Splicing Factor SRp30c Interaction with Y-box Protein-1 Confers Nuclear YB-1 Shuttling and Alternative Splice Site Selection*

Ute RaffetsederDagger §, Björn FryeDagger §, Thomas RauenDagger , Karsten Jürchott, Hans-Dieter Royer||, Petra Lynen JansenDagger , and Peter R. MertensDagger **

From the Dagger  Division of Nephrology and Clinical Immunology, University Hospital of Aachen, 52057 Aachen, Germany, the  Institute of Pathology, Charite, Humboldt University, 10099 Berlin, Germany, and the || Institute for Transplant Diagnostic and Cell Therapy, Heinrich-Heine University, 40225 Düsseldorf, Germany

Received for publication, December 9, 2002, and in revised form, February 13, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The multifunctional DNA- and RNA-associated Y-box protein 1 (YB-1) specifically binds to splicing recognition motifs and regulates alternative splice site selection. Here, we identify the arginine/serine-rich SRp30c protein as an interacting protein of YB-1 by performing a two-hybrid screen against a human mesangial cell cDNA library. Co-immunoprecipitation studies confirm a direct interaction of tagged proteins YB-1 and SRp30c in the absence of RNA via two independent protein domains of YB-1. A high affinity interaction is conferred through the N-terminal region. We show that the subcellular YB-1 localization is dependent on the cellular SRp30c content. In proliferating cells, YB-1 localizes to the cytoplasm, whereas FLAG-SRp30c protein is detected in the nucleus. After overexpression of YB-1 and FLAG-SRp30c, both proteins are co-localized in the nucleus, and this requires the N-terminal region of YB-1. Heat shock treatment of cells, a condition under which SRp30c accumulates in stress-induced Sam68 nuclear bodies, abrogates the co-localization and YB-1 shuttles back to the cytoplasm. Finally, the functional relevance of the YB-1/SRp30c interaction for in vivo splicing is demonstrated in the E1A minigene model system. Here, changes in splice site selection are detected, that is, overexpression of YB-1 is accompanied by preferential 5' splicing site selection and formation of the 12 S isoform.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Y-box protein YB-1 is a member of the cold shock protein family, which exhibits pleiotropic functions. YB-1 specifically binds to a sequence motif termed Y-box. This motif is characterized by the presence of a core ATTGG sequence, which represents the inverted CCAAT-box. YB-1 controls the transcription of numerous genes that among others include MHC class II antigen, MDR1, MMP-2, and COL1A1 (1-4). DNA binding specificity is mediated through the evolutionarily conserved cold shock domain in conjunction with the adjacent C-terminal protein residues (5, 6). Interactions of YB-1 with numerous cellular and viral transcription factors including JC virus antigen (7), AP-2 (8), Puralpha (9), CTCF (10), and p53 (11, 12) have been demonstrated. These interactions may in part explain cell-specific gene regulation, that is, stimulation and repression of transcription, even of the same gene (3). In addition, it has been proposed that YB-1 plays a role as an architectural protein by its propensity to sequence specifically unwind DNA duplexes and stabilize single-stranded templates, thereby altering sequence recognition motifs (1, 4, 8).

In addition to their role in regulating gene transcription, cold shock proteins exhibit a wide spectrum of activities by virtue of sequence-specific and -nonspecific RNA binding. YB-1 has been identified as the major component of messenger ribonucleoprotein particles (mRNPs) in mammalian cells, which constitute templates for the translational machinery (13-15). At higher concentrations Y-box proteins Xenopus FRGY2 and human YB-1 act as repressors of translation in a process called mRNA masking (13, 16-18), whereas at lower YB-1 concentrations mRNA translation is activated (19). In this regard, specific binding of YB-1 to the 5'-cap structure may be of importance as mRNA decapping and degradation is inhibited after binding (20). Sequence-specific mRNA binding by YB-1 occurs through the evolutionarily conserved cold shock domain, which contains the RNA-binding motifs RNP1 and RNP2 (21, 22). Upon binding, YB-1 regulates mRNA half-lives, e.g. of the interleukin-2 mRNA during T-cell activation (23) and granulocyte-macrophage colony-stimulating factor mRNA in activated eosinophils (24).

