From the Medical Research Service, Department of Veterans Affairs
Medical Center, San Diego, California 92161 and the UCSD Cancer Center
and the UCSD/Whittier Diabetes Research Program, Department of
Medicine, Division of Endocrinology and Metabolism, University of
California, San Diego, La Jolla, California 92093
The insulin receptor exists as two isoforms, A
and B, that result from alternative splicing of exon 11 in the primary
transcript. We have shown previously that the alternative splicing is
developmentally and hormonally regulated. Consequently, these studies
were instigated to identify sequences within the primary RNA transcript
that regulate the alternative splicing. Minigenes containing exons 10, 11, and 12 and the intervening introns were constructed and transfected into HepG2 cells, which contain both isoforms of the insulin receptor. The cells were able to splice the minigene transcript to give both A
(
exon 11) and B-like (+ exon 11) RNAs. A series of internal deletions within intron 10 were tested for their ability to give A and
B RNAs. Intron 10 contained two sequences that modulated exon 11 inclusion; a 48-nucleotide purine-rich sequence at the 5' end of intron
10 that functions as a splicing enhancer and causes an increase in exon
11 inclusion, and a 43-nucleotide sequence at the 3' end of intron 10 upstream of the branch point sequence that favors skipping of exon 11. Increasing the length of the polypyrimidine tract at the 3' end of
intron 10 caused exon 11 to be spliced constitutively, indicating that
a weak splice site is required for alternative splicing. Finally, point
mutations, insertions, and deletions within exon 11 itself were able to
regulate inclusion of the exon both positively and negatively.
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INTRODUCTION |
The human insulin receptor
(IR)1 is encoded by a single
gene that is located on chromosome 19 and composed of 22 exons. The mature IR exists as two isoforms, designated A and B, which result from
alternative splicing of the primary transcript (1-3). The A isoform
lacks exon 11, is expressed ubiquitously, and is the only isoform in
lymphocytes, brain, and spleen; the B isoform contains exon 11 and is
expressed predominantly in liver, muscle, adipocytes, and kidney
(4-6). Exon 11 is composed of 36 nucleotides that encode a 12-amino
acid segment (residues 717-728) of the carboxyl terminus of the
-subunit of IR. A number of investigators have suggested that the
isoform ratio could be altered in non-insulin-dependent diabetes mellitus (7-10), but other studies have produced conflicting results (11-14). We have found that alterations in isoform ratio in
skeletal muscle were associated with hyperinsulinemia rather than
diabetes (15). Similar results have been found in the rhesus monkey
(16). Along these lines, Sell and co-workers (17) have shown that
alternative splicing of the IR gene is regulated by insulin in the Fao
hepatoma cell line. Furthermore, we have shown that the alternative
splicing is hormonally and developmentally regulated in both the HepG2
hepatoma and 3T3-L1 adipocyte cell lines (18). The changes in splicing
were accompanied by increases in insulin sensitivity, as measured by a
number of parameters (19). These data indicate that regulation of the
alternative splicing of the IR is important for insulin sensitivity and
responsiveness.
Splicing of pre-mRNA depends on the presence of relatively short
RNA sequence elements, the 3' splice site, the 5' splice site, the
branch point sequence, and the polypyrimidine tract. In alternative
splicing, a given splice site may be selected or ignored depending on
the cell type or physiological state. This apparent flexibility of the
splicing machinery raises the question of molecular mechanisms involved
in selection of certain splice sites over others. A number of factors
have been implicated in the choice of alternative splice sites,
including RNA secondary structure (20-23), size of the exon (24, 25),
and relative strengths of the competing splice sites (26). Alterations
in the splicing pattern of a number of genes have been demonstrated during cellular differentiation (27); however, the hormonal regulation
of alternative splicing is not as common. We have shown that the
alternative splicing of exon 11 of the IR gene is modulated by
glucocorticoids in HepG2 cells, and, as mentioned above, insulin modulates splicing in Fao cells (17, 18). Insulin has also been shown
to alter the splicing pattern of the COOH terminus of protein kinase
C-
in L6 myotubes, and growth factors including epidermal growth
factor, platelet-derived growth factor, and basic fibroblast growth
factor alter the splicing of the COOH terminus of protein-tyrosine
phosphatase 1B in human fibroblasts (28, 29). Chew and co-workers (30)
have shown that splicing of the insulin-like growth factor-I mRNA
is regulated by growth hormone in HepG2 cells. The molecular mechanisms
involved in the hormonal regulation of this process are not understood.
