Abstract
One of the most regulated steps of translation initiation is the recruitment of mRNA by the translation machinery. In eukaryotes, this step is mediated by the 5′end cap-binding factor eIF4E bound to the bridge protein eIF4G and forming the eIF4F complex. In plants, different isoforms of eIF4E and eIF4G form the antigenically distinct eIF4F and eIF(iso)4F complexes proposed to mediate selective translation. Using a microarray analysis of polyribosome- and non-polyribosome-purified mRNAs from 15 day-old Arabidopsis thaliana wild type [WT] and eIF(iso)4E knockout mutant [(iso)4E-1] seedlings we found 79 transcripts shifted from polyribosomes toward non-polyribosomes, and 47 mRNAs with the opposite behavior in the knockout mutant. The translationally decreased mRNAs were overrepresented in root-preferentially expressed genes and proteins from the endomembrane system, including several transporters such as the phosphate transporter PHOSPHATE1 (PHO1), Sucrose transporter 3 (SUC3), ABC transporter-like with ATPase activity (MRP11) and five electron transporters, as well as signal transduction-, protein modification- and transcription-related proteins. Under normal growth conditions, eIF(iso)4E expression under the constitutive promoter 35 S enhanced the polyribosomal recruitment of PHO1 supporting its translational preference for eIF(iso)4E. Furthermore, under phosphate deficiency, the PHO1 protein increased in the eIF(iso)4E overexpressing plants and decreased in the knockout mutant as compared to wild type. In addition, the knockout mutant had larger root, whereas the 35 S directed expression of eIF(iso)4E caused shorter root under normal growth conditions, but not under phosphate deficiency. These results indicate that selective translation mediated by eIF(iso)4E is relevant for Arabidopsis root development under normal growth conditions.
Highlights
Translation initiation factor eIF4E binds to the cap structure (7mGpppN, where N is any nucleotide) present at the 59end of most eukaryotic mRNAs
The PHO1 protein levels were decreased in4E-1 and increased in an eIF(iso)4E overexpressing transgenic line as compared to wild type (WT). These results suggest that the presence of both isoforms, eIF4E and eIF(iso)4E, in a particular proportion is required for selective translation during normal plant growth, as well as in the phosphate limitation stress
We generated three Arabidopsis transgenic lines,4E-2, -3, and -4, expressing eIF(iso)4E fused to a GFP tag at the carboxy terminus in the Col-0 WT background (Figure S1, panels A, B)
Summary
Translation initiation factor eIF4E binds to the cap structure (7mGpppN, where N is any nucleotide) present at the 59end of most eukaryotic mRNAs. EIF4G interacts with the multi-subunit complex eIF3 bringing together the mRNA and the 43 S initiation complex formed by eIF3, the ternary complex (eIF2-MettRNAMet-GTP), the 40 S ribosomal subunit and other initiation factors [1] It recruits the RNA helicase eIF4A to unwind secondary structures in the 59 untranslated region (59UTR) of the mRNA, and the poly(A) binding protein (PABP) allowing the mRNA circularization for efficient translation re-initiation. The interaction between the cap and the translational machinery may be prevented by the binding of eIF4E to other cellular proteins through the same domain used for its interaction with eIF4G By such means cells could modulate either the global translation levels, or specific mRNA recruitment [8]
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