Genomics, Isoforms, Expression, and
Phylogeny of the MHC Class I-Related MR1
Gene
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J Immunol 1998; 161:4066-4077; ;
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References
Patricia Riegert, Valérie Wanner and Seiamak Bahram
Genomics, Isoforms, Expression, and Phylogeny of the MHC
Class I-Related MR1 Gene1,2
Patricia Riegert,* Valérie Wanner,† and Seiamak Bahram3*†
C
lassical MHC class I (MHC-I)4 molecules present short
peptide Ags to the TCR-ab of CD81 T lymphocytes (1).
They are highly polymorphic, ubiquitously expressed,
membrane-bound heterodimers formed by noncovalent association
of a 45-kDa heavy chain and the 12-kDa b2m (2). The heavy chain
is encoded by a small number of genes, i.e., HLA-A, -B, and -C in
man and H2-K, -D, and -L in mouse, located within the MHC on
human chromosome 6p21.3 and murine chromosome 17 (3, 4).
The MHC of various species contains, in addition to these welldefined classical MHC-I (also known as MHC-Ia), a rather diverse
collection of so-called nonclassical genes (also referred to as
MHC-Ib), including HLA-E (5), HLA-F (6), and HLA-G (7) in
man and the H2-Q, -T, and -M loci in mice (8 –10). These show a
restricted pattern of tissue expression, are not polymorphic, and
have not been shown to consistently interact with a defined T cell
*Basel Institute for Immunology, Basel, Switzerland; and †Centre de Recherche
d’Immunologie et d’Hématologie, Strasbourg, France
Received for publication January 30, 1998. Accepted for publication June 8, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Work in Strasbourg was supported by Association pour la Recherche sur le Cancer,
Ligues Nationales et Départementales contre le Cancer, and Hôpitaux Universitaires
et Faculté de Médecine de Strasbourg. The Basel Institute for Immunology was
founded and is supported by F. Hoffmann-La Roche (Basel, Switzerland).
2
The nucleotide sequence data reported in this paper have been submitted to the
GenBank nucleotide sequence database and have been assigned the accession numbers AF010446 (hMR1B), AF010447 (hMR1C), AF031469 (hMR1D), AF010448
(mMR1A), AF010449 (mMR1B), AF010450 (mMR1C), AF010451 (mMR1D),
AF010452 (mMR1E), AF010453 (mMR1F), AF039526 (hMR1 putative promoter
sequence), and AF035672 (mouse MR1 gene).
3
Address correspondence and reprint requests to Dr. Seiamak Bahram, Centre de
Recherche d’Immunologie et d’Hématologie, 4 Rue Kirschleger, 67085 Strasbourg,
France. E-mail address: [email protected]
4
Abbreviations used in this paper: MHC-I, MHC class I gene or protein; MHC-II,
MHC class II gene or protein; FcRn, neonatal Fc receptor; MIC, MHC class I chain
related; MR1, MHC class I related; FISH, fluorescence in situ hybridization.
Copyright © 1998 by The American Association of Immunologists
population. A long-standing body of work has, however, implicated some of these MHC-Ib genes in unique immunologic functions. These include presentation of N-formulated peptides (of bacterial or mitochondrial origin) by H2-M3 (11) and leader sequence
peptides by HLA-E (12) and Qa-1 (13); presence (and therefore
probable function) at the maternal-fetal interface for HLA-G (14)
and in the mucosa for some TL molecules (15); existence as both
glycosylphosphatidylinositol-anchored and soluble versions capable of binding a diverse peptidic array for Qa-2 (16); and, finally,
restriction of several gd T cell clones for H2-T10b (17) and H2T22b (18). Despite these apparently special features, the high degree of sequence similarity between the MHC-Ia and -Ib molecules
defines them as members of the same gene lineage, derived from
a common ancestor through gene duplication and minimal diversification, late in speciation.
The past decade has clearly shown that beside this first lineage of MHC-I genes, vertebrate genomes harbor a growing
number of highly divergent MHC-I-like structures (19). The
prototype of this category of molecules is undoubtedly CD1
(20). Initially recognized through serologic analysis, further
work characterized CD1 as a b2m-associated MHC-I-like chain
(21, 22). There are five CD1 isotypes (CD1A–E) on human
chromosome 1q21–23 (23) and two CD1 genes (CD1.1 and
CD1.2) on murine chromosome 3 (24). Despite featuring an
extracellular tri-domain structure (a1–3) like typical MHC-I
molecules, CD1 is at crossroads between MHC-I and -II at the
level of both amino acid composition and function. The molecule is equally homologous to MHC-I and -II, exhibits a tissuespecific pattern of expression, and engages Ag through an endocytic pathway as does MHC-II (20). However, in sharp
contrast to peptide presenting MHC-I and -II, CD1 molecules
have an unusually hydrophobic ligand binding groove (25) and
seem to specialize in presentation of lipidic/glycolipidic moieties of bacterial origin to TCR-ab and -gd CD42CD82 T cells
0022-1767/98/$02.00
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A growing number of non-MHC-encoded class I-related molecules have been shown to perform diverse, yet essential, functions.
