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Journal of PathologyJ Pathol 2010; 220: 152–163Published online 13 November 2009 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/path.2649

Invited Review

The pathobiology of splicingAmanda J Ward and Thomas A Cooper*Departments of Molecular and Cellular Biology and Pathology, Baylor College of Medicine, Houston, TX, USA

*Correspondence to:Thomas A Cooper, Departmentsof Molecular and Cellular Biologyand Pathology, Baylor College ofMedicine, Houston, TX77030, USA.E-mail: [email protected]

No conflicts of interest weredeclared.

Received: 1 September 2009Revised: 5 October 2009Accepted: 5 October 2009

AbstractNinety-four percent of human genes are discontinuous, such that segments expressed asmRNA are contained within exons and separated by intervening segments, called introns.Following transcription, genes are expressed as precursor mRNAs (pre-mRNAs), which arespliced co-transcriptionally, and the flanking exons are joined together to form a continuousmRNA. One advantage of this architecture is that it allows alternative splicing by differentialuse of exons to generate multiple mRNAs from individual genes. Regulatory elements locatedwithin introns and exons guide the splicing complex, the spliceosome, and auxiliary RNAbinding proteins to the correct sites for intron removal and exon joining. Misregulation ofsplicing and alternative splicing can result from mutations in cis-regulatory elements withinthe affected gene or from mutations that affect the activities of trans-acting factors thatare components of the splicing machinery. Mutations that affect splicing can cause diseasedirectly or contribute to the susceptibility or severity of disease. An understanding of therole of splicing in disease expands potential opportunities for therapeutic intervention byeither directly addressing the cause or by providing novel approaches to circumvent diseaseprocesses.Copyright 2009 Pathological Society of Great Britain and Ireland. Published by JohnWiley & Sons, Ltd.

Keywords: gene; alternative splicing; disease; intron; exon; mRNA; mutations

Introduction

The flow of genetic information has traditionally beenviewed as DNA transcribed into RNA and trans-lated into protein. Additional layers of regulation con-tinue to be discovered, greatly expanding this sim-plistic framework and revealing the complex networkthat controls gene expression. With the greater under-standing of regulated gene expression, it has becomeincreasingly clear that RNA is much more than apassive intermediate. The control of RNA process-ing is now recognized as a crucial component ofgene regulation. In addition, a variety of long andshort non-coding RNAs have recently been discov-ered to mediate regulation of gene expression at mul-tiple levels. Gene expression is modulated throughmultiple RNA-based mechanisms, including miRNA-mediated silencing, regulation by long non-codingRNAs, nonsense-mediated decay, polyadenylation siteselection, RNA editing, alternative splicing, and reg-ulation of mRNA translation efficiency, stability andlocalization. Given the integrated roles of these eventsin normal gene expression, it is not unexpected thatthese RNA-based processes are heavily involved aseither causative entities, modulating influences orcompensatory responses to disease [1].

The focus of this review is the diverse roles of splic-ing and alternative splicing in human disease. It is

estimated that 94% of human genes are alternativelyspliced and that as many as 50% of disease-causingmutations affect splicing [2–4]. Alternative splicingproduces variation within mRNAs from individualgenes, greatly increasing the diversity of transcriptsexpressed from these genes (Figure 1). The major-ity of variation is within the open reading frame,resulting in the expression of different protein iso-forms, which often have different functional proper-ties. Splicing and the regulation of alternative splicingare disrupted both by mutations within cis-acting ele-ments required for correct pre-mRNA processing aswell as by mutations that affect trans-acting com-ponents that are necessary for splicing regulation.Effects on splicing can be direct causative agents ofdisease or more subtle contributions to the determi-nants of disease susceptibility or modulators of dis-ease severity. Recently, a number of RNA-bindingproteins with roles in alternative splicing regula-tion, as well as other RNA-processing events, havebeen identified as disease-associated genes, particu-larly in neurodegenerative disorders and cancer. Herewe first review the molecular machinery requiredfor normal constitutive and alternative splicing andthen cover the mechanisms by which alterationsin splicing and its regulation create and modulatedisease.

Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.www.pathsoc.org.uk

The pathobiology of splicing 153

Figure 1. Alternative splicing patterns increase mRNA diversity. Through alternative use of exons, introns, promoters andpolyadenylation sites, alternative splicing acts to greatly increase the diversity of mRNA transcripts

Pre-mRNA splicing to produce mRNA

For each mRNA, the site of transcription initiationproduces the first nucleotide and the site of a directedendonuclease cleavage event becomes the 3! end,to which a "200 nucleotide polyA tail is added.Ninety-four percent of human genes contain intronsand exons which are spliced together post- or co-transcriptionally. The molecular machinery requiredfor intron removal and exon joining must perform ademanding task. Splicing requires extreme precisionbecause even a single nucleotide addition or dele-tion at the site of exon joining will shift the read-ing frame, with adverse consequences to the protein-coding potential. To achieve this accuracy, the splicingmachinery must efficiently recognize the intron–exonboundaries in the pre-mRNA. Splicing is performedby a multi-component machine, the spliceosome, com-posed of five small nuclear RNAs (snRNAs) pre-assembled with proteins into small ribonucleoproteins(snRNPs) and hundreds of additional proteins. Theprecision of the reaction is accomplished through acoordinated series of RNA–RNA, RNA–protein andprotein–protein interactions [5].

