminal mutants (C, CI, and CII). Binding reaction was performed as described above, and proteins were resolved by SDS-15% PAGE. 2.5 l of the input (load) used in each reaction was run in lanes 1-3. CI polypeptide did not interact with either GST⅐TBP (lane 5) or GST⅐K (lane 11); however, the binding of CII polypeptide (lanes 12 and 6) was as strong as that of a mutant (C) containing the entire C terminus of YB-1 (lanes 4 and 10). C, schematic representation of YB-1 deletion mutants used in in vitro binding experiments. than expected. This could be explained by the fact that both K protein and YB-1 have many other molecular partners.
The Role of K Protein-YB-1 Interaction in Gene Expression-One of the functional similarities between hnRNP K and YB-1 is their high affinity binding to single-stranded polypyrimidine-rich sequences. K protein and YB-1 have been shown to interact with a C-rich DNA sequence, termed the CT element upstream of the c-myc gene (24,25). Furthermore, overexpressed hnRNP K and TBP synergistically activated transcription of a CT element-dependent reporter gene in vivo (10). Recent studies revealed that the chicken homolog of YB-1 protein, chk-YB-1b, binds efficiently to the well conserved polypyrimidine tract found in ␣1(I) collagen gene promoter from rat, mouse, human, and chicken (26). Thus the promoters containing CT-rich elements could be an appropriate system to understand the significance of hnRNP-K-YB-1 interaction (Fig. 7).
Because hnRNP K and YB-1 exhibit transcriptional activities from the sequence CCCTCCCCA, known as the CT element of the human c-myc gene (10,24,25), we tested how the coexpression of K and YB-1 proteins affects the activity of a luciferase reporter gene. HeLa cells were transfected with hnRNP K and Flag-tagged YB-1 expression constructs and with a plasmid encoding the luciferase reporter gene driven by a synthetic promoter containing three repeats of the CT element of either wild-type CT3 or a mutated version, CT3mut, upstream of the minimal c-fos promoter described in Ref. 10. As shown in Fig.  7A, hnRNP K alone did not have an effect on the luciferase expression level. Flag-YB-1 slightly activated transcription from the CT3 wild type promoter element but did not affect transcription from the promoter-containing mutated CT3 element. When both proteins were coexpressed in HeLa cells, the activation effect of YB-1 from the wild-type CT3 element was reduced. Similar results were obtained in NIH3T3 cells (Fig.  7B). YB-1 activated transcription from the wild type promoter but not from the mutant. Interestingly, in this cell line hnRNP K also activated the CT element in a sequence-specific manner. The sequence-specific trans-activation effect of both K protein and YB-1 was reduced when both proteins were coexpressed. These transfection experiments show that hnRNP K and YB-1 are functionally linked. It should be noted, however, that both proteins had only a modest effect on the expression of the reporter gene. As mentioned above, K and YB-1 proteins are abundant, and the high level of the endogenous proteins may have blunted the reporter gene expression. DISCUSSION In this paper we describe the interaction between K protein and YB-1. There are many indications that hnRNP K and YB-1 are functionally similar proteins. As described in the Introduc-tion, K protein is involved in many steps of transcriptional regulation, interacting with general and sequence-specific transcription factors, and binding sequence-specific polypyrimidine-rich DNA motifs within promoter regions. It has been proposed that K protein acts as a scaffold or architectural transcription factor that promotes assembly of sequence-specific transcription factors and basal transcription machinery on the promoter elements (10). YB-1 protein possesses similar functional properties. It has been shown to modulate transcription through binding single-stranded polypyrimidine-rich DNA  4). Proteins were separated on SDS-PAGE and transferred to Immobilon-P membrane. Blots were probed with anti-K protein antibody 54.

