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Published on: May 1, 2020
Increased levels of eIF2A inhibit translation by sequestering 40S ribosomal subunits
Daisy J Grove1,2, Daniel J Levine2, Michael G Kearse1,2
1The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA.
Abstract:
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.
Insights
The eukaryotic translation factor eIF2A, when in excess, inhibits protein synthesis by sequestering 40S ribosomal subunits. This finding clarifies the enigmatic role of eIF2A in translation initiation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The precise function of eukaryotic initiation factor 2 subunit alpha (eIF2A), the first identified eukaryotic initiator tRNA carrier, remains unclear.
- eIF2A exhibits unusual non-cytosolic localization in human cancer cells, unlike typical translation initiation factors.
- Previous research faced challenges in producing sufficient soluble recombinant eIF2A for mechanistic studies.
Purpose of the Study:
- To develop an effective purification method for recombinant human eIF2A.
- To elucidate the inhibitory mechanism of eIF2A in translation.
- To investigate the interaction of eIF2A with ribosomal subunits.
Main Methods:
- Developed a novel purification strategy for recombinant human eIF2A from E. coli and insect cells, achieving significantly higher yields.
- Utilized a mammalian in vitro translation system to assess the impact of eIF2A on reporter mRNA translation.
- Performed pull-down assays to determine the binding interaction between eIF2A and 40S ribosomal subunits.
Main Results:
- Achieved substantial increases in recombinant human eIF2A yield (360-fold in E. coli, 6000-fold in insect cells).
- Demonstrated that elevated eIF2A levels inhibit translation of various mRNAs, irrespective of start codon type, before start codon recognition.
- Showed that eIF2A inhibits translation mediated by viral internal ribosome entry sites (IRESs), including those independent of initiation factors.
- Confirmed that excess eIF2A sequesters 40S ribosomal subunits, an effect reversed by supplementing with additional 40S subunits.
- Established direct binding between recombinant eIF2A and 40S subunits via pull-down assays.
Conclusions:
- Excess eIF2A inhibits translation by sequestering 40S ribosomal subunits.
- eIF2A's interaction with 40S subunits is a key mechanism underlying its inhibitory effect on protein synthesis.
- Maintaining appropriate levels of eIF2A is crucial to prevent its interference with the translation machinery.
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