The alternative splicing side of cancer

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Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology journal homepage: www.elsevier.com/locate/semcdb

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Review

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The alternative splicing side of cancer

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Giuseppe Biamonti ∗ , Morena Catillo, Daniela Pignataro, Alessandra Montecucco, Claudia Ghigna

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Istituto di Genetica Molecolare – CNR, Via Abbiategrasso 207, 27011 Pavia, Italy

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a r t i c l e

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a b s t r a c t

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Article history: Available online xxx

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Keywords: Alternative splicing Signal transduction Epithelial to mesenchymal transition Epigenetic Splicing factors

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Contents

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Alternative splicing emerges as a potent and pervasive mechanism of gene expression regulation that expands the coding capacity of the genome and forms an intermediate layer of regulation between transcriptional and post-translational networks. Indeed, alternative splicing occupies a pivotal position in developmental programs and in the cell response to external and internal stimuli. Not surprisingly, therefore, its deregulation frequently leads to human disease. In this review we provide an updated overview of the impact of alternative splicing on tumorigenesis. Moreover, we discuss the intricacy of the reciprocal interactions between alternative splicing programs and signal transduction pathways, which appear to be crucially linked to cancer progression in response to the tumor microenvironment. Finally we focus on the recently described interplay between splicing and chromatin organization which is expected to shed new lights into gene expression regulation in normal and cancer cells. © 2014 Published by Elsevier Ltd.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alteration of splicing profiles and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interplay between alternative splicing and signaling cascades . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The tight connection between SRSF1 and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternative splicing and the response to tumor microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatin organization and splicing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction The vast majority of metazoan protein-coding genes consists of a succession of short exons separated by long stretches of DNA called introns. Primary transcripts (pre-mRNAs) generated by RNA polymerase II contain both exons and introns, but only exons are retained in the mature mRNAs, which are exported to the cell cytoplasm for translation. The precise excision of introns, or splicing, is carried out by the spliceosome, a large molecular machine composed of five small nuclear ribonucleoproteins (snRNPs U1, U2, U4, U5, and U6) and more than 200 different polypeptides [1]. The fact

∗ Corresponding author. Tel.: +39 0382 546322. E-mail address: [email protected] (G. Biamonti).

that 5 and 3 splice sites at exon–intron boundaries display short and poor consensus sequences raises conceptual questions about how the spliceosome recognizes exons embedded in intron oceans. At the same time, however, this feature is the prerequisite for alternative splicing (AS) events, in which various combinations of 5 and 3 splice sites are used to generate distinct mRNAs from a single pre-mRNA. Deep sequencing experiments revealed that AS is a highly pervasive regulatory mechanism that affects more than 90% of human genes [2]. In addition to modifying protein features, AS can control RNA stability by introducing premature STOP codons that lead to mRNA degradation through the non-sense-mediated RNA decay (NMD) pathway [3]. This mechanism operates to control the homeostatic level of several RNA binding proteins (RBPs), especially splicing regulators [4], with downstream consequences on splicing profiles of numerous genes. Thus, modulation of AS is

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crucial to specifying cell identity and developmental programs and its deregulation is causatively linked to human diseases including cancer, which is one of the topics discussed in this review. Deciphering the molecular circuits that modulate AS to changes in cell growth conditions remains a major subject of investigation. Usually, alternatively spliced exons are flanked by splice sites that poorly match the consensus sequence and their recognition depends on regulatory elements, enhancers and silencers, which promote and inhibit exon splicing, respectively. These elements are present both within exons and introns [5] and form the binding sites for splicing regulators including SR factors and hnRNP proteins and a number of tissue specific RBPs [6]. Both SR factors and hnRNP proteins have a modular organization consisting of domains that mediate the interaction with RNA, such as the RNA Recognition motif (RRM), and others involved in protein interactions. Most AS events are controlled by the relative abundance and/or activity of antagonistic SR factors and hnRNP proteins through a combinatorial mechanism [7], in which multiple positive and negative factors and sequence elements influence the final outcome of the splicing reaction. Splicing regulators undergo extensive post-translational modifications [8]. However, the functional implications of these modifications are still largely unexplored and very little is known about how they are integrated with signaling pathways and cell biology. Nevertheless, the link with signaling pathways appears a decisive parameter for the role of RNA binding proteins in cancer, which is another issue addressed in this review. Finally, we will discuss the impact of chromatin organization on AS, an emerging field of investigation. Since epigenetic marks are deeply connected with gene expression programs [9], the analysis of the links with AS is expected to dramatically increase our comprehension of splicing regulation and will offer new possibilities for therapeutic intervention.

