SURVEY AND SUMMARY
Regulated functional alternative splicing
Julian P. Venables, Jamal Tazi and Franc ¸ois Juge*
Universite ´ Montpellier 2, UMR 5535, Institut de Ge ´ne ´tique Mole ´culaire de Montpellier, CNRS, 1919 Route de
Mende, 34293 Montpellier cedex 05, France
Received April 3, 2011; Revised July 4, 2011; Accepted July 24, 2011
Alternative splicing expands the coding capacity of
metazoan genes, and it was largely genetic studies
in the fruit-fly Drosophila melanogaster that estab-
lished the principle that regulated alternative splic-
ing results in tissue- and stage-specific protein
isoforms with different functions in development.
Alternative splicing is particularly prominent in
germ cells, muscle and the central nervous system
where it modulates the expression of various pro-
teins including cell-surface molecules and tran-
scription factors. Studies in flies have given us
numerous insights into alternative splicing in terms
of upstream regulation, the exquisite diversity of
their forms and the key differential cellular functions
of alternatively spliced gene products. The current
inundation of transcriptome sequencing data from
Drosophila provides an unprecedented opportunity
to gain a comprehensive view of alternative splicing.
Alternative splicing is a means by which an organism can
create multiple mRNAs from a single gene; the connec-
tion of different parts of a pre-mRNA leads to different
30-untranslated regions of mRNAs and consequently
modulate translation, stability or localization of mRNA.
Also, by modulating inclusion and exclusion of coding
exons, this process dramatically increases overall protein
diversity and is thought to account for some of the com-
plexity of higher organisms such as vertebrates and
equally that of complex tissues such as the brain. Most
importantly, alternative splicing can lead to the expression
of functionally different proteins from a single gene in a
temporal- or tissue-specific manner. About 95% of human
genes produce alternatively spliced mRNAs (1,2) but the
functional consequences of these alternatilve splicing
events are mostly unknown. Indeed, it is not obvious
that different protein isoforms produced by alternative
splicing necessarily have different functions. The answer
to this important question requires extensive in vivo
Drosophila is the geneticist’s organism of choice because
of easy manipulation, quick generation times, extensive
collections of mutants and the numerous tools available
for manipulating gene expression. Pioneer studies in
Drosophila have led to the identification of regulatory se-
quences in the pre-mRNA required for alternative splicing
regulation as well as the splicing regulators that bind to
them. These discoveries have paved the way for alternative
splicing regulation studies in other organisms. Drosophila
uses every alternative splicing strategy imaginable with an
elegance and complexity that often eclipses mammals. To
summarize these findings, we highlight several examples of
alternative splicing events illustrating the diversity of al-
ternative splicing strategies used by Drosophila. The
examples of alternatively spliced genes are organized
into different sections according to the localization of
their encoded proteins in Figure 1. For easy reference,
the examples are discussed in the same order in the text
along with their diverse biological implications, where
known. The subsequent sections highlight common
themes in the regulation and evolutionary aspects of
tissue-specific alternative splicing and the final section
assesses the implication of new technologies for our under-
standing of the systems biology of alternative splicing and
the crucial role Drosophila studies will play. We refer
throughout to the current names of genes in Flybase
(http://flybase.org/) as these may differ from those in
their original references.
The concept of alternative splicing as a major controller of
gene expression came largely from work in the 1990s on
the Drosophila sex-determination pathway. Sex determin-
ation in Drosophila is controlled by a cascade of splicing
factors that are themselves alternatively spliced, ultimately
leading to the sex-specific expression of two different
*To whom correspondence should be addressed. Tel: +33 (0) 4 34 35 96 85; Fax: +33 (0) 4 34 35 96 34; Email: firstname.lastname@example.org
Published online 8 September 2011Nucleic Acids Research, 2012, Vol. 40, No. 1 1–10
? The Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
variants of the Doublesex (Dsx) transcription factor.