These include T cell presentation of bacterially derived glycolipidic Ags by CD1, transcytosis of maternal IgG by the neonatal Fc
receptor, enriched presence and plausible function within exocrine fluids of the Zn-a2-glycoprotein, subversion of NK cytolytic
activity by the CMV UL18 gene product, and, finally, crucial involvement in iron homeostasis of the HFE gene. A recently
described member of this family is the MHC class-I related (MR1) gene. The most notable feature of MR1 is undoubtedly its
relatively high degree of sequence similarity to the MHC-encoded classical class I genes. The human chromosome 1q25.3 MR1
locus gives rise not only to the originally reported 1,263-bp cDNA clone encoding a putative 341-amino acid polypeptide chain, but
to many additional transcripts in various tissues as well. Here we define the molecular identity of all human and murine MR1
isoforms generated through a complex scenario of alternative splicing, some encoding secretory variants lacking the Ig-like a3
domain. Moreover, we show ubiquitous transcription of these MR1 variants in several major cell lineages. We additionally report
the complete 18,769-bp genomic structure of the MR1 locus, localize the murine orthologue to a syntenic segment of chromosome
1, and provide evidence for conservation of a single-copy MR1 gene throughout mammalian evolution. The 90% sequence identity
between the human and mouse MR1 putative ligand binding domains together with the ubiquitous expression of this gene favor
broad immunobiologic relevance. The Journal of Immunology, 1998, 161: 4066 – 4077.
The Journal of Immunology
4067
(20). The neonatal Fc receptor (FcRn) is the second well-characterized member of non-MHC-I-like structures. The human
chromosome 19q13.3 (murine chromosome 7) FCGRT gene encodes a highly divergent 48-kDa heavy chain bound to b2m at
the gut epithelial brush border where it acts as a transepithelial
shuttle delivering maternal IgG from the intestinal lumen into
the bloodstream, providing passive neonatal immunity (26, 27).
The Zna2gp (AZGP1) is another MHC-I like structure. A soluble single-chain 41-kDa molecule of as yet unknown function,
Zna2gp was first purified from plasma, although it is more prevalent in exocrine secretions, especially those of the mammary
gland. The gene has been mapped to human chromosome
7q22.1 and mouse chromosome 5 (28 –30). Highly divergent
MHC-I genes are not all encoded outside the MHC; in particular, a novel highly divergent multigene class I family termed
MIC (MHC class I chain related) is located within the MHC
class I region (31). The MICA gene in this family is unusually
polymorphic (32) (compared with mono-, oligomorphic
MHC-Ib genes) and encodes a highly glycosylated single-chain,
membrane-bound protein almost exclusively expressed in epithelia (33). HFE is yet another example of a drastically divergent MHC-I gene, adjacent to the MHC itself (approximately 4
megabases telomeric to HLA-F) (34). Surprisingly, the murine
HFE orthologue does not map to the H2 complex, but is located
within a paralogous locus on chromosome 13 (35, 36). A typical
class I gene by domain structure, HFE displays only 30% overall homology to typical MHC-I molecules, but requires dimerization with b2m before surface expression (37). Nevertheless
the role of an MHC-I molecule in iron homeostasis remains a
complete mystery and exemplifies yet another fascinating aspect of MHC-Ib biology. The complete sequencing of three
viral genomes, i.e., human and mouse CMV and Molluscum
contagiosum virus, has revealed the existence of a distinct
MHC-I gene within each of them (38 – 40). The significance and
possible function of these molecules are not yet understood,
although the CMV-encoded UL18 molecule has been shown to
bind endogenously derived peptides and interact with NK cell
receptors (41, 42).
In summary, a restricted collection of diverse, yet structurally
related, MHC-I genes located on various chromosomes perform
highly disparate, yet essential, functions. The latest addition to the
group of non-MHC-encoded class I genes is the human MHC class
I-related (MR1) gene (43). Discovered by degenerate PCR, MR1
was subsequently mapped to human chromosome 1q25.3. Northern blotting experiments using a number of human tissues revealed
ubiquitous expression of multiple MR1 transcripts; a cDNA clone
of 1263 bp encoding a putative polypeptide of 341-amino acid
sequence (calculated Mr of 39 kDa) resembling an authentic
MHC-I was originally reported (43). Perhaps the most interesting
feature of MR1 is that despite being located outside the MHC, the
molecule shows the highest degree of homology to the classical
MHC-I molecules (43). This opens the possibility of analogous
function for MR1 and raises enticing questions as to the scenario
of genome-wide MHC-I dispersion.