A prerequisite for mechanistic studies of hormonal regulation is a
knowledge of the RNA sequences involved in the alternative splicing
event. To that end, we have identified the regions of intron 10 and
exon 11 involved in the alternative splicing of the IR gene.
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EXPERIMENTAL PROCEDURES |
Materials--
Cell culture reagents were purchased from Life
Technologies, Inc., and fetal calf serum was from Gemini Bioproducts
(Calabasas, CA). [
-32P]dCTP (3,000 Ci/mmol) was
purchased from ICN (Costa Mesa, CA). Taq DNA polymerase
(Ampli-Taq) was purchased from Perkin-Elmer. All other
chemicals were purchased from Sigma or Fisher.
Construction of Plasmids and Site-directed Mutagenesis--
All
recombinant DNA manipulations were carried out according to standard
protocols. The minigenes were constructed by amplifying regions of the
IR gene from HepG2 genomic DNA by PCR using primers containing unique
EcoRI, BamHI, HindIII, and
BglII restriction sites to allow replacement of segments of
the minigene as cassettes. We were not able to obtain the complete
intron 11, as it is >8 kb. Consequently, we used the known intronic
sequence to amplify the two ends of the intron. Thus, all minigenes
contain approximately 180 nucleotides at each end of intron 11. In all
plasmids, the minigenes were inserted between EcoRI and
BglII sites of pSG5 vector (Stratagene, La Jolla, CA), which
contains the SV40 early promoter/enhancer, a rabbit
-globin intron
2, and an SV40 poly(A) signal after the multiple cloning site. All
mutants were generated from the complete minigene B, which was composed
of five elements (part of exon 10, entire intron 10, exon 11, a deleted
intron 11, and part of exon 12). Fig. 1
shows schematic diagram of the minigene and the sequence of the introns
and exons. Deletions and mutations were verified by dideoxy
sequencing.

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Fig. 1.
Structure of IR minigenes containing exon 11. Panel A, schematic of IR minigene. Basic minigene contains
110 nucleotides of exon 10, 2.3 kb of intron 10, 36 nucleotides of exon
11, 372 nucleotides of intron 11, and 103 nucleotides of exon 12. Intron 11 is greater than 8 kb in length; consequently, a large
internal deletion was created leaving approximately 180 nucleotides at
both the 5' and 3' ends. Corresponding amino acid residues are
indicated below the minigene. Numbers above intron 10 indicate the positions used to create the internal deletions described
in this paper. The two As indicate potential BPS upstream of
the 3' splice site. Minigenes are subcloned into mammalian expression
vector pSG5. Panel B, sequence of IR minigene. Sequence of
basic minigene is shown divided into exons and introns. The end points
of the internal deletions are indicated by numbers below the
sequence of intron 10.
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Cell Culture and Transfection--
HepG2 cells were maintained
routinely in minimum essential medium plus Earle's salts with 10%
fetal calf serum at 37 °C under 5% CO2. The cells were
plated at a density of ~2 × 106 cells/well in
six-well plates. Medium was changed every 2 days. Minigene
plasmids were transfected into HepG2 cells by the calcium phosphate
co-precipitation technique. Cells were harvested 48 h later, and
total cellular RNA was prepared using RNAzol B (Tel-Test, Inc.,
Friendswood, TX) according to the manufacturer's protocol.
Reverse Transcription and Amplification of
cDNA--
First-strand cDNA was prepared by reverse
transcription using 1.0 µg of total RNA in a volume of 20 µl (250 pmol of random hexamer primers, 1 unit of Inhibit-ACE RNase inhibitor
(5 Prime
3 Prime, Inc., Boulder, CO), 200 units of Moloney murine
leukemia virus reverse transcriptase (Life Technologies, Inc.), 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 1 mM dithiothreitol, and
1 mM dNTPs) at 42 °C for 1 h. DNA/RNA hybrids were
denatured at 95 °C for 2 min.
