Abstract
The main role of the translation initiation factor 3 (eIF3) is to orchestrate formation of 43S-48S preinitiation complexes (PICs). Until now, most of our knowledge on eIF3 functional contribution to regulation of gene expression comes from yeast studies. Hence, here we developed several novel in vivo assays to monitor the integrity of the 13-subunit human eIF3 complex, defects in assembly of 43S PICs, efficiency of mRNA recruitment, and postassembly events such as AUG recognition. We knocked down expression of the PCI domain-containing eIF3c and eIF3a subunits and of eIF3j in human HeLa and HEK293 cells and analyzed the functional consequences. Whereas eIF3j downregulation had barely any effect and eIF3a knockdown disintegrated the entire eIF3 complex, eIF3c knockdown produced a separate assembly of the a, b, g, and i subunits (closely resembling the yeast evolutionary conserved eIF3 core), which preserved relatively high 40S binding affinity and an ability to promote mRNA recruitment to 40S subunits and displayed defects in AUG recognition. Both eIF3c and eIF3a knockdowns also severely reduced protein but not mRNA levels of many other eIF3 subunits and indeed shut off translation. We propose that eIF3a and eIF3c control abundance and assembly of the entire eIF3 and thus represent its crucial scaffolding elements critically required for formation of PICs.
Highlights
The main role of the translation initiation factor 3 is to orchestrate formation of 43S-48S preinitiation complexes (PICs)
Less efficient downregulation obtained in HEK293 compared to that in HeLa cells is most probably due to differences in RNA interference (RNAi) pathway activity in these cell lines [36]
Since the mRNA levels of all reporters were unchanged in cells with knocked-down expression and control cells, we conclude that eIF3cKD has a detectable impact on the accuracy of AUG recognition. In this first complex in vivo analysis of human Eukaryotic translation initiation factor 3 (eIF3), we knocked down expression of its eIF3c and eIF3a subunits, which are both part of the octamer core of eIF3, and of eIF3j in two human cell lines and systematically analyzed their phenotypic and biochemical effects, following the fate of all 13 eIF3 subunits by several newly established techniques, which previously proved to be extremely useful in characterizing the translation initiation process in S. cerevisiae
Summary
The main role of the translation initiation factor 3 (eIF3) is to orchestrate formation of 43S-48S preinitiation complexes (PICs). Whereas eIF3j downregulation had barely any effect and eIF3a knockdown disintegrated the entire eIF3 complex, eIF3c knockdown produced a separate assembly of the a, b, g, and i subunits (closely resembling the yeast evolutionary conserved eIF3 core), which preserved relatively high 40S binding affinity and an ability to promote mRNA recruitment to 40S subunits and displayed defects in AUG recognition. Eukaryotic translation initiation factor 3 (eIF3) is a highly complex multiprotein assembly with multiple functions in translation It promotes formation of 43S and 48S preinitiation complexes (PICs) by aiding the Met-tRNAiMet and mRNA loading onto the small ribosomal subunit, and, at least in the budding yeast, it was implicated in stimulating subsequent scanning for AUG recognition (for a review, see reference 1). The observed discrepancy between the importance of yeast HCR1 and its human ortholog eIF3j for cell proliferation has been rather puzzling
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