The eIF2A knockout mouse

Abstract

Eukaryotic initiation factor 2A (eIF2A) is a 65-kDa protein that was first identified in the early 1970s as a factor capable of stimulating initiator methionyl-tRNAi (Met-tRNA^Met_i) binding to 40S ribosomal subunits in vitro. However, in contrast to the eIF2, which stimulates Met-tRNA^Met_i binding to 40S ribosomal subunits in a GTP-dependent manner, eIF2A didn't reveal any GTP-dependence, but instead was found to direct binding of the Met-tRNA^Met_i to 40S ribosomal subunits in a codon-dependent manner. eIF2A appears to be highly conserved across eukaryotic species, suggesting conservation of function in evolution. The yeast Saccharomyces cerevisae eIF2A null mutant revealed no apparent phenotype, however, it was found that in yeast eIF2A functions as a suppressor of internal ribosome entry site (IRES)-mediated translation. It was thus suggested that eIF2A my act by impinging on the expression of specific mRNAs. Subsequent studies in mammalian cell systems implicated eIF2A in non-canonical (non-AUG-dependent) translation initiation events involving near cognate UUG and CUG codons. Yet, the role of eIF2A in cellular functions remains largely enigmatic. As a first step toward characterization of the eIF2A function in mammalian systems in vivo, we have obtained homozygous eIF2A-total knockout (KO) mice, in which a gene trap cassette was inserted between eIF2A exons 1 and 2 disrupting expression of all exons downstream of the insertion. The KO mice strain is viable and to date displays no apparent phenotype. We believe that the eIF2A KO mice strain will serve as a valuable tool for researchers studying non-canonical initiation of translation in vivo.

Keywords: alternative initiation pathways; eIF2A; initiation of translation; knockout mouse; non-AUG dependent initiation.

Figure 1.

The eIF2A gene. (A) Top: Mus musculus eukaryotic translation initiation factor 2A (eIF2A) (mouse Accession: NM_001005509) gene organization and the site of insertion of Omnibank Vector 74. Bottom: Mouse genomic sequence surrounding the gene trap insertion site identified in the C57BL/6 gene trap ES cell clone IST13504C3sA4 for eIF2A. The initiating ATG codon is in red and the coding part of exon 1 is in cyan. The insertion site is denoted with an asterisk *. (B) Top: Schematic of the insertion site and the genotyping strategy. Relative positions of the primers and the expected sizes of the PCR fragments are indicated. Bottom: Genotyping results (1.5% agarose gel).

Figure 2.

The eIF2A gene expression. (A) Real-time PCR. Relative Ct values are shown; Ct values were normalized to gapdh signal and are shown relative to wild-type brain sample. Two sets of primers were used: Primer set 1, recognizing sequences within exons 1 and 2 (top) and primer set 2 recognizing sequences within exons 8 and 9 (bottom). (B) Western blotting. mRNA and protein levels were analyzed in 6 different mouse tissues heart, brain, lung, liver, kidney and pancreas, respectively.

Figure 3.

The eIF2A knockout mouse. Mouse with knockout eIF2A gene (left) and mouse with normal gene (right). Both mice are similar in size and morphology and reveal no detectable difference in their phenotype.