Báo cáo khoa học: Alternative splicing: role of pseudoexons in human disease and potential therapeutic strategies pot

Even more importantly, clinically oriented screening studies that search for splicing mutations are beginning to uncover a situation where aberrant pseudoexon inclusion as a cause of hum

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

by the spliceosome as an intron or an exon in response

to specific signals [15,16] It is now clear that these kinds of decision (What is an exon? What is an intron?) are of paramount importance in explaining genome complexity and evolutionary pathways [17–20] However, the sum of this new knowledge does not necessarily mean that we are near the goal of

Keywords

alternative splicing; antisense

oligonucleotides; mRNA; pseudoexons;

splicing therapy

Correspondence

E Buratti, Padriciano 99, 34012 Trieste, Italy

Fax: +39 040 226555

Tel: +39 040 3757316

E-mail: buratti@icgeb.org

(Received 26 August 2009, revised 15

October 2009, accepted 5 November 2009)

doi:10.1111/j.1742-4658.2009.07520.x

What makes a nucleotide sequence an exon (or an intron) is a question that still lacks a satisfactory answer Indeed, most eukaryotic genes are full

of sequences that look like perfect exons, but which are nonetheless ignored by the splicing machinery (hence the name ‘pseudoexons’) The existence of these pseudoexons has been known since the earliest days of splicing research, but until recently the tendency has been to view them as

an interesting, but rather rare, curiosity In recent years, however, the importance of pseudoexons in regulating splicing processes has been stea-dily revalued Even more importantly, clinically oriented screening studies that search for splicing mutations are beginning to uncover a situation where aberrant pseudoexon inclusion as a cause of human disease is more 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.

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

events is a prominent source of new exons in the

eukaryotic transcriptome, as schematically depicted in

Fig 1 [26,27]

However, even if we ignore this particular class of

exonization event, every in silico analysis shows that

‘false exons’ are very abundant in the intronic

sequences of most genes [with this term we refer to

any nucleotide sequence between 50 and 200–300 nucleotides in length with apparently viable 5¢ and 3¢ splice sites (5¢ss and 3¢ss) at either end] Presently, there is evidence that inclusion of many of these sequences is actively inhibited due to the presence of intrinsic defects [28], the presence of silencer elements [29–31] or the formation of inhibiting RNA secondary structures [32] Even if a combination of all these ele-ments succeeds in repressing the use of many of these pseudoexon sequences, we have to consider the possi-bility that there must be many exceptions to this rule First, it is probable that several of these pseudoex-ons may actually be recognized only in particular cir-cumstances, such as a consequence of particular external stimuli [33,34] or present in a given tissue or developmental stage Proof of this possibility is the observation that ‘novel’ exons keep being identified even in well-known and studied genes, such as the DMDgene [35]

Second, our failure to observe their use in normal conditions may also be due to the fact that their inclu-sion can intentionally lead to premature insertion of a termination codon in the mature mRNA and the con-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.

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-hensive list of more than 60 published pathological

pseudoexon events is presented in Table 1 Although

briefly reviewed previously elsewhere [38], the recent

advances in pseudoexon research warrant a second

look at several pseudoexon-related issues, especially

with regards to novel therapeutic approaches

Cis-acting sequences in pseudoexon

inclusion

As previously mentioned, most pathological

pseudoex-on inclusipseudoex-on events originate from the creatipseudoex-on of new

splicing donor or acceptor splice sites within an

intron-ic sequence, followed by the subsequent selection

of weaker ‘opportunistic’ acceptor or donor site

sequences (Fig 2A) A preliminary analysis of the

strength of donor sites activated in pseudoexon

inclu-sion events has highlighted their relatively high

strength (according to in silico prediction programs)

with respect to normally processed exons and to

cryp-tic donor sites activated following normal donor site

inactivation [39] In a slightly lower number of cases,

pseudoexon activation has been observed following the

creation of de novo acceptor sites (Table 1), whereas

branch-point creation still represents a minority

(prob-ably owing to the fact that a new branch point needs

to find both a viable acceptor and donor site nearby,

rather than just one of them)

In addition to de novo creation of strong donor,

acceptor and branch site sequences, the other most

fre-quent mechanisms that may lead to pseudoexon

activa-tion involves the creation⁄ deletion of splicing

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 strpseudoex-ongly 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-sing the differences between exonized and nonexonized Alu elements In this work, it was found that one of the major discriminating factors between these two classes of Alu elements was represented by the poten-tial availability of 5¢ss sequences in an unstructured conformation

