LIRF, a Gene Induced during Hippocampal Long-Term Potentiation as an Immediate-Early Gene, Encodes a Novel RING Finger Protein

Biochemical and Biophysical Research Communications 289, 479 – 484 (2001) doi:10.1006/bbrc.2001.5975, available online at http://www.idealibrary.com on

LIRF, a Gene Induced during Hippocampal Long-Term Potentiation as an Immediate-Early Gene, Encodes a Novel RING Finger Protein Ryota Matsuo,* ,1 Akiko Asada,* ,2 Kazuko Fujitani,* and Kaoru Inokuchi* ,3 *Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan

Received October 15, 2001

We describe here an LTP-induced gene, LIRF, which encodes a novel protein with RING finger and B30.2 domains in its N- and C-terminal portions, respectively. Each domain is encoded by one exon, suggesting that the organization of the gene was generated by exon shuffling. The amino acid sequences of the mouse, rat, and human LIRF proteins are highly conserved and contain a putative PEST sequence. LIRF is an immediate-early gene in hippocampal granule cells, and its expression is upregulated immediately after the induction of long-lasting long-term potentiation at perforant pathway– dentate gyrus synapses and returns to the basal level within 150 min. A heterologously expressed LIRF protein fused to EGFP localizes specifically to the cytoplasm in COS-7 cells. These findings suggest a possible involvement of LIRF in a limited, early phase of synaptic plasticity. © 2001 Elsevier Science

Key Words: RING finger protein; B30.2 domain; longterm potentiation; immediate-early gene; hippocampus; gene regulation.

De novo gene expression is required for the persistence of long-term potentiation (LTP), a typical form of synaptic plasticity believed to be the cellular basis of learning and memory (1). Behavioral memory also reThe rat LIRF nucleotide sequence is available from the DDBJ/ EMBL/GenBank databases (Accession No. AB070897). Abbreviations used: EGFP, enhanced green fluorescence protein; FITC, fluorescein isothiocyanate; LTP, long-term potentiation; MBP, maltose-binding protein; NMDA, N-methyl-D-aspartate; ORF, open reading frame; RT-PCR, reverse transcription–PCR. 1 Present address: Laboratory of Neurobiophysics, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 2 Present address: Department of Biological Science, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minamioosawa, Hachioji, Tokyo 192-0397, Japan. 3 To whom correspondence and reprint requests should be addressed. Fax: ⫹81-42-724-6314. E-mail: [email protected]. co.jp.

quires gene expression for its maintenance (2). Much effort has been devoted to the search for genes involved in long-term synaptic plasticity, using pharmacologically-induced seizure, or electrically-evoked LTP as models (3–12). Previously, we identified a novel gene RM3 (hereafter denoted as LIRF, LTP-induced RING finger protein), which is upregulated during longlasting LTP in the hippocampus of unanesthetized rats (9). The induction of LIRF is regulated by NMDAreceptor signaling, and correlates with the maintenance of LTP. During brain development, LIRF is expressed late after birth (around 15 days postnatally) in the cortex, cerebellum and hippocampus. In the adult rat, LIRF is expressed in the lung, liver, stomach, and small intestine as well as in the brain (9). Here we more precisely examine the LIRF mRNA expression profiles and further characterize its protein product, LIRF, which has a unique structure containing a RING finger at the N-terminus and a B30.2 domain at the C-terminus. The genomic organization of the LIRF gene is suggestive of evolutionary exon shuffling, in that each domain corresponds to one exon. By means of in situ hybridization, we examine LIRF mRNA expression profiles in the hippocampus after induction of in vivo LTP. We also examine the intracellular localization of an LIRF–EGFP fusion in cultured cells. MATERIALS AND METHODS Dentate gyrus LTP. All procedures involving animals complied with the guidelines of the National Institute of Health and were approved by the Animal Care and Use Committee of the Mitsubishi Kagaku Institute of Life Sciences. LTP was induced in anaesthetized adult male rats (400 –500 g) as described previously (9). Cycloheximide was injected intraperitoneally (40 mg/kg body wt) 40 min prior to the beginning of the high frequency stimulation. Reverse transcription–PCR. The sequences of PCR primers used for cloning the full length LIRF cDNA are as follows: 5⬘-GAGTCCGATCTGGGTAACCGAGGC-3⬘, 5⬘-GGCCCATTTATACACAAGGGAAGG-3⬘. PCR was performed in a thermal cycler (Perkin– Elmer) as follows: denaturation at 96°C for 2 min, then 27 cycles of

