MINIREVIEW
Alternative splicing: role of pseudoexons in human disease
and potential therapeutic strategies
Ashish Dhir and Emanuele Buratti
International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
Introduction
Towards the end of the 1970s, in the beginning of
pre-mRNA splicing research [1,2], defining exons and
introns was essentially based on observing the final
composition of the mature mRNA molecule. In 1978,
any sequence that was included in a mature mRNA
became tagged as an ‘exon’, whereas all the intervening
genomic sequences that were left out during the splic-
ing process became defined as ‘introns’ [3]. However,
this way of thinking did not explain what makes an
exon an exon or an intron an intron. The discovery of
the basic splice site consensus sequences during the
same years [4,5], and later on of enhancer and repres-
sor elements, has taken us a long way in the direction
of discovering exon- and intron-definition complexes
[6–8]. Nowadays, the splicing signals that define ex-
ons ⁄ introns have been greatly aided by basic research,
bioinformatic approaches and advanced sequencing
tools [9,10]. In this regard, we certainly know much
more about splicing regulation than we did 20 years
ago. Considering that several reviews have been writ-
ten recently on the subject, the reader is referred to
them for further information on the latest discoveries
[11–14]. Most important, in this respect, have been the
initial observations that in alternative splicing pro-
cesses the same nucleotide sequence could be defined

frequent than previously thought. Here we aim to provide a review of the
mechanisms that lead to pseudoexon activation in human genes and how
the various cis- and trans-acting cellular factors regulate their inclusion.
Moreover, we list the potential therapeutic approaches that are being tested
with the aim of inhibiting their inclusion in the final mRNA molecules.
Abbreviations
3¢ss, 3¢ splice site; 5¢ss, 5¢ splice site; AON, antisense oligonucleotide; LINE, long interspersed elements; NMD, nonsense-mediated decay;
PTB, polypyrimidine tract binding protein; SINE, short interspersed elements.
FEBS Journal 277 (2010) 841–855 ª 2010 ICGEB Trieste (Italy) Journal compilation ª 2010 FEBS 841
understanding most splicing decisions. Indeed, even
the latest attempts at ‘designing’ exons based on
current state-of-the-art knowledge have basically dem-
onstrated that there is still a long way to go before we
can become as good as the spliceosome in deciding
what is an exon and what is an intron [21].
Where do pseudoexon sequences come
into the story?
Central to the issue of deciding what is an exon and
what is an intron is the question of their origin, a very
much debated field to this day that basically deals with
deciding the order of appearance of introns during
evolution, whether first, early or late [22]. Whatever
the answer to this question will turn out to be, it is
now clear that many of the ‘new’ exons in our genome
originate from the insertion of transposable sequence
elements belonging to the SINE and LINE classes in
the eukaryotic genome [23–25]. In particular, exoniza-
tion of Alu elements (which are primate specific and
represent the most abundant mobile elements in the
human genome) through retrotranposition–mutation

sequent rapid degradation by nonsense-mediated decay
(NMD) pathways [36] (Fig. 1). Such an occurrence has
been described in the rat a-tropomyosin gene with a
putative pseudoexon sequence localized downstream of
two mutually exclusive exons: an upstream exon that
is included only in smooth muscle tissue and a down-
stream exon that is included in most cell types [37].
Fig. 1. The left panel shows a schematic model of Alu element exonization. The element (Alu) is inserted by retrotransposition and during
the course of evolution mutations within this sequence create viable splicing sequences. The middle panel shows the effect of the inclusion
of a nonsense exon sequence (NE) in a transcript. When this nonsense exon sequence is included, the resulting transcript is degraded by
NMD (lower diagram). The right panel shows the classical pathway of pseudoexon (PE) inclusion in human disease. In this case, a nucleotide
sequence on the brink of becoming an exon becomes activated following a number of different mutational events.
Pseudoexons in human disease A. Dhir and E. Buratti
842 FEBS Journal 277 (2010) 841–855 ª 2010 ICGEB Trieste (Italy) Journal compilation ª 2010 FEBS
Experimental analysis has shown that, when this
pseudoexon is included in the mRNA molecule
together with the ubiquitously expressed downstream
exon, the formation of a stop codon causes activation
of the NMD pathway. On the other hand, when inclu-
sion of this pseudoexon occurs with the upstream
smooth muscle tissue-specific exon, then it can still be
removed through a resplicing pathway (and a normally
processed mRNA molecule can be generated). For this
reason, the term ‘nonsense’ exon is now preferred to
define these kinds of sequence, which according to
bioinformatic analyses may be more prevalent in
human genes than previously thought [37].
Nonetheless, from a human disease point of view,
many pseudoexon intronic sequences seem poised on
the brink of becoming exons (Fig. 1) and a compre-