Recently, a novel role of YB-1 in splicing has been proposed as YB-1 binds to the A/C-rich exon enhancer element in the CD44 gene and thereby directs alternative splicing (25, 26). This A/C-rich exon enhancer element is not directly bound by serine-arginine-rich (SR)1 proteins, whereas the CD44 alternative splicing was affected through prebinding of YB-1 to this element (26). Yet, the mechanism by which YB-1 activates exon recognition in the CD44 gene is unknown. In addition it has been shown that YB-1 directly interacts with the translocation liposarcoma protein, which serves as an adapter molecule connecting gene transcription and RNA splicing and thereby regulates the adenovirus E1A pre-mRNA splicing (25). In the present study a direct in vivo interaction of YB-1 with SRp30c is demonstrated, which has fundamental effects on the subcellular YB-1 localization and is of functional relevance in the alternative splicing process.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Yeast Two-hybrid Screen-- A cDNA library derived from a human mesangial cell line (27) was subcloned into vector MAB86 (Invitrogen). YB-1 cDNA was PCR-amplified using 5'-CATGCCATGGCAATGAGCAGCGAGGCCGAGACCC-3' and 5'-GCACTAGTTCAGCCTCGGGAGCGGGAATTCTC-3' as primers and inserted into restriction sites NcoI and SpeI of pDBLeu as bait. In-frame cloning and sequences were verified by full-length sequencing using the ABI PRISM sequencing reaction (Applied Biosystems). A yeast two-hybrid screen (ProquestTM, Invitrogen) (28, 29) was performed with a total of 2 × 106 transformants being plated on 15-cm plates of Leu-, Trp-, and His- synthetic complete medium containing 40 mM 3-amino-1,2,4-triazole (Sigma). 86 His+ clones were isolated and tested for fulfillment of all five selection criteria, including growth in media containing 60 mM 3-amino-1,2,4-triazole (3-AT, Sigma), ura+ 5-fluoro-orotic-acid (Invitrogen) metabolism and beta -galactosidase expression.

Cell Culture and Transient Transfections-- HeLa and HEK-293T cells were cultured in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine at 37 °C and 5% CO2. For heat-shock experiments, cells were incubated for 1 h at 42 °C in complete medium and allowed to recover for 1 h at 37 °C before analysis by immunofluorescence. Cells were transfected with purified plasmid DNA by the calcium phosphate precipitation method as described previously (30).

Expression plasmids for YB-1 (pSG5-YB-1) and SRp30c have been described previously (1, 31). The plasmids encoding for GFP-YB-1 fusion proteins were obtained from H. D. Royer and K. Jurchat (Max-Delbrück Center, Berlin, Germany). In these plasmids full-length YB-1 cDNA was cloned into the AflII and HindIII restriction sites of vector pcDNA6/V5-His (Invitrogen) that was modified with a GFP sequence inserted at the protein C terminus. All deletion constructs of YB-1 were generated by PCR amplification of the respective cDNA sequences and subcloning into vector pcDNA6/V5-His. Sequences were verified by automatic sequencing reactions (ABI PRISM sequencing reaction, Applied Biosystems).

Immunoprecipitation-- For immunoprecipitation 2 × 106 cells were transfected with a total of 20 µg of purified plasmid DNA as indicated. 48-h posttransfection cells were harvested by trypsin digestion and centrifuged, and the cell pellet was lysed in 1 ml RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3V04, 1 mM NaF) for 15 min at 4 °C. Cell lysates were precleared for 30 min at 4 °C with 40 µl of pansorbin (Calbiochem, Darmstadt, Germany) and 4 µl of nonspecific mouse IgG1 (Dako). After centrifugation at 14,000 rpm for 10 min at 4 °C 200 µl of supernatant was incubated with the indicated antibody (monoclonal anti-GFP, Clontech, Palo Alto, CA; monoclonal M2 anti-FLAG, Sigma) at 4 °C overnight. In separate experiments RNase A (100 µg/ml, Roche Applied Science) was added to the precleared lysates for 12 h at 4 °C. To ensure complete degradation of mRNA by RNase A treatment aliquots of cell lysates were processed using the RNeasy kit (Qiagen, Hilden, Germany) and subjected to oligo(dT)-primed reverse transcription by means of superscript II (Invitrogen). The housekeeping gene GAPDH was amplified by PCR using the following primer pair: 5'-TTCCATGGCACCGTCAAGGC-3' and 5'-TCAGGTCCACCACTGACACG-3', yielding a 570-bp amplification product. To the precleared lysates 20 µl of pansorbin was added and incubated for 1 h at 4 °C. Pansorbin-bound antibodies and proteins were pelleted by centrifugation at 14,000 rpm and washed extensively with PBS on ice followed by two final washing steps using RIPA buffer. Pellets were resuspended in 50 µl of reducing sample buffer, boiled at 95 °C for 5 min, and pelleted, and the supernatant was subjected to SDS-PAGE. Proteins were transferred to nitrocellulose and detected by suitable primary antibodies anti-GFP (1:5000) or M2 anti-FLAG (1:500) and secondary peroxidase-linked anti-mouse antibody (Amersham Biosciences) using ECL (Amersham Biosciences) as chemiluminescent.

