From the Centre de Biophysique Moléculaire (affiliated with the University of Orléans), CNRS UPR4301, Rue Charles Sadron, 45071 Orléans Cedex 02, France
Received for publication, March 24, 2000, and in revised form, October 30, 2000
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ABSTRACT |
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We previously reported that alternative
transcripts were initiated within the second intron of the human
Galectin-3 gene (LGALS3). We now demonstrate that these
transcripts arise from an internal gene embedded within
LGALS3 and named galig (Galectin-3 internal gene). Tissue-specific expression of galig was assayed by
screening of several human tissues. Contrary to LGALS3,
galig appears to be tightly regulated and principally
activated in leukocytes from peripheral blood. Cloning and
characterization of galig transcripts revealed that they
contain two out-of-frame overlapping open-reading frames (ORFs).
Transfection of expression vectors encoding enhanced green fluorescent
protein (EGFP) chimeras indicated that both ORFs could be
translated in proteins unrelated to Galectin-3. The ORF1 polypeptide
targets EGFP to cytosol and nucleus whereas ORF2 targets EGFP to
mitochondria. These results revealed the exceptional genetic
organization of the LGALS3 locus.
Galectin-3 is a Regulation of Galectin-3 gene (LGALS3) expression is largely
unknown. Recently, the complete genomic sequence of human
LGALS3 has been reported, and the functional promoter
activity of the 5'-flanking region of the gene has been established
(7). Previously, we have reported that the second intron of
LGALS3 contains an internal promoter, which drives
production of alternative transcripts (8).
In this report, we have further investigated the structure of these
alternative transcripts. We demonstrate that they are preferentially
expressed in peripheral blood leukocytes. They cannot be used for
production of Galectin-3 or a modified Galectin-3 because they contain
two overlapping open-reading frames
(ORF)1 out-of-frame with the
lectin coding sequence. Upon transfection, translation of these two
overlapping ORFs target reporter proteins to distinct subcellular
compartments. These results indicate that the LGALS3 locus
exhibits an unusual genetic structure, which involves the presence of
an internal gene embedded within the Galectin-3 gene.
Primer Extension Analysis--
RNA was extracted from human
osteosarcoma HOS cells (ATCC, CRL-1543) using the
guanidium/phenol/chloroform procedure (9). One microgram of RNA was
incubated in a final volume of 20 µl containing 30 units of avian
myeloblastosis virus reverse transcriptase (Promega, Madison, WI) and
10 picomoles of 32P-end-labeled primer using T4
polynucleotide kinase (New England BioLabs, Beverly, MA) at 42 °C
for 2 h. The primer PE (5'-ACAATCACAAACATCAGAAT-3') extends from
nucleotides 334 to 315 in the second intron of LGALS3 (see
Fig. 1). The reaction products were heat-denatured and loaded onto a
6% polyacrylamide sequencing gel.
Detection of Alternative Transcripts by RT-PCR--
Two
micrograms of total cellular RNA were extracted from SVH-1, a human
smooth muscle cell line (10), and from two tumor samples from human
colon carcinoma and were reverse transcribed using 10 pmol of random
nanomer primers. Thirty PCR cycles were performed using a 3'-primer
EX6-R (5'-TCTGCCCCTTTCAGATTATAT-3'), located in the sixth exon at the
end of Galectin-3 cDNA (positions 801-780) on the human
cDNA (GenBankTM/EBI no. M36682), and I2-F
(5'-TTCTGATGTTTGTGATTGTTTTTC-3'), a 5'-primer located from nucleotides
316 to 339 in the second intron of LGALS3
(GenBankTM/EBI no. U10300). A second round of 30 cycles of
PCR was initiated using the same 5'-primer and an internal 3'-primer,
EX4-R (5'-TCTGTTTGCATTGGGCTTCACC-3') located 336 bp upstream of the
primer EX6-R in the fourth exon of the gene. All PCR was carried out
under standard conditions with an annealing step of 55 °C.
Tissue specificity of the alternative transcripts was also performed by
reverse transcriptase-PCR on various human tissues using Human
Rapid-ScanTM Gene Expression Panel (Origene Technologies, Inc.,
Rockville, MD) containing 0.25 ng or 2.5 ng cDNA. The primers used
were TSF-1 (5'-TCTGAGTAGCGGGAAGTG-3'), corresponding to nucleotides 282-299 in the second intron, and TSR-1 (5'-GGGAAAACCGACTGTCTT-3'), corresponding to nucleotides 588-605 in the fifth exon on the human
cDNA (GenBankTM/EBI no. M36682). PCR (35 cycles) was
carried out under standard conditions with an annealing step of
57 °C. Galectin-3 transcripts were also detected using the same
method with primers EX4-R and GAL25 (5' ATGGCAGACAATTTTTCGCTCC-3'),
located at nucleotides 34-55 on the second exon of the gene.
