From the Joseph P. Stokes Research Institute,
Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, the § Department of Bioscience, National Cardiovascular
Center Research Institute, Suita, Osaka 565-0871, Japan, and the
School of Medicine, University of California at San Diego,
La Jolla, California 92093-0602
Received for publication, December 22, 2000, and in revised form, April 26, 2001
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ABSTRACT |
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Alternative splicing of the 12-base exon 2 of the adenosine monophosphate deaminase (AMPD) gene is subject to
regulation by both cis- and trans-regulatory signals. The extent of
exon 2 inclusion is stage- and cell type-specific and is subject to the
physiological state of the cell. In adult skeletal muscle, a cell type
that regulates the activity of this allosteric enzyme at several
levels, the exon 2-plus form of AMPD, predominates. We have performed a
systematic analysis of the cis-acting regulatory sequences that reside
in the intron immediately downstream of this mini-exon. A complex
element comprising sequences that enhance exon 2 inclusion and
sequences that counteract this effect resides in the middle of this
intron. We demonstrate that the enhancing component is bipartite, with
more than a kilobase of sequence separating the two functional sites.
The presence of even minimal levels the mini-exon in the fully
processed AMPD mRNA requires both of these sites, neither of which
appears in any other published splicing enhancer. An RNA binding
activity derived from a muscle cell line requires both of the enhancing
sites. Mutations in either of the sites that eliminate exon 2 inclusion
abrogate this binding activity.
The vast majority of metazoan genes contain short,
information-encoding exons interspersed by relatively long stretches of noncoding introns. The processing of this information to yield a
translatable message, including the splicing of the pre-mRNA, is
subject to regulation at virtually every definable step. One such step,
including or excluding a particular exon Vertebrate exons are, on average, less than 300 nucleotides, whereas
many introns are thousands of nucleotides in length (4). Exons below an
average size of 50 nucleotides have been shown to be inherently
difficult to recognize by the splicing machinery (4, 5). One
explanation for this "masking" of small exons, originally set forth
by Berget (5), is that the initiation of the splicing reaction is
exon-centered. There is much evidence to support this model, termed
exon definition, in which ribonucleoprotein initiation
complexes recognize intron-exon boundaries and bridge across the exon.
As exon size decreases below 50 nucleotides, these complexes are
prevented from forming a productive interaction with the 3'- and
5'-splice recognition sites on the pre-mRNA or with each other,
through steric hindrance. Sequences that function either to facilitate
(enhancers) or to inhibit (repressors) the recognition of alternatively
spliced exons have been found in both introns (6-9) and exons
(9-11).
Adenosine monophosphate deaminase
(AMPD)1 catalyzes a key step
in purine nucleotide metabolism in virtually all eukaryotes. The purine
nucleotide cycle serves as the sole source (in the form of fumarate) of
citric acid cycle intermediates in contracting muscle tissue. A
deficiency in AMPD is the most prevalent genetic disease in humans, the
number of people heterozygous approaching 10% of Caucasian and
individuals of African descent (12, 13). A small percentage of
homozygous deficient individuals, nearly 2% of the affected
populations, display symptoms of chronic fatigue and lost productivity
as well as a greater predisposition to stress-related ailments,
including heart disease and stroke (12, 14). Interestingly, a mutation
in a least one AMPD allele appears to confer a protective effect on
individuals at risk for one of the most prevalent diseases of
industrialized nations, congestive heart failure. We have found that
people harboring at least one AMPD mutant allele have a significantly prolonged probability of survival after the onset of symptoms leading
to this extremely serious medical condition (15).
In muscle cells, AMPD is fully activated only when bound to myosin
heavy chain through its carboxyl terminus. This enzyme is also
influenced allosterically by cellular levels of purine nucleotides,
through a binding site near its amino terminus (12, 24-28). We have
shown that the four-amino acid peptide encoded by exon 2 influences
both of these properties in a fiber type-dependent fashion
(19). In adult, fast twitch, glycolytic myofibers, the sensitivity of
AMPD to cellular ATP/GTP levels is altered significantly by the
presence of the exon 2 domain (14, 16, 19). In the resting state, ATP
levels in the myocyte are relatively high, and a purine nucleotide
binding site near the carboxyl terminus of AMPD is occupied. The exon
2-minus isoform of AMPD, which predominates in slow twitch, oxidative
myofibers, can bind to myosin heavy chain and become activated in the
presence of relatively high concentrations of ATP (12, 19). Thus, the
essential role of AMPD in generating both fumarate (for use as a citric
acid cycle intermediate) and IMP (from the deamination of AMP) can be
retained in both fiber types, at least in part, by regulating the
alternative splicing of exon 2.
The AMPD gene is comprised of 16 exons and is regulated both
transcriptionally (13) and post-transcriptionally (16-18) in a
development- and tissue-specific manner. The primary transcript is
alternatively spliced with the exon 2-minus form predominating in all
cells prenatally and in myoblasts postnatally. This 12-base mini-exon
is largely, but not exclusively, retained in adult myotubes (17).
Recent data suggest a role for the exon 2- encoded peptide in altering
the allosteric responsiveness of AMPD to intracellular ATP levels,
clearly important for the homeostasis of muscle (19). A recently
discovered genetic lesion, a C-T transition that results in the
introduction of a nonsense mutation at the end of exon 2, is partly
responsible for human AMPD deficiency. As a consequence of alternative
splicing, however, this mutated exon is excluded in 0.6-2% of the
enzyme in adult muscle, resulting in a partial rescue of the
deficiency. Most individuals harboring this mutation are therefore
asymptomatic (14). For all of these reasons, we are very interested in
the molecular mechanisms by which the alternative splicing of AMPD is regulated.