regulatory sequences that will be discussed more in
detail below (Fig. 2B). Finally, in two individual cases,
the rearrangement of genomic regions through gross
deletions (Fig. 2C) [40] or genomic inversions
(Fig. 2D) [41] has also been described to give rise to
pseudoexon inclusion events. This has come about
either by bringing together viable splice sites that
would normally be too far away from each other on
the gene sequence or by activating exons in what
would normally have been the antisense genomic
strand.
In a few genes, a particularly interesting method of
pseudoexon activation event has also occurred follow-
ing the inactivation of naturally occurring up
stream 5¢ss (FAA, IDS, MUT) [42–45] or downstream
3¢ss (BRCA2, CFTR) [46,47] (Fig. 2E). These findings
suggest that the processivity of these mRNA tran-
scripts probably represents an element capable of
determining pseudoexon repression apart from being
capable of influencing normal splicing levels [48].
On a more general note, a still underappreciated
aspect of pseudoexon recognition that concerns the
effect of cis-acting sequences is represented by the
potential influence of RNA secondary structure on
splicing efficiency [49]. Recently, it has been shown
that donor site usage in the inclusion of two pseudoex-
on sequences in the ATM and CFTR genes is strongly
dependent on their availability in the single-stranded
region [50]. Interestingly, the same conclusion was
reached in a recent study by Schwartz et al. [51] analy-

Activating
mutation Reference DBASS3 ⁄ DBASS5 reference
a-Gal A 57 SRE creation [78] http: ⁄⁄www.som.soton.ac.uk ⁄ research ⁄ geneticsdiv ⁄ dbass5 ⁄ viewsplicesite.aspx?id=317
ATM 65 SRE deletion [56] http: ⁄⁄www.som.soton.ac.uk ⁄ research ⁄ geneticsdiv ⁄ dbass5 ⁄ viewsplicesite.aspx?id=324
ATM 137 5¢ss creation [79] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=331
b-globin 165 5¢ss creation [80] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=323
b-globin 126 5¢ss creation [81] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=336
b-globin 73 5¢ss creation [82] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=335
BRCA1 66 3¢ss creation [83] NA
BRCA2 93 Downstream
3¢ss deletion
[46] NA
CD40L 59 5¢ss creation [84] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=437
CEP290 128 5¢ss creation [85] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=342
CFTR 49 5¢ss creation [86] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=330
CFTR 84 5¢ss creation [87] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=328
CFTR 101 SRE creation [88] NA
CFTR 184 Downstream
3¢ss deletion
[47] http://www.som.soton.ac.uk/research/geneticsdiv/dbass3/view.asp?item=splice&id=31
CFTR 214 5¢ss creation [89] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=322
CHM 98 3¢ss creation [90] NA
COL4A3
a
74 3¢ss creation [91] NA
COL4A5 30 3¢ss creation [92] http://www.som.soton.ac.uk/research/geneticsdiv/dbass3/view.asp?item=splice&id=240
COL4A5 147 SRE creation [92] NA
CTDP1 95 5¢ss creation [93] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=333
CYBB 56 5¢ss creation [94] NA
CYBB 61 5¢ss creation [95] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=306