Immunofluorescence Microscopy-- HeLa cells were grown on coverslips in six-well plates (105 cells/well). At 60% confluence cells were transfected with a total of 2.5 µg of plasmid DNA/well as indicated in the figure legends by means of calcium phosphate precipitates and cultured for 48 h. After washing with PBS, cells were fixed with 4% paraformaldehyde in PBS for 30 min, washed in PBS-CM and incubated with 50 mM ammonium-sulfate in PBS-CM for 10 min. Cells were subsequently permeabilized in buffer A (0.1% Triton X-100 in PBS-CM) and blocked with 0.2% bovine albumin in buffer A. M2 anti-FLAG antibody (1:300 in buffer A) was added for 60 min at room temperature in a humidified chamber. After three washes with buffer A secondary TRITC-conjugated anti-mouse antibody (Dinova, Hamburg, Germany) was added for 60 min at room temperature (1:600). After extensive washes with buffer A and a final wash with PBS-CM coverslips were mounted with immumount (Shannon). Confocal laser scanning microscopy (Zeiss LSM 510 Meta, Zeiss, Germany) was performed at 488 nm for GFP fluorescence (detected at 500 nm < lambda FITC < 540 nm) and 543 nm for excitation of TRITC-conjugated antibody (detected at 550 nm < lambda TRITC < 600 nm).

In Vivo Splicing Analysis-- 15 µg of pMTE1A plasmid DNA containing the E1A minigene was transfected in HeLa cells (2 × 106 cells) combined with expression plasmids pSG5-YB-1, pCR-FLAG-30c or control plasmids. Care was taken that the total DNA amount was equalized to 50 µg in each reaction by inclusion of the respective amounts of control plasmid. Cells were harvested 48 h posttransfection and mRNA was extracted using the RNeasy minikitTM (Qiagen). cDNA synthesis was performed using oligo(dT) primers (Roche Applied Science) and Superscript II RNase H- reverse transcriptase (Invitrogen). For PCR the E1A-specific primers pMTE1A-sense 5'-ATTATCTGCCACGGAGGTGT-3' and pMTE1A-antisense 5'-GGATAGCAGGCGCCATTTTA-3' were used with 25 cycles of amplification (90 s at 94 °C, 120 s at 50 °C, 120 s at 72 °C). Amplification products were separated on 3% agarose gels containing ethidium bromide with band intensities being quantified by OptiQuantTM software. Band intensities of all splicing isoforms combined were set as 100%, and relative band intensities were determined.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of Splicing Factor SRp30c as Partnering Protein of YB-1-- To identify YB-1-interacting proteins a yeast two-hybrid screen was set up with full-length YB-1 as bait and a cDNA library generated from a human mesangial cell line (27) as prey. Of 2 × 106 colonies screened 86 fulfilled all five selection criteria (phenotypes: His+, 3ATR, beta -galactosidase, Ura+, and 5-fluoro-orotic-acid+), and of these two colonies encoded for full-length splicing factor SRp30c. To confirm the result of the two-hybrid screen co-immunoprecipitation studies were performed with both proteins co-expressed as tagged fusion proteins in HEK-293T cells. GFP was fused to the C terminus of YB-1 and FLAG was fused to the N terminus of SRp30c (Fig. 1A, GFP: <1 in lane 1; GFP-YB-1: <2 in lane 2, FLAG-SRp30c: <3 in lane 3). Cell lysates were subsequently used for co-immunoprecipitation studies with monoclonal antibodies directed against the respective tags. As can be seen in Fig. 1A FLAG-tagged SRp30c protein was co-immunoprecipitated with anti-GFP antibody when cells were co-transfected with GFP-YB-1 (lane 5, indicated by <4); however, not in cells expressing GFP protein alone (lane 4). Conversely, GFP-YB-1 was detected with immunoprecipitated FLAG-SRp30c (lane 8, indicated with <5); however, not in control reactions with expressed FLAG protein (Fig. 1A, lane 7). The co-immunoprecipitations were nearly quantitative and suggested a direct association of YB-1 and SRp30c. To exclude the possibility that the contact of both proteins is mediated by indirect association via mRNA binding, co-immunoprecipitation studies were repeated following RNase A treatment. RNase A treatment did not change the amount of immunoprecipitated protein, indicating direct YB-1/SRp30c partnering (Fig. 1B, compare lanes 6 and 12). In the latter experiment control reactions were set up to test for consistent and complete degradation of messenger RNA by reverse transcription and amplification of the housekeeping gene GAPDH. As can be seen in Fig. 1C untreated cell extracts contained abundant amounts of GAPDH mRNA (lanes 2-4), whereas no GAPDH mRNA was present in RNase A-treated extracts (lanes 5-7).


