Departments of 1 Internal Medicine, 2 Pathology, 3 Human Genetics, and 4 Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan 48109
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ABSTRACT |
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Na+-myo-inositol cotransport activity generally maintains millimolar intracellular concentrations of myo-inositol and specifically promotes transepithelial myo-inositol transport in kidney, intestine, retina, and choroid plexus. Glucose-induced, tissue-specific myo-inositol depletion and impaired Na+-myo-inositol cotransport activity are implicated in the pathogenesis of diabetic complications, a process modeled in vitro in cultured human retinal pigment epithelium (RPE) cells. To explore this process at the molecular level, a human RPE cDNA library was screened with a canine Na+-dependent myo-inositol cotransporter (SMIT) cDNA. Overlapping cDNAs spanning 3569 nt were cloned. The resulting cDNA sequence contained a 2154-nt open reading frame, 97% identical to the canine SMIT amino acid sequence. Genomic clones containing SMIT exons suggested that the cDNA is derived from at least five exons. Hypertonic stress induced a time-dependent increase, initially in a 16-kb transcript and subsequently in 11.5-, 9.8-, 8.5-, 3.8-, and ~1.2-kb SMIT transcripts, that was ascribed to alternate exon splicing using exon-specific probes and direct cDNA sequencing. The human SMIT gene is a complex multiexon transcriptional unit that by alternate exon splicing generates multiple SMIT transcripts that accumulate differentially in response to hypertonic stress.
human retinal pigment epithelial cells; hypertonic stress; exon splicing; osmoregulation; diabetes mellitus
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INTRODUCTION |
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THE WATER-SOLUBLE CYCLIC hexitol myo-inositol is a constituent of virtually every living cell and an essential nutrient for most mammalian cells in culture. Intracellular concentrations 50- to 1,000-fold greater than that of extracellular fluid are with few exceptions (25) ascribed to the action of membrane-associated, Na+-dependent myo-inositol cotransporters (SMITs) (2). At the cellular level, myo-inositol is an obligate and sometimes rate-limiting (19) substrate for phosphoinositide synthesis. Along with sorbitol, taurine, and betaine, myo-inositol belongs to a family of alternative nonionic organic intracellular osmolytes whose regulated accumulation and efflux in response to hypertonic stress preserve intracellular volume and tonicity without perturbing the ionic milieu (2, 5). Organic osmolyte accumulation is coordinated by the regulated expression and/or activity of osmotically responsive, osmolyte-specific genes or gene products: distinct Na+-cotransporters for myo-inositol, taurine, and betaine and aldose reductase (AR) for the synthesis of sorbitol from glucose. Gradient-dependent organic osmolyte efflux is thought to be regulated primarily but not exclusively by a nonselective, ATP-dependent, volume-sensitive organic anion channel (2, 14, 27). Thus, at any given tonicity, the relative abundance of each osmolyte depends in part on substrate availability, the abundance and activity of their transporter or biosynthetic enzyme, and their efflux or degradation. The ready availability of one osmolyte may lead to reciprocal decreases in the abundance of others. For example, hyperglycemia is associated with increased flux through AR such that sorbitol accumulates with attendant depletion of other osmolytes including myo-inositol.
Despite the ubiquitous role of myo-inositol as an osmolyte and metabolic precursor, the metabolism and transport of myo-inositol are highly cell and tissue specific. The kidney synthesizes, concentrates, excretes, and reabsorbs myo-inositol and uniquely expresses the specific mammalian cytoplasmic oxygenase for myo-inositol degradation. Brisk Na+-myo-inositol cotransport activity in the small intestine, the choroid plexus, the ocular ciliary body, and the retinal pigment epithelium (RPE) (8) accounts for rapid intestinal absorption and the higher myo-inositol levels in cerebrospinal fluid and vitreous humor (14). Human RPE cells (8) and bovine lens epithelial cells (4) demonstrate Na+-myo-inositol cotransport activities with distinct kinetics. Whether this is due to genetically diverse SMITs or due to posttranslational modification is not clear, but Na+-myo-inositol cotransport activity can be regulated posttranscriptionally in RPE cells by intermediary metabolites such as pyruvate and in RPE and in Madin-Darby canine kidney (MDCK) cells through the action of protein kinases (14, 24). Thus Na+-myo-inositol cotransport is implicated in the regulation of both cellular and systemic myo-inositol homeostasis.
