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Characterization of the Mouse PA28 Activator Complex Gene Family: Complete Organizations of the Three Member Genes and a Physical Map of the 150-kb Region Containing the - and ß-Subunit Genes^{1 ,2}

Keiko Kohda^*, Teruo Ishibashi^*, Naoki Shimbara^,, Keiji Tanaka^,§, Yoichi Matsuda^¶ and Masanori Kasahara^3,*,

^* Department of Biochemistry, Hokkaido University School of Medicine, Sapporo 060, Japan; Biomedical R&D Department, Sumitomo Electric Industries, Yokohama 244, Japan; CREST (Core Research for Evolutional Science and Technology), Japan Science and Technology Corporation, Japan; ^§ Tokyo Metropolitan Institute of Medical Science, Tokyo 113, Japan; and ^¶ Laboratory of Animal Genetics, Nagoya University School of Agricultural Sciences, Nagoya 464-01, Japan

	Abstract

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The proteasome is a multisubunit protease responsible for the generation of peptides loaded onto MHC class I molecules. Recent evidence indicates that binding of an IFN--inducible PA28 activator complex to the 20S proteasome enhances the generation of class I binding peptides. The - and ß-subunits, which constitute the PA28 activator complex in the form of an (ß)₃ heterohexamer, show significant amino acid sequence similarity to a protein, designated Ki or the -subunit, that is capable of binding to the 20S proteasome. In this study, we describe the complete nucleotide sequences of the mouse genes, Psme1, Psme2, and Psme3, coding for the -, ß-, and -subunits, respectively. The overall exon-intron organizations of the three Psme genes are virtually identical, thus providing evidence that they are descended from a single ancestral gene. The promoter regions of the Psme1 and Psme2 genes contain sequence motifs that qualify as IFN-stimulated response elements, consistent with the observation that their expression is induced strongly by IFN-. The Psme1 and Psme2 genes are located 6 kb apart with their 3'-ends pointing toward each other on bands C2 to D1 of mouse chromosome 14, supporting the idea that they emerged by tandem duplication.

	Introduction

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MHC class I molecules bind peptides produced by proteolysis of cytosolic proteins and display them on the cell surface (1, 2). The peptides are translocated from the cytosol into the endoplasmic reticulum by the TAP, where they bind to nascent class I molecules. Class I molecules bearing foreign peptides are recognized by the Ag receptor of cytotoxic T cells, thus enabling the immune system to destroy abnormal cells that synthesize viral or other foreign proteins. Accumulated evidence indicates that the proteasome is responsible for the generation of cytosolic peptides presented by class I molecules (3, 4, 5).

The 20S proteasome, which constitutes the proteolytic core of the proteasomal complex, is a cylinder-shaped particle made up of four layers of rings, each composed of seven subunits (6). The outer two rings are made up of -type subunits, while the inner two rings are composed of ß-type subunits that carry catalytic sites. The 20S proteasome is an ancient enzyme found in organisms ranging from archaebacteria to humans (7). Thus, the vertebrate immune system appears to have recruited preexisting machinery for peptide generation and have modified it to accommodate its specific needs. One of such modifications seems to be the invention of three IFN--inducible ß-type subunits known as low molecular mass polypeptide 2 (LMP2)⁴, LMP7, and PSMB10 (originally described as MECL1) (8). On stimulation with IFN-, these subunits are incorporated into the 20S proteasome by displacing the homologous housekeeping ß-type subunits (9, 10, 11, 12, 13, 14, 15). Such proteasomes appear to produce class I binding peptides more efficiently (16, 17, 18, 19). Consistent with the idea that the IFN--inducible ß-type subunits are the specialized subunits dedicated to class I-mediated Ag presentation, phylogenetic analysis (20, 21) showed that LMP2 and LMP7 become detectable first in the cartilaginous fish, the most primitive class of vertebrates in which the MHC has been identified (22). Recently, the hypothesis was put forward that the three IFN--inducible ß-type subunits emerged simultaneously in a common ancestor of jawed vertebrates as a result of chromosomal duplication involving the MHC region (23, 24).