These proteins are differentially expressed throughout
male and female bodies to control characteristically differ-
ent sets of genes (Figure 1A). The key splicing differences
between males and females are set up by the expression of
the Sex-lethal (Sxl) protein in females. The early expres-
sion of Sxl in female embryos, in response to X chromo-
some number, also allows Sxl to auto-regulate its own
productive splicing in females and thus dictate female de-
velopment [reviewed in ref. (3)]. Female Sxl protein
represses the inclusion of an alternative ‘poison exon’ con-
taining a stop codon (exon 3) in its own mRNA, allowing
the expression of full-length protein. In males, the poison
exon is spliced into the mRNA and a truncated Sxl protein
is produced. Conclusively, ectopic expression of the female
form of Sxl in male flies causes the female splicing pattern
of endogenous Sxl (4). Sxl governs multiple aspects of
female differentiation. For example, Sxl represses the ex-
pression of the Male-specific lethal (Msl2) protein, which
is involved in X-dosage compensation in males, by inhibit-
ing both splicing and translation of the msl2 transcript in
females (5). Downstream in the sex-determination splicing
cascade, Sxl interferes with the default proximal 30-splice
site in the transformer (tra) gene in females thus leading to
functional Tra protein in females only (6).
Tra is also a pre-mRNA splicing factor controlling key
alternative splicing events in females further downstream
in the sex-determination cascade and it was an early ex-
ample of combinatorial interplay between RNA-binding
proteins controlling alternative splicing. Here, Tra binds
in a complex with the Transformer 2 (Tra2) protein and
the SR protein RBP1 on the 13-nt repeat sequences in the
female-specific exon of the doublesex (dsx) gene, and with
SRp30 on the adjacent purine-rich element (7). This leads
to male and female splice forms of the Dsx transcription
factor with their own different sets of target genes that
drive sex-specific gene expression. The splicing differences
in the sex-determination pathway are found in every cell
of the male and female bodies; there are also sex-specific
splicing differences that appear only in the brain and in
germ cells, and some are controlled by other proteins than
the known sex-determination genes (8).
In the brain, another gene downstream of Tra repre-
sents one of the most striking demonstrations of the
function of alternative splicing. The fruitless locus (fru)
generates a complex set of mRNAs through the use of
alternative promoters and alternative splicing at both the
50- and 30-ends of the transcripts. One set of transcripts,
arising from the P1 promoter, are spliced differently in
males and females in order to express a specific isoform
of Fru only in males (9). The protein is not expressed in
females because female-specific splicing uses a down-
stream 50-splice site that interrupts the reading frame of
the protein (10). The female-specific 50-splice site depends
for its use on an enhancer complex containing Tra/Tra2
which assembles on the RNA 40–240nt upstream of the
female-specific splice site (11). Remarkably, male splicing
of fru is necessary for male courtship behaviour and it is
sufficient to generate male behaviour in otherwise
wild-type females (9).
Although Tra2 is ubiquitously expressed, it is most
highly expressed in testis where its high level leads to
direct repression of removal of an intron in its own
(Figure 1B) (12,13). Limiting the overabundance of Tra2
by this mechanism would appear to be important as
increasing gene dosage of Tra2 in male flies had an in-
creasingly severe effect on male fertility (14). In another
example of testis-specific splicing, TAF1 (TBP-associated
factor 1) needs two AT hooks to bind DNA, and alterna-
tive splicing of one of the hook regions comes from a 33
amino acid cassette exon that is included at much higher
levels in testis than in any other tissue (15). Tra2 is likely
involved in this testis-specific splicing event since Tra2
level affects Taf1 splicing in S2 cells (16). Splicing in the
testis is also controlled, in part, by a testis-specific homo-
logue of the U2AF large subunit (17).
A study that demonstrates the power of Drosophila
genetics to decipher the function and mechanism of alter-
native splicing involved the ovarian tumour (otu) gene,
which is required for female germ-line differentiation.
otu has a 126-base in-frame alternative exon that encodes
a Tudor domain, which is included in early stages of oo-
genesis but excluded in late stages. Inclusion of this alter-
native exon requires the activity of the pUf68 (poly U
binding factor 68kDa a.k.a. half pint) splicing factor since
pUf68 mutant flies specifically omit the exon. Remarkably,
pUf68 mutant ovaries display ovarian phenotypes that can
be rescued by correctly spliced otu, revealing a critical role
of the long Otu isoform in oogenesis (18).