A critical challenge to the spliceosome is to cor-rectly identify exons within the pre-mRNA. Exonsmake up only one-tenth of the typical pre-mRNA andtherefore must be identified within a sea of introns.The mechanism of exon recognition involves identifi-cation of a complex code of cis-acting elements withingenes [6]. It is also becoming more apparent that splic-ing of most intron-containing genes is likely to beco-transcriptional, which reduces the complexity ofintron–exon junction recognition [7]. During the pastdecade, a large research effort involving many lab-oratories has made substantial progress toward deci-phering the splicing code [6]. Every intron containsthree core sequence elements that are essential forsplicing: the 5! and 3! splice sites at the 5! and 3!

ends of the intron, respectively, and the branch pointsequence (Figure 2). These sequences are recognized

multiple times by components of the spliceosome dur-ing the processes of intron removal and exon joining.The 5! splice site is bound first by the U1 snRNPand later by the U6 snRNP. The RNA binding proteinSF1 binds the branch point sequence but is later dis-placed by the U2 snRNP [6]. In general, splice siteswith greater affinity for these recognition complexesachieve a higher splicing efficiency. The multiple andindependent recognition of these sequences by differ-ent spliceosome components contributes to the fidelityof splicing.

These three core sequences are necessary but notsufficient for defining intron–exon junctions. Thereare additional sequences located within both intronsand exons that recruit trans-acting splicing factorsto ensure inclusion of constitutive exons or modu-late the efficiency of splice site recognition, promotingalternative splicing. It is now clear that most exons,whether constitutively or alternatively spliced, con-tain cis-acting elements that affect splicing efficiency.These sequences are referred to as intronic or exonicsplicing enhancers (ISE or ESE) or silencers (ISS orESS). Although some of these elements are identi-fiable by combined use of different algorithms, it isnot yet possible to consistently predict the effectsof nucleotide substitutions on splicing [6]. As thesequences that define ESSs and ESEs are better estab-lished, exonic mutations that disrupt splicing will bemore readily recognized, based on sequence alone.Enhancer elements are bound by positive regulatorsthat increase exon inclusion, such as the SR pro-tein family, which contain at least one RNA-bindingdomain and a distinctive serine/arginine-rich domain.In contrast, silencer elements are bound by negativeregulators that decrease exon inclusion, such as theabundant hnRNP proteins which were originally iden-tified by their association with nascently transcribedpre-mRNA [8]. The critical balance of these antago-nistic regulators is necessary for controlling the levelof exon inclusion in the mRNA transcript [9]. Muta-tions within splice site sequences at the intron–exon

J Pathol 2010; 220: 152–163 DOI: 10.1002/pathCopyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

154 AJ Ward et al

Figure 2. The combination of cis-acting elements and trans-acting factors determine splice site selection. (A) Three core splicingsequences essential for splicing of all exons are the 5! splice site, beginning with an invariant GU dinucleotide, the 3! splice site,ending with an invariant AG dinucleotide, and the branch site sequence. These elements are recognized by components of thespliceosome, such as the U1 and U2 snRNPs and U2AF. (B) Additional regulatory sequences located in the intron and exonare also required for exon recognition and to modulate splice site selection of alternatively used splice sites. Exonic splicingenhancers (ESE) and silencers (ESS) are bound by positive and negative splicing regulators, such as SR proteins and hnRNP proteins,respectively. Intronic splicing enhancers (ISE) and silencers (ISS) also recruit splicing regulatory complexes. (C) Disease-causingmutations within cis-acting elements disrupt proper recognition by splicing components

junction cause approximately 10% of disease-causingmutations [10]. As described in sections below, it islikely that a larger number of disease-causing muta-tions that affect splicing are located within enhancerand silencer sequences. These mutations reduce theefficiency with which a constitutive exon is spliced oralters a critical ratio of alternative splicing patterns.