We have demonstrated recently that alternative splicing of exon 2 is
driven largely by two competing factors (20). First, the short distance
between the suboptimal 5'-donor and 3'-acceptor splice recognition
sites of this 12-base exon makes its recognition by the splicing
apparatus inherently difficult. Second, sequences located roughly in
the middle of the 5.2-kb downstream intron, the exon retention element
(ExRE), are required for inclusion of the this exon in the final
splicing product. We have shown that the strength of these two opposing
influences on exon 2 inclusion is influenced greatly by cell type. In
non-muscle cells, such as fibroblasts, exon 2 is included in slightly
less than half of the final splice products. In myoblasts, the balance
is shifted dramatically toward inclusion, about 90%, and
differentiated myotubes push this even further, to a 97% inclusion
rate (20).
We present in this report the initial characterization of sequences
residing in the ExRE of intron 2 of the AMPD gene which regulate the
extent of exon 2 inclusion in the final splicing product. We have
narrowed the ExRE to two short, novel, discreet sequences in the middle
of intron 2, separated by ~1,150 bases. We found that this bipartite
splicing enhancer functions in an orientation- and
sequence-dependent manner. In addition, there is an
absolute requirement for both of these enhancing elements in exon 2 inclusion. We go on to demonstrate the presence of a myocyte-specific
factor that is detected only when both of the enhancing sites are
present together in a binding reaction.
AMPD Mini-gene Expression Constructs
Manipulations, deletions, and mutations of intron 2 of the human
AMPD gene were made according to standard recombinant DNA protocols and
procedures (21). All constructs were sequenced by the University of
Pennsylvania Medical Center DNA Sequencing Facility. The resulting
intron 2 enhancer mutations were cloned into the Cell Lines and Transfections
Balb/c 3T3 fibroblasts and murine Soleus 8 myoblasts were
maintained in Dulbecco's modified Eagle's medium plus 10% fetal calf
serum supplemented with glutamine in an atmosphere containing 5%
CO2. Cells were transfected using a standard calcium
phosphate procedure (21). After 48, transiently transfected cells were harvested, and total RNA was isolated using a guanidinium-based procedure (21). Stable lines were established by incubating cells
48 h post-transfection in 750 µg/ml (effective concentration) Geneticin (Life Technologies, Inc.) and allowing the growth of distinct
colonies (approximately 3 weeks). RNA was isolated from pooled colonies
using the same guanidinium procedure.
Qualitative RT-PCR Analysis
Preparation of total RNA and reverse transcription reaction
conditions for the polymerase chain reaction and subsequent gel analysis of PCR products were carried out as described (20). Two sets
of primers, both specific for the human AMPD homolog, were used in
these studies. PCR products of 69 bp (exon 2-plus) and 57 bp (exon
2-minus) were generated as described (20). RT-PCR analysis yielding
products of 216 bp (exon 2-plus) or 204 (exon 2-minus) was generated
using the primer H6 (5'-GTCTGGATCTCATCCACATC-3', which bound to exon 3)
in the RT reaction, and H6 and TH2 (5'-GTCACCCCACAGTCTCCTC-3', which
bound to exon 1) in the PCR. The cycling conditions used with
the second set of primers were: 93 °C, 3 min, 1 cycle; 93 °C, 1 min, 58 °C, 1 min, 72 °C, 30 s, 30 cycles; 72 °C, 10 min; otherwise the reagents, buffers, and conditions were identical to those
described previously (20). In either case, these primers were specific
to the human homolog of AMPD and did not amplify endogenous murine
AMPD.
Generation of Gross Deletions
The plasmid p25 was constructed by cloning the 989-base
EcoRI-BamHI AC fragment from intron 2 of the
human AMPD gene (14) into the pBKS vector (Stratagene). A series of
deletions, three generated from the 5'-end of the A fragment (AC1, a
150-base deletion; AC2, a 300-base deletion; AC3, a 450-base deletion)
and two generated from the 3'-end of the C fragment (AC4, a 150-base
deletion, and AC5, a 300-base deletion) were generated via PCR using
the following primers.
AC1--
Top, 5'-CGCGAATTCCTTCCTGTGTTAATAATAGTAATCTCC-3';
bottom, 5'-CCAGGATCCAACAGAGAAGCCCACTATGTTGG-3'.
AC2--
Top,
5'-CGCGAATTCCAGTTATTATGTGGTTTGCCCAAGGC-3'; bottom, same as AC1.
AC3--
Top, 5'-CCAGGAATTCCACCTCCCGAGTTCAAGCAATTCTCC-3';
bottom, same as AC1.
AC4--
Top, 5'-CGCGAATTCCTTTGGGAGATGAAATGTGG-3'; bottom,
5'-CCAGGATCCAAATGGAACACCAAGTAAATGC-3'.
AC5--
Top, same as AC4; bottom, 5'-
CCAGGATCCAAGCAGAAGTTGGAAGAGGCTGC-3'.
All of the resulting PCR products were gel purified, digested with
EcoRI and BamHI, gel purified again, and used to
replace the wild-type AC region in the AMPD expression vector.