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Fig. 1.   A, interaction of GFP-YB-1 with FLAG-SRp30c in vivo. FLAG-tagged SRp30c plasmid was co-introduced with GFP protein or with GFP-tagged YB-1 into HEK-293T cells. Cell lysates were separated by SDS-PAGE, and expressed proteins were visualized with anti-GFP (GFP and GFP-YB-1 bands indicated by <1 and <2 in lanes 1 and 2) or M2 anti-FLAG antibody (FLAG-SRp30c band indicated by <3 in lane 3). Lysed proteins were immunoprecipitated with either anti-GFP (lanes 4 and 5) or anti-FLAG (lanes 6-8) antibody and immunoblotted with anti-FLAG (lanes 4 and 5) or anti-GFP (lanes 6-8) antibody. Co-immunoprecipitated FLAG-SRp30c is indicated by <4 in lane 5 and GFP-YB-1 by <5 in lane 8, whereas control reactions were negative (lanes 4, 6, and 7). B, the YB-1/SRp30c protein interaction is not mediated by RNA. GFP or GFP-YB-1 were co-expressed with FLAG or FLAG-SRp30c in HEK-293T cells. RNase A treatment of cell lysate was performed as indicated (lanes 7-12) followed by immunoprecipitation with the indicated antibodies and immunoblotting with anti-GFP antibody. Co-immunoprecipitated GFP-YB-1 is indicated by <1 and <2. C, determination of RNA-degradation by RNase A treatment. To assure complete degradation of mRNA by RNase A treatment extracts were subjected to reverse transcription and amplification of GAPDH cDNA by PCR. Consistent degradation of RNA was apparent (lanes 5-7) compared with untreated extract (lanes 2-4). In lane 1 the DNA ladder is given. D, endogenous YB-1 interacts with FLAG-SRp30c. Co-immunoprecipitation studies were repeated with lysates from cells that express FLAG or FLAG-SRp30c protein. Following immunoprecipitation of endogenous YB-1 with specific anti-YB-1 antibody (lane 3) FLAG-SRp30c could be co-immunoprecipitated (size indicated by <1).

As these findings indicate interaction of introduced tagged proteins FLAG-SRp30c/GFP-YB-1 additional experiments were performed to also confirm an interaction of endogenous YB-1 with FLAG-SRp30c. For this purpose HEK-293T cells were transiently transfected with either FLAG or FLAG-SRp30c expression plasmids, and co-immunoprecipitation studies were performed by means of a polyclonal anti-YB-1 antibody (32). As can be seen in Fig. 1D FLAG-SRp30c was co-immunoprecipitated with endogenous YB-1 protein (lane 3).

Mapping of YB-1 Protein Domains Involved in SRp30c-Protein Interaction-- To identify YB-1 protein domains that confer the interaction with SRp30c a panel of GFP-YB-1 deletion constructs (depicted in Fig. 2B) were co-expressed with full-length SRp30c protein in HEK-293T cells, and co-immunoprecipitations were performed using anti-FLAG antibody. As shown in Fig. 2A, the N-terminal protein domains of YB-1 containing the evolutionarily conserved cold shock domain (GFP-YB-1 Delta 1, aa 21-147) are sufficient for association with FLAG-tagged SRp30c (Fig. 2A, lanes 1 and 2, indicated by <1). In comparison to full-length GFP-YB-1 wild type a similarly high percentage of expressed GFP-YB-1 Delta 1 (Fig. 2A, lane 3) was co-immunoprecipitated by anti-FLAG antibody (Fig. 2A, lane 4, indicated by <2). After removal of the YB-1 N-terminal protein domains aa 1-147 (GFP-YB-1 Delta 3) only a weak interaction with SRp30c could be detected (Fig. 2A, lanes 7 and 8, indicated by <4). This low-level binding affinity was mapped to aa 260-317 of the C terminus (GFP-YB-1 Delta 4), whereas GFP-YB-1 Delta 2 comprised of aa 147-225 was not co-immunoprecipitated. Taken together, these results demonstrate that YB-1 strongly interacts with SRp30c via aa 21-147, whereas a low affinity binding domain resides in the C terminus (aa 260-317).


