Regulation of ascidian Rel by its alternative splice variant Narudo Kawai 1 , Masumi Shimada 1 , Hiroyuki Kawahara 1 , Noriyuki Satoh 2 and Hideyoshi Yokosawa 1 1 Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan; 2 Department of Zoology, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan The Rel/NF-jB family of transcription factors play key roles in morphogenesis and immune responses. We reported previously that As-rel1 and As-rel2 of the ascidian Halo- cynthia roretzi are involved in notochord formation. The As-rel1 protein is a typical Rel/NF-jB family member, whereas the As-rel2 protein is a novel truncated product of As-rel1 that lacks a nuclear localization signal and the unique C-terminal region. Here, we present conclusive evi- dence that As-rel1 and As-rel2 are generated from a single gene by alternative splicing. We analyzed the roles of As-rel2 using cells transfected with As-rel1 or As-rel2 or both. As-rel1 was localized in the nucleus and As-rel2 in the cytoplasm when they were transfected individually. In con- trast, when they were transfected simultaneously, both were localized in the nucleus because of the association of As-rel2 with As-rel1. In this case, the transcriptional activity of As-rel1 was suppressed by As-rel2. Ascidian IjB was found to sequester As-rel1 in the cytoplasm and suppress its tran- scriptional activity when As-rel1 and IjB were transfected simultaneously. In contrast, when As-rel1 and IjBwere transfected together with As-rel2, As-rel1 was transported into the nucleus and its transcriptional activity was rescued from inhibition by IjB, whereas As-rel2 remained localized in the cytoplasm, suggesting IjB sequestration in the cyto- plasm by As-rel2. From these findings, we conclude that the alternative splice variant, As-rel2, regulates the nuclear localization and transcriptional activity of As-rel1. Keywords: alternative splicing; IjB; nuclear transport; Rel; transcriptional activity. The Rel/NF-jB family of transcription factors contains the highly conserved Rel homology domains (RHDs) required for DNA binding and dimerization, and these transcription factors regulate the expression of downstream target genes involved in various phenomena such as morphogenesis, immune response, cell growth, and programmed cell death [1–8]. Regulatory mechanisms underlying the functions of Rel/NF-jB family members have been extensively studied, and several modes of their regulation have been proposed [5–8]. A suggested basic regulatory mechanism in the canonical Rel/NF-jB signaling pathway is based on the inhibitor protein IjB-mediated mechanism. Without extra- cellular stimuli, the Rel/NF-jB protein is sequestered in the cytoplasm through binding to IjB because IjBmasksthe nuclear localization signal of Rel/NF-jB. In response to extracellular stimuli, the IjB protein is phosphorylated by the oligomeric kinase IKKb, polyubiquitinated, and degraded by the proteasome, resulting in the release of the Rel/NF-jB protein, which moves from the cytoplasm to the nucleus, where it binds to regulatory elements of target genes to trigger their transcription. In contrast, in the noncanonical pathway, based on proteolytic processing of a mammalian Rel/NF-jB precursor, the precursor p100 binds with RelB to form a heterodimer in the cytoplasm. In response to extracellular stimuli, the p100 protein in the complex is phosphorylated by the oligomeric kinase IKKa and conver- ted into p52 via the ubiquitin-mediated proteolytic pathway. The RelB/p52 heterodimer thus formed then moves to the nucleus and functions in transcription of target genes. In addition, post-translational modification of Rel/NF-jB family members such as phosphorylation [9–11] or small ubiquitin-related modifier-1 modification [12] is an addi- tional regulatory mechanism for their transcriptional acti- vity, the phosphorylated protein thus showing enhanced transcriptional activity. On the other hand, the function of the Rel/NF-jB protein is negatively regulated by its C-terminally truncated form. For example, a homodimer of p50, a mammalian Rel/NF-jB family member that lacks a C-terminal transactivation domain, binds to the jB consen- sus element and exhibits a suppressive effect on jB-depend- ent gene expression [13]. A C-terminally truncated form of p65, which is produced by caspase-catalyzed cleavage upon apoptosis, has the nuclear localization signal but no transcriptional activity. This protein plays a suppressive role in wild-type p65-mediated transcription [14]. In a previous study [15], we found that the ascidian Halocynthia roretzi has two members of the Rel/NF-jB family: one called As-rel1 is a typical Rel/NF-jB family member that has RHD, the nuclear localization sequence, and the C-terminal region; the other called As-rel2 has a novel structure with deletion of both the nuclear localization signal and the C-terminal region. In H. roretzi embryos, it was found that the ectopic expression of As-rel1 protein led to reduction of the number of notochord cells and a defect Correspondence to H.Yokosawa,DepartmentofBiochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan. Fax: + 81 11 706 4900, Tel.: + 81 11 706 3754, E-mail: yoko@pharm.hokudai.ac.jp Abbreviations: RHD, Rel homology domain; IKK, IjBkinase; GST, glutathione S-transferase; EST, expressed sequence tag; DAPI, 4¢,6-diamidino-2-phenylindol; GFP, green fluorescent protein. (Received 20 June 2003, revised 24 August 2003, accepted 18 September 2003) Eur. J. Biochem. 