Global analysis of alternative splicing

Alternative splicing is controlled both spatially andtemporally, resulting in the expression of differentsplice variants in different tissues, in different cellswithin the same tissue, or in the same tissue at dif-ferent stages of development or in response to patho-logical processes. It is now known that most genesare alternatively spliced; however, it is also impor-tant to determine which splice variants are expressed,their relative abundance and their biological functionin order to fully understand not only the function ofa gene but also how the function is altered in dis-ease. One example of the complications introducedby alternative splicing with regard to understandingthe pathogenic mechanisms of a disease-causing muta-tion is the CACNA1H calcium T-channel. CACNA1Hgenerates a large number of functionally diverse pro-tein isoforms that have been extensively characterized[11]. It is possible that disease-associated mutationsin this gene may differentially affect the activity ofthe diverse splice variants. Missense mutations in thisgene cause familial forms of epilepsy, and a number of

studies aimed at determining the electrophysiologicaleffects of different disease-causing mutations wereperformed, using one available full-length splice vari-ant. Given the large number of functionally diverseisoforms expressed, one can imagine that the isoformstudied is not the predominant splice variant in thecells that are relevant to epilepsy. This example illus-trates the importance of knowing the functional differ-ences between each splice variant and identifying spe-cific splice variants expressed in the disease-relevantcells to understand the full impact of disease-causingmutations [11].

To study alternative splicing in a global con-text, several approaches have been developed, includ-ing specialized expression profiling arrays and high-throughput sequencing analysis. Traditional microar-rays contain oligonucleotide probes targeted primarilyto the 3! end of an mRNA to measure total transcriptlevels, but are unable to distinguish between differ-ent splice variants. More specialized exon tiling andsplice junction microarrays contain probes to individ-ual exons and exon–exon junctions. The higher cover-age allows not only the identification of transcripts thatare up-regulated or down-regulated, but also detectsdifferential use of specific exons [12]. Splice junctionprofiling was used to identify subsets of transcriptsregulated by the neuron-specific splicing factor Nova2by comparing wild-type and Nova2 knockout mousebrain tissue [13] and to identify splicing transitionsthat occur during mouse heart development [14]. Theseand other studies strongly suggest the presence of anelaborate regulatory network to coordinate splicing



transitions for specific physiological functions and dur-ing development. Most recently, high-throughput tran-scriptome sequencing has provided a highly quanti-tative analysis of mRNA abundance and differentialutilization of exons [15]. High-throughput sequencingtechnologies are developing at a rapid rate, such thattranscriptome analysis is likely to be widely availableto compare diseased and normal tissue for investiga-tive and even diagnostic purposes.

Intron mutations that alter splice siterecognition

Most of the splice site mutations that lead tohuman disease involve the invariant GT and AGdinucleotides in the 5! and 3! splice sites [10,16].These dinucleotides are essential for exon defini-tion and appropriate splicing. However, mutationsoccurring at other positions of the 5! or 3! splicesite can also lead to missplicing and typically resultin exon skipping, activation of a cryptic splicesite, or intron retention. A number of diseaseshave been reported that result from intronic muta-tions, including familial dysautonomia, neurofibro-matosis type 1, Frasier’s syndrome and atypicalcystic fibrosis, and have been previously reviewed[17–19].

Familial dysautonomia (FD) is a recessive diseaseof the sensory and autonomic nervous system causedby mutations in the IKBKAP gene and is a partic-ularly instructive example of the effects of splicesite mutations outside of the invariant first and lastnucleotides [20]. The IKBKAP gene encodes IKAP,a component of the Elongator complex thought tofunction as a transcriptional regulator [21,22]. IKAPdepletion results in reduced transcription of a num-ber of cell motility genes, opening the possibility thatimpaired cell migration in the nervous system mayunderlie the neurological dysfunction in FD patients[23]. Ninety-eight percent of the disease-causing alle-les contain a silent T-to-C transition in the sixth baseof the intron, the last nucleotide of the 5! splice siteof intron 20 [24,25]. This mutation leads to exon20 skipping, causing a shift in the reading frameand introduction of a premature termination codon.This is likely to result in nonsense-mediated decayof the mRNA and decreased expression of functionalIKAP [25,26]. In silico and in vitro splicing anal-ysis determined that the upstream 3! splice site ofintron 20 and the splicing regulatory sequences withinexon 20 are not strong enough to define the exonin the presence of the mutated 5! splice site [27].Increasing the strength of the base pairing betweenthe mutated 5! splice site and U1 snRNA, an earlystep in spliceosome regulation, restores exon 20 inclu-sion [28]. These studies demonstrate that single basepair changes in the intron can dramatically affect exoninclusion by disrupting spliceosomal recognition ofsplice sites.

