Poster abstracts
Poster number 9 submitted by Daisy DiVita
Excess eukaryotic initiation factor 2A inhibits translation initiation by sequestering the 40S ribosomal subunit
Daisy J. DiVita (Ohio State Biochemistry Program, Center for RNA Biology, Department of Biological Chemistry and Pharmacology), Michael G. Kearse (Ohio State Biochemistry Program, Center for RNA Biology, Department of Biological Chemistry and Pharmacology)
Abstract:
Canonical eukaryotic mRNA translation uses the heterotrimeric eukaryotic initiation factor 2 (eIF2) to deliver the initiator tRNA (Met-tRNAiMet) to the 40S ribosomal subunit. However, eIF2A (not to be confused with the alpha subunit of eIF2) was the first Met-tRNAiMet carrier discovered in eukaryotes, but further insights into its molecular mechanism dwindled once eIF2 was identified as the primary Met-tRNAiMetcarrier. The exact role of eIF2A in translation still remains a mystery despite it having nearly identical affinity for Met-tRNAiMet 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 recombinant eIF2A inhibits mRNA translation. In agreement with our in vitro data, overexpression of eIF2A decreased global protein synthesis in HeLa cells when assayed by puromycin labeling. To decipher which step of translation is inhibited, we used sucrose gradient ultracentrifugation along with various translation elongation 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 initiation complexes (small subunit with eIFs at start codons), we identified that excess eIF2A reduces the abundance of small subunits at start codons. Deletional analysis also identified the previously mapped eIF5B-binding site in eIF2A is required for the observed inhibition. These data support a model that eIF2A must be kept away from the translation machinery to avoid sequestering the 40S ribosomal subunit.
Keywords: Translational regulation, eukaryotic initiation factors, ribosome