FBN1 93 5¢ss creation [102] NA
FGB 50 SRE creation [63] NA
FGG 75 5¢ss creation [103] NA
FVIII 191 5¢ss creation [104] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=332
GALC 34 ND [105] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=344
GHER 69 5¢ss creation [106] NA
GHR 102 SRE deletion [57,107] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=316
GUSB
a
68 5¢ss creation [108] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=311
Pseudoexons in human disease A. Dhir and E. Buratti
844 FEBS Journal 277 (2010) 841–855 ª 2010 ICGEB Trieste (Italy) Journal compilation ª 2010 FEBS
Interestingly, repression of the tropomyosin non-
sense exon was also observed following PTB overex-
pression. PTB is a well-known and powerful splicing
modifier that plays a major role in alternative splicing
regulation [8,53]. Recently, this protein has been
reported to also downregulate the inclusion efficiency
of a pathological pseudoexon in NF-1 intron 31 inde-
pendently of the activating mutation that creates a
very strong splicing acceptor site [54] (Fig. 3B). This
finding suggests that silencer binding sites may be
Table 1. (Continued.)
Gene Size (bp)
Activating
mutation Reference DBASS3 ⁄ DBASS5 reference
HADHB 56 ⁄ 106 5¢ss creation [109] NA
HSPG2 130 5¢ss creation [110] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=346
IDS 78 5¢ss creation [111] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=329
IDS 103 Upstream 5¢ss

165 3¢ss creation [69] http://www.som.soton.ac.uk/research/geneticsdiv/dbass3/view.asp?item=splice&id=114
OAT
a
142 5¢ss creation [126] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=433
OTC 135 3¢ss creation [127] NA
PCCA 84 SRE creation [68] NA
PCCB 72 5¢ss creation [68] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=436
PHEX 50 ⁄ 100 ⁄
170
5¢ss creation [128] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=321
PKHD1 116 5¢ss creation [129] NA
PMM2 66 3¢ss creation [130] NA
PMM2 123 5¢ss creation [130,131] NA
PRPF31 175 5¢ss creation [132] NA
PTS
a
45 Branch-point
optimization
[133] NA
PTS
b
79 Py-tract
optimization
[133] NA
RB1 103 3¢ss creation [134] NA
RYR1 119 5¢ss creation [135] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=337
SOD-1 43 5¢ss creation [136] NA
TSC2 89 5¢ss creation [137] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=307
a
Alu-derived pseudoexons.

Therapeutic strategies aimed at
correcting pseudoexon inclusion in
genetic diseases
Therapeutic strategies based on antisense oligonucleo-
tide (AON) chemistry, which uses base pairing to tar-
get specific sequences in RNAs, have been extensively
employed to correct splicing disorders in human genes
[58,59]. Interestingly, apart from these therapeutic
applications, short nuclear RNAs may also play a sim-
ilar functional role to physiologically regulate exon
inclusion, such as the case of snoRNA HBII-52 in the
regulation of exon Vb inclusion in the serotonin recep-
tor 2C [60]. AONs are thought to modulate the splic-
ing pattern by steric hindrance of the recruitment of
the splicing factors to the targeted splicing competent
cis-elements, thus forcing the machinery to use the nat-
ural sites. Dominski and Kole [61] were the first to
pioneer the antisense-mediated modulation of pre-
mRNA splicing. In the earliest examples, AONs were
aimed at activated cryptic splice sites in the b-globin
and CFTR genes in order to restore normal splicing in
b-thalassaemia and cystic fibrosis patients [61,62].
Currently, however, AON strategies have been used suc-
cessfully to restore normal splicing in several disease
models.
AB
C
E
D
Fig. 2. The mutational events that determine pathological pseudoexon inclusion. The most frequent is represented by the creation