Exon mutations that disrupt splicing

Disease-causing missense mutations are commonlyassumed to disrupt protein function. Surprisingly, alarge and growing number of examples demonstratethat mutations within protein-coding exons can haveprimary disease-causing effects by disrupting splic-ing. This occurs by mutations that alter ESS or ESEmotifs. Spinal muscular atrophy (SMA) is one of thebest characterized examples of a disease caused byan exonic mutation that disrupts splicing. Survival ofmotor neurons (SMN) protein is encoded by the SMN1gene and a nearly identical SMN2 gene, created froma gene duplication event. SMN is necessary for snRNPassembly and metabolism and its loss via deletions inSMN1 results in the most common genetic cause ofinfant mortality and a disease characterized by motorneuron degeneration and progressive paralysis. SMN2is unable to compensate for loss of SMN1, owing toa silent C-to-T substitution in the sixth nucleotide ofexon 7, which promotes exon skipping and productionof a truncated, inactive protein [29–31]. Two non-exclusive models explain how the mutation promotesexon skipping, either through gain of an ESS or loss ofan ESE. In the ESS-gain model, the mutation createsan ESS binding site for hnRNPA1 which functions asa splicing repressor [32,33], whereas in the ESE-lossmodel, the mutation disrupts an ESE that is bound bythe SF2/ASF splicing activator, an SR protein [34,35].Interestingly, the mutated SMN2 exon 7 shares highsequence similarity with a disease-associated region ofthe medium-chain acyl-CoA dehydrogenase (MCAD)gene [36]. MCAD performs one of the initial cat-alytic steps in the mitochondrial fatty-acid oxidationpathway and deficiency of this enzyme results in themost common fatty-acid disorder [37]. A silent C-to-T mutation has been identified in MCAD exon5 of several MCAD-deficiency patients at the sameexonic position as the SMN2 base pair substitution,the sixth nucleotide. This mutation disrupts a puta-tive ESE binding site for SF2/ASF, resulting in exonskipping and nonsense-mediated decay of the aberranttranscript. Over-expression of SF2/ASF, but not otherSR proteins, strongly increased exon 5 inclusion, sug-gesting that the disease-associated mutation weakensbut does not totally abolish ESE recognition. When theputative ESE was replaced by the corresponding ESEsequence from SMN1 exon 7, MCAD exon 5 inclu-sion was restored. In contrast, when the disrupted ESEfrom SMN2 was used for replacement, MCAD exon5 remained predominantly skipped [36]. The propersplicing of MCAD exon 5 and SMN2 exon 7 requiresimilar exonic regulatory sequences, and disruption ofthese sequence elements alter the splicing pattern andserve as the basis of disease.

Splicing as a genetic modifier of disease

Phenotypic variability results from changes in geneexpression, including individual differences in


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splicing. Polymorphisms or mutations occurring nearsplice sites or within splicing regulatory sequencescan alter splicing patterns [4,38]. These changes, inturn, can result in the expression of different mRNAvariants that affect disease susceptibility and sever-ity. For example, splicing is a genetic modifier of twoX-linked disorders of copper metabolism, Menkes dis-ease (MD) and occipital horn syndrome (OHS) [39].Both diseases are caused by mutations in the ATP7Agene, encoding a copper-transporting ATPase [40–42].ATP7A uses energy from ATP hydrolysis to transportcopper across polarized enterocytes (intestinal absorp-tive cells), increasing its bioavailability for numer-ous cellular processes. In the absence of functionalATP7A, copper deficiency results from poor distribu-tion of copper to cells in the body [39]. Individualswith MD are affected by neurological degeneration,connective-tissue defects, distinctive kinky and brittlehair and early-childhood death. OHS is an allelic formof MD with milder symptoms, the most predominantfeature being connective-tissue abnormalities [43,44].

Several hundred disease-causing mutations havebeen identified that result in disruption of ATP7A func-tion and splice site mutations are highly represented[45,46]. Predominant among MD patients are muta-tions occurring in the invariant dinucleotides at the 5!

and 3! splice sites that abolish exon recognition by thespliceosome and eliminate or profoundly reduce theexpression of the correct splice variant. In contrast,the milder OHS phenotype results from mutations thatoccur in less conserved regions of the same splice sites.This weakens but does not prevent exon recognition,so both aberrantly and correctly spliced transcripts areproduced. The low level of functional transporter pro-tein is sufficient to prevent severe disease [47–49].This example demonstrates how splicing efficiencydetermines disease severity through different splicingmutations in the same gene.

Mutations that alter the ratioof alternatively spliced isoforms

For some genes, the ratio of two or more splice vari-ants must be properly balanced in response to changingcellular conditions. Antagonistic regulation betweenpositive- and negative-acting factors contributes to thisdelicate control and its disruption can lead to dis-ease. One particularly well-characterized example isfrontotemporal dementia and Parkinsonism linked tochromosome 17 (FDTP-17). FDTP-17 is caused bymutations in the MAPT gene that encodes tau, a pro-tein involved in microtubule assembly and stability. Anumber of disease-causing mutations disrupt alterna-tive splicing of exon 10, ultimately altering the ratioof two tau isoforms containing either three (3R) orfour (4R) repeat microtubule binding sequences. Thefinding that silent mutations in exon 10 caused diseasewas an initial clue that the primary pathogenic mecha-nism involved an effect on splicing, rather than altered

protein function [50,51]. For these mutations, changesin the 4R:3R ratio due to increased exon 10 inclusionis the causative event in tau aggregation and onset ofdisease [52].