Linker Scan Mutagenesis
The p25 plasmid (see above) was used as the starting vector from
which a series of nested deletions, averaging 50 bases, was generated
from either end of the AC fragment, using the exonuclease III-based
Erase-a-Base kit (Promega). After digesting the single-stranded 5'-overhang with S1 nuclease, double-stranded linkers incorporating an
8-base ASC restriction site (5'-GCTGACGCCCGGCG-3') were
ligated onto the resulting blunt ends. After digesting the deletions
with ASC I (New England Biolabs) and SacI (which cuts only
in the ampicillin resistance gene of the vector) they were sized on
agarose gels, and the appropriate deletion pairs were ligated. Using
the EcoRI and BamHI sites, each of the resulting
22 ASC mutations replaced the wild-type AC region in the AMPD mini-gene
expression vector.
Site-directed Mutagenesis
The mutations, designed to alter contiguous bases in groups of
four, were introduced in and around the mutation 7 and 8 ASC sites and were generated using the QuikChange site-directed
mutagenesis kit (Stratagene). The top strand of each of the 10 pairs of
double-stranded oligonucleotides used in this procedure, incorporating
the 4 mutated bases (underlined) flanked by 15 wild-type nucleotides,
were as follows (the numbers correspond to the number of the mutation in Fig. 6A).
1) 5'-GAGTCTTGCTCTGTCATTAAGGCTGGAGTCCAGT 3'.
2) 5'-TCTGTCGCCCAGGCTATTATGCAGTAGCATAATC-3'.
3) 5'-CCCAGGCTGGAGTGCTAATGCATAATCTCGGCTC-3'.
4) 5'-GAGTGCAGTAGCATATAAACGGCTCATTGCAAGC-3'.
5) 5'-AGCATAATCTCGGCTATAAGCAAGCTCCACCTCC-3'.
6) 5'-CGAGGGTGTGGAGGCTAATTCAGGCCATCGAATG-3'.
7) 5'-GGAGGCATTATCAGGTATACGAATGCATTTACTT-3'.
8) 5'-ATCAGGCCATCGAATTATATTACTTGGTGTTCCA- 3'.
9) 5'-CCATCGAATGCATTTTAAAGGTGTTCCATTTGTT-3'.
10) 5'-GCATTTACTTGGTGTATATTTTGTTCCTTCATGG-3'.
The bottom strand for each oligonucleotide is simply its complement.
All mutations were generated in the p25 construct and verified by
sequencing. As in the previous manipulations of this region, each of
the 10 mutations was subcloned using the 5'-EcoRI and
3'-BamHI sites flanking the AC region into the AMPD
mini-gene expression vector.
Preparation of Nuclear Extracts
Confluent cultures of murine Soleus 8 myoblasts were allowed to
fuse into myotubes by reducing the fetal calf serum levels in the
culture medium from 20% to 2% (23). 72 h after the medium switch, the culture dishes were cooled on ice, and the cells were lysed
in ice-cold Triton extraction buffer (T buffer) (20 mM
HEPES (pH 7.9), 10 mM NaCl, 3 mM
MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 20%
glycerol, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml
pepstatin, 10 µg/ml aprotinin) with 20 strokes of a Dounce
homogenizer (type B pestle). Nuclei were pelleted by centrifuging the
lysed cells at 3,000 × g for 15 min at
4 °C. Nuclei were washed once in T buffer, re-pelleted, and then extracted by resuspending in 1 ml of TS buffer (T buffer with
high salt, 500 mM NaCl and 0.4 M KCl)/3 × 107 cells, in a 15-ml screw-capped tube. After gently
rocking the nuclear extraction at 4 °C for 1 h, it was spun at
13,000 × g for 10 min at 4 °C, and the supernatant
was aliquoted, snap frozen in liquid nitrogen, and stored at
Preparation of Radiolabeled Transcripts for Gel Shift
Probes
The following oligonucleotides were designed with a T7 promoter
at the 5'-end (italicized) followed by 30 bases from intron 2 of the
AMPD gene. Centered in these 30 bases were the wild-type sequences
defined by either ASC mutation 7 or 18 (underlined; see Fig.
4A).
Probe 7--
Top,
5'-GTAATACGACTCACTATAGGGCCCAGGCTGGAGTGCAGTAGCATAATCTC-3';
bottom,
5'-GAGATTATGCTACTGCACTCCAGCCTGGGCCCTATAGTGAGTCGTATTAC-3'.
Probe 18--
Top,
5'-GTAATACGACTCACTATAGGCCATCGAATGCATTTACTTGGTGTTCCATT-3';
bottom,
5'-AATGGAACACCAAGTAAATGCATTCGATGGCCTATAGTGAGTCGTATTAC-3'.
Two additional pairs of oligonucleotides were synthesized, identical to
the sequences above, except that the 8 bases defining mutation 7 and 18 sites were altered to match their cognate linker scan mutations. The
mutated bases are indicated below in lowercase letters.
Mutant Probe 7--
Top,
5'-GTAATACGACTCACTATAGGGCCCAGGCTGGgGcGCgGcAGCATAATCTC-3';
bottom,
5'-GAGATTATGCTgCcGCgCcCCAGCCTGGGCCCTATAGTGAGTCGTATTAC-3'.
Mutant Probe 18--
Top,
5'-GTAATACGACTCACTATAGGCCATCGAATGCggcgcgccGGTGTTCCATT-3';
bottom,
5'-AATGGAACACCggcgcgccGCATTCGATGGCCTATAGTGAGTCGTATTAC-3'.
