Poster abstracts
Poster number 19 submitted by Daisy Grove
Increased levels of eIF2A inhibit translation by sequestering 40S ribosomal subunits
Daisy J. Grove (Ohio State Biochemistry Program, Center for RNA Biology, Department of Biological Chemistry and Pharmacology, The Ohio State University), Daniel J. Levine (Center for RNA Biology, Department of Biological Chemistry and Pharmacology, The Ohio State University), Michael G. Kearse (Ohio State Biochemistry Program, Center for RNA Biology, Department of Biological Chemistry and Pharmacology, The Ohio State University)
Abstract:
Canonical eukaryotic mRNA translation uses the heterotrimeric eukaryotic initiation factor 2 (eIF2) to deliver the initiator tRNA. However, eIF2A was the first initiator tRNA carrier discovered in eukaryotes, but further insights into its molecular mechanism dwindled once eIF2 was identified as the primary initiator tRNA carrier. The exact role of eIF2A in translation remains a mystery despite it having nearly identical affinity for initiator tRNA and being present at equal concentrations in cells as eIF2. Recent work has shown that eIF2A is required for cancer progression and proper long-term lipid metabolism in mice. Atypical of a translation factor, eIF2A primarily localizes to the nucleus, but can shuttle to the cytoplasm during cell stress. How and why eIF2A is kept away from the translation machinery is unclear but it may be a mechanism for cells to only use eIF2A in specific conditions. To answer these questions, we first supplemented mammalian in vitro translation extracts with recombinant human eIF2A and found that excess eIF2A inhibits mRNA translation. To decipher which step of translation is inhibited, we used sucrose gradient ultracentrifugation along with various translation inhibitors to capture and measure the levels of translation complexes at different stages in vitro. Using the non-hydrolyzable GTP analog GMPPNP to capture 48S translation initiation complexes, we identified that excess eIF2A reduces the abundance of small subunits at start codons. eIF2A also inhibited translation directed by all four types of viral IRESs, including those that do not require initiation factors or initiator tRNA, suggesting eIF2A sequesters the 40S ribosomal subunit. Importantly, reactions supplemented with additional 40S ribosomal subunits severely blunted the ability of eIF2A to inhibit translation. Pull-down assays using recombinant and purified components provide evidence of a direct interaction between eIF2A and the 40S ribosomal subunit. These data support a model that eIF2A must be kept away from the translation machinery to avoid sequestering the 40S ribosomal subunit.
Keywords: mRNA, ribosome, translational control