Part I Molecular Cell Biology

1 Part I Molecular Cell Biology RNA Regulation: Advances in Molecular Biology and Medicine, First Edition. Edited by Robert A. Meyers. 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

3 1 RNA Regulation in Apoptosis Christopher von Roretz and Imed-Eddine Gallouzi McGill University, Department of Biochemistry and Rosalind and Morris Goodman Cancer Research Centre, McIntyre Medical Building Room 915B, 3655 Promenade Sir William Osler, Montreal, Quebec, H3G 1Y6 Canada 1 Balancing Life and Death 6 2 The Coordinated Process of Apoptosis 6 2.1 Caspase Cascade 7 2.2 Extrinsic Activation of Caspases 7 2.3 Intrinsic Activation of Caspases 9 2.3.1 Mitochondrial Release of Cytochrome c 9 2.3.2 Activation of the Apoptosome 9 2.4 Caspase Substrates 10 3 Protein Regulators of Apoptosis 11 3.1 Regulators of Cytochrome c Release 11 3.1.1 Bcl-2 Family Proteins 11 3.1.2 BH3-Only Proteins 11 3.2 Targeting Caspase Activation 12 3.2.1 DISC Inhibition 13 3.2.2 Regulation of Apoptosome Activity 13 3.3 Caspase Inhibitor of Apoptosis Proteins 13 3.3.1 IAPs 14 3.3.2 Inhibitors of IAPs 14 3.4 Post-Translational Regulation of Apoptotic Factors 14 4 Transcriptional Regulation of Apoptosis 15 4.1 p53 and p73 15 4.2 E2F Family 16 4.3 FOXO Family 16 4.4 Additional Transcriptional Regulators of Apoptosis 16 RNA Regulation: Advances in Molecular Biology and Medicine, First Edition. Edited by Robert A. Meyers. 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

4 RNA Regulation in Apoptosis 5 Post-Transcriptional Regulation of Apoptosis 17 5.1 Splicing 17 5.1.1 Splicing to Regulate Cytochrome c Release 18 5.1.2 Splicing-Mediated Regulation of Caspases 19 5.1.3 Nuclear Export of mrna 21 5.2 Translation 21 5.2.1 cis-elements and trans-acting Factors that Regulate Translation 21 5.2.2 HuR as a Regulator of Translation 22 5.2.3 IRES-Mediated Translation and Apoptosis 23 5.2.4 micrornas as Regulators of the Translation of Apoptotic Factors 24 5.2.5 mirnas as Inhibitors of Apoptosis 24 5.2.6 Pro-Apoptotic Roles of mirna 26 5.3 mrna Turnover 27 5.3.1 The Destabilizing A/U Rich Element 27 5.3.2 ARE-Mediated Turnover of Apoptosis-Related mrnas 28 5.3.3 Alternate Destabilizing Elements 29 5.3.4 Destabilizing mirnas 29 6 Discussion 30 6.1 Rearranging Deckchairs on the Titanic? 30 6.2 All a Matter of Time 31 Acknowledgments 32 References 32 Keywords Alternative splicing Splicing of pre-mrna to generate mature mrna involves the removal of noncoding segments of messenger RNA known as introns. The factors involved in this process determine if certain splice sites are skipped or not, however, yielding different splice variants. Bcl-2 family proteins The B-cell lymphoma 2 (Bcl-2) family of proteins all contain one or more Bcl-2 homology (BH) domains. Members of the Bcl-2 family may be either pro- or anti-apoptotic, as they influence mitochondrial outer membrane permeabilization, leading to the release of cytochrome c into the cytoplasm.

RNA Regulation in Apoptosis 5 Caspase Cysteine-dependent, aspartic acid-specific proteases (caspases) are generally considered the effectors of apoptotic cell death, cleaving a variety of substrates in order to lead to the organized death of a cell. Cytochrome c Cytochrome c (cyt c) is a small polypeptide that plays a role in the electron transport chain as an electron carrier under normal conditions. When released from the mitochondria following mitochondrial outer membrane permeabilization, cytochrome c binding to Apaf-1 protein enables structural changes which ultimately permit the activation of caspase-9. IRES-mediated translation Internal ribosomal entry site (IRES)-mediated translation is the process whereby mrna translation is initiated through the recruitment of translation factors, and consequently, ribosomes, to the mrna independently of the m7g 5 cap. mirna microrna (mirna) is a single-stranded RNA of approximately 22 nt that can induce the inhibition of translation or decay of a target mrna upon binding to a mrna for which a specific mirna maintains a certain degree of complementarity. mrna turnover The turnover of messenger RNA is the stability of a particular mrna, which can be modulated by various factors that promote either mrna decay or stabilization. Post-transcriptional regulation Subsequent to the transcription of genetic material to generate messenger RNA, various mechanisms including splicing, localization, translation, and mrna turnover, can influence the expression of the gene encoded by a target mrna. The organized process of apoptotic cell death is tightly regulated, with many protein factors promoting or inhibiting the activity of its key players. A growing number of studies have shown that these numerous regulators of apoptosis are themselves regulated at the level of RNA. Through transcription, the levels of mrna encoding these factors can be increased or decreased. Less studied is the post-transcriptional regulation of expression for these pro- and anti-apoptotic players. Alternative splicing, effects on translation, and the modulation of mrna turnover can all influence protein levels of the broad cast of factors involved in apoptosis. While most of the studies delineating these mechanisms have not examined explicitly the