Trans-acting factors in pseudoexon inclusion

Not many studies have focused on identifying the role played by trans-acting factors in pseudoexon inclusion However, because of its significance, this is an area of research that would probably benefit from increased attention by researchers in the future

In the case of nonpathologically related pseudoex-ons carrying npseudoex-onsense codpseudoex-ons, the presence of splicing regulatory elements may well provide a clue with regards to the possible roles played by these sequences For example, in the case of the previously described tropomyosin pseudoexon [37], the specific binding of hnRNP H⁄ F proteins has been described

as a potential key modifier of this pseudoexon inclu-sion event [52] The fact that these proteins are partic-ularly downregulated in cardiomyocytes may explain the cell-specific repression of the downstream ‘normal’ exon 3 that is otherwise present in all cell types (Fig 3A)

Table 1 Pathological pseudoexon inclusion events in human disease NA, not available; SRE, splicing regulatory element.

Gene Size (bp)

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

3¢ss deletion

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

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

COL4A5 30 3¢ss creation [92] http://www.som.soton.ac.uk/research/geneticsdiv/dbass3/view.asp?item=splice&id=240

CTDP1 95 5¢ss creation [93] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=333

CYBB 61 5¢ss creation [95] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=306 DHPR ⁄

QDPR

152 5¢ss creation [96] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=334

inversion

inversion

inversion

inversion

inversion

inversion

DMD 58 5¢ss creation [97] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=340

DMD 89 5¢ss creation [98] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=338 DMD 90 5¢ss creation [98] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=339 DMD 95 3¢ss creation [97] http://www.som.soton.ac.uk/research/geneticsdiv/dbass3/view.asp?item=splice&id=275 DMD 147 5¢ss creation [99] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=326 DMD 149 3¢ss creation [98] http://www.som.soton.ac.uk/research/geneticsdiv/dbass3/view.asp?item=splice&id=274 DMD 172 ⁄ 202 5¢ss creation [100] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=320 DMD 46 ⁄ 132 3¢ss creation [101] http://www.som.soton.ac.uk/research/geneticsdiv/dbass3/view.asp?item=splice&id=273

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

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

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

deletion

[42,43] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=282 INI1⁄

SNF5

72 5¢ss creation [112] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=410 ISCU 86 ⁄ 100 3¢ss creation [113–115] NA

deletion

[40] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=341

or upstream 5¢ss deletion

[45,68] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=434

http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=394 NDUFS7 122 5¢ss creation [118] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=357

NF-1 172 3¢ss creation [119] http://www.som.soton.ac.uk/research/geneticsdiv/dbass3/view.asp?item=splice&id=277

NF-1 54 5¢ss creation [121] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=411 NF-1 177 5¢ss creation [70,122,123] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=318 NF-2 106 Branch-point

creation

OA1 ⁄

GPR143

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

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

PMM2 123 5¢ss creation [130,131] NA

PTS a 45 Branch-point

optimization

optimization

RYR1 119 5¢ss creation [135] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=337

TSC2 89 5¢ss creation [137] http://www.som.soton.ac.uk/research/geneticsdiv/dbass5/viewsplicesite.aspx?id=307

a Alu-derived pseudoexons b LINE-2-derived pseudoexons.

actively used by evolutionary mechanisms to decrease

the probability that random activating mutations may

determine the constitutive inclusion of pseudoexon

sequences

In this respect, one interesting molecular complex is

U1snRNP, a ribonucleoprotein complex normally

associated with 5¢ss recognition in the normal splicing

process [55] First, U1snRNP binding to an intronic

splicing processing element has been found to inhibit

pathological pseudoexon inclusion in intron 20 of the

ATM gene (Fig 3C) Inactivation of this element

through a four nucleotide deletion causes pseudoexon

inclusion and occurrence of ataxia telangiectasia in a

patient [56] In a second case, binding of hnRNP E1

and U1snRNP to a weak 5¢ss efficiently silences

pseudoexon inclusion in the GHR gene [57], preventing

the development of Laron syndrome (Fig 3D)

Finally, it should also be noted that in a variety of

pseudoexon inclusion events, the activating mutations

potentially created new splicing enhancer sequences

(Table 1) Although in very few of these cases was

trans-acting factors binding to these elements

identi-fied, in silico and experimental analyses have shown

that several of the newly created enhancer sequences

strongly correlate with potential binding to the SR

protein class of splicing regulators

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

C

E

D

Fig 2 The mutational events that determine pathological pseudoexon inclusion The most frequent is represented by the creation

of de novo functional splice sites or branch-point elements through a single or few point mutation (A) Other mechanisms include the creation

or deletion of splicing regulatory elements (B), genomic rearrangements (C, D) and inactivation of upstream or downstream splice sites (E).

