1 Graduate Institute of Microbiology, College of Medicine, No. 1, Jen-Ai Road, 1st Section, National Taiwan University, Taipei, Taiwan
2 National Health Research Institutes, Taipei, Taiwan
Correspondence
Mei-Ru Chen
mrc{at}ha.mc.ntu.edu.tw
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ABSTRACT |
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INTRODUCTION |
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EBNA-1 is a 641 aa DNA-binding protein (Fig. 1) that binds to multiple sites within the family of repeat (FR) and dyad symmetry (DS) elements that comprise the 1·8 kbp boundary of oriP (Rawlins et al., 1985
). The DNA-binding and dimerization domains of EBNA-1 have been mapped to the C-terminal 459607 aa (Chen et al., 1993
) and the crystal structure of EBNA-1 reveals a glycine-rich region that is responsible for direct DNA recognition (Bochkarev et al., 1996
). The N-terminal 50 aa region of EBNA-1 shares 50 % sequence similarity with the N-terminus of the human ribosomal protein S2, but the functional significance remains obscure (Yates & Camiolo, 1988
). EBNA-1 also contains three RGG-like motifs, repeated at aa 3356, 330350 and 354377, that have been hypothesized to be responsible for RNA binding (Snudden et al., 1994
).
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We were prompted to further characterize the RNA-binding abilities of EBNA-1, which may be mediated through multiple RGG motifs. RGG motifs were first identified in heterogeneous nuclear ribonucleoprotein (hnRNP) U, which is a member of a family of polypeptides that bind hnRNP particles and control the post-transcriptional pathways of expression of individual genes (Swanson & Dreyfuss, 1988; Kiledjian & Dreyfuss, 1992
). RGG motifs have been found in a wide variety of RNA-binding proteins that are involved in RNA processing or transport, such as herpes simplex virus (HSV) ICP27 and splicing factor TLS (Sandri-Goldin, 1998
; Lerga et al., 2001
), and even in proteins that are involved in RNA-editing activities and translation control in mitochondria of Trypanosoma brucei (Vanhamme et al., 1998
; Miller & Read, 2003
). The RGG domain within hnRNP U is essential for sequence-specific binding, whereas some RGG motifs are relatively non-sequence-specific and may cooperate with sequence-specific binding domains within the RNA-binding molecule, such as hnRNPA1 (Cobianchi et al., 1988
) and nucleolin (Yang et al., 1994
). These proteins usually prefer to bind poly(G) or poly(U) beads in RNA homopolymer binding assays. The intracellular binding substrates of RGG proteins that are identified by the SELEX protocol usually correlate with the results of RNA homopolymer binding. For example, GGUG was found to be the conserved sequence for the potential splicing factor TLS (Lerga et al., 2001
), whereas fragile X mental retardation protein recognizes GGGG (Ramos et al., 2003
).
Based on the presence of three RGG motifs in the EBNA-1 sequence, Snudden et al. (1994) demonstrated that EBNA-1 could bind to three different RNA probes: the EBV-encoded transcript EBER1, the human immunodeficiency virus (HIV)-encoded transactivation response element and an EBNA-1-coding sequence that is derived from the Fp promoter. However, it remained unclear whether all three RGG-rich motifs in EBNA-1 were functional and what the RNA-binding preference of EBNA-1 was. In this study, we addressed these issues by using RNA homopolymer-binding and gel mobility-shift assays (GMSAs). The results indicated that all three RGG motifs were able to bind to RNA and that EBNA-1 preferred to bind to RNAs with GU-rich sequences or with high degrees of secondary structure. Observation of the in vivo interaction of EBNA-1 with EBER1 further confirmed EBNA-1 as a DNA sequence-specific transactivator with strong RNA-binding activity.