Equimolar amounts of the appropriate oligonucleotide pairs were
annealed together by being placed in a boiling water bath for 1 min,
and the bath was removed to the bench top and allowed to come to room
temperature. 0.5 µg of these double-stranded oligonucleotides was
added to a prewarmed (42 °C) transcription mixture (40 mM Tris-Cl (pH 8.0), 8 mM MgCl2, 2 mM spermadine, 50 mM NaCl; 1 mM each ATP, UTP, and GTP; 60 units of RNasin (Promega); 10 mM
dithiothreitol; and 50 µCi of [ Electrophoretic Mobility Shift Assays
Binding reactions included the following components (added in
this order): 3 µl of 5 × binding buffer (25 mM
HEPES (pH 7.9), 125 mM KCl, 10 mM
MgCl2, 15% glycerol, 2.5 mM
phenylmethylsulfonyl fluoride); 1 µl of nuclear extract, water to 15 µl, and ~2.5 × 105 cpm of probe was incubated on
ice for 20 min and then loaded directly onto a pre-run 5%
nondenaturing gel (80 mM Tris-borate, 2 mM EDTA).
Inclusion of Exon 2 Requires a Complex Control Element in the
Downstream Intron--
Using a mini-gene construct comprising part of
exon 1, exon 2, intron 2, and part of exon 3 (Fig.
1A), we had demonstrated previously, in both fibroblasts and myocytes, an absolute requirement for the 5.2-kb second intron for inclusion of exon 2 in the three-exon splicing product (20). Interestingly, as the phenotype of a cell became
more muscle-like, the requirement for intron 1 for the inclusion of
exon 2 in the final splicing product dropped dramatically; that is,
when exon 1 was deleted from the mini-gene construct, 3T3 fibroblasts
excluded exon 2 totally, whereas skeletal myoblasts included exon 2 slightly greater than half of the time. When these same myoblasts
(Soleus 8 cells) were allowed to differentiate and fuse into myotubes,
greater than 90% of final splicing products included the mini-exon
(20).
As a first approach at identifying the sequences responsible for this
effect, we constructed several mini-gene constructs carrying gross
deletions of intron 2 and performed qualitative RT-PCR analysis to test
the effects of each on exon 2 inclusion. The primers employed in these
assays were specific for the human AMPD gene and did cross-hybridize
with the murine homolog (see "Experimental Procedures" and Ref.
20). Nearly half of the sequences of intron 2, those closest to the
flanking exons, could be eliminated without an effect on the wild-type
ratio of exon 2-included to exon 2-excluded splicing products.
Deletions that remove virtually all of the sequences flanking this
central 2.7 kb (Del 1/3, Fig. 1B) retain the
wild-type splicing pattern in both myoblast and fibroblast cells (Fig.
1C, Del 1/3 lanes). Located in the middle of
intron 2, this 2.7-kb fragment must, in addition, be oriented in the
wild-type 5'
Using convenient restriction sites, the 2.7-kb region was divided into
four fragments and analyzed further through their systematic deletion
(Fig. 2A). Only when the
600-bp fragment A and 400-bp fragment C were both present in the
mini-gene construct did exon 2 appear in the final splicing product in
either myoblasts or fibroblasts (Fig. 2). The 800-bp fragment
separating A and C, fragment B (the lane labeled
Distinct Regions in Fragments A and C Promote Exon 2 Inclusion--
To simplify the identification of the sequences
responsible for this effect, we began with the mini-gene splicing
substrate in which the 2.7-kb fragment in intron 2 is replaced with the positive acting A and C elements (the Systematic Mutagenesis Confirms the Number and Position of Positive
Acting Sites--
In addition to revealing that the first 150 bases of
the A fragment were dispensable for the promotion of exon 2 inclusion, the gross deletion analysis indicated approximately where two critical
regulatory sequences in A and C resided. To narrow these further, we
performed a saturation mutagenesis on the entire A-C region (minus the
5'-most 150 bases) using a standard linker scan procedure. In the
process, we introduced an ASC restriction site (5'-GGCGCGCC-3') every
38 bases, on average, for a total of 22 different mutations (Fig.
4A; the end points of the 150 base gross deletions are indicated by upward arrows). Each
of these was then tested, as before, in the context of the AMPD
mini-gene splicing substrate. In only two cases, 7 and 18, was exon 2 excluded to any extent from the final splicing product (Fig.
4B). Both of these mutations had a more subtle effect on
exon 2 inclusion than the150-bp deletions depicted in Fig. 3. The
overall results are consistent between the two approaches. One apparent
distinction is that the large deletion of 300 bases at the 5'-end of
fragment A (Fig. 3A, construct 2) reduced exon 2 inclusion by about half, whereas the individual linker scan mutations
in this same region (Fig. 4, mutations 1-6) produced no
effect on total inclusion (see "Discussion").
A Minimal Functional Distance Is Required between the Two Critical
Sites--
In the wild-type splicing substrate the sequences
surrounding ASC mutations 7 and 18 are separated by roughly 1,150 bases (including the 800-base B fragment (see Fig. 2A)). Because
both sites are required for the inclusion of exon 2, we decided to ask
directly how critical the spacing between them is. The 600-base A and
400-base C fragments catenated together promote complete inclusion of
exon 2 ( Site-directed Mutagenesis around 7 and 18 Sites Reveals Their
Uniqueness--
We then sought to narrow further still the sequences
responsible for exon 2 inclusion by generating five 4-base mutations surrounding ASC linker scan mutation 7 and a second set of five mutations surrounding mutation 18. These site-directed mutations were
designed so that every base was altered (Fig.
