Intronic alternative splicing regulators identified by comparative genomics in nematodes.

Abstract

Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in introns surrounding alternative exons. Many of the high-scoring nematode pentamer and hexamer motifs correspond to known mammalian splicing regulatory sequences, such as (T)GCATG, indicating that the mechanism of alternative splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements, and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our analysis, (T)CTATC, is important for alternative splicing regulation of the unc-52 gene.

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Intronic Alternative Splicing Regulators
Identified by Comparative Genomics
in Nematodes
Jennifer L. Kabat1, Sergio Barberan-Soler1, Paul McKenna2¤a, Hiram Clawson2, Tracy Farrer1¤b, Alan M. Zahler1*
1 Department of Molecular, Cell, and Developmental Biology and Center for Molecular Biology of RNA, University of California Santa Cruz, Santa Cruz, California, United
States of America, 2 Center for Biomolecular Science and Engineering, University of California Santa Cruz, Santa Cruz, California, United States of America
Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding
sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are
under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of
Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit
short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the
alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing
regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer
and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic
elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to
be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in
introns surrounding alternative exons. Many of the high- scoring nematode pentamer and hexamer motifs correspond
to known mammalian splicing regulatory sequences, such as (T)GCATG, indicating that the mechanism of alternative
splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements,
and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can
increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences
whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing
regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our
analysis, (T)CTATC, is important for alternative splicing regulation of the unc-52 gene.
Citation: Kabat JL, Barberan-Soler S, McKenna P, Clawson H, Farrer T, et al. (2006): Intronic alternative splicing regulators identified by comparative genomics in nematodes.
PLoS Comput Biol 2(7): e86. DOI: 10.1371/journal.pcbi.0020086
Introduction
One of the interesting lessons learned from the analysis of
the human genome is that we may possess fewer than 25,000
genes [1]. One mechanism to dramatically increase the
complexity of the human proteome from this lower-than-
expected number of genes is to allow some genes to encode
multiple proteins. This process can be accomplished by
alternative precursor messenger RNA (pre-mRNA) splicing.
Studies that use expressed sequence tag (EST) alignments to
identify alternatively spliced genes have led researchers to
predict that up to 60% of human genes are alternatively
spliced [2–5]. Alternative splicing events can be regulated in
tissue-specific, developmental, and hormone-responsive man-
ners, providing additional mechanisms for the regulation of
gene expression [6,7]. Understanding alternative splicing and
its regulation is a key component to understanding metazoan
genomes.
The current models for alternative splicing regulation are
based on the interactions of intronic or exonic RNA
sequences, known as cis elements, with splicing regulatory
proteins known as trans-acting splicing factors [8]. The
binding of splicing factors to the pre-mRNA regulates the
ability of the spliceosomal machinery to recognize and
promote alternative splicing. The role of intronic elements
in regulating splicing is well established and has been shown
to work in a combinatorial fashion based on the trans-acting
factors that are present. For example, the inclusion of the 18-
nucleotide-long, neural-specific N1 exon of the human c-SRC
gene is regulated by the downstream control sequence found
in the intron downstream of the N1 exon. This sequence
serves as a recruitment site for both constitutive and
neuronal cell-specific splicing factors such as nPTB, FOX-1,
Editor: Gary Stormo, Washington University in St. Louis School of Medicine, United
States of America
Received January 4, 2006; Accepted May 30, 2006; Published July 14, 2006
A previous version of this article appeared as an Early Online Release on June 5,
2006 (DOI: 10.1371/journal.pcbi.0020086.eor).
DOI: 10.1371/journal.pcbi.0020086
Copyright: ? 2006 Kabat et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: EST, expressed sequence tag; HMM, hidden Markov model; obs/
exp, observed over expected; pre-mRNA, precursor messenger RNA; PTB,
polypyrimidine tract binding protein; WABA, Wobble Aware Bulk Aligner
* To whom correspondence should be addressed. E-mail: zahler@biology.ucsc.edu
¤a Current address: Department of Biology and Center for Computing for Life
Sciences, San Francisco State University, San Francisco, California, United States of
America
¤b Current address: University of Virginia School of Medicine, Charlottesville,
Virginia, United States of America
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and FOX-2 [9–12]. The vertebrate RNA-binding protein
FOX-1 can also regulate muscle-specific alternative splicing
through interactions with the RNA sequence GCAUG [13],
and repeats of this sequence have been shown to be
important for alternative splicing regulation of the fibronec-
tin exon EIIIB and the rat calcitonin/CGRP exon 4 [14,15].