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Fig. 2.   Mapping of the YB-1 domain required for SRp30c interaction. A, full-length (GFP-YB-1 wild type) and truncated GFP-tagged YB-1 proteins (GFP-YB-1 Delta 1-4) were analyzed for protein interaction with FLAG-tagged SRp30c by co-immunoprecipitation of overexpressing HEK-293T cell lysates. Expressed proteins before (L) and after immunoprecipitation (I.P.) with anti-FLAG antibody were separated by SDS-PAGE and immunoblotted with anti-GFP antibody. B, schematic illustration of GFP-YB-1 constructs with amino acid residues given on top of the bars. Arrow head numbers indicate protein bands as indicated in A.

YB-1 and SRp30c Co-localize in Mammalian Cells-- Next we set out to determine the subcellular localization of YB-1 and SRp30c. For SRp30c a nuclear localization has previously been described (33), whereas YB-1 has been reported to localize both to the nucleus and the cytoplasm, depending on the cell origin and transformation (2, 6, 34). Transfections of HeLa cells were performed with expression plasmids for GFP-YB-1, FLAG-SRp30c, and tag control plasmids. In HeLa cells expressing GFP-YB-1 alone, fluorescence was predominantly localized in the cytoplasm (Fig. 3a, panel B), whereas GFP was detected diffusely in all cellular compartments (Fig. 3a, panel A). In contrast, FLAG-SRp30c protein was localized in a speckled pattern in the nucleus (Fig. 3a, panel C). A dramatic change of the subcellular GFP-YB-1 localization was detected with combined expression of both proteins, GFP-YB-1 and FLAG-SRp30c. Under these conditions GFP-YB-1 co-localized with SRp30c (Fig. 3a, panel D), while the subcellular distribution of GFP was unchanged after co-transfection with FLAG-SRp30c (Fig. 3a, panel C). The nuclear localization of both proteins in most cells was not in a speckled pattern. These findings indicate that FLAG-SRp30c confers shuttling of YB-1 to the nuclear compartment. Transient transfections of HeLa cells were repeated with expression plasmids for FLAG or FLAG-SRp30c, and the subcellular distribution of endogenous YB-1 was assessed by immunohistochemistry using a specific anti-YB-1 antibody (32). These experiments were designed to exclude any contribution of the GFP tag to the nuclear shuttling of GFP-YB-1 protein. Here, a similar shift of endogenous YB-1 from the cytoplasm to the nuclear compartment was apparent with introduced FLAG-SRp30c but not FLAG alone (Fig. 3b).


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Fig. 3.   a, GFP-YB-1 and SRp30c co-localize in the nucleus of HeLa cells. HeLa cells are co-transfected with either GFP or GFP-YB-1 expression plasmid and either FLAG or FLAG-SRp30c vector and stained with monoclonal M2 anti-FLAG antibody. The YB-1 distribution was visualized by its GFP tag fluorescence, whereas FLAG-SRp30c was detected with a rhodamine-conjugated goat anti-mouse antibody by confocal laser scanning microscopy. Co-localization of the two antigens is revealed by yellow in the merged images. Panel A, GFP and FLAG-antigen is diffusely detected throughout the cells. Panel B, GFP-YB-1 predominantly localizes in the cytoplasm, whereas FLAG-SRp30c is detected in the nucleus in a speckular pattern (panel C). With co-expression of both proteins, GFP-YB-1 and SRp30c, a nuclear co-localization is detected (panel D). b, nuclear shuttling of endogenous YB-1 with overexpressed FLAG-SRp30c. Upon transiently overexpressing FLAG or FLAG-SRp30c protein in HEK-293T cells immunohistochemistry was performed using a specific anti-YB-1 antibody. Here, a nuclear shuttling of endogenous YB-1 protein was only present with overexpressed FLAG-SRp30c protein (B) but not the FLAG alone (A).

To exclude a cell-specific effect of this protein shuttling analogous experiments were performed with HEK-293T cells yielding similar results (data not shown).

Since the N-terminal YB-1 protein domains were mapped as high affinity interacting domains with FLAG-SRp30c, the corresponding GFP-YB-1 deletion construct, GFP-YB-1 Delta 1, was tested for co-localization with SRp30c. As can be seen in Fig. 4A (images c and d) construct GFP-YB-1 Delta 1 co-localized with SRp30c in the nucleus, whereas construct GFP-YB-1 Delta 3 lacking aa 1-147 localized in the cytoplasm when co-expressed with SRp30c (Fig. 4B, images c and d). These results support the notion that the N-terminal YB-1 protein domains are responsible for the interaction with SRp30c.


