ABSTRACT
eIF2A was the first eukaryotic initiator tRNA carrier discovered but its exact function has remained enigmatic. Uncharacteristic of a translation factor, eIF2A resides in the nucleus during normal growth conditions and is shuttled to the cytoplasm during cell stress. eIF2A knockout mice have shortened lifespans and develop metabolic syndromes but show normal protein synthesis. Attempts to study eIF2A mechanistically have been limited by the inability to achieve high yield of soluble recombinant protein. Here, we developed an optimized expression and purification paradigm that produces ~360X more recombinant human eIF2A than previous reports. Using a mammalian in vitro translation system, we found that recombinant human eIF2A inhibits translation of multiple reporter mRNAs and does so prior to start codon recognition. eIF2A also inhibited translation directed by all four types of cap-independent viral IRESs, including those that do not require ribosomal scanning, initiation factors, or initiator tRNA, suggesting excess eIF2A sequesters 40S subunits. Reactions supplemented with additional 40S subunits rescued translation and pull-down assays provide evidence of direct binding between recombinant eIF2A and purified 40S subunits. These data support a model that eIF2A must be kept away from the translation machinery to avoid sequestering the 40S ribosomal subunit.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Updates references and small typographical edits