Here we present the cDNA sequences of all human and murine
MR1 isoforms and show evidence for their uniform expression in
various cell lineages and tissues. Moreover, we define the genomic
structure of MR1, localize the gene by fluorescence in situ hybridization (FISH) in the mouse genome, and study the extent of phylogenetic conservation of this molecule.
Materials and Methods
cDNA genomic cloning and sequencing
A full-length human MR1 cDNA clone was obtained by RT-PCR using
oligonucleotides derived from published sequence (43) and human intestinal total RNA (Clontech, Palo Alto, CA) as template, employing standard
procedures (44, 45). The oligonucleotides used were as follow: RT, 59TGACTCTATGGGGGACAGAG-39; and PCR, 59-GGACTATGGGG
GAACTGATG-39 and 39-CTAACGTCTAGGGAGAAAAGG-59. The
full-length cDNA clone of 1.3 kb was subsequently used as a probe to
sequentially screen approximately 106 phage clones of the following
cDNA libraries: human fetal spleen and murine (C57/BL6) spleen (Stratagene, San Diego, CA). This led to isolation of several cDNA clones from
each species, which were subsequently analyzed by sequence determination. The full-length human MR1 cDNA probe was also used to screen
approximately 6 3 105 phage clones of the 129/SvJ mouse genomic library
(Stratagene). This yielded the isolation of several genomic clones that were
subsequently characterized by blot hybridization of restriction-mapped
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FIGURE 1. Catalogue of MHC-I genomic structures. The GenBank accession number of each sequence is given below the name. Closed boxes indicate
coding exons, whereas shaded rectangles depict untranslated sequences.
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HUMAN AND MOUSE MR1 GENES
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FIGURE 2. The complete 18,769-bp MR1 genomic structure. The following exons are boxed: L (leader peptide), a1, a2, a3, TM (transmembrane), and
cytoplasmic/39 untranslated (Cyt./39 UT).
The Journal of Immunology
4069
RNA blots
All cell lines were purchased from American Type Culture Collection (Manassas, VA) and grown in the recommended medium at 37°C in an atmosphere supplemented with 5% CO2. Total RNA was isolated from approximately 107 cells after lysis in a solution containing 4.0 M guanidinium
thiocyanate, 0.1 M Tris (pH 7.5), and 1% b-ME, followed by ultracentrifugation (33,000 rpm, 16 h) on a cushion of 5.7 M CsCl/0.01 M EDTA (pH
7.5). Twenty micrograms of total RNA was subjected to electrophoresis on
a 1% agarose/formaldehyde gel (44, 45). Upon transfer onto Zeta-Probe
GT (Bio-Rad, Richmond, CA), the membrane was hybridized to a randomly [a-32P]dCTP-labeled MR1 cDNA probe (Ready z To z Go DNA Labelling Kit, Pharmacia, Uppsala, Sweden) in a solution containing 10%
dextran sulfate, 1.53 SSPE, and 0.5% SDS overnight at 65°C. The membrane was subsequently washed at 65°C in a solution containing 0.23
SSC/0.1% SDS. The wet membrane was exposed for 4 days at 280°C to
X-OMAT AR film (Eastman Kodak, Rochester, NY) opposing an intensifying screen. The mouse poly(A)1 multiple tissue Northern blot was purchased from Clontech and hybridized to a full-length murine MR1 cDNA
probe under stringent conditions according to the manufacturer’s specifications. The blot was subsequently exposed for 72 h to an intensifying screen at
270°C. The loading control probe consisted in both cases of a 2-kb human
b-actin cDNA fragment (Clontech) and required 2 h of exposure at room
temperature.
Zoo blots
FIGURE 3. Phylogeny. A, Evolutionary conservation of the MR1 locus
across mammalian evolution. Note the relative simplicity of bands given
the large genomic length. The mouse genomic DNA pattern is the result of
a double digest (see text). B: B1, Chromosomal localization of a single
copy MR1 gene within the mouse genome as evidenced by FISH. B2,
Localization of the MR1 gene on both sister chromatids is highlighted. B3,
The identity of chromosome 1 is ascertained via double labeling employing
a chromosome 1-specific marker (in B2 and B3 the sample chromosomes
are oriented head-to-head).
Eight micrograms of various high m.w. genomic DNA were digested with
HindIII (HindIII/BglII for mouse DNA) and analyzed by standard Southern
blotting employing the full-length human MR1 cDNA as a probe. DNA
from the following species were used in this study: human (a gift from Dr.