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Fig. 4.   A and B, the N-terminal region of YB-1 co-localizes with SRp30c in the nucleus. GFP-coupled deletion constructs of YB-1 (GFP-YB-1 Delta 1 or GFP-YB-1 Delta 3, compare with Fig. 2B) were co-transfected with FLAG-SRp30c in HeLa cells. SRp30c distribution was detected by staining with an M2 anti-FLAG antibody and visualized by rhodamine-conjugated anti-mouse antibody. A, confocal laser scanning microscopy revealed that GFP-YB-1 Delta 1 alone is localized in the cytoplasm (a), whereas it co-localizes with FLAG-SRp30c in the nucleus in cells overexpressing both proteins (b, TRITC; c, FITC; d, merge). B, with GFP-YB-1 Delta 3 alone a similar cytoplasmic localization is present (a), which did not change after co-expression of GFP-YB-1 Delta 3 and FLAG-SRp30c (b, TRITC; c, FITC; d, merge).

Reversal of SRp30c-dependent Nuclear YB-1 Shuttling by Heat Shock-- As the nuclear co-localization of YB-1 and SRp30c is dependent on the cellular SRp30c content, we subsequently addressed the question whether a shift of nuclear SRp30c into stress-induced Sam68 nuclear bodies (33) by heat shock treatment affects the subcellular YB-1 localization. As can be seen in Fig. 5, the localization of SRp30c (Fig. 5, panel A) and GFP-YB-1 (Fig. 5, panel B) remained unchanged after 1 h heat shock treatment at 42 °C when both proteins were overexpressed separately. Remarkably, the nuclear co-localization was no longer present with FLAG-SRp30c and GFP-YB-1 overexpressed in combination when heat shock treatment was performed. Under these conditions GFP-YB-1 was detected in the cytoplasm (Fig. 5, panel C), whereas FLAG-SRp30c remained in the nuclear compartment.


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Fig. 5.   Heat shock treatment reverses co-localization of GFP-YB-1 and SRp30c in HeLa cells. HeLa cells were co-transfected with either GFP or GFP-YB-1 combined with either FLAG- or FLAG-SRp30c vector. Cells were heat-stressed for 1 h at 42 °C and allowed to recover for 1 h at 37 °C. By confocal laser scanning microscopy GFP and GFP-YB-1 were visualized (FITC filter). At the same time FLAG- and FLAG-SRp30c protein were detected by TRITC filter. SRp30c reveals a predominant nuclear localization after heat stress (panel A) with diffuse pattern of GFP throughout the cells. The cytoplasmatic localization of GFP-YB-1 is not changed by heat stress (panel B) when co-transfected with an empty FLAG-vector. Heat shock treatment prevents nuclear GFP-YB-1/SRp30c co-localization with both proteins overexpressed in combination (panel C).

YB-1 Affects Alternative Splicing of SRp30c-- Next we tested for the functional relevance of the YB-1/SRp30c interaction in the alternative splicing process. The adenovirus E1A pre-mRNA minigene was chosen as a model system in which concentration-dependent changes of the splicing pattern by SR proteins has been described (35, 36). In vivo splicing was monitored by reverse transcription PCR analysis using the pMTE1A-sense and pMTE1A-antisense primers with mRNA collected from HeLa cells that were transiently transfected with the E1A-minigene and a combination of SRp30c expression vector and/or increasing amounts of YB-1 expression vector. Care was taken to ensure that equal DNA amounts were introduced in all transfections. The E1A minigene contains three different 5' splicing sites resulting in three major isoforms 13 S, 12 S, and 9 S (Fig. 6A). As can be seen in Fig. 6B, co-transfection of increasing amounts of YB-1 resulted in the preferential formation of the 12 S isoform, whereas the 9 S isoform decreased in a concentration-dependent manner (quantification depicted in Fig. 6C).


