Human eIF2A has a minimal role in translation initiation and in uORFmediated translational control

We apply ribosome profiling here to assess the role of eIF2A in translation initiation. For this we test the change in translation efficiency between HeLa control and eIF2A-KO cells, however we do not find any transcript to depend on eIF2A. Since eIF2A is thought to take over the function of eIF2 when eIF2 is inhibited, we also test conditions where the integrated stress response is activated, thereby leading to eIF2 inactivation. In none of our assays, however, could we detect a role of eIF2A in translation initiation. Overall design: To find, if there are any transcripts dependent on eIF2A, we generated HeLa eIF2A knock-out cell line and performed ribosome profiling on it and an isogenic control HeLa cell line. This experiment was performed in triplicate. To test if inhibition of eIF2 redirects translation initiation to eIF2A we again performed ribosome profiling on the above-mentioned cell lines in combination with tunicamycin (16 hours) treatment. This experiment was performed in duplicates.