Eukaryotic Initiation Factor eIFiso4G1 and eIFiso4G2 Are Isoforms Exhibiting Distinct Functional Differences in Supporting Translation in Arabidopsis

Abstract

The eukaryotic translation initiation factor (eIF) 4G is required during protein synthesis to promote the assembly of several factors involved in the recruitment of a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, the eIF4G isoforms in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization but both can interact with eIF4A, eIF4B, eIF4E isoforms, and the poly(A)-binding protein. Nevertheless, eIF4G and eIFiso4G from wheat exhibit preferences in the mRNAs they translate optimally. For example, mRNA containing the 5'-leader (called Ω) of tobacco mosaic virus preferentially uses eIF4G in wheat germ lysate. In this study, the eIF4G isoform specificity of Ω was used to examine functional differences of the eIF4G isoforms in Arabidopsis. As in wheat, Ω-mediated translation was reduced in an eif4g null mutant. Loss of the eIFiso4G1 isoform, which is similar in sequence to wheat eIFiso4G, did not substantially affect Ω-mediated translation. However, loss of the eIFiso4G2 isoform substantially reduced Ω-mediated translation. eIFiso4G2 is substantially divergent from eIFiso4G1 and is present only in the Brassicaceae, suggesting a recent evolution. eIFiso4G2 isoforms exhibit sequence-specific differences in regions representing partner protein and RNA binding sites. Loss of any eIF4G isoform also resulted in a substantial reduction in reporter transcript level. These results suggest that eIFiso4G2 appeared late in plant evolution and exhibits more functional similarity with eIF4G than with eIFiso4G1 during Ω-mediated translation.

Keywords: RNA; eIFiso4G; eukaryotic translation initiation; eukaryotic translation initiation factor 4G (eIF4G); mRNA stability; protein synthesis; translation; translation initiation factors.

FIGURE 1.

Expression of eIF4G isoforms in eIF4G mutants. qPCR was performed for eIF4G (white bars), eIFiso4G1 (gray bars), and eIFiso4G2 (black bars) in 8-day-old WT seedlings and eifiso4e, eif4g, eifiso4g1, eifiso4g2, and eifiso4g1/2 mutants. Expression is reported relative to the level in WT seedlings, which is set at a value of 1.

FIGURE 2.

All eIF4G isoforms are required to support Luc and Ω-LUC transcript levels. qPCR was performed for expression from 35S::Luc and 35S::Ω-Luc transgenes in 8-day-old WT seedlings in WT plants and in eifiso4e, eif4g, eifiso4g1, eifiso4g2, and eifiso4g1/2 mutants into which each transgene was crossed. In A, expression is reported relative to the level of Luc (white bars) and Ω-Luc (black bars) mRNAs in WT seedlings, which is set at a value of 1. In B, the transcript level of Ω-Luc (black bars) is reported relative to the Luc (white bars) transcript level (set at a value of 1) for WT and mutant seedlings.

FIGURE 3.

Evolution and sequence conservation of eIFiso4G1 and eIFiso4G2 in land plants. The presence of eIFiso4G1 and eIFiso4G2 genes in a species is indicated by a black dot and the number of genes for each type is indicated by the number of black dots. The evolutionary relationship of the species shown is indicated to the left. Sequence identity and similarity between eIFiso4G1 and eIFiso4G2 isoforms in each species is reported as are the identity and similarity among eIFiso4G1 isoforms and among eIFiso4G2 isoforms within a species when more than one isoform is present.

FIGURE 4.

Phylogenetic analysis of eIFiso4G1 and eIFiso4G2 isoforms in land plants. A phylogenetic tree of eIFiso4G1 and eIFiso4G2 isoforms was constructed using the maximum-likelihood method. The tree with the highest log likelihood is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Numbers on each branch denote percentages of bootstrap support. eIFiso4G isoforms from the non-vascular species, P. patens, and the early vascular species, S. moellendorffii, were used to root the tree.

FIGURE 5.

Sequence conservation among eIFiso4G1 and eIFiso4G2 isoforms. The sequence of eIFiso4G1 and eIFiso4G2 from A. thaliana (At), C. grandiflora (Cg), E. salsugineum (Es), P. vulgaris (Phv), S. lycopersicum (Sl), O. sativa (Os), and T. aestivum (Ta) were aligned. Amino acid identity is highlighted in green and amino acid similarity is highlighted in yellow. HEAT domains and binding sites for partner proteins of wheat eIFiso4G are indicated above the sequence as reported (15). eIFiso4G2-specific sequence differences that are conserved among eIFiso4G2 isoforms are indicated by asterisks.

FIGURE 6.

eIFiso4G2 isoforms differ from eIFiso4G1 isoforms within the eIFiso4E binding site. The sequences of the eIFiso4E binding site from eIFiso4G2 and eIFiso4G1 isoforms from core eudicot species were aligned and the conserved eIFiso4G2-specific sequence differences are shown. Amino acid identity is highlighted in gray and conserved eIFiso4G2-specific sequence differences are highlighted in black. The two eIFiso4G2 subgroups present in Brassicaceae are indicated to the right as are eIFiso4G1 isoforms present throughout core eudicots. The position of the eIFiso4E binding site is shown for wheat eIFiso4G at the top.

FIGURE 7.

eIFiso4G2 isoforms differ from eIFiso4G1 isoforms within the eIF4B binding site. Subsequences of the eIF4B binding site from eIFiso4G2 and eIFiso4G1 isoforms from core eudicot species were aligned and the conserved eIFiso4G2-specific sequence differences are shown. Amino acid identity is highlighted in gray and conserved eIFiso4G2-specific sequence differences are highlighted in black. The two eIFiso4G2 subgroups present in Brassicaceae are indicated to the right as are eIFiso4G1 isoforms present throughout core eudicots. The position of the eIF4B binding site is shown for wheat eIFiso4G at top.

FIGURE 8.

eIFiso4G2 isoforms differ from eIFiso4G1 isoforms within a region proximal to the eIF4A/HEAT-1 domain. The sequences of the region proximal to the eIF4A/HEAT-1 domain from eIFiso4G2 and eIFiso4G1 isoforms from core eudicot species were aligned and the conserved eIFiso4G2-specific sequence differences are shown. Amino acid identity is highlighted in gray and conserved eIFiso4G2-specific sequence differences are highlighted in black. The sequence absent in eIFiso4G2 isoforms is indicated by dashes. The two eIFiso4G2 subgroups present in Brassicaceae are indicated to the right as are eIFiso4G1 isoforms present throughout core eudicots. The region representing the sequence is shown for wheat eIFiso4G at the top.

FIGURE 9.

eIFiso4G2 isoforms differ from eIFiso4G1 isoforms within a region proximal to the eIF4A/HEAT-2 domain. The sequences of the region proximal to the eIF4A/HEAT-2 domain from eIFiso4G2 and eIFiso4G1 isoforms from core eudicot species were aligned and the conserved eIFiso4G2-specific sequence differences are shown. Amino acid identity is highlighted in gray and conserved eIFiso4G2-specific sequence differences are highlighted in black. Sequences absent in eIFiso4G2 and eIFiso4G1 isoforms are indicated by dashes. The two eIFiso4G2 subgroups present in Brassicaceae are indicated to the right as are eIFiso4G1 isoforms present throughout core eudicots. The region representing the sequence is shown for wheat eIFiso4G at the top.