To measure exogenous minigene IR transcripts, the primer pair consisted
of oligonucleotides spanning nucleotides 1022-1042 (sense primer,
5'-TAATACGACTCACTATAGGGC-3') and 1068-1088 (antisense primer,
5'-GCTGCAATAAACAAGTTCTGC-3'), which are unique sequences in the pSG5
vector just upstream and downstream of the cloning sites. To measure
endogenous IR transcripts, the primer pair consisted of
oligonucleotides spanning nucleotides 2140-2163 (sense primer, 5'-AACCAGAGTGAGTATGAGGATTCG-3') and 2424-2447 (antisense primer, 5'-TTCTCAAAAGGCCTGTGCTCCTCC-3') of the IR cDNA (numbering as in Ref. 1), which lie outside the minigene and, thus, are specific for the
endogenous gene.
Five µl of the cDNA synthesis reaction was used for PCR
amplification in a 50-µl final reaction volume (0.5 µM
each oligonucleotide primer, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.1 mM dNTPs, 2 units of Taq DNA polymerase, and 1 µCi of [
-32P]dCTP). Twenty-five cycles of
amplification were performed using a Perkin-Elmer DNA thermal cycler
(System 9600). Each cycle consisted of a 30-s denaturation at 94 °C,
a 30-s annealing at 55 °C, and a 60-s extension at 72 °C. The
number of cycles was optimized to ensure that the amplification lay
within the exponential phase. The products of the PCR amplification
were resolved by electrophoresis on 8% polyacrylamide gels. The gels
were dried and exposed to film at room temperature. The band densities
were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale,
CA).
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RESULTS AND DISCUSSION |
Intron 10 Contains All the Sequence Information for Alternative
Splicing of Exon 11--
We have shown previously that the endogenous
IR gene in HepG2 cells generates mRNA for both the A and B isoform
IR. We used this cell line to investigate which sequences surrounding
the alternatively spliced exon 11 are involved in the alternative splicing. A minigene was created from by amplifying each exon and the
adjacent intron from the known exon/intron sequences (2). This minigene
contained 110 nucleotides of exon 10, 2.3 kb of intron 10, 36 nucleotides of exon 11, 184 nucleotides of the 5' end of intron 11, 188 nucleotides of the 3' end of intron 11, and finally 103 nucleotides of
exon 12 (Fig. 2, minigene B).
Transfection of minigene B into HepG2 cells gave RNA corresponding to
both the A (
exon 11) and B (+ exon 11) isoform splicing patterns in
a 40:60 ratio (Fig. 2, panels B and C). The
endogenous gene in these cells expressed both isoform RNAs in a 50:50
ratio using the endogenous gene primer pair (data not shown),
suggesting that the minigene contained most of the information for
correct splicing. A second minigene (minigene A), containing a large
internal deletion of 2.0 kb in intron 10 (between positions 3 and 5 in
the minigene), caused an increase in the percent of B splicing isoform
(Fig. 2, compare minigenes A and B). A further deletion (to position 7)
in minigene C caused a complete loss of B isoform splicing suggesting
that sequences in this region (between positions 5 and 7) may be
important for skipping of exon 11.

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Fig. 2.
Transfection of minigenes into HepG2 cells.
Panel A, structure of basic minigene and four internal
deletions of intron 10 in order of decreasing size. Deletions are
indicated by dashed lines. Numbers above intron
10 refer to the end points of the deletions. The As indicate
potential branch point sequences. Panel B, autoradiogram of
representative gel showing splicing of transfected minigenes. Minigenes
were transfected into HepG2 cells by the calcium phosphate
co-precipitation technique. Forty-eight hours later, cells were
harvested and total RNA isolated. RNA was subjected to RT-PCR as
described under "Experimental Procedures." PSG indicates
cells transfected with the parental expression vector without a
minigene insert. Letters indicate minigene construction.