2. Alteration of splicing profiles and cancer In the last few years the contribution of AS in human disease, particularly in cancer, has been widely recognized [10]. It is now evident that the unbalanced expression of splicing variants or the failure to properly express the correct isoforms is part of the biology of cancer cells [11,12]. Interestingly, hereditary gene mutations associated with cancer predisposition may act by perturbing splicing profiles as in the case of the BRCA1/2 genes [13,14]. In addition, whole-genome approaches and transcriptome sequencing have proven that gene mutations associated with cancer-specific AS events represent a widespread phenomenon and can be potentially used as new therapeutic biomarkers [15]. More frequently, however, alterations of splicing profiles in tumors are not due to mutations in the affected genes but rather to the altered activity, expression level or even mutations of splicing regulators. An example is provided by the genes for the “splicing factor 3B subunit 1” (SF3B1) of U2 snRNP and for U2AF1, which are mutated in various hematological malignancies [16,17]. SF3B1 is the second most frequently mutated gene in chronic lymphocytic leukemia and its mutation is associated with poor prognosis. In contrast in myelodysplastic syndromes [18] and in uveal melanoma [19] SF3B1 mutations are associated with a good prognosis indicating that the final outcome depends on the cell context. Concerning U2AF1, somatic mutations are associated with aggressive types of myeloid malignancies and impact the splicing profile of 35 genes for cell cycle progression and RNA processing functions [17]. Interestingly, SF3B1 is the target of antineoplastic drugs such as Spliceostatin A (SSA). SSA prevents the interaction of SF3B1 with the pre-mRNA but, rather than a general splicing inhibition, it induces specific AS changes in transcripts for proteins

involved in cell division, including cyclin A2 and Aurora A kinase, which could account for its anti-proliferative effects [20,21]. Rarely, cancer-associated AS have a role in the initial phases of tumorigenesis. Frequently they operate during tumor progression and contribute to metastases formation that, nowadays, is the cause of approximately 90% of all human cancer casualties [22]. In general, the expression of the splicing isoform that confers advantage to tumor growth is not a pre-requisite of the cancer tissue but is detectable in normal tissues as well [23]. Frequently the function of the AS isoform is unknown, but it is plausible that maintaining a subtle balance between splicing variants is vital to cellular function and dynamics. How cell growth conditions dictate which isoform is expressed and what biological factors might influence splicing outcomes remain areas for intense further exploration. Commonly, splicing of numerous genes is altered as cells progress through the oncogenic process and is linked to the acquisition of cancer features such as a high rate of proliferation, the capacity to form new vessels, to invade extracellular matrices, to survive stressing conditions (for a review see [24]). For instance, the human tumor-suppressor TP53 gene produces 12 different isoforms through AS, alternative initiation of translation and promoter usage. Although they are expressed in normal human tissues, the balance between these isoforms is altered in a wide range of cancer types contributing to tumorigenesis and to the response to therapy [25]. Thus, deciphering the circuits that control AS of TP53 transcripts may assist in the development of personalized therapies. For instance, by manipulating the relative abundance of p53 isoforms it could be possible to switch the response to DNA damage from cell cycle arrest to apoptosis. This is exemplified by p53␤, an isoform crucial for p53-mediated senescence, whose production is controlled by splicing factor SRSF3 [26]. The production of p53␤ is promoted by caffeine that affects the expression of SRSF3 and other SR factors, including SRSF2 [27]. By targeting SRSF3 expression, caffeine can also modulate the splicing profile of several genes involved in the survival of tumor cells to hypoxia [27].

3. Interplay between alternative splicing and signaling cascades Splicing regulators undergo a wide range of post-translational modifications including phosphorylation [28], methylation [29,30] and sumoylation [31]. The impact of these modifications on protein functions, however, is far from being completely understood and we still have a fragmentary picture of how extra-cellular stimuli can be communicated to splicing decisions via specific RBPs [32]. Nevertheless, splicing regulators can be viewed as integral components of signal transduction cascades able to convey and amplify signals to post-transcriptional circuits. Below we will present few examples to illustrate this concept. One signaling cascade frequently deregulated in cancer is PI3K/Akt/mTOR. Phosphorylation of splicing factors, including SRSF1 and SRSF5, by kinases in this pathway affects splicing decisions relevant to cancer progression as in the case of fibroblast fibronectin 1 (FN1) [33], capsase 9 [34] and protein kinase C␤II genes [35]. Remarkably, phosphorylation of one factor by different kinases may have antagonistic effects on splicing decision. For example, phosphorylation of SRSF1 by Akt promotes the production of the cancer-associated EDA-FN isoform of FN1, whereas phosphorylation of the same factor by Clk or SRPK1-2 kinases has the opposite effect [36]. The analysis of the SRSF1 activities highlights how intricate the connection between AS and signaling pathway can be. In fact, by promoting the production of the oncogenic short isoform of S6K1, SRSF1 can lead to mTOR activation and cap-dependent translation