Despite its physiological importance, widespread distribution, and functional complexity, relatively little is known about the molecular regulation of Na+-myo-inositol cotransport. The functional cloning of a canine SMIT cDNA in Xenopus oocytes (16) by assaying Na+-dependent myo-[3H]inositol uptake led to the first elucidation of a SMIT primary structure. The deduced 718-amino acid polypeptide was predicted to have multiple potential protein kinase A/C (PKA/PKC) phosphorylation sites (16) and multiple hydrophobic "membrane-spanning" domains, homologous with the mammalian Na+-dependent glucose transporter (SGLT) family (12). More significantly, voltage-clamp studies of Na+-myo-inositol cotransport in Xenopus oocytes expressing the recombinant canine SMIT cDNA confirm the functional identity of the recombinant transporter through its substrate specificity and kinetic parameters (11). With the use of this cDNA as a probe, SMIT transcripts ranging from ~1.0 to >13.5 kb have been identified following hypertonic stress in Northern blots of RNA from kidney (16), lens (33), neural (21), and vascular (28) endothelial cells. The increased abundance of these transcripts in hypertonically stressed MDCK cells is at least in part due to increased transcription as detected by nuclear run-on analysis (31), but it is not clear whether these diverse transcripts are the products of a single gene or a gene family. Human genomic DNA containing a full-length open reading frame (ORF), potentially encoding human SMIT (SLC5A3), has been cloned using the canine cDNA as a probe (1). It was speculated that this intronless sequence gives rise to diverse SMIT transcripts through alternate transcript termination and polyadenylation sites. We now demonstrate that the human SMIT gene is a complex transcriptional unit comprising at least five exons, which are alternately spliced into a family of SMIT transcripts. The SMIT coding sequence is contained entirely within exon 2. The resulting transcripts differ, as a function of their exon composition, size, the presence or absence of the SMIT coding sequence, and the sequence of their 3' untranslated regions (UTR).
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METHODS |
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RNA isolation. Primary cultures of human RPE cells were established and passaged (8) in minimal essential medium (MEM) with 20% calf serum at 37°C in a humidified 5% CO2 atmosphere. Confluent cells were exposed to isotonic (295 mosmol/kgH2O) MEM (IMEM) or 300 mM mannitol-supplemented hypertonic (595 mosmol/kgH2O) MEM (HMEM) with 20% calf serum for various times, after which RNA was purified (13).
Construction and screening of the cDNA library.
Total RNA was isolated 24 h after exposure of RPE cells to HMEM. An
oligo(dT)-primed, size-selected (>1 kb) cDNA library was commercially
prepared from the RNA using a unidirectional strategy in pCDNA1
(Invitrogen, San Diego, CA) and transformed in
Escherichia coli strain MC 1061/P3 by
electroporation. The library was screened at reduced stringency using
an EcoR
I-BamH I fragment of the canine SMIT
cDNA (generously provided by H. Moo Kwon, Johns Hopkins University, Baltimore, MD; GenBank accession no. M85068; nt 724-2870,
containing most of the coding sequence and 236 nt of 3' UTR) (16)
labeled as described in Probes. Recombinants (6 × 105) were plated at high
density and lifted on nitrocellulose filters, colonies were lysed in
0.5% SDS, and the DNA was denatured before fixation in vacuo for 2 h
at 80°C. Filters were prehybridized as previously described (13)
for 4-6 h before addition of denatured probe (2 × 106 dpm/ml). After 18 h at
55°C, filters were washed at 55°C before exposure to preflashed
photographic film (X-Omat AR, Eastman-Kodak, Rochester, NY) at
70°C. Three SMIT clones [clones 1-1, 1-2 (Fig. 1) and 1-3] were
obtained. The library was rescreened at high stringency using a
Hind
III-Sph I fragment from the 5'
end of the most 5' extended clone and clone 2-1 was obtained
(Fig. 1). Additional 5' sequence was obtained by amplification of
1 µg of purified plasmid DNA representing the entire library, using
nested primers 3' flanking an
EcoR I site in clone 2-1 and a T7
primer in the pCDNA1 vector (5' flanking an
EcoR I site in the vector). The
amplification products were digested with
EcoR I and subcloned in pBluescript
(Stratagene, La Jolla, CA), and recombinant plasmids containing the
5' end of the cDNA were identified by colony hybridization with a
5' fragment of cDNA clone 2-1. The most 5' extended cDNAs were identified by EcoR I digestion,
and the largest was designated clone 3-1 (Fig. 1). Complementary DNA
clone 4-1 (Fig. 1) was obtained by specific primer extension and DNA
amplification. After reverse transcription of RNA annealed to an
oligonucleotide complementary to sequences in clone 3-1, the cDNA was
tailed using terminal deoxytransferase and dATP. This served as a
template for amplification with an oligo(dT) primer and the original
primer. Appropriately sized PCR products (see below) were
excised from a gel, subcloned in pBluescript, and identified by
nucleotide sequencing.