Letters A and B to left of the gel
show the PCR products generated from A ( exon 11) and B isoform (+ exon 11) splicing (274 and 310 base pairs, respectively). Panel
C, quantification of alternative splicing. Gels were quantified using a
PhosphorImager. Results are from four independent experiments performed
in duplicate and are expressed as percent of B isoform (+ exon 11)
splicing and show the mean ± S.E. Lowercase letters
indicate statistical significance: a, p < 0.01 vs. minigene B; b, p < 0.01 vs. minigene A; c, p < 0.01 vs. minigene B and C. Panel D, identification of
intermediate splicing products. PCR amplification was performed for 40 cycles in the absence of [32P]dCTP and analyzed on a
1.5% agarose gel. Products from fully spliced mRNA, precursor RNA
and an intermediate are indicated to the left of the
gel.
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Two potential branch point sequences (BPS) were identified in the
intron upstream of exon 11 (indicated by A in Fig. 2A). Removal of the upstream potential BPS by deletion to position 9 (minigene D) caused a large (80%) decrease in the amount of B isoform
splicing, suggesting that this BPS may be involved in splice site
selection. These deletions suggested that the regulatory sequences
might lie in the region upstream of exon 11. Therefore, intron 10 was
sequenced from the 3' end, and an Alu repetitive sequence was found
approximately 300 nucleotides upstream of exon 11. A further minigene
was created (minigene E) deleting the central portion of the intron up
to, but no further than, the Alu repeat. Transfection of minigene E
into HepG2 cells gave a splicing ratio that was identical to minigene
A; therefore, the extra sequence up to the Alu repeat did not affect
splicing.
The PCR amplification was performed for 40 cycles in an attempt to
identify low abundance intermediate splice products for these five
minigenes (Fig. 2D). The mature, fully spliced products (mature mRNA) were observed as well as the unspliced precursor RNAs
(pre-mRNA) that differed in size due to the internal deletions in
the minigenes. No precursor RNA was observed for minigene B as it is
predicted to be >3 kb and is not amplified under standard conditions.
A product of ~700 base pairs was observed for all minigenes
regardless of the size of the internal deletion in intron 10. This PCR
product was subcloned, sequenced, and found to be a splicing
intermediate lacking intron 10 but still containing intron 11. No
intermediates containing intron 10 but lacking intron 11 were isolated.
Thus, removal of intron 10 precedes removal of intron 11 in agreement
with the processive model of RNA splicing and suggests that the 3'
splice site of exon 11 is subject to selection.
Intron 10 Contains a Splicing Enhancer Sequence and an Inhibitory
Region That Causes Exon Skipping--
The increase in B isoform
splicing by the internal deletions in minigenes A, C, and E in Fig. 2
could be explained by alterations in the spacing of the splice sites.
If this were the case, then larger deletions should have an even
greater effect. Consequently, minigenes K, L, and M were constructed
with larger deletions starting 26 nucleotides downstream of the 5'
splice site of exon 10 (Fig. 3, position
1) and extending to the end points of minigenes A, C and D (Fig. 3,
minigenes K, L, and M, respectively). Surprisingly, transfection of
minigene K into HepG2 cells gave <10% B isoform splicing in contrast
to minigene A which gave >85%. It is very striking that elimination
of intronic sequences in the 5' end of intron 10 (between positions 1 and 3) caused a dramatic (75%) decrease in exon 11 inclusion. This
suggests that this region contains a sequence that enhances inclusion
of exon 11. Deletion of the sequences between positions 5 and 7 (minigene L) caused a large increase in the amount of B isoform
splicing consistent with the results from minigenes A and C (Fig. 3).
The change in splice site usage is even more dramatic for minigenes K
and L than for minigenes A and C, as a result of the absence of the upstream enhancer that favors exon 11 inclusion. These results confirm
that sequences between positions 5 and 7 cause exon skipping. Elimination of an additional 26 nucleotides to position 9 (minigene M)
caused complete loss of B isoform splicing. A similar loss in B isoform
splicing was associated with deletion of this region in minigenes C and
D. However, minigene D still showed approximately 20% B isoform
splicing, suggesting that this deletion severely weakens the 3' splice
site but partial recognition is possible in the presence of the
upstream enhancer.

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Fig. 3.