ALTERNATIVE SPLICING IN MUSCLE
Some of the first reports of regulated alternative splicing
discovered, which probably have important implications
for cellular function, came in four Drosophila myofibrillar
genes (Figure 1C). Many alternatively spliced Drosophila
genes have an elegant organization of mutually exclusively
used exons that provide subtly different coding regions to
genes. For example, the myosin heavy-chain isoforms
are encoded by a single gene (Mhc), which has in-frame
alternative cassette exons in five different regions (exons 3,
7, 9, 11 and 15) and a cassette exon (number 18) that en-
codes an alternative C-terminus. Thus, by multiplying
these independent choices, it is apparent that there are
potentially 480 different myosin isoforms in Drosophila.
An early study documented many developmental splicing
changes in Mhc in different kinds of muscle (19). For
example, exclusive use of exon 15a was observed in
thorax (20) and more recently, widespread regulation
of the Mhc exons has been shown in 33 different muscle
types from embryos (21). The major species were the
and 7(d)9(a)11(b)15(a)18(a) in fast contractile muscle
(Figure 1C). Therefore, different muscle types express spe-
cific Mhc isoforms through specific combinations of cas-
sette exons, suggesting regulated fine control of the
contractile properties of each kind of muscle. Indeed,
swapping the converter domain (encoded by the exon
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late embryo and CNS
embryo and neuroblast
Figure 1. Examples of alternative splicing patterns in Drosophila genes. Schematics illustrating regulated alternative splicing patterns of several
Drosophila genes. Exons and intervening intron size differences have been minimized to standardize formatting; therefore, genes are not depicted to
scale. For simplicity, alternative 50- and 30-terminal exons are not shown. Exons are shown as boxes, coding regions are grey tint and mutually
exclusive alternative exons are crosshatched. Splice forms are labelled according to the stage or cell type they are preferentially expressed in. (A) The
autosomal sex-specific splicing cascade. (B) Germ cell-specific splicing. (C) Muscle-specific splicing. IFM=indirect flight muscle. (D) Alternative
splicing of Dscam. Within the cluster of exon 6, ‘acceptor’ and ‘docking’ sites are shown as black dots (see the text). (E) Alternative splicing in genes
encoding cell surface molecules (F) Alternative splicing in genes encoding ion channels. (G) Alternative splicing of transcription factor genes. In Ubx,
RP3 represents a ratcheting point used for recursive splicing.
Nucleic AcidsResearch, 2012,Vol. 40,No. 13
11 cluster) from flight muscle to an embryonic exon
influenced the kinetic properties of the muscle fibres (22).
In addition to Mhc, there are indirect flight muscle
(IFM)-specific isoforms of myosin alkali light chain 1,
Tropomyosin 2 and Troponin-T. The Myosin alkali light
chain 1 gene (Mlc1) is spliced whereby the long penulti-
mate exon is skipped only in the IFM giving rise to alter-
native 12 residue C-termini (23). The intron upstream
from the alternative exon has no polypyrimidine tract,
which implies that special splicing factors might be
needed for its recognition. Tropomyosin 2 (Tm2) has a
429 base cassette, penultimate exon, used differentially
in embryo and in thoracic IFM giving rise to two highly
homologous alternative C-termini of 27 amino acids. The
two types of Tropomyosin correlate with different muscle
types and filament organization (24). In both Mlc1 and
Tm2, inclusion of the alternative exon could open the
transcript up to potential nonsense-mediated decay,
which might be an additional factor in their regulation.
A further noticeable example of muscle-specific splicing is
found in the upheld (up) gene encoding Troponin-T. up has
two mutually exclusive exons of identical size encoding
part of the C-terminal domain. The 10b exon is used in
jump muscle whereas exon 10a is almost entirely selected
in IFM (25). Moreover, in the N-terminus of Troponin-T,
three micro-exons, one being just 3nt in length, are col-
lectively skipped in IFM and jump muscle (26).