270, 4459–4468 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03838.x in tail elongation, indicating that this Rel/NF-jBprotein affects notochord formation in ascidian embryos. On the other hand, it was found that the As-rel2 protein had an antagonistic effect on the action of the As-rel1 protein. These findings imply that As-rel2, the C-terminally truncated form of As-rel1, regulates the function of As-rel1. However, the regulatory mechanism has not yet been elucidated. To determine the relationship between As-rel1 and As-rel2, we investigated the effects of As-rel2 on the localization and activity of As-rel1 by using As-rel1- transfected and As-rel2-transfected mammalian cells. As-rel2, transfected alone, was localized in the cytoplasm because of the lack of the nuclear localization sequence, whereas this protein when transfected together with As-rel1 moved to the nucleus and suppressed transcriptional activity of As-rel1, possibly because the level of activity of a heterodimer of As-rel1 and As-rel2 is lower than that of a homodimer of As-rel1. On the other hand, As-rel1 trans- fected together with the ascidian inhibitor IjBremained localized in the cytoplasm, but this protein transfected together with both IjB and As-rel2 was able to move into the nucleus, and its transcriptional activity was rescued from inhibition by IjB. These results indicate that As-rel2 regulates the function of As-rel1 in the presence of IjB. We presented conclusive evidence that As-rel1 and As-rel2 are generated from a single gene by alternative splicing. To the best of our knowledge, this is the first report of a novel regulatory mechanism for the function of the Rel/NF-jB family that is mediated by an alternative splice variant. Materials and methods Materials The proteasome inhibitor MG132 was purchased from the Peptide Institute, Inc. (Osaka, Japan). Mouse anti-(T7-tag), anti-(Flag-tag) M2 and anti-(c-Myc-tag) 9E10 monoclonal Igs were purchased from Novagen, Sigma and Santa Cruz, respectively. Horseradish peroxidase-conjugated antibodies against rabbit and mouse immunoglobulins were obtained from Amersham Pharmacia Biotech. Preparation of antibody against As-rel1 A 480-bp fragment, consisting of 460–618 amino-acid residues of As-rel1, was subcloned in-frame into the pGEX-6P-1 expression vector with the correct orientation. Escherichia coli BL21 expressing glutathione S-transferase (GST)–As-rel1 fusion protein after isopropyl b- D -thio- galactoside induction was solubilized in lysis buffer (20 m M Tris/HCl, pH 8.0, containing 1 m M EDTA and 100 m M NaCl), and the extract was applied to glutathione- immobilized agarose beads to trap the fusion protein, which was subsequently digested with PreScission protease (Amer- sham) to release the As-rel1 fragment. The isolated As-rel1 fragment was emulsified in Freund’s complete adjuvant and injected subcutaneously into rabbits followed by a four-time booster injection of the isolated fragment in Freund’s incomplete adjuvant. The antiserum was purified by affinity chromatography on Protein A-immobilized Sepharose CL-4B (Amersham), and the immunoreactivity was verified by Western blotting of the fusion protein. Preparation of the genome and genomic PCR The genome of the ascidian, H. roretzi, was prepared from muscles. A 5 mL volume of extraction buffer [2 · lysis buffer (ABI), 0.5 · phosphate-buffered saline, and 200 lgÆmL )1 proteinase K (ABI)] was added to 0.5 g muscle, and then incubated overnight at 55 °C. Phenol (5 mL) was added to the suspension, and the mixture was rotated for 1 h at room temperature and was then centrifuged at 10 000 g for 10 min. The resulting super- natant was mixed with phenol, rotated for 8 h at room temperature and centrifuged at 10 000 g for 10 min. The resulting supernatant was subjected to ethanol precipitation to give genomic DNA as a precipitate. Genomic PCR was performed with several combinations of the following primers for the As-rel sequence [genome forward F1 primer, 5¢-ATGGACAAAATGTCTA-3¢ (As-rel1 bp 47–62); genome forward F2 primer, 5¢-GGA AGCCACAAAAGTTAT-3¢ (As-rel1 bp 832–849); gen- ome forward F3 primer, 5¢-CTGCTGGATAGATGA TGC-3¢ (As-rel2 bp 941–958); genome forward F4 primer, 5¢-ACTTATTTTTCTCGCACA-3¢ (As-rel2 bp 1700–1717); genome reverse R1 primer, 5¢-TCTGATTGAGGTTAG TGG-3¢ (As-rel2 bp 1580–1563); genome reverse R2 primer, 5¢-TTTCGGTTTGTAATGTTAGT-3¢ (As-rel2 bp 1656–1647); genome reverse R3 primer, 5¢-GAATA CGAACCCAAACAA-3¢ (As-rel2 bp 2117–2100); genome reverse R4 primer, 5¢-TGTCTTCATGTGGGACAA-3¢ (As-rel1 bp 1441–1434)] using an Expand High Fidelity Taq polymerase (Roche). PCR fragments produced were cloned into the pGEM-T-vector (Promega), and the sequences were determined from each end of insert DNAs. Cell culture and transfection HEK293 or 293T cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum at 37 °C under a 5% CO 2 atmosphere. Transfection was performed using Effectene transfection reagent (Qiagen), Metafectene transfection reagent (Biontex), or FuGene 6 transfection reagent (Roche) according to the manufac- turer’s protocol. Isolation of an IjB homologue We searched the Ciona intestinalis expressed sequence tag (EST) database (Ciona cDNA resources of Kyoto Univer- sity) for an IjB homologue and found it in this database. The EST clone, cieg29i06, which is predicted to include the full length of IjB-like cDNA, was subjected to nucleotide sequence determination, and this clone, designated Ci-IjB for an IjB homologue of the ascidian C. intestinalis,was found to include the full length. Plasmid constructions To generate As-rel1 and As-rel2 expression plasmids, pCI-neo-As-rel1, pCI-neo-Flag-As-rel1, pCI-neo-c-Myc- As-rel1, pCI-neo-c-6Myc-As-rel1, pCI-neo-Flag-As-rel2, pCI-neo-c-6Myc-As-rel2 and pCI-neo-T7-As-rel2, full- length As-rel1 and As-rel2 cDNAs were inserted into the MluI–NotI sites of pCI-neo (Promega), pCI-neo-Flag, 4460 N. Kawai et al.