M.Sc Thesis

M.Sc StudentDiminsky Yaakov
SubjectQuantifying the Role of the Translation Initiation Factors
eIF4HI and eIF4HII in mRNA Unwinding and their
Relationship to Cancer Development
DepartmentDepartment of Biomedical Engineering
Supervisor PROF. Amit Meller
Full Thesis textFull thesis text - English Version


Regulation of protein synthesis plays a critical role in many cellular processes. Translation consists of four steps: initiation, elongation, termination and ribosome recycling. Translation initiation (TI) is considered to be the rate-limiting step of protein synthesis, in which most regulation takes place. During TI, the eIF4F complex associates to promote ribosome assembly on the mRNA. The eIF4AI helicase (a core member of the eIF4F complex and a DEAD-box RNA helicase) unwinds secondary structures at the 5’ un-translated region (5' UTR) of mRNAs, thereby facilitating ribosomal scanning for the start codon. Two accessory proteins eIF4B and eIF4H are known to stimulate eIF4AI’s activity. In mammalian cells there are two eIF4H isoforms, which share 91% identity. Both eIF4H isoforms exhibit an RNA recognition motif (RRM) through which they bind RNA. Upon RNA-binding, eIF4HII recruits and stabilizes the closed state of eIF4AI, thereby prolonging its residence time on the mRNA. Little is known about eIF4HI activity in vitro. Despite their similarity, recent studies suggest that the two eIF4H isoforms may be functionally distinct in vivo. Abnormality in eIF4H is linked to multiple diseases, such as predisposition to infections by Herpes simplex virus-1 (HSV-1), Williams-Beuren syndrome (WBS) and cancer. Interestingly, eIF4HI over-expression specifically, but not eIF4HII, is found in colorectal and esophageal cancers. Previous studies suggest that the expression of both eIF4H isoforms is elevated as a result of NF-kB activation. This may indicate that eIF4HI over-expression stimulates pro-survival mechanisms. In this study, we show that the RNA unwinding activity of eIF4AI in vitro is differentially stimulated by the two eIF4H isoforms. Specifically, using a fluorescence-based activity assay, we quantitatively measured the unwinding kinetics of RNA duplexes by eIF4AI in the presence of each eIF4H isoform. We found that eIF4HII elevates the unwinding activity of eIF4AI by roughly 50% more than eIF4HI. By using various concentrations and models of protein kinetics we created a titration curve, which enables better understanding of the proteins’ behavior. Importantly, the addition of loop structures, which originate from the 5 'UTR of MYCBP sequence, to the RNA unwinding reaction resulted in different modes of reaction inhibition. Hence, pointing to distinct RNA binding affinities of each eIF4H isoform.