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96°C for 20 s, 65°C for 30 s, and 72°C for 2 min, followed by 72°C for 2 min. PCR products were ligated into the cloning vector pCRIITOPO (Invitrogen) according to the manufacturer’s instructions. Vector constructions. A plasmid containing sequences encoding the full-length LIRF fused to the C-terminus of the Maltose-binding Protein (MBP–LIRF) was constructed by introducing the PCR fragment of the LIRF cDNA into the EcoRI and SalI sites of pMAL-c2 (New England Bio Labs). Vectors for the expression of the full-length LIRF with (LIRF–EGFP) or without (full-LIRF) N-terminal fusion of EGFP were constructed by introducing a PCR fragment amplified from LIRF cDNA into the XhoI and EcoRI sites of pEGFP-N3 (Clontech) and into the EcoRI and XhoI sites of pcDNA3.1 (Invitrogen), respectively. In situ hybridization. A part of the LIRF cDNA (nucleotides 801 to 1631, where the adenine residue of the initiation codon was designated as 1) was amplified by PCR and subcloned into the vector pCRII-TOPO to generate pCRII-situ. pCRII-situ was digested with BamHI or EcoRV to generate templates for in vitro transcription of antisense or sense cRNA probes, respectively. Digoxigenin-labeled antisense and sense cRNA probes were produced by transcription with T7 or Sp6 RNA polymerases, respectively. cRNA was then fragmented into molecules of about 200 bases in 44 mM NaHCO 3, 66 mM Na 2CO 3 at 60°C for 30 min. Animals were sacrificed at various time points after the induction of LTP and the brain was dissected and immediately frozen on dry ice. Cryostat sections (10-␮m thickness) were cut and mounted onto polylysine-coated glass slides. Sections were air-dried and stored at ⫺80°C until use for hybridization. After fixation of sections in 10% formaldehyde neutral buffer (pH 7.0), permeabilization with 0.3% Triton X-100 in phosphate-buffered saline (PBS) was performed followed by proteinase K treatment and by postfixation with 10% formaldehyde neutral buffer (pH 7.0). Hybridization was performed at 55°C overnight in hybridization buffer [50% formamide, 5⫻ SSC (0.75 M NaCl, 0.075 M sodium citrate, pH 7.0), 100 ␮g/ml of yeast tRNA (Boehringer Mannheim), 0.1% Tween 20, and 50 ␮g/ml heparin]. Sections were washed for 30 min at 55°C with 50% formamide in 5⫻ SSC, and then for 30 min at 55°C with 50% formamide in 2⫻ SSC. Sections were further washed twice with solution B (10 mM Tris–Cl, pH 8.0, 500 mM NaCl) at room temperature for 5 min. After treatment with RNase A (20 ␮g/ml in solution B) at 37°C for 30 min, sections were washed with 50% formamide in 1⫻ SSC for 30 min at 55°C. After equilibration with maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, pH 7.5), sections were blocked in 10% blocking reagent (Boehringer Mannheim) in maleic acid buffer, followed by reaction overnight with 1:1000 diluted alkaline phosphatase-conjugated antiDig antibody (Boehringer Mannheim) at 4°C. Sections were washed with maleic acid buffer and then equilibrated with 100 mM Tris–HCl (pH 9.5), 100 mM NaCl, 50 mM MgCl 2 (reaction buffer). Immunological color detection was done in the presence of 5-bromo-4-indolyl phosphate p-toluidine salt and 4-nitro blue tetrazolium chloride in reaction buffer. Generation of polyclonal anti-LIRF antibody. Synthetic peptide CRGLREKLAEPGARTGRRRG corresponding to the LIRF amino acid residues 89 to 107 was conjugated with maleimide-activated keyhole-limpet hemocyanin (Pierce), dialyzed according to the manufacturer’s instructions, and mixed with adjuvant (Difco). Rabbits were immunized six times with the conjugated peptide (overall approximately 3 mg). The anti-LIRF antibody was purified as follows: the antiserum was mixed with beads that were covalently attached to bacterially-expressed MBP–LIRF, then eluted with 0.1 M glycine (pH 2.5), and followed by neutralization with the same volume of 1 M Tris–HCl (pH 8.0). Cell culture and transfection. COS-7 cells were grown on dishes of 60-mm in diameter in Dulbecco’s modified Eagle medium (GibcoBRL) supplied with 10% fetal bovine calf serum (JRH Biosciences) at 37°C in 5% CO 2–95% air. LIRF–EGFP or full-LIRF