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METHODS |
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Three constructs were generated for glutathione S-transferase (GST)EBNA-1 RGG fusion proteins. The primer sets that were used for pCW12 [GSTE1(3258)] were 5'-GAAGATCTCAAAGAAGAGGGGGTGA-3' and 5'-CGGGATCCGCCCGGGGCTCCTGGTC-3' (GSTRGG1); for pCW13 [GSTE1(324360)] were 5'-GAAGATCTGCAGGAGGTGGAGGCCG-3' and 5'-GATCGGATCCTCTTTCACGTCCTCT-3' (GSTRGG2); and for pCW14 [GSTE1(351382)] were 5'-GAAGATCTGGAGGCCGCCGGGGTAG and 5'-CGGGATCCCCTGGGCCTCTTTTCTC-3' (GSTRGG3). PCRs were performed by using pRA17 (Ambinder et al., 1990) as the template and the amplified products were digested with BamHI and BglII and cloned into a derivative of pGEX-3 (Pharmacia).
To construct a recombinant EBNA-1-expressing baculovirus, the PCR product that was amplified by using primers 5'-CTAGAGATCTATGTCTGACGAGGGGCC-3' and 5'-GATCGGATCCTGTTCCACCGTGGGT-3' with p367 as the template was digested with BamHI and BglII and cloned into the BglII site of pAcHLT-B (Pharmingen) to generate pTY1. To express EBNA-1 in transfected cells, a HindIII/BamHI fragment from pRA17 was cloned into pSG5.
To generate RNA probes, pCW16 (pGEM3-EBER1, encoding 167 nt of EBER1) and pCW17 (pGEM-3Z-EBER2, encoding 172 nt of EBER2) were generated by using a B95-8 cell lysate as the template and primers 5'-GGAATTCAGGACCTACGCTCCCTA-3' and 5'-CGGGATCCTGGATGCATAAATCCCTAA-3' for EBER1 and 5'-GGAATTCAGGACAGCCGTTGCCCTA-3' and 5'-CGGGATCCTGGGTGCAAAACTAGCCA-3' for EBER2. PCR products were digested with BamHI and BglII and cloned into pGEM-3Z. pGEM-BG9 contains the cDNA of EBV DNase (BGLF5; Chen et al., 1990); the plasmid was linearized with StuI and transcribed by T7 RNA polymerase to obtain nt 83 to +72 of the DNase RNA. pLC3 (EBNA-3A intron, nt 92 58292 699 of the B95-8 EBV genome) and pLC4 (EBNA-3A junction, nt 92 57292 679) were generated by PCR using the primers 5'-CGGAATTCGTGAGCATCCCTATGGCC-3' and 5'-CGGGATCCCTGAAACCAACGGCAACA-3' for pLC3 and 5'-CGGAATTCCGCGGCTACGGTGACGAT-3' and 5'-CGGGATCCTGTCGATGCGCTGAAACC-3' for pLC4, with an EBV-positive Akata cell lysate as the template. PCR products were digested with EcoRI and BamHI and cloned into pGEM-3Z. pCL3 and pCL4 were linearized with BamHI and the RNA probes that were generated by T7 RNA polymerase were 105 and 125 nt, respectively.
For the HCV 5' non-coding region (NCR), pSP64-T7HCV (1341) (Yen et al., 1995), which contains nt 1341 of an HCV 1b isolate from Taiwan, was digested with SmaI and transcribed by T7 RNA polymerase to generate nt 1130 of the internal ribosome entry site (IRES). pGEM4Z-NCR131/278 (Yen et al., 1995
), which encoded nt 131278 of the HCV NCR, was digested with EcoRI and transcribed by T7 RNA polymerase. pSP64-C191(A)M307 contained HCV 5' NCR nt 131278 with mutations at nt 241245 (GCGAG
CGGTC).
Expression of in vitro-translated EBNA-1 mutants and RNA homopolymer-binding assays.