6A). As before, each
individual mutation was tested in the context of the mini-gene for its
ability to affect the splicing outcome. Except for the 3'-most of these
mutations (10 in Fig. 6A), all attenuated the inclusion of the mini-exon 2 to some extent. Four of these (mutations 2, 3, 8, and 9) overlapped
with the ASC mutations 7 and 18, corroborating those results. Of the
remaining five site-directed mutations, the first and seventh
(1 and 7 in Fig. 6, A-C) led to total
exclusion. Combining the results of this analysis, the stretches of
sequence having the greatest effect on the enhancement of the exon 2 inclusion are boxed in Fig. 6A.
Myocyte-specific RNA Binding Activity Requires Sequences
Surrounding Both 7 and 18 Sites--
A number of mechanisms can
account for the virtual complete inclusion of exon 2 in endogenous AMPD
splicing products in myocytes. Given the above data, an obvious one to
test is for the presence of a factor(s) in such a cell type. Our
results likewise suggest that the sites in the A and C elements
critical for this effect (the sequences defined by the ASC mutations 7 and 18, respectively) might participate in forming a complex with such
a factor. We therefore tested whether nuclear extracts from Soleus 8 myotubes harbor such a binding activity in an electrophoretic mobility shift assay. Radiolabeled, 30-base RNA probes were in vitro
transcribed, incorporating at their center either wild-type or mutant
sequences defined by ASC mutation 7 or 18 and then employed alone or in combination in the assay. As shown in lanes 2 and
4 of Fig. 7, individually,
neither wild-type sequence yielded a specific binding activity when
incubated with unfractionated nuclear extract from Soleus 8 myotubes.
Only when both wild-type probes were incubated together (lane
6) was a specific binding activity observed (arrow). In
contrast, when the sequences were altered to contain the linker scan
mutations that originally defined the 7 and 18 sites (see Fig.
4A and "Experimental Procedures"), no binding activity
could be detected, regardless of whether the probes were used
individually (lanes 8-11) or together (lanes 12 and 13) in the binding reaction.
We demonstrated previously that the 12-base mini-exon 2 of AMPD is
poorly recognized by the splicing machinery because of its small size,
and because of the presence of the suboptimal 3'-acceptor and the
5'-donor splicing sites that flank it (20). The intron-exon boundaries
of exon 2 may have evolved so that they are only weakly recognized by
the constitutive splicing apparatus, allowing it to respond to the
dynamic metabolic requirements of myocytes while preventing its
inclusion in inappropriate cell types or physiological circumstances.
Reinforcing this notion was our earlier finding that transposing exon 2 into the middle of a different intron of the AMPD gene led to its total
exclusion from the final splicing product (17). This result led us to hypothesize that sequences surrounding exon 2 (an ExRE) in the wild-type AMPD gene must act to "unmask" this exon and allow
it to be recognized by the splicing apparatus. We went on to show that
such recognition-enhancing sequences reside within the 5.2-kb second
intron and that their effect on exon 2 inclusion is most pronounced in
myocytes (20).
In the present work, we narrowed the ExRE to a roughly 2.7-kb region in
the middle of intron 2 and showed that the normal 5' When brought together outside the context of the 2.7-kb fragment, the A
and C fragments promoted total inclusion of exon 2 in fibroblasts
(Figs. 2 and 3). A systematic dissection of this 1-kb fragment, first
through gross 150-base deletions (Fig. 3) followed by linker scanning
mutagenesis (Fig. 4), narrowed the functional sequences down to two
distinct, widely spaced sites, one in A (mutation 7 from the linker
scan, Fig. 4A), the other in C (mutation 18). The locations
of these two sites are consistent with those regions from the gross
deletion analysis shown to be required for exon 2 inclusion. One
apparent exception is that removal of the 5'-most 300 bases of the A
fragment reduced the ability of the splicing machinery to recognize
exon 2 (Fig. 3B, construct 2), whereas individual
linker scan mutations 1-6 (Fig. 4) in this same region had no effect.
We hypothesize that either the linker scan mutations we generated in
this instance left critical sequences intact, or the influence of each
individually on exon 2 recognition is redundant. These sites and the
sequences immediately surrounding them were then dissected further via
site-directed mutagenesis. Only one of these mutants, centered 15 bases
downstream of the 18 site (mutation 10), had no effect on the total
inclusion of exon 2 (Fig. 6, and see below).
The wild-type intron contains the nearly 800-ase B element interposed
between the positive acting A and C fragments (Fig. 2). When we moved
the mutation 7 and 18 sites closer together by removing the B element,
recognition and inclusion of exon 2 in fibroblasts reached the levels
observed in Soleus 8 myoblasts, to 100% of the splice products (Fig.
2A, Cis-acting enhancing elements that direct the splicing machinery to
proximal, usually suboptimal, splice sites fall into two general
groups, exonic and intronic. Most exonic enhancers are comprised of
purine-rich sequences that are thought to mediate their activity mainly
through the binding of constitutive SR proteins (11, 30-32). A number
of intronic splicing enhancers from both vertebrates and invertebrates
which influence alternative splicing in a cell-specific manner have
been described (8, 33-39). In the case of the alternative splicing of
very small exons, several, including AMPD, are known to be regulated by
downstream intronic enhancers (6, 7, 40-42). In the majority of these,
two recurring types of sequence elements mediate alternative splice
site usage, polypyrimidine tracts (37, 43) and short sequence repeats (38, 39, 41, 42, 44, 45, 46). There is a polypyrimidine tract within
the A element described here (mainly Ts, between the linker scan
mutations 2 and 7, see Fig. 4A), but mutations within this
region did not produce an effect on exon 2 inclusion (Fig.
4B).