(Eur. J. Biochem. 270) Ó FEBS 2003 pCI-neo-c-Myc, pCI-neo-c-6Myc and pCI-neo-T7, respect- ively, after subcloning of full-length As-rel1 and As-rel2 containing the MluIandNotI sites by PCR using the following primers (As-rel MulI forward primer, 5¢-ACGC GTATGGACAAAATGTCTA-3¢; As-rel1 NotI reverse primer, 5¢-AGCGGCCGCTCAGTTGTAATTC-3¢;As- rel2 NotI reverse primer, 5¢-AGCGGCCGCTCATCT ATCCAGCA-3¢). The PCR products were subcloned in the pGEM-T-vector (Promega). pEGFP-C1-As-rel1 was constructed by PCR using the following primers (As-rel1 XhoI forward primer, 5¢-CTCGAGCTATTATGGA CAAAATGT-3¢; As-rel1 BamHI reverse primer, 5¢-GGAT CCGTTGTAATTCTGAT-3¢). The PCR product sub- cloned in the pGEM-T-vector was digested with XhoIand BamHI and then inserted into the XhoIandBamHIsitesof pEGFP (Clontech). pBIND-As-rel1 and pBIND-As-rel2 were constructed by PCR using the following primers (As-rel MluI forward primer, 5¢-ACGCGTTGATGGAC AAAAT-3¢; As-rel1 NotI reverse primer, 5¢-AGCGGCCG CTCAGTTGTAATTC-3¢;As-rel2NotI reverse primer, 5¢-AGCGGCCGCTCATCTATCCAGCA-3¢). The PCR products subcloned in the pGEM-T-vector were digested with MluIandNotI and then inserted into the MluIand NotI sites of pBIND (Promega). pCI-neo-Flag-Ci-IjBand pCI-neo-c-6Myc-Ci-IjB were constructed by PCR using the following primers (Ci-IjB SalI forward primer, 5¢-GTCGA CATGTCTAATAAAGCA-3¢;Ci-IjB NotI reverse primer 5¢-GCGGCCGCTCATTGTCG-3¢). The PCR products subcloned in the pGEM-T-vector were digested with SalI and NotI and then inserted into the SalIandNotIsitesof pCI-neo-Flag or pCI-neo-c-6Myc. Immunoblotting Proteins were separated by SDS/PAGE on a 10% gel and transferred to a nitrocellulose membrane (Advantec, Tokyo, Japan). The membrane was blocked with 5% nonfat milk in phosphate-buffered saline containing 0.1% Tween 20 for 1 h at room temperature, incubated with the primary antibody at room temperature for 1 h and then with a horseradish peroxidase-conjugated antibody against rabbit or mouse immunoglobulin at room temperature for 30 min, and developed with an enhanced chemiluminescence detec- tion system (Amersham). Immunoprecipitation HEK293 cells were transfected with several combinations of 1.0 lg pCI-neo-c-Myc-As-rel1 and 1.0 lg pCI-neo-Flag- As-rel1 using Effectene transfection reagent in 100-mm dishes. (The total amount of plasmid DNA was adjusted to 2.0 lg with an empty vector, pCI-neo.) After 48 h of incubation, the cells were washed with phosphate-buffered saline and disrupted by treatment with lysis buffer (50 m M Tris/HCl, pH 8.0, containing 150 m M NaCl, 0.1% Nonidet P40, and 10% glycerol) containing 10 l M MG132 and a protease inhibitor cocktail (Roche) for 30 min on ice, and the lysate was centrifuged at 13 000 g for 20 min. The resulting supernatant was pretreated with 10 lLPro- tein G-immobilized agarose (Santa Cruz) at 4 °Cfor 30 min and was then incubated with 2 lg anti-(Flag-tag) M2 Ig and 10 lL Protein G-immobilized agarose at 4 °C for 1 h. The beads were washed five times with lysis buffer, boiled for 5 min in SDS sample buffer, and subjected to SDS/PAGE and then to Western blotting with anti-(Flag- tag) M2 or anti-(c-Myc-tag) 9E10 Ig. Alternatively, HEK293 cells were transfected with several combinations of 0.2 lg pCI-neo-As-rel1 and 1.8 lgpCI-neo-T7-As-rel2. Immunoprecipitation with both 2 lg anti-(As-rel1) IgG and 10 lL Protein A-immobilized Sepharose was carried out, followed by Western blotting with antibody to As-rel1 or T7-tag. To determine the relationship between As-rel1, As-rel2 and Ci-IjB, HEK293T cells were transfected with several combinations of 1.0 lg each of pCI-neo-c-6Myc-As-rel1, pCI-neo-c-6Myc-As-rel2, and pCI-neo-Flag-Ci-IjBusing Metafectene transfection reagent in 60-mm dishes. (The total amount of plasmid DNA was adjusted to 3.0 lg with empty vectors, pCI-neo-Flag and pCI-neo-c-6Myc.) Immunoprecipitation with anti-(Flag-tag) M2-immobilized beads (Sigma) was carried out, and the beads were washed five times with lysis buffer and eluted with 100 lgÆmL )1 3 · Flag-peptide (Sigma). The eluate was subjected to a second immunoprecipitation with both 2 lg anti-(As-rel1) IgG and 10 lL Protein A-immobilized Sepharose at 4 °C for 1 h. The beads were washed five times with lysis buffer and eluted with 0.1 M glycine (pH 3.0). The eluates obtained from the first and second immunoprecipitations were subjected to SDS/PAGE and then to Western blotting with anti-(Flag-tag) M2 or anti- (c-Myc-tag) Ig. In addition, to confirm the interaction between As-rel2 and Ci-IjB, HEK293T cells were transfected with several combinations of 1.0 lgeachof pCI-neo-Flag-As-rel2 and pCI-neo-c-6Myc-Ci-IjB. Immu- noprecipitation with anti-(Flag-tag) M2-immobilized beads and elution with 3 · Flag-peptide were carried out as described above, followed