Molecular switches regulating stress granule oscillation.
Stress granules (SGs) are cytosolic droplets that separate from the cytosol by phase separation in response to various stress conditions as a result of translation suppression. We discovered that chronic infection with hepatitis C virus (HCV) leads to oscillating SG formation with ON and OFF phases of protein synthesis, which are regulated at the level of the eukaryotic initiation factor 2 alpha (eIF2α) by the antagonistic action of two main switches, the double-stranded (ds) RNA-activated Protein Kinase R (PKR) and GADD34, a regulatory subunit of Protein Phosphatase 1. Our goal is to achieve a comprehensive understanding of how such oscil- lating stress responses are established and maintained over long periods of time, and how they serve to ensure cellular survival while restricting viral replication. In the first funding period, we developed a quantitative math- ematical model that describes the stochastic behavior of HCV-induced translational oscillation. The model revealed that a stochastic switch arises when approx. 25% of total eIF2α is phosphorylated, and suggested that mRNA turnover is essential for the dynamic expression of the translation-ON switch GADD34 underlying SG oscillation. We identified an AU-rich element (ARE) in the 3’ untranslated region of GADD34 mRNA that mediates rapid decay of the mRNA, and provide evidence that proteins of the ZFP36 family bind to the GADD34 ARE. We further observed that virus infection and other stresses stabilize GADD34 mRNA and acti- vate both the p38-MAPK and Akt signaling pathways, which are known to control the activity of ZFP36 proteins. In addition, we found that the cell cycle kinase CDK1 inhibits the translation-OFF switch PKR, possibly through release of dsRNA from mitochondria. Finally, we conducted an image-based siRNA screen against all human kinases and phosphatases, which yielded candidate regulators of SG oscillation. In the second funding period, we propose (1) to determine the impact and molecular mechanism by which regulation of mRNA turnover contributes to dynamic expression of GADD34 during translational oscillation. (2) We will explore regulation of PKR mechanistically and determine how endogenous parameters such as proliferative cues and mitochondrial energy production affect the cellular stress response. (3) We will characterize newly identified regulators of SG oscillation through a step-wise systematic procedure. Taken together, the proposed work will establish molecular principles by which oscillating stress responses are achieved and serve as a cellular strategy to mount an effective anti-viral program while maintaining cellular homeostasis.