expression plasmid was transfected using Lipofectamine 2000 (GibcoBRL) according to the instruction manual. Western blotting. Cultured COS-7 cells were lysed in TNE buffer [10 mM Tris–HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1 mM Pefabloc (PENTAPHARM), 1 mM Na 3VO 4, 10 mM NaF] at 4°C and boiled in the presence of the same volume of loading buffer [50 mM Tris–HCl (pH 6.8), 4% SDS, 10% glycerol, 10% ␤-mercaptoethanol, 0.1% bromophenol blue]. The boiled lysates were electrophoresed on SDS–polyacrylamide (12%) gels and transferred electrically to nitrocellulose membranes (Pall Corp.) with a solution of 25 mM Tris, 192 mM glycine, and 20% methanol. After transfer, the membranes were incubated in T-TBS [Tris-buffered saline: 20 mM Tris–HCl (pH 7.5), 137 mM NaCl, 0.2% Tween 20] containing 5% skim milk for 1 h, and then incubated for 1 h at room temperature in the presence of anti-LIRF antibody or rat monoclonal anti-GFP antibody (provided by Dr. Fujita CS, Mitsubishi Kagaku Inst. Life Sci.). Blots were then washed three times with T-TBS and incubated for 40 min at room temperature in T-TBS containing a 1:3000 dilution of anti-rabbit (for the anti-LIRF antibody, ICN Pharmaceuticals) or anti-rat (for the anti-GFP antibody, ICN Pharmaceuticals) monoclonal antibody conjugated with horseradish peroxidase. Blots were then washed three times with T-TBS and the signals were detected with an enhanced chemiluminescence detection system (Amersham– Pharmacia Biotech) and a LAS1000 luminescence image analyzer (Fujifilm). Immunocytochemistry. Cultured COS-7 cells harboring fullLIRF-expression vector were fixed in 10% formaldehyde neutral buffer (pH 7.0) for 30 min at 4°C. After washed twice with PBS (15 min each), the dish was treated with T-PBS (PBS supplied with 0.1% of Tween 20) for 10 min at room temperature followed by twice washes with PBS for 15 min each. The dish was then blocked with blocking buffer (2.5% BSA, 2.5% goat serum in T-PBS) for 30 min, and incubated with an anti-LIRF antibody in blocking buffer overnight at 4°C. After washed with PBS three times (5 min each), the dish was incubated with 1:100 diluted FITC-conjugated anti-rabbit IgG (Chemicon) in blocking buffer supplied with 0.1 mg/ml of Hoechst No. 33258 (Sigma) for 1 h at room temperature. The dish was washed twice with PBS (15 min each), and the fluorescent signals were examined under a fluorescence microscopy (Olympus).