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Primer extension.
Analytic primer extension was performed with synthetic
oligonucleotides derived from sequences in clone 3-1 (5'-CTTAATCCACTCTCCACAAGACCATCAG-3' complementary to nt
241-268 and 5'-CTAAGTCTAGTGAAGCCTACTGTACCAAC-3' complementary to nt 173-201; see Molecular cloning of the
full-length human SMIT cDNA). HPLC-purified
oligonucleotides were end labeled with
[-32P]ATP (6,000 Ci/mmol, Amersham Life Science, Arlington Heights, IL) and annealed either to RNA from RPE cells exposed to
HMEM for 24 h or to yeast tRNA as previously described (15). After reverse transcription, the end-labeled products were analyzed on a 6%
sequencing gel and their size compared with the nucleotide sequence
obtained by sequencing cDNA clone 3-1 with the same primers.
Cloning human SMIT exons.
A human peripheral blood leukocyte genomic DNA library prepared in
-FIX was screened. Recombinant phage (1.2 × 106) were plated at high
density, and filter lifts were prepared. In an attempt to clone the
full length of the gene, filters were hybridized at high stringency
(13) with cDNA clone 3-1 and a Pst
I-Xho I fragment from cDNA clone 1-2 (corresponding to nt 3306-3570). Two distinct subsets of
nonoverlapping phage were obtained, each hybridizing to a single probe.
After plaque purification, large-scale phage DNA isolation, and partial
restriction mapping, restriction fragments hybridizing with the
original probe were subcloned, and the
exons were sequenced. To obtain the
intervening exon(s), the library was rescreened with a
Pst I fragment from cDNA clone 1-2 (nt
2330-3309). A single clone,
2-24, which did not overlap the
sequences contained within the other phage, was obtained. A 3.0-kb
Hind
III-Xba I restriction fragment was
subcloned, and the nucleotide sequence of the contained exon was
determined. The putative first exon was identified by Southern
hybridization of an EcoR
I-Xba I digest of yeast artificial
chromosome (YAC) DNA 860G11 with a fragment of cDNA clone 4-1 (nt
36-169). Appropriately sized EcoR
I-Xba I fragments were excised and
subcloned in pBluescript, and recombinant plasmids were identified by
colony hybridization. The nucleotide sequence of the putative exon 1 was determined.
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DNA sequencing. The nucleotide sequence of both strands of the overlapping cDNA clones 1-2, 2-1, 3-1, and 4-1 were determined an average of 4.5 times/base by the dideoxy chain-termination method (15). Clone 2-1 was sequenced using a modified shotgun strategy (15). The cDNA insert was purified free of vector sequences, concatenated by ligation, and randomly sheared by sonication. The resulting DNA fragments were digested with S1 nuclease and polished with the Klenow fragment of DNA polymerase before cloning into the Sma I site of pUC19. After transformation, recombinants were identified by colony hybridization and sequenced with M13 universal primers and Sequenase (United States Biochemical, Cleveland, OH) following alkaline denaturation. The nucleotide sequence was analyzed using Genetics Computer Group software. Gaps in the sequence were determined with synthetic oligonucleotide primers.
Chromosomal assignment and fluorescence in situ hybridization. PCR amplification was performed using 20 pmol of each synthetic oligonucleotide primer [5'-ACCACCACCTTTTGGTCTAAG-3' (nt 2132-2152) and 5'-TATCCTTAAGTTCATAAGGAGAAA-3' (complementary to nt 2650-2673)] and 200 ng of genomic DNA in 50 µl of 15 mM (NH4)2SO4, 2 mM MgCl2, 60 mM Tris, pH 10.0, containing 1 U AmpliTaq (Perkin-Elmer, Norwalk, CT). A single DNA fragment of appropriate size was amplified from human genomic DNA but not from murine or hamster genomic DNA. DNA (200 ng) isolated from 24 human-rodent somatic cell hybrids, each retaining one intact human chromosome (mapping panel no. 2, Coriell Institute for Medical Research, Camden, NJ) were used in 50-µl reactions. After denaturation at 95°C for 3.5 min, the DNA was amplified 30 times as follows: denaturation at 94°C for 1.5 min, annealing at 56°C for 1.5 min, and extension at 72°C for 2 min. A final elongation reaction was conducted at 72°C for 8 min. Aliquots of reactions were electrophoretically separated on a 2% agarose gel, and DNA was visualized by staining with ethidium bromide. A full-length SMIT cDNA was used for fluorescence in situ hybridization, as previously described (22).