Expression of Constructions of Reporter Plasmids Encoding EGFP
Chimeras--
Three ORFs, designated as ORF1, ORF2, and ORF3, were
detected in the alternative transcripts. Expression vectors were
constructed by insertion of the cDNA encoding EGFP at the 3'-end of
each ORF (see Fig. 3A).
EGFP cDNA was isolated from pEGFP-N1 (InVitrogen, Groningen, The
Netherlands) and modified to remove the ATG codon to avoid initiation
of translation because of potential leaky scanning process. In these
plasmids, designated as pORF1·EGFP, pORF2·EGFP, and pORF3·EGFP,
the 5'-end of the chimeric cDNA is located at position 242 in the
second intron of LGALS3. Transcription is driven by the CMV
promoter. Similar vectors were constructed in which EGFP cDNA was
replaced by the luciferase gene in pGL3basic (Promega).
Cell Transfection--
Plasmids were transfected into HOS cells
(seeded on glass coverslips at 8 × 104 cells per
12-well plate) or HEK 293 cells (ATCC, CRL-1573, seeded at 2 × 105 cells per well) using DNA-polyethylenimine complexes
(11). Briefly, 5 µg of DNA were mixed in 7.5 µl of a 10 mM polyethylenimine solution (Sigma Aldrich, France) in a
total volume of 1 ml of serum-free medium (Dulbecco's minimum Eagle's
medium, Life Technologies, Inc.). Cells were incubated for 2 h
with this solution and maintained for 24 h in fresh
Eagle's minimum essential medium (Life Technologies, Inc.) (HOS
cells) or Dulbecco's minimum Eagle's medium (HEK 293 cells)
supplemented with 10% fetal calf serum (Life Technologies, Inc.). For
EGFP detection, HOS cells were analyzed by fluorescence microscopy
using standard fluorescein filters (excitation at 488 nm and detection
at 520 nm). For mitochondrial-staining, cells were incubated for 15 min
with MitoTracker Red (50 nM, Molecular Probes, Inc. Eugene,
OR). MitoTracker Red was excited at 568 nm and detected through a > 600 nm filter. For luciferase assays, 400 ng of pRL-SV40, which
encodes Renilla luciferase (Promega), were added and used as
an internal standard of transfection efficiency and reproducibility.
Luciferase activities were assayed using Dual-Luciferase Reporter Assay
System (Promega) and an automated luminometer (Lumat LB9501, EG&G
Berthold, Badwildbad, Germany).
Preparation of Cellular Lysates--
HEK 293 cells were
trypsinized 24 h after transfection, centrifuged for 5 min at
250 × g and washed twice with phosphate-buffered saline. The pellet was suspended in 1.8 ml of lysis buffer (137 mM NaCl, 30 mM KCl, 15 mM Tris, 2 mM EDTA, 1 mM CaCl2, 0.5% Triton X-100, 1 mM phenylmethylsulfonide fluoride) containing 200 µl of protease inhibitor mixture (Sigma Aldrich, France) and
incubated 30 min on ice. The lysates were centrifuged at 15,000 × g for 5 min at 4 °C. Pelleted nuclei and membranes were
suspended in 50 µl of Laemmli buffer (125 mM Tris, 2%
SDS, 10% glycerol, bromophenol blue) and sonicated at 120 watts for
three cycles of 30 s each (Branson 5200). The supernatant was
precipitated by addition of trichloroacetic acid, and the proteins were
suspended in 300 µl of Laemmli buffer.
Gel Electrophoresis and Western Blotting--
Samples (20 µl)
were fractionated by SDS-polyacrylamide gel electrophoresis on a 15%
polyacrylamide gel under non-reducing conditions and transferred (0.8 mA/cm2 for 1 h) to nitrocellulose membrane (Schleicher
and Schuell, Dassel, Germany). Immunodetection was performed using a
murine monoclonal anti-GFP 11E5 (Molecular Probes) at a concentration of 0.5 µg/ml and the BM biochemiluminescence kit (Roche Molecular Biochemicals) according to the manufacturer's instructions.