Other proteasome subunits that might have a role specialized for class I-mediated Ag presentation are those constituting the PA28 activator complex, also known as the 11S regulator of the 20S proteasome (4, 6). The PA28 activator complex (25, 26) is a ring-shaped hexameric structure of 200 kDa, made up of alternating 28-kDa - and 28-kDa ß-subunits with a stoichiometry of (ß)₃; this complex binds to the outer -rings of the 20S proteasome and stimulates its peptidase activity in an ATP-independent manner (27, 28, 29). Recent evidence (30, 31) indicates that the PA28 activator complex enhances the generation of class I binding peptides by altering the cleavage pattern of the proteasome. Also, enhanced expression of the PA28 -subunit in virus-infected fibroblasts results in more efficient presentation of viral peptides to cytotoxic T cells (32). Like other key molecules of the class I Ag presentation machinery, expression of the PA28 - and ß-subunits is induced strongly by IFN- (33, 34, 35, 36, 37).

Recently, we showed that the PA28 - and ß-subunits are structurally related to a Ki antigen (35), a protein originally identified with autoantibodies found in sera of patients with systemic lupus erythematosus (38). Interestingly, the Ki antigen forms a homohexamer and binds to the 20S proteasome (39), suggesting that it might also modulate the proteasome activity. On the basis of its structural similarity to the - and ß-subunits and its ability to bind to the 20S proteasome, Tanahashi et al. (39) proposed that the Ki antigen should be renamed the PA28 -subunit.

As an initial step toward understanding the biologic functions of the PA28 activator complex gene family, we present here detailed structural analysis of the mouse genes, Psme1, Psme2, and Psme3, coding for the -, ß-, and -subunits, respectively.⁵ We also show that the Psme1 and Psme2 genes are located 6 kb apart with their 3'-ends pointing toward each other on bands C2 to D1 of mouse chromosome 14.

	Materials and Methods

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Isolation of phage clones containing the mouse Psme1, Psme2, and Psme3 genes

A FIX II genomic library of 129/SvJ mice (catalog no. 946309; Stratagene, La Jolla, CA) was screened sequentially using the full-length mouse Psme1, Psme2, and Psme3 cDNA clones (40) as probes. Plaque hybridization was conducted according to the standard protocol (41) at 42°C for 24 h in a solution containing 50% formamide, 1 M NaCl, 10x Denhardt’s solution, 50 mM Tris-HCl (pH 7.5), 1% Na₄P₂O₇, 1% SDS, and 150 µg/ml sheared and denatured salmon sperm DNA. After hybridization, the filters were washed twice with 2x SSC (1 xSSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0), 0.1% SDS at 60°C and once with 0.2x SSC, 0.1% SDS at 60°C. Restriction mapping of the positive clones was conducted as previously described (42). The accuracy of the maps was verified by double digestion. The restriction fragments containing the genes were cloned into the pBluescript SKII⁺ vector (Stratagene) for further characterization.

Determination of transcription initiation sites

Transcription initiation sites of the mouse Psme genes were determined by the rapid amplification of cDNA ends (RACE) essentially as described by Frohman (43) and Hirzmann et al. (44). Briefly, total liver RNA (7 µg) isolated from a 129/SvJ mouse was reverse transcribed using a gene-specific primer as described previously (42). The gene-specific primers were 5'-GTGACATCCTTGATCTCAGG-3' for Psme1, 5'-CCTCGTTCTGAGAAGTACTT-3' for Psme2, and 5'-AGGTTCATGTCTGAGTGG-3' for Psme3. The cDNA thus obtained was tailed with dATP (for Psme1) or dGTP (for Psme2 and Psme3). We tailed the Psme1 cDNA with dATP because tailing with dGTP almost exclusively produced truncated 5'-RACE products. The tailed cDNA was amplified by PCR (30 cycles of 40 s at 94°C, 1 min at 52°C, and 1 min at 72°C) using a second, more proximal gene-specific primer (5'-AGAACGCATCCAACTCTGAG-3' for Psme1, 5'-CCGTGGCAAGAAAGTGCAG-3' for Psme2, and 5'-TGCCACCAAGTCTTCTGCCTCAC-3' for Psme3) and the (dT)₁₇- or (dC)₁₂-adapter primer. The sequences of the (dT)₁₇- and (dC)₁₂-adapter primers were 5'-GACTCGAGTCGACATCGAT₁₇-3' and 5'-TTCTAGAATTCGGATC₁₂-3', respectively. The second round of PCR (30 cycles of 40 s at 94°C, 1 min at 54°C, and 1 min at 72°C, and a final extension of 10 min at 72°C) was performed with 1/25th of the material from the first round of PCR using another more proximal gene-specific primer and the (dT)₁₇- or (dC)₁₂-adapter primer. The gene-specific primers used for the second round of PCR were 5'-CACAGGTCTTCACGGAACAC-3' for Psme1, 5'-GTCTGAAGACATCCACCTG-3' for Psme2, and 5'-ACTTGTGATCCGCTCTCTG-3' for Psme3. The amplified DNA fragments were cloned into the pBluescript SKII⁺ vector and transformed into bacteria. At least six clones were sequenced for each gene.