YAC localization.
To more precisely localize the human SMIT gene, YAC DNA containing
segments of genomic DNA from the region of 21q22.13 to proximal q22.3
were screened by Southern hybridization. YAC DNAs obtained from the
University of Michigan Genomic Resources Core facility and from
Research Genetics (Huntsville, AL) were hybridized with exon-specific
probes as described below
(Probes). YACs spanning the region included YAC clones 860G11, 872B5, 874C1, 191D3, and 73D10.
After determination that all of the probes hybridized to the YAC DNA
860G11, Southern blots were prepared from human genomic DNA, YAC 860G11
DNA, and 1-36 and
1-38 phage DNA after restriction endonuclease
digestion with Hind III or
Pst I.
Northern blots.
Denatured total RNA (10 µg) from cells exposed to IMEM or HMEM for
various times, or poly(A)+ RNA (3 µg) from cells exposed to IMEM, and a 0.24- to 9.5-kb RNA ladder
(Life Technologies, Gaithersburg, MD) were resolved on 2.2 M
formaldehyde-1% agarose gels, transferred by capillary blotting to
ZetaBind filters (CUNO, Meriden, CT), stained with methylene blue to
confirm the integrity, uniformity of loading, and completeness of
transfer, and fixed in vacuo at 80°C for 2 h. Filters were
prehybridized (13) at 65°C for 4-6 h before adding denatured
probe (1 × 106 dpm/ml).
After 18 h, the filters were washed at 65°C, before exposure to
preflashed film at 70°C. Exposure was adjusted from 3 to 15 days, depending on the probe.
Probes.
Northern and Southern blots were hybridized with the 2.3-kb clone 1-2 cDNA insert or a 1.0-kb Pst I fragment
that spanned exons 2-5. Probes for exons 2 and 5 were derived from
a 1.3-kb Pst I fragment (nt
1064-2334) from cDNA clone 2-1 and a
Xho I fragment (nt 2976-3569)
from cDNA clone 1-2, respectively. Probes for exons 1, 3, and 4 were
prepared by amplification from cDNA clones using synthetic
oligonucleotides as follows: exon 1, a 134-nt fragment from cDNA clone
4-1 (corresponding to nt 36-169); exon 3, a 93-nt fragment from
cDNA clone 1-2 (nt 2680-2772); and exon 4, a 131-nt fragment from
cDNA clone 1-2 (nt 2793-2923). All amplification products were
subcloned in pBluescript and sequenced to confirm their identity. All
fragments were excised from the vector, purified from agarose or
acrylamide gels, and labeled to a specific activity of
~109 dpm/µg using random
primers (24) and
[-32P]dCTP (3,000 Ci/mmol, Amersham, Arlington Heights, IL). Labeled fragments were
separated from unincorporated label by gel filtration, denatured by
boiling, and added at a final amount of 2 × 106 dpm/ml. Northern blots were
subsequently stripped and rehybridized with human AR (24) and chicken
-actin (7) cDNA probes.
Cloning the "1.2-kb SMIT transcript." The smallest ~1.0-kb transcripts from canine kidney cortex, medulla, and MDCK cells (16) and human RPE cells (see Northern analysis) that hybridize, respectively, with canine and human SMIT cDNAs are too small to encode an ~718-amino acid SMIT protein. Exon-specific probes suggested that the "1.2-kb SMIT transcript" from human RPE cells contains sequences from putative exons 1, 4, and 5 but not exon 2 (which contains the SMIT ORF; see Cloning exons of the human SMIT gene). To clone cDNAs derived from the 1.2-kb SMIT transcript, the osmotically induced RPE cDNA library (4 × 105 transformants) was screened at high stringency with the exon 5 probe. More than 700 colonies hybridized strongly to the probe. Eighteen of these were screened by PCR using a 5' primer from exon 1 (corresponding to nt 36-169) and a 3' primer spanning exons 4 and 5 (complementary to nt 2902-2923). cDNAs derived from longer SMIT transcripts (i.e., those containing exon 2 sequence) would not be expected to amplify efficiently under the conditions employed and should yield products >2 kb in size. Six clones yielded products consistent with the predicted composition of the 1.2-kb SMIT transcript. Three of these were purified and sequenced in their entirety on both strands. To more accurately determine the size of the 1.2-kb SMIT transcript, RNA (5 µg) was resolved as described above on 2% agarose gels containing a 0.16- to 1.77-kb RNA ladder (Life Technologies), and Northern blots were probed with an exon 5 probe.