Many other examples of complex and combinatorial regu-
lation of alternative splicing through intronic cis elements
have been demonstrated, and combinatorial interactions
between proteins such as Nova-1, polypyrimidine tract
binding protein (PTB), and ETR-3, with specific cis sequences,
are important for alternative splicing regulation [16–20].
Intronic sequences are non-coding, and therefore they
should have less evolutionary selective pressure to maintain
their sequence. An exception to this should be intronic
sequences that regulate alternative splicing. In an analysis of
alternatively spliced human cassette exons, it was found that
on average, approximately 100 nucleotides of intron se-
quence, flanking either side of the exon, tend to be highly
conserved between the mouse and human genomes, with 88%
identity in the upstream sequences and 80% identity in the
downstream sequences [21]. Some clues to potential splicing
regulatory motifs arise from these studies. For example, Sorek
and Ast found that the sequence TGCATG was the second
most common hexamer in the first 100 nucleotides down-
stream of alternatively spliced exons, appearing in 18% of
these intronic regions [21]. Another study of aligned mouse/
human alternative exons found that GCATG is the most
overrepresented pentamer in the proximal conserved region
of the intron downstream of alternative exons [22]. A third
study found that TGCATG is frequently located in introns
flanking brain-enriched alternative exons, and its presence
and spacing are highly conserved in these genes from fish to
man [23].
Using the nematode Caenorhabditis elegans as a model system,
we have been working to take advantage of comparative
genomics to identify cis-acting regulators of alternative
splicing. The C. elegans gene structure, splicing machinery,
and gene expression regulation is similar to that of other
higher eukaryotes, with the exception that the average intron
size is smaller. Our lab has previously developed methods for
identification of alternatively spliced genes in C. elegans by
aligning the genome sequence with ESTs and mRNA
sequence [24]. We developed an algorithm, the Wobble Aware
Bulk Aligner (WABA), for creating interspecies genome
alignments between C. elegans and the related roundworm,
Caenorhabditis briggsae [25]. WABA employs a hidden Markov
model (HMM) to identify aligned regions as coding, high
homology, low homology, and no homology. It also factors the
AT richness of C. elegans introns into its calculations when it
defines an intronic region as ‘‘high’’ homology [25]. C. briggsae
and C. elegans diverged approximately 100 million years ago,
yet are indistinguishable by eye [26]. Alignment of these two
genomes revealed that exonic sequences are highly conserved
between these species, but intronic and intergenic sequences
are rarely conserved [25]. We found that these rare, high
homology sequences in introns are more likely to occur in the
introns flanking alternatively spliced exons than in total
introns [25]. We hypothesized that these conserved intronic
regions were cis-acting regulatory elements for alternative
splicing. This nematode alignment, with relatively limited
regions of high homology, provides the possibility for more
specific pinpointing of intronic splicing regulatory elements
than the much longer 100-nucleotide-long conserved regions
flanking alternative exons in mouse/human alignments [21].
In this paper, we present the analysis of conserved regions
of introns flanking alternatively spliced exons in C. elegans and
correlate these conserved regions with alternative splicing
regulation. We collected these conserved sequence regions
into a database and searched for overrepresented pentamers
and hexamers relative to a total intron database, similar to
the method used by Burge’s group to identify exonic splicing
enhancers [27]. This allowed us to create a table of potential
intronic alternative splicing cis-regulatory motifs. Since many
RNA recognition motif–containing splicing factors recognize
specific sequences on the order of 4–6 nucleotides in length
[11,13,18,28–32], the high-scoring motifs in this catalog may
represent specific binding sites for particular splicing factors.
Several of our highest scoring motifs in this analysis correlate
with known vertebrate splicing regulatory elements, for
example, (T)GCATG [23], but several have not been pre-
viously identified. A number of candidates identified by this
method were tested in an in vivo splicing reporter system in
C. elegans. We have used this analysis to identify and confirm a
new, highly conserved, alternative splicing regulatory ele-
ment, (T)CTATC. We show that this sequence works in
coordination with GCATG to regulate the alternative splicing
of the unc-52 gene.
Results
Identification of Highly Conserved Regions in Introns
Flanking Alternatively Spliced Exons
In order to identify alternatively spliced cassette exons in
the C. elegans genome, we used the Intronerator [24] to
generate an initial set of 1,471 putative alternatively spliced
genes. We did this by aligning over 200,000 ESTs and mRNAs
to the C. elegans genome and identifying regions where the
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Synopsis
Alternative splicing of precursor messenger RNA is a process by
which multiple protein isoforms are generated from a single gene.