Cloning of the human gene (PSME1) coding for the PA28 -subunit

The genomic DNA segment encompassing putative exons 3 to 6 of the PSME1 gene was isolated by PCR using human genomic DNA as a template. The PCR reaction mixture (50 µl) contained 0.25 µg of human genomic DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl₂, 200 µM deoxynucleotide triphosphates, 2 µM sense primer (5'-CAAGAAGATTTCTGAGCTGGATG-3'), 2 µM antisense primer (5'-TGACATCCTTGATCTCAGG-3'), 1 µl of Perfect Match enhancer (Stratagene), and 2.5 units of Taq DNA polymerase. The sense and antisense primers were designed based on the previously published human PSME1 cDNA sequence (33, 34). The conditions of amplification were 35 cycles of 40 s at 94°C, 1 min at 54°C, and 1 min at 72°C. The DNA fragment of 600 bp thus obtained was cloned into the pBluescript SKII⁺ vector and sequenced. To eliminate potential PCR artifacts such as base misincorporations, PCR was conducted three times, and two clones were sequenced for each amplification. An identical sequence was obtained from all clones.

Isolation of bacterial artificial clones (BAC)

The mouse pBeloBAC11 library (45) constructed from the embryonic stem cell line, CJ7, derived from 129/Sv mice (Research Genetics, Huntsville, AL) was screened using a mouse Psme1 genomic DNA fragment (nucleotides 2725–4099 in Fig. 2) as a probe. Screening of high density BAC filters was conducted at Research Genetics. The positive clones were sent to us in the form of bacterial colonies. BAC DNA was isolated using the standard alkaline lysis method (41).

View larger version (74K): [in this window] [in a new window]   FIGURE 2. Nucleotide and deduced amino acid sequences of the mouse Psme1 gene. Exonic and intronic sequences are written in capital and lowercase letters, respectively. The 5'-end of the previously published cDNA clone (40) is shown as the upstream boundary of the 5'-untranslated region. It does not indicate major transcription initiation sites. The conserved dinucleotides at the 5'- and 3'-splice sites (GC/AG in intron 4 and GT/AG in all the other introns) are doubly underlined. Also doubly underlined is the putative ISRE. An in-frame stop codon (TAG) preceding the ATG translation initiation codon is indicated by asterisks. The duplicated sequence in intron 5 that could function as a miniexon (nucleotides 3407–3427) is indicated by brackets. The putative polyadenylation signal AATAAA (nucleotides 4633–4638) is doubly underlined. Potential transcription factor-binding sites are also indicated. Amino acids are shown with a standard single-letter code under the nucleotide sequence. The KEKE motif located at amino acid positions 70–97 is boxed.

Restriction mapping of BAC DNA was performed as described previously (46), with minor modifications. Briefly, BAC DNA was completely digested with NotI and then partially digested with MluI, NruI, or SalI. After PFG electrophoresis and transfer to a nitrocellulose membrane, the restriction fragments were hybridized with a fluorescein-3'-end-labeled T7 or SP6 oligonucleotide primer in a solution containing 6x SET (1x SET is 150 mM NaCl, 15 mM Tris-HCl (pH 8.3), 1 mM EDTA), 0.1% SDS, 5x Denhardt’s solution, and 100 µg/ml sheared and denatured salmon sperm DNA at 40°C for 12 h. After hybridization, the membrane was washed four times for 5 min each in 6x SSC, 0.1% SDS at room temperature. Hybridization signals were detected with the CDP-star nucleic acid chemiluminescence reagent (NEN Life Science Products, Boston, MA). PFG electrophoresis was performed using the biased sinusoidal field gel electrophoresis system (47) following the instructions of the manufacturer (ATTO, Tokyo, Japan). A low range PFG marker (New England Biolabs, Beverly, MA) was included as a molecular mass size marker. Transcriptional orientation of the Psme1 and Psme2 genes and their distance were determined by long range PCR (ELONGASE amplification system; Life Technologies, Gaithersburg, MD) using the BAC DNA as a template. The primers used to measure the intergenic distance were: 5'-CTGCAATGAGAAGATTGTGGT-3' (nucleotides 3479–3499 in Fig. 2) and 5'-CTGTAGCCAAGGCTTCCAAG-3' (nucleotides 3714–3733 in Fig. 4) for primer pair A; and 5'-ATCCTGAAGAACTTTGAGAAGC-3' (nucleotides 4446–4467 in Fig. 2) and 5'-CTGTAGCCAAGGCTTCCAAG-3' (nucleotides 3714–3733 in Fig. 4) for primer pair B.

