Evidence for variation in the optimal translation initiation complex: plant eIF4B, eIF4F, and eIF(iso)4F differentially promote translation of mRNAs

Abstract

Eukaryotic initiation factor (eIF) 4B is known to interact with multiple initiation factors, mRNA, rRNA, and poly(A) binding protein (PABP). To gain a better understanding of the function of eIF4B, the two isoforms from Arabidopsis (Arabidopsis thaliana) were expressed and analyzed using biophysical and biochemical methods. Plant eIF4B was found by ultracentrifugation and light scattering analysis to most likely be a monomer with an extended structure. An extended structure would facilitate the multiple interactions of eIF4B with mRNA as well as other initiation factors (eIF4A, eIF4G, PABP, and eIF3). Eight mRNAs, barley (Hordeum vulgare) alpha-amylase mRNA, rabbit beta-hemoglobin mRNA, Arabidopsis heat shock protein 21 (HSP21) mRNA, oat (Avena sativa) globulin, wheat (Triticum aestivum) germin, maize (Zea mays) alcohol dehydrogenase, satellite tobacco necrosis virus RNA, and alfalfa mosaic virus (AMV) 4, were used in wheat germ in vitro translation assays to measure their dependence on eIF4B and eIF4F isoforms. The two Arabidopsis eIF4B isoforms, as well as native and recombinant wheat eIF4B, showed similar responses in the translation assay. AMV RNA 4 and Arabidopsis HSP21 showed only a slight dependence on the presence of eIF4B isoforms, whereas rabbit beta-hemoglobin mRNA and wheat germin mRNA showed modest dependence. Barley alpha-amylase, oat globulin, and satellite tobacco necrosis virus RNA displayed the strongest dependence on eIF4B. These results suggest that eIF4B has some effects on mRNA discrimination during initiation of translation. Barley alpha-amylase, oat globulin, and rabbit beta-hemoglobin mRNA showed the highest activity with eIF4F, whereas Arabidopsis HSP21 and AMV RNA 4 used both eIF4F and eIF(iso)4F equally well. These results suggest that differential or optimal translation of mRNAs may require initiation complexes composed of specific isoforms of initiation factor gene products. Thus, individual mRNAs or classes of mRNAs may respond to the relative abundance of a particular initiation factor(s), which in turn may affect the amount of protein translated. It is likely that optimal multifactor initiation complexes exist that allow for optimal translation of mRNAs under a variety of cellular conditions.

Figure 1.

Protein sequence alignment of Arabidopsis and wheat eIF4B. The protein sequences of the two eIF4B isoforms of Arabidopsis (At) were aligned with the wheat (Ta) eIF4B using CLUSTAL (Jeanmougin et al., 1998) and visualized graphically in MACAW v.2.0.5. The darkest blue regions indicate the greatest amino acid sequence similarity, and the lightest regions indicate no similarity. Large boxes indicate regions of high similarity between all three forms of eIF4B; small boxes indicate that there is no similarity. Gaps are introduced to maximize the alignments. The regions of α-helices (cylinders) and β-sheets (arrows) were predicted by PSIPRED (Bryson et al., 2005). RNA, eIF4A, eIF(iso)4G, and PABP binding domains were identified by Cheng and Gallie (2006).

Figure 2.

SDS-PAGE and western analysis of eIF4B preparations used in in vitro translation assays. A, Each lane contains approximately 2 μg of protein. Lane 1, Native wheat eIF4B; lane 2, recombinant wheat eIF4B; lane 3, recombinant Arabidopsis eIF4B2; lane 4, recombinant Arabidopsis eIF4B1. Molecular weight markers are as indicated. The gel was stained with Coomassie Brilliant Blue. B, The polyvinylidene fluoride blot was incubated with a 1/1,000 dilution of affinity-purified Arabidopsis eIF4B2 rabbit antibodies overnight at 4°C. A 1/25,000 dilution of goat-anti-rabbit horseradish peroxidase second antibody (Kirkegaard and Perry Laboratories) was incubated for 2 h at room temperature (Browning et al., 1990). The antibody reactive bands were visualized by chemiluminescence (SuperSignal; Pierce) and exposed to film. Lane 1, Native wheat eIF4B (0.9 μg); lane 2, recombinant wheat eIF4B (0.3 μg); lane 3, native Arabidopsis eIF4B (5 μg); lane 4, recombinant Arabidopsis eIF4B2 (0.3 μg); lane 5, recombinant Arabidopsis eIF4B1 (0.3 μg).

Figure 3.