Please cite this article in press as: Biamonti G, et al. The alternative splicing side of cancer. Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.03.016

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[37]. Interestingly, down-regulation of the short S6K1 isoform is sufficient to reverse the transforming phenotype resulting from SRSF1 over-expression [38]. To make connections even more entangled, the direct interaction of SRSF1 with mTOR and phosphatase PP2A is crucial to control 4E-BP1 phosphorylation and hence capdependent translation [39]. Another interesting example is provided by SPF45, a target of several MAP kinases, including ERKs, JNKs and p38 MAPK [40]. SPF45 phosphorylation regulates cell proliferation and adhesion by affecting the expression level of ErbB2, one member of epidermal growth factor receptor (EGFR) family, and the splicing profile of FN1 [40]. The EGFR signaling pathway is linked to the increased glucose uptake and lactate production in cancer cells (aerobic glycolysis or “Warburg effect”). This involves the expression of the embryonic PKM2 isoform of the pyruvate kinase M (PKM) gene, through AS of two mutually exclusive exons: inclusion of exon 10 leads to PKM2 expression, whereas exon 9 is present in transcripts encoding the adult isoform PKM1. The choice between the two exons is controlled by a number of splicing regulators. Poly-pyrimidine tract binding protein (PTB/hnRNP I), hnRNP A1 and hnRNPA2 inhibit inclusion of exon 9 [41,42] while SRSF3 promotes the usage of exon 10 [43]. EGFR activation leads to the coordinated transcription/splicing program responsible for up-regulation of PKM2 which involves increased transcription of both PTB and PKM genes [44]. In addition, the oncoprotein MYC also up-regulates transcription of PTB, hnRNPA1 and hnRNPA2 ensuring a high PKM2/PKM1 ratio and supporting aerobic glycolysis [42]. Additional examples illustrating how AS can impact on the physiological (or pathological) functions of oncogenic protein kinases in signaling pathways, like membrane-bound tyrosine kinases receptors or cytosolic protein kinases, have been described in a recent review [45].

4. The tight connection between SRSF1 and cancer In the last ten years several splicing regulators, including SRSF1 [38], SRSF6 [46], SRSF9 [47], hnRNP A2/B1 [48] and hnRNP H [49] have been proven to have oncogenic properties, while others, such as RBM5, RBM6 and RBM10 [50] act as tumor suppressors. So far a thorough molecular dissection of the oncogenic activity has been provided only for SRSF1, a member of the SR family of splicing factors (Fig. 1). The SRSF1 gene is up-regulated or even amplified in different human cancers [38] and its over-expression is sufficient to transform immortal rodent fibroblasts and to induce sarcomas in nude mice [38]. This factor appears to act in the same oncogenic circuit of MYC and the two onco-proteins reciprocally influence each other. Indeed, MYC activates transcription of the SRSF1 gene [51] while SRSF1, by controlling AS of BIN1 transcripts, relieves the inhibitory effect exerted by this tumor suppressor on transcription factor MYC [52]. Remarkably, up-regulation of both MYC and SRSF1 frequently occurs in lung and breast tumors [51,53] and these two proteins cooperate in transforming mammary epithelial cells, in part by activating translation factor eIF4E [53]. Finally, SRSF1 knockdown reduces MYC’s oncogenic activity, decreasing the proliferation rate and the anchorage-independent growth [51]. The tumor promoting function of SRSF1 mainly depends on its first RNA Recognition Motif (RRM1) that controls the AS of cancer-associated genes such as BIN1, MNK2 and S6K1 kinases and the apoptotic gene BIM [38]. RRM1 is also required to elevate the expression of B-Raf and to activate the MEK/ERK signaling pathway (Fig. 1). In line with this, pharmacological inhibition of MEK1 inhibits SRSF1-mediated transformation [54]. As detailed ahead the link with signaling pathways is also relevant to control both the expression and the activity of SRSF1 during the

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epithelial to mesenchymal cell transition (EMT), a crucial event in the metastatic spreading of tumors.