In vitro translation was performed by using a TNT reticulocyte lysate (Promega) in the presence of [35S]methionine. To assess binding of in vitro-translated protein to ribonucleotide homopolymers (Kiledjian & Dreyfuss, 1992), 105 c.p.m. trichloroacetic acid-precipitable translated protein was made up to a final volume of 0·5 ml with binding buffer (10 mM Tris/HCl, pH 7·6; 2·5 mM MgCl2; 0·5 % Triton X-100; appropriate concentration of NaCl). Washed agarose beads (50 µg) containing the attached ribonucleotide homopolymer (Pharmacia or Sigma) or ssDNA (Pharmacia) were added to the mixture and incubated for 60 min at 4 °C on a rocker platform. The beads were pelleted in a microfuge and washed five times with binding buffer. Bound protein was eluted in SDS sample buffer [50 mM Tris/HCl, pH 6·8; 4 % SDS; 20 % glycerol; 0·04 % bromophenol blue; 200 mM dithiothreitol (DTT)] and analysed by 10 or 12 % SDS-PAGE. Radioactive bands were detected with a phosphorimager (Storm 840; Molecular Dynamics).
Expression and purification of recombinant EBNA-1 (rEBNA-1).
Recombinant baculovirus expressing EBNA-1 was generated by co-transfection of pTY1 and baculovirus DNA with a BaculoGold Transfection kit (Pharmingen). For preparation of rEBNA-1, virus-infected Sf9 cells were incubated for 46 h at 27 °C and the cells scraped into cold PBS and harvested by low-speed centrifugation. The cells were lysed in 10 mM Tris/HCl (pH 7·5), 130 mM NaCl, 1 % Triton X-100, 10 mM NaF and protease inhibitor cocktail and incubated on ice for 45 min. After sonication and clarification at 30 000 r.p.m. (Beckman L-80, SW41 rotor) for 45 min, the supernatant was applied to a 5 ml Econo-heparin column (Bio-Rad) and eluted with eluting buffer (20 mM HEPES, pH 7·5; 0·5 mM EDTA; 1 mM PMSF; 2 mM DTT; 20 % glycerol) that contained 250, 500, 750 or 1000 mM NaCl. The fractions that contained EBNA-1 were pooled, the NaCl concentration was adjusted to 100 mM and the fractions were applied to a MonoQ column (Bio-Rad). The protein was eluted with eluting buffer that contained 100, 250, 500, 750 or 1000 mM NaCl. The purified protein produced from recombinant baculovirus will be referred to as rEBNA-1.
Expression and purification of GSTEBNA-1 fusion proteins.
To express GST or GSTEBNA-1 fusion proteins, bacterial culture at exponential phase was diluted with 10 vols fresh medium, cultured at 37 °C until the OD600 reached 0·6 and induced with 0·1 mM IPTG for 3 h. The GSTEBNA-1 fusion protein or GST was expressed in JM109 cells and purified by using a glutathione column (Chen et al., 2001).
RNA probes.
In a typical 20 µl reaction, 1 µg linearized plasmid DNA was added to 4 µl 5x transcription buffer, 1 µl 10 mM ATP, CTP and GTP, 2·5 µl 100 µM UTP, 2 µl [-32P]UTP (3000 Ci mmol1=111 TBq mmol1), 1 µl RNasin (40 U µl1) and 1 µl T7 RNA polymerase. The mixture was incubated at 37 °C for 1 h, 1 U RQ1 DNase was added and the mixture was incubated for another 15 min. The RNA product was purified on a Chroma-spin column (Clontech) and quantified by using a Beckman scintillation counter.
In vitro synthesis of RNA.
RNA was prepared by using an in vitro transcription kit (Promega). For a 100 µl reaction, 4 µg linearized plasmid was dissolved in an appropriate amount of deionized water and added to 20 µl 5x transcription buffer, 10 µl each 10 mM ATP, CTP, GTP and UTP, 10 µl BSA (1 µg µl1), 2 µl RNasin (40 U µl1), 10 µl 0·1 M DTT and 2 µl T7 or SP6 RNA polymerase (20 U µl1). After incubation at 37 °C for 1 h, 1 µl T7 or SP6 RNA polymerase was added and the reaction mixture was incubated for a further 1 h. RQ1 DNase (4 U) was added and the mixture was incubated for 15 min at 37 °C to remove the DNA template. As the plasmid pSP64C191(A)M307 was not transcribed efficiently, PCR was performed with a forward primer containing a T7 promoter sequence (shown in bold) (5'-TAATACGACTCACTATAGGATCCCCGGGAGAGCCAT-3') and the reverse primer (5'-AAGCTTTCGCGACCCAACACTACTCG-3'). All RNA products were purified by using Chroma-spin columns (Clontech). For quantification, an aliquot of RNA was denatured at 65 °C, electrophoresed on a 2 % TBE/agarose gel, stained with SYBR green II RNA stain (Molecular Probes) and analysed by using a Storm 840 phosphorimager (Molecular Dynamics) and ImageQuant by comparison with RNA standards.