View larger version (78K): [in this window] [in a new window]   FIGURE 4. Nucleotide and deduced amino acid sequences of the mouse Psme2 gene. Exonic and intronic sequences are written in capital and lowercase letters, respectively. The 5'-end of the previously published cDNA clone (40) is shown as the upstream boundary of the 5'-untranslated region. It does not indicate major transcription initiation sites. The conserved dinucleotides at the 5'- and 3'-splice sites (GT/AG) are doubly underlined. Also doubly underlined are the putative ISRE and the potential NF-B-binding site. An in-frame stop codon (TAG) preceding the ATG translation initiation codon is indicated by asterisks. The putative polyadenylation signal AATAAA (nucleotides 4319–4324) is doubly underlined. Potential transcription factor-binding sites are also indicated. Amino acids are shown with a standard single-letter code under the nucleotide sequence.

The nucleotide sequence was determined by the chain termination method (48) using an automated DNA sequencer (model 4000L, LI-COR, Lincoln, NE) and the SequiTherm Long-Read cycle sequencing kit (Epicentre Technologies, Madison, WI). Both strands of the DNA were sequenced at least once.

Fluorescence in situ hybridization (FISH)

Chromosomal localization of the mouse BAC clone containing the Psme1 and Psme2 genes was determined using the direct R-banding FISH method as described previously (49, 50). Briefly, the BAC DNA of 150 kb was labeled by nick translation with biotin-16-dUTP (Boehringer Mannheim, Mannheim, Germany) following the manufacturer’s protocol. Following denaturation and preannealing with the mouse Cot-1 DNA (Life Technologies), the biotinylated probes were hybridized overnight at 37°C to the R-banded mouse chromosomes. Hybridization signals were detected with fluoresceinated avidin (Vector Laboratories, Burlingame, CA). After washing, the slides were stained with propidium iodide (0.75 µg/ml). R- and G-bands (51, 52) were detected with Nikon B-2A (excitation wavelength, 450–490 nm) and UV-2A (excitation wavelength, 365 nm) filter sets, respectively. Kodak Ektachrome ASA100 films (Eastman Kodak, Rochester, NY) were used for microphotography.

Sequence analysis

Putative regulatory elements in the promoter regions were identified by searching the TFMATRIX transcription factor binding site profile database (release 2.4) with the computer programs TFSEARCH (version 1.3) (53) and MatInspector (version 2.1) (54). Repetitive sequences in the mouse Psme genes were identified using the RepeatMasker 2 program (http://ftp.genome.washington.edu/RM/). Putative sorting signals were analyzed with the computer program PSORT (http://www.nibb.ac.jp).

	Results

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Genomic organization of the mouse Psme1 gene

To clone the mouse Psme1 gene, we screened the FIX II genomic library of 129/SvJ mice using the previously isolated mouse cDNA probe. Approximately 30 positive clones were identified from 1 x 10⁶ plaques. Preliminary analysis of the clones revealed that they fall into at least two distinct groups, one containing the Psme1 gene; and the other containing a Psme1-like sequence without introns. The latter was found to contain a processed pseudogene thought to be derived from the Psme1 gene (data not shown). We chose one clone, designated A6-1, containing the Psme1 gene and subjected it to detailed analysis. Figure 1 shows the restriction map of this clone. Figure 2 shows the complete nucleotide sequence of the Psme1 gene. The exonic sequence of the genomic clone was identical with the cDNA sequence obtained from the C57BL/6NHsd mouse (40). Two features of the Psme1 gene are noteworthy (Fig. 2). 1) The 5'-splice site of intron 4 has GC instead of GT, thus deviating from the canonical GT/AG rule (55). This type of rare nonconsensus splice junction sequence (56) has been shown to allow the 5'-site to be accurately cleaved, albeit more slowly than the usual GT sequence (57). 2) The sequence identical with nucleotides 3455–3475 is found at nucleotides 3407–3427 (indicated by brackets). This duplication creates a potential miniexon capable of encoding five additional amino acids (PPCGP), although there is no evidence that it is actually used by alternative splicing. To examine whether these unusual features of the mouse Psme1 gene are shared in the human counterpart (designated PSME1), we isolated the corresponding region of PSME1. Figure 3 shows that the PSME1 gene also has a variant 5'-splice site in putative intron 4 but contains no duplicated sequence in the corresponding intron.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Organization and restriction map of the mouse Psme gene family. Exons are shown as solid boxes and numbered. Two overlapping clones, B2-1 and B6-1, containing the Psme2 gene are indicated by horizontal bars. The restriction maps of the Psme1 and Psme3 genes were constructed using clones A6-1 and K6-1, respectively. The exon encoding the 3'-untranslated region of the Psme3 gene is drawn in length corresponding to the longer transcript.