by Western blotting with anti-(Flag-tag) M2 or anti-(c-Myc-tag) Ig. Localization of As-rel1 and As-rel2 HEK293 cells were transfected with 0.3 lg pEGFP-C1- As-rel1 and 1.2 lg pCI-neo (empty vector) using Metafec- tene transfection reagent in 35-mm dishes. After 48 h of incubation, the cells were washed with phosphate-buffered saline and fixed with 3.7% parafolmaldehyde in phosphate- buffered saline for 30 min at room temperature. After being washed twice with phosphate-buffered saline, the specimen was incubated with 0.15% Triton X-100 in phosphate- buffered saline for 5 min at room temperature and washed again twice with phosphate-buffered saline. The specimen was then stained with 0.1 mgÆmL )1 4¢,6-diamidino-2-phenyl- indol (DAPI) in phosphate-buffered saline for 3 min at room temperature and washed twice with phosphate- buffered saline. The fixed cells were examined using a Zeiss Axiophot 2 microscope equipped with a fluorescein iso- thiocyanate filter set (488-nm excitation) for green fluores- cent protein (GFP) visualization and with a UV filter set (372-nm excitation) for DAPI visualization. For analysis of the localization of As-rel2, HEK293 cells were transfected with 0.3 lg pCI-neo-Flag-As-rel2 together with 1.2 lgpCI- neo (empty vector) using Metafectene transfection reagent. The cells were incubated with anti-(Flag-tag) M2 Ig as a primary antibody and subsequently with Alexa FluorÒ 594 Ó FEBS 2003 Regulation of Rel by alternative splice variant (Eur. J. Biochem. 270) 4461 goat anti-mouse IgG (Molecular Probes) as a secondary antibody and then visualized with a rhodamine isothio- cyanate filter set (590 nm for excitation) for Alexa. For analysis of the colocalization of As-rel1 and As-rel2, HEK293 cells were transfected with 0.3 lgeachpEGFP- C1-As-rel1 and pCI-neo-Flag-As-rel2 together with 0.9 lg pCI-neo using Metafectene transfection reagent. The cells were stained and visualized as described above. For analysis of the effect of Ci-IjB, the cells were transfected with 0.3 lg each pEGFP-C1-As-rel1 and pCI-neo-c-6Myc-Ci-IjB together with or without 0.3 lg pCI-neo-Flag-As-rel2 using Metafectene transfection reagent, and visualization was performed as described above. Luciferase assay To measure transcriptional activities of As-rel1 and As- rel2, we used GAL4 fusion proteins, GAL4–As-rel1 and GAL4–As-rel2, and a plasmid containing GAL4-binding DNA sequences and a luciferase reporter gene [16]. HEK293 cells were transfected with several combinations of 0.75 lg of the pG5/luc vector (Promega) and pBIND- As-rel1, pBIND-As-rel2, or pBIND-As-rel1 plus pCI- neo-Flag-As-rel2 using Metafectene transfection reagent in 35-mm dishes. (The total amount of plasmid DNA was adjusted to 1.5 lgwithanemptyvector,pBINDor pCI-neo-Flag.) Alternatively, the cells were transfected with the combination of 0.25 lg of the pG5/luc vector, pBIND-As-rel1 and pCI-neo-Flag-Ci-IjB with or without pCI-neo-Flag-As-rel2 using FuGene 6 transfection rea- gent. (The total amount of plasmid DNA was adjusted to 0.5 lg with an empty vector, pCI-neo-Flag.) After 48 h of incubation, the transfected cells were washed with phosphate-buffered saline and disrupted with Passive Lysis Buffer (Dual-LuciferaseÒ Reporter Assay System; Promega). Luciferase activity of the resulting lysate was measured by using a Dual-Luciferase Reporter Assay System and an AB-2000 luminescencer-PNS (Atto, Tokyo, Japan). The same experiments were repeated three times. In addition, to assess the expression levels of GAL4–As-rel1, Flag–As-rel2, and Flag–Ci-IjB, some of the transfected cells were subjected to SDS/PAGE and then to Western blotting with anti-(As-rel1) and anti- (Flag-tag) M2 Ig. Results As-rel2 is a splice variant of As-rel1 The nucleotide sequence encoding RHD of As-rel1 is completely identical with that of As-rel2 [15], indicating the possibility that As-rel1 and As-rel2 are splice variants. To understand how As-rel1 and As-rel2 mRNAs are produced, we determined the partial genomic sequence (4193 nucleo- tides) of As-rel using genomic PCR products (Fig. 1). Comparison of the genomic and cDNA sequences revealed that the genome has one sequence encoding RHD (876 nucleotides), that the As-rel1-specific sequence is located 2369 bp downstream from the RHD sequence, and that the intron (2369 nucleotides) for As-rel1 contains a stop codon and a polyadenylation signal sequence, which are located 24 and 2077 bp downstream, respectively, from the RHD sequence (Fig. 1A). These results indicate that the As-rel1 mRNA is generated from pre-mRNA by splicing at the splice sites shown in Fig. 1B, while a short mRNA of As-rel2 is generated because the intron for As-rel1, which contains a stop codon 24 bp downstream from its 5¢ end, is not excised (Fig. 1A). These results indicate that As-rel1 and As-rel2 are splice variants. With regard to the splicing for the typical Rel/NF-jB family member As-rel1, it should be noted that the sequence at the 5¢ end of the intron for As-rel1 is GC (Fig. 1B). The common sequence for the corresponding 5¢-splice site is GT, but the sequence GC has been reported in several species [17,18]. Interaction of As-rel1 with As-rel2 As-rel1 and As-rel2 have identical RHDs, which are necessary for DNA binding, interaction with IjB, and dimerization. First, to determine whether As-rel1 interacts with itself, we transiently coexpressed Flag-tagged and