Synthetic oligonucleotide synthesis.
All synthetic oligonucleotides were synthesized by the DNA Synthesis
Core Facility, a part of the Michigan Diabetes Research and Training
Center and the University of Michigan's Biomedical Research Core
Facilities, on automated DNA synthesizers (Applied Biosystems, Foster
City, CA) employing -cyanoethyl phosphoramidite chemistry on
controlled pore glass supports.
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RESULTS |
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Molecular cloning of the full-length human SMIT cDNA. Human SMIT cDNAs were cloned from a human RPE cell library prepared from RNA isolated after 24 h exposure to HMEM. The library was initially screened at reduced stringency with a fragment of the canine SMIT cDNA containing coding and 3' untranslated sequence (16). Three unique overlapping cDNAs [clone 1-1, 1-2 (Fig. 1), and 1-3] with similar restriction maps spanned some 3 kb. Limited nucleotide sequencing from the 5' end demonstrated considerable homology with the canine sequence. The nucleotide sequence of clones 1-2 and 1-3 terminated in a 3' poly(A) tail, but otherwise there was no similarity to the canine 3' sequence. Because none of the cDNAs extended to the translation initiation site, the 5' end of the most 5' extended clone was used to rescreen the library at high stringency. This resulted in a single additional clone (clone 2-1, Fig. 1), which was 100% identical in a 136-nt overlap with clone 1-2 but which failed to extend to the 5' end of the transcript. To clone more 5' sequence, PCR was performed using an anchored primer 5' flanking the polylinker in pCDNA1 and nested primers, complementary to sequences in clone 2-1, 3' to an EcoR I site. Total library DNA was amplified, digested with EcoR I, and subcloned. Nucleotide sequence analysis of the largest clone (Fig. 1, clone 3-1) was identical to clone 2-1 in a 268-nt overlap, but primer extension (see below) experiments suggested that the 5' end of the transcript was ~200 nt upstream. Because PCR amplification of the library failed to yield this sequence, a strategy of 5' rapid amplification of cDNA ends (9) was employed, using specific primer cDNA synthesis from RPE cell RNA and poly(A) tailing. Subsequent amplification, purification, and subcloning of appropriately sized PCR products yielded clone 4-1 (Fig. 1), spanning 268 nt with 100% sequence identity with clone 3-1 in a 72-nt overlap. Analytic primer extension was performed with different primers complementary to sequences in cDNA clones 3-1 and 4-1 (Fig. 2). Resolution of the primer extension products on a denaturing sequencing gel revealed little heterogeneity, and the size obtained with each primer was consistent with a single transcription initiation site only 5 nt 5' to the end of clone 4-1. No difference in the mobility of the primer extension products was noted when RNA from RPE cells in IMEM or HMEM was utilized (data not shown), suggesting that a single promoter transcribes the gene under both conditions.
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Northern analysis. Northern blots of total RNA isolated from RPE cells maintained for 24 h in IMEM or HMEM were hybridized under high-stringency conditions with the 2.3-kb SMIT clone 1-2 and the size of the SMIT transcripts determined from an RNA ladder. Six transcripts were evident in RNA from RPE cells exposed to HMEM (16, 11.5, 9.8, 8.5, 3.8, and 1.2 kb), all of which were significantly increased compared with the RNA from IMEM cells. Indeed, only the 1.2-kb transcript was readily detectable in cells in IMEM, with an 11.5-kb transcript evident only after prolonged (2 wk) exposure (Northern not shown). After transfer to HMEM, there was a time-dependent increase in SMIT transcripts (Fig. 4), with the 16-kb transcript appearing earliest and declining more rapidly than the other SMIT transcripts. SMIT transcripts <16 kb in apparent size reached a maximum abundance (40-fold increase) after 24 h in HMEM and returned to baseline after 48 h in HMEM. In contrast, AR mRNA increased at 8 h, reached a maximum at 24 h, but persisted for at least 48 h in HMEM. When a 1.3-kb Pst I fragment from SMIT clone 2-1 containing only coding sequence was used as a hybridization probe, all of the transcripts except the 1.2-kb mRNA hybridized (see Differential exon utilization and SMIT transcript diversity).
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Cloning exons of the human SMIT gene.