6 RNA Regulation in Apoptosis RNA regulation of these factors during apoptosis, a small collection of data does suggest that post-transcriptional regulation of apoptotic modulators occurs during the cell death process, thus hinting at a previously underappreciated role for RNA regulation in apoptosis. 1 Balancing Life and Death All cellular processes depend on balance. Metabolism, motility, and growth all depend on juggling factors that promote or perturb each of these events. Perhaps the most significant of cellular processes, however, is that of cell death when the decision is made either by the victim cell or by its environment that life should cease and one, possibly final, balance is tilted either to seal the fate of this cell through death, or to allow it to survive despite assaulting conditions by activating survival mechanisms. At a cellular level, with numerous factors regulating each and every pathway, balance is a matter of life or death. Several types of cell death exist [1], and while these were initially classified based on their degree of organization, during recent years such a broad-stroke classification has been challenged [1, 2]. While autophagic cell death and necrosis/necroptosis have gained attention more recently [1,3 7], the best known form of cell death is apoptosis. Derived from the Greek word for falling off, as with the leaves from a tree [8], apoptosis has been the most well-studied class of cell death, and is typically considered the default method of ending the life of a cell. In essence, apoptosis is an organized cell death whereby specific processes take place to coordinate the destruction of a cell in a practical manner, to avoid harm to neighboring cells. In fact, given the individualized focus of this process, apoptosis is often referred to as cell suicide [3], even though it can be triggered both internally by the victim cell and by external stimuli from neighboring cells or the surrounding environment. Numerous pathways are activated during apoptosis (see below) which integrate many pro- and anti-apoptotic factors to weigh in on the decision to survive or to engage death in response to a stress stimulus. While the activity of these factors has often been studied at great lengths, over the past few years evidence has been mounting to suggest that the expression of apoptotic factors is also an important determinant of apoptotic cell death. Transcription to produce mrnas encoding these factors has been studied to significant lengths. Conversely, there have been limited attempts at understanding the post-transcriptional mechanisms that influence the expression of apoptotic factors. Of these studies, only a fraction has specifically examined how these regulatory mechanisms may directly influence the progression of apoptosis. In this chapter, attention will be focused on outlining the link between RNA and apoptosis, and a summary provided of how transcription and, more specifically, post-transcriptional events may contribute to apoptotic cell death. 2 The Coordinated Process of Apoptosis Apoptosis will not occur spontaneously, and generally begins with a trigger. The diversity of sources that can activate

RNA Regulation in Apoptosis 7 apoptosis is broad, and ranges from chemical activators, endocrine signaling from neighboring cells, or internal sensors of cell damage [9 13]. Typically, the activation of apoptosis is divided into two categories: (i) that which is triggered externally, known as extrinsic-induced apoptosis; and (ii) that which results from internal signals, termed intrinsic-induced apoptosis [14]. Each of these involves a different sequence of activated factors, although the end goal is the same to enable the activation of a specific family of proteases known as caspases (Fig. 1). 2.1 Caspase Cascade Caspases are cysteine-dependent, aspartic acid-specific proteases that cleave protein substrates after an aspartate residue [15]. Caspases exist as inactive zymogens in normal cellular conditions, known as procaspases [14, 16]. Structurally, their activation may be achieved by cleaving off a precursor region, and also by cleaving their catalytic domain to yield two regions that then interact. Additionally, caspase activity depends on their dimerization. Upon the activation of one caspase, such as caspase-8 or -9, a sequential activation of other caspases can occur, commonly known as the caspase cascade [17]. These upstream caspases are referred to as initiator caspases, given that they are those initially activated in response to apoptotic stimuli, and include caspases-2, -8, -9, and -10 [16]. Each of these initiator caspases then in turn activates other caspases. For example, caspase-9 is best described as cleaving procaspases-3 and -7 to generate their active forms [18, 19]. These secondary caspases have a much broader panel of targets and, as such, are referred to as effector or executioner caspases [20]. Caspases-3, -6, and -7 are generally considered as the effector caspases in apoptosis [16]. While initiator caspases are activated by being correctly oriented following the binding to adaptor proteins (described below), effector caspases await cleavage from initiator caspases. The reason behind this is that the initiator caspases exist as monomers in the cytosol, and require platforms to allow dimerization to occur, whereas effector caspases perpetually exist as dimers but depend on cleavage to become activated [14, 21]. 2.2 Extrinsic Activation of Caspases In extrinsic-induced apoptosis (Fig. 1), an external signal is needed to act on cellular receptors at the surface of a target cell. These receptors, given their purpose, have beentermed death receptors, and include many members of the tumor necrosis factor (TNF) receptor family, such as the Fas and TNF-α receptors [22, 23]. Each of these receptors is designed to bind and be stimulated by the binding of specific substrates. For example, when Fas ligand is released by T cells, it will bind to the Fas receptor on target cells. Similarly, other secreted ligands such as TNF-α and TNF-related apoptosis-inducing ligand (TRAIL) will bind to their respective receptors [22]. The result of such binding is mechanistically similar for each of these cases. The binding of death ligands to death receptors triggers the trimerization of receptor monomers [24, 25]; this trimeric structure is then taken up by the cell through endocytosis and, in doing so, permits the recruitment of intermediary proteins, such as Fas-associated protein with death domain (FADD) [26]. FADD and other, related proteins such as death

8 RNA Regulation in Apoptosis Extrinsic Intrinsic Death ligand(ex: Trail, Fas ligand) Stress Death receptor (ex: DR5, Fas) MOMP Scaffold (FADD, DAXX) Cyt c Apaf-1 Apoptosome Casp-8 Active casp-8 Active casp-9 Casp-9 Effector casps Fig. 1 Extrinsic and intrinsic pathways of apoptosis. In the extrinsic pathway of apoptotic activation, binding of a death ligand (e.g., Fas ligand, TRAIL) to a death receptor (DR5, Fas) triggers the recruitment of an adaptor protein (FADD, DAXX) and an initiator caspase such as procaspase-8 or -10. Activated initiator caspase can then activate effector caspases such as caspase-3 or -7. Intrinsic Active effector casps Apoptotic cell death activation of apoptosis involves a stress signal which triggers the mitochondrial outer membrane permeabilization (MOMP), leading to the release of cytochrome c (cyt c) from the mitochondrion. With Apaf-1, datp, and procaspase-9 the apoptosome is formed, which allows for the activation of caspase-9. This initiator caspase can also activate effector caspases. domain-associated protein 6 (DAXX) and receptor-interacting protein-1 (RIP-1) are all capable of binding monomeric procaspases [27], in particular procaspase-8 and -10. This binding is achieved via either the death domain or the death effector domain (DED) of these adaptor proteins [28]. Collectively, the group of death ligand,

