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4 Effects of Transcription, Promoters and Chromatin on Alternative Splicing

4 Effects of Transcription, Promoters and Chromatin on Alternative Splicing

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Pre-mRNA Splicing and Disease



Integration of Splicing into Cell Signaling

and Regulatory Networks

It is considered common knowledge that transcription factors are integrated within

signaling pathways in the cell and their activation (or repression) with resulting

up-regulation (or down-regulation) of gene expression, represents one of the most

important ways in which cells respond to changes and signals from the outside

environment. However, one must also keep in mind that splice factors are also

integrated in signaling pathways, and their activity is heavily regulated. As such,

their regulation has a very similar outcome as the regulation of transcription factors

(i.e. changes in the protein repertoire occur, and therefore cell functions).

In the usual paradigm, splice factors are inactive in the cytoplasm, they are

phosphorylated by protein kinases, become active and shuttle (often chaperoned)

into the nucleus where they affect outcomes of alternative splicing. While several

protein kinases are not very specific and affect many intracellular processes and

functions, several distinct examples have emerged in the last years as being more

frequently involved in splicing regulation than other processes—e.g SR-protein

kinases (SRPKs) or cyclin-dependent like kinases (CLKs) (Zhou et al. 2012; Naro

and Sette 2013). While examples of virtually all canonical signaling pathways may

be found to be involved in splice factor regulation and isoform choice (Oltean and

Bates 2014) a recent report from Fu and colleagues in San Diego has demonstrated

a central role played by the EGFR/PI3kinase/Akt signalling axis in regulating

SRPK1 and SR-protein phosphorylation, and thus having a rather global effect on

alternative splicing modulation (Zhou et al. 2012).


Extent of Alternative Splicing Genome-Wide

and Conservation Across Species

While alternative splicing has been described more than 30 years ago and heavily

studied mechanistically, it has not been clear until recently how extensive it is

genome-wide or how conserved splicing machinery is across phyla. Two recent

studies using genome-wide RNAseq have demonstrated that more than 94 % of

genes are alternatively spliced in humans highlighting an immense combinatorial

power of ~22,000 genes (each with 7–8 exons on average) to generate incredible

protein diversity (Pan et al. 2008; Wang et al. 2008).

With the growing list of fully sequenced genomes for a plethora of plant, fungal,

vertebrate and invertebrate species, it has been clear that differences in species

phenotypes cannot be accounted for by the gene repertoire alone. Moreover, mRNA

levels of different genes measured in various organs and tissues do not vary much

during evolution of vertebrates, making unlikely that the level of gene expression is

a major driver in species diversity. It has been long postulated, and partially proven

by others, that alternative splicing is the main engine for species and organ-specific


M.R. Ladomery and S. Oltean

phenotypic differences. In a recent paper, Blencowe and coworkers (BarbosaMorais et al. 2012) have analyzed high-throughput RNAseq data from several

organs across several species and demonstrated that the further an organism is in

evolutionary terms from humans and primates the less complex the alternative

splicing repertoire. In vertebrates, the conservation of alternative splicing in neural

tissues is higher in comparison to other organs highlighting its importance in the

evolution and development of the vertebrate nervous system. Also, another important conclusion was that alternative splicing patterns that are species-specific are

coded mainly by the sequences rather than by the protein or factors in the cell that

act on them; this was demonstrated by using a partial human chromosome in mouse

cells that human sequences splice to human splice variants, even when expressed in

mouse cells (Barbosa-Morais et al. 2012). These findings have very important

implications when studying for instance alternative splicing events associated

with human disease in transgenic mice using constructs specific for the human


3 Pre-mRNA Splicing and Disease


Nature and Frequency of Splicing Mutations

While classically mutations in coding regions of genes have been studied in detail

due to the direct effect on encoded proteins and phenotype, mutations in non-coding

regions of DNA may be equally important. From a splicing perspective mutations

present in splice sites or any regulatory intronic or exonic regions may affect the

outcome of a particular splicing event, result in changes in the protein sequence or

length, change in the composition of a particular protein splice isoforms or lack of

the protein due to the RNA being targeted for degradation (for example through

nonsense-mediated decay).

Indeed, while the exact numbers vary among studies due to analysis methods and

databases used, between 10 and 15 % of pathogenic mutations involve splicingrelated elements (Lewandowska 2013; Gamazon and Stranger 2014; Hartmann

et al. 2008). Mutation at the splice donor or acceptor site are the most common

while mutations in the polypyrimidine tract or the branch point are the most rare.