Identification of splicing enhancers and
inhibitors in intron 10. Left panel, structure of minigenes
used in these experiments. Deletions are indicated by dashed
lines. Numbers above intron 10 refer to the end points
of the deletions. The As indicate potential branch point
sequences. Right panel, quantification of alternative
splicing by RT-PCR. Minigenes were transfected into HepG2 cells by the
calcium phosphate co-precipitation technique. Forty-eight hours later,
cells were harvested and total RNA isolated. RNA was subjected to
RT-PCR as described under "Experimental Procedures." Gels were
quantified using a PhosphorImager. Results are from four independent
experiments performed in duplicate and are are expressed as percent of
B isoform (+ exon 11) splicing and show the mean ± S.E. The
vertical dashed line indicates the degree of exon
incorporation observed with the parental minigene. ns, not
significantly different from the parental minigene B.
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This upstream splicing enhancer was identified by comparing minigenes A
and K. Both of these minigenes contain large internal deletions within
intron 10, which brings the enhancer closer to the 3' splice site. What
is the function of this region in the context of the full intron? This
region was deleted in the full intron (Fig. 3, minigene W). Deletion of
this region caused a 20% decrease in B isoform splicing. Consequently,
this region does function as a splicing enhancer but its effect is less
pronounced when it is >2 kb upstream of the splice site (compare
minigenes W, A, and K). The enhancer region contains a purine-rich
sequence, so two additional deletions were created to assess the
contribution of this purine-rich region. Minigene Y lacks the
purine-rich sequence, whereas minigene X lacks the remaining sequence
in this region. Only minigene Y showed a statistically significant
decrease in B isoform splicing. The purine-rich region is 75% GA over
a stretch of 60 nucleotides. Repeats of the motif GARGARGAR have been
shown to function as splicing enhancers in other genes (31). The
sequence in intron 10 does not contain such repeats, but is very
GA-rich and may function in a similar manner.
The region between positions 5 and 7 that causes exon skipping was
identified, as was the enhancer sequence, using minigenes with large
internal deletions in intron 10. What is the role of this inhibitory
region in the context of the full intron? A minigene was constructed
which lacked only the inhibitory region between positions 5 and 7 (Fig.
3, minigene F). Transfection of minigene F into HepG2 cells gave >95%
B isoform RNA compared with 65% for minigene B with the full intron.
Thus, removal of 70 nucleotides from the 2.3-kb intron causes an
increase in B isoform splicing. The inhibitory region was further
localized to a 43-nucleotide sequence between positions 6 and 7 (minigene N). Deletion of the 27 nucleotides between positions 5 and 6 had no effect on splicing (minigene S). Removal of another 26 nucleotides (minigene G) or 52 nucleotides to the 3' splice site of
exon 11 (minigene H) dramatically weakens the splice site as would be
expected. Similarly, small deletions of 26 or 13 nucleotides (minigenes
O and T) caused a dramatic drop in exon 11 inclusion, suggesting that
this 13-nucleotide sequence is necessary for the efficient use of the
3' splice site.
Mutation of the 3' Splice Site in Intron 10--
Previous internal
deletions in intron 10 had suggested that elimination of the upstream
series of adenines between positions 7 and 9 in the minigene impaired
the use of the downstream splice site and caused skipping of exon 11 (Fig. 4, minigene O). However, all
deletions eliminated other regulatory regions as well. Adenine residues
have been identified as the branch point nucleotides in many but not
all introns, so elimination of these adenines might explain the
alteration in splicing. Mutation of the four adenine residues in this
region had no effect on splicing (Fig. 4, minigene I), indicating that
this sequence cannot be the functional BPS. However, deletion of the
three adenine residues proximal to the splice site (minigene V) gave
<5% exon 11 inclusion. Although this result does not specifically
identify the branch point residue, it is likely that one of these
adenines is the functional BPS. Alignment of the most distal of the
three adenines UCCUCAA with the consensus
branch point sequence UNCURAC indicates that this residue could be the
branch point, however, accurate identification of the branch point
residue will require in vitro branch point mapping. So why
does B isoform splicing decrease when the region containing the four
upstream adenines is deleted (minigene O)? One possible explanation is
that the deletions may have impaired the function of the putative
downstream BPS. A deletion from position 8 to 9 between the two
stretches of adenines gave a similar reduction in exon inclusion
(minigene T). The 3' end of this deletion was 2 nucleotides 5' to the
BPS. All of the previous deletions were constructed by engineering
BamHI restriction sites. Therefore, a minigene was
constructed with four nucleotide substitutions to create a
BamHI site at a position analogous to the deletion mutants
(minigene U). This minigene gave <5% exon inclusion, indicating that
mutation of four residues GUCCUCAAAGG to
GGAUCCAAAGG could result in exon skipping, presumably by
impairing the function of the BPS described above. Conversely, mutation
of four nucleotides downstream of the triple adenines had little effect
on splicing (minigene AE). The 3' splice site in this intron contains a
single G residue in the middle of a stretch of nine pyrimidines. Purine residues in the center of the polypyrimidine tract have been shown to
have a detrimental effect on RNA splicing (32). Mutation of the guanine
residue to thymidine had little effect on exon inclusion (minigene AA).