Multiple Initiation Sites of Transcription in Alternative
Transcripts of LGALS3--
Transcription initiation sites of the
alternative transcripts resulting from the activity of the internal
promoter of LGALS3 were identified by primer extension
analysis (Fig. 1). Reverse transcription
was initiated from a primer located within the second intron of the
gene to avoid contaminating signals resulting from extension of
Galectin-3 mRNA. This was particularly beneficial considering the
low amount of these transcripts when compared with those produced by
the proximal promoter (8). Several signals of low intensities were
detected indicating the presence of different initiation sites (Fig.
1B). Among them, five major sites were evident. They were
all located in the second intron. Two of them were at positions 284 and
287 from the 5'-end of the second intron sequence and three were at
positions 262, 263, and 266 (Fig. 3B).
Characterization of Alternative Transcripts--
Transcripts
originating from the internal promoter were detected by RT-PCR using
forward (I2-F) and reverse (EX6-R) primers located in the second intron
and the sixth exon of LGALS3, respectively (Fig.
1A). One human smooth muscle cell line (10) and two human colon carcinomas tumor samples were tested. Positive signals, identified by a 752-bp fragment were detected after two rounds of PCR
amplification confirming the low abundance of these transcripts (Fig.
2). In one tumor sample, a second band of
lower size (around 450 bp) was detected. Both bands were cloned and
sequenced. Sequencing indicated that the larger transcript was composed
of the second intron, from the initiation transcription sites, and the
third to sixth exons of LGALS3 (Fig.
3). The smaller band presented an
internal deletion between nucleotides 95 and 389 of the large transcript. These sites correspond to consensus donor and acceptor sites for splicing events, indicating the presence of internal splicing
within these transcripts.
To avoid potential problems inherent to the RT-PCR technique and to the
low amount of transcripts detected in the cells analyzed, we have
verified that the alternative transcripts can be cloned from a cDNA
library constructed without use of PCR amplification. Because the
possible cell specificity of the internal promoter was not known, we
had screened at first the dbEST database using the sequence of the
second intron. Two clones from two different cDNA libraries were
revealed to be positive and contained sequences identical to the second
intron. The first clone (GenBankTM/EBI, AA094057)
was isolated from a human fetal heart library. The second one
(GenBankTM/EBI, R23407 and R44192) was isolated from a
human infant brain library and was completely resequenced. This clone
is another splicing variant with a donor site located at position 49 instead of 95 in the larger transcript, the acceptor site being located at position 389. This acceptor site, used in both variants, are also
used in the mature Galectin-3 mRNA and corresponds to the junction
between the second intron and third exon (Fig. 3). These results
confirmed that the structure of the alternative transcripts is not
resulting from cloning artifacts because of PCR.
Identification of Putative Overlapping ORFs--
The alternative
transcripts contain three potential ORFs (Fig. 3). Two of these ORFs,
designated as ORF1 and ORF2, have potential translation initiation
sites located in the third exon and spread over 318 and 291 nucleotides, respectively. ORF1 and ORF2 are out-of-frame with the
Galectin-3 coding sequence. The translation initiation site of the
third ORF, assigned as ORF3, is located in the fourth exon. If used,
this last ORF should produce a truncated form of Galectin-3
corresponding to the C-terminal half of Galectin-3, which contains the
complete carbohydrate binding domain of the lectin (12-13).
Translation of Out-of-Frame Overlapping ORFs--
Reporter vectors
were constructed to determine potentially translated ORFs. EGFP
cDNA was inserted in-frame at the 3'-end of each ORF (Fig.
3A). HOS cells were transfected and analyzed by fluorescence
microscopy. No fluorescence could be detected in cells transfected with
pORF3·EGFP in which the EGFP is in-frame with the putative truncated
Galectin-3, thus demonstrating that ORF3 is not translated (Fig.
4D). However, both other
vectors (pORF1·EGFP and pORF2·EGFP), produced a clear fluorescence
signal in transfected cells indicating translation of these ORFs.
Distribution of fluorescence is strikingly different for these two
vectors. The plasmid pORF1·EGFP, with an ATG located at position 394, induced a diffuse signal localized in cytosol and nucleus (Fig.
4B). This localization is similar to that one of the
nonchimeric EGFP (Fig. 4A). The plasmid pORF2·EGFP, with
an ATG codon at position 434, induced a strong fluorescence associated
with filamentous organelles and is completely excluded from the nucleus
(Fig. 4C).
Plasmids pORF1·EGFP and pORF2·EGFP were modified by replacing EGFP
cDNA with the luciferase gene. Transient transfection of HOS cells
with these vectors produced similar luciferase activity indicating that
both ORFs are translated with the same efficiency (Fig.