A comprehensive analysis of mutations affecting splice factors or proteins that

are components of the spliceosomal machinery is lacking. However, it is worth

mentioning recent published studies highlighting a high frequency of mutations in

genes involved in splicing in some hematological malignancies including myelodisplastic syndromes. Mutations in the splice factor SF3B1 are seen in 20 % of

cases, while in other types of myeloid syndromes such as acute or chronic myeloid

leukemia in less than 5 % of cases (Ogawa 2012).

Pre-mRNA Splicing and Disease



Examples of Aberrant Splicing in Cancer

Among various diseases, cancer is by far the most researched in respect to how

faulty splicing contributes to pathogenesis. There is a myriad of alternative splicing

isoforms reported to be associated (with various degrees of causality) with different

aspects of the oncogenic process (Oltean and Bates 2014; Ladomery 2013; Ghigna

et al. 2013). We will discuss a few examples that may form various paradigms for

how aberrant splicing contributes to cancer.

One of the several hallmarks of cancer (Hanahan and Weinberg 2011) and

essential property for tumours to be able to grow and survive is the ability to

form new vessels through angiogenesis. It has been known for several years that

there are two classes of molecules in the organism with opposing actions—pro- and

anti-angiogenic—that regulate a fine balance for maintaining adequate rates for

vessel formation from embryonic development to the adult life. These opposing

actions may be achieved through de-regulation of alternative splicing as well. More

than 10 years ago, an alternative splicing event in the vascular endothelial growth

factor (VEGF)—a major regulator of angiogenesis—had been described that

resulted in the VEGFxxxb family of isoforms from an 30 alternative splice site in

the terminal exon (Bates et al. 2002). At the protein level this splicing event

translates into a different sequence of amino-acids at the C-terminus, which confers

antagonistic properties and is therefore anti-angiogenic. There are numerous examples of cancers with decreased proportion of the anti-angiogenic VEGF isoforms or

studies showing that reversal of pro-to anti-angiogenic VEGF isoforms results in

decrease in tumour growth in animal models (Harper and Bates 2008).

Another essential property of cancer cells is the ability to migrate at distant sites

and metastasize. This is achieved through several mechanisms, including epithelialto-mesenchymal (EMT) transition; a plastic property of cells to switch between

epithelial, static phenotypes to mesenchymal, migratory ones. Splicing may be

again hijacked to nurture these properties as in the example of two mutually

exclusive exons in fibroblast growth factor receptor 2 (FGFR2)- exon IIIb (or 8)

found almost exclusively in epithelial cells and IIIc (or 9) found in mesenchymal

cells. The alternate exons change the coding regions in the extracellular domain of

the receptor, making it responsive to different FGF ligands. This results in different

levels of activation of the receptor and therefore differential modulation of signaling pathway and cellular properties. Again, numerous studies have shown that the

mesenchymal IIIc exon is associated with aggressive cancers (Carstens et al. 1997)

or that overexpression of the epithelial IIIb isoform decreases tumour growth in

animal models (Shoji et al. 2014; Yasumoto et al. 2004).

Apoptosis (programmed cell death) is another important hallmark of cancer; a

process through which developmentally superfluous or irreparably mutated cells are

physically eliminated. Deregulation of the signals that control apoptosis can contribute to the process of carcinogenesis. Apoptosis occurs through two pathways:

the extrinsic and intrinsic pathway, the latter mediated through mitochondria. In the

intrinsic pathway, free cytochrome c is released into the cytosol, resulting in


M.R. Ladomery and S. Oltean

accumulation of a complex known as the apoptosome. The apoptosome in turn

activates a series of proteases known as caspases. The executor caspases 3, 6 and

7 activate apoptosis. What is most striking is that several of the genes that encode

the machinery of apoptosis are alternatively spliced into pro- and antiapoptotic

(prosurvival) isoforms (Miura et al. 2012). The apoptosome itself contains two

proteins, APAF1 and caspase 9, that include alternatively spliced isoforms with

radically different properties. APAF1 (apoptosis-activating factor 1) expresses a

pro-apoptotic splice isoforms (the full length APAF1-XL) and alternative splice

isoforms (including APAF1-ALT) that impede the induction of apoptosis (Ogawa

et al. 2003). When it is predominantly expressed, the pro-apoptotic isoform of

APAF-1 activates procaspase 9. However caspase 9 is also alternatively spliced. A

series of cassette exons can be skipped in the caspase 9 pre-mRNA, resulting in the

expression of a shorter isoform known as caspase 9b, a dominant negative antiapoptotic splice variant. The regulation of caspase 9 alternative splicing has been

examined. The splice factor SRSF1 contributes to its regulation by binding to an

intronic splice enhancer (Shultz et al. 2011). The same study shows that the effect of