RNA Regulation in Apoptosis 9 receptor, DED-containing adaptor and procaspase form what is referred to as the Death-inducing signaling complex (DISC) (Fig. 1). 2.3 Intrinsic Activation of Caspases While external signals can stimulate apoptosis through the mechanisms described above, caspases can also be activated through internal sensory mechanisms. DNA damage, for example, leads to the activation of kinases such as ataxia telangiectasia mutated (ATM) kinase, which can trigger either DNA repair or apoptosis through the phosphorylation of a variety of targets [29]. One crucial target of ATM is p53, a transcription factor that regulates the expression of numerous pro-apoptotic factors (see below). Under normal conditions, p53 is bound by Mdm2, which leads to the ubiquitination and degradation of p53 [30, 31]. The phosphorylation of p53 prevents Mdm2-binding, allowing this transcription factor to promote the apoptotic response [32]. ATM can also phosphorylate Mdm2 to further interfere with its association to p53 [33]. Several other pathways, responding to various stimuli, exist to promote apoptosis. 2.3.1 Mitochondrial Release of Cytochrome c One of the key events in intrinsic-mediated apoptosis is the release of cytochrome c from the mitochondria into the cytoplasm (Fig. 1). Under normal conditions, cytochrome c plays an essential role in the electron transport chain, serving as a single electron carrier [34, 35]. Cytochrome c, a small protein of 104 amino acids, is normally located between the inner and outer membranes of the mitochondrion [36]. Specific channels that form in the outer membrane of the mitochondrion allow for cytochrome c to travel from the inter-membrane mitochondrial space to the cytoplasm; this is referred to as mitochondrial outer membrane permeabilization (MOMP) [37]. MOMP is often considered a key event in apoptosis activation, and is generally thought to be irreversible. Having reached the cytoplasm, cytochrome c plays a very different role as it is specifically capable of forming a very large complex, known as the apoptosome (Fig. 1), in view of its ability to advance apoptosis [14]. 2.3.2 Activation of the Apoptosome The core of the apoptosome is a heptameric structure comprised of seven monomers of a protein called apoptotic protease activating factor 1 (Apaf-1) [38]. Apaf-1 exists as an inactive monomer in the cytoplasm, but readily binds cytochrome c should it become available, for example following its release from the mitochondrion. When cytochrome c binds to Apaf-1 a conformational change occurs in this larger protein, allowing the association of several Apaf-1 monomers to form a seven-membered ring [39]. This structure is then capable of recruiting procaspase-9 monomers [14] that, like Apaf-1, circulate in the cytoplasm under normal conditions, without having any known activity. Once recruited to the Apaf-1 heptamer, procaspase-9 molecules are placed in close proximity, which allows for their dimerization, which is in fact sufficient for caspase-9 activation [40]. This entire process utilizes energy, however, and datp is a key ingredient for caspase-9 activation and correct apoptosome formation. datp must be recruited by Apaf-1 and converted to dadp in order for the apoptosome to assume its optimal structure capable of recruiting procaspase-9 [41].

10 RNA Regulation in Apoptosis The collective complex of cytochrome c, Apaf-1, procaspase-9, and datp is termed the apoptosome, the kinetics and regulation of the formation of which have been thoroughly reviewed [14]. 2.4 Caspase Substrates Each caspase, whether an initiator or effector, has a set of targets. In some cases, the list of known targets for a caspase is very limited. Other than its two target procaspases (-3 and -7), limited evidence has demonstrated other apoptotic targets for caspase-9 [42]. Caspase-8, on the other hand, does not solely have procaspases as its targets; indeed, caspase-8 does cleave procaspase-3 to cause its activation, but also targets a pro-apoptotic protein known as Bid [43]. Under normal conditions, Bid is a 22 kda protein that may be found in the cytosol [44]; however, upon cleavage by caspase-8, Bid is cleaved to generate truncated Bid (tbid). Numerous groups have shown that tbid is an important promoter for the release of cytochrome c from the mitochondria [43]. Hence, caspase-8 caninfactbothdirectlyactivateaneffector caspase (caspase-3) as well as activate the intrinsic apoptotic pathway, through cytochrome c release. Far more targets have been identified for effector caspases [20], which is consistent with their role as executioners of organized cell death. Ultimately, they are responsible for shutting down all functions of the target cell, anddosoinanorganizedmanner.targets of caspases-3 and -7 include poly (ADP-ribose) polymerase (PARP), DNA fragmentation factor-45 (DFF-45, also known as inhibitor of caspase-activated deoxyribonuclease; ICAD), Mdm2, protein kinase Cδ (PKCδ), actin, lamin, and fodrin [45 55]. PARP is an important enzyme for the repair of DNA breaks [56]. It is believed that, by inactivating this enzyme, caspases halt any futile, energy-requiring attempts to repair DNA damage, and commit the cells to die [57]. ICAD is an inhibitor of the cytosolic endonuclease CAD that has been shown to fragment DNA, and by subsequently inactivating the ICAD, the caspases will promote DNA degradation [47 50]. As described above, Mdm2 is an inhibitor of the apoptotic transcription factor p53; hence, the cleavage of Mdm2 further enhances transcriptional events during cell death [51]. Finally, actin, fodrin, and lamin are all structural proteins, the cleavage of which during apoptosis is believed to be responsible for morphological changes within the cell [53 55]. In addition to mediating apoptosis by cleaving downstream targets, there is substantial support that caspases also act in feedback loops to further enhance the process. For example, caspases-3 and -7 are capable of cleaving the initiator caspases-8 and -9 [58]. Collectively, through self-perpetuating caspase cascades as well as the cleavage of specific key cellular players, caspases produce a state of organized death that makes the condensed cells recognizable, ideal targets for phagocytes or other neighboring cells [59]. Unlike many other physiological processes, apoptosis is distinguished for being irreversible, given the nature of its purpose, and this provides all the more reason for a tight control to exist over the process. Caspase activation, while an important component of the pathway, is just one piece of the puzzle. The regulation of apoptosis occurs at several levels, ranging from protein protein interactions to controlling the expression of apoptotic players.