5).
Chimeric proteins from HEK 293 transfected cells were analyzed, after
cell fractionation, by Western blotting using an anti-EGFP antibody
(Fig. 6). The cytosolic fraction of
pORF1·EGFP transfected cells, showed a strong signal matching with a
38-kDa chimeric protein, whereas nuclei- and membrane-containing
fractions were weakly positive. This confirms the major cytosolic
distribution of this fusion protein. Fractions of pORF2·EGFP
transfected cells enriched in membranes and nuclei exhibited a signal
corresponding to a 38-kDa protein, the expected size if translation of
ORF2 occurred. Cytosolic fractions were negative confirming
fluorescence microscopy analysis.
Mitochondrial Localization of Fusion Protein
ORF2·EGFP--
Whereas ORF1·EGFP fusion protein has a cytosolic
distribution, the protein produced by pORF2·EGFP was associated with
organelles resembling typical mitochondria of rod-like, thread-like,
and granular structures. Therefore, cells transfected with the
pORF2·EGFP plasmid were incubated with a mitochondrial-specific dye
marker (MitoTracker Red). Confocal microscopy demonstrates that
MitoTracker Red and EGFP were colocalized in transfected cells
confirming that ORF2·EGFP was associated with mitochondria (Fig.
7). This localization was confirmed after
subcellular fractionation of cells transfected with chimeric constructs
bearing luciferase as another reporting system (data not shown).
Tissue-specific Expression of Alternative
Transcripts--
Tissue-specific transcriptional activity of the
internal promoter was analyzed in 24 human tissues by RT-PCR using a
single round of 35 cycles. The primers used were designed to amplify a
923-bp fragment. The most abundant transcripts were found in peripheral
blood leukocytes (Fig. 8) and to a lesser
extent in placenta, heart, muscle, stomach, and testis. They were
barely detectable in spleen, liver, adrenal gland, uterus, skin, and bone marrow and were negative in other tissues. Heart and muscle exhibited a high level of spliced transcripts revealed by a 629-bp fragment.
The Gene Expression Panel was also used to make semiquantitative
comparisons of alternative transcripts and LGALS3
transcripts. As expected, LGALS3 transcripts were highly
abundant in various tissues, which was confirmed by the reference to
actin transcripts. In all cases when expressed, the LGALS3
transcripts were more abundant than the alternative transcripts.
However, even if both transcripts were detected at different levels, it
should be noted that some tissues expressed only alternative
transcripts (muscle, stomach, and uterus), others produced both
transcripts (PBL, heart, placenta, testis, skin) and others produced
only Galectin-3 transcripts (kidney, lung, colon). These results
suggest the independent functioning of the two promoters.
The results presented in this study reveal the exceptional genetic
organization of the LGALS3 locus listed as the following. (i) LGALS3 contains an internal gene, which is regulated and
expressed mostly in peripheral blood leukocytes. (ii) This internal
gene contains two different overlapping ORFs, which are translated upon
transfection into proteins distinct from Galectin-3.
LGALS3 Contains an Internal Gene Named galig--
We have shown
that the mRNA transcribed from the internal promoter of
LGALS3, initiated at multiple sites located in the second intron (Fig. 1). One type of mRNA contains the second intron and exons 3-6 of LGALS3. Another type, not detected in all
cells analyzed, is produced by internal splicing, which removes most of
the second intron sequence except the first 90 nucleotides (Fig. 3).
Screening of dbEST database revealed the presence of both transcripts
in human heart and infant brain cDNA libraries confirming the
endogenous presence of these transcripts as polyadenylated mRNA in
human tissues.
These alternative transcripts share with the major mRNA of
LGALS3, the sequence spanning the third to sixth exon of the
gene. Because the ATG codon used for Galectin-3 translation is located in the second exon, a sequence not present in the alternative transcripts, none of the alternative transcripts could encode a
full-length Galectin-3 protein. The three potential ORFs, designated as
ORF1, ORF2, and ORF3, are overlapping and are each positioned in a
different reading frame. Of these three ORFs, only ORF3 would produce
if translated a truncated form of Galectin-3 restricted to the
carbohydrate recognition domain of the lectin (Refs. 12 and 13 and Fig.
3). Transfection experiments, using vectors expressing EGFP chimeras
indicated that ORF3 was not translated. Surprisingly, fluorescence was
detected in transfected cells with the two other vectors indicating
that the alternative transcripts can produce both proteins encoded by
ORF1 and ORF2 (Fig. 4). Western blotting and immunodetection using
anti-EGFP antibodies confirmed that both ORFs were translated (Fig. 6).