While MAPT exon 10 provides an example of cis-acting mutations that disrupt the balance of isoformsexpressed, the Bcl-2 protein family illustrates how dif-ferences in the trans-acting splicing environment canaffect a critical balance between splice variants. Mostmembers of the Bcl-2 family undergo alternative splic-ing to produce isoforms that either promote or preventapoptosis [53]. The relative levels of splice productsare critical for regulating apoptosis and an imbalanceof these products is an important influence in the initi-ation and progression of cancer [54]. One member ofthis family, Bcl-X, produces two protein isoforms viasplicing of an alternative 5! splice site in the first cod-ing exon. The use of the upstream splice site creates alonger isoform, Bcl-XL, with anti-apoptotic function,whereas use of the downstream splice site producesa shorter splice isoform, Bcl-XS, with pro-apoptoticfunction. The mechanistic details of how each iso-form promotes its apoptotic response is not known;however, it is clear that the alternative splicing eventcreates two splice variants with opposing activities,indicating that this process of RNA regulation is capa-ble of deciding cell fate [55]. An imbalance of thesetwo isoforms has been implicated in several cancers;the anti-apoptotic Bcl-XL is up-regulated in multiplemyeloma, small cell lung carcinoma and prostate andbreast cancer, where it is associated with an increasedrisk of metastasis [56–59]. The pro-apoptotic Bcl-XS is down-regulated in transformed cells, but itsforced over-expression sensitizes breast cancer cellsto chemotherapeutic agents [60]. These observationsindicate a change in the nuclear regulatory environ-ment associated with cancer. Some of the factors thatcan affect splicing in cancer have been identified andare described below.

Mutations in regulators of alternativesplicing

While disease-causing mutations that act in cis affectsplicing of a single gene, mutations that affect compo-nents of the splicing machinery create the potential formultiple genes to be misspliced. The relative scarcityof examples for severe loss-of-function mutations intrans-acting factors may be an indication that muta-tions with widespread consequences are lethal duringembryonic development or in individual cells. How-ever, there are several examples of diseases resultingfrom mutations in genes important for splicing, includ-ing SMA, amyotrophic lateral sclerosis (ALS) andretinitis pigmentosa [1]. In the case of SMA, whileSMN is ubiquitously required for snRNP assembly,SMN deficiency in SMA predominantly affects motorneurons, resulting in motor neuron degeneration. Inter-estingly, the snRNP repertoire was found to be altered



in an SMN-deficient SMA mouse model and was asso-ciated with widespread splicing defects in multipletissues, consistent with the role of SMN in snRNPmaturation [61]. The heightened sensitivity of motorneurons to SMN deficiency could reflect requirementsof neuron specific pre-mRNAs or specialized functionsor partners of SMN in motor neurons [1].

Disruption of other trans-acting splicing regulatorsis also thought to have significant effects on splic-ing. TDP-43, a member of the hnRNP family, hasbeen implicated in the mechanism of cystic fibro-sis, where it binds to a repetitive (UG)n element inintron 8 of the cystic fibrosis transmembrane con-ductance regulator (CFTR) gene, promoting exon 9skipping and decreased expression of functional pro-tein [62]. Moreover, a role for TDP-43 has been foundin several neurodegenerative diseases, including fron-totemporal dementia and ALS, where the protein isabnormally included in ubiquitinated protein aggre-gates in the cytoplasm of neurons and glial cells[63,64]. Partial depletion from the nucleus and, thus,a decrease in nuclear function, may be a contribut-ing factor in these diseases. Mutations in TDP-43 aswell as another RNA-binding protein, fused in sar-coma (FUS), have recently been identified in bothfamilial and sporadic forms of ALS [65,66]. The iden-tification of disease-causing mutations in TDP-43 andFUS strongly suggest that splicing abnormalities andpossibly other RNA-processing events contribute tothe neurodegeneration phenotype. A crucial next stepis to identify the RNA targets of these proteins andtheir role in motor neuron function and survival [67].