RNA-binding GMSA.
RNA-binding conditions were modified from those described by Snudden et al. (1994). Briefly, 2x104 c.p.m. [
-32P]UTP-labelled RNA probe was incubated with purified rEBNA-1 in a 20 µl reaction mixture that contained binding buffer (25 mM Tris/HCl, pH 8·0; 1 mM MgCl2; 0·4 M NaCl; 5 mM spermidine; 1 mM DTT; 5 mM EDTA; 5 % glycerol), 40 U RNasin, 0·4 µg yeast tRNA and 1 µg poly(I).poly(C) (Pharmacia) and incubated at 30 °C for 30 min. The binding complex was analysed by 4 or 5 % non-denaturing 19 : 1 PAGE containing 50 mM Tris/glycine (pH 8·8) and 5 % glycerol. After electrophoresis, the gel was dried and analysed by using a phosphorimager. In the supershift assay, 2 µl purified EBNA.OT1x (Chen et al., 1993
) or anti-His tag antibody (Qiagen) was added to the reaction and incubated for another 10 min. In competition assays, different amounts of non-radiolabelled RNA were included in the reaction.
Ribonucleoprotein immunoprecipitation (RIP) assay.
In vivo cross-linking of the proteinRNA complex was performed as described by Niranjanakumari et al. (2002). To increase the intracellular amount of EBNA-1 protein, 2x107 Akata cells were activated into the lytic cycle with 0·5 % anti-human IgG (Zetterberg et al., 2002
) and harvested at 18 h post-induction. In the epithelial system, 15 µg EBNA-1 expression plasmid pTY2 or vector was transfected into 2x107 EBV-positive NA cells (Chang et al., 1999
) and harvested at 48 h post-transfection. The cell pellets were resuspended in 10 ml PBS that contained 1 % (v/v) formaldehyde and incubated at room temperature for 10 min with slow mixing. The cross-linking reaction was quenched by the addition of glycine (pH 7·0) to a final concentration of 0·25 M and incubated at room temperature for 10 min. Cells were then harvested by centrifugation, washed twice with ice-cold PBS and resuspended in 1 ml RIPA buffer (50 mM Tris/HCl, pH 7·5; 1 % NP-40; 0·5 % sodium deoxycholate; 0·05 % SDS; 1 mM EDTA; 150 mM NaCl) in the presence of protease inhibitors. After sonication, the cell lysate was clarified by centrifugation at 14 000 r.p.m. in a microfuge for 10 min at 4 °C and the supernatant was pre-cleared with 50 µl protein ASepharose beads. The lysate was then diluted with 1 vol. RIPA buffer, mixed with EBNA.OT1x-coated beads and 40 U RNaseOUT (Invitrogen) and incubated with rotation at room temperature for 90 min. The immunocomplexes were pelleted, washed five times with high-stringency RIPA buffer (50 mM Tris/HCl, pH 7·5; 1 % NP-40; 1 % sodium deoxycholate; 0·1 % SDS; 1 mM EDTA; 1 M NaCl; 1 M urea; 0·2 mM PMSF) and suspended in 50 mM Tris/HCl (pH 7·0), 5 mM EDTA, 10 mM DTT and 1 % SDS. To reverse the cross-linking, the resuspended beads were incubated at 70 °C for 45 min. Bound RNA was extracted from these samples by using Trizol (Invitrogen) followed by digestion with 2 U DNase I (Ambion) for 30 min at 37 °C, and the DNase activity was inactivated by adding 0·1 vol. 25 mM EDTA. The RNA was reverse-transcribed with random hexamers and PCR-amplified with EBER1-specific primers (5'-CTACGCTGCCCTAGAGGTTTTGCTA-3' and 5'-ATGCGGACCACCAGCTGGTACTTGA-3').