5. Alternative splicing and the response to tumor microenvironment The tumor microenvironment has a pivotal role in cancer progression. Until recently, transcriptional programs have been considered the main targets of signaling cascades elicited by parameters in the tumor microenvironment such as acidosis, hypoxia, and serum deprivation. It is now clear that the microenvironment can also impact post-transcriptional processes including AS. This may involve changes in the expression level of splicing regulators such hnRNPA0, A1, A2, B1, and A3 [55]. One feature in the microenvironment that is particularly relevant to tumor progression is the low oxygen tension, since hypoxia affects angiogenesis, proliferation, and cell migration. Remarkably, hypoxia deeply impacts splicing decisions [56] both in cancerous and normal cells in the tumor microenvironment. This apparently involves a limited number of splicing factors such as QKI and RBFOX2 whose expression is altered in ovarian tumors [57]. One complex program activated by hypoxia is the epithelial to mesenchymal transition (EMT), a prerequisite for metastases formation. Recent studies have highlighted the involvement of “epithelial splicing regulatory proteins” (ESRPs) and “RNAbinding Fox protein 2” (RBFOX2), in this process. While ESRPs are dedicated to epithelial-specific splicing and their expression is down-regulated during EMT, RBFOX2 controls both epithelial and mesenchymal splicing events [58]. The link between RBFOX2 and mesenchymal tissue-specific splicing was first established in a study in which the splicing profiles of hundreds of cassette exons were investigated in epithelial and mesenchymal tissues obtained from human colon and ovaries. Remarkably, many splicing differences that distinguish normal epithelial from normal mesenchymal tissues matched those that differentiate normal ovarian tissues from ovarian cancer [59]. Interestingly, depletion of RBFOX2 during EMT does not prevent the activation of mesenchymal markers or changes in cell morphology, but significantly reduces the invasive potential of the cells, suggesting a role of RBFOX2 in tissue invasiveness [58]. Concerning ESRP1, its over-expression in invasive mesenchymal breast cancer cells causes morphological changes similar to the mesenchymal-to-epithelial transition (MET) characterized by a reduction in cell invasion which involves AS of the cytoskeleton regulator MENA [60]. ESRP1 also inhibits the switch in CD44 expression from the variant to the standard isoform, which is required for the execution of EMT induced by the transcriptional regulator Snail [61]. In accord with the role of ESRP1 in maintaining the epithelial status its expression is inhibited by Snail [61] and Twist [62], two transcription factors that induce EMT. Remarkably, ESRP1 over-expression can partially revert the mesenchymal phenotype induced by Twist or Snail, indicating that splicing regulation is sufficient to drive critical aspects of EMT-associated phenotypic changes [61,62]. In accord with this, EMT-dependent splicing changes occur commonly in human tumors and may represent new diagnostic and prognostic markers for the analysis of cancer progression [62]. Another trigger of EMT is the Wnt pathway, frequently deregulated in human colorectal cancer (CRC). Alterations of splicing programs appear to contribute to Wnt-mediated colonic carcinogenesis as in the case of Rac1b, a splicing isoform of the small GTPase Rac1 frequently over-expressed in breast and colorectal cancers and produced through inclusion of exon 3b [63]. This isoform is sufficient to activate EMT and is involved in tumorigenic transformation of mammary epithelial cells exposed to matrix

Please cite this article in press as: Biamonti G, et al. The alternative splicing side of cancer. Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.03.016

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Fig. 1. SRSF1 reveals the existence of complex connections between splicing regulatory factors and oncogenic signaling pathways. The tumor microenvironment triggers signaling cascades that affect both the activity and/or the expression levels of SRSF1. On the left. By promoting the expression of B-Raf, SRSF1 activates the MEK-ERK kinases. Phosphorylation of Sam68 by ERK controls the expression levels of SRSF1 through an alternative splicing event associated with the nonsense-mediated mRNA decay (AS-NMD). Center: SRSF1 phosphorylation by Akt and SRSPK kinases has opposite effects in the regulation of AS events relevant to cancer progression, such as proliferation, angiogenesis, apoptosis, EMT, invasion and metastasis. On the right. The SRSF1 gene is a transcriptional target of the c-myc proto-oncogene. The expression level of SRSF1 modulates the activity of mTOR kinase by two mechanisms: through a direct interaction and via alternative splicing of S6K1 transcripts. The final effect is a control of translation. See text for further details.