RNA Regulation in Apoptosis 11 3 Protein Regulators of Apoptosis The regulation of apoptosis involves numerous factors that directly or indirectly inhibit or activate caspases and other protein players of the process. Importantly, the expression of these factors must also be considered, as the upregulation or downregulation of any player that contributes to cell fate can tilt the balance between survival and apoptosis. 3.1 Regulators of Cytochrome c Release One of the most significant levels at which apoptosis is regulated is through cytochrome c release. As noted above, both extrinsic and intrinsic apoptosis pathways trigger the release of cytochrome c through MOMP, which has been shown to then activate caspase-9 [60]. 3.1.1 Bcl-2 Family Proteins The actual release of cytochrome c from the mitochondrion requires the formation of a channel or pore, composed of Bax and Bak monomers [61]. Importantly, Bax and Bak can be inhibited in forming this channel if they bind to proteins such as B-cell lymphoma 2 (Bcl-2), Bcl-w, Mcl-1, or Bcl-x L [62]. Structurally, the difference between the anti-apoptotic Bcl-2, Bcl-w, and Bcl-x L proteins and the pro-apoptotic Bak and Bax is not large [63], as both contain four regions termed Bcl-2 homology (BH) domains that allow dimerization of the proteins and enable each of these factors to interact with one another. During the past decade, another class of players in the regulation of apoptosis has been identified, termed BH3-only proteins [44]. This naming is based on the fact that these proteins contain only the BH3 domain homologous to those found in Bax, Bcl-2, and other members of the Bcl-2 family of proteins. 3.1.2 BH3-Only Proteins Several BH3-only proteins have been identified, including Bim, Bid, Bad, Bik, Noxa, and Puma [64 68]. These proteins have been shown as being important in the regulation of apoptosis, as their knockdown results in a resistance to apoptotic stimuli [69, 70]. The reason for this is that, with only their BH3 domains, Bad, Bim, Puma, and Noxa bind to anti-apoptotic Bcl-2 family members such as Bcl-2 and Bcl-x L, and sequester them from inhibiting Bak and Bax [64 66]. Furthermore, evidence has shown that BH3-only proteins also promote MOMP by binding Bax to enhance its integration into the outer mitochondrial membrane [71] (Fig. 2). In fact, active caspase-8 promotes intrinsic apoptosis through cleavage of the BH-3 only protein Bid to generate tbid, which can then assist in promoting Bax oligomerization at the outer mitochondrial membrane [61, 67], thus allowing MOMP (Fig. 2). The importance of BH3-only proteins in the regulation of apoptosis has been further demonstrated during recent years as several chemical agents have been developed to mimic BH3-only proteins, and have been used successfully to induce apoptosis in cancer models, whether in vitro, in vivo, or even in human clinical trials [72 78]. These intricate regulations of MOMP closely influence the release of cytochrome c, and hence, caspase-9 activation. Importantly, other proteins can promote or inhibit caspase-activation above or below the release of cytochrome c.

12 RNA Regulation in Apoptosis D th li d ( Bcl-2, Mcl-1, Bcl-x L Bcl-w (b) α 3.2 Targeting Caspase Activation The activation machineries of initiator caspases are also the targets of regulation (Fig. 2). DISC can be inhibited by several factors [79, 80], including FLICE-like inhibitory protein (FLIP), a caspase-8 mimic that contains a nonfunctional catalytic domain [81, 82]. Likewise, the apoptosome is also subject to regulation by different pro- and anti-apoptotic