NEURAL ALTERNATIVE SPLICING
Splice form diversity is most extensive in the mammalian
brain, and in Drosophila too there are many examples of
neural-specific alternative splicing events including, most
notably, cell-surface molecules and transcription factors
(Figure 1D–G). The most exquisite example of alternative
splicing controls the cell-surface diversity of the Down
syndrome cell adhesion molecule (Dscam) (Figure 1D).
Dscam is organized into four clusters of 12, 48, 33 and 2
mutually exclusive exons, thus potentially encoding over
38000 different proteins (27). The molecular bar code
provided by alternatively spliced Dscam isoforms on the
cell surface impedes self-crossing of axons and is essential
for nervous system development, as artificial restriction of
Dscam alternative splicing diversity resulted in severe dis-
organization of neural circuitry (28). Exon 4 variants are
all used in similar proportions in brains, where neurone
density is highest, whereas the second exon of the exon 4
array populates most of the transcripts in wings and legs
Among the 48 exons of the Dscam exon 6 array, only
one is used at a time. The heterogeneous nuclear
ribonucleoprotein (hnRNP) hrp36 is a major player in
preventing inappropriate inclusion of exon 6 cassettes, as
depletion of this protein in S2 cells resulted in tandem
inclusion of multiple exon 6 variants in the mature
Dscam RNA. This effect was abrogated in the absence
of the ‘SR protein’ B52 implying that a balance of these
splicing factors controls the exon 6 array (30). However,
there needs to be an additional, failsafe, mechanism to
ensure that only one exon from the region is selected. It
was observed that a sequence upstream of each exon in the
exon 6 array, termed the ‘selector sequence’, is comple-
mentary to a putative ‘docking site’ sequence downstream
of exon 5. It therefore seems likely that complementarity
between the docking site and the selected exon’s selector
sequence confers mutual exclusivity (31). Indeed, removal
of the docking site in a Dscam minigene led to widespread
exon skipping and reduced the alternative splicing diver-
sity by favouring predominant selection of the first exon of
the array (32). There is now evidence that other arrays of
multiple mutually exclusive exons in Drosophila (e.g. Mhc
discussed above) may be similarly controlled, confirming
the importance of the RNA structure in the control of
alternative splicing (33).
Other alternative splicing events affecting the neuronal
cell surface include Cadherin-N (CadN). CadN exons 7, 13
and 18 each come from a choice of a pair of exons and
their splicing is temporally regulated during the first 24h
of embryonic development, with all three variable regions
shifting in concert from the 7a13b18b form to the
(Nrx-IV) is a component of septate junctions in glial
cells, and mutually exclusive alternative exons in the extra-
cellular domain are differentially incorporated between
glial cells and neurons. Utilization of exon 4 predominates
in neurons and increases Nrx-IV binding affinity for the
glial cell surface protein Wrapper (36). Glial- versus
neuronal-specific alternative splicing is also seen in the
dystroglycan (Dg) gene. Dystroglycan links the extracellu-
lar matrix to the cytoskeleton in a variety of cell types and
its mucin-like domains, which bind to Laminin, are en-
coded by a large 795-base alternatively spliced exon. The
long form is expressed on axons while the short form,
lacking this region, is found on glial cells. There is also a
shift to the short form during embryogenesis (Figures 1E
and 2A) (37). Further studies are required to elucidate the
specific function of the short form of Dystroglycan lacking
the mucin-like domains.