As many as 60% of human genes are processed in this manner,
creating tissue-specific isoforms of proteins that may be a key factor
in regulating the complexity of our physiology. One of the major
challenges to understanding this process is to identify the
sequences on the precursor messenger RNA responsible for splicing
regulation. Some of these regulatory sequences occur in regions
that are spliced out (called introns). This study tested the hypothesis
that there should be evolutionary pressure to maintain these
intronic regulatory sequences, even though intron sequence is non-
coding and rapidly diverges between species. The authors
employed a genomic alignment of two roundworms, Caenorhabditis
elegans and Caenorhabditis briggsae, to investigate the regulation of
alternative splicing. By examining evolutionarily conserved stretches
of introns flanking alternatively spliced exons, the authors identified
and functionally confirmed splicing regulatory sequences. Many of
the top scoring sequences match known mammalian regulators,
suggesting the alternative splicing regulatory mechanism is
conserved across all metazoans. Other sequences were not
previously identified in mammals and may represent new alternative
splicing regulatory elements in higher organisms or ones that may
be specific to worms.
Intronic Alternative Splicing Regulators

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alignments are consistent with more than one way to process
a gene. The program could not distinguish between alter-
native splicing and alternative promoters, so we analyzed
each of these alignments individually to verify alternative
splicing. We found 449 examples of genes with strong cDNA
evidence for alternative cassette exons, and 454 examples of
genes with alternative promoters, which usually had unique
first exons that are spliced to common second exons. Of the
genes with alternative splicing, 162 also contained alternative
promoter usage. The remaining genes in the initial set of
putative alternatively spliced genes were mostly due to
unusual ESTs in the database that did not fit a gene model.
We also saw evidence of ESTs that showed internal deletions
at short direct repeats. To the program, these indicated the
potential for intron removal, but upon further inspection,
these did not meet the criteria of introns (start with the
dinucleotides GT or GC and end with AG) and were likely the
result of cloning artifacts of the ESTs.
Intronic regions that are highly conserved between C.
elegans and C. briggsae are rarely identified by WABA [25].
While analyzing our alternative cassette exon database, we
were struck by the fact that we could identify many examples
of WABA-defined high homology sequences in introns
flanking the alternatively spliced cassette exons, suggesting
strong evolutionary conservation. For 142 of the 449
alternatively spliced genes, WABA identified a highly con-
served sequence in flanking introns upstream or downstream
of 147 alternative cassette exons. Figure 1 shows several
examples of screen shots from the Intronerator browser of
cDNA-confirmed C. elegans alternatively spliced isoforms (in
blue) and a graphical representation of the WABA alignments
between C. elegans and C. briggsae (in purple). Dark purple
regions, indicating high homology, can be seen in these
introns either upstream or downstream of the alternative
exon. Sometimes a single conserved element is identified by
WABA, while for other cases multiple regions in the introns
are conserved.
An important question is whether these 147 C. elegans
alternative cassette exons are also alternatively spliced in C.
briggsae. Due to a lack of C. briggsae transcript data, we have no
direct evidence for alternative splicing of these exons.
Therefore, we examined the C. briggsae homologs of these
alternative exons for features of functional exons. A previous
alignment of 8 MB of the C. briggsae genome with the
complete C. elegans genome found that in coding exons there
is 79.3% overall nucleotide identity [25]. Consistent with
Figure 1. Images from the Intronerator Genome Browser Showing Alternatively Spliced Genes
Gene isoforms predicted by the Wormbase Consortium are shown in blue, and WABA homology alignments for C. briggsae to this region of the C.
elegans genome are shown in purple. Dark purple indicates a region of WABA high homology, light purple corresponds to low homology, and white
indicates no homology between species. Regions of alternatively spliced genes: (A) W01F3.1, (B) ZC477.9, (C) ZK637.8, (D) H24G06.1, and (E) C11D2.6 are
shown.
DOI: 10.1371/journal.pcbi.0020086.g001
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these findings, cross-species alignment of these 147 alter-
native cassette exons revealed an average nucleotide identity
between C. elegans and C. briggsae of 81.8%. Amino acid
identity of the open reading frame of these exons was 85.4%.
Maintenance of the reading frame for these exons also
appears to be well conserved: 57.0% of the 147 exons are the
exact same size, 34.3% differ by a multiple of three
nucleotides, and only 8.8% differ by a non-multiple of three
nucleotides (unpublished data). The high conservation of
nucleotide sequence, amino acid sequence, and maintenance
of open reading frame is consistent with these being coding
exons in C. briggsae.
