eIF4A RNA Helicase Associates with Cyclin-Dependent Protein Kinase A in Proliferating Cells and is Modulated by Phosphorylation

Eukaryotic initiation factor 4A (eIF4A) is a highly conserved RNA-stimulated ATPase and helicase involved in the initiation of messenger RNA translation. Previously, we found that eIF4A interacts with cyclin-dependent kinase A (CDKA), the plant ortholog of mammalian CDK1. Here, we show that this interaction occurs only in proliferating cells where the two proteins coassociate with 5′-cap-binding protein complexes, eIF4F or the plant-specific eIFiso4F. CDKA phosphorylates eIF4A on a conserved threonine residue (threonine-164) within the RNA-binding motif 1b TPGR. In vivo, a phospho-null (APGR) variant of the Arabidopsis (Arabidopsis thaliana) eIF4A1 protein retains the ability to functionally complement a mutant (eif4a1) plant line lacking eIF4A1, whereas a phosphomimetic (EPGR) variant fails to complement. The phospho-null variant (APGR) rescues the slow growth rate of roots and rosettes, together with the ovule-abortion and late-flowering phenotypes. In vitro, wild-type recombinant eIF4A1 and its phospho-null variant both support translation in cell-free wheat germ extracts dependent upon eIF4A, but the phosphomimetic variant does not support translation and also was deficient in ATP hydrolysis and helicase activity. These observations suggest a mechanism whereby CDK phosphorylation has the potential to down-regulate eIF4A activity and thereby affect translation

EIFiso4G augments the synthesis of specific plant proteins involved in normal chloroplast function

Copyright © 2019 American Society of Plant Biologists. All rights reserved. The plant-specific translation initiation complex eIFiso4F is encoded by three genes in Arabidopsis (Arabidopsis thaliana)-genes encoding the cap binding protein eIFiso4E (eifiso4e) and two isoforms of the large subunit scaffolding protein eIFiso4G (i4g1 and i4g2). To quantitate phenotypic changes, a phenomics platform was used to grow wild-type and mutant plants (i4g1, i4g2, i4e, i4g1 × i4g2, and i4g1 × i4g2 × i4e [i4f]) under various light conditions. Mutants lacking both eIFiso4G isoforms showed the most obvious phenotypic differences from the wild type. Two-dimensional differential gel electrophoresis and mass spectrometry were used to identify changes in protein levels in plants lacking eIFiso4G. Four of the proteins identified as measurably decreased and validated by immunoblot analysis were two light harvesting complex binding proteins 1 and 3, Rubisco activase, and carbonic anhydrase. The observed decreased levels for these proteins were not the direct result of decreased transcription or protein instability. Chlorophyll fluorescence induction experiments indicated altered quinone reduction kinetics for the double and triple mutant plants with significant differences observed for absorbance, trapping, and electron transport. Transmission electron microscopy analysis of the chloroplasts in mutant plants showed impaired grana stacking and increased accumulation of starch granules consistent with some chloroplast proteins being decreased. Rescue of the i4g1 × i4g2 plant growth phenotype and increased expression of the validated proteins to wild-type levels was obtained by overexpression of eIFiso4G1. These data suggest a direct and specialized role for eIFiso4G in the synthesis of a subset of plant proteins

A Unique 5\u27 Translation Element Discovered in Triticum Mosaic Virus

Several plant viruses encode elements at the 5\u27 end of their RNAs, which, unlike most cellular mRNAs, can initiate translation in the absence of a 5\u27 m7GpppG cap. Here, we describe an exceptionally long (739-nucleotide [nt]) leader sequence in triticum mosaic virus (TriMV), a recently emerged wheat pathogen that belongs to the Potyviridae family of positive-strand RNA viruses. We demonstrate that the TriMV 5\u27 leader drives strong cap-independent translation in both wheat germ extract and oat protoplasts through a novel, noncanonical translation mechanism. Translation preferentially initiates at the 13th start codon within the leader sequence independently of eIF4E but involves eIF4G. We truncated the 5= leader to a 300-nucleotide sequence that drives cap-independent translation from the 5\u27 end. We show that within this sequence, translation activity relies on a stem-loop structure identified at nucleotide positions 469 to 490. The disruption of the stem significantly impairs the function of the 5\u27 untranslated region (UTR) in driving translation and competing against a capped RNA. Additionally, the TriMV 5\u27 UTR can direct translation from an internal position of a bicistronic mRNA, and unlike cap-driven translation, it is unimpaired when the 5\u27 end is blocked by a strong hairpin in a monocistronic reporter. However, the disruption of the identified stem structure eliminates such a translational advantage. Our results reveal a potent and uniquely controlled translation enhancer that may provide new insights into mechanisms of plant virus translational regulation