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RESULTS |
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To compare the binding affinities of EBNA-1 for the different RNA molecules, an RNA competition assay was performed by using EBER1 as the probe in the presence of 0·2 µg EBNA-1 and various cold RNA competitors (Fig. 6a and data not shown). As demonstrated in Fig. 6b
, HCV 5'NCR(131278) appeared to be the strongest competitor, followed by pGEM-3Z, EBER2, EBER1, EBNA-3A intron, EBNA-3A junction, DNase RNA and HCV 5'NCR(1130). The G, U and G+U contents of individual probes were calculated. The stronger competitors, including HCV 5'NCR (131278), pGEM-3Z and EBER1 and EBER2, had >30 % G or 50 % G+U contents, whereas the weaker competitors had lower G or G+U contents. The stronger competitors, including HCV 5'NCR(131278), EBER1 and EBER2, are also known to contain a high degree of secondary structure.
Secondary structure of the HCV IRES RNA affects the RNA-binding ability of EBNA-1
To examine the possible effect of RNA structure on EBNA-1 binding, a mutant RNA (M307) of HCV 5'NCR(131278) was tested in the competition assay (Fig. 6c, d). This RNA contained mutations at nt 241245 (GCGAG
CGGTC), which affect the stemloop III region of the secondary structure, resulting in a predicted long hairpin instead of a multistem structure. It has a lower affinity for a cellular transcription factor (Yen et al., 1995
; Chang et al., unpublished data). In comparison with NCR(131278) of M307, wild-type HCV 5'NCR(131278) was a 10-fold stronger competitor. The predicted secondary structures of wild-type and mutant HCV 5'NCR(131278) are shown in Fig. 6(e, f)
.
Detection of intracellular EBNA-1EBER complexes by RIP
To test whether binding of RNA to EBNA-1 occurred in EBV-positive lymphocyte and epithelial cells, Akata cells were induced into the lytic cycle to increase the expression of endogenous EBNA-1 (Zetterberg et al., 2002). EBNA-1RNA complexes were cross-linked in vivo at 18 h post-induction and immunoprecipitated with EBNA.OT1x. RNA isolated from the immunoprecipitated complex was reverse-transcribed with random hexamers and detected with EBER1-specific primers by PCR. In the presence of reverse transcriptase, the EBER1-specific 157 bp product was detected in the immunocomplex pellet from the reaction by using anti-EBNA-1 antibody, but not anti-GST antibody (Fig. 7a
). The supernatant of each reaction was also examined for EBER1 to ensure the integrity of RNA molecules. In addition, EBV-positive NA cells were transfected with a plasmid expressing EBNA-1 for a similar RIP assay. The results of a 25-cycle PCR revealed that the EBER1 PCR product was only detected in the presence of transfected EBNA-1 (Fig. 7b
), whereas the EBER1 RNA bound by endogenous EBNA-1 could also be detected in a 40-cycle PCR (data not shown). Each fraction was also subjected to Western blot analysis to confirm the simultaneous presence of EBNA-1 (Fig. 7c
).
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DISCUSSION |
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It was interesting to note that EBNA-1 bound to poly(G) more efficiently in buffer that contained 0·5 or 0·75 M NaCl than in buffer that contained 0·25 M NaCl. Perhaps the folding of EBNA-1 determines the exposure of the RGG boxes and therefore regulates its RNA-binding ability. A negative regulatory domain for RNA-binding activity was observed in the C-terminus of HSV ICP27, but whether these domains are biologically relevant remains to be elucidated. We showed that all three RGG boxes of EBNA-1 could contribute additively to RNA-binding activity (Fig. 3) and that each individual box could mediate RNA binding in GMSAs (Fig. 4
). Furthermore, as the three RGG motifs can function independently of each other, large RNArEBNA-1 complexes could be formed, as observed in the GMSAs (Figs 5 and 6
).