Afibrinogenemia is caused by genetic abnormalities

within any of the three genes that encode the fibrinogen

molecule: FGA, FGB, FGG Recently, Davis et al [63]

showed that a homozygous c.115–600A>G point

muta-tion located deep within intron 1 of FGB causes

pseudo-exon inclusion In this study, pseudopseudo-exon inclusion was

corrected by targeting this mutation with an antisense

phosphorodiamidate morpholino oligonucleotide

In several forms of b-thalassaemia, two single

nucle-otide mutations (IVS2-705 and IVS2-654) in the

b-globin gene have been reported to cause pathological

pseudoexon insertion In 1993, Dominski and Kole

[61] successfully tested 2¢-O-methylribose AONs to

restore correct splicing Later, Sierakowska et al [64]

also restored correct splicing and b-globin polypeptide

production using a phosphorothioate

2¢-O-methyl-oligoribonucleotide targeted to the aberrant 3¢ss More

recently, Gorman et al [65,66] engineered the U7

snRNA gene to correct pre-mRNA splicing by

replac-ing the antihistone sequence with sequences targetreplac-ing

b-globin aberrant splice sites (Fig 4A)

The congenital disorders of glycosylation are caused

by defects in the PMM2 gene Recently, Vega et al

[130] studied a c.640–15479C>T deep intronic muta-tion that creates a new aberrant 5¢ss in intron 7 and caused pseudoexon activation Antisense morpholino 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 upstream of this element In the last case, binding of hnRNP E1 and U1snRNP to a silencer motif near a weak 5¢ss efficiently silences pseudoexon inclusion in the GHR gene, preventing the development of Laron syndrome (D).

PCCA and PCCB genes Ugarte et al [68] recently

reported the identification of three novel deep intronic

mutations in each of these genes that potentially lead

to pseudoexon activation through diverse mechanisms

Antisense therapeutics using antisense morpholino

oligomers correctly restored almost complete normal

splicing that was effectively translated

Ocular albinism type 1 involves mutations in the

OA1 gene Vetrini et al [69] identified a deep intronic

point mutation g.25288G>A that created a new

acceptor splice site in intron 7 of this gene and resulted

in pseudoexon inclusion Treatment of a patient’s

melanocytes with antisense morpholino AONs

comple-mentary to the mutant sequence rescued mRNA and

protein expression levels

Mutations in the NF-1 gene cause neurofibromatosis

type 1 Recently, Pros et al [70] identified six

neurofi-bromatosis type 1 patients carrying three different deep

intronic mutations that create new 5¢ss leading to the

activation of the pseudoexon in the mature mRNA In

this study, antisense morpholino oligonucleotides were

targeted against these newly created 5¢ss, effectively

restoring normal NF-1 splicing

All of these different therapeutic strategies are

sum-marized in Table 2

Concluding remarks

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-tide pseudoexon in intron 19 of the CFTR gene Three 2¢-O-methyl phosphorothioate oligoribonucleonucleo-tides were targeted against the splice sites and against the pseudoexonic premature stop codon sequence to rescue normal splicing Finally, (C) shows the c.6614+3310G>T splic-ing mutation that activates a 137 nucleotide pseudoexon in intron 45 of the DMD gene To restore normal splicsplic-ing, 2¢-O-methyl ribose AONs were also targeted against the donor splice site and a predicted cluster of exonic splicing enhancer sequences within the pseudoexon.

that cause some of these sequences to become exons

and, of course, vice versa

Considering that aberrant pseudoexon inclusion

events are an increasing phenomenon linked with

dis-ease, just the simple characterization of these sequences

may have some very practical consequences The

stud-ies reported in this review clearly highlight the

feasibil-ity of using AONs to correct these types of splicing

defect (even in the absence of a complete or even

par-tial understanding of the ‘basic science’ explaining

their occurrence) From a therapeutic point of view,

the major advantage of targeting pseudoexon inclusion

events is provided by the supposition that AONs

targeted against what would normally be intronic

sequences would not be expected to remain bound to

the mature mRNA (and thus interfere with later stages

of RNA processing, such as export⁄ translation)

