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Responding to stress-induced damage, eukaryotic cells activate mechanisms to save energy and repair this damage by repressing the translation of house-keeping gene transcripts which accumulate in SGs, and reciprocally up-regulating the translation of proteins required to repair the cellular insults (Anderson & Kedersha, 2008). To date, the mechanism by which stress granules assemble/disassemble is not yet fully understood, and more than 100 genes are involved in this complex process with over 80 proteins that localise to SGs (Buchan & Parker, 2009; Ohn et al., 2008). Many factors regulate and promote SG assembly such as non-translating mRNAs, post-translational modifications, protein-protein interactions, and the microtubule network (Buchan & Parker, 2009).

The first stage of SG formation is always associated with a shut-off of protein synthesis that can be caused by different pathways (Kedersha et al., 1999; Mazroui et al., 2006). Studies have demonstrated that one of the major mechanisms for triggering SG formation is the phosphorylation of the alpha subunit of eIF2 on serine residue 51 (Holcik & Sonenberg, 2005; Kedersha et al., 1999). This phosphorylation is carried out by one of four serine/threonine kinases activated in response to different types of stress (Holcik & Sonenberg, 2005)(Figure 1.1). The double-stranded RNA-dependent protein kinase (PKR) is commonly activated by RNA viruses as a part of the interferon antiviral response of cell (Maggi et al., 2000), but PKR also senses heat shock, oxidative stress and UV irradiation (Williams et al., 2001). General control non-de-repressible-2 kinase (GCN2) senses amino acid deprivation and the decrease of other types of nutrients, as well as UV irradiation (Berlanga et al., 1999). PKR-like endoplasmic reticulum kinase (PERK) responds to the ER stress caused by the accumulation of unfolded or misfolded proteins in the ER (Harding et

senses oxidative stress and heat shock (Han et al., 2001; Lu et al., 2001; McEwen et al., 2005). Activation of all of these kinases leads to phosphorylation of eIF2, thereby increasing the affinity (150-fold) of eIF2 for eIF2B, the eIF2 guanine nucleotide exchange factor, blocking the guanosine diphosphate/guanosine triphosphate GDP/GTP exchange and formation of the eIF2-GTP-tRNAMet ternary complex (TC). This prevents assembly of the 48S pre-initiation complex and results in the repression of translation initiation (Holcik & Sonenberg, 2005; Srivastava et al., 1998).

Figure 1.1 Proposed model of inducting stress granules (SG) assembly by different conditions and stimuli. The most characterised induction pathway leading to SGs assembly is the

phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by the kinases PKR, PERK, and HRI and GCN2. This blocks the formation of eIF2-GTP-Met-tRNAiMet ternary complex (TC) and in turn inhibits translation initiation to trigger the assembly of SGs. In addition SGs formation can be induced by the alteration of expression level or function of translation factors such as eIF4A, eIF4B, eIF4H, and poly (A)-binding protein (PABP).

The formation of SGs can also be induced independently of the eIF2 phosphorylation pathway via cleavage or alteration, or modification of expression level and activity, of proteins involved in translation initiation such as eIF4A, eIF4G (Anderson & Kedersha, 2009a; Bordeleau et al., 2006; Mazroui et al., 2006), eIF4B, eIF4H, as well as PABP (Mazroui et al., 2006). The activation or overexpression of key SG–nucleating proteins such as G3BP1, TIA1, TIAR, TDP-43, Caprin-1, CIRP and CBEP1 also triggers the assembly of SGs without any additional stimulus (De Leeuw et al., 2007; Gilks et al., 2004; Kedersha et al., 2013; Tourriere et al., 2003; Wilczynska et al., 2005). However, in most cases, it is the phosphorylation of eIF2 that leads to a rapid dissociation of polysomes even if other routes exist (Mazroui et al., 2006).

Therefore, the first step of SG formation can occur via both eIF2 phosphorylation- dependent and independent pathways that affect translation initiation and result in polysome disassembly whereupon the cell begins to develop foci to store the stalled translation initiation complexes (48S mRNPs) (Kedersha et al., 1999). Locally, RNA-binding proteins also localise to these small foci by their affinity for the suddenly high concentration of free mRNA in the cytoplasm (primary aggregation) (Bounedjah et al., 2014; Kedersha & Anderson, 2009). Several factors will mediate the subsequent stages of SG assembly mediated by proteins containing self-interaction domains that mediate protein-protein or protein-RNA interactions (Gilks et al., 2004; Kedersha & Anderson, 2002; Tourriere et al., 2003). Such domains are found in several RNA-binding proteins. For example, TIA-1 and TIAR contain glutamine and asparagine-rich prion-like domains mediating SG formation via their self-aggregating ability (Gilks et al., 2004; Kedersha & Anderson, 2002). G3BP1 also harbours the self-interaction (polyglutamine and asparagine rich) domains, playing a critical role in SGs assembly (Tourriere et al., 2003), and knockdown of these proteins leads to a reduced assembly of SGs (Ghisolfi et al., 2012). PABP, which associates with most

transcripts, also mediates the aggregation of SGs via its C-terminal domain (Kedersha & Anderson, 2009). Other RBPs that promote SGs assembly include FMRP/FXRI (Mazroui et

al., 2002), Staufen (Martel et al., 2010), SMN (Hua & Zhou, 2004), TTP (Stoecklin et al.,

