eIF2γ mutation that disrupts eIF2 complex integrity links intellectual disability to impaired translation initiation

Abstract

Together with GTP and initiator methionyl-tRNA, translation initiation factor eIF2 forms a ternary complex that binds the 40S ribosome and then scans an mRNA to select the AUG start codon for protein synthesis. Here, we show that a human X-chromosomal neurological disorder characterized by intellectual disability and microcephaly is caused by a missense mutation in eIF2γ (encoded by EIF2S3), the core subunit of the heterotrimeric eIF2 complex. Biochemical studies of human cells overexpressing the eIF2γ mutant and of yeast eIF2γ with the analogous mutation revealed a defect in binding the eIF2β subunit to eIF2γ. Consistent with this loss of eIF2 integrity, the yeast eIF2γ mutation impaired translation start codon selection and eIF2 function in vivo in a manner that was suppressed by overexpressing eIF2β. These findings directly link intellectual disability to impaired translation initiation, and provide a mechanistic basis for the human disease due to partial loss of eIF2 function.

Figure 1. A mutation of EIF2S3/eIF2γ causes an intellectual disability syndrome

(A) Pedigree of the family. Male family members affected by intellectual disability and microcephaly are shown as filled symbols; female carriers of the mutation are represented by a dot inside the circle; and the double line connecting I.1 and I.2 indicates consanguinity (I.1 and I.2 are first cousins). (B) Brain magnetic resonance imaging (MRI) scans of patient III.2 performed at the age of one year. Midline sagittal image demonstrates a very thin corpus callosum (arrows) and microcephaly (left). Axial image shows mild enlargement and asymmetry (arrowheads) of the lateral ventricles (right). (C) Identification of a hemizygous EIF2S3 missense variant c.665T>C (p.Ile222Thr). Sequence chromatograms showing a part of EIF2S3 exon 7 in an unaffected individual (top) and an affected family member (bottom). (D) Protein diagram of eIF2γ with its functional domains (top). The positions of the p.Ile222Thr and p.Val151Leu mutations are indicated. Amino acid sequence alignments (bottom) surrounding Ile222 in human eIF2γ and its orthologs.

Figure 2. The eIF2γ mutation impairs yeast cell growth and eIF2β binding in yeast and human cells

(A) Ribbon representation of S. solfataricus aIF2 complex (PDB code 2QMU) using PyMOL software (DeLano Scientific). The three domains of aIF2γ are colored green (G), yellow (II) and orange (III). aIF2β is colored magenta, and domain III of aIF2α is shown in cyan. The side chain of Ile181, corresponding to human Ile222 and yeast Val281, is shown in stick representation and colored red. (B) Magnification of the aIF2γ – aIF2β helix α1 interface; colored as in panel A. (C) Serial dilutions of yeast cells expressing the indicated eIF2γ mutants with or without overexpression of eIF2β were grown on minimal synthetic dextrose (SD) medium at 30 °C for 3 days. (D) Whole cell extracts (WCEs) from yeast strains expressing the indicated His-tagged eIF2γ protein, or the same strains overexpressing eIF2β, were incubated with Ni²⁺ resin, and two different amounts of precipitated proteins were subject to immunoblot analysis using antisera specific for the indicated yeast eIF2 subunits. *Immunoblot analysis using increased amounts of anti-eIF2β antiserum. (E) WCEs from HeLa cells transfected with empty vector or plasmids expressing the indicated myc- and His₆-tagged human eIF2γ protein were incubated with Ni²⁺ resin, and two different amounts of precipitated proteins were subject to immunoblot analysis using anti-myc (eIF2γ) antiserum or antisera specific for human eIF2α or human eIF2β.