Splicing and Disease

10Splicing and DiseaseEmanuele Buratti and Francisco E. Baralle

Key Concepts. An increasing number of diseases are now recognized to be caused by theselection of �wrong� splice sites.

. The selection of �wrong� splice sites can be caused bymutation in the DNA, orby changes in trans-acting factors.

. Aberrant splicing is best studied in monogenetic diseases, but is increasinglyfound in complex diseases.

10.1Introduction

In order to ensure accurate gene expression, the pre-mRNA splicing process has the taskof removing intervening sequences (or introns) from eukaryotic precursor messengerRNA (pre-mRNA) [1]. Since the pioneering studies on hemoglobin genes during theearliest days of splicing research [2–4], it has been well known that, in humans, anychangeswhich impair this processmay causediseases.However, during thepast 15 yearsor so, an increased knowledge of the pre-mRNA splicing process itself – coupledwith major advances in diagnostic screening techniques – has greatly expandedthat initial awareness [5]. Today, it is clear that splicing mutations can occur in virtuallyany human intron-containing gene, and that the resulting splicing alterations maycause disease. The pathological penetrance of these mutations may be variable, depend-ing on the individual genetic background. Until now, the most widely studied exampleshave considered only classical genetic diseases linked to alterations in a single-genesplicing regulation. However, it is becoming increasingly clear that splicing alterationsplay equally important roles in the origin and progression of complex diseases, such astumor formation or neurological defects. The aimof this chapter is to provide some basicpointers on splicing alterations and disease, and to focus especially on overviewing theconsequences of genomic variations.The complexity of the splicing process is aimed atmaintaining correct exon/intron

recognition, and is one of the essential factors that influence the shape of humangenes [6]. In keeping with this, many recent reports have consistently highlighted theobservation that even apparently neutral changes in the sequence composition ofexons may alter splicing, thus revealing evolutionary mechanisms aimed at main-taining correct splicing regulatory pathways [7–9].Both, constitutive and alternative splicing (AS) pathways are carried out by a large

ribonucleoprotein complex referred to as the spliceosome [1,10]. The assembly of thishighly sophisticated cellular machinery [11,12] in every exon–intron or intron–exonjunction is controlled by conserved (but rather degenerate) sequence elements thatinclude50 splice sites (50SS)and30 splicesites (30SS)and,upstreamof the30SS, thepoly-pyrimidine tract and the branchpoint sequence (BPS) (Figure 10.1) (& & Chapter 5Luhrmann& &). Because of their degeneracy, however, these consensus splicingsignals contain approximately half of the informationnecessary for accurate splice-siteselection [13]. The remaining information is provided by auxiliary signals in introns

Alternative pre-mRNA Splicing: Theory and Protocols, First Edition. Edited by Stefan Stamm, Chris Smith, and Reinhard Lührmann.� 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

j 119

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and exons, termed splicing regulatory elements (SREs); these may also be referred toas enhancer and silencer sequences, depending on their effect on exon recogni-tion [14–16] (&&Chapter 3 hertel&&). Inmost cases, these sequences function byinteracting with trans-acting factors, the number of which is steadily increasing overtime [17,18]. In parallel, a considerable degree of splicing regulation can also occur in aprotein-free fashionwith low-molecular-weight ligands [19], processed small nucleolarRNAs (snoRNAs) [20,21], and themodification of RNA secondary structure [22,23], allof which are capable of affecting this process. Finally, as the spliceosome andtranscription machineries are tightly linked (& & Chapter 9, Neugebauer& &),splicing can be influenced by pre-mRNAprocessing kinetics and transcription [24,25],cellular stress [26], and external extracellular signals [27]. As a result, splicingmutations may affect not only RNA processing but also transcription [28] anddownstream gene expression pathways (including translation), largely by creating oreliminating exons containing upstream open reading frames (ORFs) [29,30]. Thecombination of all the factors influencing splicing contributes to what is nowcommonly referred to as the �splicing code� [31–33] (& & Chapter 8, Smith& &).As expected from all this complexity, mutations in any of these cis-elements or factorscan dramatically alter splicing efficiency, lead to aberrant splicing and, eventually, tohuman disease, particularly in large genes with many introns [34,35].