One feature of alternative exons is that they are often
flanked by weak splice sites which allow for splicing
regulation (reviewed in [33]). If these exons are alternatively
spliced in C. briggsae we would expect that they would be
flanked by 39 and 59 splice sites of similar strength. We used
the UCSC Genome Table Browser (http://www.genome.ucsc.
edu) in an attempt to automate rapid alignment of the splice
junctions for these 147 exons in C. elegans and C. briggsae. For
the majority of these alternative exons, this automated
method was successful in identifying C. elegans and C. briggsae
59 and 39 splice sites for each exon, and we used these data to
compare the similarity of splice sites between the two species.
For 59 splice sites we examined the last three nucleotides of
the exon and first six nucleotides of the intron, as these base-
pair with U1 small nuclear RNA during initial 59 splice-site
recognition[31]. We found that these nine nucleotides were
completely identical for 47.1% of the alternative exons and
differed by only one nucleotide in 27.1% of the exons. For
the 39 splice sites, we examined the conservation of the last six
nucleotides of the intron and the first nucleotide of the exon,
as these are a binding site for the C. elegans homolog of the
heterodimeric U2 auxiliary factor (U2AF) involved in initial
39 splice-site recognition [34]. For 55.4% of the alternative
exons examined, these seven nucleotides were completely
identical, and for an additional 30.1% they differed by only
one nucleotide. This strong conservation of splice-site
sequence strength, along with regions of conservation in
flanking introns, suggests that these exons may be substrates
for alternative splicing in C. briggsae.
The Role of Conserved Elements in Alternative Splicing
Regulation
There are many examples of regions of introns flanking
alternatively spliced exons that function as cis-acting regu-
lators of alternative splicing. We hypothesized that the reason
that these high homology WABA-identified intronic elements
have been conserved over 100 million years of evolution is
that they may regulate alternative splicing. In order to test
this hypothesis, we looked at the C. elegans/C. briggsae align-
ment of the alternatively spliced region of the C. elegans
alpha(2) type IV collagen gene let-2. Let-2 has two mutually
exclusive alternative exons, exons 9 and 10, and their splicing
pattern is evolutionarily conserved as far back as the distantly
related parasitic nematode, Ascaris suum [35]. Messages
incorporate either exon 9 or exon 10 in a developmentally
regulated manner; embryos predominantly use exon 9, adults
predominantly use exon 10, and there is a gradual shift in the
usage of these two exons during the larval stages [36]. The
WABA alignment of this 400-base intron identifies four
conserved regions in the intron between exons 10 and 11
(Figure 2). To test the role of these conserved intronic
elements in alternative splicing regulation, we employed a
splicing reporter construct system containing the alterna-
tively spliced region between exons 8 and 11 that mimics the
developmental control of alternative splicing for this region
when transformed into C. elegans [37]. We mutated the first
conserved element in this intron and monitored the devel-
opmental regulation of this splicing. Deletion of this
conserved element, in which 28 of 34 bases have been
conserved between C. elegans and C. briggsae, results in a major
reduction in the usage of exon 10 in L4 animals (unpublished
data). The identical effect on splicing of an even smaller
deletion within this element, del1.2, is shown in Figure 2. For
the L4 time point, the developmental stage at which we
should detect maximal exon 10 usage, only minimal splicing
of this exon is detected. Since mutation of this element
results in only minimal exon 10 inclusion, we hypothesize that
either adults produce a splicing factor that interacts with this
element to promote exon 10 splicing or that embryos
produce a splicing repressor that inhibits exon 10 splicing.
In the past, researchers have identified splicing regulatory
elements by creating a series of mutations across an intron. In
this computational approach, the interspecies genome align-
ments led us directly to a small element required for the
developmentally regulated switch in this splicing.
Analysis of a Database of Conserved Intronic Elements
Flanking Alternatively Spliced Exons
As demonstrated in the previous section, evolutionarily
conserved elements in introns flanking alternatively spliced
exons can be important for alternative splicing. We used a
computational approach to identify sequences that are more
likely to occur in these conserved elements than in total
intron sequences. As described above, we have identified 142
alternatively spliced genes in which the introns flanking an
alternatively spliced cassette exon contain high homology
regions with C. briggsae as defined by WABA. We extracted the
highly conserved C. elegans sequences from these introns and
put them into a database. The first or last seven nucleotides of
introns were excluded from this dataset as these contain
conserved signals for the constitutive splicing machinery [38].
This dataset contained 537 conserved elements of average
length, 38.5 bases (minimum length, seven bases, longest, 231
bases, and median length, 28 bases) for a total of 20,675 bases.
See Table S1 for a list of these elements and the alternatively
spliced genes from which they were derived. We also
generated a control dataset of all introns annotated in
Wormbase genome sequence release WS 120.