A. Meyer, Strasbourg, France); primates: bonobo (Pan paniscus), gorilla
(Gorilla gorilla), and gibbon (Hylobates lar; all provided by Dr. M. Chorney, Hershey, PA); dog, bovine, porcine, rat (all purchased from Clontech);
and finally DBA/2-C57/BL6 F1 mouse (Basel Institute for Immunology
animal facility, Basel, Switzerland). Hybridization and low stringency
wash of the blot were performed as described above. The membrane was
subsequently exposed for 1 wk to Kodak X-OMAT AR films opposing an
intensifying screen.
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fragments (isolation of the bacteriophage P1 containing the MR1 gene is
detailed below). Cross-species screenings were conducted as follows. In
brief, overnight hybridization was performed at 42°C in a solution containing 50% formamide, 53 Denhart’s solution, 53 SSPE, 0.1% SDS, and
100 mg/ml of denatured salmon sperm DNA (Sigma, St. Louis, MO). The
nitrocellulose filters were subsequently treated in a final wash solution
containing 23 SSC/0.1% SDS at 45°C. High stringency screening of the
human cDNA library with an isogenic probe was performed using standard
conditions (44, 45).
The 18,769-bp full-length MR1 gene sequence was assembled as follows. Almost 13 kb was derived from the available l clones (directly or
after subcloning); the remaining 59 6 kb encoding exon 1 and a large part
of intron 1 were obtained employing the following pair of oligonucleotides,
59-GGTTGATGATGCTCCTGTTACC-39 (derived from leader peptide sequence of murine cDNAs) and 39-CGGCAATTCTCGGTCAGAGAAC-59
(pan-ultimate sequence within by then available l DNA), in a PCR reaction
using the Expand Long Template kit (Boehringer Mannheim, Mannheim,
Germany) and P1 DNA as template following the manufacturer’s recommendations (see below for P1 isolation). Finally, upstream sequence from
exon 1 was obtained using the Human GenomeWalker kit (Clontech) following the manufacturer’s recommendations and employing the following
exon 1-located nested oligonucleotides: 39-GTAACACAATTACCACT
TCGTGTCGCTA-59
and
39-CTTGACTACCGCAAGGACAATG
GAGAGT-59. Double-strand sequencing of cloned DNA fragments (plasmid, l, or P1) was performed using flanking vector sites and customsynthesized oligonucleotides (primer walking). The chain termination
method was conducted using either the Thermo Sequenase cycle sequencing kit (Amersham, Aylesbury, U.K.) and [35S]dATPaS (Amersham, UK)
or the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit
with AmpliTaq DNA polymerase, FS (Perkin-Elmer, Foster City, CA) according to the manufacturer’s protocol. In the latter technique, the reactions
were run on an ABI 373XL DNA Sequencer STRETCH (Perkin-ElmerApplied Biosystems, Foster City, CA), and the results were analyzed using
ABI PRISM Sequencing Analysis, Factura, and Sequence Navigator software (all from Perkin-Elmer-Applied Biosystems). Further sequence analysis employed the EditSeq and MegAlign (default parameters) programs of
the LaserGene Navigator (DNAstar, Madison, WI).
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HUMAN AND MOUSE MR1 GENES
Bacteriophage P1 cloning and FISH
Results
The human MR1 gene is the latest addition to the restricted family
of the non-MHC-encoded class I genes. Beside cDNA cloning, the
original report of Hashimoto et al. (43) established the localization
of the human gene to chromosome 1q25.3 and ubiquitous transcription of the molecule in various tissues. The present report
aims to define the MR1 transcriptional unit; isolate, map, and characterize the murine orthologue; investigate the extent of the phylogenetic conservation of the gene; as well as outline key architectural residues of this molecule.
To define the genomic structure of MR1, a PCR-cloned human
cDNA encompassing the entire coding sequence was used to isolate
orthologous genomic clones from a murine bacteriophage l library.
However, because of the lack of the entire transcriptional unit within
the obtained l clones, a murine bacteriophage P1 library (average
insert size, 90–100 kb) was PCR-screened employing MR1-specific
primers. Double-stranded sequence analysis identified MR1 within
18,769 bp of mouse genomic DNA (Figs. 1 and 2; GenBank accession no. AF035672). The structure of the gene is that of a typical
MHC-I gene, with respective a1, a2, and a3 extracellular domains,
and transmembrane and cytoplasmic sequences encoded by separate
exons. Particularities include an unusually large first intron of 8,857
bp. As in other MHC-I and -II genes, each domain starts with a composite codon where the first nucleotide is contributed by the previous
exon (type I splicing) (44) and is flanked by intronic sequences harboring the canonical GT/AG splice junctions. Finally, sequence analysis of 1384 bp putative promoter sequence of human MR1 gene did
not reveal any obvious similarity to other MHC-I promoters (see GenBank accession no. AF039526).