RNA Regulation in Apoptosis 13 Fig. 2 The regulated pathways of apoptotic occurs (c) and releases cytochrome c (cyt c), cell death. As noted in Fig. 1, apoptosis may be engaged either extrinsically (a) or intrinsically (b). The downstream cascades that result from external or internal apoptotic stimuli are complex and are subject to multiple levels of regulation. In particular, protein activators and inhibitors mediate both apoptotic pathways. (a) In the extrinsic pathway, FLIP and DJ-1 proteins can inhibit the activation of caspase-8 (casp-8) and caspase-10 following the binding of death ligands (such as TRAIL) to death receptors; (b) In intrinsic apoptosis, pro-apoptotic proteins Bax and Bak are inhibited by Bcl-2 family members such as Bcl-2, Mcl-1, and Bcl-xL. BH3-only members of the Bcl-2 family can block this inhibitory effect, or can activate Bax/Bak heterodimerization. Once the latter is achieved, mitochondrial HtrA2/Omi, and Smac/DIABLO. Cyt c activates Apaf-1 to form the apoptosome complex, which leads to the activation of caspase-9. Inhibitors of apoptosis proteins (IAPs) such as XIAP and survivin can inhibit caspase activity, but are themselves negatively regulated by HtrA2/Omi and Smac/DIABLO. IAPs can also target effector caspases such as caspase-3 and -7. In this figure, the solid black arrows indicate movement or processing of a factor; faint arrows indicate the activation of a process; and thicker lines represent the inhibition of a process. In addition to the regulation shown here, virtually all of these players in the regulation and execution of apoptosis are further regulated at the transcriptional, post-transcriptional, or post-translational levels. outer membrane permeabilization (MOMP) factors [83], both of which involve particular mechanisms and have undergone extensive investigation. 3.2.1 DISC Inhibition FLIP, which is the best-characterized protein regulator of extrinsic-induced apoptosis, is capable of binding the DED region of adaptors such as FADD and tumor necrosis factor receptor type 1-associated death domain (TRADD) and, in doing so, prevents the recruitment of procaspases-8 and -10 [81, 82]. Another similar inhibitor is the DJ-1 protein, which has been found to bind DAXX as well as FADD (Fig. 2), also consequentially inhibiting the activation of caspase-8 [79, 80]. 3.2.2 Regulation of Apoptosome Activity Several other factors have also been identified as regulating apoptosis, at the level of the apoptosome (Fig. 2). One such protein is prothymosin α, which inhibits the apoptosome through a mechanism that involves binding to Apaf-1, but about which nothing more has been reported [83, 84]. Prothymosin α has also been linked to cell proliferation and cancer growth [85 89]. By comparison, a positive regulator of apoptosome activity that had no previous link to cell death (putative HLA-associated protein-i; PHAPI), has been identified recently. Previously known as pp32, PHAPI is an acidic-rich protein that has been reported to inhibit oncogene-induced tumorigenesis [90 92] and, intriguingly, is also implicated in apoptosis as an activator of the apoptosome [83]. Subsequently, PHAPI, in complex with Hsp70 and a cellular apoptosis susceptibility (CAS) protein, was found to play an important part in the exchange of dadp for datp in apoptosome activation [93]. Kim et al. demonstrated that Hsp70, CAS, and PHAPI are all needed for optimal caspase-9 activation, and that the absence of CAS can produce a significant decrease in apoptotic cell death [93]. 3.3 Caspase Inhibitor of Apoptosis Proteins An entire series of apoptotic regulators exists downstream of caspase-activation,

14 RNA Regulation in Apoptosis which focuses on the inhibition or promotion of caspase activity (Fig. 2). The most well-studied molecules that aim to inhibit caspase activity are known as inhibitors of apoptosis proteins (IAPs). 3.3.1 IAPs Several members of the IAP family exist, including XIAP (X-linked inhibitor of apoptosis protein), ciap1, ciap2, and survivin [94, 95]. While each of these has unique targets and particular properties [96 99], the commonality between each is their ability to inhibit active caspases. In the case of XIAP, this is achieved through a direct interaction with caspases-9, -3, and -7 [98 102]. XIAP interacts at two sites with each of these caspases and, in doing so, blocks their catalytic sites (Fig. 2). The inhibitory mechanisms of other IAPs are less well understood; survivin has been suggested to form a complex with procaspase-9 that sequesters it from recruitment to Apaf-1 [103, 104]. It has also been suggested that IAPs may be responsible for the ubiquitination and degradation of caspases [105 107]. 3.3.2 Inhibitors of IAPs Just as BH3-only proteins exist to inhibit the inhibitors of MOMP, inhibitors of IAP proteins are also present in the cell, the roles of which are to sequester IAPs and allow normal caspase activity. A few such proteins have been described to exist in mammals during the past few years, including HtrA2/Omi and Smac/DIABLO (direct IAP-binding protein with low pi) [108 110]. These proteins were found to be released from the mitochondria during MOMP along with cytochrome c, and to promote apoptosis [13, 111], but in a distinct mechanism from the apoptosome-activating effect of cytochrome c. Both, HtrA2/Omi and Smac/DIABLO have in fact been shown to bind and block the inhibitory capacity of IAPs [13, 108, 112], thus enhancing caspase activity (Fig. 2). 3.4 Post-Translational Regulation of Apoptotic Factors The many interactions between activators and inhibitors of apoptosis described above provide a complex control over the balance between life and death. Further regulation of this balance exists within the realm of post-translational modifications, to which many of these apoptotic factors may be subjected. The best studied of these is phosphorylation, which has been found to regulate a handful of Bcl-2 family proteins by either promoting or inhibiting their activity [113 119]. Caspase-activity is also influenced by phosphorylation [120]. Caspases-2, -3, -8, and -9 have all been documented as the substrates of kinases (this subject has been extensively reviewed by Kurokawa and Kornbluth [121]). Generally, the phosphorylation of caspases results in a decreased activity, such as when caspase-9 is phosphorylated by protein kinase B (PKB) [122]. Importantly, the phosphorylation of caspases allows an additional level of regulation and fine-tuning of the apoptotic response, since phosphatases such as protein phosphatase 2 (PP2A) can be used to promote apoptosis by alleviating inhibitory phosphates from caspases [121, 123, 124]. The ubiquitination of apoptotic factors has also been found to play a role in the regulation of this process. Ubiquitin is a 76-amino acid protein that is ligated onto the lysine residues of a target protein, and polyubiquitination of a protein typically signals for the degradation of this