View larger version (45K): [in this window] [in a new window]   FIGURE 3. Partial nucleotide and deduced amino acid sequences of the human PSME1 gene. Exonic and intronic sequences are written in capital and lowercase letters, respectively. The conserved dinucleotides (GT/AG and GC/AG) at the 5'- and 3'-splice sites are doubly underlined. Amino acids are shown with a standard single-letter code under the nucleotide sequence. The amino acid sequence shown here is identical with that deduced from the previously described cDNA sequence (34).

A computer search showed that the putative promoter region of this gene has several transcription factor-binding motifs (Fig. 2). Of interest is the existence of a putative IFN-stimulated response element (ISRE) at nucleotides 1628–1641. The observed sequence (GCTTTCGCTTTCCC) contains the core sequence (TTCNNTTT) that binds IFN-regulatory factors 1 and 2 and shows 85.7% identity to the consensus ISRE motif (AGTTTCNNTTTCNY; Y = CT, N = GACT) that functions in both orientations (58, 59). This is consistent with the observation that expression of Psme1 is induced strongly by IFN- (33, 34, 35, 36). No sequence that qualifies as a -activated site (GAS; consensus sequence, TTCNNNGAA) (60) was found in the putative promoter region of the Psme1 gene.

Genomic organization of the mouse Psme2 gene

To clone the mouse Psme2 gene, we screened the genomic library using the mouse cDNA probe. Approximately 35 positive clones were identified from 1 x 10⁶ plaques. Here again, preliminary analysis of the clones revealed the existence of at least two distinct types of clones, one containing the Psme2 gene and the other containing a Psme2-like gene without introns. Two overlapping clones, B2–1 and B6-1, containing the Psme2 gene were chosen and subjected to restriction enzyme mapping (Fig. 1). Figure 4 shows the complete nucleotide sequence of the Psme2 gene. The exonic sequence of the genomic clone differed from the previously reported cDNA sequence of the C57BL/6NHsd mouse (40) by 5 bp. These nucleotide substitutions, which most likely reflect an allelic variation, are all synonymous and do not change the amino acid residues. The exon-intron boundaries of the Psme2 gene conformed to the canonical GT/AG rule without exception. The locations and the splicing phases of the exon-intron boundaries in the Psme2 gene are essentially identical with those of the Psme1 gene (Fig. 5).

View larger version (54K): [in this window] [in a new window]   FIGURE 5. Locations of the exon-intron boundaries in the mouse PA28 -, ß-, and -subunits. - and * indicate identity with the top sequence and absence of residues, respectively. indicate exon-intron boundaries. Exon-intron classes were defined as follows: class 0, splice site between codons; class 1, splice site after codon position 1; and class 2, splice site after codon position 2. The mouse sequences were taken from Figures 2, 4, and 7. The sources of the human sequences were PA28 -subunit (34), ß-subunit (35), and -subunit (38). Nuclear localization signals predicted with the PSORT program are boxed. The KEKE motif in the -subunit is underlined.

Like the Psme1 gene, expression of the Psme2 gene is induced strongly by IFN- (35, 36). Consistent with this observation, the putative promoter region of the Psme2 gene contains a consensus ISRE sequence at nucleotides 837–850. The inversely complementary sequence of nucleotides 837–850 (GCTTTCGCTTTCAC) contains the core sequence of ISRE (TTCNNTTT), and shows 85.7% identity with the consensus ISRE motif (AGTTTCNNTTTCNY; Y = CT, N = GACT). Another sequence motif that might be functionally important is the potential NF-B-binding site (consensus sequence, GGGRNNYYCC) located at nucleotides 335–344 (61). Besides the consensus ISRE sequence and potential NF-B-binding site, the putative promoter region of the Psme2 gene contains two potential SP1-binding sites at nucleotides 230–238 and 800–808 (consensus sequence, KRGGCKRRK; K = GT, R = AG; thus, a 1-bp mismatch in both sites). No sequence that qualifies as GAS was found.