SRSF1 on caspase 9 splicing can even affect the chemotherapeutic sensitivity of

non-small cell lung cancer cells. This suggests that it might be desirable to design

drugs that can alter alternative splicing patterns by targeting SRSF1 or the protein

kinases that regulate SRSF1 activity (SRPK1 and CLK1). Such drugs could be used

in conjunction with established chemotherapies.

These examples clearly illustrate how each of the hallmarks of cancer are

powerfully affected by alternative splicing. In many ways, dysregulated alternative

splicing might itself be considered a hallmark of cancer (Ladomery 2013). Accordingly, one would predict that in cancer the machinery that regulates alternative

splicing goes completely astray, much as the stability of the genome and the

regulation of cell signaling goes out of control. Metastatic cancer is thought to

arise after a series of gene mutations accumulate—a notable example being the

multistage carcinogenesis model of colorectal cancer (the adenoma-carcinoma

sequence). In this model a series of lesions occur; starting for example, with loss

of function of the APC tumour suppressor gene, followed by activation of oncogenic Ras, and inactivation of the tumour suppressor gene TP53. Note that TP53,

the most widely studied tumour suppressor, expresses a bewildering array of splice

isoforms, greatly complicating its function (Surget et al. 2013). Each of these genes

is alternatively spliced in such a way as to express isoforms that could, if

overexpressed, potentially contribute to the progression of cancer (Ladomery

2013). Hence the range of mutations that could result in colorectal cancer could

include mutations that affect splice sites of these genes, or mutations that affect the

expression and activity of key splice factors.

Pre-mRNA Splicing and Disease



Examples of Aberrant Splicing in Other Diseases

Knowing the almost global extent of alternative splicing across the human genome

it is not surprising that the number of studies describing aberrant splicing in a large

variety of diseases is continuously increasing. Among the most dramatically

affected by aberrant splicing are several neurological disorders. For example, myotonic dystrophy, one of the most common muscular dystrophies in adults, is

characterized by abnormal CUG or CUGC repeats in the mRNA of the DMPK

gene. These repeats sequester a splicing factor known as muscle-blind-like-1

(MBNL-1), and therefore affect alternative splicing of various other transcripts

targeted by MBNL-1 (Fugier et al. 2011). Spinal muscular atrophy (SMA) is an

autosomal recessive disease and the most common genetic cause of mortality in

infants. It is due to mutations or deletions that affect the SMN1 gene, which encodes

a protein with crucial functions in RNA metabolism called SMN. There is a nearly

identical gene to SMN1, called SMN2, that cannot compensate for the protein

because of a silent mutation in exon 7 that provokes exon skipping resulting in a

truncated non-functional protein (Naryshkin et al. 2014). Several reports have

identified at least six genes that have aberrant splicing and are thought to be

involved in pathogenesis of Parkinson’s disease (Fu et al. 2013).

Many genes involved in the pathogenesis of diabetes have been reported to

express splice isoforms associated with advanced diabetes: for example a splice

variant of the soluble receptor for advanced glycation end products (sRAGE) has

been reported to be associated with the severity of diabetic nephropathy (Gohda

et al. 2008); and T-cell factor 7- like 2 (TCF7L2) splice variants affect beta-cell

function (Le Bacquer et al. 2011). There is in vivo data from animal studies that

particular splice isoforms are causal to diabetes; for example soluble cytotoxic Tlymphocyte-associated protein 4 (sCTLA-4) is associated with acceleration of

progression of type 1 diabetes (when knocked down in a mouse model) (Gerold

et al. 2011).

The ever-increasing spectrum of diseases associated with aberrant splicing

includes cardiovascular diseases. For example, dilated cardiomyopathy has been

reported to be associated with aberrant splicing isoforms of titin, a protein important for the structure of the sarcomeres in striated muscles. The cause of faulty titin

splicing is mutations in RBM20, an RNA-binding protein that works as a master

regulator of alternative splicing in cardiac muscle (Guo et al. 2012).


