Increased levels of eIF2A inhibit translation by sequestering 40S ribosomal subunits.

Autor:	Grove DJ; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA.; Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA., Levine DJ; Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA., Kearse MG; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA.; Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
Jazyk:	angličtina
Zdroj:	Nucleic acids research [Nucleic Acids Res] 2023 Oct 13; Vol. 51 (18), pp. 9983-10000.
DOI:	10.1093/nar/gkad683
Abstrakt:	eIF2A was the first eukaryotic initiator tRNA carrier discovered but its exact function has remained enigmatic. Uncharacteristic of translation initiation factors, eIF2A is reported to be non-cytosolic in multiple human cancer cell lines. Attempts to study eIF2A mechanistically have been limited by the inability to achieve high yield of soluble recombinant protein. Here, we developed a purification paradigm that yields ∼360-fold and ∼6000-fold more recombinant human eIF2A from Escherichia coli and insect cells, respectively, than previous reports. Using a mammalian in vitro translation system, we found that increased levels of recombinant human eIF2A inhibit translation of multiple reporter mRNAs, including those that are translated by cognate and near-cognate start codons, and does so prior to start codon recognition. eIF2A also inhibited translation directed by all four types of cap-independent viral IRESs, including the CrPV IGR IRES that does not require initiation factors or initiator tRNA, suggesting excess eIF2A sequesters 40S subunits. Supplementation with additional 40S subunits prevented eIF2A-mediated inhibition and pull-down assays demonstrated 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 40S ribosomal subunits. (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Databáze:	MEDLINE
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