RESULTS AND DISCUSSION Using a DNA fragment isolated by reverse transcription-PCR (RT-PCR) differential display search for LTP-regulated genes (RM3, 152 bp) (9), we screened a cDNA library constructed with poly(A) RNA isolated from the hippocampus of pentylenetetrazole-treated rats (5). The longest insert (1283 bp) identified among several positive clones was used to search the DDBJ/ EMBL/GenBank databases. The search revealed that human and mouse genomic sequences homologous to the rat LIRF had been registered under Accession Nos. HSU53588 and AC005960, respectively. A putative full-length cDNA of rat LIRF was then isolated by RT-PCR, using rat hippocampal RNA as a template, with an antisense primer immediately upstream of the poly(A) signal of the rat LIRF and a sense primer corresponding to the putative 5⬘-end of the LIRF RNA as deduced from the mouse genomic sequence. Comparison of the cDNA thus isolated and the genomic sequence revealed that LIRF consists of four exons (Fig. 1A). A predicted open reading frame (ORF) spans 352 amino acids (Fig. 1B). The chromosomal

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FIG. 1. Structure of LIRF. (A) Schematic representation of LIRF genome, mRNA, and protein. Note that the scheme of the LIRF genome is based on the mouse genomic sequence. Numbers indicated on the LIRF genome show the positions of nucleotides in the mouse genomic sequence (GenBank Accession No. AC005960). (B) Alignment of the rat and mouse LIRF and human HZFw1 amino acid sequences. The mouse LIRF amino acid sequence is predicted from the genomic sequence. The putative translation initiation site is indicated by an arrow. Amino acids conserved among rat, mouse and human are depicted in bold letters. A predicted PEST sequence is underlined. RING finger and B30.2 domains are boxed. (C) Identity (%) shared by the RING finger domains and B30.2 domains in the mouse LIRF, RFP, and butylophilin.

location of LIRF is on human chromosome 6p21.3 between the telomere-proximal HLA-A locus and the centromere-proximal HLA-J locus. This region is known to be highly enriched in genes (13). We have noted that LIRF is a rat counterpart of the human HZFw1, which was recently identified by a systematic screening of nucleotide database and suggested to encode a RING finger protein (14) (GenBank Accession No. AF238315). The deduced translational start site of HZFw1 differs somewhat from that of LIRF in that HZFw1 has an extra N-terminal stretch of amino acids with respect to the LIRF protein sequence (see Fig. 1B). In-frame translations of the rat LIRF cDNA se-

quence and of the mouse LIRF genomic sequence reveal that the stop codons appear upstream of the initiation codon of LIRF proposed in this study. The methionine indicated by the arrow in Fig. 1B is therefore a more likely candidate as the actual site for translation initiation. The N-terminal part of LIRF shows homology to the RING finger domain of the RING finger-B-box-Coiledcoil (RBCC) protein RFP (15, 16), while the C-terminal part has homology to the B30.2 domain of butylophilin (17, 18) and RFP (Fig. 1C). Each domain is encoded by a single exon, which is suggestive of exon shuffling during the course of genome evolution (19). Human

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FIG. 2. In situ hybridization showing that the LIRF mRNA was induced as an immediate-early gene during dentate gyrus LTP. (A) Spatiotemporal expression profiles of LIRF and NGFI-A mRNA in the hippocampus. High frequency stimulation was delivered to the ipsilateral perforant pathway (ipsilateral). The contralateral hippocampus serves as a control. Brain was dissected at the time indicated after the beginning of high frequency stimulation. Note that even in the presence of cycloheximide LIRF mRNA was upregulated by high-frequency stimulation. Cycloheximide was preinjected intraperitoneally. CHX, cycloheximide. (B) Hybridization with the LIRF sense strand probe produced no detectable signals. (C) Magnified images of the LIRF signal in the granule cell layer of the dentate gyrus at 60 min.

