To initiate or not to initiate: A critical assessment of eIF2A, eIF2D, and MCT-1·DENR to deliver initiator tRNA to ribosomes

Abstract

Selection of the correct start codon is critical for high-fidelity protein synthesis. In eukaryotes, this is typically governed by a multitude of initiation factors (eIFs), including eIF2·GTP that directly delivers the initiator tRNA (Met-tRNA_i ^Met ) to the P site of the ribosome. However, numerous reports, some dating back to the early 1970s, have described other initiation factors having high affinity for the initiator tRNA and the ability of delivering it to the ribosome, which has provided a foundation for further work demonstrating non-canonical initiation mechanisms using alternative initiation factors. Here we provide a critical analysis of current understanding of eIF2A, eIF2D, and the MCT-1·DENR dimer, the evidence surrounding their ability to initiate translation, their implications in human disease, and lay out important key questions for the field. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Mechanisms Translation > Regulation.

Keywords: eIF; translation initiation; translational control.

FIGURE 1

Crystal structures of S. pombe and H. sapiens eIF2A N-terminus are homologous. (a) Crystal structure of C-terminally truncated S. pombe eIF2A (residues 1–424) solved at 2.5 Å. PDB: 3WJ9. (b) Crystal structure of C-terminally truncated H. sapiens eIF2A (residues 4–427) solved at 1.8 Å. PDB: 8DYS. (c) AlphaFold predicted structure of full-length H. sapiens eIF2A with specific putative binding domains colored (left). Schematic of labeled putative binding domains (right). (d) Overlay of the N-terminal β-propeller in eIF2A from panels a–c.

FIGURE 2

(a–f) eIF2A-dependent RAN translation of the CCUG repeat is downstream of eIF2α S51 phosphorylation. RAN translation of expanded CCUG repeats (encodes QAGR repeat protein) or expanded CAGG repeats (encodes LPAC repeat protein) in WT HEK293T cells, PKR KO, PERK KO, eIF2 S51A mutants, eIF2A KO, and eIF2 S51A mutant with eIF2A overexpression. Protein levels were respective to those measured in WT cells for each reporter.

FIGURE 3

BiP-FLAG reporter expression is sensitive to eIF2A protein levels. (a) UUG-encoded uORF1 and BiP-FLAG protein levels are resistant to translational repression during ER stress. (b) Upon eIF2A depletion, both uORF1 and BiP-FLAG levels decrease during ER stress (top). This decrease is exacerbated when uORF1 is mutated and cannot be translated (bottom).

FIGURE 4

MCT-1·DENR resembles eIF2D. (a) Schematic of H. sapiens eIF2D, MCT-1, and DENR showing homologous domains and location of patient-associated Mendelian susceptibility to mycobacterial disease (MSMD) mutations in MCT-1 and autism spectrum disorder mutations in DENR. DUF1947, domain of unknown function 1947; MDM2, mouse double minute 2 homolog; PUA, pseudouridine synthase and archaeosine transglycosylase; SUI, suppressor of initiator codon mutations; SWIB, SWI/SNF complex including complex B; WH, central winged helix. (b) AlphaFold predicted structure of H. sapiens eIF2D structure with domains colored as described in panel a.

FIGURE 5

ATF4 expression is sensitive to eIF2D, MCT-1, and DENR protein levels under normal growth and ER stress conditions. (a) In the presence of eIF2D, MCT-1, and DENR, expression of ATF4 by re-initiation is favored under tunicamycin-induced ER stress in HeLa cells. (b) When eIF2D, MCT-1, and DENR are deleted, re-initiation is decreased under both conditions, which results in lower levels of ATF4 protein. (c) Rescue of eIF2D, MCT-1, and DENR in their knockout cells returns ATF4 expression to being favored under tunicamycin-induced ER stress.