c-Myc-tagged As-rel1s in HEK293 cells, and the extracts of transfected cells were subjected to immunoprecipitation using anti-(Flag-tag) Ig and then to Western blotting with anti-(c-Myc-tag) Ig to detect interaction (Fig. 2A). As expected, As-rel1 was found to interact with itself to form a homodimer. Next, we carried out an experiment to determine whether As-rel1 interacts with As-rel2. As-rel1- transfected and T7-tagged As-rel2-transfected cells were subjected to immunoprecipitation using antibody against As-rel1 and then to Western blotting with antibodies against As-rel1 and T7-tag (Fig. 2B). As-rel1 was again found to interact with As-rel2 to form a heterodimer. Localization of As-rel1 and As-rel2 As As-rel1 has the nuclear localization signal and As-rel2 does not, it is reasonable to assume that the former can Fig. 1. As-rel1 and As-rel2 are splice variants. (A) Schematic repre- sentation of the structures of the As-rel genome and mRNAs of As-rel1 and As-rel2. (B) Genome sequence of splicing sites. Note that As-rel1 mRNA is generated by splicing at the sites shown by open arrowheads, whereas As-rel2 mRNA is generated without excision at the above sites. The stop codon in the alternative exon for As-rel2 is indicated by a closed arrowhead, and the polyadenylation signal sequence is indicated by an arrow. 4462 N. Kawai et al.(Eur. J. Biochem. 270) Ó FEBS 2003 move into the nucleus, whereas the latter remains localized in the cytoplasm. It would be interesting to determine whether coexpression of As-rel2 modulates the localization of As-rel1 as As-rel2 rescues the effect of As-rel1 on notochord formation [15]. To determine their localization, we first transiently expressed GFP–As-rel1 fusion protein and Flag-tagged As-rel2 in HEK293 cells individually (Fig. 3A). As expected, As-rel1 was present in the nucleus, whereas As-rel2 was in the cytoplasm. On the other hand, when GFP–As-rel1 and Flag-tagged As-rel2 were coex- pressed in HEK293 cells, both As-rel1 and As-rel2 were present in the nucleus (Fig. 3B), strongly suggesting that As-rel2, possibly as a heterodimer with As-rel1, can move to the nucleus. Suppressive effect of As-rel2 on transcriptional activity of As-rel1 It was found that the heterodimer composed of As-rel1 and As-rel2 is localized in the nucleus. We next carried out an experiment to determine whether As-rel2 modulates the transcriptional activity of As-rel1. As DNA sequences that bind As-rel1 and As-rel2 have not been determined, we employed a luciferase assay in HEK293T cells using GAL4– As-rel fusion proteins and the GAL4-binding DNA sequence. In the cells transiently expressing GAL4–As-rel1 alone, luciferase activity was enhanced, depending on the dose of GAL4–As-rel1 protein (Fig. 4A), whereas the activity was undetectable in the cells transiently expressing GAL4–As-rel2 alone (Fig. 4B). These results suggest that the C-terminal domain of As-rel1 is indispensable for transcrip- tional activity. Interestingly, in the cells transiently expressing GAL4–As-rel1 and Flag-tagged As-rel2 simultaneously, the activity of As-rel1 was moderately suppressed by As-rel2 (Fig. 4C, upper panel, luciferase assay). It should be noted that the expression level of GAL4–As-rel1 remained almost Fig. 2. Interaction of As-rel1 with As-rel2. (A) HEK293 cells were transiently transfected with the indicated combinations of Flag-As-rel1 and c-Myc-As-rel1 expression plasmids, and 48 h after transfection the cell lysates were subjected to immunoprecipitation using anti-(Flag- tag) Ig and then to Western blotting with anti-(Flag-tag) and anti- (c-Myc-tag) Igs. (B) HEK293 cells were transiently transfected with indicated combinations of As-rel1 and T7-As-rel2 expression plasmids, and 48 h after transfection the cell lysates were subjected to immunoprecipitation (IP) using an antibody against As-rel1 and then to Western blotting with antibodies against As-rel1 and T7-tag. Fig. 3. Localization of As-rel1 and As-rel2. (A) HEK293 cells were individually transiently transfected with GFP–As-rel1 (a, b) and Flag–As-rel2 expression plasmids (c, d), and fluorescence due to GFP (a) was visualized using a microscope equipped with a 488-nm excitation and fluorescein isothiocyanate filter set for GFP, while the antibody staining of Flag–As-rel2 (c) was visualized using a 570-nm excitation and rhodamine isothiocyanate filter set. DNA (b, d) was stained with DAPI and visualized using a 372-nm excitation and UV filter set. (B) GFP–As-rel1 and Flag– As-rel2 were transiently coexpressed in HEK293 cells, and fluorescence due to GFP–As-rel1 (a) and the antibody staining of Flag–As-rel2 (b) were visualized as described above. DNA (d) was stained with DAPI. Note that GFP–As-rel1 merges with Flag–As-rel2 (c). The nuclei are indicated by arrowheads. Ó FEBS 2003 Regulation of Rel by alternative splice variant (Eur. J. Biochem. 