Báo cáo khoa học: Regulation of ascidian Rel by its alternative splice variant pdf

From these findings, we conclude that the alternative splice variant, As-rel2, regulates the nuclear localization and transcriptional activity of As-rel1.. As-rel2, transfected alone, was

Regulation of ascidian Rel by its alternative splice variant

Narudo Kawai1, Masumi Shimada1, Hiroyuki Kawahara1, Noriyuki Satoh2and Hideyoshi Yokosawa1

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 IjB were 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 DNAbinding 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] Asuggested 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 IjB masks the

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-mediaconver-ted 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 transcripaddi-tional 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] AC-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, Department of Biochemistry,

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, IjB kinase;

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)

in tail elongation, indicating that this Rel/NF-jB protein

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-As-rel1-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 IjB remained

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

A480-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 mM Tris/HCl, pH 8.0, containing 1 mM EDTAand

100 mMNaCl), 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 A5 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 55C 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 DNAas a precipitate

Genomic PCR was performed with several combinations

of the following primers for the As-rel sequence [genome

(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 Taqpolymerase (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

37C under a 5% CO2 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 cDNAresources 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-pCI-neo-c-Myc-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,

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 MluI and NotI sites by PCR using the

following primers (As-rel MulI forward primer, 5¢-ACGC

GTATGGACAAAATGTCTA-3¢; As-rel1 NotI reverse

primer, 5¢-AGCGGCCGCTCAGTTGTAATTC-3¢; A

s-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

CAAAATGT-3¢; As-rel1 BamHI reverse primer, 5¢-GGAT

CCGTTGTAATTCTGAT-3¢) The PCR product

sub-cloned in the pGEM-T-vector was digested with XhoI and

BamHI and then inserted into the XhoI and BamHI sites of

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¢; A s-rel2 NotI reverse primer,

5¢-AGCGGCCGCTCATCTATCCAGCA-3¢) The PCR

products subcloned in the pGEM-T-vector were digested

with MluI and NotI and then inserted into the MluI and

NotI sites of pBIND (Promega) pCI-neo-Flag-Ci-IjB and

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 SalI and NotI sites of

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 DNAwas 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 mM

Tris/HCl, pH 8.0, containing 150 mMNaCl, 0.1% Nonidet

P40, and 10% glycerol) containing 10 lM 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 lL

Pro-tein G-immobilized agarose (Santa Cruz) at 4C for

30 min and was then incubated with 2 lg anti-(Flag-tag)

M2 Ig and 10 lL Protein G-immobilized agarose at 4C

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-anti-(Flag-tag) 9E10 Ig Alternatively, HEK293 cells were transfected with several combinations

of 0.2 lg pCI-neo-As-rel1 and 1.8 lg pCI-neo-T7-A s-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-IjB using Metafectene transfection reagent in 60-mm dishes (The total amount of plasmid DNAwas 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 4C for 1 h The beads were washed five times with lysis buffer and eluted with 0.1M glycine (pH 3.0) The eluates obtained from the first and second immunoprecipitations were subjected to SDS/PAGE and then to Western blotting with (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 lg each of 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 neo-Flag-As-rel2 together with 1.2 lg pCI-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 Fluor 594

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 lg each

pEGFP-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 rel1 and

As-rel2, we used GAL4 fusion proteins, GAL4–As-rel1 and

GAL4–As-rel2, and a plasmid containing GAL4-binding

DNAsequences 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 lg with an empty vector, pBIND or

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 DNAwas 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-Luciferase 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 (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 cDNAsequences 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 mRNAis generated from pre-mRNAby splicing at the splice sites shown in Fig 1B, while a short mRNAof 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 DNAbinding, 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-As-rel1-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.

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

(Flag-tag) Ig and then to Western blotting with (Flag-(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.

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 cDNAclones encoding ankyrin-repeat proteins from the ascidian H roretzi [19], but our attempts

to isolate a cDNAclone 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 cDNAclone, 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-IjB has 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 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-IjB was 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 0.1M 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

A s Ci-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-IjB in 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 DNAwere 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).

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-IjB in the

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

A s Ci-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-IjB was

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 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 mRNAis 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 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-IjB contains

six ankyrin motifs.

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

We tried to isolate a cDNA clone for an IjB

homologue from the ascidian H roretzi, but our attempts

were not successful Instead of H roretzi IjB cDNA, a

cDNAfor an IjB called Ci-IjB was isolated from

another ascidian, C intestinalis The Ci-IjB protein was

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.

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 rel1 mediated by rel2: (a) As-rel2, in complex with As-rel1, enters the nucleus and suppresses the transcriptional activity of 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.

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Fig 8 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 DNAwere 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 To assess the expression levels of GAL4–

As-rel1, Flag–Ci-IjB, and Flag–As-rel2, parts of transfected cells were

subjected to SDS/PAGE followed by Western blotting (WB) with

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