Date of Award
Doctor of Philosophy
Biochemistry and Cellular and Molecular Biology
Albrecht G. von Arnim
Brad M. Binder, Elizabeth M. Fozo, Mariano Labrador, Daniel M. Roberts
Translational regulation in eukaryotic cells is a key process required to maintain protein levels under various environmental conditions to regulate growth and development. One way translation can be regulated is by chemically modifying ribosomal proteins and translation factors. Ribosomal protein of small subunit 6 (RPS6) is a part of the eukaryotic 40S subunit. It is highly conserved among all eukaryotes and has been shown to undergo phosphorylation at multiple sites in its carboxy-terminal tail. The phosphorylation is regulated in response to various environmental stresses such as the level of sucrose, cold, heat shock and hypoxia and also light conditions. While RPS6 phosphorylation (RPS6-P) is conserved in several eukaryotes, its functional significance is still unclear. Recent studies in mammals and yeast have provided evidence that RPS6-P plays a role in regulating cell size, translation and ribosome biogenesis, however, very little is known about its role in plants. To better understand the potential roles of RPS6-P in plants, I investigated the regulation of RPS6-P by light and the circadian clock, as well as the role of the phosphorylation, using seedlings of the plant model species Arabidopsis thaliana. Using phospho-specific antibodies, I show that RPS6-P cycles in a diel fashion with a day peak in both wild-type and a clock-deficient strain under long-day conditions. In contrast, under continuous light, the clock drives RPS6-P with a night peak. Therefore, RPS6-P cycles are regulated jointly by light signals and the circadian clock. RPS6-P is dependent on light given that the phosphorylation is dramatically reduced and acyclic in continuous darkness. Sucrose can induce RPS6-P independent of light, and under high concentrations, sucrose also disrupts the RPS6-P cycles. I also show that RPS6 is phosphorylated in a polysomal context and the phosphorylation level correlates with the polysome level. To address the role of RPS6-P in Arabidopsis, using RPS6 knockout plants, I show that both paralogs RPS6A and RPS6B are functionally equivalent, and they are both phosphorylated in polysomes to a similar extent. I also generated transgenic lines expressing non-phosphorylatable or phospho-mimic versions of RPS6. The transgenic plants were then analyzed for growth and molecular phenotypes.
Enganti, Ramya, "Characterizing the regulation and the role of ribosomal protein S6 phosphorylation in Arabidopsis thaliana. " PhD diss., University of Tennessee, 2018.
Available for download on Friday, May 15, 2020