270) 4463 constant irrespective of the expression of Flag-tagged As-rel2 (Fig. 4C, lower panel, WB). These results indicate that As-rel2 has a suppressive effect on the activity of As-rel1, although it can enter the nucleus escorted by As-rel1. Isolation of an ascidian IjB homologue It is well known that the inhibitor IjB modulates the functions of Rel/NF-jB family members. We have been trying to isolate cDNA clones encoding ankyrin-repeat proteins from the ascidian H. roretzi [19], but our attempts to isolate a cDNA clone encoding an IjB homologue have not been successful. We therefore searched the EST database of the ascidian C. intestinalis (Ciona cDNA resources of Kyoto University) for an IjB homologue. We found in this database an EST clone, cieg29i06, which is predicted to include the full length of IjB-like cDNA, and the nucleotide sequence was determined. The cDNA clone, designated Ci-IjB, consists of 1041 nucleotides with a poly(A)-rich tail (Fig. 5A), and its single ORF encodes 347 amino acids containing six ankyrin motifs (Fig. 5B) and two consensus phosphorylation sequences (DSGXXS) (Fig. 5A). A homology search revealed that Ci-IjBhas the highest homology (56%), with human IjBa among the various IjB members hitherto reported. Interaction of Ci-IjB with As-rel1 and As-rel2 Next, we carried out an experiment to determine whether Ci-IjB is capable of forming a complex with As-rel1 or As-rel2. HEK293T cells transiently expressing Flag-tagged Ci-IjB with or without c-6Myc-tagged As-rel1 and c-6Myc- tagged As-rel2 were subjected to the first immunoprecipi- tation using anti-(Flag-tag) Ig. The eluate obtained by elution with 3 · Flag-peptide was subsequently subjected to Western blotting with anti-(c-Myc-tag) Ig. Ci-IjBwas found to be able to interact with As-rel1 or As-rel2 (Fig. 6A, middle panel, 1st IP). Next, to determine whether the Ci-IjB-containing immunoprecipitate also contains both As-rel1 and As-rel2, the above eluate was subjected to the second immunoprecipitation using antibody against As-rel1, and the eluate obtained by elution with 0.1 M glycine (pH 3.0) was then subjected to Western blotting with anti- (c-Myc-tag) Ig. Ci-IjB was found to be able to interact with both As-rel1 and As-rel2 to form a complex (Fig. 6A, lower panel, 2nd IP). Thus, Ci-IjB forms a complex with either As-rel1 or As-rel2 and also with both together. In addition, to confirm the interaction between As-rel2 and Ci-IjB, we transiently coexpressed Flag-tagged As-rel2 and c-6Myc- tagged Ci-IjB in HEK293T cells, and the extracts of transfected cells were subjected to immunoprecipitation using anti-(Flag-tag) Ig. The eluate obtained by elution with 3 · Flag-peptide was subsequently subjected to Western blotting with anti-(c-Myc-tag) Ig (Fig. 6B). It was confirmed that Ci-IjB is capable of interacting with As-rel2. As-rel2-dependent effect of Ci-IjB on As-rel1 localization AsCi-IjB is capable of interacting with As-rel1 and As-rel2, we next carried out an experiment to determine whether Ci-IjB affects the localization of As-rel1 in the presence or absence of As-rel2. We first transiently expressed GFP–As- rel1 fusion protein together with c-6Myc-tagged Ci-IjBin HEK293 cells. As expected from the cases of other Rel/NF- jB family members, As-rel1 was found to be present in the cytoplasm (Fig. 7a,b) because Ci-IjB binds to As-rel1 to Fig. 4. Effect of As-rel2 on transcriptional activity of As-rel1. Tran- scriptional activities of As-rel1 and As-rel2, transiently expressed in HEK293T cells, were measured by luciferase assay using GAL4–As-rel fusion protein and the GAL4-binding DNA sequence. The cells were transfected with expression vectors containing increasing amounts of pBIND-As-rel1 (0.18, 0.36, 0.54 and 0.72 lg of DNA) (A), increasing amounts of pBIND-As-rel2 (0.18, 0.36, 0.54 and 0.72 lg) (B), or pBIND-As-rel1 (0.18 lg) and increasing amounts of pCI-neo-Flag- As-rel2 (0.18, 0.36, and 0.54 lg) (C), together with the pG5/luc reporter vector. Total amounts of transfected DNA were kept constant (1.5 lg) by adding an empty vector (pBIND or pCI-neo-Flag vector). The level of activity was normalized on the basis of the level of activity of control Renilla luciferase. Results are expressed as n-fold induction in luciferase activity relative to control cells that had been transfected with an empty vector, pBIND. Triplicate experiments were carried out, and the error bars represent SD. To assess the expression levels of GAL4–As-rel1 and Flag–As-rel2, parts of transfected cells were sub- jected to SDS/PAGE followed by Western blotting (WB) with anti- (As-rel1) and anti-(Flag-tag) Igs (C, lower panel, WB). 4464 N. Kawai et al.(Eur. J. Biochem. 270) Ó FEBS 2003 sequester it in the cytoplasm. This result is in contrast with the result obtained from a single transfection with GFP– As-rel1 fusion protein (Fig. 3A). On the other hand, when GFP–As-rel1 and c-6Myc-tagged Ci-IjB were transiently expressed together with Flag-tagged As-rel2 in HEK293 cells, As-rel1 was found to be present in the nucleus, whereas As-rel2 was located in the cytoplasm (Fig. 7c,d,e). This situation may arise because As-rel2 sequesters Ci-IjBinthe cytoplasm to allow As-rel1 to move into the nucleus. Thus, As-rel2 regulates the localization of As-rel1 in the presence of Ci-IjB. As-rel2-dependent effect of Ci-IjB on transcriptional activity of As-rel1 AsCi-IjB can regulate As-rel1 localization, we next investigated the effect of Ci-IjB on the transcriptional activity of As-rel1. We employed the luciferase assay described above. In HEK293T cells transiently expressing GAL4–As-rel1 together with Flag-tagged Ci-IjB, luciferase activity of As-rel1 was inhibited, depending on the dose of Ci-IjB (Fig. 8A, upper panel, luciferase assay), as expected from the results on localization shown in Fig. 7. On the other hand, in the cells transiently expressing GAL4–As-rel1 and Flag-tagged Ci-IjB together with