Several ion channels are alternatively spliced in the
brain (Figure 1F). For example, the Chloride Channel-a
(ClC-a) gene has a 127 amino acid insertion in its intra-
cellular region that is thought to be a regulatory site for
channel gating and trafficking. The alternative splice
consists of four adjacent exons that are only included in
Drosophila heads (38). The slowpoke (slo) calcium-
activated potassium channel is important for neuronal
firing, and gene-product diversity is provided by 5 differ-
ent promoters and 14 alternative exons in 5 different parts
of its extracellular domain. The first alternative cassette
encodes the mouth of the ion pore, and alternative exon
selections in the first and third regions both affect calcium
ion sensitivity of the channel. Coordinated shifting of all
four alternatively spliced regions was seen between larvae
and adults. There is also higher inclusion of the final
cassette exon in brain than in other tissues (39). The para-
lytic gene (para) encodes a voltage-gated sodium channel,
and it has several regions of alternative splicing which are
differentially regulated. One alternative cassette exon
encodes 22 amino acids in the first intracellular loop
with a PKA phosphorylation site, and some individual
neurons express only the long form while others only
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have the short form and this correlates with sodium
currents in those individual cells (40). Therefore, alterna-
tive splicing of para (like dscam, see above) appears to
differentiate individual neurons from one another with
implications for CNS complexity. Recent studies have
revealed an extreme complexity of para isoforms and dif-
ferent functions for the many different variants (41,42).
ALTERNATIVE SPLICING OF TRANSCRIPTION
In addition to the Dsx transcription factor responsible for
sex-specific differentiation (see above), other transcription
factors are also alternatively spliced in Drosophila.
Homeotic genes encode transcription factors involved in
the specification of cell identities according to their
Figure 2. Visualization of alternative splicing events by RNA-Seq. All data are taken from the modENCODE website http://www.modencode.org/
(71). Different known transcripts of each gene are shown with exons as boxes and introns as lines between. RNA-Seq short reads from different
tissues, stages or conditions, are piled below. Arrows point to regions where the number of reads changes relative to the gene as a whole.
(A) Maternal Dystroglycan mRNA detected in early embryos includes an alternative exon, which is skipped later on during embryogenesis (see
the text and Figure 1E). (B) Sex-specific splicing of the zinc finger transcription factor Chorion factor 2. The male-specific splice form has two extra
zinc fingers due to the use of a downstream 5’-splice site (72). (C) In the Ovo gene, encoding a zinc finger transcription factor, inclusion of the
alternatively spliced region correlates with the use of the proximal promoter and polyadenylation sites (59). (D) The so-called Twintron of
the prospero gene has a seemingly ordinary intron in early embryogenesis but a switch occurs to a different set of borders, which are spliced by
the minor U12 spliceosome (73). This change may be caused by downregulation of the heterogeneous ribonucleoproteins Hrp38 and Hrp36 (74).
(E) The CG6084 gene was shown to be alternatively spliced in cells in which the B52 gene was knocked down, by the use of splicing-specific
Nucleic AcidsResearch, 2012,Vol. 40,No. 15
position along the antero-posterior axis. Among them,
by large genes, spanning of the order of 100kb, which
both have small alternatively spliced exons (Figure 1G).
Antp has two regions of alternative splicing directly
upstream from the region encoding the homeodomain of
the protein. Thus, four alternative splice forms can be
made due to an alternative exon of 13 codons and alter-
native 50-splices sites of the downstream exon that are four
codons apart. The form that omits both regions is
abundant in early embryogenesis and the longer form pro-
gressively accumulates in late embryos and subsequent
stages (43). There are also variations in isoforms ratio
between adult tissues (44).
Ubx is alternatively spliced to produce six mRNAs, by
the use of two 50-splice sites (a and b) at the end of exon 1
and by inclusion of two alternative micro-exons (mI and
mII). The 34 amino acids encoded by the two alternative
exons form a linker region that modulates Ubx’s
DNA-binding activity and its capacity to activate the
dpp target gene (45) or to repress Distalless gene expres-
sion (46) in transgenic assays. Skipping of mI and mII
alternative exons requires the removal of 74kb of intronic
sequence. Analysis of this splicing event led to the discov-
ery of a new concept in splicing research, namely ‘recur-
sive splicing’, whereby portions of a large intron can be
spliced out sequentially. Here, splicing of mI and mII to
the 50a site re-creates a 50-splice site each time that can
then be re-used for splicing out the remaining downstream
part of the intron (Figure 1G) (47). In a separate study,
bioinformatic analysis revealed the widespread presence of
‘zero-length exons’ (i.e. juxtaposed 30- and 50-splice sites of
the form (Y)nNCAG/GUAAGU, where ‘/’ marks the
splice junction) within large introns that can be used as
‘ratcheting points (or RP sites) for recursive splicing (48).