10.2Splicing and Disease

In recent years, the topic of splicing and disease has been reviewed several times,most recently by Tazi et al. [36], Cooper et al. [37], and Baralle et al. [5]. While placing

*

*

5'SS, 3'SSinactivating mutation

Basic Splicing Factors

GURAGUMAGYURAY (Y) NCAG G

3'SS 5'SSBPS

U1

U2

n

(a)exon skipping(single or multiple)

(b)cryptic site activation

(c)full intronretention

(d)pseudoexon inclusion

de novo splice site creation within introns

cryptic 3'SS

de novo 5'SS

cryptic 5'SS

de novo 3'SS

SF1 U2AF65U2AF35

Fig. 10.1 Classical outcomes of mutation-inducedaberrant transcripts. The upper panel shows a pre-mRNA molecule with exons (boxes) separated byintrons (lines). Splice-site consensus sequences(50SS, 30SS, polypyrimidine tract and BPS) areindicated for the central exon. Inactivation of the 50SSand 30SS sequences can lead to exon skipping (eithersingle exon skipping or multiple exon skipping). (a,b)Activationof downstreamorupstream(cryptic) splicesites; (c) Full intron retention; (d) Mutations thatcreate strong splice sites in intronic regions can leadto pseudoexon activation.

Color Fig.: 10.1

120 j 10 Splicing and Disease

different emphases on particular topics, these authors have provided an overview ofthe field in light of the latest discoveries relating to the splicing process. Hence, thereader should consult these publications to acquire a general overview of the subject.It is interesting, nonetheless, to take note of the huge amount of new information

produced each year on the relationship between splicing and disease, and which hasresulted in various reviews focused on certain types of disease. For example, startingfrom the initial overview by Venables [38] on potential connections between splicingand cancer, several other groups have followed up on this specific subject [39–44]. Ingeneral, the mechanisms through which aberrant AS can bring about a tumorigenictransformation involve rather expected events, such as the production of proteinisoformswith oncogenic properties (orwith impaired anti-oncogenic properties). Thegenes involved in these cases belong predominantly to factors that control processessuch as apoptosis, cell-cycle regulation, and angiogenesis. One important factor thatis also emerging from these studies on human cancers is the central role played byalterations in the expression of the splicing factors themselves, rather than inindividual genes being mutated in their splicing regulatory regions (as recentlyreviewed by Grosso et al. [45]). Of particular interest is the recent identification of oneof the best-studied splicing factors (SF2/ASF) as a potential proto-oncogene upon itsoverexpression in rodent fibroblasts [46]. In keeping with this conclusion, the samestudy has shown that the SF2/ASF factor is overexpressed in a variety of humantumors. The mechanism through which transformation has come about are stillunder investigation. In the same study [46], several likely targets were identified forwhich the AS patterns could be adversely affected by the upregulation of SF2/ASF,such as tumor suppressor BIN1, and theMNK2, S6K1 kinases. It was also previouslyreported that SF2/ASF expression levels can have a powerful effect on the AS processof the Ron oncogene [47]. Other well-known splicing factors, the expression levels ofwhich are altered in cancer cells, include hnRNPA1, Tra-2b, YB-1, and a host of otherfactors that were known previously just for their splicing regulatory abilities [48].Many of these connections – especially with regards to their potential functionalsignificance in tumor origin and progression – remain to be further tested. None-theless, the rapid development of the therapeutic field aimed at correcting splicingdefects, and the need to identify new targets for therapeutic treatment for this type ofailment, will undoubtedly drive the field swiftly forwards in the near future.All of these observations have increased the use ofmicroarray analysis of transcript

alterations as �biomarkers� for the diagnosis and prognosis of particular types ofcancer. For example, attempts have been made to classify, according to splicingvariations in the transcripts, diseases such as Hodgkin lymphoma [49], ovariancancers [50], leukemia cell lines [51], and human breast cancer cells [52].It should also be pointed out that human tumors are not the only complex disease