This implies an alternative initiation of translation at different AUG
codons, which can be attributed to a leaky scanning process or to an
internal entry of ribosomes (14-15). According to Kozak (16), the
leaky scanning process would be favored if the first AUG lies in a weak
context. This model is supported by the less favorable translation
context of the ORF1 start sites, which exhibit A at position +4 (16). In contrast, the AUG codon for ORF2, despite its downstream position, lies in a canonical Kozak sequence (with A and G at positions
These results justify that the structure of LGALS3 has to be
redefined. Actually, this locus contains two overlapping genes, which
could produce entirely distinct proteins. We propose to designate this
internal gene as galig (standing for Galectin-3 internal
gene). Such a genetic organization is extremely rare in mammalian
genomes and has been reported so far in only two cases, the
p16INK4A/p19ARF genes and
the growth hormone/GHDTA genes (17-18).
galig Can Be Translated into Two Distinct Proteins upon Cell
Transfection--
In addition to its noticeable genetic organization,
the most striking property of galig is the capacity to
encode two entirely distinct proteins (ORF1 and ORF2) from a single
mRNA. In this regard, galig would appear to be as yet
almost a unique example in mammals. Indeed, translation of different
proteins from a single mRNA usually occurs from the use of in-frame
AUG, resulting in proteins isoforms differing only at their N termini
(19-27). To our knowledge, among higher eukaryotes, only the chick
The few reported cases in mammalian in which two completely distinct
proteins are encoded from a single mRNA are the bicistronic mRNA producing genes (31-35). However, contrary to
galig, the ORFs are organized in tandem such as a typical
prokaryotic polycistronic mRNA.
ORF1 and ORF2 present a repetitive organization. This was expected
because these sequences are colinear with the repetitive domain of the
lectin mRNA (1). BLAST searches against ORF1 and ORF2 sequences
revealed no significant homology with any known protein.
The ORF1-predicted protein would contain 106 amino acids with an
apparent molecular mass of 11,253 Da. This sequence is highly rich in
leucine (20%), proline (13%), and glycine (12%) residues (Fig. 3).
ORF1·EGFP has a cytosolic and nuclear distribution (Figs. 4 and
6).
The ORF2-predicted protein is an 11,168 Da protein of 97 residues. The
sequence is highly hydrophobic and positively charged, because of a
large number of arginine residues (12% of total residues). A
remarkable feature is the high content of tryptophan residues. 12% of
the residues are tryptophan, the average of other human proteins is 10 times lower. This rich content in tryptophans confers hydrophobic
properties that may account for the membrane localization of the
ORF2·EGFP protein (Fig. 6). Consistent with the mitochondrial localization of the ORF2·EGFP fusion protein, this sequence exhibits the common properties of mitochondrial-imported proteins such as the
enrichment of arginine, leucine, and serine residues (36).
Tissue Specificity of galig Expression--
Detection of
galig transcripts in HOS cells and colon tumor cells
revealed a low expression level. Based on this observation, the
rationale that the appearance of galig transcripts may have resulted from a leaky transcription of a cryptic promoter rather than
from an independently functioning promoter could not be excluded. Screening of several human tissues indicated clearly that
galig is a tightly regulated gene whose expression is most
efficient in leukocytes from peripheral blood. The low level of
transcription in bone marrow indicates that galig is
specifically expressed in mature forms of leukocytes. Whereas the
precise quantification of galig mRNA has not been
addressed in these experiments, it is clear that these transcripts are
much less abundant than LGALS3 transcripts. This may not be
surprising considering that LGALS3 is known to be highly
expressed when activated (37, 38). Indeed, the amount of
LGALS3 transcripts appeared as abundant as those from actin
genes. This shows a different type of regulation by the
galig and LGALS3 promoters. In particular,
muscle, stomach, and uterus, although expressing low levels of
galig transcripts, revealed no LGALS3
transcripts, thus indicating an independent functioning of the two
promoters. For other tissues expressing both galig and
LGALS3 mRNA (heart, testis, placenta, and skin, Fig. 8),
cDNA amplification caused by infiltrating leukocytes cannot be
ruled out. As Galectin-3 is thought to regulate cell proliferation,
further investigation of a putative link between the functions
of these overlapping genes would be extremely valuable.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactoside-binding lectin involved in a
variety of biological processes (1). The expression pattern of
Galectin-3 changes during development (2) and production of the protein