Experimental validation confirmed the use of several RP
sites in vivo, such as the RP3 site found in the 52kb third
intron of Ubx, indicating that RP sites may facilitate
splicing of large Drosophila introns by sequentially truncat-
ing them during transcription (48). RP sites are also sig-
nificantly enriched in other invertebrates’ large introns but
not in vertebrate ones indicating that recursive splicing
may not be used in vertebrates (49). Finally, two adjacent
exons encoding a total of 270 amino acids in the
Grainyhead (grh) transcription factor are specifically
altering its dimerization properties (50).
and may functionby
CONTROL OF ALTERNATIVE SPLICING
While the factors that control alternative splicing in the
sex-differentiation pathway are well known (Figure 1A),
relatively little is known as yet about what controls the
other diverse splicing events that have been observed. For
the most part, alternative splicing has been considered to
result from the independent recruitment to the pre-mRNA
of RNA-binding proteins. RNA interference was used to
knockdown 250 Drosophila RNA-binding proteins in S2
cells, and reverse-transcribed RNA samples were tested by
PCRs targeting 19 alternative splicing events in three
genes; 47 of the RNA-binding proteins were thus identi-
fied as direct or indirect controllers of one or more alter-
native splice events (51). Among these were known
alternative splicing factors (i.e. proteins that are not essen-
tial for splicing generally but have substrate-specific
effects) and other proteins that had previously been con-
sidered as constitutive- rather than alternative-splicing
factors. Both alternative and constitutive splicing fac-
tors were found to regulate Taf1 alternative splicing
(Figure 1B) in response to ATR pathway activation
using the same strategy (16). These substrate-specific
effects of knockdowns of core spliceosomal components
are likely of relevance to human diseases involving consti-
tutive splicing factors. The identification of multiple
splicing factors controlling individual alternative splice
events also suggested combinatorial action of factors.
For example, Pasilla and Mub coordinately regulate
splicing of Dscam exon 4.2, whereas Rox8/dTIAR and
B52 might coordinately regulate an alternative splice site
in dADAR (51).
B52 is a ubiquitously expressed member of the ‘SR
protein’ family of splicing factors. Several strategies have
been used to identify alternative splicing events regulated
by B52, such as genomic Systematic Evolution of Ligands
by EXponential enrichment (SELEX) (52) or immunopre-
cipitation of RNA bound by a tagged-B52 overexpressed
in specific tissues (53). Analysing the phenotypes induced
by B52 overexpression also gave insights into the develop-
mental processes modulated by this protein in vivo and
therefore implicated potential target genes. For example,
B52 overexpression during early eye development led to
flies harbouring partially to totally reduced eyes, a pheno-
type that resembles the eyeless (ey) mutant phenotype. It
turned out that ey is indeed a direct target gene of B52;
B52 binds to and promotes the inclusion of an alternative
exon, which inserts 60 amino acid residues upstream of the
paired domain (Figure 1G). The Ey isoform including the
alternative exon has lower affinity for its DNA targets
in vitro and is less potent in driving eye development
in vivo (54).
To systematically study the targets of SR proteins,
Blanchette and co-workers knocked down B52 and
dASF/SF2 in S2 cells and monitored splicing of 8315 al-
ternative splicing events in 2797 genes by the use of
splicing-specific microarrays. The knockdowns affected
107 and 319 alternative splicing events, respectively (55).