Physical map of the 150-kb region containing the mouse Psme1 and Psme2 genes

We showed previously, using an interspecific backcross panel (40), that the Psme1 and Psme2 genes are tightly linked and map close to the Atp5g1 locus (a gene coding for the ATP synthase, H⁺ transporting, mitochondrial Fo complex, subunit c, isoform 1) on mouse chromosome 14. The distance between Psme1 and Psme2 was predicted to be <3.8 cM at the 95% confidence level (40). To examine the possibility that the two Psme genes are located adjacent to each other, we isolated BAC clones using the Psme1 genomic fragment as a probe. Two distinct types of BAC clones, one containing the Psme1 gene (clone 236C3) and the other containing a processed pseudogene related to Psme1 (clone 7N2), were identified. Two lines of evidence indicated that clone 236C3 contains the functional Psme1 gene: 1) the sequence identical with nucleotides 2725–4099 of the Psme1 gene (Fig. 2) was amplified by PCR from this BAC clone; 2) FISH analysis showed that clone 236C3 maps to bands C2 to D1, most likely to the proximal region of band D1 of mouse chromosome 14 (Fig. 6, A and B). This cytogenetic localization is in good accord with the map position of Psme1 obtained by interspecific backcross mapping (62). Figure 6C shows the restriction map of clone 236C3. Hybridization analysis with the cDNA probes indicated that the 32-kb SalI fragment contains both Psme1 and Psme2 genes. To establish that the gene hybridizing with the Psme2 cDNA probe is the functional Psme2 gene, we amplified from the BAC clone the 540-bp DNA fragment and confirmed that its sequence is identical with the corresponding region (nucleotides 3716–4255 in Fig. 4) of the Psme2 gene. Finally, we determined the distance and relative orientation of the two Psme genes using the primer pairs described in Materials and Methods. Primer pairs A and B produced a band of 8 and 7 kb, respectively. Therefore, taken together, Psme1 and Psme2 are located 6 kb apart with their 3'-ends pointing toward each other on bands C2 to D1 of mouse chromosome 14, indicating that they emerged by tandem duplication. Subsequent analysis of the clones, B6-1 containing the Psme2 gene and A6-1 containing the Psme1 gene (Fig. 1), revealed that their 3'-ends have an overlap of 2.5 kb, thus confirming the analysis of the BAC clone (Fig. 6, C and D).

View larger version (61K): [in this window] [in a new window]   FIGURE 6. Localization of the BAC clone to bands C2-D1 of mouse chromosome 14 and a physical map of the 150-kb region containing the Psme1 and Psme2 genes. The metaphase spreads were photographed with Nikon UV-2A (A) and B-2A (B) filters. A and B, G-band and R-band patterns, respectively. Specific hybridization signals are indicated by white arrows (B). C, Restriction map of the BAC clone (clone 236C3). The orientations of the Psme1 and Psme2 genes are indicated by arrows. Individual exons of the genes are not shown. The regions of the BAC clone covered by the clones A6-1, B2–1, and B6-1 (Fig. 1) are shown at the bottom of the figure. The BAC clone contains a 2.5-kb MluI fragment 70 to 90 kb away from the left end, the precise location of which could not be determined. D, Restriction map of the region between the Psme1 and Psme2 genes. Exons are shown as solid boxes and numbered.

We also isolated the Psme3 gene and determined its restriction map (Fig. 1) and complete nucleotide sequence (Fig. 7). Consistent with the previous observation that Psme3 is a single-copy gene (36), only one type of genomic clone was identified (data not shown). Comparison of the exonic sequence in Figure 7 and the cDNA sequence obtained from C57BL/6NHsd mice (40) revealed a total of 4 bp substitutions and an insertion of 7 bp in the genomic sequence. These substitutions (located at nucleotides 6970, 7467, 7468, and 7868 in Fig. 7) and the 7-bp insertion (at nucleotides 8312–8318 in Fig. 7) are located in the 3'-untranslated region. Thus, the PA28 -subunits of 129/SvJ and C57BL/6NHsd mice have an identical amino acid sequence. The exon-intron boundaries of the Psme3 gene obeyed the GT/AG rule. Albertson et al. (63) showed that the human gene coding for the PA28 -subunit gives rise to an alternatively spliced transcript in fetal brain tissues. This transcript encodes a larger -subunit, in which 13 additional amino acids (PSGKGPHICFDLQ) are inserted between amino acid residues 135 and 136 in Figure 5. However, this appears to be unique to the human gene because the corresponding region of the mouse Psme3 gene (intron 6) does not contain an open reading frame capable of encoding similar amino acids (Fig. 7). The size of the Psme3 gene is larger than that of the other Psme genes, mainly because the former has two large introns (introns 4 and 10). However, the overall exon-intron organization of the Psme3 gene is essentially identical with that of the Psme1 and Psme2 genes, with the exon-intron boundaries located at nearly equivalent positions and having identical splicing phases (Fig. 5).

