Caster, Stephen Z (2016-08). The Stress Response and Circadian Regulation of Translation in Neurospora crassa. Doctoral Dissertation. Thesis uri icon

abstract

  • Stress response pathways function to allow cells to adapt to changes in the environment. In Neurospora crassa, acute osmotic stress activates the conserved p38-like osmosensing mitogen-activated protein kinase (OS MAPK) pathway. When activated, the terminal MAPK, OS-2 can activate transcription factors and kinases. We show an acute osmotic stress activates OS-2, which phosphorylates and activates the conserved kinase RCK-2. RCK-2 phosphorylates and inactivates the highly conserved eukaryotic elongation factor 2 (eEF-2). To determine if this is a mechanism for translational regulation of mRNAs, I examined ribosome profiling and RNAseq data from osmotically stressed WT and ?rck-2 cultures. I found that RCK-2/eEF-2 regulate 69 constitutively expressed mRNAs at the level of translation. I also examined ribosome profiling and RNAseq data from cultures given light exposure, and found that 36 constitutively expressed mRNAs were regulated at the level of translation. In both cases, the translationally-controlled genes were enriched for metabolic processes, suggesting that rapid regulation of metabolism through translational control helps the organism overcome osmotic and light stress. The circadian clock has a profound effect on gene regulation; however, little is known about the role of the clock in controlling translation. I show that clock signaling through the OS MAPK pathway promotes rhythmic phosphorylation of RCK-2 and eEF-2 in constant conditions. Using a cell-free translation assay, I demonstrated that clock signaling to eEF-2 leads to rhythmic control of mRNA translation. To determine the extent of clock regulation of translation in vivo, I examined ribosome profiling and RNAseq data from WT cultures over a circadian time course. 637 Neurospora mRNAs showed rhythmic ribosome occupancy, and 549 of these were from constitutively expressed mRNAs. To determine which of these translationally cycling mRNAs required RCK-2/eEF-2 regulation, I examined ribosome profiling and RNAseq data from ?rck-2 cultures over a circadian time course. I found 419 of the constitutive mRNAs with cycling ribosomal occupancy required clock regulation of RCK-2/eEF-2. While the regulation of initiation was thought to be the main control point of translation, these data revealed a major role for eEF-2 activity and elongation in translation control following stress and by the circadian clock.
  • Stress response pathways function to allow cells to adapt to changes in the environment. In Neurospora crassa, acute osmotic stress activates the conserved p38-like osmosensing mitogen-activated protein kinase (OS MAPK) pathway. When activated, the terminal MAPK, OS-2 can activate transcription factors and kinases. We show an acute osmotic stress activates OS-2, which phosphorylates and activates the conserved kinase RCK-2. RCK-2 phosphorylates and inactivates the highly conserved eukaryotic elongation factor 2 (eEF-2). To determine if this is a mechanism for translational regulation of mRNAs, I examined ribosome profiling and RNAseq data from osmotically stressed WT and ?rck-2 cultures. I found that RCK-2/eEF-2 regulate 69 constitutively expressed mRNAs at the level of translation. I also examined ribosome profiling and RNAseq data from cultures given light exposure, and found that 36 constitutively expressed mRNAs were regulated at the level of translation. In both cases, the translationally-controlled genes were enriched for metabolic processes, suggesting that rapid regulation of metabolism through translational control helps the organism overcome osmotic and light stress.

    The circadian clock has a profound effect on gene regulation; however, little is known about the role of the clock in controlling translation. I show that clock signaling through the OS MAPK pathway promotes rhythmic phosphorylation of RCK-2 and eEF-2 in constant conditions. Using a cell-free translation assay, I demonstrated that clock signaling to eEF-2 leads to rhythmic control of mRNA translation.

    To determine the extent of clock regulation of translation in vivo, I examined ribosome profiling and RNAseq data from WT cultures over a circadian time course. 637 Neurospora mRNAs showed rhythmic ribosome occupancy, and 549 of these were from constitutively expressed mRNAs. To determine which of these translationally cycling mRNAs required RCK-2/eEF-2 regulation, I examined ribosome profiling and RNAseq data from ?rck-2 cultures over a circadian time course. I found 419 of the constitutive mRNAs with cycling ribosomal occupancy required clock regulation of RCK-2/eEF-2. While the regulation of initiation was thought to be the main control point of translation, these data revealed a major role for eEF-2 activity and elongation in translation control following stress and by the circadian clock.

publication date

  • August 2016