Manipulating Splicing in Therapy

As there is a growing number of chronic diseases associated with aberrant

pre-mRNA splicing, including diabetes and cancer, splicing has emerged as an

exciting new therapeutic target. Several strategies have been developed to try to

reverse faulty splicing for therapeutic purposes. The technique most employed so


M.R. Ladomery and S. Oltean

far is the use of anti-sense oligos (ASO), also called splicing-switching oligos

(SSOs). The general principle is to design ASOs that bind to regulatory sequences;

if they bind to splice sites or enhancer elements they will inhibit inclusion of an

exon; whereas if they bind splicing silencers they will activate exon inclusion. So

far SSOs have been proved very promising, with several of them in clinical trials,

including ASO-based treatments for Duchenne muscular dystrophy (DMD) and

SMA (Singh and Cooper 2012).

More recently a specialized bifunctional targeting oligonucleotide has been

designed, called targeted oligonucleotide enhancers of splicing (TOES). the typical

TOES molecule has two parts: the first is the annealing ASO that targets the splicesite, and the second is the ‘tail’ oligo, which is not complementary to the target

mRNA but instead serves to bind and recruit trans-factors to allow their local

accumulation, and thus promote the correct splicing event. This strategy has also

proved to be effective in correcting the splicing defect in SMA (Owen et al. 2011).

Use of trans-splicing has also been developed as a therapeutic tool—so-called

SMaRT method (spliceosome mediated RNA trans-splicing). The principle is to

create a hybrid RNA resulting from splicing between a 50 splice site of an endogenous RNA and 30 splice site of an artificial construct that provides a corrected

exon (Rodriguez-Martin et al. 2009).

Finally, there is a growing number of small molecule inhibitors that have been

shown to affect splicing. An interesting example is amiloride. This drug is a wellknown diuretic used to regulate the ion channels within the renal tubules of the

kidney. However, it was identified in a screen of small molecules that amiloride

potently affects splicing of several genes involved in apoptosis and moreover to be

able to decrease tumour growth in animal models (Ding et al. 2012). Recently, a

class of small molecule compounds called SPHINX, were shown to inhibit SRPK1,

the major kinase responsible for SR-protein phosphorylation, which in turn

inhibited VEGF splicing and angiogenesis in a model of ocular neovascularization

(Gammons et al. 2013) as well as melanoma xenografts growth (Gammons

et al. 2014).

4 Summary

Alternative splicing of pre-mRNA has emerged as an absolutely key process in

gene expression and cellular homeostasis. Even now, novel splice isoforms of

human genes are still being discovered and characterized. Throughout eukaryotes,

evolutionarily conserved mechanisms underlying alternative splicing allow

genomes to express a diverse variety of proteins from a relatively limited number

of genes, often having radically different biological properties. It is then not

surprising that dysregulated splicing results in disease. Aberrant alternative splicing

is now associated with cancer, neurological disorders, and diabetes. A better

understanding of alternative splicing will, without a doubt, lead to novel treatment


Pre-mRNA Splicing and Disease


Acknowledgments We would like to acknowledge Dr. Graham Dellaire (Dalhousie University,

Halifax, Canada) for his aid in editing and preparing the figures in this chapter. This work was

supported by operating grants from BBSRC (BB/J007293/1) to Sebastian Oltean and The Bristol

Urological Institute (BUI256) to Michael Ladomery.


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Acute Promyelocytic Leukaemia: Epigenetic

Function of the PML-RARα Oncogene

Julia P. Hofmann and Paolo Salomoni

Abstract Acute promyelocytic leukaemia is a myeloid neoplasm characterized by

expansion of promyelocytic progenitors. Its main driver is the oncogenic fusion

protein generated by the (t15;17) chromosomal translocation between the

promyelocytic leukaemia (PML) and retinoic acid receptor α (RARα) genes.

Being PML-RARα the primary trigger of APL, it represents an excellent model to

study neoplastic transformation in the haematopoietic system. Importantly, epigenetic changes imposed by and/or associated with PML-RARα have been implicated

not only in promoting/sustaining the tumour phenotype, but also in influencing

therapy response. In this chapter we will discuss the existing literature on chromatin

remodelling driven by PML-RARα and its impact on APL pathogenesis and therapy.

