Despite robust translation of GA
Despite robust translation of GA, only small amounts of GR and GP were translated in cultured Caspase-8 Colorimetric Assay Kit and chick embryo spinal cord neural cells from the 75 copies of G4C2 in monocistronic constructs. Interestingly, GA is also the most common dipeptide found in autopsy tissue in FALS patients with C9ORF72 HRE; however, the difference in the frequency of GA deposits compared to the other DPRs in autopsy tissue is not as extreme as in our results. Since the length of the repeat in other diseases is important in the efficiency of DPR translation (Bañez-Coronel et al., 2015; Kearse et al., 2016), small amounts of GR and GP may have been seen in our study because the repeat number of 75 is far less that the 100 s and possibly 1000s in the CNS of patients with C9ORF72 HREs. Furthermore, the presence of only 99 nucleotides 3′ to the expanded repeats may be too few to allow efficient translation of GR and GP since RNA secondary structure downstream of a non-AUG initiation codon can influence the translation efficiency (Kearse and Wilusz, 2017). It may also be the cell cultures and chick embryo neural cells lack (human neural) cell-specific IRES-transacting factors (ITAFs) that are critical for efficient translation of GR and GP. Of note, transfection of GP-nLuc and GR-nLuc constructs that had a stop codon just upstream of the HRE in the GA reading frame displayed enhanced nLuc expression. These results suggest that ribosomes that meet a stop codon after binding to the CUG initiation codon may reinitiate in the HRE. Therefore, factors that decrease CUG translation initiation or stall ribosomes, such as a paucity of a particular ITAF, can lead to inefficient translation of GA, with subsequent enhancement of downstream translation initiation of GP or GR within the HRE; it is known that translation of non-AUG uORFs can have varied effects on translation of more downstream ORFs (Young and Wek, 2016). Importantly, our results show that GA translation partly depends on eIF2A since the ratio of the first (fLuc) cistron to the second cistron (GA-nLuc) of the C9 bicistronic plasmid or of in vitro-derived transcripts from this bicistronic construct was significantly lowered in cells that had eIF2A knocked out. Knockdown of eIF2A in the chick embryo also decreased translation of GA-nLuc in chick embryo neural cells; however, the knockout of eIF2A did not completely abolish translation of GA, suggesting that GA translation initiation depends on other factors in addition to eIF2A. eIF2A is known to be used under cellular stress conditions as demonstrated in the case of translation initiation of hepatitis C virus RNA (Kim et al., 2011) and of cellular uORFs (Kwon et al., 2017; Sendoel et al., 2017; Starck et al., 2012, Starck et al., 2016). Interestingly, eIF2A has recently been implicated in the translational regulation of genes associated with cancer (Sendoel et al., 2017), presumably because of stresses in cancer cells. Cells respond to stressors, such as ER stress from misfolded proteins, oxidative stress, amino acid deprivation, and viral infections, with an ISR. The ISR triggers various kinases to phosphorylate eIF2α, leading to a decrease in cap-dependent translation. When conventional cap-dependent translation is limited by phosphorylation of eIF2α, eIF2A is able to play an increased role in translation of certain mRNAs, especially ones that have an uORF. Of note, cellular uORFs that depend on eIF2A for translation can influence the efficiency of downstream translation of the gene\'s main open reading frame, suggesting that the DPRs translated from the C9ORF72 HRE may also influence expression of the main C9ORF72 product (Kearse and Wilusz, 2017). The identification in the present study that eIF2A is important in GA translation and that ER stressors and inducers of the ISR lead to attenuation of conventional cap-dependent translation with no change in RAN translation of the G4C2 expanded repeats raised questions as to whether the ISR plays a role in translation of GA as well as the other DPRs in C9ORF72-induced disease. In fact, a possible role for ER stress in the pathogenesis of ALS has been previously proposed (Dafinca et al., 2016; Lee et al., 2016; Shahheydari et al., 2017; Taylor et al., 2016; Wang et al., 2011, Wang et al., 2014; Zhang et al., 2014), and recent studies have suggested a relationship between ISR and GA translation (Cheng et al., 2018; Green et al., 2017).