Flag-tagged As-rel2, the activity of As-rel1 that had been inhibited by Ci-IjBwas rescued by As-rel2, although to a moderate level (Fig. 8B, upper panel, luciferase assay). This finding is consistent with the results in Fig. 7 showing that As-rel1 can move into the nucleus even in the presence of Ci-IjB when As-rel2 is expressed with it. It should be noted that the expression levels of GAL4–As-rel1 remained almost constant irrespec- tive of the expression of Flag-tagged Ci-IjB and Flag- tagged As-rel2 (Fig. 8A,B, lower panels, WB). Thus, As-rel2 regulates the transcriptional activity of As-rel1 in the presence of Ci-IjB. Discussion In this study, we first determined that As-rel1 and As-rel2 are splice variants. Next, we demonstrated that As-rel2, a short splice variant, modulates the localization and tran- scriptional activity of As-rel1, a typical Rel/NF-jB family member. In the absence of Ci-IjB, As-rel2 as a heterodimer with As-rel1 enters the nucleus and suppresses the activity of As-rel1, whereas in the presence of Ci-IjB, it binds Ci-IjB and the sequestration of Ci-IjB in the cytoplasm by As-rel2 allows As-rel1 to enter the nucleus, leading to the promotion of transcription. This is a novel regulatory mechanism for the function of a Rel/NF-jB family member mediated by a short splice variant. As-rel2 is a novel short splice variant of Rel/NF-jB family proteins which lacks both the nuclear localization signal and the C-terminal region, a putative transactivation domain (Fig. 1A). Dorsal B is an alternative splice variant of Dorsal and it lacks the nuclear localization signal [20]. Dorsal B mRNA is generated because the intron for Dorsal is not excised in a manner similar to that in the case of As-rel2 mRNA generation, but, in contrast with the case of As-rel2, it functions as an activator for transcription when it can enter the nucleus because it has a C-terminal trans- activation domain [20]. As expected from the structures of As-rel1 and As- rel2, we demonstrated that As-rel1 binds to itself and to As-rel2 to form a homodimer and a heterodimer, respectively, and that As-rel1 and As-rel2 are localized in the nucleus and cytoplasm, respectively, when they are expressed individually (Figs 2 and 3). With regard to the nuclear localization of As-rel1, it should be noted that there is little interaction of mammalian IjB proteins with ascidian As-rel1 (data not shown), and this enabled us to use cultured mammalian cells for analysis of the inter- action between As-rel1 and As-rel2 even in the presence Fig. 5. Sequence and domain structure of Ci-IjB. (A) Nucleotide and deduced amino- acid sequences of Ci-IjB. The polyadenylation signal sequence is underlined by a solid line. The consensus phosphorylation sequences are underlined by dotted lines. (B) Domain structure of Ci-IjB. Note that Ci-IjBcontains six ankyrin motifs. Ó FEBS 2003 Regulation of Rel by alternative splice variant (Eur. J. Biochem. 270) 4465 of mammalian endogenous inhibitor IjB proteins. An unexpected interesting finding in this study is that As-rel2 can enter the nucleus, escorted by As-rel1, and appar- ently suppresses the transcriptional activity of As-rel1 to a moderate level (Figs 3B and 4C). This apparent suppression can be explained if it is assumed that the heterodimer formed by As-rel1 and As-rel2 has a lower level of activity than the As-rel1 homodimer. WetriedtoisolateacDNAcloneforanIjB homologue from the ascidian H. roretzi, but our attempts were not successful. Instead of H. roretzi IjB cDNA, a cDNA for an IjB called Ci-IjB was isolated from another ascidian, C. intestinalis.TheCi-IjBproteinwas demonstrated to interact with As-rel1 and As-rel2 and to suppress the nuclear transport and transcriptional activity of As-rel1 (Figs 6, 7a and 8A). Interestingly, we found that the inhibitory effect of Ci-IjB on As-rel1 is modulated by As-rel2, a short splice variant. When three proteins, a typical Rel/NF-jB family member, As-rel1, a short variant, As-rel2, and its inhibitor, Ci-IjB, were coexpressed, the short variant in the cytoplasm binds with the inhibitor, enabling As-rel1 to move into the nucleus, leading to the promotion of transcription (Figs 7 and 8B). This finding provides evidence of a short splice variant-mediated regulatory mechanism for the function of a Rel/NF-jB family member. The activity of As-rel1 was rescued from Ci-IjB inhibition by As-rel2 to a moderate level, comparable with that of the inhibited activity of As-rel1, when As-rel1 and As-rel2 were coexpressed in the absence of Ci-IjB (Figs 4C and 8B). This apparent coincidence in the levels of activity suggests that the As-rel1–As-rel2 heterodimer, but not the As-rel1 homodimer, enters the nucleus in the former case. This explanation, however, is inconsistent with the results on the cytoplasmic localization of As-rel2 in the presence of As-rel1 and Ci-IjB (Fig. 7). This discrepancy cannot be completely explained. Quantitative measure- ments of interactions between As-rel1, As-rel2, and Ci- IjB will define it. In our previous study, Northern blot analysis revealed that As-rel1 and As-rel2 mRNAs are expressed during development in H. roretzi [15]. We also showed that injection of As-rel1 mRNA interfered with H. roretzi notochord formation, resulting in a shortened tail with a reduced number of notochord cells and that H. roretzi embryos coinjected with As-rel1 and As-rel2 mRNAs