A comparative genomic Southern blot (zoo blot) including humans, nonhuman primates, and more distant species revealed the
conservation of what appears as a single-copy MR1 gene across
mammalian evolution (Fig. 3A). To map the gene within the mouse
genome, a bacteriophage P1 clone was used to FISH-localize a
single MR1 locus within bands G1–2 of mouse chromosome 1,
syntenic to human 1q25.3,5 the location of the orthologous locus
(Fig. 3B).
5
A higher degree of resolution was achieved via blastn search of GenBank STS
database using human MR1 cDNA clones as probes. This analysis revealed a com-
FIGURE 4. Ubiquitous expression of several MR1 transcripts in both
man and mouse. A, Diverse human cell lineages, i.e., Jurkat (T cell leukemia), Ramos (B cell, Burkitt lymphoma), Molt-4 (T cell, acute lymphoblastic leukemia), Raji (B cell; Burkitt lymphoma), HT-1080 (fibrosarcoma), SK-LMS-1 (vulval sarcoma), HeLa (cervical epithelial carcinoma),
U-373 (glioblastoma), and HT-29 (colon adenocarcinoma) express MR1
isoforms. B, All major murine tissues express a number of MR1 transcripts.
b-Actin intensity served as the loading control in both experiments (lower
panels). See Figure 5 for the molecular identities of these transcripts.
The original Northern blot experiment reported by Hashimoto
and colleagues (43) revealed ubiquitous expression of what appeared as multiple human MR1 transcripts in various tissues examined. However, it was not known whether this reflects universal
transcription in all cell lineages or solely within a broadly present
single cell type, e.g., endothelium, epithelium, etc. Hence, an
equivalent experiment was performed, this time using several human lineage-specific cell lines. The observation of several transcripts in all these cells confirms truly ubiquitous MR1 expression,
resembling the pattern of MHC-Ia transcription (Fig. 4A). Finally,
to ascertain a similar situation in the mouse, a multiple tissue Northern
blot revealed, as predicted, a broad range of expression for the murine
gene, once again present in multiple isoforms (Fig. 4B).
To fully understand the molecular identity of multiple MR1
transcripts, a saturation cDNA cloning strategy was clearly relevant. Extensive screening of human and mouse spleen cDNA libraries coupled with RT-PCR experiments allowed us to completely characterize the complex transcriptional activity of this
gene (Figs. 5, 6, and 7). In humans, we identified one RT-PCRcloned transcript identical with the original 1263-bp cDNA clone
plete match with STS_U22963 (locus G26705) positioned at 838.4 cR from the top
of the chromosome 1 linkage group.
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The following two oligonucleotides, 59-CAGACCTGTGTGGTGGT
GTC-39 and 39-CTAATACACCGAGTGTAGTG-59, amplifying a unique
275-bp genomic fragment within exon 3 of the murine MR1 gene, were
used to PCR screen a 129/SvJ mouse embryonic stem cell genomic P1
library (Genome Systems, St. Louis, MO) following published protocols
(45, 46, 47). This yielded isolation of two P1 clones. The identity of one of
these was ascertained through amplification and sequence analysis employing the same primer pair mentioned above. This clone, called F298, was
subsequently used to FISH-localize the MR1 locus (Genome Systems, St.
Louis, MO). In brief, DNA from clone F298 was labeled with digoxigenin
dUTP by nick translation. Labeled probe was combined with sheared
mouse DNA and hybridized to normal metaphase chromosomes derived
from mouse embryo fibroblast cells in a solution containing 50% formamide, 10% dextran sulfate, and 23 SSC. Specific hybridization signals
were detected by incubating the hybridized slides in fluoresceinated antidigoxigenin Abs followed by counterstaining with 49,6-diamidino-2phenylindole, dihydrochloride. The initial experiment resulted in specific
labeling of the distal portion of the largest chromosome pair. A second
experiment was conducted in which a probe specific for the centromeric
region of chromosome 1 was cohybridized with clone F298. This experiment resulted in the specific labeling of the centromere and the distal portion of chromosome 1. Measurements of 10 specifically hybridized chromosomes 1 demonstrated that F298 is located at a position that is 75% of
the distance from the heterochromatic-euchromatic boundary to the telomere of chromosome 1, an area that corresponds to band 1G1–2. A total of
80 metaphase cells were analyzed, with 76 exhibiting specific labeling.