where the importance of splicing is carefully evaluated. In particular, the potential roleplayed by splicing alterations in neurological diseases has also attracted much atten-tion [53,54]. For example, recent investigations have focused on clarifying the role ofNova, a neuronal-specific splicing factor (& & see Chapter 3 Allain for details& &)which regulates synapse formation during development of the human brain bycontrolling the alternatively spliced levels of several neurotransmitter receptors,adhesion molecules, cation exchangers, and scaffold proteins [55,56]. Another recentaddition to the list of splicing factors involved in neurodegeneration has been TDP-43.Althoughpreviously, this proteinwas considered to controlCFTR exon 9 splicing [57], ithas more recently been identified as a major accumulating protein in patients affectedby frontotemporal lobar degeneration and amyotrophic lateral sclerosis [58].

10.3Therapeutic Approaches

The possibility ofmodifying aberrant splicing patterns has been the subject of severalrecent reviews [59–63]. Although the strategies used to modify splicing profiles are

10.3 Therapeutic Approaches j 121

rather divergent, the most successful approaches to date have involved the use ofantisense oligonucleotides that target splicing control regions. These oligonucleo-tides can be used to inhibit the inclusion of unwanted exons, and/or to promote theproduction of a truncated but functional protein [60,64] (& & see Chapter 47Aartsma Rus& &). Antisense oligonucleotides can also be modified to contain acomplementary targeting region, as well as an effector region which can recruit ormimic splicing factor activities [65,66]. Another promising line of research that hasattracted much recent attention involves the use of small molecules that act byinterferingwith cellular signaling pathways, therebymodifying the activity of splicingregulatory proteins through an altered cellular distribution or a change in phosphor-ylation state [67] (& & see Chapter 48 denise cooper& &). For this, screeningmethods have been developed to identify small molecules from chemical librariesthat regulate a given splicing event (& & see Chapter 46 peter stoilov& &).Alternative approaches have also been described that use small interference RNA(siRNA) approaches to specifically knock down aberrant splicing isoforms, exploittrans-splicing strategies (spliceosome-mediated RNA trans-splicing; SMaRT) [68],and the use of modified U7-U1 snRNP molecules to block aberrant splice sitesequences (i.e., acting as antisense oligonucleotides) or to reverse mis-splicing bycarrying compensatory mutations in the 50 end of their U1 snRNA sequence [69–72](& & see Chapter 45 daniel schumperli & &).

10.4The Generation of Aberrant Transcripts

From a biomedical point of view, one of the most important aspects to be consideredin any research investigation iswhat type of aberrant transcriptmight be generated bya typical splicing-affecting mutation. A schematic of the possible consequences ofmutations in the basic splicing regulatory elements is shown in Figure 10.1. Amutation in the enhancer or silencer elements that leads to their disruption (orcreation) can lead to the same consequences as described for the basic regulatoryfactors.Hence, itmust be borne inmind that the creation of enhancers or silencer losscan lead to increased levels of exon inclusion (Figure 10.2).

10.5Exon Skipping

In general, the vastmajority of 50SS, 30SS and regulatory elementsmutations result inskipping of the affected exon [73] (Figure 10.1a). Although, by itself, the skipping of anexon from the pre-mRNA is a straightforward process, it should be noted that quiteoften the skipping event is not confined to the exon carrying the splicingmutation, butit can also be extended to neighboring exons (either upstream or downstream). Thishas suggested that, in all cases, the importance of the genomicmilieu should never beunderestimated [74]. It also underlines that bioinformatic predictions must bevalidated experimentally.

10.6Cryptic Splice Site Activation

Cryptic splice site activation usually occurs when the natural donor or acceptor site isinactivated or weakened by a particular mutation. In this case, depending on the localsequence context, one ormore splice sites are used that would normally be ignored bythe splicing machinery (Figure 10.1b). These events result in either the addition orsubtraction of nucleotide sequences from the original exon. In these cases, there is atwo in three chance of disrupting the reading frame by introducing aberranttranslation stop codons in the final transcript that can either cause degradation of