Another recurring downstream intronic enhancing element is the hexamer
TGCATG. This sequence has been demonstrated to function as an enhancer
in the alternative splicing of the neuron-specific 3-base N1 exon of
the mouse c-src gene (47), the 30-base N30 exon of the human
non-muscle myosin heavy chain gene (48), and exon IIIB of the
fibronectin gene (38, 39, 45, 49). The presence of one or more copies
of a TGCATG enhancing site in the intron immediately downstream of exon
IIIB of the fibronectin gene is conserved phylogenetically from frog to
human (45). Like SR-binding elements, the TGCATG hexamer and its
variants may, in certain contexts, only function in multiple copies to attract a threshold number of factors to initiate a splicing event (9).
In the nearly 1,000 bases that comprise the A and C fragments from the
AMPD intronic enhancer, the TGCATG hexamer appears twice, once about
100 bases upstream of the linker scan mutation 18 and again about 100 bases downstream of that same site (both in the C fragment; see the
asterisks in Fig. 4A). Linker scan mutation 15 overlaps (by 2 bases) the first hexamer, whereas mutation 22 is
centered 17 bases upstream of the second hexamer. Neither of these mutations, on its own, affected the splicing outcome (Fig. 4).
Partial repeats of the hexamer sequence, GCATG (once), TGCAT (twice),
and TGCA (four times) appear throughout this kilobase of sequence. One
4-base TGCA sequence is centered at the mutation 7 site, and the two
3'-most bases of TGCAT overlap the 18 site (Fig. 4A).
However, 9 out of 10 of the site-directed mutations introduced around
the mutation 7 and 18 sites disrupt the enhancing activity of the AC
element at least as well as these two linker scan mutations. This
result argues against the TGCA and TGCAT partial hexamer sites alone
having sufficient functional enhancing activity, although disruption of
the sequences flanking them may be enough to abrogate such an activity.
Sequences surrounding the 7 and the 18 sites are both required for the
inclusion, to any extent, of the mini-exon 2. The requirement for both
sites in the detection of a binding activity from muscle cells is
consistent with all of the previous results we have observed in the
detection of the AMPD mini-gene splicing products. It remains to be
proved, of course, whether the binding activity we have detected here
actually contributes to the enhancing effect.
Our survey of the literature has produced no published data that
identify the TGCATG sequence variants TGCAT or TGCA specifically as
binding sites for factors involved or potentially involved in the
regulation of splicing. Nor is there any other significant sequence
similarity between the 7 and 18 sites. Furthermore, we do not detect
any occurrence of the sequences surrounding the 7 or 18 sites in any
other published splicing enhancer. Additional mutational analysis
should reveal whether these short sequence variants are functionally
responsible for the enhancing effect on exon 2 inclusion. The
requirement for both of these sites to achieve a specific binding
activity provides additional compelling evidence that they in some way
interact with one another, perhaps through the binding of one or more
proteins (or ribonucleoproteins).
A bipartite splicing enhancer element has been identified in the intron
downstream of exon IIIB of the fibroblast growth factor receptor gene
(50). In this case, mutation of either one of the two sites alone
diminishes, but does not eliminate, the activity of this enhancing
element. This is in contrast to the data reported here in which the
disruption or deletion of sequences surrounding mutation 7 or 18 abolishes the enhancing activity completely. We have demonstrated that
both sites, normally separated by about 1,150 bases, are required for
intron 2 inclusion in all of our splicing assays as well as for the
detection of an RNA binding activity. To our knowledge, however, this
is the first report of a bipartite splicing enhancer sequence where
both of the unique sequences are required to obtain a binding activity.
It is possible that in vivo, non-muscle cells can tolerate
only minimal levels of AMPD exon 2-containing protein (19). To ensure
then that the inclusion of exon 2 was less efficient, the AMPD gene may
have evolved so that the enhancing activity of the A and C elements
works efficiently only in the context of muscle and then only under
certain physiological conditions. The presence of the B element (Fig.
2) may serve that purpose in non-muscle cells. In any case, it is clear
that cis-regulation of the alternative splicing of exon 2 is complex
and likely involves more elements, both positive and negative, than
have been demonstrated here.
How exon 2 is initially recognized by the splicing machinery to form
the 5'-exon 1-exon 2-intron 2-exon 3-3' intermediate is not known. We
do know that even in the absence of virtually all of intron 1, exon 2 can still be recognized and included in the final splicing product of
our mini-gene construct (20). We have presented evidence here that the
ultimate inclusion of this mini-exon is dependent upon a complex
regulatory region within the second intron. Included within this region
is a unique bipartite enhancing element that acts to increase the ratio
of exon 2-plus to exon 2-minus AMPD splicing products. It will be
interesting to determine whether this enhancing activity can
transferred to a heterologous splicing substrate.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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alternative splicing
provides a way to alter the functional activity of a protein
in the absence of gene duplication (1, 2). Discreet functional domains
may be added or subtracted depending on signals elaborated by the
particular needs of the cell. The study of alternative splicing further
provides an experimental tool for understanding how the participating
ribonucleoprotein complexes target intron-exon boundaries (3).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-actin-based
expression vector (22) depicted in Fig. 1A.
80 °C. Aliquots of this extract were used only once in subsequent experiments.
-32P]CTP (400-800
Ci/mmol, Amersham Pharmacia Biotech)). 10 units of T7 RNA polymerase
(Promega) was added, and the reaction was incubated at 42 °C for 30 min. After digestion of the template DNA with DNase I, the RNA was gel
purified on a 6% denaturing gel. RNA probes eluted from the gel were
resuspended in RNase-free water and used directly in the mobility shift assay.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of deletions within intron 2 on exon
recognition. Panel A, map of the vector harboring the
AMPD mini-gene construct used as the basis for all of the splicing
analysis in this paper. Panel B, different fragments of
intron 2, as defined by the indicated restrictions sites, were deleted
alone or in combination from the wild-type mini-gene as illustrated.