Different transcription initiation sites (alternate promoters) could
not readily explain the presence of diverse SMIT transcripts, since
primer extension studies with several different primers all suggested a
single initiation site. Heterogeneity at the 3' end of the
transcripts, either through the use of alternate polyadenylation sites
in the same (1) or different exons, was explored as an alternative
basis for multiple SMIT transcripts. A human genomic library screened
with fragments of the cDNA from the 5' and 3' ends yielded
two nonoverlapping clones, 1-36 and
1-38 (Fig. 5). The restriction fragments containing
the putative exons were subcloned and sequenced using synthetic
oligonucleotide primers. A single exon identified in
1-36 was 100%
identical to the sequence between nt 172 and 2675 (Fig. 3) and flanked
by appropriate splice donor-acceptor sequences (Table 1). The map of
this clone was similar to that described by Berry et al. (1), and this
exon contained the entire ORF, as reported. Clone
1-38 contained a single exon encoding a 3' UTR sequence distal to nt 2913. Rescreening the library yielded one additional clone,
2-24 (Fig. 5),
which contained a 140-nt exon encoding sequences between nt 2773 and 2912. Thus only 171 nt of 5' UTR sequence and 96 nt of 3'
UTR were unaccounted for, suggesting that the SMIT gene is encoded by a
minimum of five exons. The putative first exon was subsequently subcloned from YAC DNA 860G11 (see below).
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Chromosomal localization.
Because the description of the SLC5A3 locus (1) did not include unique
flanking sequences that could be used to confirm the identity of clone
1-36, chromosomal assignment studies were performed. A mapping panel
of human-rodent somatic cell hybrid DNAs (panel 2, Coriell Institute
for Medical Research) was screened by PCR using sequences derived from
exon 2. These primers amplified an appropriately sized product only
from GM/NA10323 (data not shown), consistent with SMIT assignment to
chromosome 21. Southern hybridization of the PCR products failed to
reveal additional bands. High-resolution fluorescence in situ
hybridization studies (data not shown) were concordant with the mapping
panel results and the localization of the gene to 21q22.1 to q22.2 (1).
Refinement of this localization was performed using YAC DNAs from this
region. Southern blots of YAC DNA were hybridized to fragments of the cDNA. YAC clone 860G11 but not flanking YAC clones 872B5, 874C1, 191D3,
and 73D10 hybridized with the cDNA probes. To confirm that this
hybridization was not spurious, Southern blots of human genomic DNA,
YAC 860G11, and
1-36 or
1-38 restricted with
Hind III or Pst I were hybridized with fragments
derived from exon 2 or 5, respectively (Fig.
6). In each case, the restriction fragments were similar in size and number. Because the most 5' untranslated sequence contained within clone 4-1 hybridized to an appropriately sized 4.0-kb EcoR
I-Xba I fragment from YAC clone
860G11, appropriately sized fragments of YAC clone 860G11 DNA were
subcloned and identified by colony hybridization and nucleotide
sequencing. The nucleotide sequence was 100% identical to clone 4-1 in
a 173-nt overlap, and the 3' end was flanked by appropriate
splice donor sequences (Table 1).
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Differential exon utilization and SMIT transcript diversity. Because little heterogeneity of SMIT transcripts was identified at the 5' end by primer extension, exon-specific probes were hybridized to Northern blots of RNA obtained from RPE cells maintained in IMEM or HMEM for 24 h (Fig. 7). As noted above, six transcripts were evident when hybridized using the 2.3-kb clone 1-2 cDNA insert that spanned exons 2-5. Only the 1.2-kb transcript was readily identified in RNA from cells in IMEM with this probe. A probe from exon 1 hybridized to all six transcripts, consistent with the lack of heterogeneity at the 5' end. However, as described above, the exon 2 probe failed to hybridize to the 1.2-kb transcript. Exon 3 sequences were preferentially represented in the 11.5-kb transcripts, whereas exon 4 and 5 sequences were preferentially represented in the 3.8- and 1.2-kb transcripts. These results demonstrate that the diverse SMIT transcripts noted by filter hybridization were due at least in part to differential exon utilization.