Western-blot analysis of wheat eIF4B elution from Superose 6. Equal amounts (15 μg) of native and recombinant wheat eIF4B (predicted M_r of 56,800) were loaded on the Superose 6 column, and fractions of 50 μL were collected. An aliquot of 20 μL of each fraction was separated by SDS-PAGE, blotted to polyvinylidene fluoride, and incubated with a 1/1,000 dilution of rabbit antisera to wheat eIF4B and further processed as described in Figure 2. A, Native wheat eIF4B fractions. B, Recombinant wheat eIF4B fractions. The peaks of the elution of the M_r standards, bovine γ-globulin (158,000) and chicken ovalbumin (44,000), were in fractions 32 and 38, respectively.

Figure 4.

eIF4B translation assays with wheat eIF4F and wheat eIF(iso)4F. A, Each 100-μL reaction contained 5 pmol of the indicated capped mRNA (barley α-amylase, rabbit β-hemoglobin, or AMV RNA 4) and 7.5 pmol recombinant wheat eIF4F (triangles) or 7.5 pmol recombinant wheat eIF(iso)4F (circles). Various eIF4B preparations were added as indicated. The left panels show native wheat eIF4B (nTa eIF4B) or recombinant wheat eIF4B (rTa eIF4B), and the right panels show recombinant Arabidopsis eIF4B1 (rAt eIF4B1) or recombinant Arabidopsis eIF4B2 (rAt eIF4B2). Each assay was performed in triplicate and the results averaged. The amount of [¹⁴C]Leu incorporated in the absence of additional eIF4B (not subtracted) was as follows: capped barley α-amylase mRNA [eIF4F, 9 pmol; eIF(iso)4F, 5 pmol]; capped rabbit β-hemoglobin [eIF4F, 33 pmol; eIF(iso)4F, 22 pmol]; capped AMV RNA 4 [eIF4F, 70 pmol; eIF(iso)4F, 87 pmol]. B, Each 100-μL reaction contained 5 pmol of the indicated capped mRNA (AtHSP21, maize ADH, wheat germin, or oat globulin) and 7.5 pmol recombinant wheat eIF4F (triangles) or 7.5 pmol recombinant wheat eIF(iso)4F (circles). Recombinant wheat eIF4B was added as indicated. The amount of [¹⁴C]Leu incorporated in the absence of additional eIF4B (not subtracted) was as follows: capped AtHSP21 [eIF4F, 32 pmol; eIF(iso)4F, 34 pmol]; capped maize ADH [eIF4F, 3 pmol; eIF(iso)4F, 4 pmol]; capped wheat germin [eIF4F, 34 pmol; eIF(iso)4F, 36 pmol]; capped oat globulin [eIF4F, 8 pmol; eIF(iso)4F, 9 pmol].

Figure 5.

Purified wheat eIF(iso)4F and Arabidopsis eIF(iso)4F1 and eIF(iso)4F2. SDS-PAGE (12.5%) of purified complexes. Lane 1, 2.8 μg of native wheat eIF(iso)4F; lane 2, 2.8 μg of recombinant wheat eIF(iso)4F; lane 3, 2.8 μg of recombinant Arabidopsis eIF(iso)4F1; and lane 4, 2.8 μg of recombinant Arabidopsis eIF(iso)4F2. The gel was stained with Coomassie Brilliant Blue.

Figure 6.

Translation assay with wheat eIF4B and wheat eIF(iso)4F, AteIF(iso)4F1, or AteIF(iso)4F2. Each 100-μL reaction contained 5 pmol of capped AtHSP21 mRNA or uncapped STNV RNA and 10.5 pmol recombinant wheat eIF4B. Recombinant wheat (iso)4F (circles), recombinant AteIF(iso)4F1 (triangles), or recombinant AteIF(iso)4F2 (squares) was added as indicated. Each assay was performed in triplicate and the results averaged. The amount of [¹⁴C]Leu incorporated in the absence of additional elF(iso)4F (not subtracted) was as follows: 7.1 pmol, AtHSP21; 8.7 pmol, STNV RNA.

Figure 7.

AteIF4B translation assays with AteIF(iso)4F1 and eIF(iso)4F2. Each 100-μL reaction contained 5 pmol of the indicated mRNA (capped AtHSP21 or uncapped STNV RNA) and 7.7 pmol recombinant AteIF(iso)4F1 or AteIF(iso)4F2. AteIF4B preparations were added as indicated, recombinant Arabidopsis eIF4B1(circles) or recombinant Arabidopsis eIF4B2 (triangles). Each assay was performed in triplicate and the results averaged. The amount of [¹⁴C]Leu incorporated in the absence of additional eIF4B (not subtracted) was as follows: AtHSP21, 29 pmol for eIF(iso)4F1 and 14.3 pmol for eIF(iso)4F2; STNV RNA, 10.3 pmol for eIF(iso)4F1 and 9.3 pmol for eIF(iso)4F2.