The Journal of Immunology
4071
reported by Hashimoto and colleagues (43), hereafter called
hMR1A, as well as three other cDNA species, respectively designated hMR1B, hMR1C, and hMR1D (Figs 5 and 6). Interestingly,
all newly described transcripts lack the Ig-like a3 domain. Moreover, the 1578-bp hMR1C transcript bears a stop codon a few
amino acids after the end of the a2 domain and therefore encodes
a putative soluble isoform, whereas the 776-bp hMR1B and the
2046-bp hMR1D remain putative integral transmembrane proteins
(Figs. 5 and 6). In the mouse, six distinct cDNA species were
delineated (Fig. 7). The mMR1A cDNA of 2406 bp encodes a
bonafide MHC-I molecule organized similar to the human orthologue. However, due to the presence of a cryptic splice donor site
77 bp inside the a1 domain and through a scheme best described
as partial exon skipping, several transcripts containing aborted
open reading frames were generated through splicing of this initial
exon 2 fragment directly to the beginning of exon 3. These include
the mMR1B, -C, -E, and -F of 2130, 1641, 1115, and 2854 bp,
respectively. The mMR1D is a 3219-bp truncated, partially spliced
transcript carrying part of intron 3 (Figs. 5 and 7). Finally, the
extensive length heterogeneity observed among both human and
murine MR1 transcripts is due to the respective sizes of their 39
untranslated regions, a fact readily visible on Northern blot experiments (see above).
Comparing individual domains of human and murine MR1
polypeptides reveals an unusually high degree of sequence similarity (i.e., a1, 90%; a2, 89%; and a3, 73%; see Figs. 8 and 9).
The same comparison including representatives of other MHC-I
molecules reveals, as expected, conservation of almost all socalled invariant residues, mostly thought to maintain the class I
scaffold (48, 49). Moreover, this analysis defines at least three
novel pan-MHC-I conserved amino acids (underlined hereafter).
These residues are P20, G26, V28, D29, S38, P47, W51, W60,
E/D61, T64, and the N86XS glycosylation site within the a1 domain; G91, H93, Q96, M98, G100, C101, G112, Y118, D119,
G120, W133, C164, L168, R170, L172, and G175 throughout the
a2 domain (Fig. 8); and P185, C203, F208, Y209, P210, P235,
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FIGURE 5. Schematic representation of human and murine MR1 isoforms. The central part of this figure reproduces the MR1 genomic organization as
shown in Figure 1. Open boxes correspond to exons. Above and below this scheme are respectively shown cartoons of all murine and human MR1 isoforms
with total cDNA length and GenBank accession number below each call sign.
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HUMAN AND MOUSE MR1 GENES
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FIGURE 6. Human cDNA clones and sequences. The first sequence is the original cDNA sequence identified by Hashimoto et al. (43); all other
sequences were uncovered during the present study and are respectively called hMR1A-D.
The Journal of Immunology
4073
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FIGURE 7. Mouse cDNA clones and sequences. The isolated cDNAs are respectively called mMR1A through -E. Only coding sequences are shown;
respective 39 untranslated sequences may be accessed within GenBank. The mMR1D could be only aligned from the a3 onward and is therefore not
represented in this alignment. Finally, both alignments employ single letter amino acid codes above codons, show nucleotide and amino acid (italic)
positions on the right, highlight exon boundaries by vertical bars and horizontal arrowed lines, and depict termination codons by an asterisk. Most of these
transcripts are present at relatively high levels, as they are readily visible on Northern blots (see Fig. 4).
D238, C259, V261, and H263 in the Ig-like a3 domain. Among
the eight residues defined by high resolution crystallographic
analysis to contact peptide termini in MHC-Ia molecules (50),
only three are conserved in MR1, i.e., Y7, T143, and W147
(Figs. 8 and 9). Finally, homologous residues to positions 223
to 229, 245, and 247 of the MHC-Ia a3 domain, indispensable
for interaction with the CD8 coreceptor and present in both
human and mouse MHC-encoded class I molecules (51, 52)
4074
HUMAN AND MOUSE MR1 GENES
(with the exception of MICA/B genes), are clearly absent from
human and mouse MR1 (Fig. 8).
Exploring the available human and murine MR1 sequences potentially originating from three and six distinct chromosomes, respectively (Refs. 43 and 53 and sequences reported here), we conclude that unlike MHC-Ia or MIC genes (2, 32) MR1 does not
seem to be overtly polymorphic, as not a single nucleotide difference was detected within respective coding sequences.
Discussion
The 90% sequence identity between human and mouse MR1 molecules is truly remarkable, not seen either in the non-MHC-I genes,
where the level of identity does not exceed 60(a1)/62(a2)% for
CD1, 59/74% for Zna2gp, 69/66% for FcRn, and 78/76% for HFE,
or in the MHC-encoded class Ia and Ib molecules, which have
average human-mouse similarities of 69/70% and 51/41%, respectively (Fig. 9). This outstanding degree of trans-species sequence
identity in MR1 should naturally include preservation of key structural/functional residues. Keeping this in mind, examination of
such positions reveals interesting information. As previously
noted, most if not all structural residues conserved across all vertebrate MHC-I molecules are also maintained in MR1, predicting
folding of the putative MR1 polypeptide chain in an analogous
three-dimensional configuration. Moreover, the N86XS glycosylation site present in all human and mouse MHC-Ia and -Ib, HFE,
and human Zna2gp, but absent from all CD1 isotypes as well as
from MICA/MICB molecules, is present in both human and murine MR1 (Fig. 8). However, the absence of over half the residues
interacting with peptide termini from the MR1 a1-a2 domains as
well as all CD8 binding residues from the Ig-like a3 domain favors
a functional digression from the peptide binding, CD81 ab T cellinteracting classical class I molecules (Fig. 8).