However, increasing the length of the pY tract to 14 pyrimidines caused
the exon to be spliced constitutively (minigene AF). Thus, the
alternative splicing of exon 11 requires a weak 3' splice site and
strengthening the site renders the exon constitutive similar to results
for other genes (33).

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Fig. 4.
Mutation of the 3' splice site. Left
panel, sequence of the intron 10 splice site mutations used in
these experiments. Deletions are indicated by dashed lines.
Point mutations are indicated by letters, and
dots indicate identity with the parental minigene.
Numbers above the sequence refer to the end points of the
deletions. The 3' splice site is indicated by the colon.
Right panel, quantification of alternative splicing by
RT-PCR. Minigenes were transfected into HepG2 cells by the calcium
phosphate co-precipitation technique. Forty-eight hours later, cells
were harvested and total RNA isolated. RNA was subjected to RT-PCR as
described under "Experimental Procedures." Gels were quantified
using a PhosphorImager. Results are from four independent experiments
performed in duplicate and are are expressed as percent of B isoform (+ exon 11) splicing and show the mean ± S.E. The vertical
dashed line indicates the degree of exon incorporation observed
with the parental minigene.
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Exon 11 Sequences Are Involved in Splice Site Selection--
To
investigate whether the alternatively spliced exon itself might be
involved in splice site recognition, we introduced mutations into exon
11 in the parental minigene B. Introduction of four point mutations in
the middle of the exon caused the exon to be spliced constitutively
(Fig. 5, minigene J). An overlapping four point mutations, however, had no effect (minigene AC). A deletion of
eight nucleotides or insertion of three thymidine residues caused an
almost complete loss of exon inclusion (Fig. 5, minigenes Z and AB).
The point mutations that rendered the exon constitutive were tested in
combination with deletions of the inhibitory region identified earlier
(Fig. 3). There was no additional effect when combined with minigenes F
and N that are deleted for the 3' inhibitory region (Fig. 5, minigenes
P and Q). Interestingly, when these exon mutations were introduced into
minigene O, which contained a deletion that impaired function of the
putative BPS, there was an increase in the amount of B isoform
splicing. Thus, the weakened BPS is able to function, albeit weakly, in
the presence of mutations in the exon. These results indicate that the
exon 11 sequences play an active role in determining the degree of exon
inclusion in both a positive and negative manner.

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Fig. 5.
Effect of mutation of exon 11 sequences.
Left panel, sequence and structure of minigenes used in
these experiments. Deletions are indicated by dashed lines. In the
upper panel, the exon is indicated by the boxed
sequence, point mutations by letters below the sequence
and an insertion by three Ts in minigene AB. In the
lower panel, numbers above intron refer to the
end points of the deletions. The As indicate potential
branch point sequences. The letters in exon 11 indicate the
four constitutive point mutations. Panel B, quantification
of alternative splicing by RT-PCR. Minigenes were transfected into
HepG2 cells by the calcium phosphate co-precipitation technique.
Forty-eight hours later, cells were harvested and total RNA isolated.
RNA was subjected to RT-PCR as described under "Experimental
Procedures." Gels were quantified using a PhosphorImager. Results are
from four independent experiments performed in duplicate and are are
expressed as percent of B isoform (+ exon 11) splicing and show the
mean ± S.E. The vertical dashed line indicates the
degree of exon incorporation observed with the parental minigene.
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Models for the Regulation of the Alternative Splicing of Exon
11--
The alternative splicing of exon 11 of the IR gene is
consistent with the models depicted in Fig.