View larger version (172K): [in this window] [in a new window]   FIGURE 7. Nucleotide and deduced amino acid sequences of the mouse Psme3 gene. Exonic and intronic sequences are shown in capital and lowercase letters, respectively. The 5'-end of the previously published cDNA clone (40) is shown as the upstream boundary of the 5'-untranslated region. It does not indicate major transcription initiation sites. The conserved dinucleotides at the 5'- and 3'-splice sites (GT/AG) are doubly underlined. An in-frame stop codon (TGA) preceding the ATG translation initiation codon is indicated by asterisks. The Psme3 cDNA occurs in two distinct forms differing in the length of the 3'-untranslated region (36, 38, 39, 40). , Poly(A) addition sites observed previously in the shorter cDNA clones (40). The putative polyadenylation signals utilized in the shorter form (nucleotides 6983–6994 and 7020–7025) and in the longer form (nucleotides 8511–8516 and 8519–8530) are doubly underlined. Amino acids are shown with a standard single-letter code under the nucleotide sequence. Potential transcription factor-binding sites in the putative promoter region are also indicated.

Previous studies showed that IFN- treatment induces a transient, modest increase in the Psme3 mRNA level (35, 36). However, the putative promoter region of the mouse Psme3 gene does not contain any sequence motifs that qualify as ISRE or GAS (Fig. 7). There is one potential NF-B-binding site at position 840–849 (1 bp mismatch from the consensus). The Psme3 gene contains no obvious TATA or CAAT box in its putative promoter.

Repetitive sequences in the mouse Psme gene family

Table I summarizes the distribution of repetitive sequences in the mouse Psme gene family. The repetitive sequences occupy 14.5, 16.6, and 16.8% of the total sequences in the Psme1, Psme2, and Psme3 genes, respectively. Thus, the three Psme genes contain almost the same proportion of repetitive sequences. With the exception of the fact that the 5'-flanking regions of the Psme1 and Psme3 genes contain the B1 repeats, none of the short interspersed nucleotide elements is shared at the corresponding positions among the members of the Psme gene family. Because these elements are thought to have emerged in the rodent lineage after its separation from the primate lineage (64), virtual absence of shared B1 or B2 sequences indicates that the duplication events that gave rise to the three Psme genes took place before mammalian radiation. This is consistent with the fact that all three members of the Psme gene family have been identified in both humans and mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The promoter regions of IFN--inducible genes often contain ISRE, GAS, or both (59, 66). Genes with a GAS element are activated rapidly by IFN-, without a need for new protein synthesis. Typically, expression of these genes is induced within 1 h and maximal mRNA induction is achieved by 6 h. Among the genes involved in class I-mediated Ag presentation, TAP1 appears to fall into this category (67, 68). In contrast, genes with ISRE, but without a GAS element, are activated by IFN- more slowly, because their induction requires the synthesis of transcription factors that bind to the ISRE. In these genes, the mRNA levels usually increase only after 6 h or more and reach the maximum between 24 and 48 h. MHC class I genes (69), and the genes coding for the ß-type proteasome subunits LMP7 (70) and PSMB10 (13) appear to be of this type. Previous studies showed that the Psme1 and Psme2 mRNAs increase gradually, attaining the maximum level in 24 to 36 h after IFN--stimulation (35, 36). Although absence of information on the half-life of the mRNA precludes us from drawing definitive conclusions, the kinetics of the response appears to suggest that Psme1 and Psme2 also fall into the latter category of the IFN--inducible genes. This is consistent with our observation that both Psme1 and Psme2 have putative ISRE, but no GAS (Figs. 2 and 4). With the possible exception of the LMP2 gene, which shares a bidirectional promoter with the TAP1 gene (71, 68) and hence might be induced rapidly by IFN-, all known IFN--inducible subunits of the proteasome appear to be activated with similar kinetics, thus presumably contributing to their coordinated expression.

