A synthetic genetic array screen for interactions with the RNA helicase DED1 during cell stress in budding yeast.

During cellular stress it is essential for cells to alter their gene expression to adapt and survive. Gene expression is regulated at multiple levels, but translation regulation is both a method for rapid changes to the proteome and, as one of the most energy-intensive cellular processes, a way to efficiently redirect cellular resources during stress conditions. Despite this ideal positioning, many of the specifics of how translation is regulated, positively or negatively, during various types of cellular stress remain poorly understood. To further assess this regulation, we examined the essential translation factor Ded1, an RNA helicase that has been previously shown to play important roles in the translational response to cellular stress. In particular, ded1 mutants display an increased resistance to growth inhibition and translation repression induced by the TOR pathway inhibitor, rapamycin, suggesting that normal stress responses are partially defective in these mutants. To gain further insight into Ded1 translational regulation during stress, synthetic genetic array analysis was conducted in the presence of rapamycin with a ded1 mutant and a library of nonessential genes in Saccharomyces cerevisiae to identify positive and negative genetic interactions in an unbiased manner. Here, we report the results of this screen and subsequent network mapping and Gene Ontology-term analysis. Hundreds of candidate interactions were identified, which fell into expected categories, such as ribosomal proteins and amino acid biosynthesis, as well as unexpected ones, including membrane trafficking, sporulation, and protein glycosylation. Therefore, these results provide several specific directions for further comprehensive studies.

Translational control by helicases during cellular stress.

Stress is inevitable, so all organisms have developed response mechanisms to allow for their survival during times of stress. Regulation of gene expression is a critical part of these responses, which allows for the appropriate cohort of proteins to be produced to counter the stress while downregulating others in order to conserve resources. Translation is both highly energy intensive and able to rapidly shift the proteome, thus making it a key target for regulation during stress. Numerous stress pathways converge on translation, and examining the regulatory mechanisms that underlie these pathways is essential for understanding the initial and long-term effects of stress on cells. A number of RNA helicases, including eIF4A, Ded1/DDX3X, and Dhh1/DDX6, have been previously linked to translation, and given their ability to dramatically alter RNA-protein interactions, they are well-positioned to play critical roles in translation regulation during stress. Therefore, assessing the role of helicases in these conditions is vital to the overall understanding of stress. Outlined below are key assays focusing on two areas: assessing cellular phenotypes in growth and survival during stress conditions, and analyzing cellular translation in the presence and absence of stress. The combination of these two approaches will begin to establish the function(s) of a given helicase in the overall stress response.

TDP-43 Proteinopathy Causes Broad Metabolic Alterations including TCA Cycle Intermediates and Dopamine Levels in Drosophila Models of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal, complex neurodegenerative disorder that causes selective degeneration of motor neurons. ALS patients exhibit symptoms consistent with altered cellular energetics such as hypermetabolism, weight loss, dyslipidemia, insulin resistance, and altered glucose tolerance. Although evidence supports metabolic changes in ALS patients, metabolic alterations at a cellular level remain poorly understood. Here, we used a Drosophila model of ALS based on TDP-43 expression in motor neurons that recapitulates hallmark features of motor neuron disease including TDP-43 aggregation, locomotor dysfunction, and reduced lifespan. To gain insights into metabolic changes caused by TDP-43, we performed global metabolomic profiling in larvae expressing TDP-43 (WT or ALS associated mutant variant, G298S) and identified significant alterations in several metabolic pathways. Here, we report alterations in multiple metabolic pathways and highlight upregulation of Tricarboxylic acid (TCA) cycle metabolites and defects in neurotransmitter levels. We also show that modulating TCA cycle flux either genetically or by dietary intervention mitigates TDP-43-dependent locomotor defects. In addition, dopamine levels are significantly reduced in the context of TDP-43G298S, and we find that treatment with pramipexole, a dopamine agonist, improves locomotor function in vivo in Drosophila models of TDP-43 proteinopathy.