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DISCUSSION |
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Intracellular concentrations of myo-inositol and the kinetic and biochemical characteristics of myo-inositol transport vary widely among and within mammalian cell types and tissues (3, 4, 6, 8, 14, 20, 21, 25, 29; Porcellati, Hosaka, Hlaing, Togawa, Larkin, Stevens, Killen, and Greene, unpublished observations), with some cells expressing more than one class of Na+-myo-inositol cotransport activity (4, 8). The effects of diabetes or hyperglycemia on SMIT regulation are equally complex (20). The maximal velocity of Na+-myo-inositol cotransport activity has been reported to be increased by experimental diabetes or hyperglycemic concentrations of glucose in renal (6) and endothelial (20) cells. The same manipulations have been reported to diminish Na+-myo-inositol cotransport activity in a variety of experimental systems including incubated endoneural preparations from streptozotocin-diabetic rats (10) and various cell culture models (3, 4, 14, 18, 32). AR inhibitors that block the formation of the intracellular osmolyte sorbitol prevent both the reported increases (6) and decreases (3, 10, 18, 32) in Na+-myo-inositol cotransport activity produced by glucose, suggesting that the regulation of Na+-myo-inositol cotransport by alternative intracellular osmolytes may be complex.
The heterogeneity of Na+-myo-inositol cotransport function and regulation and SMIT expression is poorly understood in molecular terms. A SMIT cDNA was functionally cloned in Xenopus oocytes (16) from a hypertonic MDCK cDNA library prepared from poly(A)+ RNA with a median size of 4 kb (17). Despite its origin in a 4-kb RNA pool, the "full-length" canine SMIT cDNA comprising 5' UTR, ORF, and 3' UTR hybridized to multiple transcripts ranging from 1 to 13.5 kb but most strongly to a 10.5-kb transcript (16). Subsequent studies using the canine probe (21) or probes amplified from the coding sequences of rat (29) or bovine (33) SMIT mRNA demonstrated hybridization to a similarly broad diversity of SMIT transcripts in hypertonically stressed cells in vitro (21, 28, 33) or tissues in vivo (29, 30). Although SMIT steady-state mRNA is generally induced by hypertonicity (31), the multiple SMIT transcripts consistently noted by Northern analysis (16, 21) are rapidly up- and downregulated by osmotic stress (21) in a cell-specific fashion not fully explained by tonicity alone (29). Both transcriptional regulation (31) and alterations in mRNA stability (21) have been invoked in this process.
It has been suggested that this heterogeneity may derive from distinct members of a SMIT gene family (1), yet only a single genomic sequence has been reported (1). Berry and co-workers obtained a genomic fragment localized to chromosome 21q22.1-22.2 (1) containing an intronless ORF potentially encoding a full-length SMIT polypeptide. Northern blots performed with a 1.3-kb Pst I fragment of the ORF hybridized to transcripts in a variety of tissues. SMIT mRNA was most abundant in the kidney, with multiple transcripts similar in mobility to those described by others (16, 21, 29, 30, 33). It was speculated that the diverse SMIT transcripts derived from the use of alternate polyadenylation sites in the 3' UTR. A similar intronless ORF has been noted in the canine gene (1).
Here we describe the isolation and characterization of overlapping SMIT cDNAs derived from a size-selected, tonicity-induced human RPE cell library. The 5' UTR and coding sequences are highly conserved compared with the canine sequence and the coding sequence is virtually identical with the sequence reported for the chromosome 21 ORF (1). The 3' UTR, however, diverges significantly from the canine sequence, suggesting a possible cloning artifact. However, the 3' UTR sequence was similar in two independent isolates (clones 1-2 and 1-3), and Northern blots using probes derived from the 3' UTR hybridized (with the exception of the 1.2-kb transcript) to transcripts with the same mobility as detected by probes containing the coding sequence. Isolation of genomic DNA fragments containing putative SMIT exons 1, 2, 4, and 5 suggests that the SMIT gene is more complex than previously envisioned (1) (Fig. 9). The genomic DNA clones did not overlap, but all of the sequences were contained in YAC 860G11, making it unlikely that they derive from distinct transcriptional units. Indeed, all SMIT RNA transcripts hybridized with a putative exon 1 probe and primer extension studies using multiple primers were consistent with a single transcription initiation site for the SMIT gene. These results were the same with RNA isolated from cells in IMEM or HMEM, suggesting that there is a single promoter. The ~900 bp sequence 5' flanking the putative first exon functioned to initiate transcription using chimeric reporter constructs, but its activity was not altered by hypertonic stress (Hlaing, Killen, and Greene, unpublished observations). Similar results have been obtained with the canine SMIT gene (Ref. 26; personal communication, H. Moo Kwon, Johns Hopkins University).
