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.
Highlights
Tein, eIF4G, which interacts with eIF4E, eIF4A, eIF4B, eIF3, and the poly(A)-binding protein (PABP)2 (2, 4 – 6)
As in A. thaliana, eIFiso4G is encoded by two genes in maize (GRMZM2G157061 and GRMZM2G098577) but these are more similar (90.1% identity and 92.8% similarity)
EIFiso4E, the eIF4E isoform that interacts with the eIFiso4G isoforms, is encoded by a single gene (i.e. At5g35620) in A. thaliana but the eifiso4e null mutant has no visible phenotype [20], perhaps because eIF4E can interact with eIFiso4G as a less preferred partner in the absence of eIFiso4E [13]
Summary
Plant Growth and Transformation—Arabidopsis seeds were germinated on soil and grown in a plant growth room supplemented with Sylvania Gro-Lite fluorescent bulbs (Sylvania, Danvers, MA) at a photon flux density (PFD) of 100 mol photons mϪ2 sϪ1. The primary inflorescences of Arabidopsis plants were removed, and the secondary inflorescences were allowed to initiate before infiltration. The qPCR analysis was performed using a iQ5 real time PCR detection system (Bio-Rad) in 25-l reactions containing 1ϫ SYBR Green SuperMix 500 nM forward and reverse primers and 10 ng of cDNA. Amino acid sequence alignments for examining eIFiso4G2-specific sequence differences were performed by ClustalW2 with the following parameters: pairwise gap opening penalty 10, pairwise gap extension penalty 0.1, multiple gap opening penalty 10, multiple gap extension penalty 0.2, Gonnet protein weight matrix, and no end gap separation. Initial tree(s) for the heuristic search were obtained by applying the NeighborJoining method to a matrix of pairwise distances estimated using a JTT model. Gene sequences used the analyses were from A. thaliana (AT2G24050 and AT5G57870); Arabidopsis lyrata (Al_481263, Al_495887, and Al_353746); Boechera stricta (Bs_27895s0080, Bs_26833s0467, and Bs_7867s0925); Capsella rubella (Cr_ 10022680m, Cr_10025916m, and Cr_10004122m); Capsella grandiflora (Cg_3181s0015, Cg_0380s0056, and Cg_2848s0070); Eutrema salsugineum (Es_10000058m and Es10012738m); Brassica rapa (Br_K00529, Br_H01394, Br_A00681, and Br_ B01185); Carica papaya (Cp_supercontig_46.175); Gossypium raimondii (Gr_009G009800, Gr_004G273900, Gr_013G105700, and Gr_013G241300); Theobroma cacao (Tc_1EG036897 and Tc_1EG038403); Eucalyptus grandis (Eg_C00247 and Eg_ C00248); Citrus clementina (Cc_10007494m and Cc_10007769m); Manihot esculenta (Me_1_001915m and Me_1_001934m); Ricinus communis (Rc_27455.m000039); Phaseolus vulgaris (Phv_ 003G154900 and Phv_009G207600); Glycine max (Glyma. 17G072500.1, Glyma.02G205500.1, Glyma.06G225700.1, and Glyma.04G154100.1); Vitis vinifera (Vv_GSVIVT01023638001 and Vv_GSVIVT01035980001); Solanum lycopersicum (Sl_ 07g005810.2 and Sl_12g009960.1); Solanum tuberosum (St_ PGSC0003DMT400029102, St_PGSC0003DMT400020464, and St_PGSC0003DMT400029103); Sorghum bicolor (Sb006G136400); Brachypodium distachyon (Bd_5g14687); Zea mays (Zm_ GRMZM2G098577 and ZmGRMZM2G157061); Oryza sativa (Os02g39840 and Os04g42140); Panicum virgatum (Pv_Ab02269, Pv_Aa01373, Pv_J01984, and Pv_Gb01258); Setaria italica (Si_016412m and Si_009406m); Triticum aestivum (Ta_M95747); Selaginella moellendorffii (Sm_437322); Physcomitrella patens (Pp_014G077600, Pp_014G078000, Pp_017G069800, Pp_ 017G069500, and Pp_017G069100)
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