How-ever, several factors will still need to be improved

before human application becomes a reality These

start from basic studies aimed at optimizing gene⁄ exon

specificity (that will necessarily have to be made on an

individual gene-specific basis) to the development of

appropriate carrier systems These systems will be

absolutely necessary to achieve successful delivery, low

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

References

1 Berget SM, Moore C & Sharp PA (1977) Spliced seg-ments at the 5¢ terminus of adenovirus 2 late mRNA Proc Natl Acad Sci USA 74, 3171–3175

2 Chow LT, Gelinas RE, Broker TR & Roberts RJ (1977) An amazing sequence arrangement at the 5¢ ends of adenovirus 2 messenger RNA Cell 12, 1–8

3 Gilbert W (1978) Why genes in pieces? Nature 271, 501

4 Breathnach R, Benoist C, O’Hare K, Gannon F & Chambon P (1978) Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries Proc Natl Acad Sci USA 75, 4853–4857

5 Catterall JF, O’Malley BW, Robertson MA, Staden R, Tanaka Y & Brownlee GG (1978) Nucleotide sequence homology at 12 intron–exon junctions in the chick ovalbumin gene Nature 275, 510–513

6 Black DL (2003) Mechanisms of alternative pre-mes-senger RNA splicing Annu Rev Biochem 72, 291–336

7 Berget SM (1995) Exon recognition in vertebrate splicing J Biol Chem 270, 2411–2414

8 Sharma S, Kohlstaedt LA, Damianov A, Rio DC & Black DL (2008) Polypyrimidine tract binding protein controls the transition from exon definition to an intron defined spliceosome Nat Struct Mol Biol 15, 183–191

9 Zhang MQ (1998) Statistical features of human exons and their flanking regions Hum Mol Genet 7, 919–932

Table 2 Therapeutic approaches PMO, phosphorodiamidate morpholino oligonucleotide.

Therapeutic approaches Gene and diseases caused by pseudoexon inclusion Mode of action

predicted ESE motif created by the mutation

aberrant splice sites for long-term restoration of correct splicing Antisense morpholino

oligonucleotides

PMM2, congenital disorders of glycosylation;

MUT, PCCA and PCCB, methylmalonic acidaemia and propionic acidemia; OA1, ocular albinism type 1;

NF-1, neurofibromatosis type 1

Targeting aberrant splice sites activated due to mutations AONs block access

of the splicing machinery to the pseudoexonic regions in the pre-mRNAs Antisense 2¢-O-methyl

ribose phosphorothioate

oligonucleotides;

2¢-O- methylribo-oligonucleotides

CFTR, cystic fibrosis; HBB, b-thalassaemia;

DMD, Duchenne and Becker muscular dystrophies

Targeting of antisense against aberrant splice sites, in-frame stop codon and predicted exonic splicing enhancers within pseudoexons