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the mRNA transcript through nonsense-mediated decay (NMD) or the synthesis of atruncated protein. Furthermore, even when the reading frame remains unchanged,the addition/removal of a number of amino acid residues from the resulting proteinmay well prove to be harmful with regards to its biological properties or regulation.A bioinformatic analysis of several hundred cryptic splice site activation

events [75,76] has confirmed that cryptic splice sites are, on average, intrinsicallystronger than their mutated authentic counterparts but are generally weaker thantheir authentic, wild-type counterparts [77]. However, in about 10–15% of cases, thewild-type authentic splice site was weaker than the corresponding cryptic site. Thisindicates that there are additional signals in the pre-mRNA that repress their use, andseveral experimental observations have confirmed this hypothesis. First, on thebioinformatics level, the analysis of auxiliary sequences between authentic andaberrant splice sites showed that one particular type of silencer – the putative exonicsplicing silencer (PESS) [14–16] –was particularly informative for predicting aberrantsplice site activation [78]. Second, in genes such as FGB it has been reported that anSF2/ASF binding sequence, that does not normally participate in the recognition ofthe constitutively recognized exon 7, can nonetheless profoundly influence theactivation and type of cryptic splice site sequences being used by the splicingmachinery following inactivation of the wild-type donor site [79].There are two important databases that collect disease-related, cryptic splice site

activation events following either acceptor or donor site inactivation, namelyDBASS3andDBASS5 [75,76]; both databases are freely available atwww.dbass.org.uk/. Finally,an in silico tool (Cryp-Skip; available at www.dbass.org.uk/cryp-skip/) has recentlybeen developed to predict the potential occurrence of cryptic splice site activationversus exon skipping following the introduction of mutations in any given donor oracceptor site [80] (& & see Chapter 49 dela grange,& &)

10.7Intron Retention

Intron retention events are usually defined as the retention of entire intronicsequences in the final processed mRNA (Figure 10.1c). The frequency of normal

GURAGUMAGYURAY (Y) NCAG Gn

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silencers

(a)

(b)

(c)

(d)exon skipping(single or multiple)

cryptic site activation

enhancer loss or silencer creation

enhancer creation or silencer loss

full intronretention

Splicing Regulatory Elements (SREs)

all changes in SRE composition

pseudoexon inclusion

Fig. 10.2 Mutation-induced aberrant transcriptsfollowing inactivation/activation of SRE elements.The aberrant transcripts originate mostly from exonskipping (a), cryptic splice site activation (b), fullintron retention (c), and pseudoexon inclusion (d).Mutation in enhancer or silencer elements that led totheir disruption (or creation) can lead to the sameconsequences described for the basic regulatoryfactors, with the addition that the creation ofenhancers or silencer loss can lead to increased levelsof exon inclusion.

10.7 Intron Retention j 123

intron retention events in the human genomehas been recently estimated to be about15% in a set of more than 21 000 annotated genes [81]. Although, in many cases, thebiological role of these events is currently unknown, it is known that they occurpreferentially in the untranslated region of the RNA [81,82]. Their potentiallyregulatory role, however, is established by some well-described examples, such asgeneration of the P element and Msl2 transcripts in Drosophila [83,84], in thedevelopmental regulation of the proinsulin mRNA in chicken embryos [85], in thegeneration of a novel adhesion molecule in the rat testis [86], or in controlling theexpression levels of apolipoprotein E in the central nervous system [87]. As expected,aberrant intron retention events following the introduction of mutations in splicingregulatory elements have also been shown to be associated with human disease, suchas pheochromocytoma [88], long QT syndrome [89], Leigh syndrome [90], arthro-gryposis multiplex congenita (AMC) [91], and B-lineage human cancers [92].