RNA Regulation in Apoptosis 15 protein by the proteosome [125]. Of pertinence to apoptosis, several anti-apoptotic factors have been identified as ubiquitin ligases [105 107, 126], enzymes that are capable of attaching ubiquitin to targets, consequentially signaling for their degradation. XIAP, ciap1, and ciap2 are some examples of ubiquitin ligases, and are capable of signaling the degradation of caspases [105 107]. Mcl-1, FLIP, and numerous other apoptotic players are targeted by enzymes involved in the ubiquitin pathway [127 131]. The regulation of apoptosis through protein protein interactions and modifications to prevent such interactions is a crucial facet of the tight control over the apoptotic process. One of the easiest ways to influence an imbalance in these various regulatory mechanisms is to modulate the expression of pro- and anti-apoptotic factors. Indeed, the regulation of apoptosis through gene expression is just as important a contributing factor to cell fate, if not more so, than are the interactions between activator and inhibitor proteins. 4 Transcriptional Regulation of Apoptosis Regulating apoptosis by modulating gene expression is a question of regulating mrna. From the moment it is generated until the time it is degraded, mrna is the intermediate that encodes apoptotic factors. If provided with the correct conditions, mrnas will lead to the production of pro- and anti-apoptotic players and, as such, should have a tremendous influence on the progression of this cell process. Intriguingly, only a small number of the studies characterizing the roles of mrna in influencing apoptotic factors have examined specifically what occurs in response to lethal stress, to mimic apoptotic conditions. 4.1 p53 and p73 At the level of transcription to produce mrnas, various studies have defined how transcription factors become active in response to stimuli, such as fatal triggers. While numerous apoptosis-related transcription factors have been identified, easily the most well known is p53 [132]. Although many stress signals have been shown to activate p53 function, the most common mechanism is for signals to activate kinases, such as ATM, ATR (ataxia telangiectasia and Rad3-related), and Chk2, which then phosphorylate p53 in order to reduce its association with the inhibitory Mdm2 [133]. The targets of p53 are diverse and linked to various effects on the cell. For example, p53 can halt the cell cycle by enhancing transcription of the cyclin-dependent kinase inhibitor p21 Waf1/Cip1,orbyrepressing the translation of cdc25c, which promotes mitosis [134 136]. The best-known involvement of p53, however, is through its regulation of apoptosis. Indeed, the ability of p53 to induce cell death is tied to its identification as a tumor suppressor and, given the breadth of targets that it regulates, it is not surprising that p53 is mutated or absent in more than half of all human cancers [137 140]. These targets include Bax, Bad, Bid, Fas, Apaf-1, Noxa, and Puma, the transcription of which are promoted by p53, and survivin and Bcl-2, for which transcription is inhibited [65, 132, 141 149]. p53 also promotes the expression of Mdm2, in order to exercise a tight regulation of its levels. Importantly, a relative to p53 named p73 can also promote the expression of

16 RNA Regulation in Apoptosis p21 and Mdm2 in response to stress [150]. In fact, p73 variants can also regulate apoptotic targets of p53, such as Bax and Noxa [151 153]. Similarly to p53, a complex regulation of p73 exists, and the existence of this alternate key transcriptional regulator of apoptotic factors indicates that cells possess various strategies to genetically respond to stress signals. 4.2 E2F Family The E2F family of transcription factors is also strongly linked to stress response and the induction of apoptosis. E2F1, a transcriptional promoter, is the member of this family that has been best characterized for a role in influencing apoptotic targets, such as caspase-8 and Bid [154]. Growing evidence has linked the E2F1-mediated control of expression to include a number of other apoptotic targets that are also under the control of p53, such as Apaf-1, caspases, BH3-only proteins, survivin and Smac/DIABLO [155 164]. Importantly, E2F1 also promotes the transcription of p73, and can thus promote an even broader panel of apoptotic factors [165, 166]. 4.3 FOXO Family The forkhead transcription factor family (FKHR or FOXO) contains over 90 members that influence transcription in response to upstream Akt signaling [167, 168]. FOXO3a (also known as FKHR-L1) is a member of this family that has been linked to the regulation of apoptosis. One target of FOXO3a that has been identified is the BH3-only protein Bim [149]. The upregulation of Bim by FOXO3a has been shown to occur following a decrease in cytokine levels in T lymphocytes, possibly allowing the death of these cells when there is no longer an immune signal prompting their levels [169]. FOXO3a has also been shown to repress transcription of the caspase-8 inhibitor FLIP in response to a decrease in Akt signaling [170]. Akt is capable of phosphorylating FOXO3a, which leads to its cytoplasmic retention; hence, a decrease in Akt activation allows the dephosphorylated FOXO3a to translocate to the nucleus where it can exercise its transcriptional roles. The ability of FOXO3a to promote transcription of the death ligands TRAIL and Fas ligand in different cell systems [171 173], as well as of the BH3-only protein Puma, further emphasizes the involvement of this transcription factor in the regulation of apoptosis. 4.4 Additional Transcriptional Regulators of Apoptosis One of the most widely studied stress response signaling pathways is that by which NF-κB is activated. Upon binding of TNF to the TNF receptor (TNFR), the proteins TRADD, RIP-1, and FADD associate with the receptor, and are capable of activating NF-κB. This survival transcription factor can then promote the expression of the anti-apoptotic factors ciap1 and ciap2 [174]. Expression of FLIP can also be regulated by NF-κB [175]. Other less-extensively studied transcription factors have also been described to influence the expression of apoptotic players. For example, in neurons the Brn-3a POU domain transcription factor can upregulate Bcl-2 in order to protect cells from neuronal apoptosis [176]. Bcl-6 has been shown to repress Bcl-x L transcription following its own activation by AFX (ALL1 fused gene from chromosome X), which