Some IFN--inducible genes, most notably the TAP1 and MHC class I genes, are also activated by TNF- (67). Such genes usually contain both ISRE and an NF-B-binding site in their promoters (66). Recent evidence indicates that TNF- activates NF-B by degrading its cytoplasmic inhibitor IB (61). The activated NF-B, which translocates to the nucleus and binds to the NF-B-binding site of the target genes, then interacts with the IFN--induced transcription factors that bind to the ISRE (66). This interaction results in synergistic induction of transcription by the two cytokines. In this regard, it is notable that the putative promoter of the mouse Psme2 gene contains the potential NF-B-binding site (nucleotides 335–344 in Fig. 4) besides the ISRE. Thus, expression of this gene might be induced synergistically by TNF- and IFN-.

IFN- treatment also induces a transient, modest increase in the Psme3 mRNA level (35, 36). The magnitude of induction was, however, 10% of that observed for the Psme1 or Psme2 gene (36), and the mRNA levels returned to control levels in 48 h after exposure to IFN- (35). The putative promoter region of the mouse Psme3 gene does not contain sequence motifs that qualify as ISRE or GAS (Fig. 7). Thus, it appears that the Psme3 mRNA is induced by IFN- through a pathway distinct from that mediated by ISRE or GAS. The Psme3 mRNA occurs in two forms that differ in the length of the 3'-untranslated region (Fig. 7). These two forms, which encode the same polypeptide, appear to be produced by differential transcription termination. Interestingly, the -subunit genes of humans and cows also have two mRNA forms that differ only in the length of the 3'-untranslated region and hence are presumably produced by differential transcription termination (38, 39). This observation suggests that the existence of two mRNA forms might have some functional significance.

The PA28 - and ß-subunits reside both in the cytoplasm and the nucleus, whereas the -subunit exists almost exclusively in the nucleus (37). A computer search of the deduced amino acid sequences of the -, ß-, and -subunits for sorting signals indicates that the - and -subunits have two putative nuclear localization signals, respectively (boxed in Fig. 5). One of the predicted nuclear localization signals in the -subunit is embedded in the KEKE motif (72) proposed to be involved in protein-protein interactions. Therefore, this motif might also serve as a nuclear localization signal. No nuclear localization signal was predicted for the ß-subunit. Thus, the assembly of the (ß)₃ heterohexamer might take place in the cytoplasm and this complex might be translocated to the nucleus by virtue of the sorting signal carried by the -subunit.

The structural organizations of the mouse Psme gene family described in this study provide the basic information required to create knockout mice. Given the growing evidence implicating the -subunit in the generation of class I binding peptides (30, 32), it seems reasonable to assume that disruption of the Psme1 gene will impair class I-mediated Ag presentation. In contrast, the ß-subunit alone does not stimulate peptidase activity of the 20S proteasome. However, it enhances the peptidase activity in the presence of the -subunit (28, 39), suggesting an auxiliary role for this subunit. Thus, inactivation of the Psme2 gene might impair class I-mediated Ag presentation to a lesser extent than that of the Psme1 gene. Less predictable is the outcome of the disruption of the Psme3 gene. The existence of a Psme3-like gene in the tick (65) and C. elegans (40), the organisms with no adaptive immune system, suggests that Psme3 presumably has (a) nonimmune function(s). On the other hand, the observation that expression of the -subunit is down-regulated by IFN- at the protein level (39) suggests that this subunit might also have an immunomodulatory function. Attempts to create Psme3-deficient mice are currently in progress in our laboratories.

	Acknowledgments

 
We thank Ms. Noriko Namerikawa for her secretarial help.

	Footnotes

 
¹ This work was supported in part by Grants-in-Aid for Scientific Research from The Ministry of Education, Science, Sports and Culture of Japan (to M.K., K.T., and Y.M.) and by a grant for Group Research Project "Biosystems Science" from The Graduate University for Advanced Studies, Hayama, Japan (to M.K.).

² Sequence data reported in this paper have been submitted to the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases under accession numbers AB007136 (mouse Psme1 genomic), AB007137 (human PSME1 genomic), AB007138 (mouse Psme2 genomic), and AB007139 (mouse Psme3 genomic).

³ Address correspondence and reprint requests to Dr. Masanori Kasahara at his current address: Department of Biosystems Science, The Graduate University for Advanced Studies, Hayama 240-0193, Japan. E-mail address:

⁴ Abbreviations used in this paper: LMP, low molecular mass polypeptide; BAC, bacterial artificial chromosome; FISH, fluorescence in situ hybridization; GAS, -activated site; ISRE, IFN-stimulated response element; PFG, pulsed field gel; RACE, rapid amplification of cDNA ends.

⁵ These gene symbols were approved officially by the Nomenclature Committee of The Mouse Genome Database, The Jackson Laboratory, Bar Harbor, ME.

Received for publication October 16, 1997. Accepted for publication January 16, 1998.

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