oligonucleotides that targeted the aberrant 5¢ss and
3¢ss sites achieved 100% restoration of correctly
spliced mRNA.
Pseudoexon-activating mutation 3849 + 10 kb C >
T in intron 19 of the CFTR gene has been reported to
frequently cause cystic fibrosis. In their study, Fried-
man et al. [62] reported that a cocktail of 2¢-O-methyl
phosphorothioate oligoribonucleotides against different
regions of this pseudoexon abolished pseudoexon inclu-
sion and partially restored production of normal
mRNA and CFTR processed protein (Fig. 4B).
Mutations in the DMD gene are known to cause
Duchenne and Becker muscular dystrophies. Recently,
Gurvich et al. [67] demonstrated that 2¢-O-methyl
ribose phosphorothioate AONs restored normal splic-
ing in primary myoblast cultures established from two
individual patients carrying out-of-frame pseudoexon
insertion mutations (Fig. 4C).
Methylmalonic acidaemia and propionic acidaemia
are caused by different gene defects in the MUT,
A
B
C
D
Fig. 3. A schematic diagram of the tropomyosin gene with exons 2 and 3, which are mutually exclusive (exon 3 is the predominant form in
most cell types), and the nonsense exon (NE), which causes transcript degradation following its joining to exon 3 (but not exon 2). The levels
of hnRNP H ⁄ F proteins can regulate the extent of NE inclusion. (B) shows that in the NF-1 intron, 30 pseudoexon inclusion levels are regu-
lated by silencer elements in UCUU-rich motifs that bind the PTB (hnRNP I) splicing regulator. In the ATM gene, a four nucleotide deletion
(GUAA) in the intronic region between exons 20 and 21 causes the insertion of a 65 nucleotide long pseudoexon (C). Functional analysis has
demonstrated that this deletion abolished binding of an U1snRNP molecule in this position and activated a 3¢ss lying 12 nucleotides

This review is part of a miniseries co-ordinated by
Diana Baralle [71] to look at emerging topics in splic-
ing research, such as the correct assessment of
sequence variants as pathogenic mutations [72]; the
development of novel splicing-based therapeutic agents
to treat HIV-1 infections [73]; and new methods in the
global analysis of alternative splicing profiles [74]. We
decided to examine the role of pseudoexons in recent
research, as no specialized reviews have appeared in
the past dealing with this particular kind of event.
From a basic science point of view, the possibility
for researchers to look at the splicing process on a
much more global scale than the single exon or the
individual gene will clarify the issues examined in this
review by helping to distinguish clearly between exons
and pseudoexons [19,75,76]. In turn, this will provide a
better appreciation regarding how the splicing process
has evolved to define ‘exons’, how it distinguishes them
from similar potentially pathological sequences (pseud-
oexons) and what is the preferential way it has chosen
to repress their recognition. In this respect, pseudoex-
on research will also provide us with an unparalleled
opportunity to understand evolutionary mechanisms
A
B
C
Fig. 4. A schematic representation of three different 5¢ss activating mutations in various disease-causing genes that activate pseudoexon
inclusion where therapeutic correction has been attempted with an antisense approach. (A) represents the IVS2-705 T>G splicing mutation
that activates a 126 nucleotide pseudoexon in intron 2 of the b-globin gene. In this case, 2¢-O-methyl ribose AONs and functionally modified
U7 snRNA were employed to block the acceptor and donor splice sites. In (B), the 3849+10kbC>T splicing mutation activates a 84 nucleo-

toxicity and avoidance of undesired immune responses.
Furthermore, even after achieving all of these aims,
there will still remain the need to optimize recurrent
administration protocols (this is an often overlooked
consideration, as none of these methods will cause a
permanent correction of mRNA splicing defects), and
determining their clearance ⁄ accumulation in human
organs ⁄ tissues. However, notwithstanding all of these
difficulties, AON technology [59,77] has already
entered the clinical trial stage for diseases such as
Duchenne muscular dystrophy (http://www.clinicaltri-
als.gov) and this represents a bright hope for the not
too distant future.
Acknowledgements
This work was supported by Telethon Onlus Founda-
tion (Italy) (grant no. GGP06147) and by a European
community grant (EURASNET-LSHG-CT-2005-
518238). We thank Professor F. E. Baralle for helpful
discussion.
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