modulates cell-cell or cell-matrix interactions (3-4). A large number
of studies show that expression is dependent on cellular growth
properties, correlates with neoplastic transformation (5), and confers
resistance to apoptosis (6).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin was examined as a control with primers supplied
by the manufacturer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Determination of transcription initiation
sites in mRNA issued from the internal promoter in
LGALS3. A, schematic representation of the human
LGALS3 gene. The proximal promoter is located upstream of
exon 1 (ex.1, Ref. 7), and the internal promoter is located
in the 5'-region of the second intron (int.2, Ref. 8). The
ATG translation initiation codon used for Galectin-3 production is
located in the second exon (ex.2). Introns, represented by
thick lines, are not drawn to scale. PE is a
primer used for primer extension analysis and is located at positions
334-315 in the second intron of LGALS3
(GenBankTM/EBI U10300). I2-F, EX6-R, and EX4-R are primers
used for the detection of transcripts by RT-PCR. I2-F is a forward
primer located in the second intron and EX6-R, a reverse primer located
in the sixth exon. Ex4-R is a lower primer used for internal PCR (see
"Experimental Procedures" for precise location). B,
primer extension analysis was carried out on mRNA from human HOS
cells using primer PE. The ladder was determined using a sequencing
reaction performed on a control plasmid (not shown). Numbers represent
the size of the extended products in base pairs.
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Fig. 2.
Detection of transcripts by RT-PCR.
mRNA extracted from an SV40-transformed human cell line, SVH-1
(Ref. 10, lane 1) and two tumor colon carcinomas
(lanes 2 and 3) were reverse transcribed using
random nonamers. Two successive rounds of PCR were performed on
reverse-transcribed RNA. The first PCR was initiated using primers I2-F
and EX6-R (Fig. 1). A second round of PCR was initiated using I2-F and
the internal primer EX4-R. One-tenth of the second PCR reactions were
analyzed by gel electrophoresis. The size marker is a 100-bp DNA
ladder.
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Fig. 3.
Schematic representation and sequence of the
putative overlapping reading frames. A, +1 represents
the first major transcription start site from the transcripts initiated
within the second intron of LGALS3. These transcripts
contain the last 389 nucleotides of the second intron and exons 3-6 of
LGALS3. The three ORFs are positioned and the thick
line marks the sequence 95-389 alternatively spliced out from
these transcripts. EGFP cDNA or the luciferase gene were inserted
in-frame at the 3'-end of each one of these ORFs. The resulting
chimeric recombinant cDNA were inserted within an expression vector
containing a CMV promoter. B, nucleotides 261
to +389 represent the sequence of the second intron of
LGALS3 (GenBankTM/EBI, accession number U10300).
Lowercase letters (nucleotides
261 to
1) mark the
sequence upstream of the initiation transcription start sites, which
contain the internal promoter. Uppercase letters (from nt
+1) represent the transcribed sequence. The arrow over the
bold nucleotides points out the primer PE used for primer
extension analysis (Fig. 1), and triangles point out the
major transcription start sites. The boxed nucleotides
indicate the sequence alternatively spliced out. Nucleotides 390-1156
(italic letters) are the sequence from exons 3 to 6 of
LGALS3 common to both major Galectin-3 mRNA
(GenBankTM/EBI accession number M36682) and alternative
transcripts from the activity of the internal promoter. The three
potential ORFs are translated; ORF1 with an ATG at position 393, ORF2
with an ATG at position 434, and ORF3 with an ATG at position 759. This
last ORF uses the same reading frame as the one coding the carbohydrate
recognition domain of Galectin-3. Black arrows indicate the
insertion point of EGFP or luciferase cDNA for production of fusion
proteins (see "Experimental Procedures").
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Fig. 4.
Transfection of cells with vectors encoding
EGFP fusion proteins. HOS cells were transfected with pEGFPN1, a
vector encoding EGFP (A), or fusion vectors encoding
ORF1·EGFP (B), ORF2·EGFP (C), or ORF3·EGFP
(D). Fluorescence microscopy was performed 2-days
post-transfection.
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Fig. 5.
Expression of luciferase-based fusion
proteins in transfected cells. HOS cells were transfected with
pORF1·Luc and pORF2·Luc, two plasmids in which ORF1 and ORF2 are
fused to luciferase cDNA, respectively. Cells were cotransfected
with pRL-SV40, which encodes Renilla luciferase. This last
plasmid is used as an internal standard. Luciferase activity
(Luc) was assayed 2 days after transfection and was
expressed relative to Renilla luciferase activity
(RLuc).