6. The GA-rich splicing enhancer at the
5' end of intron 10 favors inclusion of exon 11. This could be due to a
direct effect on the 3' splice site. Alternatively, as the enhancer is
>2 kb upstream of the BPS, the effect of the enhancer could be on the
adjacent 5' splice site. The proximal site (TAG:GUCAGGAC) differs
significantly from the consensus (CAG:GUAAGUAU) so the effect of the
enhancer may be to strengthen the interaction of the U1 small nuclear
ribonucleoprotein particle with the 5' splice site (34). How this might
affect alternative splicing of the downstream exon is not clear, as
this site is used whether or not the exon is included, but it may allow
the use of a suboptimal splice acceptor site. SR proteins that
recognize purine-rich enhancer sequences are known to favor use of
proximal splice sites (35-42). SF2/ASF is a member of the SR protein
family and can bind to GA-rich splicing enhancer sequences similar to
that identified in the 5' end of intron 10. Overexpression of SF2/ASF
has been shown to promote inclusion of alternatively spliced exons in
the rat clathrin light chain B and rat
-tropomyosin genes (41, 42). This is due to the ability of SF2/ASF to promote the use of a proximal
splice site, either 5' or 3', over a distal site. Interestingly, this
activity is antagonized by the hnRNP-A1 splicing factor, which favors
the use of distal splice sites over proximal. The observed choice of
splice sites reflects a balance between SF2/ASF and hnRNP-A1
activities. Both SF2/ASF and hnRNP-A1 are RNA-binding proteins. In
contrast to the SR proteins, hnRNP-A1 binds to sequences containing the
motif UAGGGA or UAGGGU (43). The 5' end of intron 10 also contains the
sequence CTTAGGGACC, which includes an hnRNP-A1 binding
site (underlined). Whether hnRNP-A1 could regulate the alternative
splicing is unknown; however, deletion of the 5' end of intron 10 in
minigene X eliminates the potential hnRNP-A1 binding site but has no
effect on the splicing. Further studies will be required to determine
if SR proteins or hnRNP-A1 can recognize the regulatory regions that we
have identified in the IR gene and modulate splice site selection.

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Fig. 6.
Potential models for splice site
selection. Figure depicts different models for splice site
recognition. SR proteins binding the purine-rich enhancer sequence at
the 5' end of the intron could exert effects at either the 3' splice
site or the proximal 5' splice site. Factors recognizing the inhibitory
sequences at the 3' end of the intron or exon 11 could repress or
stimulate recognition of the 3' splice site. Alternatively, RNA
secondary structure involving both the inhibitory sequence and the exon
could sequester the 3' splice site to regulate exon skipping.
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At the 3' end of the intron, there are two regions that influence exon
skipping. One is inhibitory and located in the intron upstream of the
BPS. Interestingly, secondary structure prediction programs based on
the Zuker algorithm indicate that this region has the potential to form
a stable stem-loop structure. Whether such a structure is formed
in vivo is not known. The role of secondary structure in
alternative splicing has been reviewed recently (23). The
polypyrimidine tract at the 3' splice site is only 9 nucleotides with a
central guanine residue. Mutation of the guanine to give a stretch of
10 contiguous pyrimidines did not have an effect on exon recognition;
however, increasing the length of the polypyrimidine tract to 14 nucleotides rendered the exon constitutive. Thus, exclusion of the exon
requires a suboptimal splice acceptor site. Similar effects have been
seen with the growth hormone and other genes (33). The second
regulatory region is contained within the alternatively spliced exon
itself and appears to have both positive and negative effects. On the
one hand, introduction of four point mutations caused the exon to be
constitutive, suggesting a negative role for the exon. On the other
hand, insertion of three thymidines or deletion of a stretch of eight
pyrimidines caused the exon to be skipped indicating a positive role.
Interestingly, the constitutive point mutations had little effect when
combined with deletions of the inhibitory region upstream. Thus these
regulatory regions could function independently to modulate recognition
of the 3' splice site (Fig. 6) or, alternatively, both sequences could
be involved in a larger regulatory region involving secondary structure
around the 3' splice site. Further studies are planned to test these
models.