mA and eIF2a-p Team Up to Tackle ATF4 Translation during Stress

Studies of translational control have benefited enormously from development of a suite of tools that allow in vivo views of ribosome positions, providing information that was difficult to extract from preexisting genetic and in vitro approaches. These new methods include ribosome profiling, which measures genome-wide translation via the sequencing of mRNA fragments bound by 80S translating ribosomes (Ingolia et al., 2012), and QTIseq (quantitative translation initiation sequencing), which precisely and quantitatively maps sites of translation initiation (Gao et al., 2015). Broad application of these methods has been powerful, often confirming defined translation mechanisms, but sometimes suggesting a need to revisit long-held assumptions about general andspecificmechanismsof translational regulation. Zhou and colleagues integrate use of these and other global approaches to reveal an added layer to the regulation responsible for stress-responsive translation of the mRNA for transcription factor ATF4 (Zhou et al., 2018). The 50 leader of ATF4 is known to contain two upstream open reading frames (uORFs). In the absence of stress, it is thought that the ATF4 ORF is poorly translated due to the translation of uORF2, which is overlapping and out of frame with the ATF4 ORF start codon (Vattem and Wek, 2004; Lu et al., 2004). Under an array of stresses, however— including starvation—the translation initiation factor eIF2a is phosphorylated (p), and the ATF4 ORF is resultantly translated. A general model for uORF-based regulation, based on elegant studies of the Gcn4 transcription factor-encoding gene in budding yeast, proposes that eIF2a-p reduces the amount of ternary complex (TC) available tomediate translation initiation. This causes a general decrease in translation initiation. However, in the case of at least a subset of genes with ORF-repressive uORFs that are translated under non-stressed conditions, the ultimate effect is increased translation of the main ORF. This is due to ‘‘leaky scanning’’ by ribosomes that lack TCs, and therefore bypass uORFs (Hinnebusch, 2014). Consistent with this model, mutation of uORF2 enhances ATF4 translation under non-stressed conditions (Vattem and Wek, 2004; Lu et al., 2004). Zhou and colleagues explored this mechanism in detail, comparing ribosome profiling and QTI-seq data for control and starved cells. They observed that, while 80S translating ribosome occupancy within the ATF4 ORF indeed increased upon starvation, there was no associated decrease in ribosome occupancy of uORF2. This result was confirmed by reporter assay and led Zhou and colleagues to investigate the possibility that ATF4 regulation involved additional mechanisms beyond the prevailing model. Mass spectrometry of proteins associated with ATF4 mRNA revealed enrichment of the RNA demethylase ALKBH5 in starved cells compared to control conditions. ALKBH5 can reverse N-methylation of adenosine (mA); therefore, the authors performed global mapping of the mA mRNA modification sites to investigate a possible link between this modification and ATF4 translation. They noted that the ATF4 mRNA showed several regions of mA modification, including one within uORF2. Intriguingly, this site in