10 Chasin LA (2007) Searching for splicing motifs Adv

Exp Med Biol 623, 85–106

11 Wang Z & Burge CB (2008) Splicing regulation: from

a parts list of regulatory elements to an integrated

splicing code RNA 14, 802–813

12 Hertel KJ (2008) Combinatorial control of exon

recog-nition J Biol Chem 283, 1211–1215

13 Cartegni L, Chew SL & Krainer AR (2002) Listening

to silence and understanding nonsense: exonic

muta-tions that affect splicing Nat Rev Genet 3, 285–298

14 Sperling J, Azubel M & Sperling R (2008) Structure

and function of the pre-mRNA splicing machine

Structure 16, 1605–1615

15 Amara SG, Jonas V, Rosenfeld MG, Ong ES & Evans

RM (1982) Alternative RNA processing in calcitonin

gene expression generates mRNAs encoding different

polypeptide products Nature 298, 240–244

16 Kornblihtt AR, Umezawa K, Vibe-Pedersen K &

Baralle FE (1985) Primary structure of human

fibronec-tin: differential splicing may generate at least 10

polypeptides from a single gene EMBO J 4, 1755–1759

17 Stamm S, Ben-Ari S, Rafalska I, Tang Y, Zhang Z,

Toiber D, Thanaraj TA & Soreq H (2005) Function of

alternative splicing Gene 344, 1–20

18 Xing Y & Lee C (2006) Alternative splicing and RNA

selection pressure–evolutionary consequences for

eukaryotic genomes Nat Rev Genet 7, 499–509

19 Blencowe BJ (2006) Alternative splicing: new insights

from global analyses Cell 126, 37–47

20 Tarrio R, Ayala FJ & Rodriguez-Trelles F (2008)

Alternative splicing: a missing piece in the puzzle

of intron gain Proc Natl Acad Sci USA 105, 7223–

7228

21 Zhang XH, Arias MA, Ke S & Chasin LA (2009)

Splicing of designer exons reveals unexpected

complexity in pre-mRNA splicing RNA 15, 367–376

22 Rodriguez-Trelles F, Tarrio R & Ayala FJ (2006)

Origins and evolution of spliceosomal introns Annu

Rev Genet 40, 47–76

23 Sorek R (2007) The birth of new exons: mechanisms

and evolutionary consequences RNA 13, 1603–1608

24 Sela N, Mersch B, Gal-Mark N, Lev-Maor G,

Hotz-Wagenblatt A & Ast G (2007) Comparative

analysis of transposed element insertion within human

and mouse genomes reveals Alu’s unique role in shaping

the human transcriptome Genome Biol 8, R127

25 Meili D, Kralovicova J, Zagalak J, Bonafe L, Fiori

L, Blau N, Thony B & Vorechovsky I (2009)

Dis-ease-causing mutations improving the branch site and

polypyrimidine tract: pseudoexon activation of

LINE-2 and antisense Alu lacking the poly(T)-tail

Hum Mutat 30, 823–831

26 Gal-Mark N, Schwartz S & Ast G (2008) Alternative

splicing of Alu exons–two arms are better than one

Nucleic Acids Res 36, 2012–2023

27 Maor G, Ram O, Kim E, Sela N, Goren A, Lev-anon EY & Ast G (2008) Intronic Alus influence alter-native splicing PLoS Genet 4, e1000204

28 Sun H & Chasin LA (2000) Multiple splicing defects in

an intronic false exon Mol Cell Biol 20, 6414–6425

29 Sironi M, Menozzi G, Riva L, Cagliani R, Comi GP, Bresolin N, Giorda R & Pozzoli U (2004) Silencer ele-ments as possible inhibitors of pseudoexon splicing Nucleic Acids Res 32, 1783–1791

30 Zhang XH & Chasin LA (2004) Computational defini-tion of sequence motifs governing constitutive exon splicing Genes Dev 18, 1241–1250

31 Fairbrother WG & Chasin LA (2000) Human genomic sequences that inhibit splicing Mol Cell Biol 20, 6816–6825

32 Zhang XH, Leslie CS & Chasin LA (2005) Dichotomous splicing signals in exon flanks Genome Res 15, 768–779

33 Blaustein M, Pelisch F & Srebrow A (2007) Signals, pathways and splicing regulation Int J Biochem Cell Biol 39, 2031–2048

34 Biamonti G & Caceres JF (2009) Cellular stress and RNA splicing Trends Biochem Sci 34, 146–153

35 Tran VK, Zhang Z, Yagi M, Nishiyama A, Habara Y, Takeshima Y & Matsuo M (2005) A novel cryptic exon identified in the 3¢ region of intron 2 of the human dystrophin gene J Hum Genet 50, 425–433

36 Maquat LE (2005) Nonsense-mediated mRNA decay

in mammals J Cell Sci 118, 1773–1776

37 Grellscheid SN & Smith CW (2006) An apparent pseudo-exon acts both as an alternative exon that leads

to nonsense-mediated decay and as a zero-length exon Mol Cell Biol 26, 2237–2246

38 Buratti E, Baralle M & Baralle FE (2006) Defective splicing, disease and therapy: searching for master checkpoints in exon definition Nucleic Acids Res 34, 3494–3510

39 Buratti E, Chivers M, Kralovicova J, Romano M, Baralle M, Krainer AR & Vorechovsky I (2007) Aberrant 5¢ splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization Nucleic Acids Res 35, 4250–4263

40 Lucien N, Chiaroni J, Cartron JP & Bailly P (2002) Partial deletion in the JK locus causing a Jk(null) phenotype Blood 99, 1079–1081

41 Madden HR, Fletcher S, Davis MR & Wilton SD (2009) Characterization of a complex Duchenne mus-cular dystrophy-causing dystrophin gene inversion and restoration of the reading frame by induced exon skip-ping Hum Mutat 30, 22–28

42 Lualdi S, Pittis MG, Regis S, Parini R, Allegri AE, Furlan F, Bembi B & Filocamo M (2006) Multiple cryptic splice sites can be activated by IDS point mutations generating misspliced transcripts J Mol Med 84, 692–700