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It seemed likely that the diverse SMIT transcripts observed by filter hybridization might derive in part from alternate splicing, as depicted in Fig. 9. Indeed, exon-specific probes selectively hybridized to transcripts of different size. Perhaps the most intriguing transcript is the 1.2-kb transcript (or, more appropriately, the "~1-kb transcript"), similar in mobility to transcripts noted in hypertonically stimulated MDCK and canine renal medulla; this transcript is too small to encode a SMIT-like molecule, and its function is unknown. This transcript was previously noted only when full-length canine SMIT probes were used, i.e., with probes containing sequence derived from the first exon (16). Others have failed to note this transcript, most likely because they employed probes derived from exon 2 coding sequence. In human RPE cells, this transcript is actually a 0.95-kb sequence derived from exons 1, 4, and 5, and it contains an ORF that shows little sequence homology with proteins within existing databases. It is, however, interesting to note that alternate splicing may not be limited to untranslated sequence. An alternate splice donor site within the SMIT ORF appears to give rise to a family of SMIT transcripts, which encode distinct SMIT isoforms different in their COOH-terminal sequence (Porcellati, Hosaka, Hlaing, Togawa, Larkin, Stevens, Killen, and Greene, unpublished observations).
The significance of the differing 3' UTR sequences derived from exons 3, 4, and 5 on the expression of the 718-amino acid SMIT is presently unknown. The largest SMIT transcript (16 kb) increases in abundance earlier and declines more rapidly than the smaller transcripts during exposure to HMEM. This is consistent with the hypothesis that alternate splicing is a regulated process and that the resulting distinct mRNA sequences could influence mRNA stability. The existence of these diverse 3' UTR sequences in RNA from RPE cells under isotonic conditions eliminates the possibility that these higher molecular weight transcripts are an artifact of osmotic stress. Preliminary studies suggest that the ~1-kb SMIT transcript devoid of exon 2 and exon 3 sequence appears to be considerably more stable than the 11.5-kb transcript, which contains these and other sequences, after inhibition of transcription (Hosaka, Larkin, Stevens, Killen, and Greene, unpublished observations). Exposure of RPE cells to isotonic hyperglycemic (i.e., 20 mM glucose) conditions simulating the diabetic milieu markedly decreases the apparent half-life of the 11.5-kb transcript (Hosaka, Larkin, Stevens, Killen, and Greene, unpublished observations). The extent to which variability in SMIT transcript stability contributes to tissue-specific differences in the response of Na+-myo-inositol cotransport activity to hyperglycemic or osmotic stress remains to be elucidated. Stability studies with specific reporter constructs will be required in order to establish the role of diverse 3' UTR sequences in these phenomena. At the very minimum, these results suggest that studies of the physiology of SMIT expression must address possible transcript-specific effects.
In summary, the studies reported in this communication confirm the reported chromosomal assignment of a human SMIT gene (1) but describe a complex multiexon transcriptional unit that gives rise to a series of transcripts by alternate splicing. The smallest SMIT transcript derives from exons 1, 4, and 5 and therefore lacks sequence from the ORF described by Berry et al. (1). Its functional significance, if any, is unclear. Exons distal to exon 2 encode a heterogeneous set of 3' UTRs that contribute to a diverse array of variously sized transcripts that may exhibit differential induction and half-life after osmotic stress. The complexity of the SMIT gene may have important but as yet unexplored implications for SMIT gene expression and the regulation of Na+-myo-inositol cotransport activity under physiological and pathophysiological conditions.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge helpful discussions of the data with David Kurnit (University of Michigan) and H. Moo Kwon (Johns Hopkins Medical School).
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FOOTNOTES |
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D. A. Greene, D. N. Henry, F. Porcellati, and M. Togawa were supported in part by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant P60-DK-20572 (Michigan Diabetes Research and Training Center). P. D. Killen was supported by NIDDK Grants RO1-DK-44848 and P50-DK-39255. D. A. Greene was supported by NIDDK Grant RO1-DK-38304. D. N. Henry was supported by National Institute of Child Health and Human Development Grant P30-HD-25520 and NIDDK Grant DK-02193 and by the Juvenile Diabetes Foundation.
Present addresses: F. Porcellati, Instituto di Medicina Interna e Scienza Endocrin e Metaboliche, Via Enrico Dal Pozzo, 06126 Perugia, Italy; M. Togawa, Shiga University of Medical Science, Third Department of Medicine, Seta, Otsu, Shiga 520-21, Japan; D. N. Henry, Michigan State University, Department of Physiology, 113 Giltner Hall, E. Lansing, MI 48814-1101.
Address for reprint requests: P. D. Killen, Dept. of Pathology, Box 0602, 1301 Catherine St., Ann Arbor, MI 48109-0602.
Received 23 January 1997; accepted in final form 16 January 1998.
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