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metalloproteinase-3 (MMP-3) [64]. Splicing of Rac1 transcripts is controlled by SRSF1 and SRSF3 that respectively promote and inhibit exon 3b inclusion. The expression of both factors is regulated by upstream signaling pathways: PI3K inhibits SRSF1 expression, while Wnt promotes SRSF3 gene transcription [65]. Another player in Rac1 splicing is hnRNP A1 that counteracts SRSF1 and prevents EMT. Remarkably an inverse correlation between expression of hnRNP A1 and Rac1b exists in normal breast tissue and breast cancer biopsies [66]. Antagonistic SRSF1 and hnRNP A1 factors determine also the choice between EMT and MET programs through AS of the Ron proto-oncogene, which encodes for a tyrosine-kinase receptor that controls cell dissociation and invasion of extracellular matrices. Skipping of exon 11 results in the production of Ron, a constitutively active isoform that promotes cell invasiveness and is frequently up-regulated in cancers [67,68]. This makes splicing of exon 11 a potential target for anti-metastatic therapeutic strategies [69]. Several splicing regulators appear to control this splicing event: SRSF1 [67], hnRNP H [49] and hnRNP A2 [48] promote exon skipping, while hnRNP A1 induces exon inclusion [70]. By binding to a splicing enhancer in exon 12, SFSR1 up-regulation leads to Ron production and activates EMT [67]. The association of SRSF1 with the enhancer is counteracted by hnRNP A1, which binds to a silencer in exon 12, promotes inclusion of exon 11 and activates the MET program [70]. In addition, hnRNP A1 downregulates the expression of hnRNP A2/B1, another positive regulator of Ron

[48], through an AS event associated with the nonsense-mediated (AS-NMD) [70]. Also the expression of SRSF1 is regulated through an AS-NMD event in the 3 UTR of its pre-mRNA. This regulatory mechanism is frequently altered in colon cancer and is controlled by the extracellular signal-regulated kinase 1/2 (ERK1/2) via phosphorylation of splicing regulator Sam68 (Src associated in mitosis, 68 kDa) [4]. Thus, a complex hierarchy of splicing factors can sense signaling pathways and integrate splicing decisions into EMT/MET programs. 6. Chromatin organization and splicing Although crucial, cis-acting regulatory elements (enhancers and silencer) along with their cognate interacting splicing factors are not the sole determinants for specifying AS profiles. The new player in this field is chromatin organization, which adds a further layer of complexity to splicing regulation and makes AS a target of the epigenetic re-organization events in tumors [71]. There are two general routes through which chromatin can affect AS (1) via nucleosome positioning and (2) through post-translational modifications (PTM) of histones. (1) Nucleosome positioning. Bio-informatics and genome-wide analyses have revealed an enrichment of nucleosomes and 5methyl-cytosine (5meC) on exons relative to introns, with a preferential positioning of nucleosomes at exon boundaries. Interestingly, in the case of alternative exons this pattern correlates

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Fig. 2. Epigenetic marks at exon may impact splicing profiles. During transcription a rearrangement of the nucleosome occurs with the displacement of histones H2A/H2B. In contrast, histones H3/H4 carrying the primary epigenetic marks are not displaced by RNA polymerase II (Pol II) in vivo. Histones associated with exons are enriched in H3K36me3 that contributes to the recruitment of splicing factors (SR) though chromatin adaptors (A). Splicing factors also interact with the phosphorylated CTD domain of RNA Pol II. Altogether these interactions contribute to exon definition and splicing choices. S: spliceosome. See text for further details.