Because many alternative splicing factors have binding
preferences for relatively short sequence motifs [11,13,18,28–
32], we decided to search the conserved intronic element
dataset for short sequence motifs that appear more
frequently here than in total introns. In order to identify
sequences that are more prevalent in the conserved intronic
regions bordering alternative exons, we counted the number
of occurrences of every possible hexamer or pentamer motif
in both our conserved intron element and total intron
datasets. For each motif in the conserved element dataset, we
determined the observed frequency, which was the count of
each motif divided by the total number of possible motifs of
that length in that dataset. We also determined an expected
frequency based on the number of counts of each motif in the
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total intron dataset divided by the total number of possible
motifs in that dataset. We then calculated an observed over
expected (obs/exp) ratio for each motif, which was the
observed frequency of each motif in the conserved element
dataset divided by the expected frequency of each motif
based on the total intron dataset. Table 1 shows the 40 top
scoring pentamer and hexamer motifs in the conserved
element dataset as determined by the obs/exp ratio value.
Several previously identified mammalian splicing regula-
tory sequences are present in our lists of high-scoring
hexamers and pentamers. This is encouraging because many
of the known mammalian splicing factors have C. elegans
homologs. For example, the sequence TGCATG and its subset
pentamer GCATG have been shown to be important intronic
regulators of splicing for the mammalian fibronectin and
calcitionin/CGRP genes [14,15,39]. The mammalian FOX-1
protein selected the GCAUG sequence in a SELEX experi-
ment, and it has been shown that the FOX-1 protein alters
alternative splicing of several GCAUG-containing genes in
transient transfection assays [13]. FOX-2, a homolog of FOX-
1, has also been shown to regulate splicing of transcripts
containing UGCAUG in neuronal cell culture [12]. FOX-1 was
originally identified in C. elegans as a splicing factor of the
transcript for the sex determination gene, xol-1 [40]. Another
well studied mammalian motif is that of intronic GU
dinucleotide repeats, which have been shown to regulate
splicing of the human CFTR gene [41] and serve as a binding
site for the ETR-3 splicing regulatory factor [42]. CU
dinucleotide repeats serve as a binding site for the PTB
family members in the intronic regions that regulate
inclusion of the N1 exon of c-src [43]. While targets for C.
elegans alternative splicing factors might be inferred from
their homology to mammalian proteins, very little exper-
imental evidence exists for nematode splicing-factor binding
sites. The C. elegans muscle-specific splicing factor SUP-12
regulates unc-60 alternative splicing. This protein interacts
with a GU-rich region, similar to the GU-rich motifs
identified in Table 1, of an unc-60 intron [44]. (For a review
of other mammalian splicing factors with C. elegans homologs
see [33].) While many of the high-scoring conserved motifs
that we identified match known mammalian splicing regu-
latory sequences, we have also identified new motifs that may
also be involved in pre-mRNA splicing regulation in
mammals as well as nematodes.
One important aspect of C. elegans introns is that they are
shorter than introns in vertebrates. Half of C. elegans introns
are below 60 nucleotides in length, too short to be spliced in
vertebrates [38]. However, many C. elegans introns are long
and resemble vertebrate introns. In our alternatively spliced
intron dataset, the median intron size is 263 bases (shortest is
43, longest is 10,719, and average is 561), indicating that these
regulated introns generally belong in the larger intron class.
We asked whether a similar analysis of the pentamers and
hexamers found in the full introns flanking alternatively
spliced exons would yield similar high-scoring motifs as our
analysis of the conserved sequence subset of these introns. We
did a pentamer and hexamer motif count of the full introns
flanking the 147 alternatively spliced exons from which we
previously extracted evolutionarily conserved elements. We
counted the occurrence of every pentamer and hexamer in
Figure 2. An Intronic Element Regulates let-2 Alternative Splicing
The top of this figure shows the alignment of C. elegans and C. briggsae sequences for the alternatively spliced region of let-2. Blue tracks indicate the
splicing for the embryonic (top) and adult (bottom) isoforms. The purple track indicates homology between the C. briggsae and C. elegans genomes as
determined by WABA. Dark purple tracks indicate regions of strong homology (.70%). The sequence of the first conserved element of intron 10 is
shown. The box indicates the part deleted and replaced with the sequence GAA in the del1.2 splicing reporter construct. The lower left part of the
figure shows the results of32P RT-PCR reactions with primers specific for the splicing reporter. Products for exon 9- or exon 10-containing messages are
indicated. In embryos, only usage of exon 9 is detected for either reporter. At the L4 stage, 34% of the wild-type reporter messages contain exon 10
while del1.2 mutant messages contain only a trace amount of this exon.
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