10.8Pseudoexon Inclusion

The term �pseudoexon� usually refers to any nucleotide sequence between 50 and300 nt in length with apparently viable 50SS and 30SS at either end. Because ofthe degeneracy of the splicing code, it is expected that many such sequences wouldbe present in most human genes. Indeed, in the hprt gene it has been estimatedthat pseudoexon sequences largely outnumber the �real� exons [93] (& & seeChapter 3 hertel& &). The evidence available to date has pointed to several factorsthat can help the spliceosome to discriminate between the real exons and these falsetargets. First, the inclusion of many of these sequences is actively inhibited due tothe presence of intrinsic defects both at level of the 50SS sequence and the poly-pyrimidine tract (despite their good agreement with the consensus) [93], the presenceof silencer elements [15,94,95], or the formation of inhibiting RNA secondarystructures [96–98].Nonetheless, the number of reported pseudoexon events involved in human

disease is steadily increasing, and this subject has been reviewed extensively [99].Usually, this situation is due to the de novo creation of classical splicing consensussequences: donor, acceptor, and branch site sequences (Figure 10.1d). Followingthese events, the second most frequent mechanism that leads to pseudoexonactivation involves the creation/deletion of splicing regulatory sequences. Finally,in two individual cases the rearrangement of genomic regions through a grossdeletion that brought near to each other two viable donor and acceptor sites [100], orgenomic inversions that have activated exons in what would normally have been theantisense genomic strand [101], has also been described as giving rise to pseudoexoninclusion events.

10.9Unexpected Splicing Outcomes Following the Disruptionof Classical Splicing Sequences

It should also be noted, that these possibilities do not rule out other types of outcome,such as those shown schematically in Figure 10.3. In this case, it has been observedthat disease-associated inactivating mutations in the 30 acceptor sequences of the TPand XPA genes not only cause skipping of the affected exon but also determine a shiftin donor acceptor usage of the preceding exon [102,103]. This type of �atypical�outcomes is not confined to 30SS sequences, as donor site inactivation in theCOL1A1andCLN6 genes has yielded very similar results [104,105]. For this reason, in order toaccurately determine any aberrant splicing events, it is always advisable to use thefull range of diagnostic possibilities (most of which are described fully elsewhere inthis book).

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10.10Conclusions

As described in this chapter, mutations that affect the splicing processmay representup to 50% of all mutations that lead to human disease. It is not surprising, therefore,that alterations at the pre-mRNA splicing level are now firmly recognized as a majorplayer in the development of human genetic diseases. The effects of these mutationsare also quite varied, ranging from straightforward exon skipping and exon creationevents to intron retention and cryptic splice site activation events. In clinical practice,the identification of which mutations are responsible for a particular splicing defecthas today become fundamentally important with regards to therapeutic and counsel-ing issues. Until now, the identification of splicing mutations has been hampered bylimited sequencing abilities and an insufficient appreciation that even very harmless-looking polymorphisms may affect the splicing outcome in unforeseeable ways.However, an increased knowledge of the splicing process and an increased availabilityof sequencing data from patients is now rapidly changing this picture. Indeed, thestage is almost set to begin identifying splicing defects from individual patients ineveryday clinical testing; these issues are described in & & Chapter 11 DianaBaralle& &.

Acknowledgments

These studies were supported by Telethon, and by the EC grant EURASNET.

3'SSGU

-31nt.

GUIVS1 IVS1

GU AGAC5'SS

3'SSGU GU AGAU

5'SS

3'SS+96nt.

GUIVS7 IVS8

GUAAA AGAG5'SS

3'SS+119nt.

GUIVS5

GUAAA AGAG5'SS

3'SS+26nt.

GUIVS4 IVS5

GUAAA AGAG5'SS

-5nt.

3'SSGU GU

IVS3 IVS4GU AGAU

5'SS-100nt.

TP gene(thymidine phosphorylase deficiency)

XPA gene(xeroderma pigmentosum)

COLIAI gene(osteogenesis imperfecta)

CLN6 gene(neuronal ceroid lipofuscinoses)

CLN6 gene(neuronal ceroid lipofuscinoses)

XPA gene(xeroderma pigmentosum)

5'SS inactivatingmutations

3'SS inactivatingmutations

Fig. 10.3 Unexpected splicing outcomesin disease. These schematic panels showsome unexpected splicing events thatmight be associated with the introductionof disease-associated mutations inclassical splicing signals, such as theacceptor or donor site of exons. Theexamples reported here have beendescribed to occur in the TP, XPA,COL1A1, and CLN6 genes,respectively [102–105].

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Keywords: Splicing; mutation; RNA; 50 splice site; 30 splice site; bioinformatics.

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