1 Acute Promyelocytic Leukaemia

Acute promyelocytic leukaemia (APL), a variant of acute myeloid leukaemia

(AML), is a neoplastic myeloid disorder that is characterized by accumulation of

undifferentiated blood cells at the promyelocyte stage in patient‘s blood samples

(de The and Chen 2010; Puccetti and Ruthardt 2004). This high number of blast

cells is accompanied by a reduced number of mature blood cells, therefore causing

a high risk of bleeding due to coagulopathy and thrombocytopenia. APL can occur

at any age with a median age-of-onset between 40 and 50 years, with peadiatric

APL occurring more rarely compared to adult APL. The increasing knowledge on

the molecular causes of APL have effectively improved therapy over the last

40 years, thus turning the previously fatal disease into a highly curable disease

with remission rates over 90 % (de The and Chen 2010). Based on molecular

analysis, APL is clinically classified by the detection of different oncofusionproteins as a result of chromosomal translocations between the RARα gene and

J.P. Hofmann • P. Salomoni (*)

Samantha Dickson Brain Cancer Unit, University College London Cancer Institute, London,

United Kingdom

e-mail: p.salomoni@ucl.ac.uk

© Springer International Publishing Switzerland 2016

D.P. Bazett-Jones, G. Dellaire (eds.), The Functional Nucleus,

DOI 10.1007/978-3-319-38882-3_4



J.P. Hofmann and P. Salomoni

the genes for PML (de The et al. 1990a), PLZF (Chen et al. 1993), NuMA (Wells

et al. 1997), NPM (Redner et al. 1996), STAT5b (Arnould et al. 1999), PRKAR1A

(Catalano et al. 2007), FIPL1 (Kondo et al. 2008), BCOR (Yamamoto et al. 2010)

or OBFC2A (Won et al. 2013). In this chapter we will focus on the PML-RARα

fusion protein and its influence on epigenetics in cancer pathogenesis.

2 The PML-RARα Oncogene

In 95 % of cases, APL blasts present the reciprocal chromosomal translocation

(t15;17) indicating that the resulting PML-RARα fusion is the likely driver of

transformation in these cells. Three different chromosomal break points for

PML-RARα are known of which the most frequently (~70 %) occurring break

point (bcr1) results in the longest PML-RARα transcript, bcr3 (~20 % of cases) in

the shortest transcript and the rarely (~10 %) occurring bcr2 in an intermediate long

transcript (Melnick and Licht 1999). All three PML-RARα isoforms contain the

functional domains of both PML and RARα and we will first give a brief overview

of the normal functions of PML and RARα to point out how both proteins

functionally contribute to APL pathogenesis.



The human PML gene is encoded on chromosome 15 and seven different isoforms

exist with variations in the central and C-terminal part of PML (Salomoni

et al. 2008; Jensen et al. 2001; Nisole et al. 2013; Condemine et al. 2006). Three

different domains of PML - the RING, B-box, and coiled-coil (RBCC) motif classify PML as a tripartite motif (TRIM) protein family member and exhibit

functional importance for the formation of higher multimers (Jensen et al. 2001;

Reymond et al. 2001). The α-helical coiled-coil domain allows self-association of

PML molecules (Burkhard et al. 2001), the RING domain and two B-boxes are

Zn-binding domains, but do not confer direct DNA binding ability to PML

(Reymond et al. 2001). Interestingly, the RING domain has E3 ligase activity

(Joazeiro and Weissman 2000). These functional domains of PML are important

for protein-protein interactions to form macromolecular structures in the nucleus,

termed PML nuclear bodies (PML-NBs) (Salomoni et al. 2008; Nisole et al. 2013;

Borden et al. 1995). More than 70 proteins—such as p53, DAXX, pRb, SP100,

HP-1—are known to be associated with PML-NBs either by direct or indirect

interaction with PML (Dellaire and Bazett-Jones 2004; Salomoni et al. 2008;

Wolyniec et al. 2013). Most importantly, post-translational modifications such as

phosphorylation, acetylation, ubiquitination and small ubiquitin-like modifier

(SUMO)-ylation regulate protein-protein interactions in PML-NBs and affect the

dynamics of PML-NB functions (Dellaire and Bazett-Jones 2004; Fu et al. 2005;

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4 Effects of Transcription, Promoters and Chromatin on Alternative Splicing

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