Identifying the role of the cap-binding complexes in the regulation of translation in Arabidopsis thaliana
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There is a fundamental gap in our understanding of the regulation of translation in plants. None of the canonical eukaryotic methods found in mammals, such as the use of 4E-Binding protein phosphorylation and inhibition of eIF2B function by phosphorylation of eIF2α, have been found in plants to date. In the very early steps of eukaryotic initiation of translation, the cap-binding complex, eIF4F, binds to the m7G of the mRNA and positions it for binding by the 43S preinitiation complex. This complex consists of the large scaffolding protein, eIF4G, and the smaller cap-binding protein, eIF4E, along with the DEAD box helicase eIF4A and the RNA binding protein eIF4B. In addition to eIF4F, plants have a well-conserved second cap-binding complex, eIFiso4F, not found in other eukaryotes and comprised of eIFiso4G and eIFiso4E, isoforms of the scaffolding protein and the cap-binding protein, respectively. Arabidopsis thaliana plants lacking eIFiso4F have stunted growth, reduced chlorophyll, reduced fertility and are more sensitive to light stress. In in vitro translation assays, there is a preference for eIF4F by capped messages. In this work, several possible methods for the regulation of translation in Arabidopsis thaliana by eIF4F and eIFiso4F are investigated. A previously discovered eIF4G wheat kinase known to phosphorylate eIF4G, eIFiso4G, eIF4B and a 60S ribosomal protein is further characterized to determine its possible role in the regulation of translation. It is found to be a calcium dependent kinase that phosphorylates eIFiso4G in the C-terminal domain most likely at a consensus sequence for calcium dependent protein kinases. Additionally, there is a conserved pair of cysteines located in the cap-binding pocket of both eIF4E and eIFiso4E, found only in plants. In an in vitro labeling assay, these cysteines are tested for their thiol reactivity in eIF4E versus eIFiso4E. eIFiso4E is found to be more thiol reactive than eIF4E, possibly indicating differences in redox activity between the two proteins. Finally, two methods for identifying specific populations of mRNA associated with plant protein synthesis initiation factors eIF4F and eIFiso4F are described, RNA immunoprecipitation and ribosome profiling.