eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition

Abstract

Recognition of the methyl-7-guanosine (m 7 G) cap structure on mRNA is an essential feature of mRNA metabolism and thus gene expression. Eukaryotic translation initiation factor 4E (eIF4E) promotes translation, mRNA export, proliferation, and oncogenic transformation dependent on this cap-binding activity. eIF4E-cap recognition is mediated via complementary charge interactions of the positively charged m 7 G cap between the negative π-electron clouds from two aromatic residues. Here, we demonstrate that a variant subfamily, eIF4E3, specifically binds the m 7 G cap in the absence of an aromatic sandwich, using instead a different spatial arrangement of residues to provide the necessary electrostatic and van der Waals contacts. Contacts are much more extensive between eIF4E3-cap than other family members. Structural analyses of other cap-binding proteins indicate this recognition mode is atypical. We demonstrate that eIF4E3 relies on this cap-binding activity to act as a tumor suppressor, competing with the growthpromoting functions of eIF4E. In fact, reduced eIF4E3 in high eIF4E cancers suggests that eIF4E3 underlies a clinically relevant inhibitory mechanism that is lost in some malignancies. Taken together, there is more structural plasticity in cap recognition than previously thought, and this is physiologically relevant.M etabolism of mRNA is a complex and highly regulated process dependent on the association of the methyl-7-guanosine (m 7 G) cap structure on the 5′ end of transcripts with appropriate proteins (1-3). In mammalian cells, the two major cap-binding proteins are the nuclear cap-binding complex (CBC) (4) and eukaryotic translation initiation factor 4E (eIF4E) (3). Specific cap recognition is key for the fate of transcripts and impacts processes such as mRNA processing and bulk mRNA export via the CBC or the nuclear export of specific transcripts as well as bulk translation by eIF4E (5). NMR and crystallographic studies reveal that m 7 G cap is specifically recognized by both the CBC and eIF4E via intercalation of the m 7 G cap moiety with the side chains of two aromatic residues, i.e., an aromatic sandwich (4, 6-9). This is an electrostatically driven process relying on the partial positive charge of the m 7 G cap and the negative π-electron clouds of the aromatic residues. The aromatic residues are completely conserved in these cap-binding proteins. This recognition motif is used almost exclusively by proteins that specifically bind the m 7 G cap including eIF4E, CBC, and vaccinia virus protein VP39 (4, 10).Dysregulation of cap-binding proteins can have striking physiological consequences. For instance through its cap-binding activity and subsequent effects on gene expression, eIF4E plays an important role in proliferation and survival (1, 3). Indeed, eIF4E is overexpressed in about 30% of human cancers and its overexpression is oncogenic in cell culture and animal models (2, 11). Mutation of the cap-binding site of eIF4E impairs its activities in translation, mRNA export, and oncogenic transform...

Tristetraprolin Recruits Eukaryotic Initiation Factor 4E2 To Repress Translation of AU-Rich Element-Containing mRNAs

“…However, our preliminary results showed that isoforms d and e also bound the cap structure analog M7-GTP (X. Tao and G. Gao, unpublished data), implying that they bind the cap structure in a manner distinct from that of isoform a. This possibility is supported by the finding that eIF4E3 binds to the cap in a manner that does not involve the Y78 equivalent residue (42). Overexpression of eIF4E2a enhanced TTP-mediated translational repression in a dose-dependent manner (Fig.…”

Activation of a GPCR leads to eIF4G phosphorylation at the 5′ cap and to IRES-dependent translation

“…In support of this assumption, our preliminary data indicate that phosphorylated eIF4G preferentially interacts with the eIF4E3 isoform in the presence of FSH. Owing to the lack of one aromatic residue to sandwich the methyl-7-guanosine cap, eIF4E3 has been shown recently to exhibit unique interaction properties with the m7-GTP-cap (Osborne et al 2013), indicating that it may compete with eIF4E1 for the same mRNA, but induce an opposite fate, such as tumor suppression. Finally, considering the rapamycin sensitivity of the initiation complex components, the phosphorylation of eIF4E has recently been shown to regulate the translation of selective mRNA (Wendel et al 2007), in an mTORdependent manner (Stead & Proud 2013).…”

Tryptophan Residues from Cap Binding Slot in eIF4E Family Members: Their Contributions to Near-UV Circular Dichroism Spectra

“…The structures of both isoforms retain the eIF4E-fold with a central curved β-sheet consisting of seven antiparallel β-stands flanked by three α-helices at its convex surfaces. The backbone rootmean-square deviation (rmsd) for a regular secondary structure is below 1.8 Å and the positions of the conserved Trp residues (except of Trp102) in the individual isoforms are conserved [45,46]. In contrast to heIF4E1a and heIF4E2, where the N-terminus of the protein is unstructured in apo form, for heIF4E3 also the C-terminus seems to be disordered.…”

“…In contrast to heIF4E1a and heIF4E2, where the N-terminus of the protein is unstructured in apo form, for heIF4E3 also the C-terminus seems to be disordered. In heIF4E1b and heIF4E2, similarly like in heIF4E1a, the N7-metylguanine of the cap is bound by traditional aromatic sandwich Trp/Trp or Tyr/Trp; however, in the case of eIF4E3 isoform, where Trp56 (according to human numbering) is replaced by Cys residue, the cap binding is executed differently using S1-S2 loop and C-terminus, which becomes much more structured after cap binding [46]. The binding studies showed that heIF4E3 binds m 7 GTP 12-fold weaker than the canonical heIF4E1a, but 18-fold stronger that its mutant with Trp56 replaced by cysteine (Table 2).…”

“…In our studies, we investigated three human eIF4E isoforms: heIF4E1b from class I, heIF4E2 (also known as 4EHP) belonging to Class II and heIF4E3 from Class III. For heIF4E2 and heIF4E3, the structures in apo and cap binding form were resolved [45,46]. The structures of both isoforms retain the eIF4E-fold with a central curved β-sheet consisting of seven antiparallel β-stands flanked by three α-helices at its convex surfaces.…”