Abstract
BackgroundEukaryotic translation initiation factor 4E (eIF4E) plays a pivotal role in the control of cap-dependent translation initiation, modulates the fate of specific mRNAs, occurs in processing bodies (PBs) and is required for formation of stress granules (SGs). In this study, we focused on the subcellular localization of a representative compendium of eIF4E protein isoforms, particularly on the less studied members of the human eIF4E protein family, eIF4E2 and eIF4E3.ResultsWe showed that unlike eIF4E1, its less studied isoform eIF4E3_A, encoded by human chromosome 3, localized to stress granules but not PBs upon both heat shock and arsenite stress. Furthermore, we found that eIF4E3_A interacts with human translation initiation factors eIF4G1, eIF4G3 and PABP1 in vivo and sediments into the same fractions as canonical eIF4E1 during polysome analysis in sucrose gradients. Contrary to this finding, the truncated human eIF4E3 isoform, eIF4E3_B, showed no localization to SGs and no binding to eIF4G. We also highlighted that eIF4E2 may exhibit distinct functions under different stresses as it readily localizes to P-bodies during arsenite and heat stresses, whereas it is redirected to stress granules only upon heat shock. We extended our study to a number of protein variants, arising from alternative mRNA splicing, of each of the three eIF4E isoforms. Our results surprisingly uncovered differences in the ability of eIF4E1_1 and eIF4E1_3 to form stress granules in response to cellular stresses.ConclusionOur comparison of all three human eIF4E isoforms and their protein variants enriches the intriguing spectrum of roles attributed to the eukaryotic initiation translation factors of the 4E family, which exhibit a distinctive localization within different RNA granules under different stresses. The localization of eIF4E3_A to stress granules, but not to processing bodies, along with its binding to eIF4G and PABP1 suggests a role of human eIF4E3_A in translation initiation rather than its involvement in a translational repression and mRNA decay and turnover. The localization of eIF4E2 to stress granules under heat shock but not arsenite stress indicates its distinct function in cellular response to these stresses and points to the variable protein content of SGs as a consequence of different stress insults.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-016-0072-x) contains supplementary material, which is available to authorized users.
Highlights
Eukaryotic translation initiation factor 4E plays a pivotal role in the control of cap-dependent translation initiation, modulates the fate of specific mRNAs, occurs in processing bodies (PBs) and is required for formation of stress granules (SGs)
In addition to the canonical eIF4E2 and eIF4E3 (eIF4E1) and eIF4E2 proteins, we cloned the coding sequences of the following isoforms: eIF4E1_3, which possesses a longer N-terminus; eIF4E2_C, which utilizes a different 3′-distal exon harboring a protein with a shorter C-terminus; and eIF4E2_CRA_a, which is not covered by any RefSeq record, even though it is available under GI: 119591413 in the GenBank
In the case of eIF4E1, which recognizes the mRNA cap during translation initiation, full functionality of the GFP fusion protein ectopically produced in human cells was confirmed by its efficient binding to the affinity resin containing an immobilized m7GTP (Fig. 2)
Summary
Eukaryotic translation initiation factor 4E (eIF4E) plays a pivotal role in the control of cap-dependent translation initiation, modulates the fate of specific mRNAs, occurs in processing bodies (PBs) and is required for formation of stress granules (SGs). The availability of eIF4E—and the formation of a functional mammalian eIF4F complex—is affected by the translational repressors 4E-BPs. Discovered as a molecular mimic of eIF4G, the 4E-BP family of proteins competes for the same eIF4E1 binding motif, thereby inhibiting the initiation of protein synthesis [7]. The substitution of W73 to a non-aromatic residue in human eIF4E1 leads to its inability to interact with 4E-BPs or eIF4G [6] and to be recruited to sites of mRNA degradation and stress-induced RNA cytoplasmic granules [8]. Interactions of eIF4E1 with its protein partners rather than its cap-binding ability seem to be essential for eIF4E1 relocalization to the stress-induced RNA cytoplasmic granules [8]. Overexpression of eIF4E1 leads to an oncogenic transformation, and increased eIF4E1 levels are observed in diverse tumor types [10]
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