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Date of Award
4-2022
Document Type
Restricted Thesis: Campus only access
Degree Name
Bachelor of Science
Department
Biochemistry & Molecular Biol.
First Advisor
Angie Hilliker
Abstract
Ded1 is a DEAD-box, ATP-dependent RNA helicase that helps initiate translation through its interactions with the eIF4F complex in Saccharomyces cerevisiae. Ded1 is necessary to resolve heavily structured 5’-UTRs of certain mRNAs to recruit the 43S ribosomal subunit to the start codon where the large ribosomal subunit then binds, and translation can begin. Ded1’s other function is to unwind any secondary structure within the 5’-UTR so the 43S subunit is not impeded on its way to the start codon. Ded1 has five arginine residues in RGG motifs that are capable of being methylated by Hmt1 (Hamey et al., 2021; Erce et al., 2013), a methyltransferase. Two of the methylatable arginine residues are found in Ded1’s C-terminal domain, which is the same region where Ded1 interacts with other monomers to trimerize (Putnam et al., 2015) and/or bind with eIF4G (Hilliker et al., 2011). Three methylation sites are found in Ded1’s N- terminal domain where Ded1 is known to bind with eIF4A and eIF4E (Gulay et al., 2020). We suspect arginine methylation may influence Ded1’s binding affinity for the translational initiation factors and serve as a mechanism of translation regulation. Co- affinity immunoprecipitation chromatography and western blotting were performed to test the influence of methylation on Ded1’s protein-protein interactions. I found that eIF4E-S-tag does not interact with 6xHis-Ded1, an unexpected result given they were shown to directly interact previously (Gulay et al., 2020; Senissar et al., 2014). I also found that Ded1 directly interacts with Dbp1, another DEAD-box ATP-dependent RNA helicase that is a paralog of Ded1; this is the first time this interaction has been observed. Future directions include testing the influence of methylation on Ded1’s ability to trimerize and on its interactions with Pab1, Xrn1, and eIF4G, as well as testing methylation’s influence on Ded1’s binding with the eIF4G-eIF4E complex.
Recommended Citation
Cook, Andrew, "Testing the Influence of Arginine Methylation on Ded1’s Protein-Protein Interactions" (2022). Honors Theses. 1629.
https://scholarship.richmond.edu/honors-theses/1629