2004), BRF1 (Kedersha et al., 2005), and CPEB (Wilczynska et al., 2005). Proteins lacking RNA-binding features can also contribute to this process via protein-protein interaction with certain mRNA binding proteins in SGs, so-called piggyback proteins (Kedersha & Anderson, 2009). For example, SRC3, FAST, PMRI are localized to SGs via interaction with TIA-1 (Yang et al., 2006b; Yu et al., 2007). In addition, TRAF2 and plakophillin 3 that bind to eIF4G and G3BP1, respectively, are recruited to SGs in a piggyback manner (Hofmann et al., 2006; Kim et al., 2005). The recruitment of specific SGs proteins components during this stage is largely associated with an excess of free mRNA (non-polysomal mRNA) and the nucleic acid/proteins imbalance may also regulate the SGs assembly (Bounedjah et al., 2014).

Protein modifications are also involved in the assembly of SGs by enhancing the recruitment of several proteins to SGs (Ohn & Anderson, 2010). Phosphorylation and dephosphorylation of several proteins play a critical role in SGs assembly, for example the phosphorylation of eIF2 plays a key role in initiation of SG formation (Kedersha et al., 1999). The overexpression and phosphorylation of G3BP also regulate SG assembly/disassembly, while the overexpression of G3BP1 induces SGs assembly; the phosphorylation of this protein at serine residue 149 inhibits SGs formation (Tourriere et al., 2003). Phosphorylation of other proteins has also been reported to regulate SGs assembly/disassembly such as phosphorylation of growth factor receptor-bound protein 7 (Grb7) by focal adhesion kinase (FAK) in heat shock–stressed cells (Tsai et al., 2008). Phosphorylation of tristetraprolin (TTP) regulates the interaction between SGs and PBs (Stoecklin et al., 2004), and phosphorylation of eIF4E-transporter (4E-T) enhances stress-

hypusination of eIF5A via a polyamine biosynthetic pathway inhibits SGs assembly (Li et al., 2010). Moreover, studies have reported that the methylation of the RGC domain of FMRP and cold inducible RNA-binding protein (CIRP) are required for SGs assembly (De Leeuw et

al., 2007; Dolzhanskaya et al., 2006). Furthermore, Ohn et al., (2008) observed that the O-

GlcNAc modification of ribosomal proteins correlates with SGs formation (Ohn et al., 2008). Finally, the cytoskeleton and associated motor proteins also participate in assembly/disassembly of SGs by transporting stalled mRNPs to sites of RNA granule assembly (Bartoli et al., 2011; Loschi et al., 2009). The depolymerisation of microtubules using nocodazole or vinblastine prevented the assembly of SGs in a mammalian CV-1 cell line that was treated with arsenite, while microtubule-stabilizing drugs such as paclitaxel promote SG formation (Ivanov et al., 2003). It was further proposed that the disruption of microtubules affects the number and size of SGs rather than inhibiting their formation (Fujimura et al., 2008; Kolobova et al., 2009; Loschi et al., 2009). In contrast, Kwon et al., (2007) reported that the stabilization of microtubules was not associated with SGs assembly (Kwon et al., 2007). In addition to microtubules, microtubule motor proteins such as dynein and kinesin localize to SGs and also contribute in SGs assembly/disassembly. Studies have reported that dynein facilitates SG assembly and the absence of this protein from cells treated with arsenite has a negative impact on the size and number of SGs (Kwon et al., 2007; Loschi

et al., 2009), while kinesin plays a role in disassembly of SGs after stress recovery (Loschi et al., 2009). According to these findings, the role of microtubules and associated motor

proteins is to facilitate coalescence of small aggregates to form large aggregates (Kolobova et

al., 2009; Tsai et al., 2009). Furthermore, pre-existing PBs may be required for the assembly

of SGs (Buchan et al., 2008). Depending on the SGs component, the mRNAs that localise to SGs can either exit SGs and re-enter translation, detach from the eIF3/40S complex and

localise to PBs for degradation via their binding to specific proteins such as TTP, or be stored in SGs for a prolonged time (Kedersha & Anderson, 2009).

The SGs disassembly process begins when the cells recover from the stress naturally or artificially (using drugs stabilising polysomes) and translation returns to normal (Anderson & Kedersha, 2008; Nadezhdina et al., 2010). Other factors such as turnover of their protein component, chaperone proteins, and autophagy are possibly involved in SG disassembly (Buchan, 2014).