RNA Regulation in Apoptosis 17 is another member of the FOXO family of transcription factors [177]. It is likely that many other factors also influence the expression of genes linked to apoptotic stress response. Furthermore, while the above-described networks influence the production of pro- and anti-apoptotic factors, investigations must still be conducted to determine the mechanisms that permit an altered transcription during the actual process of apoptotic cell death. Importantly, in order to appreciate gene expression as a means of regulating cell death, transcription cannot be a sole focus of these investigations. The journey of a transcript (mrna) following its production, up until translation is complete, includes several post-transcriptional regulatory steps at each of which expression modulation is possible. 5 Post-Transcriptional Regulation of Apoptosis The production of factors involved in apoptosis is not only regulated by transcription. In fact, during the past few years many reports have described how post-transcriptional events can also influence the levels of pro- and anti-apoptotic factors [178 180]. The splicing, translation, and turnover of numerous mrna factors have been investigated, and it has been revealed that these mechanisms may play important roles in regulating apoptotic cell death. Whilst, in general, these studies do not focus on post-transcriptional regulatory events during apoptosis, the insight that they provide on potential controls over cell death is still valuable. An mrna molecule is subject to several means of regulation before yielding the desired protein product (Fig. 3). Splicing of the message, potentially producing splice variants with differing function, can occur prior to nuclear export. The trafficking of mrna to the cytoplasm, and then to an appropriate destination within the cytosol, will also influence gene expression. The recruitment of multiple ribosomes to a message results in the formation of multi-ribosome complexes known as polysomes, and the resulting translation of a message is subject to numerous cellular influences. Finally, the protein output from one mrna copy depends on the stability of the transcript of interest, as mrna turnover can also be enhanced or perturbed through the actions of a number of factors. Collectively, these mechanisms play a crucial part in regulating the expression and the resulting actions of factors involved in the apoptotic stress response. 5.1 Splicing The splicing of mrnas and, specifically, alternative splicing provides eukaryotes with an added level of regulation for gene expression and function. The processing of RNA transcripts following transcription involves the removal of introns in the pre-mrna by the spliceosome, which is a complex of several small nuclear ribonucleoproteins (snrnps) [180]. The spliceosome recognizes splice sites with the assistance of other RNA-binding proteins such as heteronuclear ribonucleoproteins (hnrnps) and serine-arginine-rich proteins (SR proteins) [181, 182] (Fig. 3). Both SR proteins and hnrnps can manipulate the location of splicing to yield different splice variants. While this would generally suggest that alternative splicing can result in different mrnas encoding

18 RNA Regulation in Apoptosis Pre-mRNA hnrnps SR proteins Splicing NUCLEUS CYTOPLASM mrna decay Nuclear export IRES-mediated translation mrna localization Translation Fig. 3 Post-transcriptional regulation of gene expression. Gene expression may be regulated at several levels following transcription and addition of the 5 methylguanylate cap and poly(a) tail. Within the nucleus, pre-mrna undergoes splicing to remove introns, by use of hnrnps and SR proteins. Mature mrna is then exported into the cytoplasm. Once in the cytoplasmic compartment of the cell, transcripts can be localized to specific sites to enable localized translation (polarization), or proteins with varying function, alternative splicing can also produce a premature stop codon, which signals the decay of the target mrna through a process known as nonsense-mediated decay (NMD) [180]. Hence, alternative splicing can influence not only the biological effects of a protein but also its global expression levels. repression of translation. Likewise, localization of an mrna can promote or deter the decay of a message. The translation of an mrna to produce protein product is also subject to regulation, and can be mediated through cap-dependent translation, or cap-independent translation, such as IRES-mediated translation. Finally, gene expression can also be regulated through the turnover of messages, as mrna stability and decay can be influenced by a number of factors. 5.1.1 Splicing to Regulate Cytochrome c Release Numerous factors involved in apoptosis have been identified as targets of alternative splicing [180], including death ligands, death receptors, caspases, members of the Bcl-2 family, and downstream caspase inhibitors and activators (much of this regulation has been summarized

RNA Regulation in Apoptosis 19 by Schwerk and Schulze-Osthoff [180]). Included in this list are the BH3-only proteins Bid and Bim. Indeed, it has been found that three splice variants of Bid are produced, each of which contains a different region of this pro-apoptotic factor [183]. In each of these three variants, the deletion or addition of domains elicits different effects. For example, Bid S, which is a shorter version of Bid lacking its BH3 domain, inhibits the pro-apoptotic activity of tbid, while the other two extra long and extra short variants (Bid EL and Bid ES, respectively) promote apoptosis, although Bid ES also appears to counter the effects of normal tbid [183]. While Bim has had numerous splice variants identified, three principal variants exist in the cells, as Bim S, Bim L, and Bim EL [184 186]. Under normal conditions, Bim EL is the most highly expressed, which correlates with the understanding that this isoform does not possess as strong a pro-apoptotic potential as do Bim S and Bim L [187]. During apoptosis, Bim EL is actually degraded, while Bim S and Bim L are produced in larger quantities, thus realigning the purpose of Bim expression in these cells. These observations clearly suggest that alternative splicing could be active, and modulated, during the apoptotic response itself. Perhaps the best-studied Bcl-2 family member with regards to splicing regulation is Bcl-x L. The Bcl-x gene can produce two major variants, Bcl-x L and Bcl-x S [188]. As described earlier, Bcl-x L exercises an anti-apoptotic role as it can sequester Bax and Bak. By comparison, the Bcl-x S isoform lacks BH1 and BH2 domains and, as a consequence, can inhibit Bcl-2 and Bcl-x L. The regulation controlling which Bcl-x isoform is produced depends on different cellular signals and cascades. For example, Fas activation can lead to a preferential production of Bcl-x S [189]. Differing signals will determine which SR proteins or hnrnps are recruited to mediate the splicing of Bcl-x mrna, and those selected for this event will govern which variant is generated. Recently, a mechanism was characterized to explain how DNA damage signals such a variation. Under normal conditions, signaling through PKC allows the SR protein SB1 to repress the 5 splice site of Bcl-x S [190, 191], possibly by recruiting a repressor that has not yet been identified. In response to DNA damage, p53 signaling causes the removal of this repressor, permitting production of the apoptotic Bcl-x S isoform, and advancing cell death [191]. This is one of the first and only cases in which a mechanistic understanding of alternative splicing has been described in response to stress (Fig. 4). Other regulators of MOMP have also been shown to be targets of regulation via alternative splicing. While two variants of Noxa were identified, the half-lives of their protein products supported the fact that they do not likely contribute to an effect in regulating apoptosis [192]. The pro-apoptotic Mcl-1 has had one alternative splice variant identified that contains only the third Bcl-2 homology domain (BH3) of Mcl-1 [193]. Not surprisingly, this variant promotes apoptosis similarly to BH3-only proteins [194]. 5.1.2 Splicing-Mediated Regulation of Caspases Caspases and downstream regulators of caspases are also subject to the regulation of gene expression through alternative splicing. Caspases-3 and -9 can be spliced to generate dominant negative isoforms that lack the catalytic sites of these enzymes [195 199], subsequently acting as anti-apoptotic factors. Regulation of the production of these splice variants has

