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

No abstract

Several plant viruses encode elements at the 5= end of their RNAs, which, unlike most cellular mRNAs, can initiate translation in the absence of a 5= 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= 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= 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= untranslated region (UTR) in driving translation and competing against a capped RNA. Additionally, the TriMV 5= UTR can direct translation from an internal position of a bicistronic mRNA, and unlike cap-driven translation, it is unimpaired when the 5= 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. IMPORTANCEMany members of the Potyviridae family rely on their 5= end for translation. Here, we show that the 739-nucleotide-long triticum mosaic virus 5= leader bears a powerful translation element with features distinct from those described for other plant viruses. Despite the presence of 12 AUG start codons within the TriMV 5= UTR, translation initiates primarily at the 13th AUG codon. The TriMV 5= UTR is capable of driving cap-independent translation in vitro and in vivo, is independent of eIF4E, and can drive internal translation initiation. A hairpin structure at nucleotide positions 469 to 490 is required for the cap-independent translation and internal translation initiation abilities of the element and plays a role in the ability of the TriMV UTR to compete against a capped RNA in vitro. Our results reveal a novel translation enhancer that may provide new insights into the large diversity of plant virus translation mechanisms. T ranslation initiation of most eukaryotic mRNAs occurs by a scanning mechanism, where the 43S ribosomal subunit enters the mRNA from an accessible 5= end, is dependent on the 7-methyl guanosine cap structure (m7GpppG), and scans in a 5=-to-3= direction in search of the initiation codon (1). The ribosomal subunit is recruited to the 5= end by the cap-binding protein factor eIF4E, which is bound to the 5= cap. eIF4E is the small subunit and cap-binding protein in the eIF4F complex. eIF4F is also comprised of the RNA...

bCertain plus-strand RNA plant viruses that are uncapped and nonpolyadenylated rely on RNA elements in their 3= untranslated region, termed 3=-cap-independent translational enhancers (3=CITEs), for efficient translation of their proteins. Here, we have investigated the properties of the Y-shaped class of 3=CITE present in the tombusvirus Carnation Italian ringspot virus (CIRV). While some types of 3=CITE have been found to function through recruitment of translation initiation factors to the viral genome, no trans-acting translation-related factors have yet been identified for the Y-shaped 3=CITE. Our results indicate that the CIRV 3=CITE complexes with eIF4F and eIFiso4F, with the former mediating translation more efficiently than the latter. In nature, some classes of 3=CITE are present in several different viral genera, suggesting that these elements hold a high degree of modularity. Here, we test this concept by engineering chimeric viruses containing heterologous 3=CITEs and show that the Yshaped class of 3=CITE in CIRV can be replaced by two alternative types of 3=CITE, i.e., a Panicum mosaic virus-like 3=CITE or an I-shaped 3=CITE, without any major loss in in vitro translation or replication efficiency in protoplasts. The heterologous 3=CITEs also mediated whole-plant infections of Nicotiana benthamiana, where distinct symptoms were observed for each of the alternative 3=CITEs and 3=CITE evolution occurred during serial passaging. Our results supply new information on Y-shaped 3=CITE function and provide insights into 3=CITE virus-host compatibilities.

Canonical translation initiation in eukaryotes begins with the Eukaryotic Initiation Factor 4F (eIF4F) complex, made up of eIF4E, which recognizes the 7-methylguanosine cap of messenger RNA, and eIF4G, which serves as a scaffold to recruit other translation initiation factors that ultimately assemble the 80S ribosome. Many eukaryotes have secondary EIF4E genes with divergent properties. The model plant Arabidopsis (Arabidopsis thaliana) encodes two such genes in tandem loci on chromosome 1, EIF4E1B (At1g29550) and EIF4E1C (At1g29590). This work identifies EIF4E1B/EIF4E1C-type genes as a Brassicaceae-specific diverged form of EIF4E. There is little evidence for EIF4E1C gene expression; however, the EIF4E1B gene appears to be expressed at low levels in most tissues, though microarray and RNA Sequencing data support enrichment in reproductive tissue. Purified recombinant eIF4E1b and eIF4E1c proteins retain cap-binding ability and form functional complexes in vitro with eIF4G. The eIF4E1b/eIF4E1c-type proteins support translation in yeast (Saccharomyces cerevisiae) but promote translation initiation in vitro at a lower rate compared with eIF4E. Findings from surface plasmon resonance studies indicate that eIF4E1b and eIF4E1c are unlikely to bind eIF4G in vivo when in competition with eIF4E. This study concludes that eIF4E1b/eIF4E1c-type proteins, although bona fide cap-binding proteins, have divergent properties and, based on apparent limited tissue distribution in Arabidopsis, should be considered functionally distinct from the canonical plant eIF4E involved in translation initiation.

Background: Plants have a unique form of cap-binding complex. Results: Correct and mixed complexes show differential translation, and mixed complex subunits have lower binding affinity than correct complex subunits. Conclusion:The subunits of the cap-binding complexes show specificity for complex formation, and the translational efficiency is determined by the large subunit. Significance: The results suggest the potential for differential translation by the two plant cap-binding complexes.

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 59-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 downregulate eIF4A activity and thereby affect translation.

We report the structural analysis of cap-binding proteins using a chemical probe/ultraviolet photodissociation (UVPD) mass spectrometry strategy for evaluating solvent accessibility of proteins. Our methodology utilized a chromogenic probe (NN) to probe the exposed amine residues of wheat eukaryotic translation initiation factor 4E (eIF4E), eIF4E in complex with a fragment of eIF4G ("mini-eIF4F"), eIF4E in complex with full length eIF4G, and the plant specific cap-binding protein, eIFiso4E. Structural changes of eIF4E in the absence and presence of excess dithiothreitol and in complex with a fragment of eIF4G or full-length eIF4G are mapped. The results indicate that there are particular lysine residues whose environment changes in the presence of dithiothreitol or eIF4G, suggesting that changes in the structure of eIF4E are occurring. On the basis of the crystal structure of wheat eIF4E and a constructed homology model of the structure for eIFiso4E, the reactivities of lysines in each protein are rationalized. Our results suggest that chemical probe/UVPD mass spectrometry can successfully predict dynamic structural changes in solution that are consistent with known crystal structures. Our findings reveal that the binding of m(7)GTP to eIF4E and eIFiso4E appears to be dependent on the redox state of a pair of cysteines near the m(7)GTP binding site. In addition, tertiary structural changes of eIF4E initiated by the formation of a complex containing a fragment of eIF4G and eIF4E were observed.