Evidence for Variation in the Optimal Translation Initiation Complex: Plant eIF4B, eIF4F, and eIF(iso)4F Differentially Promote Translation of mRNAs1[OA]

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) α-amylase mRNA, rabbit β-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 β-hemoglobin mRNA and wheat germin mRNA showed modest dependence. Barley α-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 α-amylase, oat globulin, and rabbit β-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

The <i>Triticum Mosaic Virus</i> 5’ Leader Binds to Both eIF4G and eIFiso4G for Translation

<div><p>We recently identified a remarkably strong (739 nt-long) IRES-like element in the 5’ untranslated region (UTR) of <i>Triticum mosaic virus</i> (TriMV, <i>Potyviridae</i>). Here, we define the components of the cap-binding translation initiation complex that are required for TriMV translation. Using bio-layer interferometry and affinity capture of the native translation apparatus, we reveal that the viral translation element has a ten-fold greater affinity for the large subunit eIF4G/eIFiso4G than to the cap binding protein eIF4E/eIFiso4E. This data supports a translation mechanism that is largely dependent on eIF4G and its isoform. The binding of both scaffold isoforms requires an eight base-pair-long hairpin structure located 270 nucleotides upstream of the translation initiation site, which we have previously shown to be crucial for IRES activity. Despite a weak binding affinity to the mRNA, eIFiso4G alone or in combination with eIFiso4E supports TriMV translation in a cap-binding factor-depleted wheat germ extract. Notably, TriMV 5’ UTR-mediated translation is dependent upon eIF4A helicase activity, as the addition of the eIF4A inhibitor hippuristanol inhibits 5’ UTR-mediated translation. This inhibition is reversible with the addition of recombinant wheat eIF4A. These results and previous observations demonstrate a key role of eIF4G and eIF4A in this unique mechanism of cap-independent-translation. This work provides new insights into the lesser studied translation mechanisms of plant virus-mediated internal translation initiation.</p></div

TriMV requires eIF4A helicase activity for its translation.

<p>A) Relative luciferase activity in wheat germ extract of the TriMV 5’ UTR and m7GpppG-capped control polyadenylated mRNAs with increasing concentrations of the eIF4A-inhibiting drug hippuristanol, suspended in 5% DMSO and added to the translation reaction. The luciferase activities were standardized to the measured luciferase activities of each mRNA with no hippuristanol added. At the 0 μM concentration of hippuristanol, equal volume of 5% DMSO alone also was added to the reaction. B) Translation inhibition by 10 μM hippuristanol can be reversed by adding 15 μM recombinant wheat eIF4A protein.</p

eIFiso4F can support TriMV 5’ UTR-mediated translation.

<p>A) Translation of ApppG-capped polyadenylated TriMV RNA in wheat germ extract depleted of cap-binding factors with increasing concentrations (0 to 10 pmol) of the wheat eIFiso4F complex or its individual subunits (eIFiso4G or eIFiso4E). The assay was performed with ¹⁴C-radiolabeled leucine, and the results display the incorporation of radiolabeled leucine (picomoles) in newly synthesized proteins following TCA protein precipitation. B) Western blot against the purified recombinant wheat eIF4G or eIFiso4G proteins individually eluated from the biotinylated TriMV RNAs and controls following UV-crosslink. Unbound proteins obtained in the flow-through and washes are shown. 0.25 pmol of each of the corresponding recombinant proteins was loaded in the first lane to serve as a ladder.</p

The TriMV 5’UTR RNA binds to eIF4G and eIFiso4G with greater affinity than the cap binding proteins.

<p>Bio-layer interferometry (BLI) was used to measure the binding kinetics between purified recombinant wheat eIF4G, eIFiso4G, eIF4E, or eIFiso4E protein and the viral 5’ UTR sequence (nt 1–739). Biotin-labeled RNA was bound to a streptavidin biosensor and applied to solutions containing different concentrations of proteins. A) Relative luciferase activity in wheat germ of a m7GpppG-capped and polyadenylated vector reporter in the presence of competing free RNAs in increasing molar excess A 0- to 20-fold molar excess of the competing free RNAs corresponding to the biotin-UTP labeled and unlabeled TriMV 5’UTR sequence (1–739) and the non-functional TriMV reverse sequence (739–1) were used. In B and C are shown the BLI sensograms revealing the binding curves for eIF4G/eIF4E (B) and eIFiso4G/eIFiso4E (C). A magnification of the binding curves for eIF4E and eIFiso4E is included. A table with their corresponding kinetics values is displayed below the appropriate graphs.</p