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DEAD-box RNA helicases regulate each stage of the RNA life-cycle during gene expression. Ded1 is an essential yeast DEAD-box protein that regulates translation initiation through its effects on mRNA secondary structure and formation of pre-initiation complexes. Ded1 binding to mRNA is not sequence specific, and therefore, it relies on interaction partners for its specificity and regulatory activities during initiation. Stress conditions require large-scale changes in translation that upregulate certain stress response genes but repress most other nonstress-related genes. The target-of-rapamycin (TOR) pathway is a major regulator of these changes, and we have found that Ded1 is a critical mediator of this stress response. Interestingly, in contrast to its role in promoting translation initiation in pro-growth conditions, Ded1 plays an active role in repressing translation upon TOR inactivation. My work focuses on further characterizing the currently unknown interactions critical for Ded1’s repressive function during cellular stress. My results support a physical interaction between Ded1 and Cdc28 in stress conditions that is absent in normal growth conditions, and follow-up results suggest that this interaction may help to coordinate the cell cycle and translation during stress. Along with this clear connection to Cdc28, I conducted a large-scale screen that also shows connections of Ded1 with ATP transport, stress granule formation, cellular localization, cellular trafficking, and mitochondrial translation. All of these could play a key role in understanding how Ded1 fits into the larger picture of translational regulation during TOR inactivation and each subset seen in the annotated GO terms could be an individual area of study for future understanding of stress responses and translation.

The DEAD-Box RNA helicase family is a conserved group of enzymes that function in gene expression through ATP-dependent RNA unwinding and ribonucleoprotein (RNP) remodeling. DEAD-Box helicases function in multiple cellular processes, including pre-mRNA processing, translation, mRNA export, and mRNA decay. Although DEAD-Box proteins are critical for gene expression, much of their mechanistic activities are poorly understood. DEAD-Box proteins have increasingly been linked to tumorigenesis in humans, and better defining their activity at the mechanistic level will aid in understanding the underlying disease pathology. In this study, we used the model organism Saccharomyces cerevisiae to study the human DEAD-Box protein DDX3 orthologue, Ded1, and its role in translation initiation during cellular stress. Recently, we have found that Ded1 is an important mediator of the cellular stress response in a TOR-dependent manner. TOR regulates protein synthesis dependent on energy availability in the cell by regulating the assembly of the eukaryotic translation initiation complex. Human DDX3 has been found to interact with translation initiation complex subunit eIF4E and Ded1 has been found to interact with the translation initiation complex subunit eIF4G. In this study, we examined the purported interaction region between Ded1 and eIF4G on the C-terminus of Ded1 and found that ded1 Δ591-604 prevents eIF4G degradation under rapamycin treatment and confers resistance to rapamycin-induced growth inhibition. We also examined putative regulatory phosphorylation sites in the purported Ded1 eIF4G binding region. We propose that the Ded1/eIF4G interaction is critical for the repression of translation by Ded1 and that eIF4G degradation may be regulated by Ded1 under stress conditions.