FIG. 7. Functional interaction between K protein and YB-1 revealed by their effect on a CT element containing promoter. A,
HeLa cells were co-transfected with 2.25 g of hnRNP K and 2.25 g of Flag-YB-1 expression vectors and either 0.25 g of wild type CT3wt-cfos or mutated CT3 mut-c-fos reporter construct. 0.25 g of pSV-gal was cotransfected to normalize transfection efficiency. Data are shown as mean Ϯ standard deviation from three independent experiments. B, NIH3T3 were cotransfected with 1.6 g of hnRNP K and 1.6 g of YB-1 expression vectors together with 1.6 g of either CT3wt-c-fos or CT3 mut-c-fos. 0.25 g of pRL null plasmid was cotransfected to normalize transfection efficiency. Data are shown as mean Ϯ standard deviation from three independent experiments with different plasmid preparations. sequences and by interacting with transcription factors (10 -12). Like K, protein YB-1, is known to be involved in repressing and activating transcription (11,27). YB-1 is thought to activate transcription of human polyomavirus JC by recruiting another trans-activator (p65 or RelA) to the viral promoter (28). YB-1 can act on a single gene, the matrix metalloproteinase 2 gene, in a positive or negative manner that is dependent upon the cellular context (29). The reported transcriptional activity of YB-1 protein is strongly dependent on the intracellular environment. For example, the synergistic activity of YB-1 and Pur ␣ proteins on the JCV enhancer-promoter region has been observed in glial cells but was not detected in other cell lines (23). The transactivation properties of YB-1 on myosin light chain 2v gene promoter have been shown in cardiac myocytes, where the expression of YB-1 resulted in a 3.4 higher reporter gene activity in comparison to the control, but no activation was detected in COS-1 cells (30). A moderate level of transcription activation of matrix metalloproteinase-2 promoter by YB-1 was observed in mesangial cells but not in glomerular epithelial cells (29). Taken together, these data and our transfection experiments (Fig. 7) clearly show that transcriptional activity of hnRNP K and YB-1 is cell type-dependent. Both proteins are likely acting as scaffolds or docking platforms for multiprotein complexes that include transcription factors. Acting as docking platforms, they may respond to changes in extracellular environment at sites of nucleic acid-dependent processes (7,15).
Both proteins have multiple interactive domains. K protein contains two different types of nucleic acid binding domains, the KH domains and the RGG cluster, and the KI region responsible for protein-protein interaction. The same is true for YB-1. Safak et al. (23) have shown that YB-1 protein interacts through the region that includes the entire cold shock RNA binding domain with the transcription factor Pur ␣. Our data indicate that the N-terminal interactive domain (NIII) in YB-1 responsible for K protein binding overlaps with the Pur ␣ binding region but does not include the cold shock domain. Moreover, the CII peptide, which has a strong affinity for hnRNP K and TBP (Fig. 5B, lanes 6 and 12), does not bind Pur ␣ (23).
These data indicate that K and YB-1 proteins may simultaneously interact with more than one of their partners to form multiple protein or protein⅐nucleic acid complexes. It is further plausible that the interaction of YB-1-hnRNP K with one of their partners (nucleic acid and proteins) may serve to regulate the interaction with another partner. For example, as shown in Fig. 2B (lane 1) the interaction with poly(U) abolished the binding of K to YB-1.
When both YB-1 and hnRNP K proteins are coexpressed their transcriptional activity is reduced. HnRNP K and YB-1 interact in vitro with the general transcription factor TATAbinding protein (Figs. 4 and 5). It has been suggested that TBP-mediated activation of transcription can be regulated by a variety of gene-specific transcription factors (31). As shown previously, cotransfection of K protein and TBP resulted in activation of transcription from the CT-rich promoter element (10). Our preliminary findings indicate that coexpression of YB-1 and TBP has the same effect on the CT-rich promoter element. 2 In vitro data show that YB-1 binds K protein and TBP through the same domains (Fig. 5), suggesting that the two factors may compete for YB-1. We propose that genespecific activation of transcription, particularly from the CT element containing promoters, may be activated by increasing concentration of either hnRNP K⅐TBP or YB-1⅐TBP complexes explaining the increased reporter gene expression when either K or YB-1 was transfected. Coexpression of K and YB-1 proteins may favor formation of K⅐YB-1 complexes, which might be transcriptionally inactive, accounting for the decreased reporter gene expression. A similar effect has been shown for Sp1 and C/EBP ␤ transcription factors where the interaction with K protein abolished their trans-activation ability (11,32).
In summary, we show interaction between two functionally similar factors hnRNP K and YB-1. Because both proteins are involved in many steps of gene expression such as transcription, translation, and nuclear transport it is plausible that these processes are regulated by the interaction of hnRNP K and YB-1. Future studies will be designed to address this issue.