developed normally [15]. The results for the single overexpression of As-rel1 can be explained by its Fig. 6. Interaction of Ci-IjB with As-rel1 and As-rel2. (A) HEK293T cells were transiently transfected with the indicated combinations of c-6Myc-As-rel1, c-6Myc-As-rel2, and Flag-Ci-IjB expression plas- mids, and 48 h after transfection the cell lysates were subjected to the first immunoprecipitation (1st IP) using anti-(Flag-tag) Ig. The immunoprecipitates produced were eluted with 3 · Flag-peptide. The eluate thus obtained was subjected to the second immunoprecipitation (2nd IP) using anti-(As-rel1) Ig. The immunoprecipitates produced were eluted with 0.1 M glycine (pH 3.0). The eluates obtained from the first and second immunoprecipitations were subjected to Western blotting with anti-(c-Myc-tag) and anti-(Flag-tag) Igs. The bands of As-rel1 and As-rel2 are indicated by a closed arrowhead and an open arrowhead, respectively. (B) HEK293T cells were transiently trans- fected with the indicated combinations of Flag-As-rel2 and c-6Myc-Ci- IjB expression plasmids, and 48 h after transfection the cell lysates were subjected to immunoprecipitation (IP) using anti-(Flag-tag) Ig. The immunoprecipitates produced were eluted with 3 · Flag-peptide and the eluate thus obtained was subjected to Western blotting with anti-(c-Myc-tag) and anti-(Flag-tag) Igs. Fig. 7. Localization of As-rel1 and As-rel2 in the presence of Ci-IjB. HEK293 cells were transiently transfected with GFP-As-rel1 and c-6Myc-Ci-IjB expression plasmids with (c, d, e) or without Flag- As-rel2 expression plasmid (a, b). Fluorescence due to GFP-As-rel1 (a, c), DAPI staining of DNA (b, e), and the antibody staining of Flag-As-rel2 (d) were visualized as described in Fig. 3. The nuclei are indicated by arrowheads. 4466 N. Kawai et al.(Eur. J. Biochem. 270) Ó FEBS 2003 promotion of target genes that are not expressed normally, leading to a defect in development, and the antagonistic effect of As-rel2 on As-rel1 can be explained by the results of the present study showing that As-rel2 has a suppressive effect on the transcriptional activity of As-rel1. An H. roretzi IjB homologue has not been isolated, but Ci-IjB is expressed in embryos of another ascidian, C. intestinalis (data not shown). If an IjB homologue is expressed in H. roretzi embryos, As-rel2, in the presence of the IjB protein, will modulate the function of As-rel1 in the same manner as that found in this study. The isolation of an H. roretzi IjB homologue will lead to clarification of the relationship between As- rel1 and As-rel2 and their functions in the development of H. roretzi. In conclusion, we propose the following novel regula- tory mechanism of As-rel1 mediated by As-rel2: (a) As- rel2, in complex with As-rel1, enters the nucleus and suppresses the transcriptional activity of As-rel1; (b) As- rel2 binds to IjB in the cytoplasm, resulting in an increase in the nuclear translocation and transcriptional activity of As-rel1. Acknowledgements We thank Dr Hiroki Takahashi of the National Institute for Basic Biology for helpful discussion. This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, Culture, and Technology of Japan. References 1. Verma, I.M., Stevenson, J.K., Schwarz, E.M., Van Antwerp, D. & Miyamoto, S. (1995) Rel/NF-jB/IjBfamily: intimate tales of association and dissociation. Genes Dev. 9, 2723–2735. 2. 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Effect of Ci-IjB on transcriptional activity of As-rel1. Tran- scriptional activity of As-rel1 in the presence of Ci-IjB and/or As-rel2 was measured by luciferase assay as described in Fig. 4. HEK293T cells were transfected with expression vectors containing pBIND- As-rel1 (0.05 lg) and increasing amounts of pCI-neo-Flag-Ci-IjB (0.05, 0.1, and 0.15 lg) (A) or pBIND-As-rel1 (0.05 lg), Flag-As-rel2 (0.05 lg) and increasing amounts of pCI-neo-Flag-Ci-IjB (0.05, 0.1, and 0.15 lg) (B), together with the pG5/luc reporter vector. Total amounts of transfected DNA were kept constant (0.5 lg) by adding an empty vector (pCI-neo-Flag vector). The level of activity was nor- malized, and results are expressed as fold induction in luciferase activity as in Fig. 4. Triplicate experiments were carried out, and the error bars represent SD. 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Kondoh, M., Kasai, T., Shimada, M., Kashiwayanagi, M. & Yokosawa, H. (2003) cDNA cloning and characterization of an osmotically sensitive TRP channel from ascidian eggs. Comp. Biochem. Physiol. B132, 417–423. 20.Gross,I.,Georgel,P.,Oertel-Buchheit,P.,Schnarr,M.& Reichhart, J.M. (1999) Dorsal-B, a splice variant of the Drosophila factor Dorsal, is a novel Rel/NF-jB transcriptional activator. Gene 228, 233–242. 4468 N. Kawai et al.(Eur. J. Biochem. 270) Ó FEBS 2003 . FEBS 2003 Regulation of Rel by alternative splice variant (Eur. J. Biochem. 270) 4467 15. Shimada, M., Satoh, N. & Yokosawa, H. (2001) Involvement of Rel/ NF-jB in regulation of ascidian notochord. GFP–As -rel1 merges with Flag–As -rel2 (c). The nuclei are indicated by arrowheads. Ó FEBS 2003 Regulation of Rel by alternative splice variant (Eur. J. Biochem. 270) 4463 constant irrespective of. Ig. Results As -rel2 is a splice variant of As -rel1 The nucleotide sequence encoding RHD of As -rel1 is completely identical with that of As -rel2 [15], indicating the possibility that As -rel1 and As -rel2