Fragment 2 was excised and reversed (Rev). Qualitative
RT-PCR analysis was performed from RNA isolated from pooled colonies of
stably transfected Balb/c 3T3 cells or Soleus 8 myoblasts as described
under "Experimental Procedures." Panel C, radiolabeled
PCR products were separated on a nondenaturing polyacrylamide gel and
visualized by autoradiography. Restriction enzymes are as follows:
RV, EcoRV; RI, EcoRI;
B, BamHI; Kp, KpnI.
3' direction because flipping it 180° caused a total
elimination of exon 2 in the final splicing product (Fig. 1,
panels B and C, Rev). A
myoblast/myocyte-specific enhancement of exon 2 inclusion was observed
consistently throughout these studies.
A
C
D) cannot, by
itself, promote exon 2 inclusion to any extent measured by this assay.
Interestingly, in the absence of the B, the presence of A and C
together promoted the inclusion of exon 2 to greater than 95% in
fibroblasts (over double the rate in the presence of fragment B),
suggesting that B may function to attenuate that effect in these cells
(see "Discussion"). Taken as a whole, these data indicate that the
regulation of exon 2 inclusion in the final splicing product is
complex, involving both positive and negative influences. We chose to
focus our efforts on narrowing down those sequences in A and C
responsible for the enhancement of exon 2 inclusion in the final
splicing product.
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Fig. 2.
Inclusion of exon 2 requires a complex
control element in the downstream intron. Panel A,
using the indicated restrictions sites, the 2.7-kb region of intron 2 was divided into four large fragments, A, B, C, and D, which were then
systematically deleted as depicted in the schematic. Panel
B, densitometer analysis of typical gel results of Soleus 8 and
3T3 cell transfections. Numbers shown are the average of
three separate sets of RT-PCR/gel analysis. Panel C, gel
analysis of the qualitative RT-PCR analysis from Soleus 8 myoblasts.
Restriction enzymes are as follows: RI, EcoRI;
Bs, Bsu36I; Hp, HpaI;
B, BamHI.
B
D
construct in Fig. 2A). In this context, the combined 1,000 bases (approximately) of sequence promotes virtually total inclusion of
exon 2, independent of cell type (see Fig. 2B). Using
PCR-generated fragments, we created a series of progressive deletions
in A and C in ~150-base increments (the end points of which are
demarcated by upward arrows in Fig. 4A). Thus,
the 600-base A fragment was subjected to four incremental
deletions, starting from its 5'-end, whereas the 400-base C fragment
had three, proceeding in the opposite direction (Fig. 3A). These deletions were then
used, in turn, to replace the wild-type A and C elements for subsequent
splicing analysis. The results in Fig. 3B clearly show that
the sequences responsible for the enhancing activity of A reside within
the second and third deletion fragments (labeled 2 and
3 in the schematic in Fig. 3A), whereas the
positive acting sequences in fragment C lie within 3'-most 150 bases of
that fragment (labeled 5 in Fig. 3A).
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Fig. 3.
Distinct regions defined through gross
deletions in fragments A and C promote exon 2 inclusion.
Panel A, schematic of the deletions made in the fused AC
fragment. The size of the A fragment is 591 bases; the C fragment is
402 bases. Deletions were made in either the 5' or 3' direction by, on
average, successive 150-base increments, depicted here by the
filled boxes (deletion end points in the sequence are
indicated by upward arrows in Fig. 4A). The
numbers of each deletion correspond to the lane
numbers in panel B. Each deletion was used, in turn, to
replace the wild-type AC fragment in the AMPD mini-gene construct and
stably transformed into Balb/c 3T3 cells or Soleus 8 myoblasts. Total
RNA from pooled G418-resistant colonies was then analyzed by RT-PCR
assay. Panel B, results of the qualitative RT-PCR assay from
myoblast RNA reveal that sequences near the middle of the A element
(lanes 2 and 3) and toward the 3'-end of the C
element (lanes 4 and 5) are each required for the
complete inclusion of the 12-base mini-exon 2 in the final splicing
product. The results obtained from 3T3 fibroblasts and the myoblasts
were identical. nt, nucleotides.
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Fig. 4.
The positions of two critical linker scan
mutations through A and C are consistent with the gross deletion
data. Panel A, sequence of the human 989-base AC region
showing the position of the 22 linker scan-generated mutations. The
downward arrow denotes the junction of the A and C
fragments, as defined in Fig. 2; the upward arrows demarcate
end points of the 150-bp deletions used in Fig. 3. An alteration in a
given base in the linker scan mutations is denoted by the presence of
at least part of the ASC restriction enzyme recognition site (GGCGCGCC)
above the sequence. TGCATG hexamers are highlighted in
boldface, underlined, and marked with an
asterisk. Pentamer and 4-base partial repeats of that
hexamer sequence are highlighted in boldface and
underlined. The two linker scan mutations that,
individually, disrupted total inclusion of exon 2 (see panel
B), mutation 7 and 18 sites, are boxed. Panel
B, results of the qualitative RT-PCR analysis of the linker scan
mutations from RNA isolated from pooled colonies of stably transfected
3T3 cells. AC fragments harboring, in turn, each of the individual
mutations depicted in panel A were cloned in place of the
wild-type AC region in the mini-gene construct and tested for their
ability to affect the complete inclusion of exon 2. Only two, mutations
7 and 18, had any appreciable affect, disrupting exon 2 inclusion by
greater than 50%. B2 is the mini-gene construct that
harbors the entire wild-type sequence of intron 2 depicted in Fig.