The genomic organization of MHC class I genes parallels their
multidomain polypeptide chain configuration. Indeed, each extracellular domain is encoded by a separate exon, preceded by the
leader sequence and followed by transmembrane, cytoplasmic, and
39-untranslated sequences (one or more depending on the locus;
Fig. 1) (44). Examination of the large number of genomic structures available reveals, with the exception of CD1, a clear dichotomy between the structurally homogeneous HLA-A-G genes
(mouse H2-K, L, D, Q, T, and M) and those encoding the divergent
(both functionally and structurally) MICA/B, HFE, FcRn, Zna2gp,
and MR1 molecules; this is regardless of their physical location in
the genome, i.e., whether they are located within the MHC proper
(Fig. 1). Indeed, the former group averages a total gene length of
3 to 5 kb, whereas the latter easily exceeds 6 kb, with 6.8 kb for
HFE, 10 kb for Zna2gp, 11 kb for FcRn, 12 kb for MICA/B, and
close to 19 kb for the MR1 gene reported here (Fig. 1). With the
exception of FcRn, where this is due to a lengthy fourth intron
(between exons 3 and 4), the extra length is due to an unusually
large first intron preceding the a1 domain, reaching a peak stretch
of 8857 bp in MR1 (Fig. 2). Whether this unusual length has any
functional repercussions, possibly at the transcriptional level, remains to be seen. Finally, it is noteworthy that large MHC-I genes
are not exclusive to mammals, as, for instance, several carp genes
have been documented with first introns up to 14 kb (54).
Alternative splicing has long been documented for MHC class I
genes (55–57) and involves mainly transmembrane and cytoplasmic sequences. The true physiologic contribution of various isoforms, however, is not known, especially given the fact that they
are severely under-represented compared with the canonical transcript; the majority are only detectable upon PCR amplification.
Thus, high expression of a plethora of MR1 isoforms in both human and mouse, all readily visible on Northern blots, is unique
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FIGURE 8. MR1 structures within the MHC-I gene family. Sequence comparison of human and mouse MR1 molecules with other key members of the
MHC-I family. Residues identical with human MR1 sequence are boxed. Dashes indicate gaps. Each domain is separately numbered. Triangles depict
locations of a2 and a3 conserved cysteine residues. Closed circles indicate locations of residues involved in binding of peptide termini in classical MHC-I
molecules, whereas stars point to residues conserved across MHC-I evolution. The rectangle in the a1 domain indicates the location of the X86 N-linked
glycosylation site, whereas those in the a3 domain outline key residues enabling CD8 coreceptor engagement.
The Journal of Immunology
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FIGURE 9. Sequence distance among members of the MHC-I family. Domain-by-domain sequence analysis of representatives of human and murine
classical and nonclassical MHC-I genes was performed, based upon sequence alignment employing default parameters of the Clustal method, MegAlign
program, Lasergene Navigator (DNAstar). Relevant comparisons are highlighted (box and arrow) and additionally bold-italicized with regard to MR1.
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These are IL6RA, Ly9, CD48, CD3z, CD64, LYAM1, ELAM1,
OX40L, IL10, CD34, CRP, and a constellation of Fc receptors, i.e.,
FCGR1A, FCER1A, FCER1G, FCGR, and FCGR3A and -B (65).
The relatively high and truly ubiquitous transcription of MR1 is
unique among the so-called nonclassical MHC-I genes (Fig. 4).
Human CD1a, -b, and -c, for instance, are mainly expressed on
cortical thymocytes and Langerhans cells, whereas both human
and mouse CD1d seem to be mainly transcribed in intestinal epithelial cells (20). The expression pattern of neonatal FcRn is selective to the intestinal brush border and placental syncytiotrophoblast (26). Zna2gp is highly prevalent in breast fluids (28), and
HFE expression appears to be confined to the gut (36). Even
among the MHC-encoded nonclassical genes and with the exception of human HLA-E (5), there is an evident degree of compartmentalization, e.g., epithelia for MICA/B (31), trophoblasts for
HLA-G (14), intestine or thymocytes for most TL genes (15), and
so on. Within this context the wide cellular expression of MR1 is
clearly reminiscent of ubiquitous classical MHC-I molecules and
is therefore in favor of a general physiologic function.