The same group also knocked down four hnRNPs. In
parallel to their splicing analysis, a global analysis of
pre-mRNA regions bound by these proteins was per-
formed using RNA-immunoprecipitation followed by hy-
bridization to tiling arrays. All four hnRNPs affected a
similar number of splicing events, which overlapped sig-
nificantly with the targets they bound; however, there were
interesting differences. For example, hrp36 and hrp38 had
significant overlap in their splicing targets, suggesting a
redundant function in vivo, and bound to introns signifi-
cantly more than hrp48, which bound mostly to exons
(56). The binding patterns of hnRNPs to genes offered
insights into the different mechanisms of hnRNP action,
including the ‘looping out’ model whereby hnRNP
binding at either end of an intron brings splice sites
6 Nucleic AcidsResearch, 2012,Vol. 40,No. 1
closer together, and also the nucleation model whereby
specific binding to RNA by an hnRNP is followed by
multiple binding and spreading of hnRNPs over a large
region (56). Recently, the targets of Pasilla (PS), the
homologue of mammalian NOVA1/2 splicing factors,
have been investigated by new generation full transcrip-
tome sequencing (RNA-Seq) after depletion of PS by
RNAi in S2 cells (57). This approach identified 405 alter-
native splicing events in 323 genes. Interestingly many
splicing events modified by PS depletion were previously
unknown; this discovery of previously unannotated splice
junctions highlights an important advantage of RNA-Seq
over pre-designed microarray methods.
Despite the valuable mechanistic insights these studies
have provided, simple up- or downregulation of splicing
factors in cells cannot fully uncover the complex network
underlying alternative splicing, as the activity of splicing
factors is controlled by the tissue-specificity of upstream
signals at different levels. For example, the Tra, Tra2 and
RBP1 splicing factors, involved in dsx alternative splicing,
are phosphorylated by the Darkener of apricot (Doa)
kinase, and in a sensitized genetic background for tra or
tra2, Doa mutants females display altered splicing of dsx
and an intersex phenotype (58). Tra2 protein level can also
be controlled by proteasomal degradation as shown in S2
cells in response to ATR signalling pathway activation
Many of the genes with multiple regions of alternative
splicing highlighted above have correlated alternative
splices between different regions within the gene, that
is to say the alternative splicing choice in one region is
often linked to alternative splicing in the other regions
and not independent. Examples include Mhc, para, slow
and CadN. In other genes like up, ClC-a and grh, sev-
included (Figure 1). In addition, there is sometimes a
clear correlation between alternative splicing and specific
promoter and polyadenylation site use as seen in the
ovo gene (59) (Figure 2C). Further studies are required
to establish if these linked changes within a gene are
due to coordinated action of trans-acting factors or
whether they are controlled at the level of transcription.
The prevailing theory is that fast transcription fa-
vours alternative exon skipping and slow transcrip-
tion allows time for alternative exon selection (60).
Co-transcriptional effects are likely to play an especially
important role in genes with long introns that can take
many minutes to be transcribed (e.g. Antp and Ubx,
collectively skipped or
CONSERVATION OF REGULATED FUNCTIONAL
There are big differences in gene structure between
Drosophila and mammals. For example, in humans
introns are usually much bigger than exons (87% of
introns are >250nt), whereas in Drosophila the reverse is
true (66% of introns are <250nt) (61). There are also
several unique facets of splicing in Drosophila that are
not found in mammals. For example, multiple variants
of mutually exclusive exons and recursive splicing are
not found in mammals. Another non-mammalian phe-
nomenon is that of trans-splicing, which is widespread in
Caenorhabditis elegans and also found in some Drosophila
genes (62). However, within mammals on the one hand
and insects on the other, there is extensive conservation
of alternative splicing patterns.
Several examples of regulated alternative splicing are
Cadherin-N has a broadly conserved exon structure
across 300 million years of arthropod evolution with the
orthologues of the ‘a’ and ‘b’ exons in other species being
far more conserved than the paralogous ‘a’ and ‘b’ exons
within the Drosophila CadN gene. Importantly, the central
pair of mutually exclusive exons is differentially expressed
in embryonic mesoderm and neurons in both Drosophila
and beetles (35). However, several attempts to discern the
functional differences between CadN isoforms have failed,
but clearly the conservation of the regulation entails an
important functional difference. Similarly, in the sex-
determination cascade, dsx and fru splicing are controlled
by Tra in diverse insects and this is the subject of recent
reviews (63,64). Conservation of sex-specific splicing has
led to the development of bio-control technologies that in-
duce the sex-specific gene expression of toxic genes in
insect agricultural pest species. For example, in the
Mediterranean fruit-fly Ceratitis capitata, it has been pos-
sible to exploit female-specific splicing controlled by en-
dogenous Tra/Tra2 to engineer sterilization genes into
females (65). Alternative splicing is also currently being
used to engineer flightless female mosquitoes, thus allow-
ing release of sterile males to control Dengue virus
Spliceosome dynamics and composition are very similar
between human and Drosophila (67) and most known
mammalian splicing factors have orthologues in flies
(51). These include members of the SR protein family
(8,30,55,56) and specific alternative splicing factors such
as Tra2 (7,14,58), TIAR/Rox8 (5,51), PUF60/pUf68 (18),
Fox proteins (68) and Nova/Pasilla (57,69). A recent study
compared the action of homologues of the mammalian
brain-specific splicing factors of the Nova family from
six different deuterostome and protostome species. This
showed, consistent with an earlier study (57), that the
mechanism of Nova/Pasilla proteins in enhancer- and
inhibitor-dependent splicing was conserved across the
animal kingdom, but in contrast, that the exons that
Nova controls are only conserved within the vertebrates.