chromosome 6p21.3 is also known to be enriched with genes encoding RING finger and B30.2 domains (19 –21). Next, we examined the spatial and temporal expression profiles of the LIRF mRNA during long-lasting LTP by in situ hybridization (Fig. 2). Similar to what has been observed for NGFI-A (also called zif268, Egr1, or krox24), which is known to be an immediate-early gene, the LIRF mRNA was up-regulated rapidly after LTP induction in granule cells of the dentate gyrus in the stimulated side of the hippocampus. The level of LIRF mRNA returned to the basal level within 150 min. When the protein synthesis inhibitor cycloheximide was preinjected, superinduction was observed at 150 min, a time by which the amount of mRNA completely returns to the basal level in the absence of cycloheximide. These data indicate that LIRF is an immediate-early gene whose transcription does not require de novo protein synthesis.

Finally, we examined the intracellular localization of the LIRF protein fused to the N-terminus of EGFP (LIRF–EGFP) and expressed heterologously in COS-7 cells (Fig. 3). Western blot analysis demonstrates that an intact LIRF–EGFP protein is present (ca. 70 kDa) (Fig. 3C). When EGFP alone was expressed, EGFPspecific fluorescence was observed both in the cytoplasm and the nucleus of COS-7 cells (Fig. 3A). In contrast, when LIRF–EGFP was expressed fluorescence was observed almost exclusively in the cytoplasm (Fig. 3B). A similar result was obtained with PC12 cells (data not shown). The cytoplasmic localization of LIRF was further confirmed by immunocytochemical analysis. Using a polyclonal antibody raised against purified LIRF, we observed immunoreactivity of the heterologously-expressed LIRF protein mainly in the cytoplasm of COS-7 cells (Fig. 3D). Expression of the fulllength LIRF protein was confirmed with this polyclonal antibody by Western blotting (Fig. 3E, left). Preincu-

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FIG. 3. Intracellular localization of heterologously-expressed LIRF–EGFP and LIRF in COS7 cells. Confocal images of (A) EGFP and (B) LIRF–EGFP. (C) Western blotting of cell lysates prepared from COS-7 cells transfected with EGFP or LIRF–EGFP probed with an anti-GFP antibody. (D, left) Fluorescence microscopic images of immunostaining by anti-LIRF antibody of COS7 cells transfected with the LIRF expression vector. (D, middle) Hoechst No. 33258 staining. (D, right) Superimposed image. (E) Western blotting with the anti-LIRF antibody of cell lysates prepared from LIRF-transfected COS-7 cells. Scale bar, 20 ␮m.

bation of the antibody with amylose resin coated with bacterially-expressed MBP–LIRF causes the signal to disappear (Fig. 3E, right). An apparent shift in size from the predicted molecular weight (38 kDa) is probably due to the high content of basic amino acids (13% arginine and lysine) and proline residues (10%) in LIRF. The cytoplasmic localization of LIRF in COS-7 cells indicates that its function might be distinct from that of the transcriptional regulator RFP (22), whose RING finger domain is highly homologous to that of LIRF (Fig. 1C), although this does not necessarily exclude the possibility that regulated nuclear translocation might occur in some circumstances. The polyclonal antibody generated in this study failed to recognize endogenous LIRF protein from the stimulated (LTP) or unstimulated hippocampus as indicated by Western blotting analysis (data not shown), probably due to the low expression level of LIRF and/or its rapid turnover. In fact, there is a candidate PEST sequence in the middle of the LIRF ORF (Fig. 1B). We have shown previously that the up-regulation of LIRF mRNA correlates with the persistence of hippocampal LTP in unanesthetized rats (9). In addition, the fact that the LIRF gene is upregulated rapidly and transiently during long-lasting LTP suggests that LIRF might function for a limited period in long-term persistence of hippocampal LTP.

ACKNOWLEDGMENTS We thank Akiko Murayama for assistance in cDNA library screening, Dr. Yugo Fukazawa for technical advice, and Dr. Shinobu C. Fujita for providing anti-GFP antibody. This work was partly supported by Special Coordination Funds for Promoting Science and Technology of the Science and Technology Agency of the Japanese Government to K.I.

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