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Fig. 6.   A-D, YB-1 affects alternative splicing of the E1A minigene. An in vivo splicing assay was performed using the adenovirus E1A minigene model system. A, schematic presentation of the alternative spliced isoforms of the E1A pre-mRNA. The major isoforms 13 S, 12 S, and 9 S are generated by three different 5' splice sites. B, the E1A minigene was introduced into HeLa cells, and splicing was analyzed by quantitative reverse transcription PCR. cDNA was analyzed by electrophoresis in 3% agarose ethidium bromide gels and quantified densitometrically. C and D, results are expressed as relative intensities of corresponding bands compared with minigene alone, the intensities of which was set as 1. Given are means of a representative experiment that was confirmed in seven independent experiments. B and C, upon overexpression of increasing YB-1 amounts a concentration-dependent change in splicing pattern toward the 12 S isoform is detected. At the same time the 9 S isoform is reduced. B and D, overexpression of SRp30c alone leads to preferential formation of the 13 S isoform, whereas combined expression of YB-1 at increasing concentrations and SRp30c at a fixed concentration leads to a minor reduction of 13 S formation.

In contrast to this finding overexpression of SRp30c alone lead to the preponderance of 13 S transcripts with concomitant decreased appearance of the 9 S isoform (Fig. 6B, lane 5), as has been described previously (33, 36). When both expression plasmids were introduced in combination, that is SRp30c at a fixed and YB-1 at increasing concentrations, the relative intensity of the 12 S isoform was increased, whereas 13 S transcripts were slightly decreased at lower YB-1 concentrations. At the same time isoform 9 S was decreased (Fig. 5D). Taken together these changes in splicing indicate YB-1-dependent selection of splice site selection that is a shift from the 13 S toward the more distal 12 S splicing site. Thus, YB-1 influences alternative splice site selection of the adenovirus E1A pre-mRNA in vivo and interferes with the SRp30c-dependent splicing pattern in a concentration-dependent manner.

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Here we report the identification of SRp30c as an interacting protein of YB-1 by a two-hybrid screen against a human mesangial cell cDNA library. The identified SRp30c clones encoded for full-length protein. Co-immunoprecipitation studies with tagged endogenous proteins expressed in HEK-293T and HeLa cells confirm this protein-protein interaction. Although both proteins, SRp30c and YB-1, have been shown to specifically bind to mRNA sequences (26, 37), binding properties of SRp30c and YB-1 to each other were independent of the presence of mRNA, as shown by RNase A digestion. Interestingly, two independent protein domains of YB-1 are involved in SRp30c partnering, a high affinity binding region in the N terminus and a low affinity binding domain in the C terminus. Similar findings have been reported for YB-1 partnering with heterogeneous nuclear ribonucleoprotein K (hnRNP K) (38).

SRp30c is a member of the SR protein family, that has multiple functions in constitutive and alternative splicing processes. It is expressed in most tissues (36) and exhibits structural similarities with the more intensely studied alternative splicing factor ASF/SF2. SRp30c may directly contact RNA via two independent RNA recognition motifs that are linked by a glycine-hinge. This protein family has been named SR-proteins due to arginine/serine dipeptides within the C terminus.

Previously, interaction of SRp30c with nuclear proteins involved in nuclear architecture maintenance has been reported, that is, nuclear and nucleolar protein (Nop30) (39), heterogeneous nuclear ribonucleoprotein A1-interacting protein HAP (33, 40), and src activated during mitosis (Sam68) like mammalian protein (SLM2) (31). For HAP and SLM2 a function in the organization of the nuclear architecture and RNA-trafficking has been described, and these are localized in Sam68 nuclear bodies (41). By changing local concentrations of SRp30c or by recruiting SRp30c to other nuclear compartments, e.g. stress-induced bodies (33), splicing patterns are regulated, as has been determined with the E1A minigene (33).

Changes in local concentrations of splicing factors are a common regulatory mechanism. From this standpoint it is an important observation that YB-1 shifts from its cytoplasmic localization to the nucleus when the cellular SRp30c content is increased by protein overexpression. It cannot be ruled out that this effect is mediated by another protein or an alternatively expressed isoform(s) due to elevated SRp30c concentrations. However, some data support the notion that a direct YB-1/SRp30c interaction is responsible for this shift. First, the same protein domains of YB-1 that interact with SRp30c are required for the nuclear YB-1 localization. Secondly, heat shock treatment, a condition under which SRp30c is recruited to stress-induced Sam68 nuclear bodies (33), leads to a reversal of the nuclear YB-1 shift within 1 h.