Unstable nucleotide repeat expansions

Short (#10 nucleotide) repetitive sequences are locatedthroughout the genome and their expansion beyond apathogenic threshold is responsible for a class of dis-orders called microsatellite expansion disorders [68].When repeats are located in an open reading frame,expansions result in both loss and gain of proteinfunction, such as in Huntington’s disease and severaltypes of spinocerebellar ataxias. However, when theexpanded repeats occur in non-coding regions of thegene, disease can result not only from protein loss-of-function but also from a gain-of-function of the repeat-containing RNA transcribed from the expanded allele.The best characterized example of an RNA gain-of-function resulting from repeat expansion is myotonicdystrophy (DM). This adult-onset neuromuscular dis-order is characterized by multi-systemic clinical fea-tures, including cardiac arrhythmias, skeletal musclewasting, cataracts, myotonia, insulin resistance andneuropsychiatric dysfunction [69].

There are two types of DM, which differ in thesequence and location of the expansion but share acommon pathogenic mechanism. DM type 1 (DM1)is caused by a CTG trinucleotide expansion in the3! untranslated region of the DMPK gene [70,71],

whereas a generally milder DM type 2 (DM2) iscaused by a CCTG tetranucleotide expansion locatedwithin the first intron of the ZNF9 gene [72]. A molec-ular hallmark of DM is the accumulation of CUG- orCCUG-repeat RNA in nuclear foci [72,73]. The alter-native splicing regulator MBNL1 has a high affinityfor expanded CUG- and CCUG-repeat RNA and co-localizes with the nuclear foci, depleting MBNL1 fromthe nucleoplasm [74]. The role of MBNL1 depletionin DM was demonstrated by a Mbnl1 knockout mouseline that recapitulated many clinical features of the dis-ease, including myotonia, cataracts and skeletal muscleabnormalities [75]. A second alternative splicing reg-ulator, CUGBP1, is aberrantly up-regulated in DM1heart and skeletal muscle tissue [76,77]. CUGBP1up-regulation is due to protein kinase C-mediatedhyperphosphorylation and stabilization of CUGBP1induced by CUG-repeat RNA [78]. Functional lossof MBNL1 and gain of CUGBP1 are thought tobe primarily responsible for the widespread disrup-tion of developmentally regulated alternative splic-ing events in DM tissues. Several disease featurescan be directly attributed to misregulation of individ-ual splicing events, including myotonia and insulinresistance. Myotonia is due to aberrant inclusion ofmuscle-specific chloride channel (CLNC1 ) exon 7a inadults resulting in nonsense-mediated decay of CLNC1mRNA and reduced chloride conductance in mus-cle [79,80]. Correction of the Clcn1 splicing defectreversed the myotonia phenotype in DM mouse mod-els [81]. Insulin resistance in DM directly correlateswith decreased inclusion of insulin receptor (IR) exon11 and predominant expression of a lower-signallingIR isoform with decreased insulin sensitivity [77]. Thismulti-systemic disease demonstrates the widespreadconsequences that result from a trans-dominant muta-tion that affects alternative splicing regulation.

Splicing in cancer

Different splice variants are commonly found to beenriched in cancer tissue compared to the normal sur-rounding tissue. The splicing change can result frommutations within intronic or exonic splicing elementswithin the genes relevant to cancer, such as onco-genes or tumour suppressors. In many cases, however,the aberrantly spliced genes are not mutated, indicat-ing that the defects involve a change in the nuclearenvironment that regulates splice site choice [82].Recently, high-throughput transcriptome sequencingof cancer cells has led to the identification of tran-script chimeras between neighbouring genes, calledread-throughs, potentially due to trans-splicing or toco-transcription and intergenic splicing [83]. Futurework should determine the mechanism responsible forread-through formation and their causal roles, if any, incancer. The relevance of splicing to cancer raises sev-eral questions, including: (a) do the splicing changesinitiate and/or promote cancer progression; (b) do the


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splicing changes increase the oncogenic potential ofthe proteins expressed from these variants; and (c) canan alternative splicing signature be used to identifycancer subtypes, predict clinical outcome or aid inidentification of the most effective treatments? Theroles of splicing in cancer progression, diagnosis andtreatment have been addressed in several excellentreviews [84,85].