MHC-Ia molecules show an extraordinary degree of polymorphism, with more than 300 alleles collectively defined for human
HLA-A/B/C genes (2). In clear contrast, MHC-Ib genes, with the
important exception of the MIC loci (32), are not polymorphic, a
situation that has led some authors to postulate their presentation
of invariant ligands (68), for instance glycolipidic moieties by CD1
molecules to CD4, CD8 double-negative ab, or gd T lymphocytes
(20). Following this rationale, the apparent monomorphism of
MR1 favors possible interaction with a putative invariant ligand.
In summary, MR1 is a broadly expressed, phylogenetically conserved, invariant class I molecule located within an MHC-paralogous locus. Although at this point any suggestions regarding MR1
function within the immune system are highly speculative, possible interaction with a growing family of orphan leukocyte Ig superfamily receptors, whose founding members interact with a diverse set of MHC-I molecules, warrants further investigation (69).
Acknowledgments
S.B. thanks Georges Hauptmann for encouragement. We thank Nassima
Fodil for help with genomic cloning, and Drs Marco Colonna, Louis Du
Pasquier, and Susan Gilfillan for critical reading of this manuscript.
References
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among all MHC-I (Fig. 4). In man, the apparent over-representation of transcripts lacking the Ig-like a3 domain may have functional relevance, although this can only be verified once specific
Abs to various extracellular domains are available.
MHC-I genes constitute diverse multigenic families in various
vertebrate species (58 – 60). On the other hand, non-MHCencoded class I genes are, in general, more simply arranged, although the human Zna2gp locus contains a pseudogene in addition
to the functional locus (28), and the CD1 loci are present in various
configurations in different species (20). Within this context, both
the low complexity of genomic bands seen in our zoo blot experiments and especially observation of single hybridization spots in
both human and mouse FISH favor the existence of a single MR1
locus throughout mammalian evolution, in clear contrast therefore
to most MHC-encoded and to some non-MHC-I-encoded genes.
Given the strong sequence conservation between such phylogenetically distant species as Homo sapiens and Mus musculus, these
data are largely in favor of strong selective pressure maintaining
MR1 structure. Finally, the location of MR1 on human chromosome 1 might not be fortuitous, as the relative proximity of the
CD1 locus is intriguing. Indeed, capitalizing on theories first forwarded by Ohno, who proposed creation of the present day vertebrate genomes via tetraploidization (61), a process recently verified through sequence analysis of the complete yeast genome (62),
Kasahara and colleagues identified another MHC-like gene cluster
outside chromosome 6 in man and chromosome 17 in mouse
(9q33–34 in man and chromosome 2 in mouse) harboring elements
resembling those found in the MHC itself, e.g., ATP-binding cassette transporters (such as the MHC-encoded TAP molecules), proteasome Z subunit (such as MHC-encoded LMP2 and -7), Grp78
stress protein (such as the MHC-encoded 70-kDa heat shock protein genes), complement C5 gene (such as the MHC-encoded C2/
Bf/C4 molecules), etc. (63). Through similar observations, this
time regarding several nonimmune genes (NOTCH and PBX),
Katsanis and co-workers added the long arm of chromosome 1 as
yet an additional paralogous locus (64). However, the true MHC
signature, i.e., MHC-I and -II genes, has not been found or noted
on either 9q34 or 1q. In this light, the relatively close proximity of
human CD1 and MR1 loci might parallel for the first time the
human 6p21.3 (mouse 17) MHC-II/I topology and therefore define
an authentic MHC paralogous locus. In this respect, it is interesting
to note that through many functional/structural aspects (see above)
CD1 is more closely related to MHC-II than to MHC-I. Given
these facts, CD1 could be considered the chromosome 1-encoded
MHC-II paralogue and MR1 an authentic MHC-I equivalent.
Moreover, several other key genes chart this genomic segment and
therefore contribute to its emergence as yet another MHC paralogous locus. These are 1) the retinoid X receptor g located between
MR1 and CD1; interestingly, the two other RXRs genes are located, respectively, in the MHC (retinoid X receptor b) and chromosome 9q34 (retinoid X receptor a) (65); 2) the recently mapped
proteasome b subunit PSMB4 to 1q21 (66), in comparison to other
b subunits encoded within 9q34 for Z (PSMB7) (63) and the MHC
for LMP2/7 (PSMB9/8) (3); 3) the 1q25-q32-located tenascin-R
gene (67) paralogous to MHC-encoded tenascin-X molecule (3);
and 4) the so-called regulator of the complement activation (RCA)
gene cluster comprising complement receptors type 1 and 2, decay-accelerating factor, and C4 binding protein, closely linked on
1q32 (65); reminiscent perhaps of a somewhat similar organization
within the MHC, where the complement genes C2, BF, and C4 are
sandwiched between MHC-I and -II loci (3) and 9q34, the location
of yet another complement subunit, C5 (65). Finally, a close inspection of the other loci neighboring MR1 and CD1 reveals the
existence of an unusually large number of immune-related loci.
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