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Fig. 6.
Immunodetection of chimeric proteins in
pORF1·EGFP and pORF2·EGFP transfected cells. Twenty-four hours
post-transfection, cells were fractionated. Membranes and
nuclei-enriched fractions (lane M) and cytosolic fractions
(lane C) were run on a polyacrylamide gel and transferred
and incubated with a murine monoclonal anti-EGFP antibody.
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Fig. 7.
Mitochondrial localization of
ORF2·EGFP. Left, confocal fluorescence image of HOS
cells transfected with a vector encoding ORF2·EGFP. Right,
same cells were costained with MitoTracker Red. Mitochondria in
non-EGFP-transfected cells are also stained with MitoTracker Red.
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Fig. 8.
Detection of alternative transcripts in
various human tissues. Human Rapid-ScanTM Gene Expression Panel
was used to detect the alternative transcripts in various human tissues
using RT-PCR. The primers used were designed to amplify a 923- or
629-bp fragment. LGALS3 transcripts were detected as a
457-bp DNA and actin transcripts as a 640-bp DNA. PCR was performed
using 0.25 ng or 2.5 ng (10×) template cDNA.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 and + 4, respectively). To assess experimentally the efficiency of initiation
at these different AUG, the expression vectors were modified by
replacing the EGFP cDNA with the luciferase gene. Upon
transfection, the same luciferase activity was detected with both
vectors, suggesting that the two ORFs were translated with the same efficiency.
2(I) and type III collagen genes might present a situation similar
to galig. These genes contain internal promoters in introns
2 and 23, respectively. The resulting transcripts exhibit several
overlapping ORFs, which appeared to be out-of-frame with the collagen
coding sequences (28-30). However, in both cases, the question of the
potentially translated ORFs has not been addressed.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. C. Hughes (NIMR (National Institute for Medical Research), London, United Kingdom) and Dr. M. Tiberi (LRI (Loeb Health Research Institute), Ottawa, Canada) for critically reviewing the manuscript.
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FOOTNOTES |
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* This work was supported by a grant from the Ligue Nationale Contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF266280.
Supported by fellowships from the Ministère de
l'Enseignement Supérieur et de la Recherche and the Fondation
pour la Recherche Médicale.
§ To whom correspondence should be addressed. Tel.: 33 2 38 25 55 36; Fax: 33 2 38 25 78 07; E-mail: legrand@cnrs-orleans.fr.
Published, JBC Papers in Press, November 3, 2000, DOI 10.1074/jbc.M002523200
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ABBREVIATIONS |
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The abbreviations used are: ORF, open-reading frame; galig, Galectin-3 internal gene; RT-PCR, reverse transcriptase-polymerase chain reaction; bp, base pair(s); GFP, green fluorescent protein; EGFP, enhanced GFP; CMV, cytomegalovirus; SV40, simian virus 40.
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REFERENCES |
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---|
1. |
Barondes, S. H.,
Cooper, D. N.,
Gitt, M. A.,
and Leffler, H.
(1994)
J. Biol. Chem.
269,
20807-20810 |
2. | Fowlis, D., Colnot, C., Ripoche, M. A., and Poirier, F. (1995) Dev. Dyn. 203, 241-251[Medline] [Order article via Infotrieve] |
3. |
Bao, Q.,
and Hughes, R. C.
(1995)
J. Cell Sci.
108,
2791-2800 |
4. |
Bao, Q.,
and Hughes, R. C.
(1999)
Glycobiology
9,
489-495 |
5. | Perillo, N. L., Marcus, M. E., and Baum, L. G. (1998) J. Mol. Med. 76, 402-412[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Yang, R. Y.,
Hsu, D. K.,
and Liu, F. T.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
6737-6742 |
7. | Kadrofske, M. M., Openo, K. P., and Wang, J. L. (1998) Arch. Biochem. Biophys. 349, 7-20[CrossRef][Medline] [Order article via Infotrieve] |
8. | Raimond, J., Rouleux, F., Monsigny, M., and Legrand, A. (1995) FEBS Lett. 363, 165-169[CrossRef][Medline] [Order article via Infotrieve] |
9. | Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve] |
10. | Legrand, A., Greenspan, P., Nagpal, M. L., Nachtigal, S. A., and Nachtigal, M. (1991) Am. J. Pathol. 139, 629-640[Abstract] |
11. | Boussif, O., Lezoualc'h, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J.-P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7297-7301[Abstract] |
12. |
Hsu, D. K.,
Zuberi, R. I.,
and Liu, F. T.