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with splicing outcomes [72] through a process that involves the effect of positioned nucleosomes on the elongation rate of RNA Pol II [73]. The reduced rate of RNA synthesis, in turn, would expand the window of opportunity for using weak instead of strong splice sites [74]. Similarly to positioned chromosomes, the Brm subunit of the chromatin remodeling factor SWI/SNF [75] creates a crosstalk between transcription and RNA processing by decreasing RNA Pol II elongation rate and facilitating the recruitment of the splicing machinery to variant exons with suboptimal splice sites. (2) Histone PTMs. Splicing decisions may be also influenced by the non-random distribution of some PTMs of histones associated with exons (Fig. 2). Genome wide analyses have proven that exons are enriched in histone H3 trimethylated on K36 (H3K36me3) or monomethylated on K79 (H3K79me), monomethylated histones H4 on K20 (H4K20me) and H2B on K5 (H2BK5me) and mono, di, and trimethylated H3 on K27 [72,76–78]. Some PTMs can change the RNA polymerase II elongation rate, others can mediate the interaction with splicing factors and the splicing machinery either directly [79] or with the help of chromatin-splicing adaptor complexes [80,81]. A few papers have recently established the role of H3K36me3 in AS. This PTM is enriched at exons, particularly those of highly expressed genes [82], and its level at alternatively spliced exons correlates with their inclusion. Interestingly, this pattern appears to reflect the existence of a two-way communication between chromatin organization and AS regulation. Splicing, in fact, is required to establish and to maintain this and other epigenetic marks [83,84]. The first molecular dissection of the link between H3K36me3 and splicing has been provided by Tom Misteli and colleagues who analyzed the effect of epigenetic marks on AS of the fibroblast growth factor receptor 2 (FGFR2) transcripts. This gene contains two mutually exclusive exons: exon IIIb is included in normal epithelial cells whereas in human mesenchymal stem cells exon IIIc is exclusively used [85]. Splicing factor PTB has a major role in this choice and represses inclusion of exon IIIb by binding to silencing elements in the flanking introns. The recruitment of PTB depends on H3K36me3, which is enriched over the FGFR2 gene in mesenchymal cells, where exon IIIb is repressed. Similar epigenetic re-organization events occur in a subset of genes whose splicing profile is controlled by PTB, including tropomyosin 2 (TPM2) and the pyruvate kinase type M2 (PKM2) genes. The interaction of PTB with H3K36me3 is mediated by the histone tail-binding protein MORFrelated gene 15 (MRG15), a component of the retinoblastoma binding protein 2 (RBP2)/H3-K4 demethylase complex. Manipulation of the MRG15 level is sufficient to modulate splicing of PTB-dependent exons but not of the control PTB-independent CD44

exon v6. Genome wide analysis point to MRG15 as a modulator of alternative exons that are weakly regulated by PTB [85]. H3K36me3 can control also the recruitment of SRSF1 as recently proved by the group of Wendy Bickmore [86]. This involves the short splicing isoform (p52) of the chromatin-associated protein PC4 and SF2 interacting protein 1 (Psip1) also called Ledgf (lens epithelium-derived growth factor). Only the p52 isoform of Psip1 can interact with SRSF1 and splicing factors and can module AS. Binding of Psip1/Ledgf to chromatin depends on the PWWP domain which belongs to the Tudor (Royal) family of protein domains that bind methylated lysines in a number of proteins including histones [87]. Interestingly, the PWWP domains of Brpf1, Dnmt3a, MSH6, NSD1, NSD2 and N-PAC have been shown to specifically bind H3K36me3. Other chromatin-splicing adaptors include CHD1 [88] and Gcn5 [89] that mediate the recruitment of U2 snRNP, while HP1 assists the association of hnRNP proteins to H3k9me3 in Drosophila [90]. It is likely that additional adaptors will be discovered in the next future reinforcing the notion that integrated complexes exists between chromatin, transcriptional apparatus and splicing factors with reciprocal influences among players. In this perspective it is not surprising that defects in splicing factors may lead to DNA damage mediated by the transcriptional apparatus [91].

7. Conclusion Although the relevance of AS to cancers biology is no longer disputed, we are still far from deploying its potentiality in diagnosis and therapy. In the last few years, the growing awareness of the role played by deregulated AS programs in human pathologies has fostered the development of promising new therapeutic strategies. These include the identification of small molecules to target components of the splicing machinery [92] or the exploitation of antisense oligonucleotides to manipulate splicing decisions [93]. A great impulse to this field is expected by the development of genome wide RNA sequencing studies and bio-informatic analyses that will help us to incorporate splicing programs in the complex regulatory networks operating in cells and organisms. Even though we are just beginning to appreciate the intricacy of this system, recent studies have already unveiled the existence of a tight interplay between molecular processes so far viewed as completely unrelated. Thus, the AS machinery is involved in a number of twoway connections with signal transduction pathways and epigenetic mechanisms operating in response to a wide range of stimuli and stressing conditions. Deciphering these connections will be the

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challenging task of the next future with the potential to provide us with a better comprehension of the logic of living organisms. Conflict of interest None declared. Acknowledgements This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC) to G.B. and C.G., from the Association for International Cancer Research (AICR-UK) to C.G. and from Flagship project Epigen CNR-MIUR to G.B. References

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