eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation

Abstract

Eukaryotic mRNAs contain a 5' cap structure critical for recruitment of the translation machinery and initiation of protein synthesis. mRNA recognition is thought to require direct interactions between eukaryotic initiation factor 4E (eIF4E) and the mRNA cap. However, translation of numerous capped mRNAs remains robust during cellular stress, early development, and cell cycle progression1 despite eIF4E inactivation. Here we describe a new cellular cap-dependent pathway of translation initiation that relies on a previously unknown cap-binding activity of eIF3d, a subunit of the 800-kilodalton eukaryotic initiation factor 3 (eIF3) complex. A 1.4 Å crystal structure of the eIF3d cap-binding domain reveals unexpected homology to endonucleases involved in RNA turnover, and allows modeling of cap recognition by eIF3d. eIF3d makes specific contacts to the cap, as exemplified by cap analog competition, and these interactions are essential for assembly of translation initiation complexes on eIF3-specialized mRNAs2 such as the cell proliferation regulator c-Jun. The c-Jun mRNA further encodes an inhibitory RNA element that blocks eIF4E recruitment, thus enforcing alternative cap recognition by eIF3d. Our results reveal a new mechanism of cap-dependent translation independent of eIF4E, and illustrate how modular RNA elements work in concert to direct specialized forms of translation initiation.

A Unique ISR Program Determines Cellular Responses to Chronic Stress

“…Reduction in eIF3d levels prevented translational recovery during chronic ER stress (Figure 4D), while not altering the effects of Tg or PERKi on eIF2B activity (Figure 4G). Consistent with earlier observations that eIF3d can selectively affect translation (Lee et al, 2016), depletion of eIF3d strongly inhibited polysomal association of the ATF4 mRNA even in unstressed cells (Figures S3E vs. 1D), but had only a marginal effect on GADD34 mRNA translation (Figures S3E vs. S2). Furthermore, the eIF3d-RNA co-immunoprecipitation experiments revealed a decrease in association of eIF3d with mRNAs that are translationally repressed ( e.g.…”

“…Under specific conditions, it has been reported that eIF3 may directly bind a subset of mRNAs and recruit them to the 43S complex in an eIF4F-independent manner (Lee et al, 2015; Lee et al, 2016). We therefore tested whether selected eIF3 subunits show differential binding to mRNAs in control vs. chronically stressed cells.…”

“…Almost 500 mRNAs with supposedly related structural elements in their 5’ UTR were PAR-CLIPed to eIF3a, b, d and g subunits in human 293T cells and proposed to be regulated in an eIF3-specific, cap-dependent manner (Lee et al, 2015). Using in vitro binding assays, purified eIF3d (as part of the multisubunit eIF3 complex) was then shown to directly contact the 5’ cap of the c-JUN mRNA which was used as a model to support this translation initiation mechanism (Lee et al, 2016). It was proposed that in order to contact the m 7 GTP cap, the eIF3d cap-binding pocket needs to be allosterically activated by other eIF3 subunits and/or by the c-JUN 5’ UTR-specific secondary structure.…”

“…RNA immunoprecipitation was analyzed as previously reported (Lee et al, 2015; Lee et al, 2016) with following modifications. To isolate cytoplasmic fractions, cells were incubated in hypotonic buffer A (10 mM HEPES-KOH at pH 7.9, 10 mM KCl, 1.5 mM MgCl 2 , 0.5 mM DTT) supplemented with EDTA-free protease inhibitor (Roche Applied Science) and PhosSTOP phosphatase inhibitor (Roche Applied Science) on ice for 15 min, followed by the addition of IGEPAL (Sigma-Aldrich) to the final concentration of 0.25% for 10 min.…”

Biochemistry and Molecular Biology of Flaviviruses

“…230 This model was based on the assumption that cap recognition is always mediated by eIF4E, but this assumption has been disproven by important studies that demonstrate that eIF3D, a subunit of the eIF3 protein complex, can recognize the cap and mediate cap-dependent translation initiation in some mRNAs with structured 5′UTRs. 231 We propose that DENV2 employs noncanonical mechanisms to mediate cap-dependent translation initiation, possibly via direct recruitment of the eIF3 complex.…”