Stein et al. (34) recently reported on a nuclear shift of YB-1 with heat stress treatment, whereas in our experiments a mainly cytoplasmatic localization of YB-1 was observed. These differences may be explained by a cell-specific regulation of YB-1 or by differences in the experimental setup. Stein et al. (34) have used two human colon carcinoma cell lines that possess different sensitivities to multiple drug resistance-related drugs. By means of immunofluorescence microscopy, YB-1 was detectable in the cytoplasm of most cells under stress-free conditions; however, it was also observed in the nuclei of untreated cells exhibiting higher intrinsic drug resistance, which corresponds to higher intrinsic expression of MDR1 and MRP1. It was concluded that hyperthermia-induced effects are cell type-specific and dependent on the intrinsic resistance status (34). Given our observations of a nuclear YB-1 shift that depends on the cellular SRp30c content it may be of interest to determine the SRp30c content in human carcinoma cell lines to correlate this with multiple drug resistance.

To further determine the functional meaning of the YB-1/SRp30c interaction an in vivo splicing assay using the adenoviral E1A minigene was performed. Both YB-1 and SRp30c are reported to affect the splicing pattern of the E1A minigene (25, 33, 36). For YB-1 an increase of the major 13 S isoform was found by RNase protection assay in NIH/3T3 cells but not in Ewing sarcoma cells (25). In our hands a similar increase of the 13 S and even more the 12 S isoform could be detected by in vivo splicing assay. According to results by Screaton et al. (36) SRp30c overexpression also enhances the 13 S isoform. A similar change of the splicing pattern was observed with overexpression of FLAG-SRp30c, suggesting that the FLAG tag does not affect the splicing properties of SRp30c (Fig. 6, B and D). With combined overexpression of FLAG-SRp30c and increasing amounts of YB-1 a concentration-dependent increase of the 12 S isoform was observed. This result reflects a change in splice-site selection from the most proximal 5' splice site toward a more distal splice site and may hint at a role for YB-1 as an adapter protein linking RNA binding and splicing.

Further evidence for YB-1 acting as an adapter protein that recruits splicing factors is provided by Stickeler et al. (26) who described YB-1 binding to exonic splicing enhancers in the human CD44 gene and observed exon inclusion. The function of YB-1 may thus resemble the one of a slightly different member of the SR protein family, Tra 2, found in Drosophila melanogaster (26) or human Tra2beta that strengthens the binding of SRp30c to AG-rich exonic splicing enhancers within exon 7 of the human survival motoneuron gene to increase exon 7 inclusion (37).

In summary our findings reveal a direct interaction of the multifunctional protein YB-1 with splicing factor SRp30c. This interaction may have a direct influence on the subcellular YB-1 localization. A nuclear co-localization is found in SRp30c-overexpressing cells with subsequent changes in the splicing pattern. However, the splicing pattern was also changed in cells overexpressing only YB-1, most likely due to minute amounts of protein being shuttled to the nucleus. These results suggest a model in which YB-1 acts as an adapter molecule between RNA splicing recognition motifs and splicing factor(s). Furthermore, given the other known functional properties of YB-1, which include gene regulation and mRNA masking in the cytoplasm, a link between transcription and mRNA-processing events may be provided. YB-1 as well as SRp30c are strongly regulated by cellular stress events (33, 42-44). It will be of great interest to unravel the function of this interaction in cellular stress responses as this interaction may contribute to oncogenetic and metastatic processes. Future experiments will also address the potential role of YB-1 in mRNA export due to its presence in the cytoplasmic and nuclear compartment as well as to its regulating functions in transcription, splicing, and translation. A common feature of proteins involved in the formation of ribonucleoprotein complexes shuttling between the nucleus and cytoplasm are alternating basic and acidic protein residues. Such alternating residues are present in Y-box proteins (22), although little is known about the mechanisms underlying the YB-1 shuttling observed under certain conditions, e.g. after exposure to UV irradiation or anticancer agent exposure (44).

    ACKNOWLEDGEMENTS

We are grateful for plasmid donations by S. Stamm, Max-Planck Institute of Neurobiology, Martinsried, Germany (pCR-FLAG-30c) and A. R. Krainer, Cold Spring Harbor Laboratories, New York, NY (E1A minigene pMTE1A). Expert technical assistance was provided by Marina Wolf El-Houari.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft SFB 542, Project C4 (to P. R. M.)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.

§ Both authors contributed equally to this work.

** To whom correspondence should be addressed: Medizinische Klinik II, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstrasse 30, 52057 Aachen, Germany. Tel.: 49-241-8089532; Fax: 49-241-8082446; E-mail: pmertens@ukaachen.de.

Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M212518200

    ABBREVIATIONS

The abbreviations used are: SR, serine-arginine-rich; GFP, green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; CM, conditioned medium; TRITC, tetramethylrhodamine isothiocyanate; aa, amino acids; FITC, fluorescein isothiocyanate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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