Cis-acting mutations in tumour suppressorsand oncogenes

The best documented examples of the role of splicingin cancer involve alterations in known tumour sup-pressors and oncogenes. KLF6, a Kruppel-like zincfinger transcription factor, is a tumour suppressorthat inhibits cell growth via trans-activation of thecyclin-dependent kinase inhibitor p21 and throughp21-independent mechanisms [86–89]. A splice vari-ant of KLF6, KLF6-SV1, is generated by an alternative5! splice site in exon 2, producing a protein isoformthat lacks the zinc finger DNA-binding domains butretains most of the activation domain [90]. There-fore, KLF6-SV1 antagonizes wild-type KLF6 func-tion in a dominant-negative manner, promoting cellproliferation and migration [91]. A prostate cancer-associated single nucleotide polymorphism near theintron–exon boundary creates a binding site for theSR protein SRp40, which leads to increased expres-sion of the KLF6-SV1 isoform [92]. The associationof this polymorphism and prostate cancer suggests thataltered splicing and KLF6-SV1 over-expression leadsto increased cancer risk. KLF6-SV1 over-expressionin vivo accelerates prostate cancer progression andmetastasis, whereas its knockdown by RNAi inducesapoptosis and decreases tumour growth [93]. Cumu-latively, these studies demonstrate the potential roleof the KLF6-SV1 oncogenic splice variant in can-cer progression. Another interesting example con-necting splicing of tumour suppressors to cancer isthe CDKN2A gene locus, which encodes two tumoursuppressors, p14ARF and p16INK4a, with alternativereading frames. CDKN2A is a high-risk locus formelanoma formation through the loss of p14ARF andp16INK4a [94]. In a family with melanomas, neurofibro-mas and multiple dysplastic naevi, a novel splice sitemutation has been detected in the splice acceptor siteof intron 1, which promotes exon 2 skipping in boththe p14ARF and p16INK4a transcripts [95]. Inactivationof both tumour suppressors may have combinatorialeffects in the development of these diseases.

The receptor tyrosine kinase KIT provides an exam-ple of a proto-oncogene activated by aberrant splic-ing. KIT gain-of-function mutations have been doc-umented in gastrointestinal stromal tumours (GISTs)and are sufficient for GIST development. Small dele-tions encompassing the 3! splice site of intron 10 havebeen identified in multiple GISTs [96,97]. Surpris-ingly, this deletion does not result in exon 11 skipping,

but rather creates a novel intra-exonic 3! splice sitewithin exon 11. The deleted region is critical for inhi-bition of KIT kinase activity in the absence of ligandand structural analysis of the novel KIT splice variantindicates that the kinase adopts a constitutively activeconformation [96]. These data suggest that aberrantpre-mRNA splicing of the proto-oncogene KIT couldplay a key role in the development of GISTs.

Trans-acting alterations in cancer that affectsplicing

SR proteins control alternative splicing events inproto-oncogenes and tumour suppressor genes, fre-quently modifying their cellular activity. Many SRproteins are up-regulated in tumours, as exemplified bythe high levels of SRp20, SF2/ASF and SC35 in ovar-ian cancer [98] and SF2/ASF in tumours of the colon,thyroid, small intestine, kidney and lung [99]. A recentstudy showed that SF2/ASF over-expression can trans-form immortalized cells and SF2/ASF-expressing cellsare tumourigenic in nude mice. The transformed phe-notype of these cells and a lung cancer cell lineexhibiting SF2/ASF up-regulation could be reversedby shRNA-mediated knockdown of SF2/ASF, indicat-ing that splicing regulators can indeed act as onco-genes [99]. SF2/ASF alters splicing of the BIN1tumour suppressor, which binds and suppresses thec-Myc oncogene. BIN1 exon 12A, the inclusion ofwhich is increased by SF2/ASF, disrupts Myc bindingand abolishes its tumour suppressor function [100].SF2/ASF also induces oncogenic isoforms of theMNK2 and S6K1 kinases and the RON tyrosine kinasereceptor [99,101]. Importantly, the SF2/ASF-inducedisoform of S6K1 is sufficient for cell transformation,demonstrating the potency of trans-acting alterationsin cancer [99]. Changes in the expression of additionalsplicing factors are documented in cancer developmentand progression, but the splicing defects they induceand their relation to cellular transformation remain tobe elucidated [82].

Novel therapeutic approaches for diseasesassociated with mis-splicing

Small molecules

The activities of factors that directly regulate alterna-tive splicing are modulated in response to a variety ofcellular signalling pathways, and commonly involvepost-translational modifications. Small molecules thateither promote or inhibit these signalling events cansignificantly alter the splicing patterns of a sub-set of transcripts [102]. The best-documented post-translational modification affecting splicing is thephosphorylation of SR proteins. The phosphorylationstate of an SR protein controls multiple features ofits function ultimately affecting its ability to enhanceexon recognition [103]. GSK3 is one of several kinases



Figure 3. Antisense oligonucleotide (AON) based therapies. Modified AONs that avoid RNase H-mediated degradation of targetpre-mRNAs can modulate splicing in several ways by (A) binding to splice sites, (B) splicing enhancer elements or (C) splicingsilencer elements and therefore abolish control by the corresponding splicing regulatory proteins. (D) AONs designed to bind anddegrade the target pre-mRNA by endogenous RNase H can block expression of potentially harmful mutant mRNAs. (E) AONsthat bind pathogenic RNA can prevent interactions with RNA binding proteins causing release of both the proteins and the RNA

that phosphorylate SR proteins and therefore inhibitionof GSK3 has the potential to alter SR-regulated splic-ing events. SR proteins regulate the inclusion of tauexon 10, mis-splicing of which can cause FTDP-17.Small molecule kinase inhibitors against GSK3 restoretau exon 10 inclusion, illustrating the ability of smallmolecules to alter splice site selection and potentiallytreat disease [104]. A drawback with this approachis the potential for numerous off-target effects. Mul-tiple SR proteins and their respective targets will beaffected by GSK3 inhibition. In addition, GSK3 has

many substrates, including metabolic, signalling andstructural proteins [105]. The future development ofsmall molecule modulators of splicing will dependupon how well the specificity of the effects can beoptimized.