1A, giving the partial exon 2 inclusion splicing pattern
seen here.
B
D construct, Fig.
2A). Beginning with this construct (in which the 7 and 18 sites are separated by 431 bases (labeled wt AC in Fig.
5A), we systematically reduced
the distance between the centers of ASC mutations 7 and 18 and found that the two sites could be separated by as little as 150 bases and
still give complete inclusion of the mini-exon (Fig.
5B).
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Fig. 5.
The critical sites in A and C necessary for
exon II inclusion require a minimal distance between them.
Panel A, schematic of the constructs derived by removing
increasing amounts of sequence between 7 and 18 sites. AC1 and AC5 are
controls derived from the 150-base gross deletions depicted in Fig. 3
(called 1 and 5, respectively, in that figure).
AC1 gives total exon 2 inclusion in the final mini-gene splicing
product, AC5 gives total exclusion. B2 is the mini-gene
construct that harbors the entire wild-type sequence of intron 2 depicted in Fig. 1A. The asterisks in panel
A mark the relative positions of the ASC linker scan mutations 7 and 18. Numbers to the left of each construct and
above the lanes in panel B are the amount of
sequence removed from between these two sites. Panel B,
results of the qualitative RT-PCR analysis (3T3 cells) using each of
the constructs depicted in panel A. When 7 and 18 sites are
separated by a distance of between 150 and 110 bases, exon 2 is
completely excluded in the final splicing product. wt,
wild-type.
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Fig. 6.
Site-directed mutagenesis around 7 and 18 sites further narrows the sequences critical for exon
inclusion. Panel A, sequences surrounding 7 and 18 sites. The positions of the ASC mutations (top) and the 10 site-directed 4-base mutations (bottom) are indicated.
Panel B, results of the qualitative RT-PCR analysis of RNA
from pooled colonies of 3T3 fibroblasts stably transformed with
mini-gene constructs individually harboring one of each of the 4-base
mutations. Numbered lanes correspond to the number of the
mutation depicted in panel A. Panel C, summary of
the effect of each site-directed mutation on complete inclusion of exon
2; +++, a total loss of exon 2 inclusion; ++, roughly 50:50 inclusion
to exclusion ratio; +, roughly 66:33 inclusion to exclusion ratio; ,
no effect on total inclusion.
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Fig. 7.
Separate oligoribonucleotides incorporating
either the site of 7 or 18 are required together to bind an activity
from myotube nuclear extracts. Electrophoretic mobility shift
analysis on a nondenaturing polyacrylamide gel of the products of the
incubation of nuclear extracts prepared from Soleus 8 myotubes.
Radiolabeled RNA probes used in this experiment are as follows.
Lanes 1-6 are all wild-type (wt) sequence
probes. Lanes 8-13 are all mutant sequence probes. Each
radiolabeled probe is 30 bases long and has at its center the 8 bases,
either wild-type or mutated, defined by ASC mutations 7 or 18 depicted
in Fig. 4A (these sequences are described in detail under
"Experimental Procedures"). Lane 7 is empty. Samples in
lanes 1, 3, 5, 8,
10, and 12 contained radiolabeled probe with no
nuclear extract. Samples in lanes 2, 4,
6, 9, 11, and 13 contained
probe incubated with myotube nuclear extract. Lanes 1 and
2, wild-type 7 sequence alone. Lanes 3 and
4, wild-type 18 sequence alone. Lanes 5 and
6, 7 and 18 together in the reaction. Lanes 8-13
follow the same scheme as lanes 1-6 except, as indicated,
all probes contained the mutated sequences. The arrow points
to a specific binding activity seen in lane 6, only in the
presence of both wild-type 7 and 18 sequences. Free probe is indicated
by the asterisks.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3' orientation
of this sequence is critical (Fig. 1). When this 2.7-kb fragment is
replaced with an unrelated sequence of similar size, the mini-exon is
again absent from the final splicing product (data not shown). Dividing
this 2.7-kb region using convenient restriction sites (Fig.
2A), we showed that two fragments, A and C, were both
necessary and sufficient for the enhancing activity of the ExRE (Fig.
2, B and C).
B construct and
B
D construct). Total inclusion
continued to be observed as the two sites were moved progressively
closer together. At the transition from 150 to 110 bases the enhancing
activity is lost completely. Because both sites are required for exon 2 inclusion, one possibility, based on the spacing data, is that
torsional constraints, imposed by RNA secondary structure, prevent a
required interaction between them below a separation of about 150 nucleotides. Looping of the RNA in this region may bring together two
positive acting sites that, individually, cannot promote exon 2 inclusion (11).
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ACKNOWLEDGEMENTS |
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We thank D. Fassler and A. Soderman for excellent technical assistance and the staff at the University of Pennsylvania Medical Center Sequencing Facility for continued support.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK12314 (to E. W. H.).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.
¶ Recipient of a fellowship from the American Heart Association.
** To whom correspondence should be addressed: Office of the Dean, School of Medicine, University of California at San Diego, Rm. 1313 Basic Science Bldg., 9500 Gilman Dr., La Jolla, CA 92093-0602.
Published, JBC Papers in Press, April 30, 2001, DOI 10.1074/jbc.M011637200
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ABBREVIATIONS |
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The abbreviations used are: AMPD, adenosine monophosphate deaminase; kb, kilobase(s); ExRE, exon retention element; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s).
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