We tested eight different RNA probes for rEBNA-1 binding in GMSAs. The results indicated that EBNA-1 bound to a broad spectrum of RNA, except for HCV 5'NCR(1130), and that the binding affinity correlated with the G or G+U contents of the different probes (Fig. 6). The stronger competitors had 3035 % G or 5060 % G+U contents, whereas the weaker competitors had 2325 % G or 4348 % G+U. Interestingly, the probe with highest affinity, HCV 5'NCR(131278), contained a region that is bound by several unidentified cellular factors (Yen et al., 1995
) and the La antigen (Ali & Siddiqui, 1997
). The possible contribution of the secondary structure of the RNA probe to EBNA-1 binding was analysed by comparing wild-type HCV 5'NCR(131278), which was predicted to form a multistem structure, with HCV 5' NCR-M307, which harbours a long hairpin-like structure and showed much weaker binding (Fig. 6
). How the altered RNA structure affects the binding of EBNA-1 remains to be determined. The RNA-binding abilities of EBNA-1 reported here support the idea that many transactivators, including p53 and STAT1, are able to bind specific DNA sequences and RNA molecules [reviewed by Cassiday & Maher (2002)
].
Although lacking sequence homology, we found that the arrangement of RNA-binding motifs and the RNA-binding characteristics of EBNA-1 share some similarities with the La antigen, which is also composed of three separate RNA-binding domains with a hinge region in between [reviewed by Maraia & Intine (2001)]. Similar to EBNA-1, the La antigen binds to most RNAs and the affinity increases as the number of terminal Us increases. The La antigen was found to bind many pol III transcripts, including EBER1 and -2, VAI and VAII of adenovirus and the NCR of HCV (Maraia & Intine, 2001
). Functionally, the La antigen is required for the processing and maturation of precursor tRNA (Van Horn et al., 1997
; Pannone et al., 1998
). It also binds to some IRESs in viral and cellular mRNAs and has been shown to stimulate IRES-mediated translation (Belsham et al., 1995
; Holcik & Korneluk, 2000
). A stimulatory effect of rEBNA-1 on HCV IRESluciferase has also been observed in our laboratory (data not shown).
The in vivo EBNA-1EBER1 interaction detected by the RIP assay indicated that EBNA-1 can form ribonucleoprotein complexes in EBV-infected cells. The biological impact of EBNA-1 RNA binding should be considered in relation to previously mapped functional domains that overlap the RGG motifs. For instance, it is possible that the EBNA-1-interacting protein EBP2, which is involved in rRNA processing and EBNA-1 chromosome binding (Kapoor & Frappier, 2003), associates with metaphase chromosomes through an RNA-mediated mechanism. This might be similar to the scaffold attachment factor A (SAF-A), which binds to a specific DNA sequence and a non-coding RNA, XIST. The RGG box of SAF-A is required for its binding to the inactive X chromosome (Helbig & Fackelmayer, 2003
). In addition, the transactivation function of EBNA-1 could also involve RNA binding. This suggestion is based on the similarity of EBNA-1 to the human papilloma virus E2 protein, which also contains an RGG motif and interacts with p32 and was found to turn on gene expression by functioning as a transcription transactivator and as a pre-mRNA processing modulator (Li et al., 1998
).
The RNA-binding properties of EBNA-1 characterized here seem to be affected by the secondary structure of the RNA (Fig. 6). Therefore, we searched for sequence similarity with the dsRNA-binding domain (dsRBD), a 6570 aa sequence/structure motif that mediates dsRNA interaction, in EBNA-1 by using the PIR Domain Similarity Search and Pairwise Alignment tool (http://pir.georgetown.edu). However, no dsRBD consensus sequence was observed in EBNA-1. Insight into the significance and structural specificity of the EBNA-1RNA interaction is most likely to come from identification of physiological RNA ligands of EBNA-1 in EBV-infected cells. The RIP protocol described in this report could be used to search for the most abundant RNA species bound by EBNA-1 in EBV-positive cells, as has been described previously for identifying the substrates of HSV US11 (Attrill et al., 2002
).
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ACKNOWLEDGEMENTS |
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Received 25 April 2004;
accepted 14 June 2004.
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