(1992)
J. Biol. Chem.
267,
14167-14174 |
13. |
Agrwal, N.,
Sun, Q.,
Wang, S. Y.,
and Wang, J. L.
(1993)
J. Biol. Chem.
268,
14932-14939 |
14. | Kozak, M. (1992) Annu. Rev. Cell Biol. 8, 197-225[CrossRef] |
15. | Gray, N. K., and Wickens, M. (1998) Annu. Rev. Cell Dev. Biol. 14, 399-458[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Kozak, M.
(1997)
EMBO J.
16,
2482-2492 |
17. |
Labarriere, N.,
Selvais, P. L.,
Lemaigre, F. P.,
Michel, A.,
Maiter, D. M.,
and Rousseau, G. G.
(1995)
J. Biol. Chem.
270,
19205-19208 |
18. | Quelle, D. E., Zindy, F., Ashmun, R. A., and Sherr, C. J. (1995) Cell 83, 993-1000[Medline] [Order article via Infotrieve] |
19. |
Voss, J. W.,
Yao, T. P.,
and Rosenfeld, M. G.
(1991)
J. Biol. Chem.
266,
12832-12835 |
20. |
Aoki, M.,
Hamada, F.,
Sugimoto, T.,
Sumida, S.,
Akiyama, T.,
and Toyoshima, K.
(1993)
J. Biol. Chem.
268,
22723-22732 |
21. | Calligaris, R., Bottardi, S., Cogoi, S., Apezteguia, I., and Santoro, C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11598-11602[Abstract] |
22. | Vagner, S., Gensac, M. C., Maret, A., Bayard, F., Amalric, F., Prats, H., and Prats, A. C. (1995) Mol. Cell. Biol. 15, 35-44[Abstract] |
23. | Packham, G., Brimmell, M., and Cleveland, J. L. (1997) Biochem. J. 328, 807-813[Medline] [Order article via Infotrieve] |
24. | Akiri, G., Nahari, D., Finkelstein, Y., Le, S. Y., Elroy-Stein, O., and Levi, B. Z. (1998) Oncogene 17, 227-236[CrossRef][Medline] [Order article via Infotrieve] |
25. | Okazaki, S., Ito, T., Ui, M., Watanabe, T., Yoshimatsu, K., and Iba, H. (1998) Biochem. Biophys. Res. Commun. 250, 347-353[CrossRef][Medline] [Order article via Infotrieve] |
26. | Yang, X., Chernenko, G., Hao, Y., Ding, Z., Pater, M. M., Pater, A., and Tang, S. C. (1998) Oncogene 17, 981-989[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Ayoubi, T. A.,
and Van De Ven, W. J.
(1996)
FASEB J.
10,
453-460 |
28. |
Bennett, V. D.,
and Adams, S. L.
(1990)
J. Biol. Chem.
265,
2223-2230 |
29. |
Nah, H-D.,
Niu, Z.,
and Adams, S. L.
(1994)
J. Biol. Chem.
269,
16443-16448 |
30. |
Zhang, Y.,
Niu, Z.,
Cohen, A. J.,
Nah, H-D.,
and Adams, S. L.
(1997)
Nucleic Acids Res.
25,
2470-2477 |
31. | Lee, S. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4250-4254[Abstract] |
32. | Szabo, G., Katarova, Z., and Greenspan, R. (1994) Mol. Cell. Biol. 14, 7535-7545[Abstract] |
33. | Ritchie, H., and Wang, L. H. (1997) Biochem. Biophys. Res. Commun. 231, 425-428[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Sloan, J.,
Kinghorn, J. R.,
and Unkles, S. E.
(1999)
Nucleic Acids Res.
27,
854-858 |
35. | Stallmeyer, B., Drugeon, G., Reiss, J., Haenni, A. L., and Mendel, R. R. (1999) Am. J. Hum. Genet. 64, 698-705[CrossRef][Medline] [Order article via Infotrieve] |
36. | Horwich, A. (1990) Curr. Opin. Cell Biol. 2, 625-633[Medline] [Order article via Infotrieve] |
37. | Hamann, K. K., Cowles, E. A., Wang, J. L., and Anderson, R. L. (1991) Exp. Cell Res. 196, 82-91[Medline] [Order article via Infotrieve] |
38. | Nangia-Makker, P., Ochieng, J., Christman, J. K., and Raz, A. (1993) Cancer Res. 53, 5033-5037[Abstract] |