20 RNA Regulation in Apoptosis Pre-mRNA hnrnps SR proteins Modified hnrnps SR proteins Splicing Alternative splicing Protein product Altered Protein product Promoter of apoptosis Inhibitor of apoptosis ex: Smac, ex: Bcl-x L, Bim EL caspase -9s Normal conditions Altered promotor of apoptosis Promotor of apoptosis ex: Smac3, ex: Bcl-x s, Bim s /Bim L caspase -9 Apoptotic conditions Fig. 4 Alternative splicing as a regulator of apoptosis. Several modulators of apoptosis are regulated at the post-transcriptional level of splicing. For certain of these factors, evidence suggests that alternative splicing produces protein variants that exercise altered effects on been investigated, and in the case of caspase-9, has implicated hnrnp L as well as SR proteins such as SRp30a and SRSF1 [199 201]. Other caspases, such as -2 and -10, are also susceptible to alternative splicing, and as with what is seen for many of the proteins described above, opposite effects are mediated by these altered isoforms [180, 202, 203]. Consistently, certain alternate splicing isoforms of the caspase inhibitor survivin act in pro-apoptotic manners. In fact, four alternative splicing isoforms of survivin exist and, depending on the missing structural components, these can either inhibit the anti-apoptotic effect of full-length the progression of apoptosis. Under apoptotic conditions, alternate or modified splicing factors can produce variants that have opposing functions, or enhanced functions. References for the examples given may be found in the text. survivin or they can still succeed to inhibit cell death [204 208]. Importantly, mutations in p53, which regulates survivin expression, correlate with an increase in the production of anti-apoptotic survivin isoforms, without affecting the pro-apoptotic variants [209]. In the case of FLIP, the splice variant actually possesses a more potent effect than does the full-length protein [210], rather than producing an isoform with an opposing effect. Comparably, the Smac3 splice variant of Smac/DIABLO contributes to apoptosis by interfering with XIAP activity, similar to Smac/DIABLO, though possibly via a different mechanism [211].

RNA Regulation in Apoptosis 21 The importance of splicing as a mechanism of post-transcriptional regulation that affects the apoptotic response has been emphasized by experiments in which an oligonucleotide successfully blocked the 5 splice site of Bcl-x L, resulting in an upregulation of the pro-apoptotic Bcl-x S [212, 213]. Recently, a similar approach has been used to induce death by enhancing expression of the pro-apoptotic isoform of Mcl-1 [194]. Clearly, this post-transcriptional process has important implications in regulating the factors that modulate apoptotic cell death. While some studies have described how stress signals can trigger alternative splicing to influence apoptosis [190, 191], the cellular mechanisms that enable differential regulation through this post-transcriptional event remain largely unexplored [180]. 5.1.3 Nuclear Export of mrna Following transcription and the splicing of pre-mrnas, a target message is covered with many RNA-binding proteins, several of which allow for the nuclear export of a message (Fig. 3). A handful of mrna-export pathways exist [214 216], though each is under the control of regulatory mechanisms. Controlling the export of messages is another means of influencing gene expression, as mrna translation occurs in the cytoplasmic compartment of the cell. To date however, there is no evidence supporting the involvement of the nuclear export of mrnas encoding apoptotic players in the regulation of apoptosis. The conversion of RNA transcripts to polypeptides via translation is quite the opposite case, and many modulators of apoptosis may have their translation promoted or perturbed,[178, 179]. This demonstrates that post-transcriptional mechanisms are conceivable targets for influencing apoptosis; hence, the possibility exists that in the future mrna export will prove likewise to be targeted. 5.2 Translation Considered the ultimate purpose of most mrnas, translation converts genetic material into functional proteins that can execute countless cellular functions (Fig. 3). Whilst many of these functions in apoptosis have been described above, it is particularly important to appreciate how translation permits the production of these apoptotic factors, and how this imperative process is regulated. Similar to the case of alternative splicing, the expression of numerous apoptotic factors has been shown to undergo regulation at the level of translation, although the investigations made to date on this process during apoptosis are few in number [217 220]. 5.2.1 cis-elements and trans-acting Factors that Regulate Translation In order to specifically alter the translation of one message or one group of messages, regions in the 5 or 3 untranslated region (UTR) of the mrna frequently encode cis elements to regulate this effect. This is certainly the case for the translation of Bcl-2, whereby the 5 UTR of Bcl-2 mrna has been shown to regulate its translation [221]; more recently, it was also shown that this involves the presence of a G-quadruplex element in the 5 UTR of this message [222]. These authors noted that this element could reduce the translation of its message, potentially because of a need for helicases to enable an effective translation. It was not clear how the presence of this element factored into the regulation of Bcl-2 expression, however. It has also been shown that, under certain conditions, Bcl-x L and