Consistent with this, Nova expression is specific to the
nervous system in chordates, but not in flies (69). The
synopsis of these findings is that splicing factors and
their action are conserved across the animal kingdom
but their location, targets and therefore their developmen-
tal functions have changed. This means that studies in flies
are highly relevant to the mechanisms of alternative
splicing in humans and that we need to keep flies in the
bigger picture to understand the function of alternative
splicing in development and the way that evolution
adapts it to its needs.
Nucleic AcidsResearch, 2012,Vol. 40,No. 17
Recently, two RNA-Seq studies have been published
characterizing the full transcriptome of multiple embry-
onic, larval, pupal and adult time-points for both
Drosophila sexes (34,70). A full analysis of these new
data over the coming years will likely greatly impact
splicing, its mechanisms and its biological impact. For
example, the study of Graveley et al. (34) reports 11457
intron retention events, 3548 alternative 30-splices sites,
3457 alternative 50-splice sites, 1844 cassette exons, 831
multiple cassettes events and 145 mutually exclusive
splicing events that shift significantly between one cell
type and another. These data broadly confirm past
findings (Figure 2) but also identify a huge number of
new potential regulated functional alternative splice
events. In addition, to gain general insights into alterna-
proteins have been knocked down by RNAi in S2 cells
and analysed by RNA-Seq and also made available
through modencode (Figure 2E). When fully assimilated,
this information will certainly reveal further insights into
the systems biology and combinatorial control of alter-
One way of understanding this new mountain of RNA-
Seq data is to group alternative splice events that shift
with similar time courses in development (34). Gene
ontology classifications can also give indications of what
the function of alternative splicing programmes are.
Similarly, bioinformatic searches in the sequences of co-
regulated genes can identify RNA target motifs and hence
what splicing factors might control these changes. Finally,
one important way to distill the data will be to compare
alternative splicing in Drosophila with RNA-Seq data
from multiple tissues of other arthropods as these become
available, on the assumption that most important regu-
lated and functional alternative splicing events will be con-
served to some extent. In summary, as with classical
genetics, the easy manipulation of Drosophila has led to
the foundation of our view of how alternative splicing is
used to control development of higher organisms. Studies
in this model organism will likely continue to give key in-
sights into the complex questions now facing the splicing
field, such as how organisms exploit combinatorial control
by multiple splicing factors and what are the functions of
programs of multiple co-regulated alternatively spliced
diversity of alternative
targets, 26 RNA-binding
Thanks to Philippe Fort for useful discussions and
comments on the manuscript.
J.P.V. and J.T. were supported by the European Alter-
native Splicing Network of Excellence (EURASNET,
FP6 life sciences, genomics and biotechnology for
health) and a grant from Canceropo ˆ le and l’INCa 2009-
1-RT-10-CNRS13-1. J.T. was supported by Institut
Universitaire de France as a senior member. Funding
for open access charge: Agence Nationale de la Recherche
(ANR – 05 –BLAN – 0261 – 01); European Alternative
Splicing Network of Excellence (EURASNET, FP6 life
sciences, genomics and biotechnology for health).
Conflict of interest statement. None declared.
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