Ajuste polin´ omico uniforme

The compendium of genes that have been associated with ALS have shown a high enrichment for RNA-binding proteins, indicating that changes to RNA processing in motor neurons is a major factor to the disease progression (Andersen and Al-Chalabi, 2011; Chen et al., 2013; Therrien et al., 2016). Further, many of the ALS-associated RNA-binding proteins are responsible for the formation, assembly, and disassembly of stress granules—transient ribonucleoprotein (RNP) granules that form during periods of cellular stress and contain translationally silent mRNA (Anderson and Kedersha, 2008; Aulas and Vande Velde, 2015; Buchan, 2014). ALS-linked RNA-binding proteins associated with stress granules include: TDP- 43, FUS, ATXN2, hnRNPA1, hnRNPA2B1, TAF15, EWSR1, RBM45, C9ORF72 and TIA1 (Andersson et al., 2008; Colombrita et al., 2009; Kim et al., 2013; Li et al., 2015; Mackenzie et al., 2017; Maharjan et al., 2017; Nonhoff et al., 2007). This list contains many of the ALS- related RNA-binding proteins that are involved in miRNA biogenesis mentioned in the previous section. Based on this information, it is widely believed that stress granules may be the seed to pathological aggregate formation of RNA-binding proteins ultimately leading to changes in miRNA processing in ALS.

When cells face adverse conditions within their environment (e.g. oxidative, osmotic, or proteasomal stress), cells start to produce structures called stress granules (Gilks et al., 2004; Kedersha et al., 2000; Kedersha et al., 1999). The purpose of these granules is to act as a triage for mRNA, where mRNA can be either stored within the stress granule, transferred to processing bodies (p-bodies) for degradation, or go back into polysomes to be translated (Anderson and Kedersha, 2008; Aulas and Vande Velde, 2015).

Phosphorylation of eIF2α appears to be an early trigger that initiates stress granule assembly; however, there are cases of stress granule assembly that are independent of eIF2α phosphorylation (Kedersha et al., 2002; Kim et al., 2007; McEwen et al., 2005; Shenton et al., 2006). The phosphorylation of eIF2α results in reduced formation and activity of translational machinery (Shenton et al., 2006). This is followed by an accumulation of RNA-binding proteins and RNA molecules into granules that lack a membrane structure (Anderson and Kedersha, 2008; Baradaran-Heravi et al., 2020; Gilks et al., 2004). TIA-1 and G3BP1 are two RNA-binding proteins that early on were thought to be critical for proper stress granule formation. However, as we learn more about stress and stress granules, we are starting to understand how truly dynamic and diverse these structures are. Thus, while some proteins are critical for the formation of stress granules for certain stresses, they might not be critical for others (Aulas et al., 2015; Gilks et al., 2004; Kedersha et al., 2016; Markmiller et al., 2018). For example, G3BP1 is critical for the formation of stress granules during oxidative stress, but it is not needed for stress granule

assembly during periods of osmotic stress (Kedersha et al., 2016; Solomon et al., 2007). Further, proteomics of stress granules has shown different stresses and different cell types result in different protein profiles (Markmiller et al., 2018). This suggests that stress granule formation and proteomic profile are highly dynamic and context dependent.

After cellular stress disappears, stress granules start disassembling as the cell goes back to its homeostatic state allowing translation of previously silent mRNA to begin again (Molliex et al., 2015). Several mechanisms have been implicated in stress granule disassembly, including: reduced stress results in the formation and increased activity of translational machinery

destabilizing the granule structure; inhibition of RNA helicases that promote assembly and maintenance of stress granules; heat shock proteins disassemble RNP complexes; and, ubiquitination of proteins via VCP allowing for stress granules to be targeted by autophagy pathways (Buchan et al., 2013; Cherkasov et al., 2013; Jain et al., 2016; Kroschwald et al., 2015; Meyer and Weihl, 2014; Protter and Parker, 2016; Wheeler et al., 2016). All these pathways likely work together to allow for efficient disassembly of stress granules. It is also clear that stress granule disassembly is an active process that requires ATP hydrolysis (Jain et al., 2016; Meyer and Weihl, 2014; Protter and Parker, 2016).

In all of this, LCD-containing proteins appear to be critical for the formation of stress granules by allowing them to phase separate and form liquid droplets, which produces dynamic interactions with p-bodies, other stress granules and the cytosol of the cell (Aulas et al., 2015; Gilks et al., 2004; Kato et al., 2012; Mackenzie et al., 2017; Molliex et al., 2015; Murray et al., 2017; Patel et al., 2015). Structurally, this is because the LCD participates in β-pleated sheet formation and forms amyloid-like interactions similar to what is seen in prion disease (Molliex et al., 2015; Murakami et al., 2015; Patel et al., 2015). These interactions are critical for the

formation of RNA granules which lack a membrane to contain its components (Baradaran-

Heravi et al., 2020; Taylor et al., 2016). Therefore, phase transition into reversible liquid droplets is a critical mechanism for the formation of stress granules. While the LCD’s are necessary for quick assembly of stress granules, it can also be a detriment to the cells if proteins are left to

accumulate, risking the formation of hydrogels (Kato et al., 2012; Li et al., 2013; Molliex et al., 2015).

1.5.2 Hydrogels

LCD-containing proteins in both cell-free and cell in vitro models have been shown to form hydrogels (Kato et al., 2012; Molliex et al., 2015; Murakami et al., 2015; Murray et al., 2017). Hydrogels are defined by the aggregation of proteins leading to phase transition into liquid droplets and eventually irreversible gelatin structures (Murakami et al., 2015). The formation of these hydrogel structures is primarily driven by the LCD. (Kato et al., 2012; Molliex et al., 2015; Murakami et al., 2015; Murray et al., 2017).

As discussed, several ALS-related proteins are LCD-containing proteins, including: TDP- 43, FUS, hnRNPA1, hnRNPA2B1, TAF15, EWSR1, RBM45 and TIA-1 (Baradaran-Heravi et al., 2020). ALS-linked mutations within the LCD of TDP-43, FUS and hnRNPA1 have been shown to increase the rate at which these proteins phase separate and form hydrogel-like

structures (Conicella et al., 2016; Kim et al., 2013; Molliex et al., 2015; Murakami et al., 2015). Since LCD-containing ALS-related proteins tend to form insoluble aggregates, several

researchers have focused on therapeutics that prevent the accumulation of these proteins. However, little research has been done on the dysregulation of miRNAs in ALS, and potential changes to their regulation on the expression of ALS-related genes that could be a major contributor to the accumulation of these proteins.