Antisense oligonucleotides

Short antisense oligonucleotides (AONs) complemen-tary to specific regions within target pre-mRNAsare being used in multiple approaches to reverse or


160 AJ Ward et al

circumvent disease processes: (a) promote exon skip-ping by blocking 5! and/or 3! splice sites; (b) promoteexon skipping or inclusion, respectively, by blockingbinding of cognate factors to splicing enhancers orsuppressors; (c) promote degradation of mutant tran-scripts; and (d) prevent harmful interactions betweenproteins and pathogenic RNA (Figure 3). An unmod-ified DNA AON hybridized to RNA will inducedegradation of the RNA that is base-paired with theDNA by ribonuclease RNase H, which is ubiquitouslyexpressed. The functionality of AONs can be changedby chemical modifications that prevent activation ofRNase H [106]. Modified AONs targeted to particu-lar regions within a pre-mRNA are used to preventaccess to factors that recognize specific cis elements.For example, AONs that bind to splice sites induceexon skipping, and this approach is being successfullyapplied to restore the reading frame of mutated genes.

One example of an application in clinical practice isin Duchenne’s muscular dystrophy (DMD), a neuro-muscular disorder in which two-thirds of the mutationsare deletions within the massive (2 Mb) dystrophingene producing mRNAs that are out of frame, resultingin severe loss of function. Two observations suggestedthat retention of the reading frame, despite internaldeletions, would produce partially functional protein.First was the observation of rare dystrophin-expressingskeletal muscle fibres in DMD muscle tissue that arethought to arise from aberrant splicing, resulting inexon skipping and expression of in-frame protein con-taining internal deletions [107,108]. Second was therealization that the milder Becker muscular dystrophy(BMD) is caused by dystrophin deletions that retainan open reading frame. These observations supportedthe idea that in-frame deletions producing partiallyfunctional protein could reduce the severity of DMD.AONs directed at splicing sites that restore the dys-trophin reading frame by induced skipping of selectedexons have been successfully applied to DMD patients[109,110]. In these initial studies, AONs are deliv-ered by direct injection into skeletal muscle tissueand a clinical trial for systemic delivery is ongoing(http://clinicaltrials.gov/ct2/show/NCT00844597).

A different application for AONs has been devel-oped as a therapeutic strategy for treating DM1. Thecurrent model for DM1 pathogenesis is that expandedCUG repeats result in a toxic RNA gain-of-function.Two recent studies used modified CAG-repeat AONsto hybridize to the CUG repeats and disrupt theRNA–protein complexes that assemble on CUGrepeat-containing RNA in cultured cells from individ-uals with DM1 and in DM1 mouse models [111,112].The CAG AONs neutralized the toxic RNA bydecreasing the level of CUG repeat-containing RNAand by disrupting nuclear foci, releasing sequesteredMBNL and allowing transport of the mutant RNA tothe cytoplasm [112]. A major advantage of AONs isthe ability to target specific sequences, overcoming theobstacle of target specificity encountered by the useof small molecules to modulate splicing. The issues

for AONs as a therapeutic approach include efficientdelivery to the appropriate tissues and the duration ofthe effect. Despite these challenges, AONs remain apromising application for research studies and clinicaltherapeutics.

Summary

Alternative splicing is a highly coordinated processthat relies on a combination of positive and negative-acting factors, intronic and exonic sequence elementsand temporal and spatial signalling pathways forproper control. Mutations that disrupt any of thesecritical features, either in cis or trans, may alter thesplicing patterns of one or multiple transcripts, dis-rupting the production or functions of the encodedproteins. Large numbers of splicing-related diseaseshave been documented; however, this number is likelyto be substantially underestimated because the effectsof mutations on splicing are often not pursued as aprimary cause of disease. A complete understandingof the splicing code, as well as the factors that deci-pher the code, will allow accurate predictions of whichnucleotide changes affect splicing and identificationof diseases for which altered splicing is the primarydefect. Modulation of splicing provides a potent ther-apeutic approach to either address the main cause ofdisease or circumvent a disease process toward molec-ular normalcy.

Teaching materials

PowerPoint slides of the figures from this revieware supplied as supporting information in the onlineversion of this article.

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