|Ph.D Student||Masoud Rula|
|Subject||Quantification of the Initiation Factors eIF4AI|
|Department||Department of Nanoscience and Nanotechnology||Supervisor||Professor Amit Meller|
Eukaryotic translation initiation (TI) is a highly regulated process leading to protein synthesis. It begins with the recruitment of a ribosome to the start codon of the transcript. This process is facilitated by a set of translation factors, collectively called eukaryotic initiation factors (eIFs). eIF4F, the core complex for TI, consists of three proteins: eIF4E, which binds the cap- structure at the 5’-end, eIF4G, a scaffold protein, and eIF4A, a DEAD box RNA helicase.
Secondary structures within the 5'-UTR pose a barrier to TI process. eIF4A, is required to unwind these secondary structures, thereby facilitating ribosome recruitment to mRNA. To date, eIF4A has two known accessory proteins, eIF4H and eIF4B, which stimulate its helicase activity. However, the molecular mechanism of their activity is still unclear.
Mammalian cells express three eIF4A isoforms: eIF4AI, eIF4AII and eIF4AIII. Both, eIF4AI and eIF4AII are cytoplasmic proteins showing ~90% sequence identity, whereas eIF4AIII is nuclear and shares only 65% identity with the other two isoforms. In this research we aim to elucidate the biochemical differences of eIF4AI and eIF4AII. Previous in vitro studies have suggested that eIF4AI and eIF4AII are functionally interchangeable within the eIF4F complex, but recent studies highlighted differences between the two isoforms in live cells.
Understanding eIF4A’s properties is principle for developing strategies to modulate TI and provide novel therapeutic solutions to target faulty regulated translation in cancer cells. Although the components involved in each step in the TI process are known, there is no detailed description of the dynamical bimolecular processes.
With this in mind, we sought to compare eIF4AI and eIF4AII in a quantitative manner to elucidate potential subtle differences between their biochemical activities. Specifically, we measured their dsRNA unwinding kinetics and ATPase activities using fluorescent tools. Since both isoforms show comparable helicase and ATPase activities in vitro, we analyzed the effect that each eIF4B and/or eIF4H, has on these activities. Furthermore, we carried out single-molecule FRET assays to determine the substrate specificity of each eIF4A isoform in the absence and presence of the accessory proteins.
Surprisingly, our results reveal that these two similar isoforms, eIF4AI/II, have different substrate specificity and that their biochemical activities are differently affected by eIF4H/eIF4B. We found that while the lone factors (eIF4AI/eIF4AII) exhibit similar basal RNA unwinding levels, the degree to which each of them could be stimulated by eIF4B and/or eIF4H markedly differed. Moreover, eIF4AI has a very low binding affinity to the RNA substrates, but this binding was substantially increased in the presence of either of eIF4H/eIF4B. In contrast, eIF4AII demonstrated relatively high binding affinity to dsRNA substrates (but not ssRNA), regardless of the presence of eIF4B and/or eIF4H. In addition, we found that eIF4H, but not eIF4B can stimulate the ATPase activity of eIF4AI, while in a case of eIF4AII there was no effect of eIF4H/eIF4B on its ATPase activity. Furthermore, eIF4AI, but not eIF4AII, interacts directly with eIF4H. These results pinpoint significant differences between the two isoforms that are mediated by the presence of the accessory proteins.